EP1444266A1 - Use of abalone processing waste - Google Patents

Abstract

The use of process waste from abalone food processing operations as a source for protein and other natural products.

Description

Use of abalone processing waste

Technical Field

The present invention is concerned with the use of waste from the processing of abalone as a source for natural products, particularly proteins.

Background Art

Oceanic organisms are of enormous scientific interest, for two major reasons. First, they constitute a major share of the Earth's biological resources. Second, marine organisms often possess unique structures, metabolic pathways, reproductive systems, and sensory and defense mechanisms because they are adapted to extreme environments ranging from the cold polar seas at -20°C to the great pressures of the ocean floor.

The ocean therefore offers abundant resources for research and development . Yet the potential of this domain as the basis for new biotechnology remains largely unexplored.

Marine biotechnology will generate advanced technologies for producing new pharmaceuticals, biomaterials, and other products; developing and improving bioprocessing; and understanding of biological processes in the oceans. Bioprocessing enables the translation of research discoveries into commercial products with unique and highly desirable characteristics and offers new production opportunities for a wide range of items:

• Improved high value products such as naturally occurring non-toxic pharmaceuticals and flavoring agents

• Polymers for films and coatings, and use in food and beverage processing and other specialty uses

• Commodity chemicals such as enzymes and proteins • Cosmetic ingredients such as collagen

• Medical products such as collagen and gelatin Bioprocessing offers a level of specificity, predictability and productivity that otherwise would not exist in the manufacture of these products. Moreover, when the raw material contains certain molecules with complex structures, bioprocessing enables the isolation of products that cannot be made by any other means. Combined, these capabilities provide for new process designs that are cost effective, energy efficient, and product specific . Thus bioprocessing requires an understanding of the biological system employed (such as the marine organism) , isolation and purification of a product, and translation of the product into a stable, efficacious, and convenient form. Bovine spongiform encephalopathy (Mad Cow

Disease) , or BSE, is an extremely serious disease of cattle, considered to originate from infected meat and bone meal in cattle feed concentrates. BSE is transmissible in cattle, and was first identified in United Kingdom in 1986. It is invariably fatal. There is no treatment and it is difficult to detect. Recent research indicates that humans who eat infected meat could develop Creutzfeldt-Jacob Disease (CID) , the human equivalent of the cattle disease. At least 10 CID patients in Britain are believed to have contracted the disease from eating beef. Most people who develop CID are aged between 50 and 70.

Currently the culling of the cattle is of primary importance in the United Kingdom and Europe to safeguard the herd, and hence to the future supply of bovine meat and dairy products. This strategy is also important to maintain supply of bovine by-products used in the pharmaceutical, medical and cosmetic industries.

However, at present pharmaceutical makers across Japan, UK, Europe and other countries have stopped using

British beef and beef products in the manufacture of pharmaceutical, medicine and cosmetics products to prevent the spread of "Mad Cow" disease to humans . Also imports of medicine and cosmetics containing substances from British cattle have stopped.

The most widely used beef products are collagen and gelatin, the spongy substances derived from beef skin or bones, used as an additive in foods, as a cosmetic ingredient and in medical applications.

The variety of drugs derived from cattle parts is diverse and includes : • Growth hormones from the cattle pituitary glands

• Adrenalin products for hay fever, asthma and other allergies, from the adrenal gland

• Cortizone, for arthritis, asthma, shock, also from the adrenal gland • Insulin, for diabetes, from the pancreas

• Tissues used as patches during heart bypass surgery, from bovine pericardial tissue

• Thromboplastin, a blood coagulant used in surgery, from the brain • Drugs for the treatment of stomach ulcers

• Gelatin capsules for many drugs are made from cattle tissues

Spongiform encephalopathies occur also in land animals other than cattle. For example, Transmissible mink encephalopathy (TME) is a comparatively rare disease of farmed mink that has been observed in the mid-western United States since 1947. n some outbreaks, most or all adult mink on a ranch have eventually died. The source of infection is contaminated foodstuffs, and it is generally assumed that outbreaks occur after feeding mink with carcass material from scrapie-infected sheep. Chronic wasting disease (C D) , which occurs in mule, deer and Rocky Mountain elk in Colorado and Wyoming in the United States, has been recognised since 1967. Cases of Spongiform encephalopathy have been reported in six species of antelope in British zoological parks. About 55 cases of Spongiform encephalopathy have been diagnosed in domestic cats in the United Kingdom since 1990. It is possible that these originated from BSE-contaminated meat meal incorporated in commercial cat food. In early 1992, spongiform encephalopathy was diagnosed in an adult cheetah in a zoological park in Western Australia. The animal had shown characteristic signs of a slowly progressive neurological disease.

There is already consumer resistance to beef products in European countries, and other land animals also appear to give rise to some risk of prion infection and development of Spongiform encephalopathies if used as a source of food. Therefore, there exists a need for an alternative source of proteinaceous products.

Abalone are marine snails belonging to the class Gastropoda of the phylum Mollusca and assigned to the family Haliotidae and genus Haliotis. Molluscs form the second largest phylum in the world, and there are over 100 species in the genus, Haliotis of which 10% are commercially important. Abalone are slow-growing herbivores which move by means of a broad muscular foot. In Australia abalone are usually named for the colour of the foot, thus Haliotis ruber is called 'black-lip', Haliotis conicopora is called *brown-lip' and Haliotis laβvigata is *green-lip' . The abalone is one of the most primitive gastropods in form and structure (Cox 1960) .

In temperate and cool regions abalone are found on rocky reefs around headlands or offshore, to the depth limit of marine plants (approximately 80 to 100 m) . In the southern parts of Australia the abalone survives well on untouched natural reef . Adult abalone feed on seaweed .

The abalone industry is very important to Australia and has three commercially valuable large abalone species. They are found along the southern coast of mainland Australia and Tasmania between 30 and 50° south with water temperatures from 11 to 23° C (Ino, 1980; Imai 1977; and Shepherd 1975). *Haliotis ruber

H. ruber, black-lip abalone or red-ear shell, is the most important and common species in Australia ( mai 1977) . Individuals are usually 12 to 14 cm in length but some grow to 20 cm. Black-lip abalone are generally found in 1 to 10 metres of seawater. Large animals will generally produce 500 grams of edible meat (Harrison 1969) . This species are found on the southern and eastern coasts of the Australian mainland and Tasmania. In Tasmania the black-lip constitutes 90% of the annual catch and is found from tidal areas to depths in excess of 30 metres on all coasts south of about 41 degrees south.

*Haliotis laevigata H. laevigata, green-lip abalone or mutton fish, commonly grows between 13 and 14 cm, and has white flesh (Harrison 1969) . Individuals occur at depths of 5 to 40 metres in moderate to rough water (Imai 1977; Harrison 1969) . The green-lip abalone is also found on the southern and eastern coasts of the Australian mainland and Tasmania but in a smaller quantity (10%) than the black-lip. In Tasmania the green-lip is found mainly in Bass Strait.

*Haliotis roei H. roei, Roe's abalone, grows to between 7 and 8 cm in length, and is the smallest of the commercial Australian species (Shepherd and Laws 1974) . Individuals are found in less than 5 metres of water (Shepherd 1976) . This species is found along the Western Australian coast. Australia's annual abalone catch is about 5500 tonnes. In fact, Australia accounts for 50% of global production of abalone. The white flesh of the abalone muscular foot may weigh up to 500 grams and is highly valued by several Asian cultures for its unique flavour. The Chinese and Japanese are the main consumers of abalone. Disclosure of the Invention

Processing of abalone for food on an industrial scale gives rise to a large amount of waste. The non- utilised portion is declared process waste and then discarded. If these wastes are dumped they may cause pollution and emit an offensive odour. It has now been found that abalone waste material can be processed to give a number of valuable by-products.

Accordingly, in a first aspect of the present invention there is provided the use of process waste from abalone food processing operations as a source for protein and other natural products.

The abalone waste items may be their gonads, oseophagus, stomach, anus, mouth tissue, gills, gut, shell, head, frills, blood or meat or muscle tissue pieces, and the term "process waste" is intended to encompass all of these.

The waste generated from one black-lip abalone (total wet weight =750 gm) is 67%. Blood =17%,

Guts, anus, frills, gills, oseophagus, muscle tissues, gonad = 17%,

Shell = 33%

Abalone muscle tissue, which is the portion usually consumed, is also a source of such products.

Intact muscle may be processed, for example, to isolate collagen and/or gelatin, but fragments of meat or muscle also occur as waste (eg when the abalone breaks up during processing) and are proposed to be used in the present invention in view of the very high market value of intact muscle.

Preferably, the species used are the green-lip abalone (Haliotis laevigata) , the brown-lip abalone (Haliotis conicopora) , Roe's abalone (Haliotis roei ) and the black-lip abalone (Haliotis ruber) , most preferably the black-lip. There is a large potential for recovery of valuable products from abalone process waste and there are several reasons for the need for protein products from this source: • Abalone waste material could provide an alternative source for the production of by-products such as collagen which are currently being produced mainly from land animals.

• There is continued demand for new sources of proteins and enzymes to cut costs, to find proteins with novel properties and even totally new protein activities and applications.

The protein products from abalone process waste may be used as a substitute for existing products isolated from land animals, such as collagen and gelatin, or maybe novel proteins. Indeed, the substitutes for existing proteinaceous products may be different in structure from the corresponding product isolated from a land animal.

The abalone may be any of the one hundred or so species in the genus Haliotis, but is conveniently one of the commercial species, and the waste from processing the muscular foot for use as food is utilised.

Advantageously, the protein products are haemocyanin from abalone blood and collagen and/or gelatin from waste or intact muscle tissue. Abalone collagen has unique properties, as discussed in International Application No. PCT/AU01/00708 entitled "Process for Obtaining Native Collagen", the contents of which are incorporated herein by reference, and is likely to be useful as a substitute for collagen-based products derived from land animals such as cattle. Gelatin products may also be derived from abalone collagen by heating abalone collagen. Isolated collagen may be boiled, but heating to temperatures as low as 40°C can effect degradation of the collagen chains to gelatin. The collagen isolated from abalone is Type I collagen, and may be isolated by the process described in the International Patent Application No. PCT/AU01/00708. Gelatin may also be prepared without first isolating abalone collagen, as described in the above-mentioned co-pending patent application.

The protein may also be haemocyanin (He), and this is a novel protein. The protein is described in International Patent Application No. PCT/AU01/00710 entitled "Novel Haemocyanin", in which the process of isolation from abalone blood is also described, and which is incorporated herein by reference. In addition, novel proteins and replacements for existing land animal proteins may be isolated from other waste material such as the frill, mouth tissue and gills, and there is an expectation that novel amino acids and polysaccharides may also be derived from these sources . Abalone gut and gonads may be a source of enzymes, proteins and lipids, and the abalone shell is a source of novel proteins, lipids, polysaccharides and proteoglycans .

Brief Description of the Drawings Preferred embodiments of the present invention will now be described, by way of example only, with reference to the accompanying Figures, in which:

Fig 1 is a chromatogram showing separation of He from East Coast Abalone on Phenyl HIC; Fig 2 is the absorbance spectrum of East Coast

Abalone He column load;

Fig 3 is a chromatogram showing separation on Phenyl HIC for batch 1 in Example 3;

Fig 4 is a chromatogram showing separation on Phenyl HIC for batch 2 in Example 3;

Fig 5 is an SDS-PAGE gel of the diafiltration retentates of batches 1 and 2 of Example 3 and resuspensions of batches 1 and 2 of Example 6 in which the lanes are as follows: Lane 1 - molecular weight marker

4 - large scale batch 1 retentate

5 - large scale batch 2 retentate

6 - batch 1 (freeze drying trial) 7 - batch 2 (freeze drying trial); Fig 6 is a chromatogram showing chromatographic separation of He on Phenyl HIC after freezing and then thawing; Figure 7 is a cross-sectional view of the abalone muscle showing

(A) dorsal surface of foot (epipodium)

(B) hard part of foot (pedal sole)

(C) soft part of foot (pedal sole) (D) middle part of adductor (columellar) muscle

(E) upper part of adductor (columellar) muscle; Figure 8 is an SDS-PAGE gel of the various native abalone collagen fibrils (Parts A, B, C, D, D* and E) ; which are located in the following lanes:

Lane 1 - molecular weight standard

2 - Part A

3 - Part B 5 - Part C 7 - Part D 8 - Part D^*

9 - Part E

10 - calf skin collagen and

Figures 9A and 9B are SDS-PAGE gels showing abalone collagen and calf skin collagen in which Figure 9A has the following lanes:

Lane 1 - molecular weight standard

2 - calf skin collagen

3 - calf skin collagen 4 - 1^st Extract

5 - 1^st Extract incubated at 27°C for 17 hrs

6 - 1^st Extract incubated at 27°C for 48 hrs; and

Figure 9B has the following lanes: Lane 1 - molecular weight standard 2 - calf skin collagen

3 - 2^nd Extract; and

Figure 10 is an SDS-PAGE gel of abalone gelatin in which lane 1 is a molecular weight standard, lane 2 is the gelatin and lane 3 is collagen 1^st extract.

Modes of Performing the Invention

Haemocyanin (He) is the blue, copper-containing respiratory protein of many molluscs and arthropods. Haemocyanins are always found freely dissolved in the blood (or hemolymph) . The molluscan haemocyanins have an entirely different structure and arrangement of subunits compared to arthropod haemocyanins. Aerobic metabolism of abalone is supported by gas exchange through gills found in the respiratory cavity. The blood pumped through the gills, via a low-pressure open circulatory system, contains haemocyanin which transports oxygen to respiratory tissues. In open systems blood flows from arteries into the tissue spaces and finally into venous sinuses before being collected in veins and returned to the heart . Oxygenated blood ranges from pale to strong blue depending on the degree on oxygenation, haemocyanin concentration and species of animal. Dimeric copper pairs in the haemocyanin provide reversible sites for the binding of one oxygen molecule. Haemocyanin is also a source of copper that may lead to inorganic and organic blueing reactions in abalone food processing.

Haemocyanins are arranged into multi-subunit proteins which carry as few as six or as many as several hundred oxygen molecules. Molluscan haemocyanins are extremely large macromolecules having molecular masses of around 4 million dalton (Da) . Molluscan haemocyanins have subunits containing seven or eight oxygen binding functional units. Each globular functional unit is of about 50 kDa and they are arranged like a string of beads. Ten such subunits assemble to form cylindrical decameric whole molecules and in gastropods multiples of two or more decamers may be found. The wall of the decamer has sixty oxygen binding units, and the remaining units form the so- called collar which lies in the centre of the cylinder and, in the case of gastropod haemocyanin, offset to one end. The association of haemocyanin subunits requires divalent cations, either Mg²⁺ or Ca²⁺, as well as competent monomers (Mangum, 1983) .

The copper content of molluscan haemocyanins averages around 0.25%, corresponding to 1 gram atom per 25000 daltons of protein. Haemocyanins are potent immunogens which induce the synthesis of large amounts of specific antibodies. The He may exist in associated or dissociated forms (Bartell and Campbell, 1959) . In addition to containing associated or dissociated He molecules, various preparations may contain a number of other immunologically distinct proteins. For instance the hemolymph of the crab may contain at least 5 distinct proteins as well as two electrophoretically distinct He (Horn and Kerr, 1969) . During fishing, studies have shown that even with extreme care, abalone are cut at least 12% of the time when pried off a rock. Since abalone have no blood clotting agent they will usually bleed to death.

When abalone are processed for food, they are shucked by inserting a knife between the shell and the foot. The guts are removed and the head area cut away. Only the foot is retained for sale as meat with the remainder being discarded as waste. Due to the absence of blood clotting agents, blood loss is very significant in the abalone food processing industry where animals are sold on a weight basis. Up to 17% of the body weight may be lost as fluid during processing. This represents not only an economic loss but also results in a loss of flavour from the meat. Unless stated otherwise, all experiments in

Examples 1 to 5 were performed at 15°C or on ice. Example 1

Isolation and Purification of Haemocyanin from East Coast

Abalone (WEC)

Step 1. Abalone Fishing, Storage and Transport Black-lip abalone (Haliotis ruber) were fished from Storm Bay on the east coast of Tasmania. These animals were shipped to Brisbane, Queensland without tank storage at the process plant in Tasmania.

Seven live abalone (batch 1) were air-freighted in March 2001 from Tasmania to Brisbane, Queensland in sealed, oxygen-filled bags at 4°C.

Step 2. Live Holding Tank

On arrival, the live animals were transferred to a live holding tank. It measures 1430 mm long X 430 mm wide X 450 mm high, giving a volume of approximately 280 litres. A pump circulates the water through a filter and aeration system while a refrigeration unit controls the water temperature at 10°C. The tank is sited in a separate room for quarantine purposes and is protected from fluctuations in the external environment. The status and movements of the animals were closely monitored and feeding of seafood pellets was conducted once a week. The abalone have been kept in the live holding tank for over two months with zero mortality. Water filtration is quite efficient and so the tank requires little cleaning.

Step 3. Shucking and Method of Blood Collection Each animal was washed under cold running water to remove slime and sand. The animal was turned upside down and shucked by sliding a broad spatula under the foot at the flat region of the shell until the attachment of the foot to the shell was cut. Care was taken not to rupture any internal organs . The spatula was then run gently around the inside edge of the shell to detach the internal organs . The whole animal was then able to be tipped out of the shell.

The guts and other organs (visceral part) were carefully separated from the foot using a scalpel. Care was taken not to rupture any internal organs so as to prevent possible contamination of the blood. The internal organs were further dissected, bagged separately and stored at -20°C for other protein extraction. The mouth area was cut away from the front of the foot with a scalpel, bagged and stored at -20°C.

The foot was rinsed with water and weighed. Several deep incisions were made in the front area of the foot with a scalpel and the foot suspended over a strainer to allow the blood to drain to a collection vessel. Care was taken to avoid bacterial contamination. This was done at 4°C with an initial collection after 1 hour and a further collection after 6 hours.

The foot was either processed immediately or stored at -20°C for the extraction of collagen by the process described in Examples 6 to 9.

Any remaining organic material was scraped from the inside of the shell, which was rinsed with water and left to dry at room temperature. The shells were stored at room temperature for future work (extraction of proteins) . The blood was centrifuged at 12000 x g for 10 minutes at 4°C. The supernatant was decanted, the volume measured, and stored at 4°C in clean, sterile containers. The pellets were discarded.

Step 4. Isolation and Purification of He

Method

A 14 ml Phenyl HIC column was run on a

Perseptive Biosystems BioCAD 700E at a flowrate of 1.0 ml/min. The equilibration buffer contained 50mM potassium phosphate, 1M NaCl, lmM MgCl₂, ImM CaCl₂, at pH 6.0. The elution buffer contained 50mM potassium phosphate, ImM MgCl₂# Im CaCl₂# at pH 6.0. The cleaning in place solution was 0.5M NaOH.

Sample Preparation 1. The load sample was prepared the day before the chromatography on the BioCAD.

2. 0.5 ml of 4M NaCl was slowly added to 2 ml of centrifuged blood supernatant with constant mixing.

3. 2.5 μl of 1M MgCl₂ + 2.5 μl of 1M CaCl₂ were then added and mixed.

Chroma t ography

1. The resin was equilibrated with 5 column volumes of equilibration buffer. 2. 2 ml of sample (equivalent to 1.6 ml blood) was loaded onto the column.

3. Flow through fractions were collected (4 ml per tube) .

4. The resin was washed with 2 column volumes of equilibration buffer.

5. Step elution was with 100% elution buffer for 4 column volumes .

6. Elution fractions were collected (4 ml per tube) . 7. Cleaning in place was performed with 2 column volumes of 0.5M NaOH.

8. Cleaning in place fractions were collected (4 ml per tube) .

Dialysis

The cleaning in place fractions were pooled and extensively dialysed against de-ionised water to remove traces of sodium hydroxide.

Protein Estimation Protein concentrations of the chromatography fractions were carried out using absorbance measurements at 340 nm.

1. The absorbance of the load sample, pooled flow through and pooled elution fractions at 340 nm was measured against an elution buffer blank. The load sample required an initial dilution of 1/20 with elution buffer.

2. The haemocyanin concentration is calculated from the extinction coefficient for abalone haemocyanin (ε^1%ι _cm =0.223). A Biorad Smart Spec 3000 spectrophotometer was used with a quartz UV grade cuvette.

Absorbance Spectrum

An absorbance spectrum was scanned from 240 to 460 nm for column load He samples using a Biorad Smart Spec 3000. Samples were initially diluted 1/15 with de- ionised water.

Amino Acid Analysis

Amino acid analysis of He samples (pooled flow through and elution fractions) were done using a Waters amino acid analyser. Samples containing approximately 5 μg of protein were prepared for amino acid analysis.

Results

Table 1 - Appearance of Centrifuged He

Table 2 - Yield of He from Phenyl HIC Chromatography (Fig. 3)

Table 3 - Amino Acid Analysis of Chromatography Fractions

51 - WEC flow through

52 - WEC elution

Example 2

Isolation and Purification of Haemocyanin from Green-Lip

Abalone

Step 1. Abalone Fishing, Storage and Transport A single green-lip abalone (Haliotis laevigata) was fished from King Island in Bass Strait and tanked at the process plant for 2 days. The time in the crate (from catch to tank storage) was around 8 hours. The maximum time out of water was 14-15 hours. The animal was air-freighted in April 2001 from

Tasmania to Brisbane, Queensland as described in Example 1. Step 2. Live Holding Tank

On arrival, the live animal was transferred to a holding tank as described in Example 1. On the 14^th day the green-lip abalone was able to be slid easily along the floor of the tank and so was removed and shucked.

Step 3. Shucking and Method of Blood Collection This is described in Example 1.

Step 4. Isolation and Purification of He

Method A 5 ml Phenyl HIC column was run on a Biologic

LP at a flowrate of 1.5 ml/min. The equilibration buffer contained 50mM potassium phosphate, 3M NaCl, ImM MgCl₂, ImM CaCl₂, at pH 7.0. The elution buffer contained 50mM potassium phosphate, ImM MgCl₂, ImM CaCl₂, at pH 7.0. The cleaning in place solution was 0.5M NaOH.

Sample Preparation

1. The load sample was prepared immediately prior to the chromatography on the Biologic LP. 2. 2 ml of 6M NaCl was slowly added to 2 ml of centrifuged blood supernatant with constant mixing.

3. 4 μl of 1M MgCl₂ + 4 μl of 1M CaCl₂ were then added and mixed.

Chromatography

1. The resin was equilibrated with 6 column volumes of equilibration buffer.

2. 2 ml of sample (equivalent to 1.0 ml blood) was loaded onto the column. 3. Flow through fractions were collected (4 ml per tube) . 4. The resin was washed with 3 column volumes of equilibration buffer.

5. Step elution was with 100% elution buffer for 5 column volumes . 6. Elution fractions were collected (4 ml per tube) .

7. Cleaning in place was performed with 2.5 column volumes of 0.5M NaOH.

8. Cleaning in place fractions were collected (4 ml per tube) .

Protein Estimation

Protein concentrations of the chromatography fractions were carried out using absorbance measurements at 340 nm as described in Example 1.

Results

Table 4 - Appearance of Centrifuged He

Table 5 - Binding of He to Phenyl HIC Chromatography Resin

The percentage bound is calculated as 100 X (He elution + He CIP) / (He flow through + He elution + He CIP) . This table indicates good binding of green lip abalone haemocyanin to the resin under the conditions tested. The He purification for the green-lip abalone was similar to the east coast animals, with a similar % binding as seen in Table 5.

Example 3

Validation of Purification of Haemocyanin by Phenyl HIC

Chromatography

The He purification process was scaled up to a development stage. Two litres of abalone blood was processed using a one litre column of Phenyl HIC resin.

Step 1. Abalone Fishing, Storage and Transport

Black-lip abalone (Haliotis ruber) were fished from Storm Bay (April 2001) on the east coast of Tasmania. These animals were shipped directly to the abalone process plant in Tasmania and shucked immediately. A total of 140 abalone (weighing around 90 kg) produced around 4.5 litres of blood. This blood was collected as aseptically as possible in 1000 ml sterile containers. The blood was air-freighted to Brisbane in an esky and kept at 4°C. Upon arrival, the blood was immediately centrifuged at 12000 X g for 10 minutes at 4°C and the pooled supernatant aliquoted into sterile 500 ml containers. 2.3 litres were retained for purification of He and the remainder stored at -20°C for validation of long-term storage.

Step 2. Isolation and Purification of He

Method

A 1000 ml Phenyl HIC column was run at a flowrate of 30 ml/min. The equilibration buffer contained

50mM potassium phosphate, 3M NaCl, ImM MgCl₂, ImM CaCl₂, at pH 7.0. The elution buffer contained 50mM potassium phosphate, ImM MgCl₂, ImM CaCl₂, at pH 7.0. The cleaning in place solution was 0.5M NaOH.

le Preparation Two litres of abalone blood was purified in two batches on the Phenyl HIC column.

1. The load sample was prepared immediately prior to the chromatography.

2. 1200 ml of 5M NaCl was slowly added to 1000 ml of centrifuged blood supernatant with constant mixing.

3. 2.2 ml of 1M MgCl₂ + 2.2 ml of 1M CaCl₂ were then added and mixed.

Chroma tography 1. The resin was equilibrated with at least 5 column volumes of equilibration buffer until a pH between 6.9 and 7.1 is reached.

2. 2200 ml of sample (equivalent to 1000 ml blood) was loaded onto the column. 3. Flow through fractions were collected (400 ml per tube) . 4. The resin was washed with at least 4 column volumes of equilibration buffer until the absorbance of the fractions reached baseline.

5. Step elution was with 100% elution buffer for at least 5 column volumes until the absorbance of the fractions reached baseline.

6. Elution fractions were collected (400 ml per tube) .

7. Cleaning in place was performed with 2 column volumes of 0.5M NaOH.

8. Cleaning in place fractions were collected (400 ml per tube) .

Protein Estimation Protein concentrations of the chromatography fractions were carried out using absorbance measurements at 340nm as described in Example 1.

Molecular Weight and Purity The molecular weight and purity of abalone haemocyanin was evaluated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) . A 4-20% Gradipore iGel precast Tris glycine gel was used. SDS-PAGE was performed according to the method of Laemmli (1970) . The haemocyanin standard was initially diluted

1/50 in deionised water. All samples were then diluted to half concentration with Gradipore Glycine sample buffer.

The samples were then placed into a boiling water bath for 3 minutes then allowed to cool. The gel was assembled in a Biorad Mini-Protean 3 electrophoresis cell.

The inner chamber was filled with SDS glycine running buffer and the samples loaded with an autopipettor and standard yellow tips. The total protein load per well was 2 μg. A molecular weight marker (Biorad broad range prestained marker) was run with each gel. The outer chamber was filled with running buffer to the level of the wells. The running conditions were 150V constant voltage over 60 minutes with an approximate start current of 50 mA. The gel was then removed from the casing and rinsed with water for around 30 seconds. The gel was stained with around 50 ml of

Gradipore Gradipure stain (based on colloidal G-250 Coomassie blue) overnight with gentle shaking. The gel was destained with frequent changes of water. Bands were generally visible after 5 minutes with about a day required for complete destaining.

Permanent storage of gels was achieved by drying between cellophane sheets. The destained gels were soaked in a drying solution of 20% methanol and 2% glycerol with gentle shaking for 15 minutes. Two cellophane sheets per gel were wetted in the drying solution for around 30 seconds. The trimmed gel was clamped between the cellophane sheets in a drying frame and left to stand in an open container at room temperature for 2 days . The gel was then pressed for a number of days prior to display.

Ultra filtration and Diafiltration

A Millipore Prep Scale TFF cartridge was used for the initial concentration and diafiltration steps. A Vivascience Vivaflow 50 TFF cartridge was used for the final concentration step. The diafiltration buffer contained 83mM sodium phosphate, 150mM NaCl at pH 7.2.

1. The pooled elution was concentrated from 3.6 litres down to approximately 400 ml using the Prep Scale TFF cartridge with a cross-flow rate of 1200 ml/min. 2. This was followed by 5 x volume diafiltration using Prep Scale TFF cartridge. The cartridge was drained and rinsed to retentate.

3. The protein concentrations of retentate and permeate were checked. 4. The retentate was concentrated to approximately 50 mg/ml using the Vivaflow 50 cartridge. The cartridge was drained and rinsed to retentate. 5. The protein concentrations of retentate and permeate were rechecked.

6. The retentate was sterile filtered through a 0.2 μm filter capsule into sterile container. The protein concentrations of retentate and permeate were checked. The filter was rinsed through with diafiltration buffer to give the required final volume for a He concentration of 30 mg/ml.

7. The He sample was aliquoted into 10 ml sterile vials for dispatch.

Amino Acid Analysis

Amino acid analysis of He samples (batch 1 and 2 diafiltration retentates) was done using a Waters amino acid analyser. Samples containing approximately 5 μg of protein were prepared for amino acid analysis.

Results

Table 6 - Appearance of Centrifuged Blood

Table 7 - Yield of He from Phenyl HIC Chromatography (Figs. 3 and 4)

The percentage yield is calculated as 100 X (He elution) / (He flow through + He elution + He CIP) . This table indicates good binding of He to the resin.

Table 8 - Recovery of He following Ultrafiltration / Diafiltration / 0.2μm Filtration

Table 9 - Amino Acid Analysis of Final He Product from Large Scale Process

Thus, a novel, commercial scale purification procedure for He from waste abalone blood with high yield and purity has been developed. This procedure is simple and cost effective and has been successfully validated. No initial processing steps such as removal of contaminants were required prior to a single chromatography step on Phenyl HIC. This process was robust enough to give consistent separation on scale up with a high yield of 10 g of He from 2 L of east coast abalone blood (Tables 7 and 8) . The electrophoretic mobility of the He showed a single band at 250 kDa and a purity of 99% (Figure 5) . The amino acid composition of He from the large scale purification was analysed (Table 9) and was comparable to the small scale east coast He (Table 3) .

Example 4

Freeze / Thawing Trial of WEC Abalone Blood

Step 1. Abalone Fishing, Storage and Transport This is described in Example 1.

Step 2. Live Holding Tank This is described in Example 1.

Step 3. Shucking and Method of Blood Collection This is described in Example 1.

Step 4. Isolation and Purification of He Method

A 5 ml Phenyl HIC column was run on a Biologic LP at a flowrate of 1.5 ml/min. The equilibration buffer contained 50mM potassium phosphate, 3M NaCl, ImM MgCl₂, ImM CaCl₂, at pH 7.0. The elution buffer contained 50mM potassium phosphate, ImM MgCl₂, ImM CaCl₂, at pH 7.0. The cleaning in place solution was 0.5M NaOH.

Sample Preparation An aliquot of centrifuged blood supernatant was frozen at -20°C for a period of 7 days. The sample was thawed and purified.

1. The load sample was prepared immediately prior to the chromatography. 2. An aliquot of centrifuged blood supernatant was taken.

3. An appropriate volume of concentrated NaCl was slowly added with constant mixing to give a final salt concentration of 3M. 4. 1M MgCl₂ and 1M CaCl₂ were then added to give a final concentration of ImM and mixed.

Chroma tography

1. The resin was equilibrated with 8 column volumes of equilibration buffer.

2. 2 ml of sample was loaded onto the column.

3. Flow through fractions were collected (4 ml per tube) .

4. The resin was washed with 4 column volumes of equilibration buffer.

5. Step elution was with 100% elution buffer for 4 column volumes.

6. Elution fractions were collected (4 ml per tube) . 7. Cleaning in place was performed with 3 column volumes of 0.5M NaOH. 8. Cleaning in place fractions were collected (4 ml per tube) .

Protein Estimation Protein concentrations of the chromatography fractions were carried out using absorbance measurements at 340 nm as described in Example 1.

Molecular Weight and Purity The molecular weight and purity of abalone haemocyanin was evaluated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) . A 4-15% Biorad precast Tris glycine gel was used. SDS-PAGE was performed according to the method of Laemmli (1970) as described in Example 3.

Results

Table 10 - Effect of Freeze / Thawing on Purification of

He

The percentage bound is calculated as 100 X (He elution + He CIP) / (He flow through + He elution + He CIP) .

The data from Table 10 (east coast abalone) suggest that freeze/thawing does not adversely affect the chromatographic performance of the haemocyanin in terms of reduced binding. This indicates the stability of the protein and the consistency of purification of He. Hence abalone blood can be stored at -20°C with no deleterious effects on He purification. Example 5

Freeze Drying Trial of WEC Abalone Blood

Method

1. Samples (2 ml) of the diafiltration retentates from batches 1 and 2 were freeze dried for 12 hours. The drying samples were continuously monitored so that they could be removed as soon as drying was complete.

2. The freeze dried samples were resuspended in de-ionised water to their original concentrations and analysed by BCA protein assay (as described in Example 1) and SDS-PAGE.

Results Table 11 - Appearance of Freeze Dried and Resuspended He

The freeze dried product showed a single band at 250 kDa in gel electrophoresis, and a purity of 99% (Figure 5) . Unless otherwise stated, all steps in Examples 6 to 10 were carried out at 4° C or on ice. All solvents and water used were pre-chilled at 4°C. This minimizes bacterial growth, enhances the solubility of native collagen, and ensures the retention of native conformation on the part of the solubilised collagen. Example 6

Novel Method for Removal of Pigmentation from Abalone

Tissue

The abalone foot is covered by skin where the mucus-secreting glands are located. The skin also contains cells that give colour. The colour varies with species type. The black-lip has black pigmentation. Currently abalone food processors have difficulty in the removal of pigment from abalone foot without breaking the meat up. The process used in the abalone food industry involves the forcing of a jet of warm water through a rumbler containing the abalone in order to remove the pigment, however this process is likely to convert collagen to gelatin thereby softening the meat and breaking it into pieces. This also changes the texture of the meat. A collagen molecule is transformed into gelatin by heat denaturation above body temperature.

It is highly preferable that the native collagen product described above be white, with absence of any black pigment. A process to remove the pigmentation without any thermal denaturation to the collagen is described in detail below, by way of example only.

Step 1. Abalone Fishing, Storage and Transport . Black-lip abalone (Haliotis ruber) was fished from Port Davey on the west coast of Tasmania. The abalone were air-freighted in March 2001 from Tasmania to Brisbane, Queensland. The abalone were transported from Tasmania to Queensland as a dry consignment. The abalone were placed in sealed, oxygen filled bags with wet foam to keep the humidity high. The animals were held vertically in a head down position by attachment to waxed cardboard sheets. This allows waste products to flow away from the animal. At all times during transport the animals were kept in an insulated container to maintain a constant temperature of 4°C. Step 2. Shucking and Method of Tissue Preparation.

All animals were shucked immediately on arrival . The animal was first washed under running water to remove any slime and sand. The animal was turned upside down and shucked by sliding a broad spatula under the foot at the flat region of the shell until the attachment of the foot to the shell was cut. Care was taken not to rupture any internal organs. The spatula was then run gently around the inside edge of the shell to detach the internal organs. The whole animal was then able to be tipped out of the shell.

The guts and other organs (visceral part) were carefully separated from the foot using a scalpel. Care was taken not to rupture any internal organs so as to avoid contamination of the foot tissue. The internal organs were further dissected, bagged separately and stored at -20°C for other protein extraction. The mouth area was cut away from the front of the foot with a scalpel, bagged and stored at -20°C. The foot was rinsed with water and weighed.

Several deep incisions were made in the front area of foot with a scalpel and the foot suspended over a strainer to allow the blood to drain to a collection vessel. Care was taken to avoid bacterial contamination. This was done at 4°C with an initial collection after 1 hour and a further collection after 6 hours. The blood could be used for the preparation of haemocyanin as described in Examples 1 to 5, and the remaining tissue for the extraction of collagen. Any remaining organic material was scraped from the inside of the shell, which was rinsed with water and left to dry at room temperature.

Step 3. The weight of the abalone muscle tissue was measured and found to be 100 gms.

Step 4. The tissue was soaked in 0.2M acetic acid overnight with slight agitation. Step 5. The tissue was washed under running cold tap water which removed the pigmentation from the outer areas of the epipodoium, the hard part of the foot (pedal sole) and the upper part of the adductor (columellar) muscle

(Figure 10) .

It will be appreciated that a simple and efficient process for the removal of pigment from the abalone foot area and adductor area has been developed. Soaking the tissue in 0.2 M acetic acid causes swelling and softens the tissue, thus allowing easy removal of black pigmentation from the outer regions of the foot (pedal sole) epidermis. Too much swelling is a disadvantage since it results in a rigid and stiff tissue. This process will be of value to the abalone food processing industry as well as aiding in the extraction of white collagen fibrils.

Example 7

Extraction of Native Collagen from the Individual Parts of the Muscle Tissue Using Acetic Acid

The presence, quantity and quality of collagen from the different parts of the abalone muscle were determined. The abalone muscle was divided into foot (pedal sole) , the dorsal surface of foot (epipodium) , and adductor (columellar) muscle (see Figure 10) . The foot and adductor muscle were further separated into soft and hard parts, and upper and middle parts, respectively.

Step 1. After removal of the pigments from the tissue the various muscle parts (A, B, C, D, and E) were dissected using a scalpel and the individual parts weighed: Part A = 4 gm

Part B = 28 gm

Part C = 7 gm Part D = 47 gm Part E = 3 gm

Each individual part was treated separately as follows.

Step 2. The tissue was further cut into smaller pieces using a scalpel.

Step 3. 0.5 M acetic acid solution (pH 3.0) was added to the tissue.

Part A = 50 ml

Part B = 100ml

Part C = 50 ml

Part D = 200 ml Part E = 200 ml

Step 4. The individual suspensions (part A, B, C, and E) were stirred overnight.

Part D was not stirred and allowed to stand overnight. The supernatant (D*) was retained for analysis to determine if collagen was extracted without any agitation to the tissue. A further 200 ml of 0.5 M acetic acid was added to the remaining part D tissue.

Step 5. The suspensions were homogenised using a hand held blender.

Step 6. The pH of the slurry was adjusted to 3.5 with a small volume of 1.0 N HCl .

Step 7. The slurry was stirred overnight to extract collagen fibrils.

Step 8. The stirrer was turned off and the solids were permitted to settle out. Step 9. The solution was centrifuged at 3,000 rpm, for 20 minutes to remove tissue particulates.

Step 10. In order to precipitate the native collagen fibrils the supernatant was brought to 0.3M sodium chloride by gradually adding solid sodium chloride to the supernatant with constant stirring. Visible white collagen fibrils precipitated within 2 minutes.

Step 11. The solution was allowed to stir overnight to further extract the native collagen fibrils.

Step 12. The solution had a high viscosity indicating the presence of collagen.

Step 13. The native collagen fibrils were collected by centrifugation at 5,000 rpm at 4° C for 30 minutes.

Step 14. The native collagen fibrils from parts A, B, C, D, D* and E were each dissolved in a minimum quantity of de-ionised water.

Step 15. The native collagen fibrils were extensively dialysed against de-ionised water to remove any salt.

Step 16. The native collagen fibrils from parts A, B, C, D, D*, and E were transferred into separate freeze drying bottles and frozen in liquid nitrogen.

Step 17. The samples were freeze dried for approximately

16 hours.

CHEMICAL ANALYSIS

1. Protein Estimation

Protein estimation was carried out using the

Pierce BCA assay. This method is based on the reduction in alkaline conditions of Cu²⁺ to Cu¹⁺ by protein (biuret reaction) and the colourimetric detection of Cu¹⁺ using bicinchoninic acid (BCA) . An appropriate amount of working reagent was prepared by the mixture of 50 parts of reagent A and 1 part of reagent B. For each sample, 2 ml of working reagent was aliquoted into Johns 5 ml polystyrene tubes .

The freeze dried abalone collagen samples (A, B,

C, D, D* and E) and calf skin collagen (Sigma Chemicals) were resuspended with de-ionised water to a concentration of 1 mg/ml. Then 0.1 ml of each sample was added to a tube and mixed by gentle inversion. A blank was prepared using 0.1 ml de-ionised water. The tubes were placed in a preheated water bath at 37°C for 30 minutes, then allowed to cool on the bench for 10 minutes.

A standard curve was prepared by diluting a stock solution of BSA to a range of concentrations from 25-2000 μg/ml and assaying as described above.

The samples were read on a Biorad Smart Spec 3000 spectrophotometer using the inbuilt BCA protein assay function. This allows the storage of standard curves and automatic calculation of sample concentration. Disposable UV grade PMMA cuvettes were used for absorbance measurement at 562 nm.

2. Molecular Weight, Purity and Chain Type Composition Determination

The molecular weight, purity and chain type composition of abalone collagen from each part (A, B, C,

D, D* and E) was evaluated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) . 8% Gradipore iGel precast Tris glycine gels was used. SDS- PAGE was performed according to the method of Laemmli (1970) .

Freeze dried abalone collagen (samples A, B, C, D, D* and E) and calf skin collagen were dissolved at 1 mg/ml in deionised water. Samples were then diluted to half strength with Gradipore Glycine sample buffer.

The samples were then placed into a boiling water bath for 3 minutes, then allowed to cool. The gel was assembled in a Biorad Mini-Protean 3 electrophoresis cell. The inner chamber was filled with SDS glycine running buffer and the samples loaded with an autopipettor and standard yellow tips. The total protein load per well was 2 μg. A molecular weight marker (Biorad broad range prestained marker) was run with each gel. The outer chamber was filled with running buffer to the level of the wells.

The running conditions were 150V constant voltage over 60 minutes with an approximate start current of 50 mA. The gel was then removed from the casing and rinsed with water for around 30 seconds. The gel was stained with around 50 ml of Gradipore Gradipure stain (based on colloidal G-250 Coomassie blue) overnight with gentle shaking. The gel was destained with frequent changes of water. Bands were generally visible after 5 minutes with about a day required for complete destaining.

Permanent storage of gels was achieved by drying between cellophane sheets. The destained gels were soaked in a drying solution of 20% methanol and 2% glycerol with gentle shaking for 15 minutes. Two cellophane sheets per gel were wetted in the drying solution for around 30 seconds. The trimmed gel was clamped between the cellophane sheets in a drying frame and left to stand in an open container at room temperature for 2 days. The gel was then pressed for a number of days prior to display. 3. Solubility Determination

The solubility of freeze dried material from parts A, B, C, D, D^*, and E were tested.

Test Method:

1. To around 10 mg of each freeze dried sample, de- ionised water was added to 1 mg/ml and swirled.

2. 20 μl of 1M HC1 was added with moderate swirling.

3. The tubes were left on their side for gentle swirling on an orbital shaker, then stood upright and allowed to settle. The clarity of the solution was observed.

RESULTS

Table 12 shows the total Weight of Freeze Dried Native Abalone Collagen Fibrils (Parts A, B, C, D, D* and E) and Their Appearance.

Table 12

Table 13 shows Native Abalone Collagen Fibril Extraction Yield. Table 13

Table 14 shows Protein Content of Native Abalone Collagen Fibrils (Part A, B, C, D, D* and E) .

Table 14

Table 15 shows the Solubility of Native Abalone Collagen Fibrils (Parts A, B, C, D, D* and E)

Table 15

A large amount of collagen could be extracted from the different parts of the abalone tissue when treated with 0.5 M acetic acid. When the collagen fibrils in a tissue are treated with 0.5 M acetic acid at pH 3.5 the hydrolysis of unstable cross-links releases into solution ^Λacid-soluble' native collagen.

Approximately 91% of native abalone collagen was extracted from muscle part C and 60% from part E, while part A, B, and D and D* were 10%, 7.1%, 3.1% and 6.8% respectively. Thus abalone contains large amounts of collagen in the muscle, which vary depending on muscle parts.

Analysis of D* sample indicated that 7% protein was extracted from non-homogenised and non-agitated raw material (Part D) . However the freeze dried material from D* was cream coloured and rubbery with poor solubility as compared with the Part D sample which was white. This could be due to soaking the D sample in 0.5 M acetic acid overnight .

When examined by SDS-PAGE (Figure 8) parts A, B, C, D* and E contained two major bands at 123.9 kD and 110.6 kD. These bands could be the αl and α2 chains. Part D had just one single broad band at 105 kD. The molecular weight of native abalone collagen was significantly different from calf skin collagen which showed two main bands at 204 kD and at 138.5 kD.

The electrophoretic behaviour of abalone collagen has clearly demonstrated the occurrence of two types of alpha chain. Thus abalone native collagen is Type

I, being the main protein constituent of abalone muscle tissue. The ratio of αl and α2 in the different parts varied. In parts A and B there was a higher level of α2 than αl chains. In parts C, D and E there were equal amounts of αl and α2 chains. Part D had just one single broad band which is currently being analysed to determine if this protein is collagenous or non-collagenous.

Example 8A

Extraction of Native Abalone Collagen Fibrils From the

Whole Muscle Tissue (1^st Extract)

Step 1. Abalone Fishing, Storage and Transport

Wild East Coast Abalone (WEC)

Black-lip abalone (Haliotis ruber) were fished from Storm

Bay on the east coast of Tasmania. These animals were shipped directly to Brisbane, Queensland without tank storage at the abalone food process plant in Tasmania.

Seven live abalone (batch 1) were air-freighted in March 2001 from Tasmania to Brisbane, Queensland (as described in Example 6) .

Step 2. Live Holding Tank

On arrival, the live animals were transferred to a holding tank. It measures 1430 mm long X 430 mm wide X

450 mm high, giving a volume of approximately 280 litres. A pump circulates the water through a filter and aeration system while a refrigeration unit controls the water temperature at 10°C.

The tank is sited in a separate room for quarantine purposes and is protected from fluctuations in the external environment. The status and movements of the animals were closely monitored and feeding of seafood pellets was conducted once a week. Abalone have been kept in the live holding tank for over a month with zero mortality. Water filtration is quite efficient and so the tank requires little cleaning. Step 3. One abalone was removed from the tank after one day of storage.

Step 4. The total weight of the live abalone including the shell was weighed = 450 gm.

Step 5. Shucking and Method of Tissue Preparation. The method is as described above for Example 6.

Step 6. The weight of the abalone muscle tissue was measured (146 gm) .

Step 7. The pigmentation from the foot area and adductor area was removed as described in Example 6.

Step 8. The muscle tissue was re-weighed (127 gm) .

Step 9. The whole muscle tissue was cut into smaller pieces using a scalpel.

Step 10. 1000 ml of 0.5 M acetic acid solution (pH 3.0) was added to the tissue.

Step 11. The mixture was stirred for 2 hours.

Step 12. The mixture was further homogenised using a hand held blender.

Step 13. The pH of the slurry was adjusted to 3.5 with a small volume of 1.0 N HC1.

Step 14. The slurry swelled and therefore another 500 ml of 0.5 M acetic acid solution (pH 3.0) was added.

Step 15. The slurry was stirred overnight to extract native collagen fibrils. Step 16. The mixture was centrifuged at 3,000 rpm, for 20 minutes to remove tissue particulates. The pelleted tissue was retained for further extraction.

Step 17. In order to precipitate the native collagen fibrils the supernatant was brought to 0.3M sodium chloride by gradually adding solid sodium chloride to the supernatant with constant stirring. Visible white collagen fibrils precipitated within 2 minutes.

Step 18. The mixture was allowed to stir overnight to further extract native collagen fibrils.

Step 19. The solution had a high viscosity indicating the presence of collagen.

Step 20. The native collagen fibrils were collected by centrifuging at 5,000 rpm at 4° C for 30 minutes.

Step 21. Solid sodium chloride was added to 1250 ml of supernatant (2^nd extraction) to give a final concentration of 0.3 M.

Step 22. The solution was allowed to stir overnight,

Step 23. The solution was clear and not viscous.

Step 24. The solution was centrifuged at 5,000 rpm to pelletise the native collagen fibrils. Very little pellet was present in the second extraction.

Step 25. The collagen pellets obtained from Step 20 and Step 24 were pooled.

Step 26. The native collagen fibrils were dissolved in a minimum quantity of de-ionised water. Step 27. The native collagen fibrils were extensively dialysed against de-ionised water to remove salt.

Step 28. The native collagen fibrils were then dialysed against 0.1 M acetic acid. The dialysis medium was replaced frequently by fresh acid until the pH of the solution inside the dialysis bag reached 3.5.

Step 29. The native collagen fibrils were transferred into freeze drying bottles and frozen in liquid nitrogen.

Step 30. The sample was freeze dried for approximately 16 hours .

Step 31. The freeze dried collagen samples were weighed.

Example 8B

Re-extraction of Collagen Fibrils (2^nd Extract) from 1^st Extract Pellet Tissue,

Step 1. The pellet (110 gm) obtained in Step 16 of Example 8A was re-extracted with 1500 ml of 0.5 M acetic acid.

Step 2. The mixture was stirred overnight to extract native collagen fibrils.

Step 3. The mixture was centrifuged at 5,000 rpm, at 4°C for 20 minutes to remove tissue particulates.

Step 4. Solid sodium chloride was added gradually to the 1480 ml of the supernatant with constant stirring to give a final concentration of 0.3 M.

Step 5. The solution was allowed to stir overnight to precipitate native collagen fibrils. Step 6. The solution did not have a high viscosity.

Step 7. The solution was centrifuged at 5,000 rpm at 4° C for 30 minutes.

Step 8. The native collagen fibrils were dissolved in a minimum quantity of de-ionised water.

Step 9. The native collagen fibrils were extensively dialysed against de-ionised water to remove salt.

Step 10. Then the native collagen fibrils were dialysed against 0.1 M acetic acid until the pH of the solution inside the dialysis bag reached pH 3.5.

Step 11. The collagen sample was transferred into freeze drying bottles, frozen in liquid nitrogen and freeze dried for 16 hours.

Step 12. The freeze dried collagen samples were weighed.

CHEMICAL ANALYSIS

1. Protein Estimation

The freeze dried abalone collagen samples (1^st and 2^nd extracts) and Sigma Calf Skin collagen were resuspended with de-ionised water to a concentration of 1 mg/ml .

Protein estimation was carried out using the

Pierce BCA assay as described in Example 6. A standard curve was prepared by diluting a stock solution of BSA to a range of concentrations from 25-2000 μg/ml and assaying as described above.

The samples were read on a Biorad Smart Spec

3000 spectrophotometer using the inbuilt BCA protein assay function. This allows the storage of standard curves and automatic calculation of sample concentration. Disposable UV grade PMMA cuvettes were used for absorbance measurement at 562 nm.

2. Molecular Weight, Puri ty and Chain Type Composition Determination

The molecular weight, purity and type composition of abalone collagen (1^st and 2^nd extracts) and calf skin were evaluated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). 12% (1^st extract) and 8% (2^nd extract) Gradipore iGel precast Tris glycine gels were used. SDS-PAGE was performed according to the method of Laemmli (1970), as described in Example 6.

3. Amino Acid Analysis

Amino acid analysis of collagen samples (abalone 1^st and 2^nd extracts and calf skin) was done using a Waters amino acid analyser. Samples containing approximately 5 μg of protein were prepared for amino acid analysis from l g/ml resuspensions of freeze dried collagen. Calf Skin Collagen standard : (SI) 1^st Extract Abalone Collagen: (S2) 2^nd Extract Abalone Collagen: (S3).

4 . Incubation of Collagen at 27 °C - Stability Trial

1^st Extract freeze dried native collagen was incubated in an oven at 27° C. Samples were taken after 17 and 48 hours and analysed by SDS-PAGE as described above.

5. Solubility Determination

The solubility of freeze dried material from the 1^st and 2^nd abalone collagen extracts and the calf skin collagen were tested. Test Method:

To around 10 mg of both abalone freeze dried collagen samples de-ionised water was added to 1 mg/ml and swirled.

To around 1 mg of calf skin freeze dried collagen, de-ionised water was added to 1 mg/ml and swirled.

10 μl of 1M HC1 was added to the 1 extract and calf skin samples, with moderate swirling.

20 μl of 1M HC1 was added to the 2nd extract sample, with moderate swirling.

The tubes were left on their side for gentle swirling on an orbital shaker, then stood upright and allowed to settle. The clarity of the solution was observed.

RESULTS

Table 16 shows the Total Weight of Freeze Dried Native Abalone Collagen Fibrils (1^st Extract and 2^nd Extract) and Calf Skin Collagen and Their Appearance.

Table 16

Table 17 gives the Protein Content of Freeze Dried Native A Abbaalloonnee CCoollllaaggeenn FFiilbrils (I^s Extract and 2ⁿ Extract) and Calf Skin Collagen. Table 17

Table 18 describes the Solubility of Native Abalone C Coollllaaggeenn Fibrils (1^st and 2^nd extracts) and Calf Skin Collagen

Table 18

Table 19 gives the Amino Acid Composition of Native A Abbaalloonnee CCoollllaagen Fibrils (1^st and 2^nd Extracts) and Calf Skin Collagen.

Table 19

SI - calf skin collagen

52 - 1 extract abalone collagen

53 - 2^nd extract abalone collagen

Example 8C

All extraction steps were repeated this time at room temperature. The native collagen fibrils were prepared and analysed as discussed in Example 8A, with essentially no difference in the results achieved.

Examples 8A to 8C show that native acid-soluble collagen fibrils are advantageously extracted with 0.5 M acetic acid and separated by sodium chloride precipitation from the supernatant. Extraction with 0.5 M acetic acid solubilised a large amount of the total collagen in contrast to vertebrate collagens which do not contain any acetic acid soluble collagen. Solubilising 1 kg of calf skin with pepsin only yields 0.025% collagen (Laurain et al 1980) . Most of the collagen was extracted in the first extraction (1^st extract, Table 16) .

Thus native solubilised collagen fibrils extracted from abalone muscle tissue lead to purified Type I native collagen. Furthermore, the solubility of the native abalone collagen samples produced a clear solution while the calf skin collagen (mammalian source) produced a turbid solution (Table 18) .

The SDS-PAGE gels exhibited two main bands at 123.9 and 110.6 kD (Figures 9A & 9B) . The ratio of αl and α2 chains of the 1^st and 2^nd extract were similar. Individual collagen chains were easily separated on the SDS-PAGE without column purification. Most type I collagens are composed of a heterotrimer of two αl(I) and α2 (I) chains which corresponds to the upper and lower chain bands respectively. The electrophoresis experiments conducted on calf skin collagen showed a main band at 204 kD (β chain) and bands at 138.5 and 132 kD, corresponding to αl and α2 chains respectively (Figure 9A) .

Chemical composition and physical properties of fish meat show seasonal variations causing variations in texture (Dunajuski, 1979) . Seasonal variations in free amino acids of seafood have also been reported on levels of glutamic acid, glycine, and taurine in lemon sole (Jones, 1959) . Little study has been reported on whether chemical components in abalone meat show seasonal variations. The tough texture of abalone is considered important since abalone is usually consumed raw in Japan. During summer, abalone meat texture is not tough and this is related to the low collagen content. Thus customarily, summer is the best and winter is the worst season for abalone meat toughness.

The imino acids, proline and hydroxyproline are both stabilising factors, so that the melting temperature of collagen from many animals is proportional to the imino acid content (Jose and Harrington, 1964) . The amino acid analysis of abalone native collagen fibrils is given in (Table 19) . The hydroxyproline content of abalone collagen was low and this could be related to the seasonal catch as the abalone analysed in our work were summer abalone. There were variations in some of the residues between calf skin and abalone collagen, particularly a lower imino acid residue content in abalone collagen, indicating a lower denaturation temperature compared to calf skin collagen.

Glycosylation of hydroxylysine is related to extrusion of soluble collagen into the extracellular matrix. Large amounts of hydroxylysine residues may influence the structure of collagen fibrils (Blumenkrantz, et al 1969) .

Collagen in the abalone meat may be important in energy storage and may have some effect on muscle metabolism before the spawning season, in order to make the gonads grow. An extraordinarily large growth of gonad index in abalone in spawning seasons has been reported (Webber 1970), thus abalone need much energy around spawning season. If abalone stored energy in muscle, storage of collagen might be reasonably expected because collagen is mainly composed of non-essential amino acids. Synthesis and decomposition of collagen might occur largely around spawning season. In summer such turnover might not be so active.

Following incubation at 27°C for 17 and 48 hours, abalone native collagen fibrils had similar electrophoretic mobility as those stored at 4°C (Figure

9A) . This indicates that abalone collagen has good thermal properties.

Example 9 Extraction of Abalone Collagen from the Whole

Animal by Pepsin Digestion Step 1. The pellet (wet tissue =110 gm) obtained in Step 16 of Example 8A was solubilised in 500 ml of 0.5 M acetic acid.

Step 2. To the solution was added 0.1 gm of pepsin (Sigma) .

Step 3. The pH of the solution was adjusted to 2.8 with a small amount of 1 N HCl.

Step 4. The solution was stirred at room temperature for 8 hours then at 4°C overnight for further extraction.

Step 5. The tissue was completely solubilised.

Step 6. The pH of the solution was changed from 2.8 to 6.0 with a small of amount of 1 M sodium hydroxide to stop the enzymatic action of the pepsin.

Step 7. The solution was centrifuged at 10,000 rpm at 4°C for 1 hour.

Step 8. The collagen pellets were dissolved in a minimum quantity of de-ionised water and pooled.

Step 9. The collagen samples were transferred into freeze drying bottles, frozen in liquid nitrogen and freeze dried for 16 hours.

Step 10. The freeze dried collagen samples were weighed.

CHEMICAL ANALYSIS

1. Molecular Weight, Purity and Chain Type Composition Determination

The molecular weight, purity and type composition of pepsin-solubilised abalone collagen was evaluated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) . 8% Gradipore iGel precast Tris glycine gels was used. SDS-PAGE was performed according to the method of Laemmli (1970), as described in Example 10.

2. Solubility Test

Test Method:

1. To around 10 mg of freeze dried sample, add de- ionised water to 1 mg/ml and swirl for 15 minutes.

2. Add 20 μl of 1M HCl and give moderate swirling for around 2 hours.

3. The tubes were left on their side for gentle swirling on an orbital shaker, then stood upright and allowed to settle. The clarity of the solution was observed.

RESULTS

Table 20 shows the Total Weight of Freeze Dried Pepsin- Solubilised Abalone Collagen and Its Appearance.

Table 20

Due to the lack of solubility of the pepsin- solubilised collagen, the collagen bands were not visible on the SDS-PAGE gel (gel not shown) . Therefore, analysis of the molecular weight, purity and chain type composition did not prove to be possible. Table 21 gives the Solubility of Native Abalone Collagen Fibrils (pepsin-solubilised collagen) .

Table 21

In contrast to the process of the invention, the use of pepsin to solubilise abalone muscle tissue produces a yellow coloured final product. It will not be cost- effective to use this process on an industrial scale as pepsin is an expensive agent and furthermore the final product does not retain the native structure of collagen. The poor solubility of the freeze dried sample could be due to prolonged freeze drying. Solubilisation of pepsin- digested collagen results from hydrolysis of peptide bonds within the telopeptides between the cross-linking sites and the triple helix. Nevertheless, abalone is a hitherto unexpected source of collagen.

Example 10

Isolation of Gelatin

Step 1. The pigment from the abalone tissue was removed as described in Example 6 Steps 4-5.

Step 2. The tissue (50 gms) was homogenised and to the slurry was added 200 ml 0.5 M acetic acid (pH 3.5) to extract the gelatin. The extraction was carried out in a water bath at 40°C.

Step 3. The slurry was centrifuged at 3,000 rpm for 30 minutes, 25°C to remove tissue particles. Step 4. The gelatin solution was transferred into a freeze drying bottle, frozen in liquid nitrogen and freeze dried for 16 hours.

Step 5. The freeze dried gelatin sample was weighed.

Chemical Analysis

Method

1. Molecular Weight and Purity

The abalone gelatin sample was analysed as discussed in Example 6. A native abalone collagen sample was also included for comparison.

2. Solubility The freeze dried material was dissolved at 1 mg/ml in de-ionised water as discussed in the collagen method section (Example 6) .

3. Gelling Properties of Abalone Gelatin 2 ml of abalone gelatin was left at room temperature to observe the gelling properties.

Results

Table 22 shows Total Weight of Freeze Dried Abalone Gelatin and Its Appearance

Table 22

Table 23 gives Solubility of Abalone Gelatin Table 23

The abalone gelatin had a molecular weight of 110 kD on SDS-PAGE (Figure 10) and exhibited good solubility (Table 23).

The abalone gelatin gelled at room temperature in a matter of minutes which shows good gelling properties compared to calf skin gelatin which would require 4°C overnight in order to set .

Thus, there has been developed a novel, simple and cost effective process for the extraction of gelatin from abalone waste tissue. Alternatively gelatin could be prepared from isolated collagen by heating, as would be well understood by the person skilled in the art .

Industrial Applicability

The invention is useful since the process waste from the processing of abalone for food is used as a source for novel compounds, rather than being treated as a disposal problem.

References

The following references have therein disclosure incorporated herein by reference:

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Cox, K. W. , Review of the abalone in California, Calif. Fish Game 46 (4).381, 1960.

Dunajuski E. (1979) J Texture Studies 10, 301.

Ino,T., 1980, Fisheries in Japan. Abalone and Oyster. Japan Marine Products Photo Materials Assoc, Tokyo, 165.

Imai, T., Aquaculture, 1977, in shallow seas: progress in shallow sea culture. Amerind Pub, New Delhi, Part IV. Jones, N.R. (1959) J. Sci. Food Agric. 10; 282.

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Laemmli U.K (1970) Nature 227, 680-685.

Laurain G, Delvincourt T, and Szymanowicz A.G. (1980) FEBS Letter, 120, 44.

Shepherd, S. A., 1975, Distribution, habitat and feeding habits of abalone. Aust. Fish. 34(1), 12.

Shepherd, S. A., 1976, Breeding, larval development and culture of abalone, Aust. Fish. 35(4), 7. Harrison, A. J., 1969, The Australian abalone industry, Aust Fish. 28(9), 1.

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879.

Claims

Claims :

1. The use of process waste from abalone food processing operations as a source for protein and other natural products.

2. The use as claimed in claim 1 wherein the abalone process waste is selected from the group consisting of gonads, oesophagus, stomach, anus, mouth tissue, gills, gut, shell, head, frills, blood, meat or muscle tissue pieces.

3. The use as claimed in claim 2 wherein haemocyanin is isolated from blood.

4. The use as claimed in claim 2 wherein collagen-based products are derived from meat or muscle tissue pieces.

5. The use according to any one of claims 1 to 4 wherein the abalone is selected from the group consisting of the green-lip abalone (Haliotis laevigata) , the brown-lip abalone (Haliotis conicopora) , Roe's abalone (Haliotis roei) and the black-lip abalone (Haliotis ruber) .

6. A method of processing abalone comprising the steps of:

(1) separating intact abalone muscle tissue from process waste;

(2) processing intact muscle tissue for food; and

(3) isolating protein or other natural products from the process waste.

7. A method as claimed in claim 6 wherein the process waste is selected from the group consisting of gonads, oesophagus, stomach, anus, mouth tissue, gills, gut, shell, head, frills, blood, meat or muscle tissue pieces.

8. A method as claimed in claim 7 wherein haemocyanin is isolated from blood.

9. A process as claimed in claim 7 wherein collagen- based products are derived from meat or muscle tissue pieces.

10. A method as claimed in any one of claims 6 to 9 wherein the abalone is selected from the group consisting of green-lip abalone (Haliotis laevigata) , the brown-lip abalone (Haliotis conicopora) , Roe's abalone (Haliotis roei) and the black-lip abalone (Haliotis ruber) .

11. Haemocyanin when isolated from abalone process waste.

12. A collagen-based product when derived from abalone process waste.