THE UNIVERSITY OF NEW SOUTH WALES FACULTY OF APPLIED SCIENCE SCHOOL OF BIOLOGICAL TECHNOLOGIES DEPARTMENT OF FOOD SCIENCE AND TECHNOLOGY
UTILISATION OF THE AUSTRALIAN JELLYFISH CATOSTYLUS SP. AS A FOOD PRODUCT
Presented as a Thesis for the degree of
Doctor of Philosophy
by
Lucita G. Suelo B.Sc.(VISCA), M.Sc.(UPLB), M.App.Sc.
Submitted
Sydney, October 1986 The candidate, Lucita G. Suelo hereby declares that none of the work presented in this thesis has been submitted to any other University or Institution for a higher degree. i
ACKNOWLEDGEMENT
I wish to express my heartfelt thanks and profound
gratitude to the following persons/entities who have extended
their support while I was doing my postgraduate study:
Dr Michael Wootton, my supervisor, for his guidance,
unlimited help and valuable suggestions during the experimental
and preparation of this thesis;
The Australian Government through the Australian
Development Assistance Bureau (ADAB) for granting the scholarship
under the Colombo Plan and the University of New South Wales which made this study possible;
The Philippine Government through the Panay State
Polytechnic College for granting me a study leave to pursue a postgraduate degree;
The UNSW, Department of Food Science and Technology
staff headed by Prof. R.A. Edwards for their assistance during
the duration of my stay in the University;
The School of Zoology through Dr Macintyre for allowing
the use of the boat for fishing;
Prof. McGilchrist of the School of Mathematics for his assistance in the statistical analysis;
Dr Penny Farrant of the School of Botany for her help in the light microscopy and Sergei Kouprach of the SEM unit for his assistance in freeze-fracture and photoelectron microscopy;
Mrs Rose Varga of the Dept. of Biotechnology for her tutorship in microtechniques and use of microtome;
The Fishing Industry Research Committee for partial financial support of this research; ii
Ms Cherelle Czerney for typing this thesis;
My fellow postgraduate students.at the Department
of Food Science and Technology especially Phaisan for his
help in graphing, Lubis in statistical analysis, Pairat, Made,
Hari, Redi, Kartini, Sutardi, Haryadi, Noryati, Elli and many
others who made my stay a pleasant experience;
My parents, sisters and brothers for their love,
understanding and prayers while I was undertaking this study; and
Libor, for his patience, unlimited help, love and
understanding, this work is humbly dedicated. iii
TABLE OF CONTENTS Page no Acknowledgement i
Table of Contents iii
Abstract ix
1. Introduction 1
2. Literature Review 6
2.1 Zoological Classification of Jellyfish 6 2.1.1 Phylum Cnidaria 6 2.1.2 Taxonomy 8 2.1.3 Semaeostomae and Rhizostomae species found in Australian waters 10 2.1.4 Life cycle 11 2.1.5 Regeneration 13 2.1.6 Ecology/Adaptation 13 2.1. 7 Availability/Distribution 14 2.1.8 Seasonal distribution of jellyfish in Australian waters 18
2.2 Studies on the Processing of Jellyfish 23
2.3 Utilisation as Food 24
2.4 Chemical Properties of Unprocessed Jellyfish 26 2.4.1 Moisture content 26 2.4.2 Protein and amino acids 27 2.4.3 Lipid 34
2.5 Preservative Mechanism of the Traditional Jellyfish Processes 38 2.6 Disposal of Brine for Jellyfish Processing 39
3. Materials and Methods 40
3.1 Materials 40 3.1.1 Jellyfish samples 40
3.2 Methods 41 3.2.1 Processing of jellyfish 41 3.2.1.1 Traditional method 41 3.2.1.2 Shortened method 42 iv
Page 3.2.1.2.1 Brining and drying technique 42 3.2.1.2.2 Blanching and drying technique 42 3.2.2 Analysis of the yield, chemical and organoleptic properties of processed jellyfish 42 3.2.2.1 Yield 42 3.2.2.2 Chemical properties 43 3.2.2.2.1 Moisture content 43 3.2.2.2.2 Water activity 43 3.2.2.2.3 Protein 44 3.2.2.2.4 Fat (acid hydrolysis) 45 3.2.2.2.5 Ash (dry ashing) 46 3.2.2.2.6 pH 46 3.2.2.2.7 Sodium chloride (Volhard method) 46 3.2.2.2.8 Aluminium 48 3.2.2.2.9 Total volatile acidity 49 3.2.2.2.10 Titratable acidity 49 3.2.2.2.11 Total volatile bases (TVB) 49 3.2.2.2.12 Trimethylamine (TMA) 50 3.2.2.2.13 Trimethylamine oxide (TMAO) 50 3.2.2.2.14 TCA soluble nitrogen 51 3.2.3 Organoleptic properties 51 3.2.3.1 Panel evaluation of texture and flavour 52 3.2.3.2 Flavour, texture and overall acceptability evaluation 52 3.2.4 Solubility studies 53 3.2.4.1 Solubility on O.lM potassium chloride 53 3.2.4.2 Solubility on heating in sodium dodecylsulphate + B-mercaptoethanol 53 3.2.4.3 Solubility of protein in sodium chloride solution 53 3.2.4.4 Solubility of protein using enzymes 54 3.2.4.4.1 Pepsin solubility 54 3.2.4.4.2 Trypsin solubility 54 3.2.4.4.3 Pepsin-trypsin solubility 54 3.2.4.5 Solubility of sample in water 54 V
Page 3.2.5 Extractability studies 55 3.2.5.1 Effect of sodium chloride on extractability of protein in jellyfish 55 3.2.5.2 Effect of temperature on extractability of protein in jellyfish 55 3.2.5.3 Effect of pH on protein extraction efficiency 56
3.2.6 Digestibility of jellyfish protein by pepsin 56
3.2.7 Isoelectric focusing 57 3.2.7.1 Materials 57 3.2.7.2 Reagents 58 3.2.7.3 Sample preparation 58 3.2.7.4 Methods 59 3.2.7.4.1 Casting the gel 59 3.2.7.4.2 Moulding 60 3.2.7.4.3 Running the gels 60 3.2.7.4.4 Fixing and staining 61 3.2.7.4.5 Determination of isoelectric point (pi) 61 3.2.7.4.6 Densitometric analysis 62
3.2.8 Amino acid analysis in jellyfish 63 3.2.8.1 Acid hydrolysis 63 3.2.8.2 Sample preparation for chromatography 63 3.2.8.3 Reagent preparation 64 3.2.8.3.1 Ninhydrin 64 3.2.8.3.2 Buffer A (trisodium citrate dehydrate) 65 3.2.8.3.3 Buffer B (trisodium citrate dehydrate) 65
3.2.9 Structural examination of jellyfish tissues 66 3.2.9.1 Light microscopy 66 3.2.9.2 Freeze-fracture technique and electron photomicroscopy 66
3.2.10 Brine microbiological assay 67 vi
Page 3.2.11 Brine recycling 67 3.2.12 Microbial changes during spoilage of fresh jellyfish 68
4. Results and Discussion 69 4.1 Chemical Composition and Properties of Fresh Jellyfish 69 4.1.1 Proximate analysis 69 4.1.2 Total amino acids 71 4.1.3 Isoelectric focusing of proteins 74 4.1.4 Soluble solids, solubility and digestibility of proteins 76 4.2 Preliminary Study of the Traditional Processing Method 76 4.2.1 Effect of alum and salt as preservatives 76 4.2.1.1 Alum alone 78 4.2.1.2 Salt alone 78 4.2.1.3 Alum-salt combination 79 4.2.2 pH of jellyfish and preservatives used during processing 84
4.3 Optimisation of Salt Levels 85 4.3.1 Moisture content, a and weight loss w 86 4.3.2 Protein 87 4.3.3 pH 88 4.3.4 Salt and aluminium levels 88 4.3.5 Organoleptic properties 91 4.3.5.1 Texture 92 4.3.5.2 Flavour 92 4.3.6 Protein in brine 94
4.4 Effect of Processing Time on the Properties and Yield of Jellyfish 96 4.4.1 Changes during the first brining step 97 4.4.2 Changes during the second brining step 97 4.4.3 Drying time 100 4.4.4 Organoleptic evaluation of jellyfish 100 vii
Page
4.5 Approaches for Rapid Processing of Jellyfish 102 4.5.1 Dry salting of jellyfish 103 4.5.2 Artificial drying 103 4.5.3 Brine properties and recycling 104 4.5.4 Artificial drying of unsalted jellyfish 108 4.5.5 Recommended rapid process for jellyfish preservation 109 4.5.6 In vitro digestibility of jellyfish proteins 111
4.6 Solubility Properties of Processed Jellyfish Proteins 112 4.6.1 Protein extractability 113 4.6.1.1 Influence of pH 113 4.6.1.2 Influence of salt concentration 115 4.6.1.3 Influence of temperature 117 4.6.2 Solubilisation by O.lM KCl 119 4.6.3 Solubilisation by SDS + B-mercapto ethanol 119 4.6.4 Solubilisation by pepsin 120 4.6.5 Amino acid composition of processed jellyfish 122 4.6.6 Isoelectric focusing analysis of proteins from processed jellyfish 126 4.6.1.1 Isoelectric focusing analysis of protein from brine 132
4.7 Physical Structure of Jellyfish 135 4.7.1 Fresh jellyfish 136
4.7.1.1 Freeze~fracture and electron microscopy 136 4.7.1.2 Light microscopy of fresh jellyfish 137 4.7.2 Effects of processing on jellyfish tissues 146 4.7.2.1 Effects of alum alone and drying 146 4.7.2.2 Effects of salt alone and drying 149 4.7.2.3 Effects of salt-alum combination and drying 149
4.8 Chemical and Microbial Changes During Storage and Processing of Fresh Jellyfish 152 viii
Page 4.8.1 Fresh jellyfish 155 4.8.1.1 Trimethylamine oxide, trimethylamine, total volatile base and pH 155 4.8.1.2 Total plate count 157 4.8.1.3 TCA soluble nitrogen 159
4.8.2 Changes in TMA and TVB during the rapid processing of jellyfish 160
4.8.3 Total volatile acidity and titratable acidity of processed jellyfish 160 4.8.3.1 Rapid process jellyfish 161 4.8.3.2 Traditional process jellyfish 163
5. Conclusion 165
6. Appendix 168
7. Bibliography 180 ix
ABSTRACT
This study explored the feasibility of utilising the Australian jellyfish Catostylus sp. as food. The major aims of this study were to develop a processing technique that requires less time and labour commitments but produced salted products comparable in chemical and organoleptic properties to existing commercial products and to evaluate the products in terms of chemical, physical and storage qualities.
The traditional 35-day process produced salted jellyfish which were somewhat inferior in organoleptic qualities while the process itself was time and labour intensive. Even though it could be shortened considerably to 10 days, it was still not suitable under Australian conditions because of time and labour costs. A rapid process based on brining in 15% salt and 5% alum for 48 h followed by drying at 30°C for 9-12 h was developed.
This method produced salted products comparable in chemical and organoleptic attributes to those of commercial products. Salt and alum were both necessary to obtain products of desirable structure and texture. Brines produced during rapid processing could be recycled thus reduced preservative~ cost and waste disposal problems. Blanching and drying of untreated jellyfish were not successful techniques for jellyfish processing.
Rapid processing had only slight effects on protein solubility and amino acid content of jellyfish but improved its in vitro digestibility. The product had excellent stability during storage under ambient conditions. This important feature is attributed to a combination of salt content (22.7%), low water activity (0.72) and low pH (2.6). X
Isoelectric points of proteins, determined by isoelectric
focusing, were affected by alum and salt-alum in combination
as preservatives but not by salt alone. Jellyfish proteins,
collagenous in nature as shown by amino acid analysis and solubility
properties, are thus low in essential amino acids.
Fresh jellyfish deteriorated more rapidly under ambient conditions in summer than in winter due to the effects of temperature.
This spoilage of fresh jellyfish is prevented once the product
is treated with salt and alum. Trimethylamine, total volatile
base, trichloroacetic acid soluble nitrogen and total plate count were good indices of spoilage in fresh jellyfish. During production of processed jellyfish, changes in trimethylamine, total volatile , bases and titratable acidity could not be related to quality however, total volatile acidity may be a useful tool. This aspect needs further investigation despite the excellent storage stability of the processed product. 1
1. INTRODUCTION
Jellyfish is a marine organism which is unfamiliar to many as a source of food. It is an invertebrate belonging to Phyhum Cnidaria of which only seven species are reported to be edible. Four of these species are eaten by the Japanese
(Morikawa, 1984) while the Chinese have the large species
Rhizostomae as an important part of their cooking (Omori, 1981).
Jellyfish have been exploited commercially along the coast of
China for more than a thousand years as food and for medicinal purposes (China Pictorial, 1982).
Fishing for jellyfish has been carried out in a number of Asian countries. In Japan, about 1,000 tonnes of raw jellyfish are being caught every year around Ariake Bay, however, no statistical report on domestic production is available. The Japanese are one of the leading consumers of jellyfish, but their domestic production is rather small necessitating importation of salted products from other countries to meet the high consumer demands.
Malaysia leads the jellyfish exporting countries, followed by
China, Indonesia, Burma and the Philippines (Morikawa, 1984).
In 1980, Japanese imports from these countries amounted to more than $US40 million (Omori, 1981). Korea is believed to be another majQiV importer of jellyfish although precise statistics for this country are not available. Other exporting countries are
Hong Kong, South Korea, North Korea and the United States of
America (Morikawa, 1984). In Australia, processed jellyfish is imported from Hong Kong, Malaysia and Taiwan. In May 1986 such products retailed for $A3.25/454g. 2
Jellyfish can be found in almost all waters throughout
the world. Population dynamics of the different species of
jellyfish had been reported in many countries (Table 1))
Table 1. Regions of research into jellyfish population dynamics
Location Authors Date published
Australia Kv.amp 1965
Pope 1949,1953
Azov Sea, USSR Moshina 1974
Baltic Sea Moll er 1980
Barentsea Zenkevitch 1956
Ceylon Panikkar 1944 China Omori 1981
Cuba Krumbach 1925 Florida McGraw 1975 French Polynesia Yasumoto et al. 1980
Georgia Kraeuter and Setzler 1975 Germany Moll er 1980 Gulf of Mexico May er 1910 Hawaii Edmondson 1930
Iceland Kramp 1939 Japan Uehida 1954
Yasuda 1970,1971,1975, 1976
Omori 1981 Maine, Chesapeake Bay Larson 1976 Mid North Atlantic Ocean Winkler & Van Soest 1981 Mississippi Phillips & Burke 1970 3
Location Authors Date published
New England Waters Beatty & Beatty 1974
North Atlantic Ocean Russell 1978
Norway Lid 1979
Ohio (North) Dexte~,Surrarrer & Davis 1949
(Rivers) Beckett & TtiFanchik , 1980
Ohio, Kentucky & Indiana Beitman 1986
Port Arkansas Hoese, Copeland & 1964 Miller
Philippines Mayer 1915,1917
Puerto Rico Larson 1980
Red Sea Schrnidt 1971
Scotland Krumbach 1926
Texas Coast Whitten, Rosene & 1950 Hedgpeth
Gunther 1956
Sirnrnons 1957
Guest 1959
In spite of their wide commercial availability little systematic research and background information has been published on processing and utilisation of jellyfish for human consumption.
China was the first country to produce jellyfish for food with other South-East Asian countries following (Movikawa, 1984).
Japanese eat the manubrium (bell) pickled in alum and served with 4
vinegar (Firth, 1969). The salted products are cut into strips, scalded and served in a dressing composed of oil, soy sauce, vinegar and sugar (Morikawa, 1984).
In Indonesia, a study on the use of salt and alum for processing has been conducted by Iljas and Arifudin (1971).
A similar technique was employed by Wootton, Buckle & Martin
(1982) using the Catostylus species of jellyfish common in Australian waters. Soonthonv.ipat ~976) reported a processing technique used in Thailand which also utilised salt and alum as preservative agents. The Japanese have two different methods of processing salted jellyfish which are also salt-alum based (Monikaw&,l984).
The different techniques mentioned above all utilise salt and alum but vary somewhat in the concentration and method of application of these two materials.
An interest in the processing of jellyfish was motivated by the abundance of this marine animal during certain periods of the year in Australian estuaries and coastal waters. Most ofte~ jellyfish pose a problem to fishermen during trawling.
They clog trawl nets and comprise a great bulk of the catch when in season. They are either returned to the sea or left to die on the shore. According to Moller (1980), jellyfish also clog power plant systems and sting swimmers - but the more significant ecological and economic effect is predation on larval fish and their food resources. Because of these existing situations, the processing and utilisation of jellyfish as food offers an attractive and practical activity. 5
This study is focused on the processing of Catostylus mosaicus, an Australian species of jellyfish for utilisation as food. Specifically, the aims are to:
1. Study the processing techniques to be adopted;
a) evaluate the effects of salt, alum, salt and
alum in combination as preservatives;
b) determine the effects of salt concentration
on the physical, chemical and organoleptic
properties of the finished product;
c) examine the effects of processing time using
traditional methods on the chemical and
organoleptic properties of jellyfish;
d) explore the feasibility of shortening the
processing time involved in traditional
techniques;
e) compare the effects of blanching, curing and
drying on the chemical properties of jellyfish;
2. Evaluate the effects of salting and drying on the physical
structure of jellyfish;
3. Study the proteins and amino acids in jellyfish;
4. Correlate the effects of salt, alum and salt-alum mixture
on muscle structure and protein properties in jellyfish
during processing;
5. Explore the feasibility of recycling brine;
6. Examine spoilage of fresh jellyfishatambient temperatures. 6
2. LITERATURE REVIEW
2.1 Zoological Classification of Jellyfish
2.1.1 Phylum Cnidaria
Jellyfish belongs to the Phylum Cnidaria which also
includes sea anemones and corals. Hydras are among the few fresh water species (Villee, Walker & Barnes, 1978). Cnidarians are world-wide in distribution and occur in an immense variety of
forms and colours. There arre about 10,000 species. The smallest
are microscopic but the largest jellyfish weighs almost a tonne and has tentacles over 30m long. Cnidarians oftenform large
colonies but may live singly (Morris, 1975).
There are three classes in phylum Cnidaria, namely;
Hydrozoa (hydroids and sea firs), Scypho?oa (large jellyfishes), and Anthozoa (sea anemones and corals). These animals all have a low grade of organisation; simpler structures that are rourid only in protozoans, mesozoans and sponges (Bertin, 1973).
Cnidarians are radially symmetrical with the mouth and surrounding tentacles located at one end of the radial axis.
Some Cnidarians are columnar in shape (polypoid form) and live attached to the bottom, with the oral end directed upward.
Only few organs are present and the body wall is composed of two principal layers of cells known as ectoderm and endoderm separated by a layer of non-cellular material, the mesoglea.
The coelenteron (gastro-vascular cavity) is the single internal cavity which has both digestive and circulatory functions. The mouth is its only opening to the exterior and is generally surrounded by tentacles. Both tentacles and general ectoderm bear nematocysts 7
(cnidae or thread capsules) which are used for stinging or capturing prey (Bertin, 1973; Villee et al., 1978).
Most cnidarians are carnivorous and cnidocytes are important in prey capture. In the digestive cavity, digestion is both extracellular and intracellular. The nervous system is commonly in the form of a net with receptor cells dispersed over the body surface. The only cnidarian sense organs are the statocysts and ocelli of jellyfish (Villee et al., 1978).
Cnidarians are either hermaphroditic or the sexes are separate. The gonads are only aggregations of developing gametes, and there are no gonoducts. Fertilisation is usually external and development leads to a free-swimming planula larvae.
Reproduction is usually asexual in the polyp stage and always sexual in the medusa stage (Villee et al., 1978; Morris, 1975).
Scyphozoans are the cnidarians most frequently referred to as jellyfish. In this class, the medusa is the dominant and conspicuous individual in the life cycle. It may range from a few cm to over 1 m in diamter, consisting mostly of jellyfish mesoglea; the polyp stage is minute or lacking. Coloration is often striking. The gonads and other internal structures which may be dee_:p orange, pink, or other colours are visible through the colourless or more delicately tinted bell. The umbrella-shaped body of a jellyfish is fringed with tentacles interrupted by 8 pairs of lappets. Around the mouth are 4 grooved oral arms, edged with nematocyst& Off the gastro-vascular cavity are four pouches containing the gonads, and a system of digestive canals branches through the body to a marginal ring canal. Between 8
each pair of lappets is a sense organ containing a sensitive eyespot, hollow statocysts with limy granules serving for equilibrium, and two sense pits probably aiding in recognition of food (Starer et al., 1968; Barnes, 1980)(Figure 1).
2.1.2 Taxonomy
The following classification of phylum Cnidaria was adapted from Barnes (1980), VQllee et al. (1978); Bertin (1973);
Cockrum and McCauley (1965); Morris (1975) and Grzimek (1974).
Phylum Cnidaria
Class 11 Scyphozoa
Order 1 Stauromedusae
Order 2 Cubomedusae
Order 3 Coronatae
Order 4 Semaeostomae
Order 5 Rhizostomae
In the first order, Stauromedusae are sessile scyphozoans attached by a stalk on the aboTal side of the trumpet-shaped body. These are chiefly found in cold littoral waters. The second order Cubomedusae has bells with four flattened sides and simple margins and is mostly found in tropical and sub-tropical oceans. In the third order Coronatae, the bells of medusae have a decep groove or constriction, the coronal groove, extending around the exumbrella. Many deep sea species belong to this order.
The fourth order, Semaeostomae, has scyphomedusae with bowl- shaped or saucer-shaped bells having scalloped margins. Gastrovascular cavity with radial canals or channels extending from central stomach to bell margin. It occurs throughout the oceans of the Enteron
canal
Ring
Mesoglea Gastrodermis Lappet
Tentacles
Figure 1. Structure of jellyfish
1.0 10
world (Barnes, 1980; Villee et al., 1978). The fifth order Rhizostomae lacks tentacles. Oral arms and manubriums are branched and bearing deep folds into which food is passed. Folds or secondary mouths lead into arm canals. Arm canals of the manubrium pass into stomach. The original mouth was lost through fusion of oral arms. These are tropical and sub-tropical shallow water scyphozoans
(Barnes, 1980; Villee et al., 1978).
2.1.3 Semaeostomae and Rhizostomae species found in Australian waters
The following list was cited by Pulley (1971) and taken frem Kramp (1961).
Pelagia noctiluca - Forskal (1775)
Cyanea capillata - Einne (1758)
Aurelia aurita - Linne (1738)
Aurelia coerula - von Lendenfeld (1884)
Cassiopea andromeda - Forskal (1775)
Cassiopea ndrosia - Agassiz & Mayer (1889)
Cassiopea oronata - Haeckel (1880) von Lendenfeld (1884)
Cephea cephea - Forskal (1775)
Mastigias albipunctatus - Stiasny (1920)
Ma$tigias andersoni - Stiasny (1926)
Mastigias papua - Lesson (1830)
Mastigietta palmipes - Haecke1 (1880)
Phyllorhiza punctata- von Lendenfe1d (1884)
Versu:tiza;: ana~:yomene- Maas ( 1903)
Thysanostoma thysamura - Haecke1 (1880)
Psuedorhiza aurosa - von Lendenfeld (1884) 11
Pseudorhiza haeckeli - Haacke (1884)
Catostylus mosaicus - Quoy & Gaimard (1824)
Crambione cooki - Mayer (1910)
2.1.4 Life Cycle
The life cycle of scyphozoan cnidarian includes several developmental stages. These have been described by Schwab (1977),
Cockrum & McCauley ( 1965), Starer et al. ( 1968) " Grzimek ( 197 4),
Morris (1975) and Kakinuma (1975).
The large medusae produce sperm or eggs that unite in the gastric cavity of the female jellyfish. The fertilised egg or zygote emerges from the female's mouth and settles on the arms surrounding it, where it develops into a planula larva.
This larva escapesandswims freely before settling in the sea floor where it develops into a polyp known as scyphistomae.
Scyphistomae feed and produce other scyphistoma by budding. Seasonally, the scyphistoma produce new medusae by transverse fission. A series of grooves form perpendicular to the oral aboral axis and the segments formed metamorphose:iJ:tnta · larval medusae called ephyrae.
Development of ephyrae proceeds in an aboral direction on the polyp, the most oral segment is the most advanced and is the first to be released as a free eph¥r~ This process is termed strobilation and the transversely segmented polyp of the strobila, where segmentation has not occured, develops tentacles.
When all the ephyral segments have been released the remaining polyp feeds and repeats the strobilation process the following year. The released ephyrae develop into large, sexual medusae, completing the life cycle (see Fig. 2). Male
/' ...~.-~':.·.
s~x o~ {JAL REPROOUCTl
p l".ssr Early oft( embryology
Strobila~
Cyp~ ~ ~Settles
Figure 2. Life cycle of jellyfish
f-' N 13
2.1.5 Regeneration
Cnidarians possess fairly strong powers of regeneration and are capable of growing various missing parts. According to Curtis and Cowden (1972), the remarkable regenerative capacities are particularly apparent in cases in which small fragments of a single layer of an individual animal first replace the missing layer or layers and then progressively develop into complete animals possessing the symmetry, polarity, and cellular organisation of the original.
In Hydra, Normandin (1960) discovered that the entire animals could regenerate from small fragments of the gastrodermis.
In another study, Haynes and Burnett (1963) found that the explants initially lacking interstitial cells and possessing only two types of cells, gland cells and digestive cells, could develop into complete animals. These findings were confirmed by Davis et al. (1966) and Davis (1970). Other researchers who conducted comparable experiments (Lowell and Burnett, 1969; Zwilling, 1963;
Diehl, 1969; Gilchrist, 1937; and Steinberg, 1963) obtained similar results except that in these cases, complete animals developed from small isolated fragments of the epidermis.
2.1.6 Ecology/Adaptation
Cnidarians are considered conformers to environmental change because they lack many of the mechanisms found in higher invertebrates and because their body tissues are in intimate contact with the environment (Dillon, 1977).
The environmental factors affecting the occurrence 14
of scyphozoans are temperature (Mayer 1912; Spangenberg, 1965;
Thi11, 1937; Moller, 1980; PuJley, 1971; Gohar & Eisawy, 1960), oxygen (Mangum, Oakes & Shick, 1972); salinity (Dillon, 1977;
Mayer, 1914; Spangenberg, 1967; Lukanin, 1976); pressure (Rice,
1964; Digby, 1967), and predation (Phillips, Burke & Keener,
1969; Thomas, 1963; Herrnkin~,Halusky & Kanciruk, 1976; Powell and Gunter, 1968; Farr, 1978; Russell, 1970; Stephenson, 1970;
Goha~ and Eisawy, 1960).
2.1.7 Availability/Distribution
Jellyfish can be found in both tropical and temperate waters of the world. The appearance of this invertebrate animal is primarily governed by the different temperature requirements during its life cycle.
Medusae (mainly scyphomedusae) from Australian coastal waters were reviewed by Kramp (1965) based on communication from
Dr R.V. Southcott of the South Australian Museum, Adelaide. 19 species of Australian jellyfish were reported and classified as follows:
Order Coronatae:
Family Linuchidae - 1 species
Order Semaeostomae
Family Pelagiidae - 1 species
Family Cyaneidae 2 species
Family Ulmaridae 1 species
Order Rhizostomae
Family Cassiopedae 1 species
Family Cepheidae - 2 species 15
Family Mastigiidae - 3 species
Family Lynchnorhizidae - 1 species
Family Catostylidae - 2 species
Order Leptomedusae
Family Eirenidae - 3 species
Order Limnomedusae
Family Olindiadidae - 2 species
This was a representative collection of macroscopic material however, the order Cubomedusae was omitted. It was
reported earlier that studies on Australian Cubomedusae, including
new genera and species apparently harmful to man were conducted.
Southcott (1955) examined a series of large cubomedusae from
northern Australian seas around Darwin and north Queensland.
He found the specimens to belong to a single species described
as Chironex fleckeri, gen. nov., sp. nov.
The species of Scyphomedusae were collected from west to
east on southern coasts, northwards along the eastern coast and westwards along the coast from Cape York towards Darwin in the
Northern Territory. Among the species represented, nine have an entirely tropical distribution in other regions (Linuche ungniculata,
Cyanea buitendijki, Cassiopea ndrosia, Cephea octoslyla, Netrostoma
coerulences, Mastigias papua, Mastigias ocellatus, Acromitoides purpurus and the hydromedusa Helgicirrha danduensis). In Australiii:l,
these species are restricted to the coasts of Queensland or the
Northern Territory. Phyllorhiza punctata was taken in several
Queensland localities, while some specimens were also found near
Fremantle in Western Australia (Kramp, 1965). Lendenfeld (1884) originally described this species from Port Jackson, New South Wales. 16
According to Kramp (1965), there are only two species of Rhizostomae, Pseudorhiza haeckeli and Catostylus mosaicus, that are widely distributed and very common in Australian waters.
These species seemed to be endemic in this area. The three species of Semaeostomae, Pelagia noctiluca, Cyanea capillata and Aurelia aurita which have an almost world-wide distribution, seemed to occur in all Australian waters.
Table 2. Geographical distribution of some medusae (mainly
Scyphomedusa) found in Australian coastal waters*
Species Locality
Linuche ungiculata (Swartz, 1783) Tropical parts of the Indian and Pacific Oceans and the Western Atlantic
Pelagia noctiluca (Forskal) Oceanic in the upper strata of all warm and temperate seas
Cyanea capillata (L.) Coastal waters, mainly in aretic and temperate seas, less frequent in tropical regions
Cyanea buitendijki (Stiasny) Amboina in the Malayan Archipelago
Aurelia aurita (L.) Coastal waters
Cassiopea ndrosia Agassiz and Australian localities are all Mayer (1899) on the coasts of Queensland, Fiji Islands and New Caledonia
Cephea octostyla (Forskal) Red Sea, Philippines
Netrostoma coerulescens (Mass, Arabian Sea, Philippines, Japan 1903)
Mastigias papua (Lesson, 1829) Tropical coastal waters in the Malayan Archipelago and Western Pacific to the Fiji Islands and Japan
* Kramp (1965) 17
Table 2. (cont'd)
Species Locality
Mastigias ocellatus (Modeer, Andaman Islands and Merghi 1791) Archipelago, Malayan Archipelago, Philippines, Hong Kong
Phyllorhiza punctata (Lendenfeld Gulf of Siam, Southern 1884) Japan
Pseudorhiza haeckeli (Haacke, Coasts of South Australia, 1884) Arnhem Land, Rottnest Island, Cockburn Sound, Koombana Bay, Fremantle and Bunbury Coast
Acromitoides purpurus (Mayer, Philippines 1910)
Catostylus mosaicus Australia and the Philippines (Quay and Gaimard, 1824)
Eirene menoni (Kramp, 1583) Southeast Africa, India, Macassar Strait to Chekiang Coast in China, Cook Islands in Polynesia
Helgicirrha danduensis Maldive Islands, Nicobar (Bigelow, 1904) Islands, Vietnam (doubtful record)
Phialopsis diegensis (Torrey, Mainly oceanic. Common in 1909) the Atlantic Ocean, from the Irminger Sea to the Cape of Good Hope; off east-coast of Africa; California and Southwest of Galapagos Islands in the eastern Pacific. The occurrence in South Australia bridges the gap between the east Pacific and the western part of the Indian Ocean
Olindias singularis (Brown, 1905) Warm coastal waters of the Indian Ocean from the Iranian Gulf to the Malayan Archipelago; Low archipelago in Polynesia, about 135°W
Gonionemus hamatus n.sp. Eastern shore of St Vincent Gulf, South Australia 18
2.1.8 Seasonal distribution of jellyfish in Australian waters
There is limited literature on the seasonal distribution
of jellyfish particularly in Australia. So far, only Pulley
(1971) has examined_this aspect of the occurrence of jellyfish
in Lake Macquarie and the Tuggerah Lakes. Locations are shown
in maps 1-3.
In this survey, scyphomedusae were common in the lakes
from January to September. During the period of the survey from
January to October, the species presented in Table 3 were sighted.
Species of the semaeostome Cyanea were also sighted in Botany Bay and the Georges River during September and October, and in Sydney Harbour in October. It is believed to be common in the immediate offshore waters of the region during early spring
The resident species in Lake Macquarie is Aurelia coerula, however it was not sighted until spring.
In Tuggerah Lakes, the rhizostome Catostylus mosaicus, although not the only scyphozoan observed, was present in very large numbers during summer season. The medusae were abundant throughout the lake for the first four months of the year. The surface population however, decreased from autumn into winter.
In late May, no specimen was sighted and the final disappearance was in mid-Ju~~ It was further observed that the appearance of the free swimming larval stage, the ephyra, starts in early April and in the later part of the year the subadults were sampled with a mid-water trawl.
Catostylus species occurred in large numbers on the bottom of the lake (Cleland and Southcott, 1965). The medusae 19
MAP 1 THE COAST OF NEW SOUTH WALES, SYDNEY TO NEWCASTLE.
·Lake Macquarie
The Tuggerah Lakes
Broken Bay
8 otany Bay and Georges Rtver:~'""'
Lake Illawarrah
0 10 20 JO miles o 50 km 20
MAP 2 LAKE MACQUARIE Cockle Creek
• - Hydrology Station
Bolton Point
Dora Creek
Mannering Creek o 1 2 3 Miles.
o 1 2 3 4 5 6 Km 21
MAP 3 THE TUGGERAH LAKES
Munmorah Power Station
Wallerah Creek
+ Hydrology Statioh SCALE •12 0 1 2 J Miles 0 1 2 J 4 5Km
Wyong Creek
Tuggerah Lake 22
Table 3. Jellyfish sighted in Lake Macquarie during the survey (January to October 1971) *
Species Date Size (cm) Method of detection
Catostylus mosaicus 20-4-71 20 Visual, not captured
Phyllorhiza punctata 30-4-71 1.3-4.0 Plankton net
Cyanea capillata 11-9-71 15 Visual, captured
26-9-71 28 Visual, captured
Aurelia coerula 11-9-71 23 Plankton net
* Adapted from Pulley (1971)
when active and healthy tend to move into deeper water in response
to rainfall and strong winds. On sunny, calm days, the surface
layer is preferred (Pulley, 1971). A similar observation was made by Russell (1970).
Adult Catostylus were not randomly distributed over
the lakes. They aggregated into groups, containing several hundred
individuals when the population was high and occupied well-defined
areas. Between these groups, few specimens could be seen. A
similar phenomenon was observed in February at Smiths Lake, in
March at Botany Bay and in May at Lake Illawara (Pulley, 1971). 23
2.2 Studies on the Processing of Jellyfish
Among previous published work on jellyfish preservation
are papers by Iljas and Arifudin (1971), Soonthonvipat (1976),
Wootton et al. (1982) and Morikawa (1984).
In Indonesia, Iljas and Arifudin (1971) studied the
effects of alum and salt on preservation of jellyfish of unspecified
type. Several concentrations of salt and alum were tried until
an acceptable product was obtained. Salt and alum used singly
in the processing of jellyfish did not have any preservative effect.
Extensive liquifaction of the tissues occurred in the absence
of salt while disagreeable odours developed in the absence of
alum.
On the other hand, combinations of salt and alum exerted
a preservative effect on jellyfish. Products obtained had moisture
content 67-70% and salt content from 22-23% with yields varying
from 3.90-5.10%. There was a tendency to decreased yields while
the texture and crispness of the product deteriorated and thickness was affected when the alum levels were increased.
Wootton et al. (1982) conducted a study on the processing
of an Australian species of jellyfish (Catostylus mosaicus).
They followed the processing method described by Iljas and Arifudin
(1971). As the previous workers found, salt and alum when used
singly did not have any preservative effect on jellyfish. Brining with salt (25-35%) and alum (2-10%) produced a preserved product
of satisfactory properties. The composition of the final product
is compared with that of fresh and commercially processed jellyfish
in Table 4 (Wootton et al •• 1982). The researchers concluded 24
that salted jellyfish can be satisfactorily prepared from the
Catostylus genus. A 5 week process using salt and alum gave a product which compared favourably in flavour and texture with a Malaysian commercial product.
Another method of processing jellyfish of unnamed species was described by Soonthonvipat (1976). This method was introduced by a Japanese merchant in Thailand with the intension of producing an export product for Japan. The method, kept secret initially, was also based on salt and alum but with lower proportions of alum gave a yield of about 30% of bell weight and :18% of live weight. This dried jellyfish product is divided into two grades for export. Grade A product contains those jellyfish with bell diameters of 30 cm or more. Grade B contains those with the diameters of less than 30 cm.
In Japan, two different methods of processing jellyfish were reported. One was murasakizuke which required oak leaeves for aroma but is rarely used at present. The other was myobanzuke and is currently employed by most jellyfish processors. This method also uses salt and alum. Alum was added initially in higher proportion with a total of 10% or more being required during the entire process (Morikawa, 1984).
2.8 Utilisation as Food
Little information has been reported on the utilisation of jellyfish as food for human consumption. However, jellyfish have been commercially exploited by the Chinese for more than
1000 years. Large jellyfish of the order Rhizostomaeare important 25
Table 4. Comparison of fresh and processed jellyfish (as is basis)
Proportion Jellyfish (%) sample Moisture content Protein Salt Alum Ash+
Fresh 95.8 1.3 2.1 * 2.5 Experimental 66.2 6.7 25.0 0.07 27.4 (salted) Commercial 73.1 4.0 18.4 * 22.3 (salted)
Dried 68.2 9.3 14.8 0.08 24.8
* None detected + Includes salt and alum
food in Chinese cooking (Omori, 1981).
Jellyfish may be eaten as a salad, or mixed with chicken or other types of meat. Firth (1969) described the Japanese method of preparing it by pickling in alum, then rinsing in fresh water, cutting into thin strips of about 3mm wide by 80mm long and serving with vinegar as a small dish at rather an elaborate meal. The Chinese have more complex ways of preparing the processed jellyfish. Some recipes have been described by Peimei (1979).
Wootton et al. (1982) prepared a chicken and jellyfish dish from dried and salted jellyfish by following the recipe described by
Ma (1968).
In Thailand, Soonthonvipat (1976) reported that before cooking, the dried jellyfish is sliced into small thread like noodles. These threads are washed several times to remove salt and are dipped in hot water. The dipped jellyfish is ready for 26
use in various recipes. Jellyfish is not served as a main dish in Thailand and in other Asian countries, but with other food items as hors d'oeuvres or appetizers and is now very popular in Chinese restaurants throughout Southeast Asia (Soonthonvipat,
1976).
2.4 Chemical Properties of Unprocessed Jellyfish
2.4.1 Moisture content
Scyphozoan medusae are for the most part, fragile animals of very high water content. Joseph (1979) reported that Aurelia aurita collected in Nova Scotian waters had 98.9% water. Similar values have been found in medusae of the Chesapeake Bay system:
Cbrysaora quinquecirrba, 98.6%; Cyannea capillata, 98.0%. A south east Atlantic coast medusae, Stomolophus meleagris, locally known as the 'Cannon Ball' is considerably more dense and contained
92.9% water.
A water content of more than 98% was recorded for Aurelia aurita from the Baltic Sea near Greifswald where salinity was only 0.7%, whereas the same medusa has a moisture content of 95.56% in sea water of 3.5% salinity. In another report, Rhizostoma,
Aurelia and Cbrysaora contain about 95-97% water (Chapman, 195~).
According to Hatai (1917), the water content of Cassiopeia mesoglea is 94.6% and the cellular part is 93.8%. Lowndes (1942) found that the water content of Aurelia of 10 cm diameter is 95.56%; but Thill (1937) states that it may rise to 98% in brackish water.
Catostylus species were reported to contain 95.8% water by Wootton et al. (1982).
Medusae are generally considered to be the animal highest 27
of all in water content. Despite fantastic figures in some reports,
Ludwig (1977) believed that marine medusae can contain at the most 94-96.5% water. He is in agreement with Thill (1937) that higher water contents are found in medusae living in brackish water as they are poikilo-osmotic in different salinities. In fresh water, he found that the medusa Craspedacusta sowerbyi contained 99.26% water.
Since jellyfish are in approximate osmotic equilibrium with sea water which contains about 3% salt (Robertson, 1939) and since they contain about 95-98% water, Chapman (1953) opined that their content of solids other than salts must be extremely small, probably not greatly above 1% and may be less.
2.4.2 Protein and amino acids
Aside from water, jellyfish also contains protein.
Wootton et al. (1982) found 1.3% protein in fresh Catostylus sp.
In a very early report, Agassiz (1862) noted that the washed and dried residue of Cyanea capillata Linne, presumably insoluble proteins, was just 0.2% of the total body weight. Chapman
(1953) reported levels of about 0.1% organic material by weight for Chrysaora but subsequently reported 1% organic and 3% inorganic matter to be general levels in medusae (Chapman, 1966).
Jones (1956) found that the organic component is derived from fibres, interstitial matrix and living cells. Amino acid analyses of various medusae and Anthozoa showed a mesogleal composition comparable with that of vertebrate collagen yielding high proportions of hydroxyproline and hydroxylysine. The collagen fibres appear to be associated with mucopolysaccharides and the 28
whole tissue has the character of an organic hydrophilic colloid.
In hydra, amino acid analysis showed that glycine is the most abundant amino acid in the mesoglea hydrolysate (Table
5). Glutamic acid and aspartic acid are next in quantity and of special significance is the presence of hydroxyproline, proline and hydroxylysine. Due to the presence of these amino acids and large amount of glycine, it was concluded that the mesoglea contains a protein belonging to the collagen group (Barzansky, Lenhoff and Bode, 1975; Spiro, 1970). Mesoglea from other Cnidarians were found to contain a collagen-like protein (Piez and Gross,
1959; Gosline and Lenhoff, 1968; Nordwig and Hayduk, 1969; Katzman and Jeanloz, 1970).
The amino acids of protelliriin' the extra-cellular fluids of Physalia physalis and Aurelia aurita were studied by Lane,
Pringle and Bergere (1965). They found that the extracellular fluid in Physalia physalis occurs chiefly in the gastrovascular cavity and in Aurelia aurita it permeates the loosely organised mesoglea. The total nitrogen content of extracellular fluid in
Aurelia aurita and Physalia physalis are 0.06% and 8.0% respectively.
The amino acids comprising the protein moiety of the total solids are shown in Table 6 with hydroxylysine and hydroxyproline undetermined.
Lane et al. (1965) opined that the presence of a protein rich fluid in the gastrovascular space of Physalia physalis may help to explain why freshly captured undamaged specimens of this organism placed in aquaria soon begin to deteriorate. The water in the tank becomes cloudy, tentacles begin to fragment, the colour fades and the pneumatophore crest deflates. Within an hour, the 29
Table 5. Amino acid composition of the mesoglea of hydra *
Amino acid Residues/1000 residues
Hydroxyproline 30
Aspartic acid 96
Threonine 38
Serine 61
Glutamic acid 121
Proline 57
Glycine 229
Alanine 57
Valine 27
Half cystine 15
Methionine + 12
Isoleucine 24
Leucine 56
Tyrosine 20
Ph en ylal,and:ne 23
Hydroxy lysine 40
Lysine 43
Histidine 13
Arginine 38
* Adapted from Barzansky et al. (1975) + Corrected value 30
Table 6. Concentration of amino acids in proteins of extracellular fluid (mg/ml of whole fluid) *
Aurelia aurita Physalia physalis
Cystine 0.0003 0.12
Aspartic acid 0.0049 2.05
Threonine 0.0030 1.24
Serine 0.0034 1.43
Glutamic acid 0.0071 3.71
Proline 0.0014 0.99
Glycine 0.0049 1.68
Alanine 0.0032 1.59
Valine 0.0022 0.75
Cystine Trace 1.01
Methionine Trace 0.64
Isoleucine 0.0018 1.16
Leucine 0.0036 2.02
Tyrosine 0.0015 0.76
Phenylalanine 0.0028 1.32
Ammonia 0.0019 0.42
Lysine 0.0039 2.62
Histidine 0.0012 0.77
Arginine 0.0025 1. 75 Taurine 0.0035 0.16
* Adapted from Lane et al. (1965) 31
water in such aquaria becomes ninyhdrin-positive, suggesting
that a considerable loss of protein may be associated with the
damage which is observed.
Of the free amino acids (FAA), Webb, Schimpf and Olmon
(1972) found that glycine, structurally the simplest amino acid, was the most abundant in jellyfish. Likewise, Von Halt (1968)
indicated that among FAA of coral tissue from Zoanthus flos marinus
35% of a total of 10 ]..IM/mg protein nitrogen is glycine. Raum (1970)
found glycine to be 78% of the FAA pool in normal, and 84% in
starved, polyps of Aurelia aurita obtained from Massachusetts region of the Atlantic coast, and cultured at salinity of 3.2% and l9°C. Glycine was also the most concentrated individual amino acid in Aurelia mesogleal fluid on a molar basis comprising 15.8% of the total (Lane et al. 1965).
Table 7 shows the absolute amounts of acids and neutral
free amino acids in Texas Aurelia aurita polyps. It can be seen that glycine predominates and taurine and B-alanine are present in substantial amounts. Values for total ninhydrin positive substances
(NPS) are also given (Shick, 1976). Hydroxylysine and hydroxy-
proline were not determined in this work but are among the basic amino acids.
The relative molar percentages for FAA in the Virginia
Institute of Marine Science (V .I.M.S.) strain of laboratory-grown
A. aurita polyps at salinities from 1.0 to 3.5% are presented in Table 8. At the higher salinities, glycine, B-alanine and taurine were the most prolific amino acids in agreement with the findings of Shick (1976). Hydroxylysine and hydroxyproline no doubt comprise part of the unidentified amino acids. The relative 32
Table7. Absolute amounts (n moles/mg dry weight) free amino
acids (FAA) in fed (2 day) and starved (14 day) Corpus
Christi, Texas Aurelia aurita polyps at 3% salinity
Amino acid Fed Starved
Taurine 29 49
Aspartic acid 4 6
Threonine 10 8
Serine 23 36
Glutamic acid 5 6 Proline 22 Tr
Glycine 89 154
Alanine 17 14
Valine 25 12
Methionine Tr Tr
Isoleucine 15 6
Leucine 21 4
Tyrosine 13 Tr
Phenylalanine 10 Tr
B-alanine 23 48
Total FAA ** 306 343 Total NPS 353 364
Tr - trace amounts * - Adapted from Shick (1976) ** - Excluding undetermined basic amino acids 33
Table 8. Molar percentage of FAA in laboratory grown A. aurita polyps from Chesapeake Bay starved for 48 hr *
Salinity (%) 1.0 1.5 2.0 2.5 3.0 3.5
Cysteic acid
Taurine 4.2 7.8 11.3 14.2 10.4 9.7
Aspartic acid 1.1 0.6 0.7 0.4 0.7 0.6 Threonine 1.1 0.7 0.7 0.7 0.9 1.0 Serine 1.1 1.5 1.6 1.4 1.8 2.4
Glutamic acid 20.5 8.5 7.6 6.1 5.2 5.3 Proline 0.4 0.8 1.0 2.1 4.2
Glycine 9.4 31.6 44.2 46.2 41.1 33.0
Alanine 2.1 1.4 1.8 1.2 1.5 2.2
Valine 0.6 1.1 1.3 1.5 1.9 2.2
Cystine
Methionine 0.6 0.4 0.3 0.6 0.4 0.4
Isoleucine 0.4 0.8 0.8 0.9 1.1 1.1
Leucine 0.5 0.8 0.9 0.9 1.0 0.8
Tyrosine 0.6 0.6 0.7 0.8 0.9 Phenylalanine 0.6 0.4 0.5
B-alanine 1.3 6.7 7.7 16.4 21.7 Ornithine 0.6 0.4 0.3 0.3 0.4
Lysine 14.4 4.1 2.0 1.2 1.1 0.9 Tryptophan 0.3 0.3 0.3 0.4
Histidine 0.9 0.4 0.3 0.3 0.3 0.5
Arginine 2.0 1.1 1.1 1.2 1.6 1.4
Unidentified 41.2 35.7 16.2 12.9 11.0 10.8
Total nM per 65 polyps** 442 1394 1965 2932 2775 2267 nM/mg extracted dry weight 14.8 78.0 118 299 296 * Adapted from Webb et al. (1972) ** Tissue volume about 0.1 ml 34
proportions of glutamic acid, lysine and unidentified fractions declined with increasing salinity (Webb et al., 1972).
2.4.3 Lipid
The lipid content of schyphozoan medusae as percent of the wet weight is very low. These organisms contain no visible lipid deposits except in relatively well-developed gonads during the reproductive cycle (Joseph, 1979). On a wet weight basis, lipid contents from 0.0046-0.2% have been reported (Yasuda, 1974;
Hooper and Ackman, 1973). The non polar lipids of lyophilized medusae comprised 31.1% of the total lipid present (Schmidth and Joseph, 1971). Sipos and Ackman (1968) obtained a very similar value and in addition found that the non-polar fraction has the following composition: hydrocarbons, 1.7%; steryl and wax ester,
4.5%; triacylglycerols, 14.0%; fatty acids, 7.3%; sterols, 47.8%; mono-and-dia~ylglycerols, 13.9%; uncharacterized components,
10.8%. These findings were supported by Goldner et al. (1969).
Alkyl- and/or alk-1-enyl diaeylg1ycerols were also detected (Joseph,
1979).
Table 9 shows the simplified total lipid fatty acid analyses of the four species of scyphozoan medusae. The fatty acid compositions are generally similar. The medusae from Canadian waters, C. capillata and A. aurita, have significantly more monoenoic acids particularly 20:1 and 22:1 but less 20:4 (n-6) than Charleston
Harbour medusae, Stomolophus meleagris and C. quinquecirrha.
The differences in percentage of monoenes could be attributed to differences in the diets of the medusae (Joseph, 1979). 35
In another report, White and Hager (1977) found that fatty acid chlorohydrins are lipid components of A. aurita.
Identification on fatty acids of edible jellyfish showed six chlorohydrin acids, namely; 9-chloro-10-hydroxypalmitic acid,
10-chloro-9-hydroxypalmitic acid, 9-chloro-10-hydroxystearic acid, 10-chloro-9-hydroxystearic acid, 11-chloro-12-hydroxy stearic acid, 12-chloro-11-hydroxystearic acid.
The sterols of cnidarians have been studied by a number of researchers. Haurowitz and Waelsch (1926) isolated cholesterol from the hydrozoan jellyfish, Velella spirans (little snail).
Middlebrook and Lane (1968) found cholesterol in Physalia physalis and van Aarem, Vonk and Zandee (1964) also found that
Scyphozoan jellyfish of Rhizostoma species contained cholesterol as a major sterol. Furthermore, cholesterol, 24-methylenecholesterol and B-sitosterol had already been isolated from Anthozoa,
Gorgonacea (horny corals) and Actinaria (sea anemones) (van Aarem et al., 1964).
Sterols of other species of Cnidarians were also examined and found to be the complex mixtures of c27 - to c30 - sterols
(Ciereszko et al., 1968). Similar findings were obtained by
Bergmann, McLean and Lester (1943) in the Gorgonacea, Plexaura flexuosa; and Gupta and Scheuer (1969) in three species of Athozoa.
The sterol compositions of four species of Cnidarians are presented in Table 10. 36
Table 9. Major fatty acids of representative scyphozoan medusae *
Species c. capillata A. aurita S. meleagris c. quin- quecirrha
Fatty acids Weight percent composition
14:0 L5 3.3 1.7 0.7
16:0 14.1 16.0 12.1 10.0
18:0 7.1 6.4 8.7 10.9
20:0 2.0 1.2 0.5 1.9
16:1a 5.2 4.7 2.9 3.2
18:1a 7.0 11.8 3.4 3.9
20:1a 12.2 13.7 0.4 0.7
22:1a 2.0 4.5 0.1 0.4
18:2(n-6) 0.2 0.8 0.9 0.5
20:4(n-6) 6.2 6.7 15.5 22.4
22:4(n-6) 2.0 0.6 2.6 6.3
22:5(n-6) 1.5 0.3 1.2 6.7
18:3(n-3) 0.4 0.4 0.5 0.4
18:4(n-3) 0.2 0.1 1.1 0.1
20:5(n-3) 9.8 8.5 19.1 7.6
22:5(n-3) 4.3 1.6 3.1 3.6
22:6(n-3) 13.3 7.0 15.9 11.9 t16:1(n-10) n.r. 2.0 n.r. n.r. 7M7Hb n.r. 0.3 0.5c 0.3c
~Sum of cis isomers 7-methy1-hexadecenoate cTentative1y identified in hydrogenated esters as 7-methy1-hexadecanoate *Adapted from Joseph (1979) 37
Table 10. Sterol compositions of Cnidarians (percent)**
Spirocodon Aurelia Stomolophus Anthopleura
c26-sterol 1.9 5.7 1.8 0.6 22-Dehydro- cholesterol 1.4 1.3 Trace Trace
Cholesterol 88.8 72.2* 78.3 87.8
Desmosterol 4.9 9.8
Brassicasterol Trace 0.7 8.2 2.2
24-Methyl- cholesterol 1.3 Trace 1.2 2.9
24-Methylene- cholesterol 0.5 4.8 8.7 4.5
24-Ethyl- cholesterol 0.9 2.4 3.9 2.1
24-Ethylidene cholesterol 0.3 3.0 1.9
* Mixture of cholesterol and cholestanol
** Adapted from Yasuda (1974) 38
2.5 Preservative Mechanism of the Traditional Jellyfish Processes
The traditional method of processing jellyfish involves brining and air drying. Previous publications have not dealt with the mechanisms of preservation. Obviously, brining and drying remove a large percentage of moisture while appropriate proportions of salt and alum are also required to produce desired structure and texture (Iljas and Arifudin, 1971). Reduction of moisture content from 96% in fresh jellyfish to about 66% in salted forms with corresponding decreases in water activity
(a ) from 1.00 to approximately 0.73 contribute to stability w of the product at ambient temperature.
Salted jellyfish also contains high levels (15-25%) of salt (Wootton et al. 1982) which aids in keeping the product microbially stable. However, no-one has reported yet on the spoilage patterns of fresh and processed jellyfish. This aspect needs to be examined in order to improve processing methods.
The quality of the product obtained from the traditional method of processing has not been assessed as far as protein quality is concerned. There is no published work concerning solubility, extractability, digestibility and IEF patterns of protein in jellyfish as to this date. These aspects need to be studied in relation to the processing technique employed. Knowing the qualities of jellyfish protein will enable the processor to optimise handling of the product prior to,and during,processing as well as treatment of the final product. 39
2.6 Disposal of Brine for Jellyfish Processing
Discharging spent brine for jellyfish processing directly
into waterways could pose an environmental problem. Further, direct
discharge of jellyfish brine could lead to loss of fluids that
may be rich in nutrients. Thus, disposal of brines must be regarded
as an important issue.
Procedures for brine treatment include a system described
by Welsh and Zall (1984) using an ultrafiltration/activated carbon
process where spent brines were first ultrafiltered using a 30,000 MW
cut off membrane and then treated with activated coconut charcoal.
Another method involved precipitation of protein from the waste
water with 10 N H (Hang, Woodams and Parsons, 1980). Similar 2so4 work was undertaken by Toma and Meyers (1975) in order to isolate
and evaluate protein from cannery effluent.
In processing jellyfish, a large volume of spent brine
is expected because of its high water content and the high levels
of salt and alum used. Therefore, it seems necessary to examine
the feasibility of recycling from the point of view of both environ mental safety and for economic reasons. 40
3. MATERIALS AND METHODS
3.1 Materials
3.1.1 Jellyfish samples
Jellyfish of Catostylus species were captured by hand net in Botany Bay near the Captain Cook bridge or in Lake Illawarra neartheTallawarra power station, New South Wales. Bell diameters varied from 200-400 mm.
The fresh jellyfish were placed in SOL plastic containers with sea water, transported to the laboratory and stored at 4°C for up to 24 h prior to treatment. Only the bells (caps) were used for processing since the tentacles (oral arms inthecase of
Catostylus species) contained most of the nematocysts (toxin containing capsules). The tentacles (oral arms) were cut off and discarded during processing.
One batch of jellyfish samples was utilised in each set of experimentsconducted, and in every trial a new batch of samples was collected.
3.1.2 Chemicals/reagents
The chemicals/reagents used were of AR grade, purchased from the different chemical companies within or outside Australia. 41
3.2 Methods
3.2.1 Processing of jellyfish
3.2.1.1 Traditional method
Jellyfish tentacles were removed and discarded. The bells were cleaned by removing the slime by hand and placed in glass jars or plastic containers. Salt and alum of varied concentrations were mixed thoroughly before spreading uniformly on top and bottom of jellyfish bells. The treated jellyfish were turned over periodically during processing.
The salting process was carried out in three stages.
Stage I was the addition of 40% and 50% of the total salt and alum respectively, to jellyfish bells. After 48 h, the samples were washed thoroughly with tap water and allowed to drain for
2 h.
Stage 2 was the addition of 50% salt and 50% alum to samples. These were left in the curing solution for 19 days with periodic turning to allow uniform salt and alum penetration. The samples were then rinsed with tap water and allowed to drain for
2 h.
Stage 3 was the addition of the remaining 10% salt as saturated solution. The samples were left for 48 h in the saturated salt solution, washed thoroughly and allowed to dry on wire racks for 14 days at ambient temperature. 42
3.2.1.2 Shortened method
3.2.1.2.1 Brining and drying technique
Cleaned jellyfish bells were weighed and treated with varied
concentrations of salt and alum. A mixture of salt and alum was rubbed on
top and bottom of the bells. After 24 h, jellyfish were turned
over and let stand in the curing solution for another 24 h. After
the brining period (48 h), the bells were washed with tap water anddrained for 2 h prior to mechanical drying. Drying was carried
out in a cross draught dehydrator at 30° and 50°C to a moisture
content of about 60-65% and water activity (a ) around 0.70. The w drier had an air flow rate of 2.5 m/sec. Relative humidity conrol
was not possible with this equipment.
3.2.1.2.2 Blanching and drying technique
Cleaned jellyfish bells were blanched in steam for 5
min and tray dried at 30° and 50°C for 12 and 4 h respectively.
3.2.2 Analysis of the yield, chemical and organoleptic properties
of processed jellyfish
3.2.2.1 Yield
The loss in weight of jellyfish bells was measured after
each stage of salting and drying. Percent yield was calculated as
follows: Final weight % yield = Original weight X 100 43
3.2.2.2 Chemical properties
3.2.2.2.1 Moisture content
Jellyfish bells were cut into cubes and placed in a low speed electric blender and blended for 1 min to obtain a homogeneous mixture.
100 g of the homogeneous mixture was weighed into a aluminium drying dish, 80 mm diameter, provided with tight-fit cover. The samples were partially dried in an air oven because of high moisture content. Then the loosely covered dish was transferred in a vacuum oven at 100°C, under a pressure of 70-80 kPa. Drying was carried out to constant weight (36-48 hA During drying, a slow current of air dried by passing through concentrated H so was admitted 2 4 into the oven. Percent moisture was calculated as follows:
% Moisture = Loss in weight of dish plus sample after drying x lOO Weight of wet sample + weight of dish
Note: On processed jellyfish samples, lOg was used for moisture
determination.
3.2.2.2.2 Water activity (awL
Jellyfish samples were cut into small pieces using the high-speed blender for approximately 1 to 2 min, stopping between blends until the desired size was obtained. The prepared samples were placed in small closed plastic containers and allowed to equilibrate for at least 24 h.
Saturated salt solutions covering the range of the samples were prepared in similar closed plastic containers and equilibrated similarly to the samples. 44
A Novasina AG hygrosensor (Model en ZFBA-3(4)ePP) was used throughout this study. The sensor operates through adjustable plug-in units (EZFB-3(4) within a range of 0.50-1.00 a • Other w ranges can be obtained by substituting appropriate plug-ins.
Aw measurements were made by placing a small, plastic container (40 mm diam x 12 mm deep) containing a sample in a special holder. The sensor, mounted in a gasketed, and enclosed fitting, was then used to cap the sample holder. Readings were taken directly from the 50-100% equiibrium relative humidity (ERH) meter scale and converted to a w by division by 100. Readings were made to two decimal places.
Before measurements, the sensor was calibrated by using a range of saturated standard salt solutions with ERH 53-97%.
3.2.2.2.3 Protein
Jellyfish bells were diced and blended in a low-speed electric blender for 30-60 sec. The homogenised samples were immediately weighed into 6 mL sample bottle~ The weighed samples were stored in a freezer if any delay occurred prior to analysis.
Dried samples were cut into 10 mm squares and ground in an electric coffee grinder. The fine particles were then weighed onto nitrogen-free paper ready for analysis.
About 2 g wet sample or 1 g dry sample was used for analysis, using a Kjel-Foss automatic protein analyser 16210-A/SN which gave readings based on N x 6.25.
Read-out x 1000 % Protein = Weight of sample (mg) 45
3.2.2.2.4 Fat (acid hydrolysis)
Jellyfish bells were cut into cubes and blended in a high speed electric blender for 1 min to obtain a homogeneous mixture.
The prepared samples were kept in sealed jars and chilled if any delay in the determination occurred.
Wet sample (approximately 2 g) was weighed into a 50 mL beaker. 2 mL alcohol was added and stirred to moisten all particles to prevent bumping when acid is added. 7N HCl (10 mL) was then added and mixed well. The beaker with a watch glass cover was placed on a steam bath and heated for 40 min with occasional stirring.
10 mL alcohol was added and the mixture allowed to cool.
A 250 mL clean, dry Erlenmeyer flask containing some glass beads was weighed and a filter funnel packed with cotton wool was placed in the flask.
The sample in the beaker was transferred to a Mojonnier fat-extraction flask and was rinsed with diethyl ether(25 mL) added in three portions. The flask was stoppered and shaken vigorously for 1 min. Petroleum spirit (25 mL) was added, the flask shaken vigorously for 1 min and allowed to stand until the upper layer was practically clear. The clear liquid was decanted and filtered through a cotton wool into the weighed Erlenmeyer flask. The liquid remairHmg:in the Mojonnier flask was re-extracted twice with 15 mL of each solvent. The flask was shaken well on addition of solvent and the clear liquid was decanted into the same flask as before.
The funnel was then washed with a few mL of mixture of equal parts of each solvent. The washings were combined with the upper layers and slowly evaporated on a steam bath in a fume cupboard. The
Erlenmeyer flask was placed in an air oven at 100°C for 90 min, 46
let stand in air for exactly 30 min and then weighed. The weight was corrected by a blank determination on reagents used. Percent crude fat was calculated as follows:
Weight of fat % fat in wet sample = X 100 Weight wet sample
3.2.2.2.5 Ash (dry ashing)
Approximately 2 g of dry;homogenised sample was weighed into a pre-weighed crucible. The sample was then charred on a hotplate in a fume cupboard. The crucible with the lid on was then placed in a muffle furnace at 550°C for 48 h. It was then removed, placed in a desiccator to cool, and weighed. Percent ash was calculated as follows:
Weight residue in crucible after ashing % Ash = X 100 Weight wet sample
3.2.2.2.6 .E!!. Jellyfish bells were blended in a high-speed electric blender for 1 min and the homogenised mixture (100 g) was used for measurement. The pH meter was calibrated with a known buffer and the pH of the sample was measured.
Dry samples were ground and 10 g blended with distilled water ( 100 mL) •
3.2.2.2.7 Sodium chloride (Volhard Method)
Jellyfish bells were cut into cubes and blended in a high speed electric blender for 1 min to obtain a homogeneous mixture. 47
1-2 g of mixed solid and liquid portions was taken for determination.
Dry samples were ground in an electric coffee grinder and 10 g portions blended with 100 mL distilled water for 2 min and then filtered. 5 or 10 mL of the filtrate (depending on the
NaCl concentration in the sample) was used for analysis.
The following reagents were used: Silver nitrate standard solution (0.1 N): 16.987 g of AgN0 in 1 L distilled water was standardised 3 against 0.1 N NaCl containing 5.844 g of pure dry NaCl/1.
Potassium thiocyanate standard solution (0.1 N): 9.718 g
KSCN was dissolved in 1 L distilled water. The worki:gg :titer was determined by titrating 50 mL standard AgN0 solution with added 3 saturated ferric alum solution (2 mL) and 5 mL HN0 (l+l)"with the 3 thiocyanate solution until the solution appeared pale rose after vigorous shaking.
Ferric alum indicator-saturated solution of FeNH ) .12H 4(so4 2 20. 1-2 g of sample was weighed and transferred into a 500 mL
Erlenmeyer flask. 50-70 mL of 0.1 N AgN0 and 20 mL HN0 were added. 3 3 This was boiled gently on a hot plate on heating mantle for 20-30 min. After cooling, 50 mL distilled water and 5 mL indicator were adaedrandthe mixture titrated with 0.1 N KSCN solution until a permanent light brown colour appeared. mL 0.1 N KSCN used was subtracted from mL 0.1 N AgN0 added and the difference was calculated as NaCl. 3 Percent NaCl was calculated as follows:
mL of AgN~ x Normality of AgN0 x MW of NaCL x 100 % NaCl = 3 3 Weight of sample x 1000 48
3.2.2.2.8 Aluminium
5 solutions containing 0, 200, 400, 600 and 800 ~g/mL
Al were prepared from a stock solution containing 1.00 mg Al/mL and kept in polyethylene bottles. Potassium nitrate containing a final concentration of 2000 ~gK/mL was added to suppress aluminium ionization in the nitrous oxide-acetylene flame during the determination.
Crucibles containing added samples were placed on a hotplate and enough 6 M HCl added to wet the ash. It was brought to a point of dryness at approximately 60°C, then 10 mL 3 M HCl was added and the mixture heated to just below boiling point. It was cooled and filtered into a 50 mL volumetric flask. 5 mL (10 mg/mL) caesium chloride solution was added and the solution made up to volume with distilled water.
An Atomic absorption spectrophotometer, type AA-5, SeriaL
No. 632 of Varian Techtron Pty Ltd was used under the following conditions.
Working conditions:
Lamp current 10 mA
Fuel Acetylene
Support Nitrous oxide
Flame stoichiometry Reducing; red cone
10-20 mm high
Wavelength 237.3 nm
Spectral band pass 0.5 nm
Working range 200-800 ~g/mL
Typical sensitivity 3.9 ~g/mL
Burner height 10 mm
Slit width 200 ~
Detection limit 0.02 ~g/mL 49
Standard solutions were read first, then samples and standards again. A calibration curve was drawn and the concentration of aluminium in the sample was calculated as follows:
sample volume -4 % Aluminium = ug/mL Al X X d X lQ sample weight where d = dilution factor
3.2.2.2.9 Total volatile acidity
10 g samples were blended for 1 min with 100 mL deionized water and filtered through Whatman filter paper (#541). Carbon dioxide was removed from the filrate under low vacuum for 5-10 min.
25 mL of freshly prepared sample was pipetted into the inner chamber of the steam distillation flask and stoppered. About
300 mL was distilled into the Erlenmeyer flask and 0.5 mL of phenoph thalein indicator was added to the distillate. This was titrated rapidly with O.lN NaOH until the pink colour persists for 15 sec.
Results were expressed as g HOAC/100 mL. TVA = mL O.lN NaOH x 0.006 x 4
3.2.2.2.10 Titratable acidity
10 g samples were blended with 100 mL deionized water for 1 min and the suspension was titrated with O.lN NaCH to a pH of 8.1. Results were expresed as mL NaOH used as titrant.
3.2.2.2.11 Total volatile bases (TVB)
10 g samples were blended with 20% trichloroacetic acid
(10 mL) and filtered through No 41 Whatman frlter paper. The volume was made up to 50 mL with distilled water.
,10 mL of the TCA extract was placed into the Parnas- so
Wagner Still, lOM NaOH (10 mL) added and distilled into a S mL
saturated boric acid with 3 drops of methyl red-methylene blue
indicator. Approximately 20 mL of distillate was collected and
titrated with O.OOSM H using a S mL piston burette. Analysis 2so4 was carried out in triplicate.
3.2.2.2.12 Trimethylamine (TMA)
Samples for TMA analysis was extracted at the same time
with TVB sample. 10 mL aliquot of TCA extract was placed into
the distilling unit, l.S mL neutralized AR grade formalin added,
followed by 10 mL lOM NaOH. Succeeding steps were as described
for TVB. TMA and TVB were expressed as mg N/100 g flesh.
3.2.2.2.13 Trimethylamine oxide (TMAO)
Homogenized sample (10 g) was weighed and placed into aporcelain mortar. About 3S mL ethyl alcohol (96%) was added
in small portions while the sample was ground with the pestle.
The paste was transferred to a SO mL volumetric flask and made
up to the mark with ethyl alcohol. Mortar and pestle were rinsed
with ethyl alcohol. The flask was shaken thoroughly and filtered
into a SO mL Erlenmeyer flask.
The solution (10 mL) was transferred to the distillation
apparatus, S mL saturated Ba(OH) was added (to avoid foaming) 2 and 10 mL 2N NaOH was introduced. The sample was steam distilled
and the distillate taken up in an excess of 0.02N HCl (standardized
in 50% ethanol-water against 0.02N NaOH with rosolic acid as indicator)
and titrated with 0.02N NaOH using the same indicator. 3S% formaldehyde
(neutralized with powdered MgC0 ) was added (5 mL/SO mL solution) 3 51
and the amount of acid liberated determined by renewed titration
with 0.02N NaOH.
To a further 10 mL of the alcoholic filtrate, TiC1 3 solution (10-15%) was added in slight excess (greyish-purple colour
of the solution). After 10 min, the sample was transferred to
the distillation flask, steam distilled and titrated as described
above. TMAO was expressed as mg TMAO - N/100 g flesh. The method
was based on that described by Ronold and Jacobsen (1947).
3.2.2.2.14 TCA soluble nitrogen
Samples were prepared in conjunction with total volatile
base and trimethylamine determination. The filtrate was centrifuged
at 0°C for 20 min at 8,000 rpm. 2 mL of this was used for total nitrngenanalysis using the Kjel-Foss automatic protein analyser.
3.2.3 Organoletpic properties
Processed samples were rehydrated for 3 days and then
cut into strips of about 3 mm wide. A salad-type recipe was followed
in its preparation which is described as follows:
Ingredients:
100 g shredded jellyfish
50 g shredded turnips
22.5 mL vinegar (white)
22.5 mL soy sauce (light)
2.5 g salt
10 mL sesame oil
5 g sugar 52
Procedure:
Shredded jellyfish were scalded in boiling water for
3 sec and soaked in cold water for over 3 h and drained. Shredded turnips were soaked in salt solution for 15 min, then squeezed dry. Drained jellyfish were squeezed dry and placed in a big bowl with turnips. A mixture of vinegar, soy sauce, salt, sesame oil and sugar was prepared, added and blended well with jellyfish and turnips.
3.2.3.1 Panel evaluation of texture and flavour
Five samples were coded and presented to the test panel for evaluation. Panelists were asked to rank the samples based on preference as to texture and flavour. The most preferred sample was ranked Number 1 and the least preferred was Number 5.
The ranks were tabulated and converted to scores according to the Method of Fisher and Yates (1942). Score given to each rank was determined by using the Table of Scores for Ranked Data
(Larmond, 1982). The scores were then subjected to analysis of variance in order to determine if there were differences among treatments. Tukey's Test was used to test significance of any differences observed be_tween samples (Larmond, 1982).
3.2.3.2 Flavour, texture and overall acceptability evaluation
In another set of organoleptic evaluations, three coded samples were presented to a panelist who was asked to rate for preference based from flavo"ur, texture and overall acceptability.
An incomplete block design was used to analyse the results using the Cyber computer program of the School of Statistics, UNSW. 53
A total of 30 panelists evaluated the 6 samples.
3.2.4 Solubility studies
3.2.4.1 Solubility on O.lM potassium chloride
Fresh or processed jellyfish bell (1 g) was suspended
in 10 mL O.lM Kel for 30 min and filtered. The total nitrogen
contents of both filtrate and residue were determined using the
Kjel-Foss automatic protein analyser (Obanu, Ledward and Lawrie,
1975).
3.2.4.2 Solubility on heating in sodium dodecyl sulphate + B-mercapto
ethanol
Fresh or processed jellyfish (1 g) was suspended in 50 mL
3% SDS + 1% B-mercaptoethanol for 30 min; heated in a boil±ng water
bath for the same period of time and centrifuged warm at 8000 rpm for 20 min. The nitrogen contents of both the clear supernatant and residue were determined ~banu et al., 1975).
3.2.4.3 Solubility of protein in sodium chloride solution
Fresh and processed tissues (5 g) and 250 mL of sodium chloride solution (5%) at 4°C) were homogenised three times for
60 sec with two intervals each of 120 sec. The homogenate was transferred into 100 mL centrifuge tubes and centrifuged for 20 min at 3500 rpm. The protein in the supernatant was determined using the Kjel-Foss automatic analyser. 54
3.2.4.4 Solubility of protein using enzymes
3.2.4.4.1 Pepsin solubility
100-500 mg pepsin in 100 mL O.lN HCl was mixed with 4 g of jellyfish sample in a 200 mL conical flask. The mixture was tncubated with shaking at 37°C for 24 h, filtered and nitrogen in the filtrate was determined. NPN was determined by adding an equal volume of 20% TCA to 5 mL filtrate and left for 24 h at 4°C. NPN was analysed using the Kjel-Foss automatic protein analyser.
3.2.4.4.2 Trypsin solubility
100-500 mg trypsin in 100 mL distilled water were mixed with 4 g samples then incubated with shaking at 40°C for 24 h. The mixture was filtered and NPN analysed as for pepsin digests.
3.2.4.4.3 Pepsin-trypsin solubility
One mg/mL pepsin and trypsin solutions were prepared as above and temperature and length of time of incubation were the same as described for samples above. Aliquots were taken after
1, 2.5, 5, 10, 20 and 30 min then 3, 6, 9, 12 and 24 h. Nitrogen was determined in the filtrate, then 5 mL filtrate was added with an equal volume of 20% TCA, left for 24 h at 4°C. The mixture was centrifuged at 8000 rpm for 20 min andnitrogen determined in the clear supernatant.
3.2.4.5 Solubility of sample in water
Samples (5 g) were weighed and 25 mL distilled water added.
This was held for 3 h with frequent shaking and then centrifuged.
The liquor was decanted into a weighed stainless steel evaporating 55
dish and evaporated to dryness on a water bath. The extraction
procedure was repeated with two further amounts of 25 mL distilled
water but held for 1 h at 40-50°C. The combined residue was dried
in an oven at 105°C for 3 h and weighed. Percent solubility was
calculated from the weight of residual matter (Lees, 1968).
Weight of residual matter % Solubility = X 100 Weight of sample
3.2.5 Extractability studies
3.2.5.1 Effect of sodium chloride concentration on extractability of
protein in jellyfish
Frozen samples were crushed into smaller pieces using an
electric blender. 50 g of the ground samples were added with 100 mL
of distilled water containing various levels of sodium chloride.
Samples were homogenised using the Ultra-Turrax homogeniser and
then centrifuged for 30 min at 10000 rpm. The protein contents
in the supernatant and residue were determined.
3.2.5.2 Effect of temperature on extractability of protein in jellyfish
Frozen samples were cut into cubes and ground in a blender.
50 g of these samples were mixed with 100 mL distilled water and
stirred for 20 min at temperatures between 0°C and 100°C. Ionic
strength and pH of the mixtures were not adjusted. After stirring
for 20 min, samples were homogenised and centrifuged for 30 min
at 10000 rpm. The protein contents were determined in the super natant and residue using the Kjel-Foss automatic protein analyser. 56
3.2.5.3 Effect of pH on protein extraction efficiency
Frozen samples were cut into cubes and ground in an electric blender. 50 g of these were mixed with 100 mL distilled water and pH adjusted using lN NaOH or lM HCl. The samples were then homogenised and centrifguged for 20 min at 8000 rpm. Protein in the supernatant was determined using the Kjel-Foss automatic analyser.
3.2.6 Digestibility of jellyfish protein by pepsin
1 mg/mL pepsin solution in 0.075N HCl was heated to 42-
450C. 100 mL of the prewarmed pepsin solution was added to 4 g of samples in a 250 mL conical flask which was stoppered and incubated with constant agitation for 16 h at 45°C.
Individual sheets of filter paper (Whatman No. 42) were dried in moisture dishes for 12 h in vacuum oven at 70°C, cooled in a desiccator and weighed (W ). 1 After digestion, flasks were removed from incubator and the residue allowed to settle. The weighed filter paper was placed in a Buchner funnel, moistened with water and suction was applied.
Pepsin hydrolysate was filtered rapidly and the remaining residue in the flask was removed by washing with 15 mL acetone. Washing was repeated thrice if necessary.
The filter paper was then transferred to a mosture dish, dried at 70°C in a vacuum oven, cooled and weighed as before (W ). 2 Percent indigestible residue was calculated as follows:
(W2-Wl) X 100 % Indigestible residue = g sample
Indigestible protein was determined using the Kjel-Foss automatic protein analyser. Percent protein was calculated as in 57
Section 3.2.2.2.3. The above result represents percent indigestible
protein in a sample. This was converted to % indigestible protein content as follows:
% Indigestible protein in sample Indigestible protein = · x 100 % Total crude protein in sample
The method described is based on AOAC Method No. 7.055;
7.058-7.059 modified to suit the sample used.
3.2.7 Isoelectric focusing
3.2.7.1 Materials
Electrophoresis Unit (Flat Bed Apparatus FBE-3000)
Constant power supply (ECPS 2000/300)
Agarose IEF
Pharmalyte carrier ampholyte
Gel casting frame
Agarose IEF Accessory kit containing;
Gelbond (114x225 mm)
sample applicators
electrode strips (6xl0 mm)
filter paper
Levelling table
Spirit level
Spring clips
Scalpel
Hair dryer 58
3.2.7.2 Reagents
Gel composition
0.3 g Agarose IEF
3.6 g Sorbitol
27.0 mL distilled water
1.9 mL Pharmalyte (pH 3-10)
Electrode solutions
Cathode (-) lM NaOH
Anode (+) O.OSM H 2so4 Fixing solution
5% Sulphosalicylic acid and
10% Trichloroacetic acid in distilled water
Destaining solution
35% Ethanol and
10% Acetic acid in distilled water
Staining solution
0.2% Coomassie Blue R250 in destaining solution
3.2.7.3 Sample preparation
Fresh or frozen samples were cut into cubes and homogenized
twice using an electric blender for 1 min with an interval of 2 min.
The homogenized solution was then centrifuged for 30 min at 10000
rpm at 0°C. The supernatant was poured into dialysis tubing and
dialysed against distilled water at 4°C for at least 48 h or until
sodium chloride could not be detected by testing the dialysate with
O.lN silver nirate. The dialysed solution was concentrated using
25% polyethylene glycol 6000. When the required volume was reached,
this was redialysed against distilled water. The sample was 59
further concentrated using a 5000 MW cut off membrane (YMS, Amicon) to obtain the desired volume of about 1 mL. This sample is ready for isoelectric focusing.
Processed samples were also cut into cubes and homogenized with cold 1% Triton X-100 (Ajax) at a ratio of 1:1 w/v solid: liquid using an electric blender as described above. The mixture was further homogenized for 20 min using an Ultra-Turrax homogenizer. The sample being homogenized was immersed in iced water to prevent denaturation of protein. Dialysis and concentration procedures as described above were then used.
Brine samples were filtered, dialysed and concentrated using a 30000 MW cut off membrane (YM30, Amicon) to a volume of
1-2 mL.
3.2.7.4 Methods
3.2.7.4.1 Casting the gel
The agarose IEF and sorbitol were dissolved by simmering in water. While the agarose was being dissolved, the leveling table was made horizontal using a spirit level. About 2 mL of distilled water was poured onto the leveling table and a sheet of gelbond with the hydrophilic surface on top was placed onto the leveling table. The film was rolled flat using a glass tube to remove excess moixture and air bubbles beneath it. The plastic film and surroundings were dried with tissue paper. The gel casting frame was placed in position over the plastic filmanu fastened down with spring clips.
The leveling table and plastic film were prewarmed to approximately
45°C using a hair dryer. 60
3.2.7.4.2 Moulding
After all the agarose IEF had been dissolved, the mixture
was allowed to cool to around 75°C. Pharmalyte was added and mixed
thoroughly before pouring onto the mould. The gel was allowed to
set for 15 min and a scalpel was run around the edge of the casting
frame and the frame and gel were removed from the leveling table.
The prepared gel was left for 1 h at 4°C or overnight at room
temperature to harden fully before use.
Samples homogenized with 1% Triton X-100 (non-ionic
detergent) were spotted on gels containing a similar concentration
of the detergent. TX-100 was added just before pouring the gel mixture onto the mould.
3.2.7.4.3 Running the gels
About 2 mL of distilled water was poured onto the cooling
plate and the gel was put over it, ensuring that the water spread
to a thin film under the gel plate. The excess was removed with
paper towel.
Electrode strips were soaked in appropriate electrode
solutions and blotted on filter paper for about 80 sec to remove excess liquid. Electrode strips for gels with detergent were soaked
in electrode solutions containing 1% TX-100.
25 ~L samples were applied using the paper sample applicators. The constant power supply was set to deliver a maximum of 15 Wand 1500 V, current unlimited. The experiment was run for
I! h with a coolant temperature of 8-l0°C. The sample applicators were removed after 45 min. 61
3.2.7.4.4 Fixing and staining
The gel was fixed immediately in the fixing solution for
30 min and washed in two lots of destaining solution for 45 min each. It was then dried by placing three layers of filter paper on top of it, followed by a glass plate and a weight of about 1 kg.
After 15 min, they were removed and the gel was dried with a hair dryer. It was then placed in the staining solution for 30 min and destained until the back-ground is clear (overnight). Finally, the film was dried again with a hair dryer.
Gels with detergent were fixed in 10% TCA, 33% ethanol in water for 30 min. They were then washed in aqueous 5% TCA and
33% ethanol twice for 45 min each or until the precipitate is dissolved.
After washing, the gels were placed in the destaining solution of methanol: acetic acid: distilled water (3:1:6) for 30 min, stained and destained in similar manner ?S those gels without the detergent.
Staining solution was methanol: acetic acid: distilled water (3:1:6).
Methods in gel casting, moulding, fixing and staining were adopted from Agarose IEF leaflet by Pharmacia Fine Chemicals with slight modification to suit the samples used.
3.2.7.4.5 Determination of isoelectric point (pi)
Pharmacia Fine Chemicals broad pi kit (pH 3-10) made up of 11 proteins was run routinely with all gels. The migration distances of protein bands of both the samples and calibration standards were measured from the cathode end of the gel to the centre of the band
(middle of the peak). Measurements were made from the densitometric scan of the gel except those samples having very poor separation where scanning was not possible. Protein migration was expressed 62
as distance from the cathode.
The pH gradient profile curve was constructed by plotting the distance from the cathode of the standard proteins against their corresponding pi. The pi of each protein fraction of jellyfish was measured from the standard pH gradient profile plot (Appendix
1).
3.2.7.4.6 Densitometric analysis
The electrophoretic patterns obtained from isoelectric focusing analysis were scanned using a Kenic Desitometer (Atago
Co. Ltd., Tokyo, Japan) with a recorder (Omniscribe Series D5000,
Houston Instrument, Houston, Texas, M-D5216-15) attached. The operating conditions used were:
Wavelength: 590 nm
Slit dimensions:
Width: 0.5 mm
Length: 4.0 mm
Range: 1.5-2.0
Chart speed: 5 cm/min
The protein bands to be scanned were aligned carefully above the slit on the scanning platform of the desitometer. Then, a clear portion of the gel was put directly above the slit to adjust the zero output of the desitometer and to set the baseline of the recorder. After this, the darkest band was selected and the attenuation of the recorder was adjusted to attain full scale pen movement.
Scanning was started from the cathode end of the plate and moved towards the anode side. 63
3.2.8 Amino acid analysis in jellyfish
3.2.8.1 Acid hydrolysis
0.5 g freeze-dried sample was placed into a glass culture tube fitted with screw cap with Teflon liner (Pyrex No. 9825 and
9826). The amount of sample analysed was equivalent to 50 mg protein and 2 mL of 6N HCl was added. Air was removed from the tube by nitrogen flushing for 1 min and the sample was subsequently frozen in acetone-dry ice. The tube was tightly sealed and hydrolysis was carried out in an air oven at ll0°C for 24 h. After hydrolysis, the solution was dried on a rotary film evaporator to remove the
HCl. The residue was resuspended in trisodium citrate dihydrate buffer (Buffer A, see below) and the hydrolysate filtered through
Whatman No. I filter paper to remove humin. The sample was transferred to a 25 mL volumetric flask and made up to volume with buffer A.
3.2.8.2 Sample preparation for chromatography
A Sep-pak cartridge (Waters Assoc.) was activated c18 with two 10 mL volumes of methanol and then washed with two 10 mL volumes of double distilled water. This was further washed with
10 mL water : methan61~80:20). 1 mL sample was mixed with 2 mL water:: methanol (70:30) and then passed through the Sep-pak cartridge.
The first 1 mL was discarded and the next 2 mL were collected.
20 wL of this sample was injected on a Waters HPLC Amino Acid
Analyser comprised of the following systems; Ninhydrin AAA system consisting of 2M510 pumps, a dual channel M440 absorbance detector,
M710B sample processor, M680 automated gradient controller, M730 data module, column heater, post-column reaction system, and a specially designed reactor and heater. The conditions used during 64
the analysis were as follows:
Column: Waters AAA, 4.6x250 mm Part No. 80002
Column temperature: 55°C
Reactor temperature: ll0°C
Eluents: A 0.066 M Trisodium citrate, pH 3.08
B 0.066 M Trisodium citrate and l.OM
Sodium chloride, pH 6.45
Reagent: Ninhydrin prepared as described below
Wavelength: 546 nm (436 nm for proline)
Elution time: 100 min
Gradient used:
Time Flow % A % B Curve
0 0.5 100 0
45 0.5 20 80 8
60 0.5 0 100 8
84 0.5 0 100 6
86 0.5 100 0 6
An amino acid standard mixture (0.5 uM/ml) was injected in a similar manner as the samples. Amino acid concentration was expresed as g amino acid/16 g N.
3.2.8.3 Reagent preparation
3.2.8.3.1 Ninhydrin
750 mL of DMSO (Pierce 20687) was poured into a 2 L amber glass bottle and sparged for 10 min with high purity, ammonia free, nitrogen. 18 g of ninhydrin (Pierce 21003) was added and 65
sparging continued until all materials were dissolved. 1 g hydrindantin (Pierce 24000) was added and sparging continued.
Then 250 mL 4 M lithium acetate buffer pH 5.2 (Pierce 27203) was added, sparging continued for 15 min. The reagent was stored under nitrogen for 12 h prior to use.
3.2.8.2.2 Buffer A (Trisodium citrate dihydrate)
800 mL of distilled water purified by the Milli Q system to remove ammonia was added with 19.6 g of Trisodium citrate dihydrate.
After dissolution, 3 drops of Pierce buffer preservative 27207
(5.0 mg/mL of pentachlorophenol in ethanol) was added and pH adjusted to 3.08±0.02 with 11 mL nitric acid GR. This was made up to 1 L with water and final pH readjusted to 3.08 if necessary. The buffer was then filtered through Millipore Durapore filter HVL
P04700, sparged for 20 min with high purity nitrogen and blanketed with the same until required.
3.2.8.3.3 Buffer B (Trisodium citrate dihydrate)
800 mL of distilled water purified by the Milli Q system to remove ammonia was added with 19.6 g of Trisodium citrate dihydrate and 58.4 g of sodium chloride (Analar). After dissolution, 3 drops of Pierce buffer preservative 27207 was added and the solution made up to 1 L. Nitric acid was added dropwise to adjust pH to
6.45. The buffer was filtered using a Millipore HVL P04700 filter, sparged for 20 min with high purity nitrogen then blanketed with the same until required for use. Reagent preparation and instrument operationwereadapted from Instruction Manual and leaflets by
Waters Associates. 66
3.2.9 Structural examination of jellyfish tissues
3.2.9.1 Light microscopy
Small pieces of jellyfish (fresh and processed samples) were preserved in 3% glutaraldehyde in 0.05M cacodylate buffer with pH 7.1 at 4°C for approximately 12 h. They were dehydrated in alcohol and xylol and embedded in paraplast then sectioned to 4-5 ~m (Standard Method in Drury and Wallingto~ 1980). The sections were stained with Mallory-Heidenhain Stain (Cason, 1950), and mounted on slides. The sections were dried and photographed in black and white using a Leitz compound photo-microscope in the School of Botany, University of New South Wales.
3.2.9.2 Freeze-fracture technique and electron photomicroscopy
Fresh jellyfish were given a starvation treatment in 20%
glycerol for at least 4 h at 4°C. They were then frozen on gold planchettes in liquid nitrogen under vacuum (-210°C). The replicas were made with a Balzer BHF 400 equipped with electron gun beam. Freeze-fracturing were performed at -170°C and freeze etching at -100°C for 2 min at a vacuum of 2xl0-6 torr. They were shadowed uni-directionally (45° angle) and freeze-fractured specimens replicated with platinum/carbon.
After the specimens have thawed out, the gold planchette was slowly immersed at a slight angle in distilled water and transferred in 70% sulphuric acid. After 24 h, the replica was transferred to distilled water and washed for 5 min three times.
The washed replica was recovered with a specimen support grid.
The replica was examined using a Philips 300 electron microscope 67
and photomicrographs taken. Details of this method were supplied by the freeze-fracture section of the Electron Microscopy Unit,
UNSW).
3.2.10 Brine microbiological assay
Brine samples were taken from storage buckets using sterile flasks. Serial dilutions were made by aseptically transferring
100 mL brine samples into a stomacher bag and homogenized (Lab-
Blender 400) for 30 sec. The spread plate technique with 0.1 mL of each dilution was used for all assays. Analyses were done in duplicate for each sample.
The standard plate count was obtained using malt extract agar incubating at 20°C for 5 days before counting.
Salt and alum used for processing were likewise assayed.
Salt and alum mixture (10 g) was dissolved in 90 mL 0.1% sterile peptone and homogenised in a stomacher bag.
3.2.11 Brine recycling
Spent brine (first batch) obtained from salt-alum treated samples was used in curing a new batch of jellyfish. The ratio of jellyfish to brine was 1:1, w/v.
The brine (second batch) produced during this stage was again used to cure another batch of fresh jellyfish. This was repeated for a third batch and the fourth cycle was a combination of one part each of second and third batches brine. Moisture content, water activity, salt, pH and aluminium were determined on each batch after 48 h brining and 9 h drying at 30°C. 68
3.2.12 Microbial changes during spoilage of fresh jellyfish
Jellyfish were left in an open container on a bench at ambient temperature. Samples (10 g) were taken at intervals up to 48 h at which point the product was putrid.
The samples were homogenised with 90 mL 0.1% sterile peptone in a stomacher bag for 30 sec. Serial dilutions were made by aseptically transferring 1 mL of the homogenised sample to the 9 mL of 0.1% sterile peptone in test tubes. These were shaken thoroughly and 0.1 mL of this was spread on plates with plate count agar containing 3% salt. All assays were done in duplicate. The inoculated plates were incubated at 25°C for 24 h prior to colony counting. 69
4. RESULTS AND DISCUSSION
4.1 Chemical Composition and Properties of Fresh Jellyfish
4.1.1 Proximate analysis
As baseline data for succeeding determinations, proximate
analysis of fresh jellyfish was required. Table 11 shows that
moisture content of fresh jellyfish ranged from 96.04-96.27% with
a mean value of 96.13% and did not vary significantly with size.
This value is close to that found by several previous workers
(Wootton et al. 1982; Ludwig, 1977; Lowndes, 1942; Sipos & Ackman,
1968; Chapman, 1953) for various species. Moisture content of
medusa have however been reported to be as high as 98.8% and as
low as 92.9% depending on species and salinity of the waters (Joseph,
1979). This latter factor was investigated by Ludwig (1977) who
reported that A. aurita collected from the Baltic Seas near Greifswald where salinity was only 0.7% had moisture content of more than
98% whereas, the same medusa contained 95.56% moisture in sea water of 3.5% salinity. Craspedacusta sowerbyi, a fresh water medusa contained 99.26% moisture (Ludwig, 1977).
Protein content had a mean value of 1.12%, close to
1.3% as by Wootton et al. (1982), and ranged from 0.91% in small
specimens to 1.34% in larger ones although these results were not statistically significant. Jellyfish proteins have been fuond
to contain collagen-like components to a large extent (Piez & Gross, 1959; Gosline and Lenhoff, 1968; Nordwig & Hayduk, 1969;
Rigby and Hafey, 1972).
The fat content of Catostylus sp. was very low, ranging
from 0.06-0.23% with a mean value of 0.13%. This is within the 70
Table 11. Chemical composition (as is basis) of fresh jellyfish
of different sizes +
Small Medium Large Mean (250mm) (320mm) (410mm) (330mm)
Moisture (%) 96.27 96.04 96.09 96.13
Protein (%) 0.91 1.11 1.34 1.12 Fat (%) 0.06 0.11 0.23 0.13 Salt (%) 1.68 2.51 2.49 2.23 Ash (%) 1.36 1. 74 3.14 2.08 pH (%) 7.31 7.48 7.59 7.46
Analysis of variance:
Moisture - NS (not significant)
Protein - NS
Fat - * (P<0.05)
Salt - **(P Ash - NS + Small - 1200g Medium - 1700g Large - 3500g Mean - 2133g 71 range (0.0046-0.2%) reported by Yasuda (197~) and Hooper and Ackman (1973). Fat content in this study varied (P that C. capillata, contained only a low level of lipids but its fatty acid composition are characterized by a high proportion of monoenoic and polyunsaturated fatty acids and less saturatd fatty acids. A similar finding was reported by Joseph (1979) on the fatty acid composition of C. capillata, collected in Terence Bay, Nova Scotia. Salt content of fresh jellyfish ranged from 1.68-2.49% with a mean of 2.23%. This is comparable to 2.1% as reported by Wootton et al. (1982). Size significantly (P Ash levels ranged from 1.36-3.14% with a mean value of 2.08%, close to 2.5% as found by Wootton et al. (1982). No relation of ash content with size was apparent. pH of fresh jellyfish varied slightly between the samples but fell close to neutrality. No report on jellyfish pH has been published previously. 4.1.2 Total amino acids Table 12 shows the amino acids in untreated jellyfish after acid hydrolysis. It can be seen that glutamic acid, glycine, aspartic acid and lysine are the most abundant amino acids present. Next in quantity are threonine, proline, alanine, leucine and arginine. Of special significance is the presence of hydroxylysine, proline and hydroxyproline. However resolution of the last amino acid 72 Table 12. Concentration of amino acids in proteins of untreated jellyfish compared with collagen Amino acid Concentration Jellyfish Collagen* (gAA/16gN) (gAA/lOOg protein) Aspartic acid 7.8 6.0 Threonine 4.4 2.2 Serine 3.4 4.2 Glutamic acid ll.4 11.0 Proline 5.2 13.0 Glycine 9.2 26.1 Alanine 5.2 9.9 Cystine 0.9 0 Valine 3.8 2.3 Methionine 1.6 0.8 Isoleucine 3.2 1.4 Leusine 4.9 3.2 Tyrosine 1.9 0.5 Phenylalanine 2.2 2.0 Hydroxy lysine 1.7 1.3 Lysine 7.4 3.6 Ammonia 13.8 0.6 Histidine 1.2 0.3 Arginine 5.6 8.3 Hydroxyproline + 13.6 * Adapted from Haurowitz (1963). Soluble collagen extracted from cafskin + Detected but not quantified 73 was poor so its quantification was not possible. (A typical chromatogram of the amino acid in jellyfish protein hydrolysate may be found in Appendix 2). The presence of these latter amino acids and the large amount of glycine indicate that jellyfish contains proteins of the collagen group. These findings are in agreement with those of Spiro (1970); Barzansky et al. (1975); Kalyani (1973); Chapman (1953); Rigby and Hafey (1972); Quensen and Black (1974-75) who reported collagenous materials in the mesoglea of Cnidarians. Other researchers who found that mesoglea of Cnidarians contains collagen-like protein include Piez and Ross (1959); Gosline and Lenhoff (1968); Nordwig and Hayduk (1969); Katzman and Jeanloz (1970). An interesting feature was the presence of cystine in Catostylus. According to Kalyani (1973), apart fromCnidarians, the only group in which cystine was reported is Annelida (Rhinodrilus). He opined that the presence of cystine might indicate the presence of disulphide links in the collagen molecule. The essential amino acids in jellyfish are present in low proportions when compared with the non essential amino acids. Lysine however, is an exception being quantitatively comparable to aspartic acid. No tryptophan was detected but since this amino acid is labile, any present in jellyfish may have been destroyed during acid hydrolysis. Hultin (1976) however reported that tryptophan is almost totally absent from collagen, tending to support the conclusion that jellyfish contains collagen-like proteins. It should be noted that there were unidentified components detected in the chromatogram. 74 The amino acid pattern of Catostylus mosaicus was similar to those of Aurelia aurita and Physalia physalis (Lane et al. 1965), Hydra ehrysaora quinquecirrha, Aurelia coerula and Virgularia (Barzansky et al. 1975; Quensen and Black, 1974-75; Rigby and Hafey, 1972; Kalyani, 1973). This was especially so for levels of glutamic acid, glycine and aspartic acid. It should also be noted that Cnidarians contained proline, hydroxyproline and hydroxylysine (Barzansky et al. 1975; Rigby and Hafey, 1972; Kalyani, 1973; Chapman, 1953). The amino acid composition of collagen is also presented in Table 12 to allow comparison with that of jellyfish protein. Obvious similarities occur between the two sets of data. Collagen is an unusual protein in that glycine comprises over 25% of the total amino acids and is unique in being the only protein that has large amounts of hydroxyproline, proline and hydroxylysine. Collagen however, has a poor spectrum of amino acids (Hultin, 1970); tryptophan being almost totally absent. On the other hand, the partially denatured product of collagen, gelatin, is a useful ingredient in many food products since it serves as the basis for temperature-dependent gel type desserts. 4.1.3 Isoelectric focusing of proteins Jellyfish proteins were separated and their isoelectric points (pi) determined using an agarose gel isoelectric focusing technique. Results are presented in Table 13. It can be seen that 15 protein fractions were separated from fresh jellyfish with pi ranging from 5.0-8.65. Of this number, 8 were acidic and the remaining 7, basic. The resolution of protein fractions 75 Table 13. Isoelectric point of protein fraction in fresh jellyfish separated by isoelectric focusing Number * pi 1 8.65 2 8.50 3 8.45 4 8.30 5 8.10 6 7.70 7 7.50 8 6.95 9 6.75 10 6.20 11 6.00 12 5.30 13 5.20 14 5.10 15 5.00 * Number 1 represents the protein fraction farthest from the cathode end and number 15, the nearest. 76 Gfi fresh jellyfish on agarose gel is shown in Plate No. 1 and its densitometric scan in Appendix 3. 4.1.4 Soluble solids, solubility and digestibility of proteins The water solubility of jellyfish solids was determined using the method described by Lees (1968) in Section 3.2.5.4.6. The fresh jellyfish contained very low soluble solids (4.8%). This was expected since the fresh jellyfish contain over 95% water and its solids are comprised mainly of proteins and mineral matter (Table 11). Solubility of proteins was also evaluated using different solubilising agents. With SDS+l% B-mercaptoethanol, the solubility was 27% of total protein, O.lM KCl 5.4%, pepsin 32.4%, and 5% salt solution also 5.4%. These results show that KC! and salt solution are not as effective solubilising media as SDS and pepsin. This will be discussed further in Sections 4.6.2 - 4.6.4. The indigestible proteins in fresh jellyfish was also determined by pepsin digestion and comprised about 6% of the total protein. 4.2 Preliminary Study of the Traditional Processing Method 4.2.1 Effect of alum and salt as preservatives The traditional process consists of three brining steps: 2 days with 40% salt and 50% alum, 19 days with SO% salt and the rest of the alum and 2 days in the remaining 10% of the salt as a saturated solution. These steps are interspersed with draining for 2 hand are followed by air drying. Based on this process, the preservative effects of salt and alum were evaluated individually and in combination. 77 l!tl Cl : F· Plate 1. Electrophoretic patterns of protein from fresh jellyfish separated by IEF of pH 3-10 on 1% agarose gel. 78 4.2.1.1 Alum alone In this initial study, three concentrations of alum were used (2,6,10%) for processing. 50% of each concentration was added for the first 48 h of processing and the second half duringtne second stage of the process. After 48 h in alum, the samples had very soft and lumpy texture. Jellyfish treated with the highest alum concentration had the softest texture. At the end of the second stage of processing, the tissues started to disintegrate and undesirable odour could be detected for all treatments. These observations were in agreement with the findings of Iljas and Arifudin (1971) and Wootton et al. (1982). The third stage of processing was not carried out on these samples due to liquifaction. On subsequent storage, mould growth developed on the surface of the liquid mixture as observed previously (Wootton et al., 1982). Alum has been widely used in food processing and preservation with various effects being attributed to it (Windholz, 1976; Mark et al., 1978; Winter, 1978; Etchells and Jones, 1942, 1944; Etchells, 1938). However, when used alone it is not effective as a preservative agent for jellyfish. 4.2.1.2 Salt alone Three different concentrations of salt (25,30,35%) were added to jellyfish samples. 40% of the total salt was added for the first 48 h of processing and the remaining amount at the second (50%) and third stages (10%) of the process. 79 Salt treated jellyfish had also very soft texture after 48 h brining and at the end of the second stage of processing, extensive liquifaction was observed. There was not much difference on the degree of tissue disintegration as a function of salt concentration, but a very disagreeable odour was detected in all treatments. Off odours were also detected by Wootton et al. (1982) who used 30% salt alone. No mould growth developed on the surface of brine produced during processing. The beneficial effects of salt as preservative of fishery and other products is widely known. Salt is a preservative at high concentrations and levels of 18 to 25% in solution will generally prevent all growth of microorganisms (Potter, 1968). This was manifested by the absence of mould growth on the brine produced during processing in this study. However, it is not effective when used alone in preserving jellyfish due to tissue breakdown and off-odour development. 4.2.1.3 Alum-salt combination Various mixtures of salt and alum (25, 30, 35% salt combined with 2, 6, 10% alum) were used in this initial study. Jellyfish treated with a mixture of salt and alum had an intact structure throughout processing. It appears that both are important components in order to produce desirable texture and structure in salted jellyfish. Weight loss at various stages of processing and final yield are presented in Table 14. After the first 2 days of brining, weight loss ranged from 60-64%, rose to 73-85% at the end of the 80 Table 14. Weight loss and yield of salt-alum treated jellyfish during the stages of processing Treatment % weight loss after processing stage: (salt %, alum %) 2 days 19 days 35 days Final yield (%) 25, 2 63.0 77.5 94.3 5.7 25, 6 62.2 83.0 94.2 5.8 25, 10 60.2 75.3 92.5 7.5 30, 2 61.6 84.8 94.9 5.1 30, 6 64.2 77.2 92.6 7.4 30, 10 59.8 72.8 91.0 9.0 35, 2 60.7 74.2 93.2 6.8 35, 6 60.2 73.4 92.2 7.8 35, 10 60.8 82.4 93.2 6.8 Analysis of variance Treatment ** ** (Significant at P The highest yield was obtained from samples treated with 30% salt mixed with 10% alum and the lowest from samples treated with the three levels of salt mixed with 2% alum. This effect of alum on yield is the reverse of the findings of Iljas and Arifudin (197l).who found that the higher the level of alum used in processing, the lower the yield. Differences in values obtained could perhaps be attributed to environmental factors (RH, temperature) and 81 jellyfish species employed during processing. The yield obtained (5-9%) are lower than those of Wootton et al. (1982) (23-25%) but are higher than those reported by Iljas and Arifudin (1971) (4-5%). The low yield in this study was a consequence of high moisture loss due to low RH during air drying. Salted jellyfish obtained using the traditional 35 day process had a very brittle texture with salt crystals aggregating on the surface of the finished product. The colour changed from the very light brown before air drying to yellowish brown after 14 days drying at room temperature. The finished product likewise, became very thin as indicated by the very low yield. The chemical composition of jellyfish treated with various levels of salt and alum mixtures is shown in Table 15. Jellyfish treated with 30% salt and 2% alum had the lowest moisture content and the highest was that of samples treated with 35% salt and 6% alum. Moisture content of the 9 treatments ranged from 10-27%. These values are very much lower than those reported by Iljas and Arifudin (1975) and Wootton et al. (1982). This could be attributed to ambient drying conditions alluded to above. The moisture content of the final product was significantly (P The water activity (a ) of salted jellyfish ranged from w 0.59-0.68 and varied significantly (P The highest a was also that of sample having the highest moisture w content and the lowest was that of 25% salt and 6% alum-treated samples. Salt, alum and the two combined had highly significant (P Table 15. Chemical composition (dry basis) of salted jellyfish treated with different concentrations of salt and alum mixture Treatment Moisture a w Protein Salt Aluminium (salt %, alum %) (%) (%) (%) (%) 25, 2 16.9 0.60 8.4 53.4 0.2 25, 6 14.2 0.59 6.8 56.9 0.1 25, 10 17.6 0.60 6.8 55.2 0.2 30, 2 10.4 0.62 6.2 47.0 0.1 30, 6 19.6 0.66 6.0 58.0 0.1 30, 10 20.5 0.65 6.0 62.0 0.1 35, 2 15.0 0.65 5.6 54.0 0.1 35, 6 26.6 0.68 7.0 70.4 0.2 35, 10 20.2 0.63 6.1 60.8 0.1 Analysis of variance: Treatment ** ** ** ** ** Salt * ** ** ** ** Alum ** ** ** ** NS Saltxalum ** ** ** ** *i~ ** (Significant at P Protein of salted jellyfish ranged from 6-8%. Jellyfish treated with the lowest salt and alum levels (25% salt, 2% alum) gave the highest protein and the lowest was that of samples treated with the highest salt and lowest alum levels (35% salt, 2% alum). The protein levels obtained in this study were similar to those of Wootton et al. (1982) on the same species and Iljas and Arifudin (1975) on an unspecified species of jellyfish. Protein content of the 9 treatments varied significantly (P (P Products of:all treatments had salt levels ranging from 47-70%. The final products did not inrease in salt concentration in parallel with that of salt levels used for treatment. However, statistical analysis showed that salt content was significantly (P Salt content in this initial study was almost twice those reported by Iljas and Arifudin (1975) and Wootton et al. (1982) and almost certainlyreflectslower moisture content. As with salt, the aluminium level also varied among the treatments. It ranged from 0.1-0.2% and was significantly influenced ~P 4.2.2 pH of jellyfish and preservatives used during processing pH of fresh jellyfish is approximately neutral (Table 11) however, during processing, it decreases substantially. In order to investigate this decrease, the influence of salt alone, alum alone and salt/alum on the pH of jellyfish was examined. These results are shown in Table 16 which also includes pH of the salt, alum and salt/alum solutions of concentrations equivalent to levels applied to the jellyfish (15%, 5% and 15%, 5% respectively). It can be seen that salt alone is close to neutrality while alum andthesalt/alum mixtures had a pH of approximately 3. Salt and alum were used singly and in combination to treat separate batches of jellyfish over a 48 h period and it is evident that alum is responsible for the decrease in pH of jellyfish whether alone or together with salt. The salt/alum mixture gave a slightly lower pH than alum alone in line with the lower pH of a solution of the mixture as compared to alum alone. Thus, alum not only has important effect on the texture of the product but causes a reduction in pH. When combined with salt, the effects of this must greatly enhance the microbial stability of the salted product even when stored at ambient temperature. Acidity is widely used in food preservation and most often synergistically with a wide range of other factors including reduced water activity, low temperatures and salt. It has also been implicated in affecting the texture of muscle foods such as meat and fish (Dunagski, 1979; Kelly et al., 1966; Cowie and Little, 1966; Love et al., 1974). 85 Table 16. pH of salt, alum, salt-alum mixture, jellyfish flesh and brine after 48 h curing Treatment pH Salt (15%) 6.24 Alum (5%) 3.09 Salt-alum mixture (15x5%} 2.55 Salt-cured: flesh 6.37 brine 6.13 Alum-cured: flesh 3.14 liquid 3.03 Salt-alum cured: flesh 2.63 brine 2.59 Control (untreated): flesh 7.36 liquid 6.77 4.3 Optimisation of Salt Levels Results presented inSection4.2.1.3 clearly show that salt and alum must be used in combination to preserve jellyfish. Further work was carried out in order to establish optimum levels of salt forthetreatment of Catostylus. Alum levels of 5% were adopted as standard because this quantity was deemed sufficient to ensure the desirable changes in texture and adequate decreases in pH. Levels between 2-10% did not cause significant variations in product properties even allowing for variations in raw material. Salt concentration ranging from 15-35% with 5% alum 86 were used to treat jellyfish using the traditional process. Changes in chemical composition, yield and organoleptic properties of the product during processing were evaluated. 4.3.1 Moisture content, a and weight loss 'W Changes in moisture content and bell weight using various salt levels during the 35 day process are shown in Table 17. After the first 2 days of brining, bhe mo±sture content of the 5 treatments ranged from 88-92% with samples treated with 20% salt being the highest, and samples treated with 30% salt, the lowest. At the conclusion of brining, samples treated with the two highest salt concentrations had the lowest moisture content and samples treated with the lowest salt (15%) concentration were highest in moisture content. This was to be expected since greater proportions of salt employed during brining should lead to faster and more complete loss of moisture. ;ffihiis has been demonstrated for other foods, for example Narayanaswamy, Rao and Govindan (1980) showed that higher salt levels accelerated absorption of salt and shedding of moisture during salting of fish. After the samples were air-dried for 7 days and 14 days respectively, moisture contents did not vary significantly between treatments. These samples also had similar a (approximately w 0.70 and 0.45) paralleling their moisture contents. The values obtained after 14 days drying were very much lower than those reported by Wootton et al. (1982) and Iljas and Arifudin (1975). This is attributed to the lower relative humidity prevailing at the time of the present study. It is obvious that 7 days only was needed to achieve a sufficiently low a under these conditions. w 87 Table 17. Changes in moisture content and bell weight during processing with 5% alum and different salt levels Salt level Moisture content (%) and % original bell weight after 0 day 2 days 19 days 28 days 35 days (first (second (7 days (14 days (fresh) brining) brining) drying) drying) 15 96.1,100 91.9,53.4 80.6,23.5 45.2,5.5 4.3,3.7 20 96.1,100 92.3,51.6 77.4,24.6 44.2,7.5 4.5,4.6 25 96.1,100 90.0,57.4 76.0,27.0 34.8,8.4 3.9,5.1 30 96.1,100 87.7,49.8 74.2,22.9 35.6,7.3 4.6,4.4 35 96.1,100 87.8,49.6 74.2,20.8 41.7 ,6.9 3.8,4.6 Analysis of variance: Moisture - NS (not significant) Bell weight - NS Thus, salt levels did not have a significant effect on the moisture content of the finished product. These changes in moisture content are paralleled by decreases in bell weight during processing. Clearly, much weight loss, predominantly water, occurs during all stages of the process but does not appear to be influenced by salt level. 4.3.2 Protein The protein content of jellyfish treated with various concentrations of salt was determined after the second brining and 14 days of drying. These values ranged from 1.96-2.48% and 88 6.76-9.71% respectively. Protein content differed significantly (P had somewhat higher protein contents than those of 5-6% and 6.7% found by Iljas and Arifudin (1975) and Wootton et al. (1982) respectively. This is again attributed to the low moisture content of the final product. 4.3.3 .£!! The changes in pH of jellyfish during processing are shown in Figure 3. It can be seen that after the first 2 days of brining the pH of the 5 treatments ranged from 3.4-3.8. At conclusion of the brining stages (21 days), pH of the 5 treatments ranged from 2.3-3.1 with the 15% salt treatment giving the higest pH. In the final products, the pH of all treatments were identical having increased to approximately 4.0. This is attributed to the decrease in alum between the conclusion of the second brining and the final product. This is discussed in Section 4.3.4. Results indicated that salt concentration did not influence the pH of the final product. The low pH observed is no doubt an important contribution to the stability of this material against microbial attack. This aspect has been discussed in Section 4.2.2. 4.3.4 Salt and aluminium levels Salt and aluminium levels in jellyfish at various stages of processing with different salt levels are shown in Table 18. After the first brining, salt content ranged from 5-7% with samples treated with the lowest salt concentration (15%) being lowest and those with highest (35%) containing the greatest salt levels. 89 5 KEY 4. 5 -&--&- 15% salt +-+- 20% salt ~25% salt -a---Er 30% salt +---+- 35% salt 4 IJ) Cll :J ...... c > 3. 5 :::c a.. 3 2. 5 2~~~~--~--~--~--~--~~--~---L--~--~--L---~~--~ 0 5 10 15 20 25 30 35 40 Tima (days) Figure 3. pH changes of jellyfish during processing 90 Table 18. Salt and aluminium contents in salted jellyfish during processing Treatment % salt and Al in jellyfish after processing stage (salt %, Al %) (1) (2) (3) 15, 5 5.32 0.03 7.66 0 .. 58 60.77 0.18 20, 5 5.85 0.04 11.86 0.38 72.22 0.12 25, 5 6.42 0.03 13.80 0.38 69.90 0.14 30, 5 6,80 0.03 15.53 0.40 69.58 0.09 35, 5 7.14 0.03 17.80 0.46 67.21 0.16 Analysis of variance: Salt - ** (P A similar trend was obtained after the second brining as expected. No definite pattern could be seen in the dried product although those treated with 15% salt still had the lowest salt content. The 5 treatments differed significantly (P All products had salt crystals aggregating on the surface, and texture was very brittle. It is likely that some loss from the surface could have occurred and affected these results. Only one concentration of alum (5%) was used in this experiment. After the first brining stage, the aluminium contents ranged from 0.03-0.04% (Table 18). After the second brining, there was a substantial increase in aluminium content which now ranged from 0.38-0.58%. Samples treated with 15% salt were 91 the highest, and samples treated with 20 and 25% salt, the lowest in aluminium content. The final products had less than one-third of the aluminium content of those after the second brining. This could be due to loss during the final short soaking in saturated brine with no alum added. The values ranged from 0.09-0.18% with samples treated with 15% salt being the highest. These figures are high compared to those obtained by Wootton et al (1982) and Iljas and Arifudin (1975). Variations in results could be attributed to lower mo~sture content of the products in this study. Although only one level of alum was used for treatment, statistical analysis showed that the aluminium content of the final products varied significantly (P<0.05) in relation to salt level used in processing. Thus, it appears that salt levels used for the treatment of jellyfish have a significant influence on the salt and aluminium levels of the final dried product. Regardless of the salt levels used, salt and aluminium contents were high compared to those reported by Iljas and Arifudin (1975) and Wootton et al. (1982), no doubt reflecting the lower moisture content obtained in the present study. 4.3.5 Organoleptic properties Salted jellyfish produced using various salt levels during 35-day processing were evaluated by taste panel for texture and flavour. Details ofthemethods could be found in Sections 3.2.3-3.2.3.1. 92 4.3.5.1 Texture Mean values of rank scores in texture are presented in TabLe 19. A significant variation was detected among the five treatments. It was found that samples treated with 15% salt are significantly preferred (P<0.05) in texture compared to the other samples. This treatment had a slightly elastic but crispy texture; a desirable attribute for this particular product. Other samples were quite rubbery and hard in texture. Jellyfish is eaten mainly for its texture thus, in processing, the most important consideration that a processor should be aware of is to attain the acceptable texture in a product. Results revealed that salt concentration had a significant effect on texture of salted jellyfish with the lowest salt level producing preferred products. 4.3.5.2 Flavour Table 19 also shows the mean values of rank scores in flavour of jellyfish. Significant variations occurred (P Results showed that salt concentration caused a significant variation in flavour of salted jellyfish. The product has a bland flavour which may be enhanced by addition of sauces or mixing it with other foods like meat, chicken and vegetables. 93 Table 19. Mean values of rank scores in texture and flavour of jellyfish salad Treatment Texture Flavour (salt %, alum %) 15, 5 +0.51 +0.33 20, 5 -0.22 +0.60 25, 5 0 +0.10 30, 5 -0.61 -0.58 35, 5 +0.32 -0.45 Analysis of variance: Samples * ** Panelists NS NS * (significant at P<0.05) ** (significant at P Thus, based on this study of the effects of salt levels on yield, composition and organoleptic properties, a combination of salt (15%) and alum (5%) was chosen as optimal for the processing of Catostylus sp. 94 4.3.6 Protein in brine Results described above have shown that a level of 15% salt inthetraditional process gave the best dried salted jellyfish product in terms of its texture and flavour when Catostylus sp. was used. Because this process embodies three brining steps of 2 days, 19 days an 2 days respectively, there exists the possibility that material, most likely protein, may be extracted from the jellyfish into these brines. This is of great importance because of its implications in terms of mass balance, it complicates waste disposal and there may be potential uses for such material. Hence, the brines from the three stages were analysed for protein content for each of the five salt levels used. Table 20 shows the protein contents of each brine for the five salt level experiments.The first brines had protein contents ranging from 0.09-0.13% which donotvary markedly among the 5 treatments. Of the second brines, that with 15% salt had the highest (0.11%) protein followed by 20% salt-treated samples (0.08%). The rest of the treatments all contained only 0.06% protein. This was paralleled by protein levels in the third stage brines. It can be seen that the lowest .sa[t concentration resulted in the highest protein levels in the brine. When these data were anlysed statistically, significant differences were found between salt levels. However, there appeared to be no advantage in selection of salt level for the purpose of minimising loss of protein into the brines. Although the amount of protein in the various brines is quite low, it could contribute to a waste disposal problem for the processor since the traditional process, using solid salts 95 Table 20. Protein at various stages of brining with 5% alum and different salt levels Salt level % protein in brine after brining stage: (1) (2) (3) 15 0.13 0.11 0.09 20 0.13 0.08 0.05 25 0.09 0.06 0.04 30 0.09 0.06 0.04 35 0.12 0.06 0.04 Analysis of variance: Treatment ** ** (Significant at P<0.001) is not readily amenable to brine recycling. Such low levels do not seem to offer much potential for reclamation and subsequent use. 4.3.7 Chemical composition of jellyfish as consumed Commercially available salted dried jellyfish contains approximately 5% protein, 21% salt, 0.05% alum, and 69% water. Quite obviously this composition is not ideal for a food product especially in terms of salt content. The product is typically prepared for consumption by rehydration, shredding and blanching. Presented in Table 21 are analytical data for salted, dried jellyfish 96 Table 21. Chemical composition of salted, dried jellyfish before and after preparation for eating Sample MC pH Protein Salt Aluminium (%) (%) (%) (%) Salted product 23.7 0.68 3.94 10.42 55.30 0.16 Prepared product 90.0 0.99 4.20 6.80 0.26 0.11 before and after preparation for eating as described in Section 3.2.3. It can be seen that salt level in particular has decreased to approximately 0.3%. In terms of protein level, it is similar to foods such as pasta and boiled rice. Moisture content and a w have shown expected increases while pH has also increased. Alum level remained relatively unchanged and is apparently bound to the matrix of the tissues in some way. Thus, as consumed, salted dried jellyfish even if not a nutritionally valuable material, does not appear to offer any harmful effects. 4.4 Effect of Processing Time on the Properties and Yield of Jellyfish The traditional 35-day process employed in previous experiments is probably inappropriate for Australian conditions due to the time and labour commitment involved. Thus, samples were taken at different intervals during the first and second brining steps and dried for various periods under ambient conditions. This investigation would allow evaluation of the necessity for 97 the number and duration of brining steps, and of drying times, in terms of product quality. 4.4.1 Changes during the first brining step Shown in Table 22 are changes in original bell weight and chemical composition during the first brining step. During this step, moisture content decreased to 89.4%, a to 0.95 and w pH to 3.41 with corresponding increases in salt level to 6% and protein content to 0.50%. Because of the changes shown and the relative shortness of the first brining step, there seems little potential for decreasing the length of this process. 4.4.2 Changes during the second brining step Samples were taken after 0, 6, 12 and 19 days of the seconrl brining stage. Changes in % original bell weight and chemical composition are shown in Table 23. It can be seen that during the first 6 days of this step, pronounced decreases in·maisture, aw, pH and remaining bell weight occurred while salt and protein concentrations increased. Subsequently, pH changed little with moisture content and a decreasing slightly. Both salt and protein w contents increased as the second stage brining progressed. This brining step is the longest stage in the process, and changes in composition were relatively slow after the init:tcH 6 days. Thus, samples were taken after 6 and 12 days and dried for periods of 2-14 days in air. The composition of these dried samples are shown in Table 24. It can be seen that moisture content was somewhat lower and protein content somewhat higher for each respective drying time, in the samples brined for the longer period, On the other hand, no substantial differences existed in aw, pH 98 Table 22. Chemical composition and yield of jellyfish bells during the first brining step Brining time MC A pH Salt Protein Yield (h) (%) w (%) (%) (%) 0 96.1 0.99 7.28 2.39 0.35 100 12 93.8 0.98 3.85 3.98 0.37 72.1 24 90.5 0.95 3.36 5.65 0.46 57.1 48 89.4 0.95 3.41 6.00 0.50 41.8 Table 23. Changes in chemical composition and yield of jellyfish bells during the second brining stage Brining time MC A pH Salt Protein Yield w (day) (%) (%) (%) (%) 0 89.4 0.95 3.41 6.00 0.50 41.8 6 80.9 0.86 2.80 10.04 0.68 25.2 12 80.4 0.85 2.69 11.63 0.83 22.8 19 76.6 0.83 2.98 15.18 1.18 20.5 99 Table 24. Changes in chemical composition of jellyfish bells brined for 6 and 12 days and air-dried for 2-14 days Drying time MC A pH Salt Protein (days) (%) w (%) (%) 6 days brining 0 80.9 0.86 2.80 10.04 0.68 2 64.6 0.71 3.71 21.79 1.26 5 45.6 0.70 3.80 34.99 3.26 7 40.2 0.70 3.88 41.18 4.00 10 11.7 0.54 3.83 59.58 5.90 14 10.8 0.53 3.82 67.22 8.54 12 days brining 0 80.4 0.85 2.69 11.63 0.83 2 66.9 0.71 3.90 22.08 2.97 5 39.6 0.70 4.04 44.32 6.04 7 7.8 0.55 4.01 62.49 7.67 10 7.2 0.54 4.10 62.53 8.80 14 6.5 0.52 4.04 69.71 10.23 100 and salt content between the different brining times. Protein content and pH of the final products in both drying time were close to those found earlier but moisture content, a w and salt content were substantially higher. These results indicate that the traditional process may be shortened by reducing the second brining stage to 6 days at least in terms of chemical parameters. It is also apparent that air drying for 2 days under ambient conditions existing at the time of this experiment was sufficient to produce a w and moisture content equivalent to commercial samples (0.73 and 69.3% respectively). 4.4.3 Drying time The final stage of brining was carried out as in the traditional process since it is a relatively short step. At the conclusion of this step, the product was dried for 2-14 days in air. The properties of the product after drying for various times are shown in Table 25. It can again be seen that 2 days drying gave a product of desiredmoisturecontent and water activity. When compared with dried products from the second brining step, these were similar in most respects (Table 24) although somewhat higher in protein. 4.4.4 Organoleptic evaluation of jellyfish Sensory evaluation in terms of texture, flavour and overall acceptability was carried out on the dried samples in the form of jellyfish salad. Statistical analysis of differences on means are presented in Table 26. 101 Table 25. Changes in chemical composition of jellyfish bells after the final brining step and drying for 2-14 days. Drying time MC A pH Salt Protein w (day) (%) (%) (%) 0 75.6 0.81 2.89 16.26 2.10 2 64.4 0.71 3. 77 23.44 4.29 5 47.1 0.70 4.06 39.40 6.16 7 23.7 0.68 3.94 55.30 10.42 10 8.6 0.54 3.86 63.46 12.27 14 ~s;;.4 0.51 3.87 64.51 12.67 Table 26. Mean values of texture, flavour and overall acceptability of jellyfish salad Treatment * Texture Flavour Acceptability 0 2.87 2.98 2.93 2 4.90 4.95 5.84 5 4.18 5.31 4.80 7 4.99 5.13 5.69 10 5.98 5.27 5.34 14 4.31 4.44 4.43 Analysis of variance: NS+ NS NS * Number of days air-dried + Not significant 102 It can be seen that drying time did not influence texture, flavour and overall acceptability significantly. However, overall, samples dried for 2 days appear to have best general acceptance. These samples had superior colour and appearance compared to the other samples. The chemical attributes are comparable to commercial samples. It can therefore be seen that the traditional 35 day process can be shortened by reducing the second brining stage from 19 days to 6 days,and depending on ambient conditions, the drying stage may be reduced from 14 days to 2 days. This represents a total potential decrease from 35 days to 10 days. Howeve~ it should be noted that there is little scope for reducing the labour commitment and the decrease in drying time is subject to variation because of changes in ambient conditions of temperature and relative humidity. 4.5 Approaches for Rapid Processing of Jellyfish Results of experiments described above have demonstrated that although the lengthy, labour intensive traditional process for jellyfish preservation may be shortened, it is still probably not economically viable under Australian conditions. Hence, fresh approaches to both the salting and drying operations were sought. Such approaches must decrease both the time and labour commitments but, at the same time, yield a product which is similar to existing preserved jellyfish. 103 4.5.1 Dry salting of jellyfish Attempts were made to replace the3steps, 21 day brining of the traditional process with a single salting procedure. The method adopted involved treatment of fresh bells with the required amounts of dry salt and alum (15% and 5% of bell weight respectively). Details are described in Section 3.2.1.2. The 48 h brining was chosen based on the previous work of Wootton et al. (1982). It should be noted that within 48 h, the curing salts had dissolved in water from the bells. At the conclusion of brining, the amount of brine present amounted to approximately 480g/kg of fresh bell weight. In this brine which contained 18% salt and had pH of 2.59, the bells remained stable even after long storage. Thus, a period of 48 h at ambient temperature (20-25°C) was chosen as the brining time for this accelerated process. 4.5.2 Artificial drying The drying procedure of 14 days under ambient conditions used in the traditionalprocesswas unsatisfactory not only because of its length but also because of the variation in product yield and quality in response to existing temperatures and relative humidity. Hence, the use of artificial drying of the salted product was investigated. Earlier work (Wootton et al., 1982) showed that 50°C produced an unacceptable dried product from salted jellyfish while at 35°C the product, though acceptable, was less preferred than the traditional one. Hence, this work was restricted to a drying temperature of 30°C. 104 Shown in Table 27 are moisture content and a of salted w jellyfish bells during artificial drying at 30°C for up to 24 h. It can be seen that a and moisture content had decreased to 0.72 w and 65.5% respectively after 12 h. These values correspond to those found in commercially dried product. Thus, it appears that a process embodying brining with dry salt ami alum for 48 h followed by artificial drying at 30°C for 12 h gave products of suitablemois'ture content and water activity. It should be noted that subsequent work showed that, depending on ambient conditions, drying times as low as 9 h were sufficient to decrease moisture and a to required levels. This occurred because the drier used w had no facility for relative humidity control although both temperature and air flow rate could be specified. Chemical properties of this rapid process product may be compared to those of commercial and traditional process products in Table 28. It is obvious that in these terms at least, the rapid process product was satisfactory. This product was also comparable with commercial product in texture, colour and general appearance. These indicate that the rapid process product compared favourably with commercial product hence, the rapid process outlined above was deemed successful in terms of product composition and quality. 4.5.3 Brine properties and recycling As mentioned 'im Section 4,5 .1, the rapid process yields as a by-product, 20% of fresh jellyfish, 480g of brine/kg of fresh bell weight containing 18% salt, 5% alum at pH 2.59. This material represents a waste disposal problem for the processor. It also 105 Table 27. Changes in moisture content and water activity of salted jellyfish during artificial drying at 30°C Moisture A Drying time w (h) (%) 0 90.6 0.95 3 89.8 0.94 6 74.4 0.82 9 72.2 0.78 12 65.5 0.72 15 63.1 0.71 18 61.4 0.71 21 57.8 0.71 24 55.5 0.69 Table 28. Comparison of the chemical properties of rapid process, commercial and traditional process products Treatment Moisture A Salt Protein w (%) (%) (%) Rapid process product 65.5 0.72 22.7 5.2 Commercial product 69.3 0.73 21.3 4.9 Traditional process product 8.4 0.51 64.5 12.7 106 provides an economic loss since most of the curing salts are present in this brine. Therefore, further use of this brine was investigated. In these experiments, fresh jellyfish bells were salted using the brine produced from dry salting with 15% salt and 5% alum (first brine) and this was reused (second brine) and reused once more (third brine). In a further experiment, jellyfish was brined in a mixture of the second and third brines. Proportions of brine and jellyfish were chosen such that there would be sufficient salt and alum in the brine to provide the required levels of 15% salt and 5% alum on the basis of bell weight. The moisture, aw, salt, aluminium and pH of the products were determined after brining for 48 h and after drying at 30°C for 9 h. These results are shown in Table 29 along with comparative data for fresh, and dry salted jellyfish. These data show that slight differences occurred in mosture, a , salt and pH of samples treated with recycled brines w after 48 h and aluminium levels remained the same throughout the recycling process. On the other hand, marked changes occurred in moisture, a and salt content after drying of jellyfish treated w with recycled brines. It can be seen also that products of second brine have chemical properties close to those of dry salted jellyfish suggesting that brines could be recycled once only in jellyfish processing. Changes in brine composition with recycling are shown in Table 30. These results clearly show the decreased concentration in salt and increased pH accompanying recycling and explain the inadequacies of products obtained by recycling more than once. Thus, these results indicate that the brine from dry salting can be reused once in a 48 h brining of jellyfish bells 107 Table 29. Composition of jellyfish products from recycled brines Treatment Moisture A Salt pH Aluminium (%) w (%) (%) Fresh 96.4 0.99 2.5 7.5 0.002 Dry salted (undried) 89.6 0.95 7.4 3.4 0.07 First brine (undried) 90.6 0.97 6.0 3.5 0.07 Second brine (undried) 92.3 0.96 5.8 3.5 0.07 Third brine (undried) 93.4 0.98 4.1 3.6 0.07 Second & third brine (undried) 92.9 0.98 4.8 3.7 0.06 Dry salted (dried) 65.5 0.72 22.7 2.6 0.05 First brine (dried) 62.7 0.73 20.7 2.6 0.05 Second brine (dried) 66.0 0. 74 22.9 2.7 0.06 Third brine (dried) 77.2 0.84 13.7 2.9 0.07 Second & third brine (dried) 74.4 0.80 17.5 2.6 0.06 Table 30. Changes in salt and pH of brine during recycling Treatment Salt pH (%) First brine 17.8 2.8 Second brine 13.6 2.9 Third brine 8.7 3.2 Second & third brines 7.8 3.4 108 to obtain a satisfactory product. This is an important finding in that it effectively reduces both the cost of curing salts and waste disposal problems by approximately SO%. It seems likely that addition of more salt and alum to the reused brine may enable even further efficiencies to be achieved. This aspect would however, be best explored in long term commercial operation. It may be that build-up of protein and other material from the process may impose some limitation on extended re-use of brine. As a final step in this investigation of brine recycling, the products and brines were examined for their microbial loads. It was found that fresh jellyfish contained approximately 200 organisms/g but were sterile after 48 h in all brines. No bacteria could be detected in any of the brines although 10 yeast cells/mL were found in the first brine. These low microbial numbers are not surprising in view of salt levels and pH prevailing in the samples. These data indicate that no problems of a microbial nature are likely to occur as a result of brine recycling. 4.5.4 Artificial drying of unsalted jellyfish The experiments described in Sections 4.5.1-4.5.3 would enable a rapid method with comparatively low labour requirements for the preservation of jellyfish. However, a 48 h brining stage is required with attendant expense of salt and alum together with some waste disposal problems. Thus, experiments on artificial drying of unsalted jellyfish bells were undertaken. Drying of unsalted jellyfish has been explored earlier (Magno-Orejana, 1983) using 7 hr at 52°C when a thin, brittle 109 brownish product was obtained. Thus, in the present work, untreated jellyfish were dried at 30°C. It was hoped this lower temperature would avoid problems encountered by Magno-Orejana (1983). Another experiment was conducted by blanching jellyfish bells and then drying them at 30°C and 50°C. The blanching process may have assisted the drying procedure by decreasing the water holding capacity of the tissue and by deactivating enzyme systems which could cause colour and texture problems. Presented in Table 31 are changes in moisture content and a during drying of untreated and blanched samples. It can w be seen that blanching had in fact greatly slowed rate of moisture loss at 30°C to an unacceptable level. After 4 h drying at 50°C, the blanched product had a soft texture but the outer layers were hard with a burnt appearance and hence were unacceptable. The untreated jellyfish, although faster drying at 30°C than the blanched sample, still yielded a brittle, brown coloured product and hence was not acceptable. These approaches to drying jellyfish in the absence of a brining step were obviously not successful and hence were discontinued. 4.5.5 Recommended rapid process for jellyfish preservation Outline of the process: - Remove slime from jellyfish bells; - Treat with 15% salt and 5% alum for 48 h; - Wash with tap water and drain for 2 h; - Dry at 30°C for 9-12 h using the mechanical dryer; - Quality control measures. (see below Table 31) 110 Table 31. Changes in moisture content and water activity of untreated and blanched jellyfish during drying Moisture A Drying time (%) w (h) Untreated Blanched Untreated Blanched 30°C 0 95.1 96.1 0.97 0.98 3 93.9 94.1 0.97 0.98 6 91.6 93.2 0.97 0.97 9 89.5 92.3 0.96 0.95 12 86.3 86.5 0.94 0.93 50°C 0 96.1 0.98 1 95.4 0.98 2 93.2 0.98 3 93.0 0.98 4 90.7 0.97 The progress of the drying may be followed by measuring the a and moisture content as in-process quality control parameters. w Water activity measurement is more rapid and simple than moisture determination and therefore, is recommended in this role. Excessive drying leads to yield loss as the most important drawback but if greatly excessive may also damage product quality. Product quality control parameters suggested are water activity, moisture content and pH. 111 4.5.6 In vitro digestibility of jellyfish proteins In this series of experiments, the effects of brining for 48 h in 15% salt, 5% alum alone and in combination, and drying at 30°C for 9 h on pepsin digestibility were examined. These results are presented in Table 32 along with the protein content for each sample studied. It is apparent that some reduction in the level of indigestible protein had occurred as a consequence of the various processing steps. Although small, differences observed are statistically significant (P 7 treatments, samples treated with salt alone and dried gave the lowest indigestible protein and the highest was that of the untreated and undried samples. Thus, drying at 30°C obviously improved the digestibility of jellyfish proteins. This is consistent with reported increases in digestbility of fish protein dried at 60°C (Sheikh and Shah, 1974). The total protein contents of the 48 h brined jellyish were similar regardless of the preservatives applied and close to that of the untreated samples. Slight increases occurred when these samples were dried but the salt-alum treated samples showed substantial increase in protein after drying for 9 h, as a result o:f moisture loss. 112 Table 32. Indigestible protein and total protein in jellyfish treated with 5% alum, 15% salt and salt-alum mixture for 48 h and dried at 30°C for 9 h Indigestible protein Total protein Treatment (% of total protein) (%) Fresh (untreated) 5.9 0.7 Alum, 48 h cure 4.4 0.9 Alum, 9 h dry 3.8 1.3 Salt, 48 h cure 4.4 0.9 Salt, 9 h dry 3.0 1.2 Salt-alum, 48 h cure 3.2 1.1 Salt-alum, 9 h dry 3.5 2.2 Analysis of variance: Indigestible protein - ** (P 4.6 Solubility Properties of Processed Jellyfish Proteins It is clear from earlier results (Section~l.l- 4.1.2) that jellyfish proteins contain collagenous material and as such, may have potential for exploitation as an alternative source of gelatin. In order to explore this potential, studies were conducted on the influence of pH, salt concentration, temperature and solubilising agent's on the solubility of processed jellyfish proteins. 113 Rapid processed jellyfish were examined in this context because the stability of the processed product may offer advantages to the manufacturer. In addition, production of gelatin from conventional sources involves the use of acid conditions in some processes. Hence, the low pH of the brine used in the rapid process may increase protein extractability. Furthermore, dissolution of the proteins is essential for electrophoretic studies, hence this aspect was examined in some detail. 4.6.1 Protein extractability 4.6.1.1 Influence of pH The solubility of dried salted jellyfish at pH between 3 and 12 is shown in Figure 4. These data show that pH has affected the solubility of processed jellyfish proteins. In the fresh state 30% of the total protein is soluble at its natural pH. For processed jellyfish protein solubility was minimal at pH 5-6, only 7% of the total protein being soluble in this pH range. However, at pH ~extractable protein comprised 27% of the total, approaching the level observed for fresh jellyfish. Unlike pure proteins such as B-lactoglobulin where solubility rises at specific levels of pH, jellyfish proteins did not display a marked change in solubility even if pH was increased. Betwe:enpH 7-11, proteins had lowest solubility no doubt reflecting isoelectric points of individual components. This point:l5discussed further in Section 4.6.6. Thus pH 3 is the most suitable level for extracting proteins from jelly fish. It is interesting to note that the pH of the rapid processed product is also approximately 3. "'...c Cll ~ 0 (. Q. .... 0 ~ 0 ~ ~ 0 .....,M ...c Cll ~ 0 '- Q. 10 ....Cll .D 0 ~ 0 0 s '- ~ X w 0 0 2 4 6 8 10 12 pH Figure 4. Effect of pH on the extractability of proteins in jellyfish 115 4.6.1.2 Influence of salt concentration The solubility of processed jellyfish proteins in salt solutions of concentration 0.1-1.0% is shown in Figure 5. Solubility was highest at the lowest salt level, and as concentration increased, it decreased to a minimum at 0.6%. Solubility at this point was only 5% of the total protein; less than half of the highest level observed. The maximum solubility obtained from the processed jellyfish was only one-third of the protein solubility of the fresh product, indicating that the process had decreased protein solubility. It should be noted that even at 5% salt concentration, protein solubility of processed jellyfish remained at 5%. Thus it was concluded that salt solution is not an efficient solvent for extracting proteins from processed jellyfish. The solubilising effect of salt depends upon its concentration, the charge of the ions, and their effect on the structure of water. Ions affect protein solubility because the charged groups on proteins interact with inorganic ions and solubility is enhanced (salting-in) whereas, at higher ion concentration, water activity is reduced and protein-protein interactions ensue with concomitant insolubilisation (salting out) (Kinsella, 1982). Salting-in depends more on surface charge distribution and polar interactions with the solvent whereas, salting-out is largely influenced by hydrophobicity of the protein (Scopes, 1984). These results indicate the jellyfish proteins are somewhat hydrophobic in nature. 116 ~c ~ ~• 0 L a. a ~ 0 ~ ~ 0 w V c -• ~ 0 L a. 4 • ~ -a ~ 0 a 2 L ~ X w Salt concentration Figure 5. Effect of salt concentration on the extractability of jellyfish proteins 117 4.6.1.3 Influence of temperature Figure 6 shows the effect of temperature on solubility of processed jellyfish proteins. Overall, processed jellyfish yield more soluble protein as extraction temperature increases. This effect is greater than that observed in the fresh material (Section 4.6.1.1). This is attributed to the low pH of the processed productwhichmay lead to some hydrolysis of the protein during extraction at higher temperatures. At 50°C, 36% of the total protein was extracted, increasing to 42% at 100°C. The results obtained are exceptions to the general observation that although protein solubility increases as the temperature is increased between0° and 40°C, above 40°C, proteins become increasingly unstable and begin to denature and become less soluble (Lehninger, 1970; Anglemier & Montgomery, 1976). However, jellyfish proteins belong largely to the collagen group as discussed earlier (Section 4.1.1). Collagen molecules are made up of 3 chains of a-helices which are intertwined and held in position by covalent cross- links and other weaker links. When collagen is treated with hot water, and acid or alkali, weak cross-links are broken to an extent depending on the harshness of treatment. The resulting product is gelatin which is soluble and :contains a range of protein molecules. Commercially, this is dried to about 12% water and contains 1% mineral salts (Fox & Cameron, 1983). Collagens are also solubilised by boiling with water (Haurowitz, 1963). This is reflected in the increase in solubility as temperature increased. Thus, in extracting protein from jellyfish higher temperature should be employed for optimum recovery and the low pH of the processed product also appears to be advantageous. 118 "c Cll -+) 0 (. a. 25 c Cll 20 -+) 0 (. Cl. 15 ....Cll .0 0 10 +) 0 0 (. +) 5 X w 10 20 30 40 so 60 70 80 90 100 Temperature (°C) Figure 6. Effect of temperature on the extractability of proteins in jellyfish. 119 4.6.2 Solubiisation by O.lM KCl After processing, some denaturation would be expected in jellyfish proteins. O.lM KCl has been used to determine the amount of soluble (undenatured) protein and salt soluble NPN in other systems (Obanu et al., 1975). Thus, the effectiveness of O.lM KCl in extracting proteinaceous material from jellyfish was evaluated. This would also allow some evaluation of the extent of denaturation caused during processing by comparison of the soluble protein yields from fresh and processed jellyfish. Solubility of jellyfish proteins from both fresh and processed samples in O.lM CKl was only 5% of the total in both cases, similar to that in 5% salt solution. These results indicate that O.lM KCl is a poor solvent for extracting jellyfish proteins and do not confirm that the process caused protein denaturation. O.lM KCl has also been shown to be a poor solvent for proteins in intermediate moisture beef (Obanu et al., 1975). 4.6.3 Solubilisation by 3% sodium dodecylsulphate (SDS) + 1% B-mercaptoethanol In order to enhance extraction of jellyfish proteins, SDS + B-mercaptoethanol were investigated as these have been reported to be effective in the solubilisation of certain denatured proteins and protein aggregates especially those which tend to be more hydrophilic (Gordon, 1969). The poor solubility of jellyfish proteins in salt solutions indicated their hydrophobicity and hence these agents were expected to enhance solubility. In the present study, the protein solubility of fresh and processed jellyfish in SDS + B-mercaptoethanol was about 27% 120 and 26% of total protein respectively. These levels are comparable to those obtained by hot water extraction. Thus, SDS and B-mercaptoethanol do not offer any quantitative advantages over hot water extraction. However, in the context of IEF studies, this combination proved to be valuable (Section 4.6.6). Since heat is not required, denaturation resulting from boiling water treatment is avoided. SDS + B-mercaptoethanol have been used previously to solubilise denatured proteins for electrophoretic studies (Obanu et al., 1975). 4.6.4 Solubilisation by pepsin Solubility of jellyfish proteins after incubation for various periods with pepsin is shown in Table 33. The enzyme was employed because of low protein solubility in other extraction media used. Jellyfish proteins are collagenous in nature and collagen is extremely resistant to proteolytic enzymes (Reutersward, 198Sr). Several workers studied the degradation of native collagen by enzymes in connection with gelatin manufacture (Balian & Bowes, 1977; Johns & Courts, 1977); biochemistry and medicine (Mandl, 1961; Weiss, 1976; Bailey & Etherington, 1980); tenderness of meat (Sorensen, 1981) and the leather industry (Gustavson, 1956). Native collagen is generally accepted to resist digestion (Cheftel, 1977; Prandl, 1980; Kies, 1981; Ashgar & Henrickson, 1982) so, in order to become digestible, wet heat treatment is necessary resulting in the formation of gelatin (Rogowski, 1980). Thus, cooking makes collagen susceptible to enzymes (Paul, 1972; Ashgar & Henrickson, 1982). 121 Table 33. % soluble nitrogen in jellyfish cured for various times after pepsin digestion % soluble nitrogen after pepsin digestion of Hydrolysis time samples cured for: (min) 0 (fresh) 12 h 24 h 48 h 1.0 27.0 20.0 28.6 21.7 2.5 27.0 22.0 34.0 23.9 5.0 27.0 22.0 34.3 26.1 10.0 32.4 26.0 34.3 28.3 20.0 32.4 26.0 34.3 28.3 30.0 32.4 26.0 34.3 28.3 Some in vitro studies on enzymatic degradation of highly colagenous materials have been conducted using pepsin and HCl at 37°C (Neuman & Tytell, 1950; Franke, 1964; Loewit, 1967, Harkness, Harkness & Venn, 1978; Reutersward, 1985). These studies concentrated on solubilisation of collagen from different types of tendon and pigskin. Thus, solubilisation of jellyfish proteins by pepsin was investigated as a potential means of maximising protein recovery. In initial studies, levels of 1-5 mg/mL pepsin and trypsin alone and in combination were tested. No advantage was found in using enzyme levels greater than 1 mg/mL during 24 h incubation. Trypsin did not offer any advantages over pepsin alone and hence was not used in subsequent studies. Shown in Table 33 are the changes in protein solubility during incubation with pepsin for 122 up to 30 min of samples brined for various periods in salt (15%) and alum (5%). Maximum solubilisation was attained after 10 min incubation for all samples. It can be seen that maximum solubilities ranged from 26-34% between samples suggesting that processing does not substantially affect the susceptibility of jellyfish proteins to pepsin. These results are consistent with data on in vitro digestibility of fresh and processed jellyfish (Section 4.5.6) which showed that brining and drying had little effect on this parameter. 4.6.5 Amino acid composition of processed jellyfish Samples prepared for in vitro digestibility studies described in Section 4.5.6 were analysed for their amino acid composition. These results are presented in Table 34 and typical chromatograms in Appendices 4-5. The high levels of glycine, glutamic acid, aspartic acid and hydroxylysine and the absence of tryptophan are characteristic of collagen type proteins. The presence of these fn,jellyfish has been discussed at several points above. It is apparent that jellyfish contained low levels of essential amino acids as compared with the non-essential amino acids. The four highest in proportion were glutamic acid, glycine, aspartic acid and lysine. Tryptophan was absent. When jellyfish were cured in 5% alum, 15% salt and salt-alum in combination, slight decreases in amino acid values occurred although these are not statistically significant. Drying somewhat increased levels of most amino acids but statistical analysis again showed this to have no significance, Processing significantly (P<0.05) increased the proportion of glycine, a non essential amino acid, 123 Table 34. Comparison of the amino acid composition of treated and untreated jellyfish (g AA/16g N) Amino Fresh 5% Alum 15% Salt 15% Salt, 5% Alum acid Cured Dried Cured Dried ! · Cured Dried Asp 7.8 7.6 8.8 7.8 8.4 7.8 8.2 Thr 4.4 3.6 4.1 3.9 4.2 4.5 4.5 Ser 3.4 3.2 4.1 3.4 3.7 3.9 4.0 Glu 11.4 10.7 12.5 10.6 11.4 10.4 11.1 Pro 5.2 5.3 6.7 6.0 6.4 5.8 6.0 Gly 9.2 12.0 13.4 12.2 11.0 10.3 10.6 Ala 5.2 5.4 6.2 5.8 5.6 5.2 5.5 Cys 0.9 0.8 1.1 0.9 0. 7 1.8 1.1 Val 3.8 2.7 3.6 3.1 3.3 3.9 3.9 Met 1.6 1.1 1.7 1.4 1.6 1.9 1.4 Ileu 3.2 3.9 3.0 2.6 2.8 2.2 2.5 Leu 4.9 4.0 4.6 4.3 3.7 4.4 5.0 Tyr 1.9 1.4 2.1 3.0 1.7 1.5 1.7 Ph en 2.2 1.2 1.9 1.4 1.4 1.3 1.4 Hylys 1.7 2.5 2.5 3.2 2.2 2.0 2.3 Lys 7.4 5.6 6.2 5.7 6.2 6.1 6.9 NH 13.8 10.1 13.3 12.1 11.1 10.4 3 10.6 His 1.2 1.0 1.0 1.0 1.0 1.0 1.1 Ang 5.6 5.3 6.1 5.7 4.9 5.8 5.8 Analysis of variance: Glycine - * (significant at P<0.05) Valine - * Lysine - *~~ (significant at P but decreased those of the essential amino acids valine (P<0.05) and lysine (P The amino acid composition of jellyfish processed by the traditional technique was also determined (Table 35). Slight differences in amino acid proportions occurred between rapid process and traditional process jellyfish. Of the 8 essential amino acids determined, 5 of these have higher proportions compared to that of rapid process product whereas, the non-essential amino acids have also similar number of slightly higher proportions. Marked difference was apparent between the aspartic acid content of the two products. These variations could be attributed to the technique employed in hydrolysing the samples. Rapid process products were hydrolysed for 24 h at ll0°C whereas, conditions of 4 h at 145°C were used for the traditional product. In addition, artificial drying of the rapid process product could have also contributed to loss of some amino acids. These two factors may have contributed to differences observed between the two products. Jellyfish proteins, although collagenous in nature still contain at least 20 amino acids present in muscle proteins. Of this number 8 essential amino acids are present although in lesser proportion compared with the non essential amino acids. Jellyfish proteins are not considered high quality protein because tryptophan is absent and there is low proportion of essential amino acids. High quality protein contains essential amino acids in ratios commensurate with human needs. However, jellyfish is not eaten alone but mixed or eaten with other foods. Thereby, in a mea~ amino acid balance could also be met. 125 Table 35. Amino acid composition of traditional process jellyfish Amino acid g AA/16 g N Asp 3.7 Thr 5.0 Ser 4.1 Glu 10.7 Pro 8.3 Gly 10.4 Ala 6.9 Cys 0.5 Val 4.6 Met 1.7 Ileu 3.3 Leu 5.1 Tyr 2.0 Ph en 2.8 Hylys 3.2 Lys 5.7 NH 8.5 3 His 0.4 Arg 7.8 126 4.6.6 Isoelectric focusing analysis of proteins from processed jellyfish Further investigation of the effects of processing on the properties of jellyfish proteins was carried out by determining isoelectric points (pi) of proteins by isoelectric focusing (IEF) in 1% agarose IEF gel containing Pharmalyte 3-10. Samples examined were previously treated with salt (15%), alum (5%) and combination of both for periods up to 48 h followed by drying at 30°C for 9 h. This work posed numerous problems since jellyfish are high in moisture and low in protein content thus, concentration of sample solution was necessary. Samples also contained salt both naturally and as a result of processing. Dialysis was required since the agarose gels are broken down by salt which also affects electric mobility of proteins. The effects of salt on the mobility of proteins are of two kinds; one is concerned with the ion atmosphere surrounding the proteins and the other with the charge of the protein due to a more intimate interaction of the protein with the ions of the salt. This could be a result of chemical processes such as ion pair formation, or adsorption of the ion on the protein (Abramson, Moyer & Gorin, 1964). Sample preparation was so lengthy that during the process, some protein components may have been denatured or lost. Solubility of processed jellyfish proteins was the biggest problem. Solubility of jellyfish proteins was low whether fresh or processed. To optimise solubility, agents such as urea, sodium chloride, potassium chloride, SDS (an ionic detergent) plus B-mercaptoethanol and Triton X-100 (non-ionic detergent) were used. Urea and SDS may have been effective (treated proteins 127 rendered more hydrophilic)(Gordon, 1969) as solubilising solutions, however, the agarose gel appeared to have been 'cooked' by urea and SDS and gel appearance was like that of cooked albumin. Wiggin and Krzynowek (1983) reve~led that urea weakened the strength of agarose gel, making it watery and non-adherent to the gel bond. Dense background staining in urea-based gels makes the protein bands hard to distinguish. Thus, with jellyfish protein no distinct bands appeared on urea-based gel with wide smearing occurring on the gel surface. Since it was reported that urea disrupts the structure of agarose (Pharmacia Fine Chemicals, 1982), this approach was discontinued. Experiments with various concentration up to 5% of sodium chloride and O.lM potassium chloride gave poor extraction of protein and hence were of no use for jellyfish (Sections 4.6.1.2, 4.6.2). The beneficial effects of Tr~n X-100 (TX~lOO) on protein recovery and zone resolution have been pointed out by Hearing et al. (1976). They reported that TX-100 had excellent protein solubilisingproperties with no denaturation of protein, fairly accurate molecular size determinations and allowed rapid fixation and staining. Moreover, its non-ionic nature allowed the intrinsic charge of the proteins to be a factor in electrophoretic migration. Dulaney and Touster (1970) also expressed their preference of TX-100 to solubilise proteins compared to reagents such as urea and acids. The detergent could be incorporated in the gel and the electrophoresis buffer. However, the drawback is that some proteins are insoluble in TX-100. This last feature may have been a major problem with jellyfish protein as no distinct protein bands were separated. Instead, there was trailing of samples 128 across the gel which resulted in a fuzzy pattern thereby, densitometric scanning was not possible. Other factors involved in this poor performance of TX-100 could include precipitation of the sample, denaturation, poor solubility with precipitation at the point of application, high molecular weight of the sample and interference from atmospheric C0 (Pharmacia Fine Chemicals, 2 1982). Several attempts were made to remedy these problems however, this approach was ultimately abandoned. The extraction procedures found most effective for fresh jellyfish was homogenisation using the electric blender for 2 min with an interval of 2 min and then the homogenised samples were centrifuged for 30 min at 1000 rpm at 0°C. The cured and dried jellyfish were initially blended with cold 1% Triton X-100 (1:1 w/v) using the electric blender and then homogenised further with the Ultra-Turnax homogeniser for 20 min and centrifuged as for fresh jellyfish (Section 3.2.7.3). SDS + B-mercaptoethanol and urea were also tested as extractants for dried products but resolutions were poor (reasons described previously) so densitometric scanning was not possible. Using the TX-100 procedure for cured jellyfish and blending for fresh jellyfish protein extracts were well resolved using IEF. Their electrophoretic patterns are shown in Plate No. 2,and densitometer scans in Appendices 6-8. The pi of proteins from the 48 h cured jellyfish treated with 5% alum, 15% salt and salt-alum combined are shown in Table 36. There were 8 protein components detectedfrom alum-treated jellyfish with pi (isoelectric point) ranging from 6.95 to 8.65, only 1 being acidic. In salt treated samples, 12 protein components were separated. Half 129 A Plate No. 2. Electrophoretic patterns of protein from 48 h cured jellyfish treated with 15% salt (S), 5% alum (A) and salt-alum in combination (SA), separated by IEF of pH 3-10 on 1% agarose gel. Also shown are those of fresh (F) samples and standard (Std) proteins. 130 Table 36. Isoelectric point of proteins in 48 h cured jellyfish Sample cured for 48 h in: Band No.* Alum (5%) Salt (15%) Salt-alum (15%,5%) 1 8.65 8.65 ~2 8.62 8.60 3 8.60 8.58 8.55 4 8.55 8.50 5 8.50 8.45 8.45 6 8.15 7 8.05 8 7.85 9 7.25 10 7.15 7.05 11 6.95 6.85 12 6.60 6.70 13 6.30 14 6.10 6.15 15 5.90 16 5.70 5.75 17 5.00 Total 8 12 9 *Band numbers were assigned on the basis of position and relative intensities of stained bands on the gel. 131 of these were basic and the rest acidic with pi ranging from 5-8.65. With salt-alum treated samples, 9 protein fractions weTe detected with pi ranging from 5.75-8.55. Four of these were acidic and 5 basic. It is apparent that with alum and salt-alum in combination as curing salts, less protein fractions were detected than in fresh samples where 15 protein components were separated. The disappearance of some components could be attributed to denaturation by alum or salt-alum in combination. Aluminium ions are triply-charged and could affect the solubility of proteins by various mechanisms. The alum also induces low pH into the system which may also influence the proteins. Salt has been known to denature protein (Duerr and Dyer, 1952; Linko and Nikkila 1961). However, salt alone as curing medium resulted in more protein fractions being detected compared to alum suggesting that jellyfish proteins are less affected by salt than by alum. The separation of protein components by IEF is essentially dependent on pi of proteins in the sample. pi of proteins in jellyfish appears to have been affected by the preservatives used in processing. As seen in Table 36, treatments with alum.and salt-alum combined, resulted in the detection of fewer protein bands and alteration to pi. The presence of interfering bands could be seen (Plate No. 2). Salt also affected both the number and pi of protein bands although to a much more limited extent. This can be seen by comparing data for salt processed jellyfish (Table 36) with those from fresh jellyfish (Table 37). These changes were further examined by studying pi 132 of jellyfish proteins as a function of brining time in salt (15%) and alum (5%). These results are presented in Table 37 and the densitometric scans in Appendices 9-10. It is obvious that as brining time progressed, fewer protein components were detected. After 12 h brining, 12 out of 15 in fresh jellyfish were detected with pi ranging from 6.55-8.65. Of this number, only 2 were acidic and the rest were basic. Brining for 24 h decreased the number of bands to 11 with pi ranging from 5.40- 8.65 and at the conclusion of brining, only 9 fractions were detected. Their pi ranged from 6.15-8.55. It is apparent that decreases in acidic pi fractions occurred as brining progressed and there was a lack of change in basic proteins. The relatively unchanged protein solubility between samples indicates that the variations in numbers of band and pi were not simply due to loss of solubility. These changes were observed mainly in acid proteins and their exact nature is very much in the area of speculation. 4.6.6.1 Isoelectric focusing analysis of protein from brine The pi of protein components recovered from brine produced during processing was also determined (Table 38). With alum as the curing agent, 5 protein fractions were detected and only 1 was acidic. The pi ranged from 6.95-8.65 reflecting the low pH of alum. Basic protein components would be extracted by the acidic solution. The densitometric scan can be found in Appendix 11. When salt alone was used, the same number of components asalumwere separated from the brine but 2 fractions were acidic 133 Table 37. Isoelectric point of jellyfish proteins Treatment times with 15% salt, 5% alum: Band No.* 0 h 12 h 24 h 48·h 1 8.65 8.54 8.65 2 8.50 8.55 8.55 8.55 3 8.45 8.50 8.50 8.50 4 8.45 8.45 8.45 5 8.30 8.30 6 8.20 8.15 7 8.10 8.00 8 7.70 7.75 7.65 7.85 9 7.50 7.40 10 7.25 11 6.95 7.05 7.10 7.05 12 6.75 6.65 6.70 13 6.55 6.50 14 6.30 15 6.20 6.15 16 6.00 17 5.65 5.75 18 5.30 5.40 19 5.20 20 5.10 21 5.00 Total 15 12 11 9 * Band numbers were assigned on the basis of position and relative intensities of stained bands on the gel. 134 Table 38. Isoelectric point of proteins in brine produced during processing Sample treated for 48 h with: Band No.* Alum (5%) Salt (15%) Salt-alum (15%,5%) 1 8.55 2 8.45 8.48 3 8.40 8.40 4 8.15 5 8.05 8.10 6 7.75 7 7.45 8 7.25 9 7.10 10 6.80 6.80 11 6.65 6.70 12 6.45 6.55 13 5.45 Total 5 5 9 * Band numbers were assigned on the basis of position and relative intensities of stained bands on the gel. 135 and 3 basic (Table 38). The pi ranged from 6.45-8.40 and the densitometric scan is shown in Appendix 11. With salt-alum brine, 9 protein components were detected with pi ranging from .5.45-8.55. Five of these proteins were basic and 4 acidic (Table 38). The densitometric scan is seen in Appendix 12. Protein extraction by curing brines is of importance not only because of its impact on product composition and quality, but also since it may complicate the problem of effluent disposal. However, although differences exist between protein components extracted by the different brines, the salt/alum combination is essential for adequate product quality. It does not appear that leaching of individual proteins into the brine is the explanation for changes in IEF pattern during processing since no apparent correspondence between bands disappearing from the product during processing and those detected in the brineswasnoted. 4.7 Physical Structure of Jellyfish The physical structure offfresh and processed jellyfish was examined microscopically. The freeze-fracture technique was used to enable scanning electronmicroscopyof fresh jellyfish and light microscopy for both samples. This was undertaken in order to determine the effects of salting and drying on the physical structure of jellyfish because of the great importance of the texture of the dried product. The structure and functions of the various tissues have been discussed earlier (Section 2.1.1). 136 4.7.1 Fresh jellyfish 4.7.1.1 Freeze-fracture and electron microscopy Because of the high moisture content of jellyfish, fixation and dehydrationofthe material as a prelude to electron microscopy would lead to extensive changes in physical structure. This would render electron micrographs largely meaningless. The use of conventional thin sectioning procedures on jellyfish was also likely to result in major destruction of the tissues due to its very high moisture content. Thus, such an approach followed by freeze drying was also discounted. In freeze-fracturing, moisture is not removed but, instead, is frozen hence leading to better retention of tissue structure. Fresh jellyfish were cut into 4 sections; upper layer, middle layer, bottom layer and margin of the bell. Details of sample preparation and microscopy are found in Section 3.2.9.2. Electron micrographs of these sections are shown in Plate Nos. 3-7. These 4 sections correspond respectively to the ectoderm (outermost layer), mesoglea (thick, gelatinous layer), endoderm (gastrodermal layer), and the structure at the edge (possibly nerve net) of the jellyfish bell. The structure of whole jelly fish is found in Section 2.1.1, and a typical epidermal cell is seen in Plate No. 3. The outer layer of cells, the epidermis, covers the outer surface of the body of jellyfish. The body iscomposed mainly of musculo-epithelial cells which are found only in the Cnidarians (Villee et al., 1978; Bertin, 1973). The mesoglea lies between the two cell layers of the body wall and is largely non-cellular as can be seen in Plate No. 4. In medusae, the mesoglea constitutes the bulk of the jellyfish. It is composed of a protein similar to collagen and 137 presumed to be produced mainly by the cells of the epidermis. It forms as a base upon which the muscle system can operate and as a skeleton which prevents excessive deformation of the body (Morris, 1975; Barnes, 1980; Curtis and Cowden, 1972; Bertin, 1973). The interior cavity of jellyfish is lined by gastrodermis. The structures shown in the micrograph could be part of a gastro dermal cell (Plate Nd. 5). Only the gastrodermis contains extensive muscle fibres in the column and longitudinal fibres in the septa (Starer et al., 1968). These structures however, are not visible in the micrograph. The features found at the margin of the bell may be a nerve net which made up the neuron system of jellyfish. In Plate No. 6, it can been seen that it is composed of a large number of multipolar nerve cells - cells with many dendrites or connecting links. Nerve cells have been positively identified only in medusae and anthozoans. A nerve net serves to coordinate the contractions of the bell for movement and also feeding actions (Bertin, 1973; Morriss, 1975; Villee et al., 1978). Plate No. 7 shows the part where the nerve net on tihe bell margin and gastro dermal layer meet. 4.7.1.2 Light microscopy of fresh jellyfish Fresh jellyfish were also examined by light microscopy. Sample preparation and microscopy details are found in Section 3.2.9.1. The light micrographs are shown in Plate Nos. 8-9. 138 Plate No. 3. Electron micrograph of an epidermal cell from fresh jellyfish prepared by freeze-fracture technique (42.160x) 139 Plate No. 4. Electron micrograph of a part of mesoglea from fresh jellyfish prepared by freeze~fracture technique (32.640x) 140 Plate No. 5. Electron micrograph of a part of gastrodermis of fresh jellyfish prepared by freeze-fracture technique (24.480x) 141 Plate No. 6. Electron micrograph of a part of the margin of fresh jellyfish showing structure which could possibly be a nerve net (20.400x) 142 Plate No. 7. Electron micrograph showing structures where gastrodermal layer and margin of fresh jellyfish meet ( 15. 640x) 143 The dark aggregation of cells found at the uppermost section of the micrographs is the epidermal layer taken near the margin of jellyfish (Plate No. 8 a-b). The epidermis is separated from the mesoglea by a distinct basement membrane. This is more apparent in the upper micrograph. The rounded-whitish spot at the lower micrograph appears to be a part of the gastrovascular cavity which is enclosed by the gastrodermal cells. In Plate No. 9, the mesoglea shows ameboid cells and fibrous structures. The latter are also visible in freeze-fracture electron micrographs (Plate No. 4). In the upper micrograph, algal cells are apparent. They are greyish-white, rounded and apparently subdivided cells. In jellyfish, algal cells are concentrated in the mesoglea and are generally confined to the periphery of this layer. They appear to be trapped in locules of the mesogleal matrix (Kevin, Zahl & McLaughlin, 1966). The presence of algal cells in jellyfish mesoglea was noted by Dakin, Bennett and Pope (1979) who commented on the remarkable colour differences between jellyfish from the Sydney area and Brisbane or Melbourne. This was due to the presence of algal cells which resulted in Sydney jellyfish being cream to brownish while those from Brisbane and Melbourne may be blue in colour. It should be noted that the fibrous structures and mesogleal ameboid cells in the mesoglea of the fresh specimen are fairly well apart. These features of mesoglea are used later for comparison between samples that have been treated with different curing agents and dried. 144 ,. . . # • Plate No. 8. A light micrograph of structures showing the (a) margin of the bell and (b) part of mesoglea of the fresh jellyfish (SO ~m, ~) 145 B Plate No. 9. A light micrograph of a part of mesoglea from fresh jellyfish showing (a) structures of the outer most layer of mesoglea and (b) structures of the inner most layer of mesoglea (50 urn,~). 146 4.7.2 Effects of processing on jellyfish tissues During exposure to salt andalumand subsequent drying, the appearance and texture of the bells undergo major changes in size, shape and texture. Because of the great importance of the texture of dried jellyfish to its market acceptability, the effects of the brining agents and drying on its structure were studied. In this section, light microscopy was used to examine the material , since the high levels of salt in the product precluded any attempts at electron rnicroscopy. 4.7.2.1 Effects of alum alone and drying When jellyfish were cured in 5% alum for 48 h, it can be seen (Plate No. 10) that the fibrous structures of the mesogleal layer had shrunk together compared to the fresh form (Plate No. 9). The more prominent fibrous structures came into alignment while the ameboid mesogleal cells appeared to have decreased in size. This is attributed to the collapse of the layer due to loss of moisture duringcuringand the attendant contraction of collagenous material. Some effect of the alum itself in terms of protein denaturation, and pH decrease in the system may also make an important contribution. When these samples were dried, further compression of mesogleal fibrous structures occurred (Plate No. 11). This shows that heat complemented the effect of alum so that enhanced collapse of jellyfish structure was observed, no doubt due to further moisture loss. 147 . ' . " ,f \', :' ""-<:i ~, '( \~ ' ., ~: .. A B Plate No. 10. A light micrograph of 48 h alum-treated jellyfish showing (a) structures of the outer most part of mesoglea and (b) structures of the inner most layer of mesoglea (50 ~m,~) 148 A B Plate No. 11. A light micrograph of alum-treated jellyfish, dried for 9 hr at 30°C showing (a) structures of the outer most layer of mesoglea and (b) inner most layer of mesoglea (50 urn,~) 149 4.7.2.2 Effects of salt alone and drying With 15% salt as the curing agent, the physical structure of jellyfish was affected in a noticeably different way to that caused by alum. In 48 h-salted samples, the mesogleal fibrous structures have aggregated closely while maintaining a random arrangement similar to the fresh samples (Plate No. 12). This is in contrast to the alignment of structures caused by alum (Plate No. 10). The aggregation of fibrous structures is most likely due to shrinkage of mesoglea because of moisture loss caused by salt. The different effects between salt alone and alum alone are attributed to the effects of alum in terms of protein denaturation and pH decreases in the system. After drying, the mesogleal fibrous structures disappeared to a considerable degree (Plate No. 13) with few prominent fibrous structures still present. The number of ameboid cells may have decreased. Further loss of moisture and denaturation of protein due to salt and heat could have been responsible for the above phenomenon. It is obvious that heat has a dramatic effect on the physical structure of jellyfish in addition to changes caused by alum and salt alone. 4.7.2.3 Effects of salt-alum combination and drying The physical structure of salt-alum treated jellyfish can be seen from the micrographinPlate No. 14. It is similar to that of the fresh materal (Plate No. 9). There was apparently less aggregation of mesogleal fibrous structures as a result ofthecombination of salt and alum than with either alone. More uncollapsed cells are present. 150 "' •.#-~....._""'="' ~~- r ,...... _ ,. ~ '""'':.' ~ -..._ ~"'··' A B Plate No. 12. A light micrograph of 48 h-brined jellyfish showing structures of (a) outer layer of mesoglea and (b) inner layer of mesoglea (SO ~m,~ 151 ••.,. [. '-·. . ' ~'--- ~.· A B Plate No. 13. A light micrograph of salted and dried (9 h at 30°C) jellyfish showing structures from (a) outer layer of mesoglea and (b) inner most layer of mesoglea (50 urn,~ 152 After drying, the salt-alum treated samples had changed slightly in physical structure with features found in fresh jellyfish still apparent (Plate No. 15). Thus, the salt-alum combination is a requirement for obtaining the structure of the final product and, hence, its textural properties. This is of course in addition tothenecessity of this combination in achieving preservative effects as established earlier (Section 4.2.1.3). It should be noted that microscopy of jellyfish entailed a number of problems due to its very high moisture content. Fixation and dehydration, basic in sample preparation, could result in collapse of structure leading to artefactual changes that could complicate interpretation of microscopic examination. 4.8 Chemical and Microbial Changes During Storage and Processing of Fresh Jellyfish The stability of jellyfish both in the fresh state andduring processing are of obvious importance. The fresh product may deteriorate rapidly when removed from sea water undergoing both liquifaction and putrefaction. Storage of the processed product for periods in excess of 6 months under ambient condition led to no deterioration in odour, appearance or texture. This is expected from its a and low pH. However, it is essential w that the feasibility of spoilage during the process be evaluated. Mechanisms of deterioration of fresh jellyfish are especially important to the processor since this greatly restricts the time and handling procedures between capture and brining. Some study of chemical changes during processing is also important to the processorandpurchaser of such products. 153 l • . -..-./ I ' ~ ...,-·• A B Plate No. 14. A light micrograph of 48 h salt-alum treated jellyfish showing structures of (a) outer layer of mesoglea and (b) inner most layer of mesoglea 154 B Plate No. 15. A light micrograph of salt-alum treated and dried (9 h at 30°C) jellyfish showing structures of (a) outer part of mesoglea and (b) inner most part of mesoglea (50 ~m,~) 155 Changes in the chemical properties and microbial status of fresh jellyfish during spoilage and processing were evaluated using methods described in Sections 3.2.2.2.9-14 and 3.2.12. Jellyfish collected in winter 1985 and summer 1986 were examined for changes in trimethylamine oxide (TMAO), trimethylamine (TMA), total volatile base (TVB), pH, trichloroacetic acid (TCA) soluble nitrogen and total plate count (TPC) during spoilage. Results are presented in Table 38. 4.8.1 Fresh jellyfish 4.8.1.1 Trimethylamine oxide (TMAO) The TMAO content of jellyfish collected in both winter and summer seasons was the same when fresh (approximately 28 mg/ lOOg) but decreased during subsequent storage at ambient temperature (Table 39). Rate of decrease was more rapid for the samples collected in summer. TMAO occurs in a large number of fish and shellfish. Generaly, it is highest in the elasmobranchs and negligible in the freshwater fish (Dyer, 1952; Groninger, 1959; Ruiter, 1971; Harada, 1975). There has been no report on the TMAO content of scyphozoans but sea anemones of the tribe Hexactiniae of the phylum Cnidaria contain 5.7-27 ~M of TMAO/g of body weight (Norris and Benoit, 1945) This level is comparable to that found in Catostylus sp. in this study. 156 Trimethylamine (TMA) The TMA content of jellyfish, slightly higher in the summer samples increased upon exposure at ambient temperature at similar rates in both (Table 39). Sulphide odours however, were detected in jellyfish collected in summer after 24 h but not in winter collected samples until 48 h had elapsed. At the conclusion of the experiment (48 h exposure), the summer collected samples were putrid. The increase in TMA is used as measure of spoilage in fish (Connell, 1980) and constitutes a warning of incipient spoilage by bacterial action (Dyer and Mounsey, 1945). About 94% of the TMA is derived from TMAO naturally present in fish muscle which is reduced to TMA during spoilage (Beatty, 1938). In the present study, TMA increases were accompanied by TMAO decreases indicating that TMAO conversion into TMA had occurred. Total volatile bases (TVB) TVB, another index of spoilage in fish was also determined in spoiling jellyfish. Jellyfish collected in summer was initially slightly higher in TVB than winter samples. TVB increased during storage at ambient temperature in both samples but occurred much more rapidly in the summer samples. As noted previous!~ after 24 h exposure, sulphide odours could be detected in summer samples buft was not apparent intnewinter samples until 48 h had elapsed. At these times, the TMA and TVB levels in the samples were both approximately 10 mg/100 flesh. This suggests that this level of TMA on TVB could be used as a maximum level for quality control purposes in fresh jellyfish. This value is lower than those proposed for other fish and the reason for this is the very high 157 moisture content of jellyfish. It is also apparent that TVB exceeds TMA by a small amount only, indicating that, under the conditions used in this experiment, TMA is the predominant volatile amine produced during jellyfish spoilage • .Pl! The pH of fresh jellyfish (Table 39) was found to be near neutrality and did not vary greatly during storage up to 24 h. After 48 h exposure, jellyfish were putrid and pH had risen somewhat. This rise in pH is attributed to production of basic nitrogenous compounds such as TMA and ammonia (Connel, 1980). 4.8.1.2 Total plate count Changes in the number of microorganisms in jellyfish stored at room temperature are shown in Table 39. Total plate counts were low for both summer (20 organisms/g) and winter (10 organisms/g) samples. These increased during subsequent storage at a much faster rate in the summer samples. The higher ambient temperature in summer than in winter no doubt led to the more rapid growth and multiplication of spoilage organisms. The rate of spoilage is related to time and temperature (Ayres, Mundt and Sandine, 1980).and this is clearly shown by results in Table 39. After 48 h exposure, the total plate counts of summer 4 collected samples had risen to 10 organisms/g. This is much less than bacterial numbers in spoiled fish flesh (109/g) although the material was putrid. This again no doubt reflects 158 Table 39. Chemical and microbial changes during spoilage of fresh jellyfish Treatment TMAO TMA TVB pH TPC TCA soluble N (mg/lOOg) (mg/lOOg) (mg/lOOg) organisms/g (% total N) Fresh 1 (summer) 28.0 3.0 3.2 6.97 2.0xl0 5.9 12 h 11.6 5.7 5.1 6.98 l.OxlO3 6.5 4 24 h+ 0 9.9 10.2 6. 72 1.8xl0 5.3 4 48 h 0 43.6 48.3 7.62 3.0xl0 8.7 Fresh (winter) 28.9 1.6 2.2 l.OxlO 1 1.2 2 12 h 11.2 3.1 3.2 l.lxlO 2.4 3 24 h 9.7 5.4 5.7 2.0xl0 2.6 48 h+ 8.0 8.2 + Putrid odour noticed - Not determined the high moisture content of jellyfish. Spoilage microbial flora in jellyfish may not have the same pattern of changes as in spoiling fish however, no attempt was made to identify the organisms involved in jellyfish spoilage. 159 4.8.1.3 TCA soluble nirogen The TCA soluble nitrogen in spoiling jellyfish was determined (Table 39). Nitrogenous constituents not classified as protein include TMAO, urea, taurine, peptides, amino acids, nucleotides and related purine-based compounds (Konosu, Watanabe and Shimizu, 1974). Many of these compounds have been correlated with flavour and freshness and are used as spoilage indices in fish (Spinelli, 1971). TCA soluble nitrogen was deemed especially important in jellyfish due to its liquefaction out of water which implies protein breakdown. The TCA soluble nitrogen in jellyfish was approximately 6% of total nitrogen in summer samples and 1% of total nitrogen in winter samples. These levels increased during storage reaching 8.7% in summer samples compared to 2.6% in the winter samples. These increases in TCA soluble nitrogen showed a similar trend to that reported by Kawamura et al. (1981) in fish. It is clear from data in Table 39 that the samples collected in summer (1986) deteriorated more rapidly than those collected in winter (1985). This is reflected in all indices measured except for pH which varied little in summer samples and was not determined in the winter samples due to shortage of samples. This difference in spoilage rate is attributed mainly to difference in ambient temperature between summer (~25°C) and winter (~l7°C)., The somewhat higher microbial load of the summer samples may also have contributed to its more rapid spoilage. 160 4.8.2 Changes in TMA and TVB during rapid processing of jellyfish Results from Section 4.8.1 showed that changes in TMA and TVB are good indicators of spoilage. In particular, i"n .both winter and summer samples putrefaction first appeared at TMA and TVB levels of approximately 10 mg/100 g flesh. Thus, samples were taken at various times during rapid processing of jellyfish and their TMA and TVB levels determined. These results are shown in Table 40. It is apparent that decreases in TMA and TVB levels occurred during the first 24 h brining. These could be attributed to leaching of these compounds into the brine. Slight increases occurred thereafter up to 48 h brining, no doubt reflecting decreases in moisture content. Further increases in TMA and TVB levels of the final product were again attributed to subsequent moisture loss during drying. The bases at this stage of processing would be non volatile due to low pH and hence are not lost during drying. These results indicate that no major increase occurred in TMA and TVB during processing reflecting minimal spoilage in the rapid process product as expected from brine and product pH and salt contents. 4.8.3 Total volatile acidity and titratable acidity of processed jellyfish Volatile acid value has been suggested as a tool for quality evaluation in fish (Venugopal, Lewis and Nadkarni, 1981; Quaranta and Curzio, 1983). Titratable acidity was also found to increase during cooking, processing and storage (Madovi, 1980) and may be used as quality index in fish. In this study, titratable 161 Table 40. Total volatile bases and trimethylamine during rapid processing of jellyfish Treatment TVB TMA (mg/100 g flesh) (mg/100 g flesh) Fresh 1.6 0.8 12 h cure 1.1 0.8 24 h cure 0.8 0.7 48 h cure 2.7 1.6 48 h cure, 9 h dry 7.9 7.2 acidity may have an important influence on texture and flavour of salted jellyfish as well as providing an additional quality index. For these reasons, volatile acidity and titratable acidity of fresh and processed jellyfish were determined. 4.8.3.1 Rapid process jellyfish As shown in Table 41, both volatile acidity and titratable ~ acidity in fresh jellyfish were low but increased substantially as drying progressed. Titratable acidity is of course greatly influenced by the alum content of the product which will increase during processing, especially on drying. Results in Table 41 show this. Thus, titratable acidity is of limited value for this product because of the effect of alum. Volatile acidity on the other hand, is not likely to be affected by alum and hence may give a useful index of quality deterioration. While 162 Table 41. Titratable acidity and total volatile acidity of rapid process jellyfish Treatment Titratable acidity Total volatile acidity (mL O.lN NaOH/lOg (g HOAC/100 mL sample jellyfish) solution) Fresh 0.45 0.00024 24 h cure 8.65 0.00090 48 h cure 12.15 0.00078 3 h dry * 31.50 0.00204 6 h dry 39.60 0.00282 9 h dry 52.40 0.00174 * Dried at 30°C no maximum limits appear to have been set for volatile acidity, it has been reported that a correlation exists between this parameter and loss in organoleptic acceptability of stored fish (Venugopal et al., 1981; Quaranta and Curzio, 1983). The use of this parameter therefore needs further study to establish whether an upper limit can be set for volatile acidity in jellyfish. Increases irrvolatile acidity have been attributed to non-enzymic browning reactions (Madovi, 1980) and hence may be a valuable parameter in process control. 163 4.8.3.2 Traditional process jellyfish The volatile acid and titratable acid values of jelly fish processed by the traditional technique are presented in Table 42. It can be seen that titratable acidity continued to increase as processing time progresse~ The impact of alum on the titratable acidity has been described in the preceding section where it was deemed to be of little value for salted jellyfish quality assessment. Volatile acid values tended to increase during the process although considerable fluctuations are apparent. As discussed in the section above, volatile acidity may have some value as a process control parameter but needs further evaluation in this role. 164 Table 42. Titratable acidity and total volatile acidity of traditional process jellyfish Treatment Titratable acidity Total volatile acidity (mL O.lN NaOH/10 g (g HOAC/100 mL jellyfish) sample solution) Fresh 0.45 0.00024 24 h cure 8.65 0.00090 48 h cure 12.15 0.00078 8 days cure 29.40 0.00057 14 days cure 29.90 0.00114 21 days cure 28.10 0.00036 8 D cure, 2 D dry 54.80 0.00036 14 D cure, 2 D dry 44.90 0.00048 21 D cure, 2 D dry 106.90 0.00108 165 5. CONCLUSION The potential for utilisation of the Australian jellyfish Catostylus sp. as a food product has been investigated. The effectiveness of salt and alum alone and in combination as preservatives for jellyfish were evaluated. Salt and alum in combination were essential to obtain desirable structure and texture of the final product. Alum is important for its role in texture development and lowering of pH of the processed jellyfish. A traditional 35-day process was applied to Catostylus sp. and proved to be time and labour intensive. Despite shortening of the process to 10 days, it was still regarded as economically non-viable under Australian conditions. In addition, organoleptic properties of the traditional process product were inferior to those of imported commercially produced salted jellyfish. New approaches included first blanching and drying of the untreated jellyfish which proved unsuccessful. A new process involving dry salting of jellyfish with 15% salt and 5% alum for 48 h and artificially drying for 9-12 h at 30°C was suitable for processing jellyfish under Australian conditions. This rapid process produced salted products that are comparable in chemical and organoleptic attributes to commercial product; in yields of approximately 20% of bell weight. Processing time is only 2t days compared to 35 days of the traditional process, thus, time and labour commitments are greatly reduced. The products obtained from the rapid process were evaluated in terms of nutritional and storage quality. Jellyfish proteins are comprised by collagenous materials and cannot be considered high quality because the essential amino acids are of low levels 166 and tryptophan was not present. Rapid processing did not have an apparent effect on protein solubility but digestibility of processed jellyfish was significantly better than fresh samples. Amino acids of fresh and processed jellyfish differed significantly only in glycine, lysine and valine indicating that processing in general did not have detrimental effect on amino acid content of jellyfish. On the other hand, the isoelectric points (pi) of proteins were affected by processing depending on the preservative employed. The more acidic proteins were affected by alum but not by salt. As processing time progressed, fewer protein fractions were detected by IEF and migration rate of proteins was likewise affected due to changes in pi. Rapid processed jellyfish were stable at room temperature even after 6 months storage. This is attributed to low pH and water activity, and high salt content of the product. The effects of preservatives used for rapid processing on the physical structure of jellyfish were studied microscopically. Jellyfish treated with salt-alum in combination had structures similar to those found in fresh samples therefore, these in combination were essential in order to obtain desirable structure and texture of the final product. Brine produced during the rapid process could be recycled once yielding products of comparable qualities to those of the dry salted jellyfish. Recycling brine therefore, minimises environmental pollution and at the same time is economically advantageous to the jellyfish processor due to savings in curing chemicals. 167 Spoilage of fresh jellyfish stored at ambient temperature was found to be rapid in summer and after 24 h exposure it became putrid. In winter, such deterioration becomes detectable only after 48 h exposure. Of parameters examined, TMA and TVB appeared to correlate well with onset of spoilage in fresh jellyfish while total volatile acidity may provide an index of processed jellyfish quality but still needs further investigation on this vole. 168 6. Appendices Appendix 1 3~~~--~-L~~~~--L-~~--~_L~~~_. __L_~~~~_J 0 10 20 30 40 so 60 70 80 90 100 Distance from cathode (mm) Standard curve for determination of pH gradient profile using the broad pi calibration kit on a 1% agarose gel containing pharmalyte 3-10. 169 Appendix 2 8.IV 1-i c:u Cl) 1>. M tU Q tU < < (.) •rl +J tU E! 0 .1-1 :::s £HN tU Cl) 1-i SA'1 c:u .1-1 ;:.:tU 1>. SA1AH ..0 '1:1 c:u .1-1 tU 1-i tU ...... 0.. Q c:u ·M Cl) ....._,E! ..c: Cl) mn c:u ·M na1I E! \H ·rl 1>. E-t M ".laW M c:u ·r; P~A '1:1 s.& c:u ·rl 1-i '1:1 I B1V c:u N c:u c:u .&1D 1-i \H I '1:1 O.ld c:u .j.J tU c:u 1-i n1D .j.J Q .I aS :::s \H .ll{J, 0 c:u dsv r-i ·r-1 \H 0 1-i 0.. "'d •r-1 u tU 0 Q ·r-1 E! < asuodsa.I .Iap.Io:la"H 170 Appendix 3 ~ I 00 ~ I 0 ~ ~ ~ ~ ~ 00 ~ ~ ~ H ~ ~ ~ ~ H 0 0 ~ ~ ~ ~ ~ < H ro 0 u u ~ ~ 20 40 60 80 Distance from cathode (mm) Densitometric scan of the protein fractions separated by IEF in untreated jellyfish 171 Appendix 4 8.IV 1-< Q) U} >. r--im m~ < < u ·r-1 S1H .j.Jm E3 0 .j.J ::s £HN m U} SA1 1-< Q) .j.J ::s:et! >. SAlAH ..c "1:) Q) .j.J m 1-< et! 0.. Q) Cl) ...... n•n ~ J:: •r-1 Cl) nan E3 ·r-1 ...... 4-l >. :J-aW Q) r--i E3 r--i •r-1 Q) E-t ...., TB .I\ "1:) Q) SA:) .j.J et! Q) BlV 1-< Al~ .j.J E3 ::s .-lm I .j.J O.ld r--i et! lll~ Cl) J:: .I as 00 .llJ:J. ...;t 4-l dsv 0 Q) r--i ·r-1 4-l 0 1-< 0.. "t:: •r-1 u m 0 ~ ·r-1 E3 < asuodsa;1 .Iap.Io:>aN 172 Appendix 5 8.IV 1-< Q) Cll 0 >. CX) r-i CO ~ CO < < S1H u ·r-1 ,.....0 .j..) eCO £HN 0 .j..) ::I SA1 CO Cll 1-< Q) 0 .j..) SATAH \0 ~ >. ..0 '"d Q) .j..) 0 """'~ ctl If) ·r-1 1-< e CO '-" 0.. na1 Q) eQ) Cll nan ·r-1 .c E-t Cll :law ·r-1 4-l >. r-i 1BA r-i SA:) .Q)...., '"d Q) ·r-1 BlV 1-< Al~ '"d .c1-< Q"\ '"d O.Id ~ CO fll~ '"d Q) .j..) .I as r-i CO .Il.J:.L Cll 4-l dsv 0 Q) 0 r-i r-i ·r-1 4-l 0 1-< 0.. '"d ·r-1 u CO 0 ~ ·r-1e asuGdsaN .Iap.IoJaN < 173 Appendix 6 "0 ~ ~ ~ "0 00 ~ ~ ~ ~ 0 "0 ~ ~ 0 00 "0 ~ ~ 0 < ~ ~ ~ ~ ro ~ u ~ ~ 0 u ~ ~ 20 40 60 Distance from cathode (mm) Densitometric scan of the protein fractions separated by IEF in 48 h alum-treated jellyfish 174 Appendix 7 ~ 00 ~ ~ ~ ~ 0 ~ ~ ~ ~ 00 ~ ~ ~ ~ ~ ~ 0 0 ~ ~ ~ < ~ ~ ~ ro ~ u 0 u ~ ~ 20 40 60 Distance from cathode (mm) Densitometric scan of the protein fractions separated by IEF in 48 h salt-treated jellyfish 175 Appendix 8 w ~ 00 ~ ~ w 0 ~ w 00 ~ ~ w ~ 0 ~ w <~ ~ w w ~ I ~ 0 ~ ~ 0 ~ u ro w u ~ I 20 40 60 Distance from cathode (mm) Densitometric scan of the protein fractions separated by IEF in 48 h salt-alum treated jellyfish 176 Appendix 9 (]) (/)c:: 0 0.. "'0 (/) c:: '"d (]) (]) c:: 1-< (]) (]) 1-< '"d (]) (]) 0 '"d "'0 ..c:: 0 1-< oi.J 0 ctl c:: tJ u < (]) p:: 20 40 Distance from cathode Densitometric scan of the protein fractions separated by IEF in 12 h salt/alum-treated jellyfish 177 Appendix 10 ~ ~ ~ ~ ~ 00 ~ ~ ~ 0 ~ ~ o- ~ ~ 00 0 0 ~ ~ ~ H ~ ro < H u ~ ~ H 0 u ~ ~ 20 40 60 Distance from cathode (mm) Densitometric scan of the protein fractions separated by IEF in 24 h salt/alum-treated jellyfish 178 Appendix 11 m 00 ~ c ~ c 0 c m ~ m 00 m m m ~ ~ ~ 0 0 c ~ ~ < m ~ ~ ~ ~ u 0 um ~ 20 40 60 Distance from cathode Densitometric scan of the protein fractions separated by IEF in alum brine m 00c 0 ~ m00 m ~ ~m ~ ~ 0 u m 20 40 60 80 ~ Distance from cathode (mm) Densitometric scan of the protein fractions separated by IEF in salt brine 179 Appendix 12 Q) "Cl "Cl Ul !=: Q) !=: Q) !=: 0 p, Q) Q) Ul "Cl "Cl Q) 0 0 1-< ..c: !=: +J < 1-< C\1 Q) u "Cl 1-< 0 (.) Q) p:; 20 40 Distance from cathode (mm) Densitometric scan of the protein fractions separated by IEF in salt-alum brine 180 7. BIBLIOGRAPHY ABRAMSON, H.A., MOYER, L.S. and GORIN, M.H. Electrophoresis of proteins and the chemistry of cell surfaces. New York: Hafmer Publishing Co., Inc.; 1964. AGASSIZ, L. Contributions to the natural history of the United States of America. 4. Boston; 1862. ANGLEMIER, A.F. and MONTGOMERY, M.W. Amino acids, peptides and proteins. Fennema, O.R., ed. Principles of food science: Food chemistry. New York: Marcel Dekker, Inc.; 1976: 205-78. AOAC. Official methods of analysis. Association of Official Analytical Chemists. 12th ed. Washington, D.C. AOAC; 1975. ASHGAR, A. and HENRICKSON, R.L. Chemical, biochemical, functional, and nutritional characteristics of collagen in food systems. Adv. Food Res. 28: 231-72; 1982. AYRES, J.C., MUNDT, J.O. and SANDINE, W.E. Microbiology of foods. Schweigert, B.S., ed. San Francisco: W.H. Freeman and Co.; 1980. BAILEY, A.J. and ETHERINGTON, D.J. Metabolism of collagen and elastin. Florkin, M. and Stotz, E.H., eds. Comprehensive biochemistry 19B:l. Amsterdam: Elsevier Scientific Publishing Co.; 1980: 299-431. BALIAN, G. and BOWES, J.H. The structure and properties of collagen. Ward, A.G. and Courts, A., eds. The science and technology of gelatin. London: Academic Press; 1977. BARNES, R.D. Invertebrate zoology. 4th ed. Philadelphia: Saunders Co.; 1980. 181 BARZANSKY, B., LENHOFF, H.M. and BODE, H. Hydra mesoglea: similarity of its amino acid and neutral sugar composition to that of vertebrate basal lamina. Comp.Biochem.Physiol. SOB: 419-24; 1975. BEATTY, S.A. Studies of fish spoilage. II. The origin of trimethylamine produced during the spoilage of cod muscle press juice. J.Fish.Res.Bd.Can. 4: 63-8; 1938. BEATTY, G.H. and BEATTY, A.F. Marine invertebrates of New England waters. Proceeding of the Pennsylvania Academy of Science. 48: 54; 1974. BECKETT, D.C. and TURANCHIK, E.J. Occurrences of the freshwater jellyfish Craspedacusta sowerbyi in the Ohio river. Ohio J.Sci. 80: 95-6; 1980. BEITMAN, R.E. M.S. thesis. Univ. of Cincinati. 1975. cited by Beckett, D.C. and Turanchik, E.J. Occurrence of the freshwater jellyfish Craspedacustasowerbyi in the Ohio river. Ohio J.Sci. 80: 95-6; 1980. BERGMANN, W., MCLEAN, M.J. and LESTER, D. Contributions to the study of marine products - XIII. Sterols from various marine invertebrates. J.Org.Chem. 8: 271-81; 1943. BERTIN, L. ed. Larousse encyclopedia of animal life. London: The Hamlyn Publishing Group Ltd; 1973. CASON, J.E. A rapid one-step Mallory-Heidenhain stain for connective tissue. Stain Technol. 25: 225-26; 1950. CHAPMAN, G. Studies of the mesoglea of Coelenterates. Q.J. Micros.Sci. 94: 168-75; 1953. CHAPMAN, G.- The structure and function of the mesoglea. Rees, W.J., ed. Cnidaria and their evolution. New York: Academic Press; 1966: 147-68. 182 CHEFTEL, J.C. Chemical and nutritional modifications of food proteins due to processing and storage. Whitaker, J.R. and Tannenbaum, S.R., eds. Food proteins. Westport, Connecticut: The AVI Publishing Co. Inc.; 1977. CHINA Pictorial. The life of jellyfish. 2: 24-5; 1982. CLELAND, J.B. and SOUTHCOTT, R.V. Injuries to man from marine invertebrates in the Australian region. Nat. Health & Med. Res. Foundation Spec. Rep. Ser. 12. Canberra, A.C.T.; 1965. COCKRUM, L.E. and McCAULEY, W.J. Zoology. Philadelphia: W.B. Saunders; 1965. CONNELL, J.J. Control of fish quality. England: Fishing News Books Ltd.; 1980. COWIE, W.P. and LITTLE, W.T. The relationship between the toughness of cod stored at -29°C and its muscle protein solubility and pH. J.Food Technol. 1: 335; 1966. CURTIS, S.K. and COWDEN, R.R. Regenerative capacities of the Scyphistoma of Cassiopea (Phylum, Coelenterata; class, Scyphozoa). Acta Embry. Exp. Suppl., 429-54; 1972. DAKIN, W.J., BENNETT, I. and POPE, E. Jellyfishes, anemones, 'blue-bottles' and corals. Austarlian Seashores, Sydney: Angus & Robertson Publishers; 1979. DAVIS, L.E. Cell division during dedifferentiation and redifferentiation in the regenerating isolated gastrodermis of Hydra. Exp.Cell Res. 60: 127-32; 1970. DAVIS, L.E., BURNETT, A.A., HAYNES, J.F. and MUMAW, V.R. A histological and ultra-structural study of dedifferentiation and redifferentiation of digestive 183 and gland cells in Hydra viridis. Dev. Biol. 14: 307-29; 1966. DEXTER, R.W., SURRARRER, T.C. and DAVIS, C.W. Some recent records of the freshwater jellyfish Craspedacusta sowerbyi from Ohio and Pensylvania. Ohio J.Sci. 49: 235-41; 1949. DIEHL, F.A. Cellular differentiation and morphogenesis in Cordylophora. Wilhelm Roux Archiv. 162: 309-35; 1969. DIGBY, 1967. cited by PULLEY, K.J. The biology of the Scyphozoa. Kensington, NSW. The University of New South Wales. 1971 B.Sc. honours thesis. DILLON, T.M. Effects of acute changes in temperature and salinity on pulsation rates in ephyrae of the scyphozoan Aurelia aurita. Marine Biol. 42: 31-35; 1977. DRURY, R.A.B. and WALLINGTON, E.A. 'Carleton's histological technique' 5th ed. Oxford: Oxford University Press; 1980. DUERR, J.D. and DYER, W.G. Protein in fish muscle. IV. Denaturation by salt. J.Fish.Res.Bd.Can. 8: 325-31; 1952. DULANEY, J.T. and TOUSTER, 0. The solubilisation and gel electrophoresis of membrane enzymes by use of detergents. Biochim.Biophys.Acta. 196: 29-34; 1970. DUNAJSKI, E. Texture of fish muscle. J.Texture Studies. 10: 301-18; 1979. DYER, W.J. Amines in fish muscle. VI. Trimethylamine oxide content of fish and marine invertebrates. J.Fish. Res.Bd.Can. 8: 314-24; 1952. 184 DYER, W.J. and MOUNSEY, Y.A. Amines in fish muscle. II. Development of trimethylamine and other amines. J. Fish.Res.Bd.Can. 6: 359-67; 1945. EDMONDSON, C.H. New Hawaiian medusae. Occ.Pap.Bernice Bishop Mus. Honolulu. 9: 1-16; 1930. ETCHELLS, J.L. Rate of heat penetration during the pasteurization of cucumber pickles. Fruit Prod.J. 18: 68; 1938. ETCHELLS, J.L. and JONES, I.D. Procedure for pasteurizing pickle product. Glass Packer. 23: 519; 1944. FARR, J.A. Blue crab predation on jellyfish. Florida Sci. 41: 217-19; 1978. FIRTH, F.E. ed. The encyclopedia of marine resources. New York: Van Nostrand Reinhold Co.; 1969. FISHER, R.A. and YATES, F. Statistical tables for biological, agricultural and medical research. 3rd ed. London, England: Oliver and Boyd Ltd.; 1942. FOX, B.A. and CAMERON, A.G. Food science: A chemical approach. London: Hodden and Stoughton; 1983. FRANKE, B. Dissertation. Vet.Med. 1964. cited by Reutersward, A.L. Solubilization of pigskin and bovine tendon after pepsin and pancreatin treatment. Effect of incubation conditions and age of animal. J.Food Technol. 20: 129-43; 1985. GILCHRIST, F.G. Budding and locomotion in scyphistomae of Aureli& Biol.Bull.Mar.Biol.Lab., Woods Hole. 72: 99-124; 1937. GOHAR, H.A.F. and EISAWY, A.M. The biology of Cassiopea andromeda with a note on the species problem. Publ.Mar.Biol.Stat. Ghardaqa (Red Sea) Nr. 11: 3-39; 1960. 185 GOLDNER, R., BURNETT, J.W., STONE, J.S. and DILAIMY, M.S. The chemical composition of sea nettle nematocysts. Proc.Soc.Exp.Biol.Med. 131: 1386-8; 196~. GORDON, A.H. Electrophoresis of proteins in polyacrylamide and starch gels. Amsterdam: North Holland Publishing Co.; 1969. GOSLINE, J.M. AND LENHOFF, H.M. Kinetics of incorporation of 14 c-proline into mesogleal proto-collagen and collagen of the sea anemone Aiptasia. Comp.Biochem.Physiol. 26: 1031-9; 1968. GRONINGER, H.S. The occurrence and significance of trimethylamine oxide in marine animals. U.S.Fish and Wildl.Serv. Spec.Rep.Fish. 233; 1959. GRZIMEK, B. Ed. Grzimek's animal life encyclopedia. New York: Van Nostrand Reinhold Co.; 1974. GUEST, W.C. The occurrence of thejellyfishChiropsalmus quadrumanus in Matagorda Bay, Texas. Bull.Mar.Sci. Gulf and Carib. 9: 79-83; 1959. GUNTER, G. Seasonal population changes and distribution as related to salinity of certain invertebrates of the Texas coast including the commercial shrimp. Publ.Inst.Mar.Sci.Univ.Tex. 1: 8-51; 1950. GUPTA, K.C. and SCHEUER, P.J. Zoanthid sterols. Steroids. 13: 343-56; 1969. GUSTAVSON, K.H. The chemistry and reactivity of collagen. New York: Academic Press; 1956. HANG, Y.D., WOODAMS, E.E. and PARSONS, G.F. Isolation and chemical evaluation of protein from clam wash water. J.Food Sci. 45: 1040-1; 1980. 186 HARADA, K. Studies on enzyme catalysing the formation of formaldehyde and trimethylamine in tissues of fishes and shells. J.Shimonoseki Univ.Fish. 23: 163-241; 1975. HARKNESS, M.L.R., HARKNESS, R.D. and VENN, M.F. Digestion of native collagen in the gut. GUT. 19: 240-3; 1978. HATAI, S. On the composition of Cassiopeia xamachana and the changes in it after starvation. Washington: Carnegie Inst.Pub. No. 251; 1917. HAUROWITZ, F. The chemistry and function of proteins. New York: Academic Press; 1963. HAUROWITZ , F. and WAELSCH, H. Uber die chemische Zusammensetzung der Qualle Velella spirans. Ann. 161: 300-17; 1926. HAYNES, J. and BURNETT, A.L. Dedifferentiation and redifferentiation of cells in Hydra viridis. Sci. 142: 1481-83; 1963. HEARING, V.J., KLINGHER, W.G., EKEL, T.M. and MONTAGUE, P.M. Molecular weight estimation of Triton X-100 solubilised protein by polyacrylamide gel electrophoresis. Anal. Biochem. 72: 113-22; 1976. HERRNKIND, W., HALUSKY, J. and KANCIRUK, P. A further note on phyllosoma larvae associated with medusae. Bull.Mar. Sci. 26: 110-12; 1976. HOESE, H.D., COPELAND, B.J. and MILLER, J.M. Seasonal occurrence of Cyanea medusae in the Gulf of Mexico at Port Aransas, Texas. Texas J.Sci. 16: 391-3; 1964. von HOLT, C. Uptake of glycine and release of nucleoside polyphosphates by zooxanthellae. Comp.Biochem.Physiol. 26: 1071-9; 1968. HOOPER, S.N. and ACKMAN, R.G. Distribution of trans-6- hexadecenoic acid, 7-methyl-7-hexadecenoic acid 187 common fatty acids in lipids of the ocean sunfish Molamola. Lipids 8: 509-16; 1973. HULTIN, H.O. Characteristics of muscle tissue. Fennema, O.R., ed. Principles of food science: Food Chemistry. New York: Marcel Dekker Inc.; 1976: 577-614. ILJAS, A. and ARIFUDIN, R. Eksperimen pendahuluan pada pengolahan uburubur. Jakarta: Lembaga Teknologi Perikanan; Laporan Peneliltian No. 1; 1971. ILJAS, A. and ARIFUDIN, R. Pengolahan uburubur (asin) III. Perbandingan optimum garam dan tawas pada pengolahan 'Salted jellyfish'. 1: 1-17; 1975. JOHNS, P. and COURTS, A. Relationship between collagen and gelatin. WARD, A.G. and COURTS, A., eds. The science and technology of gelatin. London: Academic Press; 1977. JONES, W.C. Colloidal properties of the mesoglea in a species of Leucosolemia. W.J.Micros.Sci. 97: 269-85; 1956. JOSEPH, J.D. Lipid composition of marine and estuarine invertebrates. Porifera and Cnidaria. Prog.Lipid Res. 18: 1-30; 1979. KAKINUMA, Y. An experimental study on the life cycle and organ differentiation of Aurelia aurita ~amarck. Bull.Mar. Biol.Stat. Asamushi. 15: 101-13; 1975. KALYANI, M. A note on the amino acid composition of the collagen of Virgularia sp. Current Sci. (Bangalore) 42: 829-30; 1973. KATZMAN, R.L. and JEANLOZ, R.W. The carbohydrate chemistry of invertebrate connective tissue. Balazs, E.A., ed. Chemistry and molecular biology of the intercellular matrix. New York: Academic Press; 1970: 217-27. 188 KAWAMURA, Y., NISHIMURA, K., IGARASHI, S., DOI, E. and YONEZAWA, D. Characteristics of autolysis of antarctic krill. J.Biol.Chem. 45: 93-100; 1981. KELLY, K.O., JONES, N.R., LOVE, R.M. and OLLEY, J. Texture and pH in fish muscle related to 'cell fragility' measurements. J.Food Technol. 1:9; 1966. KEVIN, P., ZAHL, P.A. and MCLAUGHLIN, J.J.A. In situ studies of the alga Symbiodinium microadriaticum and the sea anemone, Condylactis, and in the jellyfish, Casseopeia. J.Protozool. (Suppl.) 13: Abstract No. 82; 1966. KIES, C. Bioavailability: a factor in protein quality. J.Agric. Food Chem. 29: 435-40; 1981. KINSELLA, J.E. Relationships between structure and functional properties of food proteins. Fox, P.F. and Condon, J.J., eds. Food proteins. London: Applied Science Publishers; 1982: 51-103. KONOSU, S., WATANABE, K. and SHIMIZU, T. Distribution of nitrogenous constituents in the muscle extracts of eight species of fish. Bull.Jap.Soc.Sci.Fish. 40: 909-15; 1974. KRAEUTER, J.N. and SETZLER, E.M. The seasonal cycle of scyphozoa and cubozoa in Georgia estuaries. Bull.Mar.Sci. 25: 66-74; 1975. KRAMP, P.L. Medusae, siphonophore and ctenophora. Zool.Iceland. 2: 1-37; 1939. KRAMP, P.L. Some medusae (mainly scypho-medusae) from Australian coastal waters. Trans.Roy.Soc.S.Australia. 89: 257-78; 1965. 189 KRUMBACH, T. Scyphozoa. Handb. Zool. 1: 522-686; 1925. LANE, C.E., PRINGLE, E. and BERGERE, A.M. Amino acids in extracellular fluids of Physalia physalis and Aurelia aurita. Comp.Biochem.Physiol. 15: 259-62; 1965. LARMOND, E. Laboratory method for sensory evaluation of food. Can.Dept. of Agric.Publ. 1637; 1983. LARSON, R.G. Marine flora and fauna of the Northeastern United States. Cnidaria: scyphozoa. Nat'l. Ocean Atmos.Adm. Tech.Rep.Nat'l.Mar.Fish.Service Circ. 397: 1-18; 1976. LARSON, R.G. A new stauromedusa, Kishinouyea corbini (scyphozoa, stauromedusae) from the tropical Western Atlantic. Bull.Mar.Sci. 30: 102-7; 1980. LEES, R. Laboratory handbook of methods of food analysis. London: Leonard Hill Books; 1968. LEHNINGER, A.L. Biochemistry. New York: Worth Publishing Inc; 1970. LENDENFELD, R. VON. The scyphomedusae of the southern hemisphere. Proc.Linn.Soc.N.S.W. 155-306; 1884. LID, G. The occurrence of jellyfish (scyphozoa) along the Norwegian coast. Fauna. 32: 129-36; 1979. LINKO, R.R. and NIKKILA, O.E. Inhibition of the denaturation of salt of myosin in Baltic herring. J.Food Sci. 26: 606-10; 1961. LOEWIT, K. Zur frage der verdaulichkeit von bindegewebe. Archiv fur lebensmittelhygiene. 18: 178-90; 1967. LOVE, R.M., ROBERTSON, I., SMITH, G.L. and WHITTLE, K.J. The texture of cod muscle. J.Texture Studies. 5: 201-12; 1974. 190 LOWELL, R.D. and BURNETT, A.L. Regeneration of complete Hydra from isolated epidermal explants. Biol.Bull. 137: 312- 20; 1969. LOWNDES, A.G. Nature. 150: 234; 1942. cited by Ludwig, H.W. 99.26% water content in the freshwater medusa, Craspedacusta sowerbyi. Z. Naturforsch. 32C: 1011-2; 1977. LUDWIG, H.W. 99.26% water content in the freshwater medusa, Craspedacusta sowerbyi. Z. Naturforsch. 32C: 1011-2; 1977. LUKANIN, V.V. Adaptive response to changing water salinity in the larvae of the scyphoid jellyfish Aurelia saurita (L Zh.Obshch.Biol. 37: 776-87; 1976. MA, N.C. Mrs. Ma's favourite Chinese recipes. New York: Kodansha International; 1968. MACPHERSON, N.L. The dried codfish industry. Ann.Rep.NFD.Fish Res.Comm. 2: 8; 1933. MADOVI, P.B. Changes in the free-NH , free-C0 H and titratable 2 2 acidity of meat proteins. J.Food Technol. 15: 311-18; 1980. MAGNO-OREJANA, F. Drying of jellyfish (Aurelia aurita). Preliminary Rp~Inst.Fish Dev.Res., Coll.Fish, U.P., Phil.; 1983. MANDL, I. Collagenases and elastases. Adv.Enzymol. 23: 162-264; 1961. MANGUM, C.D., OAKES, M.J. and SHICK, J.M. Rate-temperature responses in scyphozoan medusae and polyps. Mar.Biol. 15: 298-303; 1972. MARK, H.F., OTHMER, D.F., OVERBERGER, C.G. and SEABORG, G.T., eds. Encyclopedia of chemical technology. 3rd ea. New York: John Wiley & Sons; 1978. 191 ·MAYER, A.G. Medusae of the world. Washington D.C.: Carnegie Inst.; 1910. MAYER, A.G. Ctenophores of the Atlantic coast of North America. Publ.Carnegie Inst. 162: 1-58; 1912. MAYER, A.G. The effects of temperature upon tropical marine animals. Publ.Carnegie Inst. 183: 1-24; 1914. MAYER, A.G. Medusae of the Philippines and of Torres Straits. Pap.Tortugas Lab. 8: 157-202; 1915, cited by Kramp, P.L. Some medusae (mainly scyphomedusae) from Australian waters. Trans.Roy.Soc. S.Aust. 89: 258-78; 1965. MAYER, A.G. Report upon the scyphomedusae collected by the U.S. Bureau of Fisheries Steamer 'Albatross' in the Philippine Islands and Malay Archipelago. Bull.U.S.Nat.Mus. 171-233; 1917. McGRAW, K.A. Two aberrant form of the moon jellyfish, Aurelia aurita (Linne), in the Northeastern Gulf of Mexico. Chesapeake Sci. 15: 55-6; 1975. MIDDLEBROOK, R.E. and LANE, C.E. Cholesterol in Physalia physalis. Comp.Biochem.Physiol. 24: 507-10; 1968. MOLLER, H. Scyphomedusae as predators and food competitors of larval fish. Meeresforschung. 28: 90-100; 1980. MORIKAWA, T. Jellyfish. Infofish Marketing Digest. 1: 37-9; 1984. MORRIS, D. Coelenterates: Larouse Encyclopedia of the animal world. New York: Larouse & Co.Inc.; 1975. MOSHINA, N.I. Mass development of Rhizostoma pulmo and Aurelia aurita discomedusae in the Axee Azov Sea, USSR. Zool.Z. 53: 1248-57; 1974. 192 NARAYANASWAMY, D., RAO, C.V. and GOVINDAN, T.K. Penetration of sodium chloride during prolonged salting of fish. Fish Technol. 17: 63~4; 1980. NEUMAN, R.A. and TYTELL, A.A. Action of proteolytic enzymes on collagen. Proc.Soc.Exp.Biol.Med. 73: 409-12; 1950. NORDWIG, A. and HAYDUK, U. Invertebrate collagens: isolation, characterization and phylogenetic aspects. J.Molec. Biol. 44: 161-72; 1969. NORMANDIN, D.K. Regeneration of hydra from the endoderm. Sci. 132: 678; 1960. NORRIS, E.R. and BENOIT, G.J.Jr. Trimethylamine oxide. I. Occurrence of trimethylamine oxide in marine organisms. J.Biol.Chem. 158: 433-8; 1945. OBANU, Z.A., LEDWARD, D.A. and LAWRIE, R.A. The protein of intermediate moisture meat stored at tropical temperature. 1. Changes in solubility and electrophoretic pattern. J.Food Technol. 10: 657-66; 1975. OMORI, M. Edible jellyfish (scyphomedusae: rhizostomeae) in the Far East Waters. A brief review of the biology and fishery. Bull.Plank.Soc.Jap. 28: 1-11; 1981. PANIKKAR, N.K. Occurrence of a stauromedusa on the Indian coast. Current Sci.Bangalore. 13: 238-9; 1944. PAUL, P.C. Food theory and application. Paul, P.C. & Palmer, H.H., eds. New York: John Wiley & Sons; 1972. PEIMEI. Peimei's Chinese Cook Book. Taipei, Taiwan; 1978. PHARMACIA FINE CHEMICALS. Isolectric focusing, principles and methods. Sweden: Ljungforetagen AB; 1982. PHILLIPS, P.J. and BURKE, W.D. The occurrence of sea wasps (cubomedusae) in Mississippi Sound and the Northern Gulf of Mexico. Bull.Mar.Sci. 20: 853-9; 1970. 193 PHILLIPS, P.J., BURKE, W.D. and KEENER, E.J. Observations on the trophic significance of jellyfishes in Mississippi Sound with quantitative data on the associative behaviour of small fishes with medusae. Trans.Amer.Fish.Soc. 98: 703-12; 1969. PIEZ, K.A. and GROSS, J. The amino acid composition and morphology of some invertebrate and vertebrate collagens. Biochim.Biophys.Acta. 34: 24-39; 1959. POPE, E.C. A large medusa in Sydney Harbour. Aust.Mus.Mag. 10: 14-6; 1949. POPE, E.C. Sea lice or jellyfish. Aust.Mus.Mag. 11: 16-21; 1953. POTTER, H.N. Food Science. Connecticut: The AVI Publishing Co.Inc.; 1968. POWELL, E.H.Jr. and GUNTER, G. Observations on the stone crab Menippe mercenaria Say, in the vicinity of Port Aranas, Texas. Gulf Res.Rep~ 2: 285-99; 1968. PRANDL, 0. Meat in nutrition and health. Franklin, K.R. and Davis, P.N., eds. Chicago, Illinois: Nat'l. Livestock & Meat Bd.; 1980. PULLEY, K.J. The biology of the scyphozoa. Kensington, NSW. The University of New South Wales; 1971. BSc. Honours Thesis. QUARANTA, H.O. and CURZIO, O.A. Total volatile acids content as a quality index of irradiated hake (Merluccius merluccius Hubsii) fillets. Food Chem. 11: 27-30; 1983. QUENSEN, J.M. and BLACK, R.E. Changes in amount and composition of gelatin from developmental stages of the scyphozoan jellyfish, Chrysaora quinquecirrha. Proc. 47: 59; 1974-75. 194 RAUM, W.J. Glycine metabolism in the scyphistoma of Aurelia aurita. cited by Webb, K.L., Schimpf, A.L. and Olmon~. J. Free amino acid composition of scyphozoan polyps of A. aurita, C. quinquecirrha and C. capillata at various salinities. Comp.Biochem.Physiol. 43B: 653-63; 1972. REUTERSWARD, A.L. Solubilisation of pigskin and bovine tendon after pepsin and pancreatin treatment. Effect of incubation conditions and age of animal. J.Food Technol. 20: 129-43; 1985. RICE, 1964. cited by Pulley, K.J. The biology of the scyphozoa. Kensington, NSW. The University of New South Wales; 1971. B.Sc Honours Thesis. RIGBY, B.J. and HAFEY, M. Thermal properties of the collagen of jellyfish (Aurelia coerulea) and their relation to its thermal behaviour. Aust.J.Biol.Sci. 25: 1361-3; 1972. ROBERTSON, J.D. The inorganic composition of the body fluids of three marine invertebrates. J.Exp.Biol. 16: 387-97; 1939. ROGOWSKI, B. Meat in human nutrition. World Rev.Nut.Diet. 34: 46-101; 1980. RONOLD, O.A. and JAKOBSEN, F. Trimethylamine oxide in marine products. J.Soc.Chem.Ind. 66: 160-6; 1947. RUITER, A. Trimethylamine amine and the quality of fish. Voedings. Voedingsmiddelen Technol. 2: 1-10; 1971. RUSSELL, F.S. The medusae of the British Isles. II. Pelagic scyphozoa. Cambridge Univ.Press.; 1970. RUSSELL, F.S. Scyphomedusae of the North Atlantic. 2. Families Pelagiidae, Cyaneidae, Ulmaridae, Rhizostomatidae. Fiches Identif.Zooplancton. 158: 1-4; 1978. 195 SCHWAB, W.E. The ontogeny of swimming behaviour in the scyphozoan, Aurelia aurita 1. Electrophysiological analysis. Biol.Bull. 152: 233-50; 1977. SCOPES, R.K. Protein purification: principles and practice. New York: Springer-Verlag; 1984. SHEIHK, A.B. and SHAH, F.H. Effect of heat on the vitro digestibility of fish protein. Pakistan Sci.Ind.Res. 17: 136-8; 1974. SHICK, J.M. Free amino acids in Aurelia aurita scyphistomae from Corpus Christi, Texas.Comp. Biochem. Physiol. 53B: 1-2; 1976. SIMMONS, E.G. Ecological survey of the upper Laguna Madre of Texas. Publ.Inst.Mar.Sci.Univ.Tex. 42: 156-200; 1955. SIPOS, J.C. and ACKMAN, R.G. Jellyfish (Cyanea capillata). Lipids: Fatty acid composition. J.Fish.Res.Bd.Can. 25: 1561-9; 1968. SOONTHONVIPAT, V. Dried jellyfish. Tieros, K., ed. Fisheries resources and their management in South-east Asia. Proc.Int'l. Seminar Nov-Dec 1974. German Foundation for Int'l.Dev.; 1976: 149-51. SORENSEN, J.B. Thesis. Royal Veterinary Agric.Univ.; Kobenhavn. 1981 cited by Reutersward, A.L. Solubilisation of pigskin and bovine tendon after pepsin and pancreatin treatment. Effect of incubation conditions and age of animal. J.Food Technol. 20: 129-43; 1985. SOUTHCOTT, R.V. Studies on Australian cuba,:medusae, including a new genus and species apparently harmful to man. Aust.J.Mar.Freshwater Res. 7: 254-79; 1955. 196 SPANGENBERG, D.B. Cultivation of life stages of Aurelia aurita under controlled conditions. J.Exp.Zool. 159: 303-18; 1965., SPANGENBERG, D.B. Iodine induction of metamorphosis in Aurelia. J.Exp.Zool. 165: 441-50; 1967. SPINELLI, J. Biochemical basis of fish freshness. Process Biochem. 6: 36-7, 54; 1971. SPIRO, R.G. Glycoproteins. Ann.Rev. Biochem. 23: 599-638; 1970. STEINBERG, S.N. The regeneration of whole polyps from ectodermal fragments of scyphistoma larvae of Aurelia aurita. Biol.Bull. 124: 337-43; 1963. STEPHENSON, 1970. cited by Pulley, K.J. The biology of the scyphozoa. Kensington, NSW. The University of New South Wales; 1971 BSc. Honours Thesis. STORER, T.I., USINGER, R.L., NYBAKKEN, J.W. and STEBBINS, R.C. Elements of Zoology. New York: McGraw-Hill Book Co.; 1968. TRILL, H. Beitrage zur kenntnis der Aurelia aurita (L). Z.Wiss.Zool. 150: 51-96; 1937. THOMAS, L. Phyllosoma larvae associated with medusae. Nature 198: 208; 1963. TOMA, R.B. and MEYERS, S.P. Isolation and chemical evaluation of protein from shrimp cannery effluent. J.Agric.Food Chem. 23: 632-5; 1975. UCHIDA, T. Distribution of scyphomedusae in Japanese and its adjacent waters. J.Fac.Sci.Hokkaido Univ., Ser 6. Zool. 12: 209-19; 1954. 197 VAN AAREM, H.E., VONK, H.J. and ZANDEE, D.I. Lipdd metabolism in rhizostoma. Arch.Int.Physiol.Biochim. 72: 606-14; 1964. VANHOFFEN, E. Acraspedae. Nordisches Plankton. 6: 40-64; 1906. VENUGOPAL, V., LEWIS, N.F. and NADKARNI, G.B. Volatile acids content as a quality index of Indian mackerel (Rastrelliger kanagurta). Lebensm.-Wiss.U.Technol. 14: 39-42; 1981. VILLEE, C.A., WALKER, W.F.Jr. and BARNES, R.D. General zoology. Philadelphia: W.B. Saunders Co.; 1978. WATERS ASSOCIATES Instruction Manual. Ion Exchange Background. WEBB, K.L., SCHIMPF, A.L. and OLMON, J. Free amino acid composition of scyphozoan polyps of Aurelia aurita, Chrysaora quinquecirrha and Cyanea capillata at various salinities. Comp.Biochem.Physiol. 43B: 653-63; 1972. WEISS, J.B. Enzymic degradation of collagen. Hall, D.A. and Jackson, D.S., eds. International reviews of connective tissue. London: Academic Press; 1976. WELSH, F.W. and ZALL, R.R. An ultrafiltration activated carbon treatment system for renovating fishery refrigeration brines. Can.Inst.Food Technol.J. 17: 092-096; 1984. WHITE, R.H. and HAGER, L.P. Occurrence of fatty acid chlorohydrins in jellyfish lipids. Biochem. 16: 4994-8; 1977. WRITTEN, H.L., ROSENE, H.F. and HEDGPETH, J.W. The invertebrate fauna of Texas coast jetties; a preliminary survey. Publ.Inst.Mar.Sci.Univ.Tex. 1: 54-87; 1950. WIGGIN, K. and KRZYNOWEK, J. Identification of frozen, cooked shellfish species by agarose isoelectric focusing. J.Assoc.Off.Anal.Chem. 66: 118-22; 1983. WINDHOLZ, M. ed. 'The Merck Index'. An encyclopedia of chemicals and drugs. New York: Merck & Co., Inc.; 1976. 198 WINKLER, J.TH., VAN SOEST, R.W.M. First record of the scyphomedusa Deepstaria enigmatica Russell, 1967, from the Mid Atlantic Ocean (coelenterata, scyphozoa). Bull. Zool.Mus. 8: 33-8; 1981. WINTER, R. A consumer's dictionary of food additives. New York: Crown Publishing Inc.; 1978. WOOTTON, M., BUCKLE, K.A. and MARTIN, D. Studies on the preservation of Australian jellyfish (Catostylus sp.). Food Tech.Aust. 34: 398-400; 1982. YASUDA, S. Sterol compositions of jellyfish (medusae). Camp. Biochem.Physiol. 48B: 225-30; 1974. YASUDA, T. Ecological studies on the jellyfish, Aurelia aurita in Urazoko Bay, Fukui Prefecture V. Vertical distribution of the medusa. Ann.Rep.Noto Mar.Biol.Lab.Kanazawa Univ. 10: 15-22; 1970. YASUDA, T. Ecological studies on the jellyfish, Aurelia aurita (Linne) in Urazoko Bay, Fukui Prefecture. VI. Seasonal change in the vertical distribution of the medusa. Jap.J.Ecol. 21: 115-9; 1971. YASUDA, T. Ecological studies on the jellyfish, Aurelia aurita (Linne) in Urazoko Bay, Fukui Prefecture. XI. An observation on ephyra formation. Publ.Seto Mar.Biol.Lab. 22: 75- 80; 1975. YASUDA, T. Ecological studies on the jellyfish, Aurelia aurita (Linne) in Urazoko Bay, Fukui Prefecture. XIII. Vertical distribution of ephyrae and metephyrae. Bull.Jap. Soc.Sci.Fish. 42: 743-52; 1976. 199 YASUMOTO, T., INOUE, A., OCHI, T., FUJIMOTO, K., OSHIMA, Y., FUKUYO, Y., ADACHI, R. and BAGNI, R. Environmental studies on a toxic dinoflagellate responsible for ciguatera. Bull.Jap.Soc.Sci.Fish. 46: 1397-1404; 1980. ZENKEVITCH, L. Biology of the seas of the USSR. Engl.transl: Botcharskaya, S. London: Allen & Unwin Ltd.; 1956. ZWILLING, E. Formation of endoderm from ectoderm in cordylophora. Biol.Bull. 124: 368-78; 1963.