DETERMINATION OF ALUMINUM CONTENT IN FOOD … This study analyzed texture properties and mineral profiles of jellyfish products, which can provide valuable information for utilizing

DETERMINATION OF ALUMINUM CONTENT IN FOOD PRODUCTS AND TEXTURE

PROFILE OF JELLYFISH PRODUCTS

by

CHAO XU

(Under the Direction of Yao-wen Huang)

ABSTRACT

This study analyzed texture properties and mineral profiles of jellyfish products, which

can provide valuable information for utilizing jellyfish as a potential food resource, as well as

developing appropriate strategies to control the product quality. Desalting cured jellyfish in

water is a critical step to create jellyfish a desirable texture. Inorganic elements in processed

jellyfish were also under investigation. Fresh jellyfish are processed with mixture of salt and

alum, and then the cured jellyfish are desalted in water before consumption. Very little is known

about the inorganic constituents of jellyfish. In this study desalted jellyfish were examined for 7

elements, including Al, Ca, K, Mg, Na, Fe, and Zn, using inductively coupled plasma optical

emission spectrometry. High amount of aluminum was found in cannonball jellyfish samples.

High performance liquid chromatography (HPLC) with spectrophotometric detection using

quercetin is developed to determine aluminum content.

INDEX WORDS: Jellyfish, Texture, Mineral Profile, Aluminum, HPLC, Instrumental

Texture Properties


OF JELLYFISH PRODUCTS

by

CHAO XU

B.S., China Agricultural University, China 2010

A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment

of the Requirements for the Degree

MASTER OF SCIENCE

ATHENS, GEORGIA

2013

© 2013

Chao Xu

All Rights Reserved


OF JELLYFISH PRODUCTS

by

CHAO XU

Major Professor: Yao-wen Huang

Committee: William L. Kerr

Robert L. Shewfelt

Electronic Version Approved:

Maureen Grasso

Dean of the Graduate School

The University of Georgia

August 2013

iv

DEDICATION

I would like to dedicate this to my parents for their love.

v

ACKNOWLEDGEMENTS

I would like to thank Dr. Yao-wen Huang for his great guidance and encouragement

throughout this project. He has been a great source of inspiration throughout my education. I

would like to thank Dr. William L. Kerr and Dr. Robert L. Shewfelt for their support and advice.

Dr. Kerr gave me a lot of guidance at every step of my research. I am lucky to have Dr. Shewfelt

as my committee member. He is extremely helpful in completing my research.

I would like to extend my appreciation to Huiping Huang, Lu Shen, Jing Chen, Xiaomeng

Wu, Carl Ruiz and Long Zou for their technical assistance and help. I would also like to thank Dr.

Kong for letting me use the instrument in his lab. I would definitely want to thank Chi Zhang.

Thank you for giving me the warmth and happiness I needed in difficult days. Last, but certainly

not least, I would like to thank my parents and grandmother for their love and encouragement.

vi

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS .............................................................................................................v

LIST OF TABLES ........................................................................................................................ vii

LIST OF FIGURES ..................................................................................................................... viii

CHAPTER

1 INTRODUCTION .........................................................................................................1

2 LITERATURE REVIEW ..............................................................................................6

3 TEXTURE PROFILE OF JELLYFISH PRODUCTS .................................................34

4 INORGANIC CONSTITUENTS IN PROCESSED JELLYFISH AND

DETERMINATION OF ALUMINUM CONTENTS BY HPLC ...............................64

5 SUMMARY AND CONCLUSIONS ..........................................................................93

vii

LIST OF TABLES

Page

Table 3.1 Mean values of force peaks and areas for single-blade shear test and moisture content

for five commercial products .............................................................................................47

Table 3.2 Mean values of springiness (mm) for TPA ....................................................................48

Table 3.3 Correlation matrix between multiple-blade shear test and single-blade shear test of

jellyfish ..............................................................................................................................49

Table 3.4 Mean values for single-blade shear for newly-processed sample under different heating

conditions ...........................................................................................................................50

Table 3.5 Mean values for single-blade shear for refrigerator-stored sample under different

heating conditions ..............................................................................................................51

Table 4.1 Calibration curve, LOD and LOQ data (n=5) ................................................................76

Table 4.2 Intra- and inter-day precision and accuracy for the quantitative determination ........... 77

Table 4.3 Changes of dry matter of jellyfish sample over 24 h .....................................................78

Table 4.4 Element concentrations of processed jellyfish samples (bought from Dalian, China) ..79

Table 4.5 Element concentrations of processed jellyfish samples (bought from Nantong, China)80

Table 4.6 Element concentrations of commercial jellyfish products bought from China .............81

Table 4.7 Element concentrations (mg/kg) of processed jellyfish samples (newly-processed) ....82

viii

LIST OF FIGURES

Page

Figure 2.1 Diagram of jellyfish. .....................................................................................................22

Figure 3.1 Multiple-blade attachment with 16 blades (a) and a view of newly-processed sample

placed in perpendicular direction to the attachment (b).....................................................52

Figure 3.2 Five different kinds of commercial products collected on the local markets. ..............53

Figure 3.3 Visual comparison between processed jellyfish and deslated jellyfish. .......................54

Figure 3.4 Moisture content of two kinds of samples soaking in water ........................................55

Figure 3.5 Force-time curves of samples soaking for 0, 2, 4, 6, and 8 h generated by single-blade

attachment. .........................................................................................................................56

Figure 3.6 Single-blade shear force at the different locations on a sample ...................................57

Figure 3.7 Single-blade shear force (a) and area (b) of newly-processed sample and refrigerator-

stored sample after soaking in water for 8 h. .....................................................................58

Figure 3.8 Multiple-blade shear force (a) and area (b)of newly-processed sample and

refrigerator-stored sample after soaking in water for 8 h. .................................................59

Figure 3.9 Tension force (a) and area (b) of newly-processed sample and refrigeratore-stored

sample after soaking in water for 8 h. ................................................................................60

Figure 4.1Typical chromatogram for Al-quercetin chelate ...........................................................83

Figure 4.2 Aluminum calibration curve; n=5. ...............................................................................84

Figure 4.3 Aluinum concentrations in newly-processed sample (a) and refrigerator-stored sample

(b) soaking in water and 2% vinegar solution. ..................................................................85

ix

Figure 4.4 Aluminum concentrations in dried refrigerator-stored sample soaking in water and 2%

vinegar solution. .................................................................................................................86

Figure 4.5 Aluminum concentrations in soaking solutions. ..........................................................87

1

CHAPTER 1

INTRODUCTION

Jellyfish is the common name of certain invertebrate animals of the phylum Cnidaria,

which belongs to the Class Scyphozoa (Gibson and Barnes 2000). Several species with mild

stings are edible, among which, Rhopilema esculentum Kishinouye is the most abundant and

important species in the Asian jellyfish fishery (Hsieh, Leong and Rudloe 2001, Yu et al. 2006b).

They have symmetrical soft bodies consisting of a gelatinous umbrella-shaped bell and trailing

tentacles.

Millions of jellyfish are found along the stretch of coast in the United States especially

during the summer season. Jellyfish are regarded as a complex problem set by fish farmers since

they sting swimmers, clog up fishing boats and interfere with fishing, aquaculture and power

plant operations (Pastino 2007, Carpenter 2004, Owen 2006). Although in the water they are

considered as a nuisance, several species of scyphozoan jellyfish with mild stings, about 12 of

the approximately 85 species, are used as food in Asian countries for a long time owing to their

unique textures (Huang 1988). Jellyfish production is a multi-million dollar business with an

increase in the demand together with a reduction in the stock size (Hsieh et al. 2001).

A large portion of the jellyfish body is called umbrella, composed of mesogloea and outer

skin, and the upper surface is called the exumbrella and the lower surface is called subumbrella.

The mouth is on the undersurface of the umbrella and is protected by oral arms, commonly

known as legs (Omori and Nakano 2001).

2

China is the first country utilizing jellyfish as food sources, therefore, a variety of

processing methods are conducted to achieve standardization of product. Owing to its

perishability, jellyfish is treated immediately with a dehydration processing with a mixture of salt

(NaCl) and alum (Al2[SO2]·14H2O). Traditional processing method is a low-cost operation but

requires intensive labor and lasts long time. The salt and alum is used to reduce the water content,

decrease the pH, and firm the texture (Li and Hsieh 2004). In addition to salt and alum small

amount of soda is also added during the jellyfish processing in Malaysia and Thailand. The pH is

reduced from 4.5-4.8 to 6.6 in fresh and processed jellyfish, respectively (Huang 1988). The

lowered pH inhibits the microbial growth and prolongs the shelf life. The final processed

jellyfish contain 60-70% moisture, 5.5% protein and 16-25% salt, while more than 60% of the

tissue protein was found to be collagen (Miura and Kimura 1985). The processed jellyfish has a

stable shelf life up to 6 months to one year at ambient temperature.

The treated jellyfish needs to be desalted and rehydrated by soaking in water for hours even

overnight before consumption. Asian countries have various methods for preparing jellyfish.

After being rinsed sliced and served in a dressing, they are popular due to its medicinal value and

its dietary characteristics. Currently, the evaluation of jellyfish texture mainly replies on the

experience of technical personnel at the processing plant. The lack of description of objective

textural attributes stimulates debate on the commercialization of the product.

The addition of alum to jellyfish product may introduce consumers to higher dietary

aluminum exposure. Considering that aluminum can produce toxicity to the central nervous,

skeletal and hematopoietic systems, increasing attention is paid on use of aluminum food

additives of processed jellyfish (Wong et al. 2010, Ogimoto et al. 2012).

3

The objectives of the present study are as follows:

1. To study the textural properties of jellyfish with single-blade shear test, multiple-blade shear

test, tension test and texture profile analysis (TPA).

2. To study the effect of soaking treatment on the textural properties of processed jellyfish.

3. To study the effect of heating process on the textural properties of processed jellyfish.

4. To determine the inorganic constituents in processed jellyfish with inductively coupled

plasma atomic emission spectrometry (ICP-AES).

5. To determine aluminum content in processed jellyfish with high performance liquid

chromatography (HPLC).

4

REFERENCES

Carpenter, B. (2004) Feeding the sting: Warming oceans, depleted fish stocks, dirty water--they

set the stage for a jellyfish invasion. U.S. News.

Gibson, R. & M. Barnes (2000) Management of jellyfish fisheries, with special reference to the

order Rhizostomeae. Oceanography and Marine Biology, An Annual Review, 38, 85-156.

Hsieh, Y. H. P., F. M. Leong & J. Rudloe (2001) Jellyfish as food. Hydrobiologia, 451, 11-17.

Huang, Y. W. (1988) Cannonball jellyfish (Stomolophus meleagris) as a food resourceE. Journal

of Food Science, 53, 341-343.

Li, J. R. & Y. H. P. Hsieh (2004) Traditional Chinese food technology and cuisine. Asia Pacific

Journal of Clinical Nutrition, 13, 147-155.

Miura, S. & S. Kimura (1985) Jellyfish mesoglea collagen - characterization of molecules as

alpha-a-alpha-2-alpha-3 heterotrimers. Journal of Biological Chemistry, 260, 5352-5356.

Ogimoto, M., K. Suzuki, J. Kabashima, M. Nakazato & Y. Uematsu (2012) Aluminium Content

in Foods with Aluminium-Containing Food Additives. Food Hygiene and Safety Science,

53, 57-62.

Omori, M. & E. Nakano (2001) Jellyfish fisheries in southeast Asia. Hydrobiologia, 451, 19-26.

Owen, J. (2006) Jellyfish invasion puts sting on Europe Beaches. National Geographic News.

Pastino, B. d. (2007) Blue jellyfish invade australia beaches. National Geographic News.

Wong, W. W. K., S. W. C. Chung, K. P. Kwong, Y. Y. Ho & Y. Xiao (2010) Dietary exposure

to aluminium of the Hong Kong population. Food Additives and Contaminants Part a-

Chemistry Analysis Control Exposure & Risk Assessment, 27, 457-463.

5

Yu, H. H., X. G. Liu, R. E. Xing, S. Liu, Z. Y. Guo, P. B. Wang, C. P. Li & P. C. Li (2006) In

vitro determination of antioxidant activity of proteins from jellyfish Rhopilema

esculentum. Food Chemistry, 95, 123-130.

6

CHAPTER 2

LITERATURE REVIEW

1. Biology of the jellyfish

Jellyfish is the common name of certain invertebrates of the phylum Cnidaria, consisting of

umbrella-shaped bell and trailing tentacles, all aquatic and nearly all marine. Cnidarians occur in

two forms: polyps such as sea anemones and corals, and medusas such as jellyfish. The phylum

Cnidaria is divided into four classes; Hydrozoa, Scyphozoa, Cubuzoa and Anthozoa (Pechenik

2000). Jellyfish are a life stage exhibited in some species of the phylum Cnidaria. Medusa

jellyfish belong exclusively to Medusozoa, the clade of cnidarians which excludes Anthozoa

(e.g., corals and anemones). Jellyfish belonging to class Scyphozoa are referred to as the ‘true

jellyfish’, more than 200 species and all marine (Sugahara et al. 2006). Scyphozoan jellyfish can

be roughly distinguished from hydrozoan jellyfishes by their large size and by the absence of a

velum (Buchsbaum 1987). Examples of True Jellyfish include Moon Jellies, Mediterranean

Jellyfish, Sea Nettles, Lion's Mane Jellyfish, Blue Jellies, and many other lesser known species.

Although there are only about 200 species of Scyphozoa, they are sometimes the most

abundant members of coastal planktonic communities and play an important ecologic role. They

can be found everywhere in oceans living at depths up to 3000 m or more (Meglitsch 1991).

Jellyfish can swim relatively fast, as their muscles work against the much expanded mesoglea,

which is elastic and springy. Mesoglea provides the buoyancy that enables the animal to float.

Storage of oxygen is another function of the gel, assisting vertical movements and survival at

low oxygen levels (Moore and Overhill 2006).

7

Medusa jellyfish (showed in figure 2.1) consist of two basic cell layers and the mesoglea,

folded to form an umbrella-shaped structure, with a mouth centered in the flatter side of the

umbrella. The mouth, commonly known as legs, is ringed by oral arms or tentacles; they are used

in feeding and defense and sometimes for locomotion (Westheide 2004). The umbrella is formed

of thick, gelatinous mesoglea which makes it buoyant. The convex surface consists of concave

(exumbrella) and the concave (subumbrella). Some jellyfish have a stinging apparatus injurious

to humans. Many species are considered health hazards and can appear individually or in swarms

(Burnett 2001).

Jellyfish are commercially exploited for human consumption in Asian countries, especially

in China and Japan. Only 8-12 species within the order Rhizostomeae are cultured and harvested

as food, including Lobonema smithi, Lobonemoides gricilis, Rhopilema esculentum, Rhopilema

hispidum, Stomolophus meleagris, and Stomolophus numurai (Morishige et al. 2011). They are

preferred because they are larger and have more rigid bodies than other scyphozoans and their

toxins are harmless to humans (Kawahara et al. 2006).

Cannonball (Stomolophus meleagris) belonging to the class Scyphozoa and order

Rhizostomeae, is one of several rhizostome medusae used for human consumption. The

hemispherical bell reaches 20 to 25 cm in size and the color is brown. Small prey is trapped by

its protruding oral arms at the base of the bell (Calder 1982). Cannonball jellyfish have been

found from New England to Brazil in the western Atlantic (Larson 1976), from southern

California to Ecuador in the eastern Pacific and from the Sea of Japan to the South China Sea in

the western Pacific. Their ability to grow rapidly makes them one of the most abundant

scyphomedusae year round along the southeastern coasts in the United States (Hsieh, Leong and

Barnes 1996). The cannonball has great potential value as a food item in the world market. It is

8

considered a delicate appetizer in Japan, China and other Asian countries. Japan imports about

10,000 tons of jellyfish annually, valued more than 25 million dollars (Omori and Nakano 2001).

Jellyfish fisheries exist in 15 countries, including China, India, Indonesia, Japan, Malaysia and

the Philippines, with export industries in Australia and the USA (Richardson et al. 2009). Interest

in cannonball jellyfish from the United States increased recently due to the rising demand in Asia

(Hsieh et al. 2001). Fishery for cannonball jellyfish in Georgia has been permitted since 1998

(Landers 2011).

2. Processing of jellyfish

Live jellyfish are cleaned and processed immediately after being caught, otherwise they

spoil very quickly. Processing jellyfish is a low-cost but labor-intensive operation. Typically, it

requires a multi-phase processing procedure using a mixture of salt (NaCl) and alum

(KAl[SO4]2·12H2O or [NH4]Al[SO4]2·12H2O) to lower the pH, reduce the moisture content, and

firm the texture. Live jellyfish are harvested and rinsed with seawater, scraped to remove mucus

membranes and gonadal material (Li and Hsieh 2004). Umbrella and oral arms, also known as

legs, are left for further processing.

Methods for processing jellyfish vary according to species and locations (Hsieh and Rudloe

1994). The most common method consists of six phases and can be completed in 19 days

(Resources et al. 1976). Huang modified this method and two different methods were developed.

In Huang’s method, a salt mix containing about 7.5% salt and 2.5% alum is used for the initial

slating of cannonball jellyfish (Stomolophus meleagris). Salted jellyfish are then soaked in the

brine for 2 - 4 days, followed by several transfers to another container with fresh mix containing

higher content of salt and a smaller amount of alum. At the end salted jellyfish are drained and

dried mechanically with a closed system heat pump dryer. The entire process lasts 20 to 40 days

9

to produce a salted product with 60 - 70% moisture content and 16 - 25% salt (Huang 1988).

Liquefaction of the body tissue occurred without salt added whereas unpleasant odor is given off

in the absence of alum.

Processed jellyfish consists of 5.5% protein, 25% salt and 68-70% water. Typically, the

processed product is desalted and dehydrated in water before it is consumed. The rehydrated

jellyfish contains about 90% water 0.26% salt, 6.8% protein and 0.11% alum, like that of fresh

jellyfish (Hsieh et al. 2001). Umbrella parts and leg parts are usually separately processed and

independently analyzed, given that legs are worth half the price of the umbrella (Morais et al.

2009). The leg part of jellyfish is less favorable than the umbrella part in the market due to their

irregular size and shape and sting of venom in tentacles (Yu et al. 2006a).

3. Chemical composition and venom of jellyfish

3.1. Moisture content

Fresh jellyfish usually contain superior 95% water, varying with species of medusa (Hsieh

and Rudloe 1994). The dry weight of jellyfish ranged from 1.12 to 10.53% of wet weight, with

the mean value of 5.49% (Lucas 2008). The content of solids other than salts (2.90%) is very low

(Morinaga et al. 2010). Ash-free dry weight (AFDW) was estimated to range from 25.19 to

34.89% of dry weight (DW) (Youngbluth and Bamstedt 2001), or 25.19% in legs and 14.4% in

umbrella (Morais et al. 2009). The composition of jellyfish varies with species, season, and

location. In jellyfish Atolla wyvillei from the Southern Ocean, mean contents as a percentage of

wet mass were 0.83% protein, 0.21% lipid and 0.08% carbohydrates (Lucas 2008), while for a

large edible jellyfish, Stomolophus nomurai, caught in Japan, it contains about 0.40% proteins,

0.02% lipids and 0.58% carbohydrates (Huang 1988).

10

3.2. Lipids content and fatty acids composition

According to Joseph (Joseph 1979), jellyfish contain no visible lipid deposits, except in

relatively well-developed gonads during the reproductive cycle. Therefore, jellyfish products

contain little amounts of lipids, due to the removal of mucus and gonads during the processing

procedure. Crude fat content in rehydrated jellyfish products is less than 0.01% or lower (Hsieh

et al. 2001, Hsieh and Rudloe 1994). Kariotoglou and Mastronicolis found relatively high

content of lipids in Aurelia aurita, an abundant but harmless Aegean jellyfish. Total lipids of A.

aurita were 0.031 – 0.036% of fresh tissue (Kariotoglou and Mastronicolis 2001).

Morais et al. (Morais et al. 2009) identified thirty-seven fatty acids in Catostylus tagi.

Polyunsaturated fatty acids (PUFA) were found to be the most abundant, followed by saturated

fatty acids (SFA) and monounsaturated fatty acids (MUFA). PUFA accounts for 48.3% and

51.87% of total FA in umbrella and legs, respectively. The n-3/n-6 ratio of fatty acids found for

C. tagi has been considered an advantage in reducing the risk of many inflammatory chronic

diseases, with 3.7 and 3.2 in umbrella and legs, respectively (Morais et al. 2009). According to

Hsieh & Rudloe (Hsieh and Rudloe 1994), the cholesterol content of whole fresh jellyfish is less

than 0.35 mg/100g on the wet basis, thus, jellyfish can be considered as a fat-free and

cholesterol-free food.

3.3. Inorganic compounds

Fresh unprocessed jellyfish are rich in minerals, such as Ca, Na, K, Mg and Fe, whereas,

these macro elements are rather low in dehydrated products. Aluminum concentrations were

found significantly high in processed jellyfish (leg, 671 µg/g; umbrella, 449 µg/g) than fresh

jellyfish (leg, 0.85 µg/g; umbrella, 0.55 µg/g), due to contribution of curing agent (Liu et al.

2011). Higher Aluminum concentration in legs could be associated with the firmer and crispier

11

texture of the legs compared to the umbrella. Hsieh et al. (Hsieh et al. 1996) concluded that for

fresh jellyfish, no significant difference of mineral composition was found between the umbrella

part and the leg parts. However, significantly higher concentrations of Al, Ca, Mg, K, Na were

found in the legs than in the umbrella. The macro elements, Ca, Mg, K, and Na, which were very

high in fresh jellyfish, were significantly reduced in dehydrated processed-jellyfish. The authors

suggest that these elements were removed by dehydrating, indicating that these elements are

exchangeable or soluble rather than tissue-bound. Elements such as Ba, Co, Zn and Cu showed

no significant difference between fresh and processed jellyfish (Hsieh et al. 1996). Morais et al.

(Morais et al. 2009) evaluated the mineral composition of Catostylus tagi. The 7 more abundant

elements (Cl, Na, P, Mg, K, Ca, S) coincided with the macro minerals essential for human

nutrition. It needs to be noted that high boron (B) content, the 8th mineral, may help explain the

antiarthritic properties attributed to jellyfish in Asia.

The biological accumulation of mineral elements has been studied. Five species of

Scyphozoa and two species of Cubozoa were collected and both cubozoan and scyphozoan

species accumulate elements above ambient water concentrations. The extent of accumulation

varied among species, depending on the species of elements. Their use as biomonitor should be

investigated further (Templeman and Kingsford 2012).

3.4. Carbohydrates

The sum of water, lipids, protein and ash accounts for 100% approximately, indicating that

jellyfish contain trace levels of carbohydrates (Lucas 1994). Barzansky et al. (Barzansky,

Lenhoff and Bode 1975, Barzansk.B and Lenhoff 1974) analyzed the mesoglea of Pelmatohydra

pseudoligactis to identify carbohydrates. The results showed that neutral sugar accounted for

6.7% and glucosamine for 1.4% of the total dry weight of mesoglea. Large amounts of glucose

12

and galacotose and small amounts of fucose and rhamnose were identified in hydra mesoglea by

thin layer chromatography, as was the amino sugar glucosamine. The glucose and galactose are

joined as a dimer with an alkali-stable bond to the polypeptide backbone of the collagen.

Polysaccharides of Chrysaora quinquecirrha were extracted and hydrolyzed. The presence of

neutral sugars, uronic acids and amines indicated the carbohydrates are bonded to the gelatinous

matrix of the mesoglea (Gardner and Zubkoff 1978). The carbohydrate moiety of C. tagi native

collagen was estimated to be 3.7 ± 0.4% in relation to the protein total mass (Calejo, Morais and

Fernandes 2009).

3.5. Collagen

Jellyfish are made of almost of entirely protein and water. Protein content in Aurelia aurita

ranged from 2.34 to 8.31% and from 4.38 to 22.98% of dry mass in umbrella tissue and in

gonadal tissue, respectively (Lucas 1994). High protein content (mean 63.71 mg/dry mass)was

also observed in species Periphylla periphylla (Lucas 2008). Gorbatenko et al. (Gorbatenko et al.

2009) investigated the composition of large jellyfish on the West Kamchatka shelf and found

protein content range from 7.1 to 14.6% on the dry basis. Protein contents of processed jellyfish

tend to be highly variable due to the difference of species and processing methods. Cannonball

jellyfish products were analyzed to determine the protein content, with results ranging from 5.5%

to 6.8% of total wet, for a Malaysian product and a Chinese product, respectively (Huang 1988).

While lower values (4.7% in umbrella and 5.6% in legs) were found in other ready to eat

cannonball jellyfish products (Hsieh et al. 2001).

More than 60% of jellyfish mesoglea component is collagen used to retain a large amount

of water (Nagai et al. 1999, Barzansky et al. 1975). Collagen is composed of a triple helix,

consisting of two identical chains (α1) and an additional chain which differs a little in its

13

chemical composition (α2). The structure of collagen is stabilized by intra- and inter-chain

hydrogen bonding, a product of repeat of the Gly-X-Y sequence (Gómez-Guillén et al. 2011). In

most cases, X is proline and Y is hydroxyproline. The collagen extracted from jellyfish

Rhopilema esculentum is similar to type I collagen (Zhuang et al. 2009a); collagen in jellyfish

Catostylus tagi is similar to vertebrate type V/XI (Calejo et al. 2009). Vertebrate type V collagen

has been grouped with collagen types l, II, Ill and XI as a fibrillar collagen and is a minor

constituent in a number of tissues (Hsieh and Rudloe 1994).

Amino acid analysis shows that glycine, glutamic acid, proline and aspartic acid are the

most abundant amino acids; high proportion of hydroxyproline and hydroxylysine are present

(Barzansk.B and Lenhoff 1974, Nagai et al. 1999, Morais et al. 2009, Zhuang et al. 2010,

Zhuang et al. 2009b). However, the protein quality of certain jellyfish species is low due to the

absence of tryptophan (Nagai et al. 1999).

4. Function of collagen

Gelatin is a soluble protein compound made by partial hydrolysis of collagen, which is

normally done by heating insoluble collagen in water at temperatures higher than 45℃. A

chemical pre-treatment is needed in prior to break non-covalent bonds, disorganize the protein

structure (Stainsby 1987). Two types of gelatin are obtained after both the pre-treatment and the

warm-water extraction process. Type-A (isoelectric point at pH 8 - 9) and type-B gelatin

(isoelectric point at pH 4 - 5) (Montero et al. 1990).

The classical food, cosmetic and pharmaceutical application of gelatin depends largely on

its gel-forming and viscoelastic properties (Stainsby 1987). In recent decades, there is a boom of

scientific literature about different alternative sources and new function of gelatin and collagen.

14

On the other hand, a lot of studies have been carried out, investigating enzymatic hydrolysis of

collagen or gelatin for the production of bioactive peptides.

4.1. Gelling and water binding properties

The largest single use of gelatin in food industry is in water gel desserts, due to its unique

melting properties. Water gel desserts made from various gelatins possess different textural

properties and melting behavior. Desserts from fish gelatins would be similar to desserts made

from high bloom pork skin by increasing the gelatin content or by using gelatin mixtures. In

addition, the lower melting temperature in fish gelatin desserts may accelerate flavor release

(Zhou and Regenstein 2007). Introducing a gas phase into gelatin-gel-based products, such as

fruit jellies or marshmallows, can provide food products new textures and appearance (Zúñiga

and Aguilera 2009). Gelatin hydrogels is formed by dissolving gelatin or collagen chains in

solution, with the ability to absorb large quantities of water. As an inexpensive, biodegradable

and versatile natural material, hydrogels would be applied to medicine, pharmacy, agriculture

and biodegradable food packaging (Farris et al. 2009). Collagen of marine sources can also be

utilized as additives due to its water-holding capacity, such as preventing denaturation of surimi

protein subjected repeat freeze-thaw cycles (Kittiphattanabawon et al. 2012) and improving

water retention in surimi processing (Hernández-Briones et al. 2009).

4.2. Surface properties

In collagen and gelatin, the presence of both the hydrophilic and hydrophobic amino acids,

tends to migrate towards surface in solution, hence reducing the surface tension of aqueous

systems (Schrieber and Gareis 2007). Gelatin surface properties make gelatins suitable oil-in-

water emulsifiers (Dickinson and Lopez 2001).Gelatin and acid-soluble. Collagen also shows

15

suitable foaming properties. For example, farmed giant catfish skin gelatin with high viscosity

would facilitate foam formation and stabilization (Townsend and Nakai 1983).

4.3. Film-forming and microencapsulation properties

There have been extensive studies on gelatin film-forming ability. It can be used as an outer

film to protect food from drying (Avena-Bustillos et al. 2006) and biodegradable films to replace

plastic products (Tharanathan 2003). Gelatin is also one of the widely-used materials as a carrier

of bioactive components (Young et al. 2005, Gómez-Guillén et al. 2009).

5. Bioactive properties of hydrolysates

Collagen and gelatin are potential sources of biologically active peptides, which could be

obtained during gastrointestinal digestion, fermentation or food processing. In recent decades,

more attention has been paid to collagenous material from by-products of poultry and fish, such

as chicken bone and squid skin (Cheng et al. 2008, Cheng et al. 2009, Nam, You and Kim 2008),

and other novel material such as bullfrog, sea cucumber and jellyfish (Zhou, Wang and Jiang

2011, Qian, Jung and Kim 2008, Nam et al. 2008, Zhuang et al. 2010).

5.1. Antioxidant activity

A lot of studies have been carried out to study the antioxidant properties of hydrolysate and

peptide. Collagen from fish products draw a great deal of attention because of the vast mass of

waste produced during the processing. The mechanism of anti-oxidation of peptides is still not

known. However, it is assumed that peptides would inhibit lipid peroxidation and scavenge free

radicals and transition metal chelators (Kim et al. 2001). A peptide isolated from jellyfish

(Phopilema esculentum) has high antioxidant activity and was able to inhibit tyrosinase activity,

which might result in melanogenesis inhibition (Zhuang et al. 2009b)

16

5.2. Antihypertensive activity

Antihypertensive peptides are peptide molecules which may lower blood pressure when

ingested through inhibition of vasoactive enzymes such as the angiotensin converting enzyme

(ACE) (Gómez-Guillén et al. 2011). Potent ACE inhibitory hydrolysates have been prepared

from the powder of the jellyfish Stomolophus nomurai, and further fractionated by high

performance liquid chromatography (Morinaga et al. 2010).

5.3. Antimicrobial activity

A few studies indicate that hydrolysate separated from peptides or collagen shows

antimicrobial property (Jus, Kokol and Guebitz 2009).

6. Textural and sensory characteristics of jellyfish

Texture is by definition a sensory parameter that only a human being can perceive, describe

and quantify (Hyldig and Nielsen 2001). Instrumental texture assessment on meat is made by

means of a texturometer, a device that allows tissue resistance both to shearing and to

compression to be measured.

Fresh jellyfish spoil very easily, hence the addition of alum and salt is an essential step to

obtain the desired texture. Processed jellyfish need to be dehydrated in water for certain period of

time before consuming, while the temperature and time of this step would be also related to the

texture of final ready to eat product. Textural studies have been reported for processed jellyfish

(Hsieh et al. 2001, Hsieh and Rudloe 1994, Huang 1988, Kimura et al. 1991a, Kimura et al.

1991b). The information obtained indicates that textural and sensory properties of jellyfish are

affected by numerous aspects, such as their species variety, composition of salt mix, processing

methods and so on.

6.1. Texture studies on muscle of processed jellyfish

17

Alum, used to brine fresh jellyfish, is a class of chemical compounds, potassium aluminum

sulfate and ammonium aluminum sulfate. Brine treatment eliminates the compounds responsible

for the sting and gives jellyfish products a unique crunchy texture property, which is one of the

most important reasons why customers pursue it (Armani et al. 2012). Adding alum to fresh

jellyfish is the most crucial step to both preserve the product and to get the satisfactory texture

attributes.

6.1.1. Aluminum used in jellyfish

Aluminum is used as a direct food additive as a firming agent, carrier, coloring agent,

anticaking agent, buffer, neutralizing agent, dough strengthener, emulsifying agent, stabilizer,

thickener, leavening agent, curing agent and texturizer (A.Yokel 2012). In the case of jellyfish

products, aluminum is a firming agent interacting with the carboxyl groups of collagen side

chains (He et al. 2012). From the analysis of the IR and UV spectra, the peak of the band for

collagen has been found shifted to higher frequency, indicating that the peptide chain of collagen

has extended (Li et al. 2003). There is no published literature focusing on the relationship of

alum concentration and the sensory texture of jellyfish. However, aggregation of collagen is

observed when it is treated with a solution of higher aluminum concentration (He et al. 2012). It

is reasonable to assume that higher content of alum added the crunchier the product is.

6.1.2. Structure studies on fresh jellyfish

Fresh jellyfish cannot be consumed immediately after being caught, due to its perishability.

Thus, they are usually considered as a suitable natural source of collagen supplement. Studies on

fresh jellyfish structure have been focused on biochemical and biological structure of collagen,

isolated from fresh jellyfish (Addad et al. 2011, Calejo et al. 2009, Miura and Kimura 1985,

Nagai et al. 1999). Previous studies revealed an α1α2α3 heterotrimer, similar to vertebrate type

18

V/XI, i.e. type I collagen, with a denature temperature of 26.0-29.9℃. These results indicate that

jellyfish collagen is a good candidate in selected biomedical applications.

6.1.3. Texture studies of processed jellyfish

Retailers or customers need to soak jellyfish in water to remove excess amount of salt both

from the outside and the inside of the product. The soaking time varies from couple of hours to

overnight. In some countries, desalted jellyfish are also dipped in hot or boiling water to obtain a

crunchier texture. Then the jellyfish can be served as an appetizer or a salad by adding different

seasonings.

The rheological properties of 6 kinds of processed jellyfish were analyzed by stress-

relaxation measurement and rupture strength measurement. Samples were boiled at the certain

temperature, i.e. 30℃, 40℃, 50℃, 60℃, and 80℃ for 30 min, respectively. Then, samples were

dipped in water at 5℃ for 9 days, and texture test was conducted for these samples every day.

The texture of samples was determined by two factors, heating temperature and the standing time

in water. It is concluded that the elastic modulus increased in the first 2 days, and then decreased.

Values of elastic modulus and viscosity increased at 50℃ or 60℃, while rupture strength rose

with heating temperature from 30℃ to 50℃ and then fell at 60℃ (Kimura et al. 1991b). The

samples were also examined by observation with a scanning electron microscope and with an

ordinary microscope. The samples became shortened in the direction along the surface of the

skin by boiling at 80℃ for 30 min, thus, the thickness which was perpendicular to the surface

increased, which is caused due to the shrinkage of collagen in the umbrella. Structural damage

was observed after the heated sample was dipped in water for 6 days (Kimura et al. 1991a).

However, since little is known about the composition except for collagen, it is difficult to

conclude that only collagen shrinkage affects the texture of cooked jellyfish.

19

6.1.4. Sensory studies on processed jellyfish

There are few studies to evaluate the sensory attributes of processed jellyfish. A sensory

study was conducted to compare the color, texture and overall preference of laboratory processed

cannonball umbrella and leg products with a commercial Malaysian product. Experienced

panelists evaluated cannonball product that had been stored for one year in a refrigerator as a

crunchier product than the Malaysian sample. They also prefer the cannonball products over the

Malaysian samples, indicating a relationship between product crunchiness and preference of

customers (Leong 1995). Huang also conducted a sensory test of shredded umbrellas of

cannonball jellyfish and commercial products; appearance, color, texture and overall

acceptability were evaluated. For the lab processed samples, they have been stored as 10℃ for

one month and six months after processing. The results showed that no significant difference in

texture was found (Huang 1988).

6.2. Other texture studies on similar products

Numerous studies have been published focused on sensory and instrumental texture of meat

and fish products. Some of the most important sensory attributes of meat are hardness,

springiness, crunchiness and juiciness. In the case of jellyfish products, crunchiness is the

essential factor that determines whether customers buy it or not. However, there are few studies

on methodologies of crunchiness assessment.

Quantitative descriptive analysis was recognized as a reliable technique to measure sensory

properties including crunchiness. The analysis is performed with small group of trained panelists

(8-10 people) and they are asked to score the samples with comparison to a given reference.

Usually, the evaluations include a training session, with the objective to reach a consensus

among panelists on the meaning of every attribute. However, if the definition provided is not

20

effective or even misleading, it may lead assessors to evaluate some other sensory attributes.

Knowing this, sensory leader helped them discuss together and reach a consensus about the

meaning of the property, instead of providing panelists with a definition but (Roudaut et al.

2002). Studies on crunchiness were conducted with various products, such as fish stick, apple,

cracker and squid, utilizing descriptive profiling analysis (Albert et al. 2012, Albert et al. 2009,

Costa et al. 2011, Fillion and Kilcast 2002, Martinez et al. 2002, Raffo et al. 2005, Vaz-Pires and

Seixas 2006, Zdunek et al. 2011). Although sensory analysis provides a more complete

description, developing instrumental tests to assess crunchiness is an interesting area. The most

common types of measurements are based on rheological principles: shear strength, puncture,

and compression (Hyldig and Nielsen 2001).

7. Aluminum in foods

Aluminum content in fresh jellyfish is quite low (leg, 0.29 µg/g; umbrella, 1.63 µg/g).

However, processed jellyfish contains high value of aluminum (leg, 688 µg/g; umbrella, 271

µg/g) due to the addition of alum during the processing (Hsieh et al. 1996). The side effects of

aluminum on health have been noted. Elevated levels of aluminum might have serious

consequences for biological communities. Analytical methods used for aluminum determination

are voltammetry, UV-vis spectrophotometry and fluorometry, flame/electrothermal atomic

absorption spectrometry (AAS), direct current plasma-atomic emission spectrometry (DCP-AES)

and neutron activation analysis (Albendin et al. 2003, Tria et al. 2007).

8. Aluminum and diseases

Aluminum enters into the body from the environment and from diet and medication, since it

is widely distributed in the environment and is extensively used in modern life. In the past few

decades, there has been no conclusion about the impact of aluminum on biological systems

21

(Campbell and Bondy 2000). The distribution of aluminum to various tissues has been reported

as unequal, based on the data collected from normal, aluminum-exposed humans and aluminum-

treated experimental animals (Yokel and McNamara 2001). In the general population, about half

of the total amount of aluminum of the body is found in the skeleton and about one-fourth is in

the lungs; the brain is an important accumulation site of aluminum in the body (Ganrot 1986).

The aluminum levels of lungs, liver, kidneys, and brain were found to be increased with

increasing age (Associates et al. 1992).

Some studies suggest that aluminum shows toxic to plants, some aquatic animals, and

humans (Ganrot 1986, Nayak and Chatterjee 2001, Pineros and Kochian 2001). Numerous

disturbances in organism are induced by aluminum ion, which could replace Mg+2

and Fe+3

.

Besides the disorders in intercellular communication, secretory functions, and cellular growth,

the severest danger of aluminum toxicity presents in its neurotoxicity (Barabasz et al. 2002).

Aluminum-evoked changes in neurons are similar to degenerative lesions observed in patients

suffering from Alzheimer’s disease (Kawahara, Kato and Kuroda 2001). Though many studies,

the mechanisms of aluminum toxic effects on humans have not been fully elucidated (Han and

Dunn 2000, Levesque et al. 2000).

However, preventive actions should be undertaken to limit the exposure of aluminum to

humans. Awareness of aluminum in food is the primary factor in preventing aluminum-induced

toxicity.

22

Figure 2.1 Diagram of jellyfish

23

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34

CHAPTER 3

TEXTURE PROFILE OF JELLYFISH PRODUCTS1

___________________________

1 Xu, C., Huang, Y.W. and W. Kerr. To be submitted to Journal of Food Science.

35

Abstract

Texture is one of the most important attributes of jellyfish products, and desalting cured

jellyfish in water is a critical step to create jellyfish a desirable texture. However, objective

instrumental methods to assess textural attributes in this product have not been studied

sufficiently. Crunchiness is a major quality determinant of jellyfish, which is usually evaluated

by instrumental methods such as shear test. The present work was conducted to evaluate a

method for the determination of jellyfish crunchiness. Jellyfish received 5 treatments: 0-h, 2-h, 4-

h, 6-h and 8-h soaking in tap water to yield a vast array of crunchiness levels. Assessment of this

attribute was measured with a Texture Analyzer by tension, texture profile analysis (TPA),

single-blade (SB) shear, and multiple-blade (MB) shear. Peak force and area read from the

curves for tension and shearing were analyzed. The SB attachment could predict the key texture

attribute (hardness) of jellyfish samples without damage the integrity of the sample. Our results

showed that, while the moisture content increased from 83.39% to 96.34%, both the peak force

and area of tension and shearing were significantly affected by soaking time. Unheated processed

sample could obtain similar textural properties of ready-to-eat products after being soaked for

about six hours; similar results could also be obtained by dipping salted samples in boiling water.

Introduction

Most edible jellyfish belongs to phylum Cnidaria, class Scyphozoa, order Rhizostomese

(Sugahara et al. 2006). Live jellyfish are cleaned and processed immediately after being caught,

otherwise they spoil very quickly. Processing jellyfish is a low-cost but labor-intensive operation.

Typically, it requires a multi-phase processing procedure using a mixture of salt (NaCl) and alum

(KAl[SO4]2·12H2O or [NH4]Al[SO4]2·12H2O) to lower the pH, reduce the moisture content, and

firm the texture. Live jellyfish are harvested and rinsed with seawater, scraped to remove mucus

36

membranes and gonadal material. Umbrella and oral arms, also known as legs, are left for further

processing (Li and Hsieh 2004). Treatments may vary from place to place even with some

confidential procedures. Jellyfish products are so popular in China that some artificial jellyfish

foods have been made from sodium alginate.

Processed jellyfish is a tasty and crunchy seafood and often served as an appetizer or salad.

It is very popular in Asian countries, such as China, Japan, and Malaysia, due to its unique

sensory attributes, low cost, excellent long storage stability, and effect in weight loss (Huang

1988). However, most of the studies have been focused on collagen extracted from jellyfish and

its biochemical applications (Addad et al. 2011, Calejo et al. 2009, Ding et al. 2011, Wang,

Wang and Brown 2011). The potential of jellyfish as a food resource have been barely explored.

Sensory properties are determined by texture, appearance and flavor (Hsieh et al. 2001). Sensory

analysis requiring trained panelists is considered the preferable method for measuring the quality

of processed jellyfish (Hsieh et al. 2001). However, sensory methodology is time-consuming and

expensive due to the panel recruitment, training and the validations of each step of the methods

using the appropriate statistical tools (2011). Accordingly, instruments have been developed to

evaluate the texture of jellyfish. The texture changes of cooked jellyfish have been studied by

stress-relaxation experiment and rupture strength measurement (Kimura et al. 1991b). In addition,

the structure change of the processed jellyfish during cooking has been studied with a scanning

electron microscope and with an ordinary microscope (Kimura et al. 1991a). Since there are

various pretreatment and cooking methods for processed jellyfish, research about their effects on

the texture change of jellyfish is of great importance. One of the most commonly used methods

to desalt jellyfish is to leave them standing in tap water. In several countries, desalted jellyfish is

also dipped in boiling or hot water. The texture characteristics of cooked jellyfish depend on the

37

heating temperature and the standing time in water. However, there are few studies about the

relationship between changes of texture of jellyfish and the treatment conditions. The most

common types of measurements are based on rheological principles: shear strength, puncture,

and compression (Lepetit and Culioli 1994). In shearing test, to measure firmness of fish

product , the important features for the shear device are blade thickness and its orientation to the

muscle fiber (Smith and Fletcher 1998). The benefit of using the less destructive and small

incision (8.9 mm wide) blade has been justified by the shear test in broiler breast fillets. A

previous study validated a novel multiple-blade shear using sensory analysis, and compared this

device with Allo-Kramer (AK) attachment in broiler breast fillets (Cavitt et al. 2005), while

similar device was also used in fish fillets (Aussanasuwannakul et al. 2010). The authors

concluded that multiple-blade is accurate and less destructive in fillet texture analysis. In this

study, a similar shear device was also employed in order to compare the sensitivity of different

devices. The novel attachment with 4 rows of 16 blades allows a wide and deep incision that

captures a full range of texture variation. Accordingly, this study was an investigation the

crunchiness property of jellyfish. In addition, effects of heating process on the crunchiness test

for processed jellyfish were determined.

Materials and methods

Materials

Two kinds of cured cannonball jellyfish (Stomolophus meleagris) were shipped to our

laboratory in polythene barrel for analysis. One was treated 20 years ago and has been stored in a

4℃ refrigerator ever since (refrigerator-stored sample), and the other kind was newly processed

and stored at room temperature (newly-processed sample). All the samples were cut into two

portions, umbrellas (the cap-shaped part) and manubriums (the legs), cleaned with sea water and

38

treated with salt (sodium chloride) and alum (ammonium aluminum sulfate) before they were

sealed in the barrels. The processed jellyfish, ca, 15 cm in diameter for the part of the umbrella,

were split into 5 equal wedges from the center and then were soaked in tap water (material: water

ratio is 1:20) for 8 hours. During the procedure, the water was changed every two hour. Each

desalted jellyfish swelled to about 1.5 times the length. A set of 6-10 samples is collected at 0, 2,

4, 6, and 8 hour respectively.

Some of the desalted sampled were dipped in the hot water. The procedure was described as

follows: each piece was dipped in water at the designated temperature, i.e. 60℃, 80℃, and 100℃

for 10 s, 30 s, and 60 s, respectively. Both the heated and unheated samples were then wiped

with a paper towel for several seconds and packaged in ziplock bags.

Five commercial products, coming from the umbrella-part of jellyfish, were purchased from

the local food market. All products were brought to our laboratory and labeled as C1, C2, C3, C4,

and C5. They are evaluated with single-blade shear test.

Moisture analysis

All of the samples were wiped with a paper towel for several seconds, cut into strips and

ground into slurry by a miller. The moisture content was determined by a standard oven drying

method (105℃ for 48 h) in triplicate (Chen 2003).

Instrumental analysis

Samples were analyzed in a TMS-PRO Texture Analyzer (Food Technology Corporation,

Sterling, VA, USA) fitted with a 5 kg load cell at room temperature. For each attachment,

parameters, determined from the plot of force compared with time consisted of the maximum

shear force (N) and area under the curve (N mm).

Single-blade shear evaluation

39

This test measures the force of single blade needed to cut through a piece of sample. The

test cell consisted of a 0.635 mm-thick sharpened blade (12.7 mm x 25.4 mm) and was fitted

through a slit on the base platform. The sample to test was placed on the platform, under the

blade, and was cut through as the blade moved down with a constant speed (pre-test speed: 2.0

mm/s; test speed 1.0 mm/s; post-test speed: 2.0 mm/s) through the slit on the plate. Down stroke

distance was: 10.0 mm to make sure the probe could cut the sample thoroughly. The parameter

recorded was the maximum shear force, which is the maximum resistance of the sample to

shearing. Each sample was assessed in duplicate.

Multiple-blade shear evaluation

Test settings and acquired parameters were the same as single-blade shear evaluation except

that the base platform was removed so that the blade could cut through the sample completely

(Figure 3.1). The whole piece of square-shape sample, which was held by hands, was used

without cutting to make sure the sample surface area could fit the cutting area.

Texture profile analysis (TPA)

This test measures the compression force with a double bite cycle test. All TPA tests were

performed up to 50% compression of the original portion height with a 50 mm-diameter

aluminum plate. The probe returned to its original point of contact with the sample and stopped

for a set period of time (2 s), before the second compression was conducted. Force-time

deformation curves were acquired with a cross-head speed of 1 mm/s. TPA parameter was read:

springiness (mm), distance over which the material recovers its height between the end of the

first bite and the start of the second bite.

40

Tension evaluation

The tension test measures the maximum resistance to extension (N) when a sample is pulled

apart. The attachment comprises 2 grips and was conducted at a grip speed 1.0 mm/s. One grip is

fixed to the base of the texture analyzer, while the other was attached to the load cell. Initial grip

separation was 3 mm and test cell speed was 1.0 mm/s until rupture. In general, samples were cut

in a dumbbell shape, approximately 6 cm long and 1.5 cm wide and were placed between both

grips on the texture analyzer.

Statistical analysis:

The moisture content and textural properties of samples were evaluated using analysis of

variance (ANOVA) of SAS system, version 9.2 (SAS Institute Inc., 2008). Pearson product

regression analysis (at 95% of confidence level) was performed to determine the relationships

between data obtained by tension test, shear test, and TPA. Data were presented as the mean of

each sample and the standard deviation (SD) of the mean.

Results and discussion

Textural properties of commercial products:

All of the five commercial and ready-to-eat products were packaged with plastic envelopes

(showed in Figure 3.2) and collected from the shelves in a refrigerator. They are imported from

southeastern China, which were already processed and desalted for the purpose of direct

consumption. Mean values of force peaks of single-shear test for these five jellyfish products

ranged from 4.39 to 11.16 N, with the mean values of area ranging from 11.38 to 30.20 N mm

(listed in Table 3.1). The highest force needed to cut through the samples was 2.5 times the value

for lowest one. The areas increased with force peaks accordingly. Moisture content showed less

variation among samples compared to the textural characteristics, varying from 93.91% to

41

96.55%. As soon as the single-blade shear force of samples falls within 4.39 and 11.16 N, the

sample will be considered as acceptable.

Moisture contents of processed jellyfish:

Rehydration of processed jellyfish was a crucial step to remove the excessive salts within

the samples and to make it ready-to-eat. Changes of the appearance of the desalted samples were

showed in Figure 3.3.

Moisture content of processed and desalted jellyfish is showed in Figure 3.4. When the

soaking time was extended, the moisture content increased due to the rehydration. Newly-

processed samples (newly-processed) contain higher water content (77.34%) than that (83.39%)

of the old samples (refrigerator-stored) at the beginning. However, extending the soaking time to

2 h, the difference was small between these two samples. According to a previous research,

lower moisture content in the muscle is caused by a lower capacity of the myofbrillar proteins to

retain water. The lower water holding capacity in turns produces a tougher meat texture. These

results suggest that the protein composition of both species causes the differences in toughness

(Kimura et al. 1991b).

Instrumental analysis of processed jellyfish

Single-blade shear test

Strength and toughness are different mechanical properties that can be expressed by

different parameters (Rosenthal 1999). Strength could be determined by peak force and

toughness could be determined by area. In this work, these two parameters were consistent with

the changes. All the variables measured decreased with the soaking time.

Force-time curves of single-blade shear test were recorded every two hour to determine the

maximum force and area under the curve (Figure 3.5). Every curve in the graph represented the

42

changes of shear force for one single test. When the maximum of shear force was reached, the

curve dropped immediately within the first six hours. However, curve tended to be flat as the

soaking time increased. The jellyfish umbrella, containing about 70% protein on the basis of ash-

free dry weight, was separated into the two tissues of a mesoglea and skins (Barzansk.B and

Lenhoff 1974). This may explain the second peak of the 2-h curve, which was caused by the

second outer skin of jellyfish umbrella.

Maximum shear force of different positions on the sample was also determined and showed

in Figure 3.6. Every piece of sample was considered as a circle and shear force was relevant to

the distance of the test position from the center; the distance of the test location from the center

was set as 1 cm, 3 cm and 5 cm, respectively. The center of the sample was more sensitive and

firmer than the edge part, varying from 18.95 to 9.03 N, whereas the shear force of 5-cm-position

ranged from 13.30 to 11.39 N. Therefore, following tests were conducted near the center of the

samples (1 cm).

The shear force of desalted samples decreased from 20.25 to 9.79 N and from 21.74 to 9.97

N (Figure 3.7(a)) in newly-processed sample and refrigerator-stored sample, respectively. Trends

of single-blade shear force and area (Figure 3.7) were similar: the change of this two samples

were both ascended quickly at the initial stages, then decreases were slow after 6 h. Shear force

of the 8-h refrigerator-stored sample was 45.9% of the original shear force. The result indicates

that the hardness of desalted jellyfish is affected by soaking time rather than moisture content of

the samples. Compared with the results of five commercial products, newly-processed sample

was acceptable after soaking at tap water for 6 h with shear force of 11.19 N and area of 31.11 N

mm, while refrigerator-stored sample was comparable in shear force (9.97 N) and area (26.98 N

mm) after 8-hour soaking.

43

Single-blade Shear force has often been used as an objective measurement of meat

tenderness (Aussanasuwannakul et al. 2010, Caine et al. 2003, Cavitt et al. 2005, Cavitt et al.

2004).

Multiple-blade shear test

Shear force of newly-processed sample and refrigerator-stored sample ranged from 26.29 to

40.66 N and from 27.93 to 41.73 N (Figure 3.8(a)), respectively. Results showed that this

method had low variance, which could be explained by the fact that multiple-blade captured the

features of the whole piece of jellyfish sample.

Tension test

The tension force decreased from 6.78 to 2.42 N and from 7.63 to 3.51 N (Figure 3.9(a)) in

newly-processed sample and refrigerator-stored sample, respectively. Refrigerator-stored sample

had higher resistance to tear apart. However, tension force showed no significant difference (p <

0.05) after 2 h according to the statistical analysis. Figure 3.9(b) revealed that tension area for

newly-processed sample and tension area for refrigerator-stored sample exhibit closest

agreement.

Texture profile analysis

The springiness of two kinds of samples fell between 5.71 and 4.49 mm and 3.54 and 2.46

mm, respectively (Table 3.2). For newly-processed sample, most of them showed springiness in

the range of 4.24 and 4.87 mm; for refrigerator-stored sample, this range is 2.46 and 2.87 mm.

Springiness of old samples from TPA was lower than that of newly-made ones.

Correlation between multiple-blade shear and single-blade shear

Correlations were obtained between multiple-blade shear test and single-blade shear test to

examine possible relationships between these two methods (Table 3.3). The area of multiple-

44

blade test showed strongly positive correlation (r = 0.78 and 0.83) for area and force of single-

blade test. The correlation coefficient of force of multiple-blade test also represented high

relation with area of single-blade (r = 0.71) and with force of multiple-blade (r = 0.75),

respectively. The high correlation indicted that multiple-blade attachment could predict the key

texture attribute (hardness).

Textural properties of jellyfish under different heating conditions

In order to compare rheological properties of commercial products with treated samples in

our lab, single-blade shear test were also used to evaluate the heated samples. Both the results of

newly-processed sample and refrigerator-stored sample were generated in Table 3.4 and Table

3.5. The sample shortened in the direction along the surface of the umbrella skin by heating, so

that the perpendicular thickness of the umbrella increases.

Shear force of newly-processed sample reached the maximum (29.91 N) when dipping in

100℃ water for 10 s without soaking and the minimum (3.87 N) when dipping 100℃ water for

30 s after being soaked for 8 h; for refrigerator-stored sample, it reached the maximum (28.20 N)

when dipping in 100℃ water for 10 s without soaking, and reached the minimum (2.17 N) when

dipping in 100℃ water for 60 s after being soaked for 6 h. The shear force stayed relatively

steady when it was dipped in water of 60℃. However, it decreased dramatically both in 80℃ and

100℃ water. Therefore, temperature is a crucial factor having effect on textural properties of

jellyfish. Processed samples without being soaking in water previously showed the toughest

textural property. High temperature heating and long-time desalting is the main factors to soften

samples. However, if the sample was heated in 100℃ for a long time, such as 60 s, the strength

decreased. This may be explained by the structure of collagen was damaged. Being heated on

60℃ for 10 s, shear force decreased from 16.04 to 4.59 N and 13.16 to 8.86 N for newly-

45

processed sample and refrigerator-stored sample, respectively. However, when the treating

temperature increased to 100℃, the ranges of shear force for newly-made and old samples were

from 29.91 to 4.61 N and from 28.20 to 3.17 N, respectively.

Regardless of heating conditions, newly-processed sample and refrigerator-stored sample

showed desirable textural properties (shear force < 11.16 N) when they had been soaked for

more than 6 hours and 8 hours, respectively. The fast ways to make a ready-to-eat jellyfish

products from newly-processed sample were dipping samples in 100℃ water for 60 seconds after

soaking in tap water for 2 hours; for refrigerator-stored sample, dipping samples, having been

desalted for 2 h, in 100℃ water for 60 seconds would produce satisfactory textural properties.

The binding force between tissues in the umbrella became weaker by the both heating and

swelling of the samples. The result may be due to the destruction of the network structure by

thermal denaturation of the collagen in the jellyfish.

Conclusion

Commercial products showed various properties. Average shear force ranging from 4.39 to

11.16 N and average area ranging from 11.38 to 30.20 N mm were found in shear test of ready-

to-eat jellyfish commercial products. The results of tension test, shear test and TPA all showed

the strength of processed jellyfish samples decreased rapidly during the first two hours and the

newly processed samples showed more elastic than the samples that have been stored for a long

time. Unheated newly-processed sample and unheated refrigerator-stored sample could obtain

similar textural properties of ready-to-eat products after being soaked for 6 hours and 8 hours,

respectively. The moisture content is not a crucial parameter to evaluate the textural properties of

jellyfish.

46

Different positions of the samples showed different textural properties, and the center part is

firmer and more sensitive to the soaking processing. The multiple-blade attachment could predict

the key texture attribute (hardness) of jellyfish samples in which the test locations are no longer a

concern.

Heating process is very important to the changes of textural properties, in which the heating

temperature and heating time are the key factors to determine the final product quality and

texture.

47

Table 3.1 Mean values of force peaks and areas for single-blade shear test and moisture content for five commercial products

Sample ID Single-blade shear test (n=20)

Moisture content (%) (n=3) Area (N mm) Force (N)

C1 30.20 ± 8.35a 10.81 ± 3.33

a 93.91 ± 0.06

c

C2 27.46 ± 6.73a 11.16 ± 3.17

a 94.02 ± 0.06

c

C3 22.12 ± 5.46b 7.31 ± 1.81

c 95.35 ± 0.07

b

C4 25.74 ± 8.89ab

8.89 ± 2.05b 95.27 ± 0.08

b

C5 11.38 ± 2.44c 4.39 ± 1.28

d 96.55 ± 0.09

a

a,b,c,d Means in columns with the same letter indicate no significant difference (p < 0.05); values are means ± standard deviation.

48

Table 3.2 Mean values of springiness (mm) for TPA

Soaking time (hour)

Sample ID 0 2 4 6 8

Newly-processed 5.71±0.82a 4.87±0.28

ab 4.24±0.83

b 4.48±1.02

b 4.49±0.36

b

Refrigerator-stored 3.54±0.59a 2.82±0.21

b 2.83±0.24

b 2.60±0.34

b 2.46±0.20

b

a,b Means in rows with the same letter indicate no significant difference (p < 0.05); values are means ± standard deviation of 20

replicates.

49

Table 3.3 Correlation matrix between multiple-blade shear test and single-blade shear test of jellyfish

Multiple-blade--Area Multiple-blade--Force Single-blade--Area Single-blade--Force

Multiple-blade--Area

Multiple-blade--Force 0.9228*

<0.0001

Single-blade--Area 0.7767*

<0.0001

0.7084*

0.0005

Single-blade--Force 0.8286*

<0.0001

0.7512*

0.0001

0.9500*

<0.0001

* Numbers are correlation coefficients greater than r=0.7, n=20.

50

Table 3.4 Mean values for single-blade shear for newly-processed sample under different heating conditions

Heating conditions Soaking time (hour)

Temp. (℃) Time (s) 0 2 4 6 8

60 10 13.16±5.07a 12.34±3.47

a 12.45±3.58

a 10.73±4.17

ab 8.86±3.22

b

30 10.56±1.80bc

15.71±4.91a 11.22±3.21

b 8.59±2.47

c 8.65±3.16

c

60 13.85±3.21ab

14.67±6.52a 11.25±2.96

bc 9.82±2.15

c 6.82±2.11

d

80 10 26.93±4.97a 14.37±3.77

b 8.75±4.57

c 6.71±2.68

c 6.55±2.27

c

30 25.45±6.21a 13.20±4.38

b 9.11±1.89

c 5.35±2.09

d 4.59±1.92

d

60 19.51±4.67a 14.74±3.70

b 10.49±2.74

c 5.27±1.57

d 4.00±2.49

d

100 10 29.91±6.85a 15.88±4.83

b 10.72±3.71

c 7.11±3.12

d 4.61±1.82

d

30 24.54±4.45a 12.55±1.78

b 12.00±2.75

b 6.86±2.29

c 3.87±1.86

d

60 12.93±4.77a 8.84±2.30

b 6.14±1.55

c 6.31±2.30

c 3.94±1.92

c

a,b,c,d Means in rows with the same letter indicate no significant difference (p < 0.05); values are means ± standard deviation of 20

replicates.

51

Table 3.5 Mean values for single-blade shear for refrigerator-stored sample under different heating conditions

Heating conditions Soaking time (hour)

Temp. (℃) Time (s) 0 2 4 6 8

60 10 16.04±6.41a 10.92±5.69

b 8.03±3.06

bc 7.84±1.78

bc 4.59±1.92

c

30 11.30±3.63a 12.69±6.02

a 7.87±1.47

b 6.53±3.28

b 5.41±4.01

b

60 18.18±6.11a 17.81±3.29

a 7.01±3.96

b 7.16±3.98

b 5.08±3.11

b

80 10 24.88±6.14a 18.37±3.67

b 7.17±2.89

c 3.64±1.91

d 4.18±2.13

cd

30 25.42±7.53a 16.95±4.43

b 6.23±4.10

c 4.49±3.17

cd 2.10±1.52

d

60 19.51±4.67a 12.18±4.79

b 3.55±2.15

c 3.76±1.47

c 2.92±1.74

c

100 10 28.20±8.31a 11.69±5.01

b 6.81±3.06

c 2.83±2.01

c 3.17±1.83

c

30 20.83±3.24a 10.51±5.12

b 5.74±3.76

c 2.41±1.25

d 2.65±1.24

d

60 16.31±3.18a 9.59±3.78

b 6.53±1.63

c 2.17±1.13

d 2.63±1.32

d

a,b,c,d Means in rows with the same letter indicate no significant difference (p < 0.05); values are means ± standard deviation of 20

replicates.

52

Figure 3.1 Multiple-blade attachment with 16 blades (a) and a view of newly-processed sample placed in perpendicular direction to

the attachment (b).

a b

53

Figure 3.2 Five different kinds of commercial products collected on the local markets.

C5

C1 C2 C3

C4

54

Figure 3.3 Visual comparison between processed jellyfish and deslated jellyfish.

55

Figure 3.4 Moisture content of two kinds of samples soaking in water. NP:

newly-processed samples; RS: refregirator-stored samples.

56

Figure 3.5 Force-time curves of samples soaking for 0, 2, 4, 6, and 8 h generated by single-blade

attachment.

57

Figure 3.6 Single-blade shear force at the different locations on a sample; the distance of test

location to centern of the sample are 1 am, 3 am and 5 cm, respectively; n=20.

58

Figure 3.7(a) Single-blade shear force of newly-processed sample and

refrigerator-stored sample after soaking in water for 8 h. NP: newly-

processed samples; RS: refregirator-stored samples.

Figure 3.7(b) Single-blade shear area of newly-processed sample and



59

Figure 3.8(a) Multiple-blade shear force of newly-processed sample and



Figure 3.8(b) Multiple-blade shear area of newly-processed sample and



60

Figure 3.9(a) Tension force of newly-processed sample and refrigerator-

stored sample after soaking in water for 8 h. NP: newly-processed samples;

RS: refregirator-stored samples.

Figure 3.9(b) Tension area of newly-processed sample and

refrigeratore-stored sample after soaking in water for 8 h. NP: newly-


61

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Relating instrumental texture, determined by variable-blade and Allo-Kramer shear

attachments, to sensory analysis of rainbow trout, Oncorhynchus mykiss, fillets. J Food

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64

CHAPTER 4

INORGANIC CONSTITUENTS IN PROCESSED JELLYFISH AND DETERMINATION OF

ALUMINUM CONTENTS BY HPLC1

___________________________

1 Xu, C. and Y.W. Huang. To be submitted to Journal of Food Science.

65

Abstract

Cannonball jellyfish, Stomolophus meleagris has been under investigation. Fresh jellyfish

are processed with mixture of salt and alum, and then the cured jellyfish are desalted in water

before consumption. Very little is known about the inorganic constitute of jellyfish. In this study

desalted jellyfish were examined for 7 elements, including Al, Ca, K, Mg, Na, Fe, and Zn, using

inductively coupled plasma optical emission spectrometry. High amount of aluminum was found

in cannonball jellyfish samples. In order to determine aluminum levels, high performance liquid

chromatography (HPLC) with spectrophotometric detection using quercetin is developed.

Introduction

Cannonball jellyfish, Stomolophus meleagris, belong to the class Scyphozoa and order

Rhizostomeae, is one of the most common kinds of jellyfish for human consumption. Cannonball

jellyfish have been found from New England to Brazil in the western Atlantic, from southern

California to Ecuador in the eastern Pacific and from the Sea of Japan to the South China Sea in

the western Pacific (Larson 1976). It has great potential value as an export food in the world

market, due to its popularity in Asian countries. Japan imports about 10,000 tons of jellyfish

annually, valued more than 25 million dollars (Omori and Nakano 2001). Their ability to grow

rapidly makes them one of the most abundant scyphomedusae year round along the southeastern

coasts in the United States (Hsieh et al. 1996).

Fresh jellyfish are cleaned and processed immediately after being caught, due to their

perishability. Typically, it requires a multi-phase processing procedure using a mixture of salt

(NaCl) and alum (KAl[SO4]2·12H2O or [NH4]Al[SO4]2·12H2O) to lower the pH, reduce the

moisture content, and firm the texture (Huang 1988). Captured jellyfish are separated into two

parts: umbrella, the bell-shape part, and legs, also known as the oral arms. The entire process

66

lasts 10-40 days, varying with the methods and the species of jellyfish. Liquefaction of the body

tissue occurred without salt added whereas unpleasant odor is given off in the absence of alum.

Although the history of consuming jellyfish as a food has been more than a thousand years,

little is known about the inorganic compounds in processed jellyfish. Most research so far has

focused on the biochemical and medical applications of collagen extracted from jellyfish;

jellyfish has certain effects to anti-fatigue, anti-oxidation, and replacing human collagen in

selected biomedical applications (Ding et al. 2011, Morais et al. 2009, Morishige et al. 2011,

Addad et al. 2011).

Since processed jellyfish have been treated with the mixture of salt and alum, advanced

analytical methods are used to analyze the mineral profile of the samples. Elements in biological

tissues can be determined by a variety of methods, such as atomic absorption spectrometry

(AAS), atomic emission spectrometry (AES), atomic fluorescence spectrometry, inductively

coupled plasma mass spectrometry laser-induced breakdown spectroscopy, X-ray spectrometry,

and secondary ion mass spectrometry (Butler et al. 2013). Among these methods, inductively

coupled plasma (ICP) technique is widely used to investigate the trace elements of foods due to

high selectivity and sensitivity, low analytical limits, and multi-element capability (Shimbo et al.

1999, Kohlmeyer et al. 2003, Nardi et al. 2009, Husáková et al. 2011, Zand et al. 2012).

Aluminum concentrations were found significantly high in processed jellyfish (leg, 671

µg/g; umbrella, 449 µg/g) than fresh jellyfish (leg, 0.29-0.85 µg/g; umbrella, 0.55-1.63 µg/g),

due to contribution of curing agent (Liu et al. 2011, Hsieh et al. 1996). Higher Aluminum

concentration in legs could be associated with the firmer and crispier texture of the legs

compared to the umbrella. Although there is no direct relationship between aluminum intake and

disease, the side effects of aluminum on health have been noted (Miu and Benga 2006, Zatta et al.

67

2003). Elevated levels of aluminum might have serious consequences for biological communities.

Analytical methods used for aluminum determination are voltammetry, UV-Vis

spectrophotometry and fluorometry, flame/electrothermal atomic absorption spectrometry (AAS),

direct current plasma-atomic emission spectrometry (DCP-AES) and neutron activation analysis

(Albendin et al. 2003, Tria et al. 2007). High performance liquid chromatography (HPLC) has

been popularly used for determination of aluminum due to the advantages of powerful separation,

good reproducibility and wide calibration curve (Sato et al. 1996, Remenyi et al. 2011, Yang,

Yin and Shao 2011). Several reports have been published on RP-HPLC separation and

determination of aluminum with the use of chelating agents such as 8-hydroxyquinoline (Lian et

al. 2001, Lian et al. 2003, Lian et al. 2002). Recently a simple, direct and direct method for

determination of aluminum with HPLC has been conducted. Lian et al. (Lian et al. 2004a)

utilized bioactive quercetin as the chelating reagent, complexing with aluminum ion. Quercetin

(3,5,7,3’,4’-pentahydroxyflavone) is a ubiquitously phenolic compounds in photosynthetic plants.

This quercetin/ RP-HPLC method may be an effective technique to determine aluminum content

in jellyfish samples.

The objectives of this study were: 1) to determine the inorganic constituents in desalted

processed cannonball jellyfish using ICP-AES; 2) to determine the aluminum concentrations in

cannonball jellyfish with quercetin/ RP-HPLC; 3) to analyze the effect of water desalting on the

concentrations of inorganic elements in cannonball jellyfish.

Materials and methods

Materials

Two kinds of cured cannonball jellyfish were shipped to our laboratory in polythene barrel

for analysis. One of them has been treated more than 20 years ago and has been stored in a 4℃

68

refrigerator (refrigerator-stored sample) and the other samples were newly processed (newly-

processed sample). All the samples have been cut into two portions, umbrellas (the cap shaped

part) and manubriums (the legs), cleaned with sea water and treated with salt (sodium chloride)

and alum (ammonium aluminum sulfate) before they were sealed in the barrels. The processed

jellyfish, ca, 15 cm in diameter for the part of the umbrella, were split into 5 equal wedges from

the center and then were soaked in tap water (material: water ratio is 1:20) for 8 hours. During

the procedure, the water was changed every two hour. Each desalted jellyfish swelled to about

1.5 times the length. A set of 6-10 samples was collected at 0, 2, 4, 6, and 8 hour respectively.

They were then wiped with a paper towel for several seconds, cut into strips and ground into

slurry by a miller. The slurry was sealed in ziplock bags and stored in a 4℃ refrigerator. Newly-

processed sample were also sent to the Soil, Plant, and Water Laboratory in the University of

Georgia, where the samples were analyzed with ICP-AES to get a mineral profile.

Two batches of processed jellyfish samples were purchased in the retail food market settled

in Chinese cities of Nantong and Dalian, respectively, with both of the umbrella and legs. Four

commercial jellyfish products, which are ready to eat, were also bought from the local groceries

in China. All these samples were sent to the laboratory in Nanjing Agricultural University and

were analyzed to get the mineral profile with inductively coupled plasma-atomic absorption

spectroscopy (ICP-AES) detection.

Reagents and chemicals

All reagents used in this study are analytical-reagent grade unless stated otherwise. Water

(>18MΩ cm) was used for all solutions and mobile phase. Quercetin and aluminum chloride

solution were purchased from Sigma-Aldrich Chemical Company (St Louis, MO, USA).

Methanol (HPLC grade) was obtained from Aldrich Chemical Company (Milwaukee, WI, USA).

69

Perchloric acid, acetic acid, hydrochloric acid, and ammonium hydroxide are all obtained from

Fisher Scientific Chemical Company (Suwanee, GA, USA).

Quercetin stock solution was prepared by dissolving quercetin powder in methanol to a

concentration of 0.015M and stored in a 4℃ refrigerator. 20 ml of aluminum chloride standard

was transferred to a 100 ml volumetric flask and then diluting to the mark with water, to make a

1.5×10-3

M stock solution. Ammonium acetate-acetic acid buffer (pH 4.5) was made to 1.0 M.

Tap water used to soak jellyfish samples were from the supply system of University of Georgia.

Apparatus

The HPLC system consists of a pump, an autosampler, and a UV-Vis detector (Thermo

Fisher Scientific, West Palm Beach, FL, USA). An Aquasil C18, 5 μm, 150 mm × 4.0 mm i.d.

(Thermo Fisher Scientific, West Palm Beach, FL, USA) is used. The aluminum contents in

Chinese samples were determined with ICP-AES in a chemistry laboratory in Nanjing University

(Nanjing, Jiangsu, China); the aluminum contents in American samples were determined with

ICP-AES in an analytical laboratory in University of Georgia.

Procedure

The general procedure refers to a previous study with slight modifications (Lian et al.

2004b). A suitable amount of Al is added into a 25 ml volumetric flask. 1.25 ml of 1.0 M

ammonium acetate-acetic acid buffer (pH 4.5), 2.5 ml of quercetin solution and 7.5 ml of

methanol are added, and the solution is made up to the volume with methanol. A part of the

mixture is filtered through a cellulose membrane with 0.45 μm micropore and 10 μl of the

solution is injected onto the column. The mobile phase is a mixture of acetonitrile and water.

Water is adjusted to pH 1.0 with perchloric acid. The flow rate is 1.0 ml/min. The detection

70

wavelength is 415 nm. The blank test is necessary and the determination of Al concentration is

performed by calibration curve based on area measurement.

The proposed method was applied to determination of total aluminum in jellyfish samples. 5

g jellyfish samples, which had been milled into sludge, were dispersed in 4 mg/ml pepsin

solution and agitated magnetically for 20 min. The temperature of the magnetic stirrer was set at

35℃ at a moderate speed to obtain well-proportioned and stable sample solutions. After the

samples dissolved completely, all the solutions were transferred to a 50 ml volumetric flask and

water was added to the mark. The following procedures of analyzing samples were the same as

that of the general procedure.

HPLC method validation

The linearity of the method was evaluated by a calibration curve consisting of 6

concentrations in the range of 15-150 μM aluminum.

The limit of detection (LOD) is the lowest concentration of analyte can be detected, and

quantitation (LOQ) is defined as the lowest concentration of the analyte that can be determined

with acceptable conditions of the method. LOD was calculated based on the SD of the response

and the slope (S) of the calibration curves according to the formula: LOD=3.3(SD/S). In this

work, the SD of the response was determined based on the SD of the blank tests. LOQ was

determined according to the formula: LOQ=10(SD/S) (Shabir 2003).

Intra-day precision and inter-day precision of this method were investigated by injections of

spiked samples on three consecutive days with five replicates (Deng, West and Jensen 2008).

The accuracy, as well as the recovery, was determined by spiking the samples with aluminum

chloride standard solution at specific concentrations and detected with the HPLC system.

71

Results and discussion

HPLC analysis and method development

Chromatogram

The typical chromatogram of quercetin and its chelate of aluminum was showed in Figure

4.1(a). Al-quercetin chelate was eluted at about 4.95 min at flow rate of 1 ml/min.. Excessive

quercetin was eluted at the time of 27 min. Figure 4.2(b) shows the chromatogram of quercetin

and jellyfish samples.

HPLC method validation

The standard curve generated with aluminum standard solution from 0 to 150 μM was linear

with a correlation coefficient R2=0.9993 (Figure 4.2).

The LOD and LOQ were 0.204 and 0.620 μM, respectively (Table 4.1). Intra-day precision

and inter-day precision of this method, expressed as percent RSD, ranged from 1.3% to 3.0% and

from 2.0% to 6.2%, respectively (Table 4.2). Recovery rate ranged from 98.5% to 103.9%.

Aluminum content in jellyfish samples

Jellyfish were soaked in tap water and vinegar for 24 h and the samples were collected for

determination of aluminum content with HPLC. According to previous studies, pH value and

ionic strength will affect the aluminum speciation (Wang et al. 2010, Devoto and Yokel 1994,

Scancar and Milacic 2006). Therefore, the effect of vinegar on the aluminum concentrations in

jellyfish was studied. Aluminum concentrations declined significantly after being soaked for 24

h, both for the newly-processed sample and refrigerator-stored sample (Figure 4.3). Samples

treated with vinegar solution retained lower level of aluminum than that being dipped just in tap

water. Aluminum concentration in newly-processed sample soaked in water dropped from 466.4

to 129.8 mg Al/kg, while samples soaked in 2% vinegar solution only detained 45.1 mg/kg

72

aluminum. For refrigerator-stored sample both soaked in water and 2% vinegar solution,

aluminum levels showed less decline, ranging from 343.5 to 206.1 mg Al/kg and from 343.5 to

183.4 mg Al/kg, respectively. Since vinegar is one of the commonly used seasonings added to

the jellyfish dishes, this proposed pretreatment could be useful to lower the aluminum

concentration in jellyfish products in real world.

However, the decrease of aluminum levels could be caused by rehydration effect. In order

to explore the changes of aluminum contents on the dry basis, samples were dried and then

analyzed. Refrigerator-stored sample was desalted and was divided into two parts. Aluminum

levels in one part of the samples were determined with HPLC directly; the other part was dried

with an oven at 110℃ for 48 h before the analysis was conducted to remove the moisture. Data

of aluminum concentrations, collected in Figure 4.4, showed that aluminum content on the dry

basis increased from 2491 to 5165 mg Al/kg, and the samples soaked in the 2% (v:v) vinegar

solution showed lower aluminum levels, ranging from 2491 to 1466 mg Al/kg. In addition,

aluminum content dissolved in the water and vinegar solution showed a slight increase over time

(showed in Figure 4.5), from 29.8 to 46.1 mg/kg and from 17.8 to 29.0 mg/kg, respectively.

Every 100 g processed sample contained 22.23 g dry matter at the beginning, whereas after

being soaked in water for 24 h, it dropped to only 6.25 g (showed in Table 4.3). It indicates the

dry matter within the sample would be removed dramatically over the soaking time. That would

explain the increase of aluminum levels in the dried samples in Figure 4.4.

Mineral profiles of jellyfish

Mineral profiles of jellyfish purchased in China

The concentrations of 7 elements in 2 kinds of processed jellyfish are listed in Table 4.4 -

4.5. Generally, significantly higher concentrations of most elements were found in the legs than

73

in the umbrellas. However, for the macro elements, Mg, K, and Na, which presented

significantly lower concentrations in umbrella part than that of the leg parts in the unsoaked

samples, after being soaked for 8 h, there was no significant difference. Elements Al, Fe and Zn

increased over the soaking time, which could also be explained by the previous findings (showed

in Table 4.3). The rising element levels on the dry matter appear to be due to the lower dry

matter of the sample. Apparently, the macro elements were removed with water during the

desalting procedures. Results indicated that, these elements are exchangeable or soluble in the

fresh jellyfish. Interestingly, only aluminum concentrations increased significantly in both the

umbrella and leg parts, ranging from 0.28% to 1.58% and from 0.21% to 1.75% in umbrella and

legs, respectively. According to a previous study, aluminum ion bounds with the hydroxyl

groups in the peptide chain of collage (Li et al. 2003). Generally, the crispiness of cured jellyfish

is attributed to the protein precipitation of the firming agent, alum, and processed legs have a

crispier and firmer texture than the processed umbrella. It may indicate that the higher aluminum

content in legs could cause firmer and crispier texture of the legs.

Another kind of processed jellyfish purchased in China was also analyzed and the

information was generated in Table 4.5. They presented similar mineral profiles as the first

sample. Fe in both of the two samples showed great variation, however, it was significantly

higher in in leg parts than that of the umbrella. Increased Fe in processed jellyfish could be

attributed to the impurities in the curing chemicals, tap water, and processing equipment during

processing.

Elements such as Cd, Co, Cr, Cu, Mn, Ni, and Pb were present at very low levels that

cannot be detected. Heavy metal contamination appears not to be a problem in the cannonball

jellyfish.

74

The concentrations of minerals in four commercial products were listed in Table 4.6.

Mineral profiles varied in different products. The Ca and Mg concentrations in commercial

products were much lower than that of the processed jellyfish. However, the aluminum levels of

these products are about 50% of the processed ones.

Mineral profiles of jellyfish purchased in the USA

Mineral profiles of samples processed, purchased and analyzed in the USA (Table 4.7) were

generated and showed similar trends as that of the samples purchased and analyzed in China.

However, the levels of these elements determined by two different laboratories were

significantly different. Aluminum concentrations of Chinese products were from 2100 to 17500

mg/kg, while samples in the USA only ranged from 763 to 1443 ppm. Difference in jellyfish

species, processing methods, and detection devices might contribute to this result.

In 2006, the provisional tolerable weekly intake (PTWI) level of aluminum from all sources

was reduced from 7 mg/kg body weight to 1 mg/kg body weight. An adult with 70-kg body

weight could intake 70 mg aluminum every week. Given the data in this work, aluminum intake

from one portion of jellyfish product would be about 30 to 75 mg, assuming the serving size

as150 g. Therefore, consuming jellyfish would greatly elevate the aluminum exposure.

Conclusion

This study offers a simple, direct and reliable method for determination of total aluminum

contents. This method could be applied to other biochemical samples with accuracy and

precision. Aluminum levels in salted jellyfish remain relatively steady and could be lowered by

soaking in vinegar solution. Jellyfish product could be an elevated source of aluminum exposure

for human being.

75

In addition, all aluminum concentrations got from ICP-AES were determined on dry matter

basis, which means more information will be needed to estimate the aluminum exposure from

jellyfish samples, i.e. water content. Therefore, it is one of the advantages of this proposed

chromatographic method to determine aluminum in samples without drying procedure.

76

Table 4.1 Calibration curve, LOD and LOQ data (n=5)

Linearity range (µM) Calibration equation LOD (µM) LOQ (µM) Correlation coefficient

15.0-150.0 y= 19827x-36285 0.204 0.620 0.9996

77

Table 4.2 Intra- and inter-day precision and accuracy for the quantitative determination

Standard concentration (µM) Concentration found (µM) Accuracy (%) Precision, CV (%)

Intra-day (n=5)

30 30.97±0.64 103.2 2.1

60 59.11±0.74 98.5 1.3

150 150.52±4.49 100.3 3.0

Inter-day (n=5)

30 31.17±0.62 103.9 2.0

60 59.51±3.66 99.2 6.2

150 152.92±8.58 101.9 5.6

78

Table 4.3 Changes of dry matter of jellyfish sample over 24 h

Soaking time(hour) Dry matter (%) (n=3)

0 22.23±0.25a

24 6.25±0.56b

a,b Means in columns with the same letter indicate no significant difference (p < 0.05); values are means ± standard deviation.

79

Table 4.4 Element concentrations of processed jellyfish samples (bought from Dalian, China)

Element (mg/kg)

Soaking time (h) Sample Al Ca K Mg Na Fe Zn

0 Legs 2800±200d 2700±800

a 2600±300

a 5800±1100

a 277800±366

a 280.6±433.9

ab 18.7±20.0

b

Umbrella 2100±700d 1900±900

ab 1700±600

b 4200±1800

b 208900±783

b 52.1±20.3

c 20.9±10.5

b

2 Legs 13000±1500c 1900±300

ab 700±2

c 2800±10b

c 73000±221

c 93.0±3.1

c 22.9±9.2

b

Umbrella 12500±600c 1800±100

ab 700±1

c 2700±2

c 87500±91

c 223.8±16.9

ab 30.7±2.3

b

4 Legs 15200±1300b 1200±100

bc 200±0

cd 700±100

d 9300±600

d 231.51±99.1

ab 31.7±8.5

b

Umbrella 15500±800ab

900±100c 200±100

d 600±100

d 6400±2800

d 319.7±19.2

ab 31.3±9.7

b

8 Legs 15800±2200ab

1800±500b 300±0

cd 400±0

d 1500±100

d 119.3±12.8

c 75.1±23.1

a

Umbrella 17500±600a 1300±100

bc 300±100

cd 300±0

d 2000±600

d 571.5±320.2

a 62.3±22.5

a

a,b,c,d Means in columns with the same letter indicate no significant difference (p > 0.05) of three replicates; values are means ±

standard deviation; samples were bought from Dalian, China.

80

Table 4.5 Element concentrations of processed jellyfish samples (bought from Nantong, China)

Element (mg/kg)

Soaking time (h) Sample Al Ca K Mg Na Fe Zn

0 Legs 2100±500d 700±100

d 1300±500

a 1100±200

a 240600±28800

b 32.5±8.5

c 22.6±11.8

bc

Umbrella 4700±400c 800±100

d 1500±300

a 1200±300

a 285900±4600

a 245.4±195.5

ab 32.9±17.1

bc

2 Legs 11800±800ab

1300±100ab

500±100b 1200±200b

a 104100±20700

c 163.0±10.2

bc 22.7±9.7

bc

Umbrella 10400±800b 1000±100

c 500±100

b 400±0

b 27800±24300

c 326.2±109.6

ab 15.0±4.0

c

4 Legs 14500±2900a 1300±200

ab 300±0

b 500±100

b 19300±3600

d 278.5±45.9

ab 35.0±1.6

a


1200±100c 300±0

b 400±100

b 16600±3700

d 362.6±109.1

a 24.1±5.1

bc

8 Legs 12800±1900ab

1200±100ab

200±100b 200±1000

b 1500±700

d 171.2±40.2

bc 54.4±12.2

a


1400±100a 300±0

b 300±0

b 1000±200

d 406.7±58.4

a 64.9±7.1

a


standard deviation; samples were bought from Nantong, China.

81

Table 4.6 Element concentrations of commercial jellyfish products bought from China

Elements (mg/kg)

Sample ID Al Ca K Mg Na Fe Zn

A 6800±500b 300±0

c 7800±100

b 100±0

b 95000±1800

b 96.8±5.6

c 19.8±11.9

a

B 6000±400b 1200±100

a 3900±200

c 400±0

a 97300±3900

b 185.3±26.1

b 15.5±6.3

a

C 4700±600c 600±100

b 9400±1100

a 300±0

a 155100±9300

a 341.1±39.0

a 37.9±26.0

a

D 7800±300a 600±100

b 1800±0

d 100±0

b 104100±3400

b 91.0±4.4

c 16.9±4.6

a


standard deviation; samples were bought from China.

82

Table 4.7 Element concentrations (mg/kg) of processed jellyfish samples (newly-processed)

Soaking time (hour)

Elements 0 2 4 12 24

Al 1443 763 989 825 1017

B <1 <1 <1 <1 <1

Cd <0.5 <0.5 <0.5 <0.5 <0.5

Ca 272 39.4 55.4 74 66.8

Cr <0.5 <0.5 102 <0.5 <0.5

Cu <0.5 <0.5 0.59 <0.5 <0.5

Fe 108 44.4 411 72.3 107

Pb <2.5 <2.5 <2.5 <2.5 <2.5

Mg 242 15.5 20.1 25.5 25.1

Mn <0.5 <0.5 3.13 <0.5 <0.5

Mo <0.5 <0.5 <0.5 <0.5 <0.5

Ni <1 <1 26.8 <1 <1

P 241 155 211 194 196

K 183 <20 50 68.6 74.3

Si 138 103 139 146 122

Na 9697 3308 3790 3612 3814

S 822 202 231 204 216

Zn 9 <0.5 <0.5 <0.5 <0.5

83

Figure 4.1 Typical chromatogram for Al-quercetin chelate. (a) pure Al-quercetin (4.5×10-5

M Al:1.5×10-3

M quercetin); (b) sample

after digestion with pepsin. Column: Aquasil C18, 5 μm, 150 mm × 4.0 mm i.d.; mobile phase: 30:70 methanol/water adjusted to pH

1.0 with HClO4; flow rate: 1.0 ml/min; injection volume: 10 μl; wavelength: 415 nm.

84

Figure 4.2 Aluminum calibration curve; n=5.

85

Figure 4.3(a) Aluinum concentrations in newly-processed sample soaking in water

and 2% vinegar solution.

Figure 4.3(b) Aluminum concentrations in refrigerator-stored sample soaking in

water and 2% vinegar solution.

86

Figure 4.4 Aluminum concentrations in dried refrigerator-stored sample soaking in

water and 2% vinegar solution.

87

Figure 4.5 Aluminum concentrations in soaking solutions.

88

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CHAPTER 5

SUMMARY AND CONCLUSION

This study analyzed textural properties and mineral profiles of jellyfish products, which can

provide valuable information for utilizing jellyfish as a potential food resource, as well as

developing appropriate strategies to control the product quality.

Texture is one of the most important attributes of jellyfish products, and desalting cured

jellyfish in water is a critical step to create jellyfish a desirable texture. However, objective

instrumental methods to assess textural attributes in this product have not been studied

sufficiently. Crunchiness is a major quality determinant of jellyfish, which is usually evaluated

by instrumental methods such as shear test. The present work was conducted to evaluate a

method for the determination of jellyfish crunchiness. Jellyfish received 5 treatments: 0-h, 2-h, 4-

h, 6-h and 8-h soaking in tap water to yield a vast array of crunchiness levels. Assessment of this

attribute was measured with a Texture Analyzer by tension, texture profile analysis (TPA),

multiple-blade shear, and single-blade shear. Peak force and area read from the curves for

tension and shearing were analyzed. The Multiple-blade attachment could predict the key texture

attribute (hardness) of jellyfish samples without damage the integrity of the sample. Our results

showed that, while the moisture content increased from 83.39% to 96.34%, both the peak force

and area of tension and shearing were significantly affected by soaking time. Unheated newly-

processed sample and unheated refrigerator-stored sample could obtain similar textural

properties of ready-to-eat products after being soaked for 6 hours and 8 hours, respectively;

similar results could also be obtained by dipping salted samples in boiling water.

94

Inorganic elements in processed jellyfish were also under investigation. Fresh jellyfish are

processed with mixture of salt and alum, and then the cured jellyfish are desalted in water before

consumption. Very little is known about the inorganic constitute of jellyfish. In this study

desalted jellyfish were examined for 7 elements, including Al, Ca, K, Mg, Na, Fe, and Zn, using

inductively coupled plasma optical emission spectrometry. High amount of aluminum was found

in cannonball jellyfish samples. HPLC offers a simple, direct and reliable method for

determination of total aluminum contents. This method could be applied to other biochemical

samples with accuracy and precision. Aluminum levels in salted jellyfish remain relatively

steady and could be lowered by soaking in vinegar solution. Jellyfish product could be an

elevated source of aluminum exposure for human being.

DETERMINATION OF ALUMINUM CONTENT IN FOOD … This study analyzed texture properties and mineral profiles of jellyfish products, which can provide valuable information for utilizing

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