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Critical Reviews in Environmental Science and
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ISSN: 1064-3389 (Print) 1547-6537 (Online) Journal homepage: http://www.tandfonline.com/loi/best20
Marine shells: potential opportunities for

extraction of functional and health-promoting
materials
Yakun Hou, Amin Shavandi, Alan Carne, Adnan A. Bekhit, Tzi Bun Ng, Randy
Chi Fai Cheung & Alaa El-din A. Bekhit
To cite this article: Yakun Hou, Amin Shavandi, Alan Carne, Adnan A. Bekhit, Tzi Bun Ng,
Randy Chi Fai Cheung & Alaa El-din A. Bekhit (2016): Marine shells: potential opportunities for
extraction of functional and health-promoting materials, Critical Reviews in Environmental
Science and Technology, DOI: 10.1080/10643389.2016.1202669
To link to this article: http://dx.doi.org/10.1080/10643389.2016.1202669
Accepted author version posted online: 20

Jun 2016.
Published online: 20 Jun 2016.
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Marine shells: potential opportunities for extraction of functional and health-promoting
materials
YAKUN HOUa, AMIN SHAVANDIa*, ALAN CARNEb, ADNAN A. BEKHITc, TZI BUN
NGd, RANDY CHI FAI CHEUNGd, ALAA EL-DIN A. BEKHITa*
a
Department of Food Science, University of Otago, Dunedin, New Zealand
Downloaded by [University of California Santa Barbara] at 06:53 25 June 2016
b
Department of Biochemistry, University of Otago, Dunedin, New Zealand
c
Faculty of Pharmacy, Alexandria University, Alexandria 21521, Egypt
d
School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong,
Hong Kong, China
* Corresponding author
amin.shavandi@postgrad.otago.ac.nz; Ph: ++64 3 479 5463,
aladin.bekhit@otago.ac.nz; Ph: ++64 3 479 4994
Abstract
Marine shell waste is a very rich source of several bioactive compounds and materials, such as
calcium, chitin, pigments and proteins. Currently, this waste material is greatly underutilized and
contributes to significant environmental problems due to off odour and concentration of minerals
in landfill. The main objective of this review is to highlight the potential to value-add to and
maximize the utilization of this waste stream. Therefore, this review provides up to date
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information on various compounds available in marine shells that are generated as waste
co-product from commercial processing operations and their potential uses. Methods are
described for extraction of these compounds for use in food and pharmaceutical applications.
Keywords
Waste shells, Chitin, Hydroxyapatite, Pigment

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Introduction
Seafood is a rich source of diverse bioactive compounds, including proteins, unsaturated fatty
acids, vitamins and essential amino acids. As a result, aquaculture became the fastest growing
animal-food producing sector [1] and a wide variety of products are being produced including
finfish, and crustaceans such as shrimps, prawns, crabs, and shellfish such as clams, oysters and
mussels, as well as seaweeds and other aquatic plants. World capture production (from open
water) of fish, crustaceans and molluscs reached 91 million tonnes in 2012 and in addition 66.6
million tonnes were produced by aquaculture around the world [2]. More than 70% of the total
production is utilized in further processing. The large quantity of processed aqua-cultured
species, in addition to the processed harvest from open water has led to large amounts of marine
by-product waste, such as crustacean shells, viscera, heads, skins, fins, trimmings and shellfish
waste being generated and discarded annually. It is estimated that about 25% of the total weight
of annual marine production is discarded as waste, which is equivalent to more than 20 million
tonnes [3]. These wastes are normally disposed of in landfill, processed into feed stock or used as
a fertilizer. Land filling of waste shells can cause many environmental issues such as generation
of off odours, contamination of air and soil pollution and damage to the marine ecosystem [4].
There is considerable potential for conversion of this waste into value-added products to resolve
some of the issues associated with environment pollution and cost of disposal. Research in the
area of renewable and marine by-products has increased significantly over the past decade.
Shells account for a significant proportion of seafood processing waste which is not being widely
utilized and hence provides significant opportunities for development of value-added products.
As a result, there is increasing interest in conversion and utilization of these waste materials. The
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majority of marine shells are composed of more than 90% calcium carbonate, which can be
converted to different calcium containing products such as calcium phosphate and calcium citrate
with biomedical and food industry applications. Shrimp and crab shells are composed of high
protein content (40%), calcium carbonate (30%), chitin (20-25%), lipid and pigments. The
current application of crustacean waste is mainly focused on production of chitin using chemical
methods that require the use of large amounts of hazardous chemicals (NaOH and HCl) that are
released into the environment. During this chemical process the protein component in the shells
is not recovered. The use of enzymatic processing methods can solve this issue and proteins,
chitin and pigments can be recovered. The main focus of this review is on shell material, such as
that derived as a waste stream from processing of mussels, oysters and sea urchins, which are
normally dumped in a landfill and are regarded as having no economic value. This review
discusses the various products that can be obtained from waste marine shells and describes
various methods that can be used to produce these products with the aim of highlighting
opportunities to add-value to this waste stream, opportunities for novel sources of bioactive
compounds for health, the potential for reduction of impact on the environment, and improving
the sustainability of shellfish production.
Processing of shells
All waste material generated from seafood processing has to be processed prior to disposal which
presents a challenge for the manufacturer. Shells normally have some residual organic tissue
material present on the shell after sea-food processing. When non-shell containing animal
by-product waste is being disposed of through the food chain in some form, it requires
processing according to animal by-product regulations such as EC Regulation 1069/2009. After
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seafood processing, the shell material usually has residual organic materials attached which can
be removed using different processes, including mechanical or heat treatment, high pressure
washing, enzyme hydrolysis and acid/alkali washing. The process choice depends on the type of
shell and processing site with mechanical and high pressure water washing being the most
common processes due to their ease of operation and cost effectiveness, however this can
generate an environmental waste water issue. The shell waste, once cleaned and verified free of
any other attached material can be used in low-risk product applications such as gardening, land
drainage and roading construction, and as a buffering agent for ponded mine water. The isolation
of proteins and pigments from shells has been developed, but requires investment in technologies
for scaling up the processes.
Chitin, chitosan from waste shells
Chitin is an important abundant natural polymer with annual production of about 2200 tonnes
with a value of about 2 billion US$ [5] and with China currently as the top producer. Chitin is an
oligomer and is composed of α-(1-4)-linked 2-acetamido-2-deoxy-D-glucose [6-9]. Chitin is
produced by crustaceans, molluscs, insects, fungi, yeasts, cell walls and green algae. However,
crustacean shells are the principal sources for commercial chitin [10]. In 2010, capture fisheries
and aquaculture supplied the world with 158 million tonnes of fish, crustaceans and molluscs
[11]. Around 40% of the shrimp weight becomes waste, which is composed of mainly chitin,
lipid, meat offcuts, calcium carbonate and pigments.
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Properties and applications of chitin
The three different polymorphic chitin structures (α-, β-, and γ-) each have a different spatial
arrangement of their oligomer chains. In α-chitin, the oligomer chains are arranged in a parallel
structure and this is the most common type of chitin in nature, especially in shrimps and crab
shells. In squid pen, which is constructed of β-chitin, the oligomer chains are arranged in an
antiparallel structure. -chitin is formed from a mixture of α- and β-chitins and is found in beetle
cocoon and loligo squid stomach [12-14]. α-chitin can be converted to β-chitin chemically by
alkali treatment [13]. It has been reported that chitin with different degrees of deacetylation (DD)
and molecular weight have different antibacterial, water uptake, water retention, fat binding,
solubility, viscosity and biodegradation ability [15, 16]. Chitosan is derived from chitin via
chemical or enzymatic deacetylation processes [17] and so chitin can be differentiated from
chitosan using the DD. Chitin has a DD less than 50% and is not soluble in water which is due
to its robust intra- and inter-hydrogen bonding. On the other hand, chitosan is soluble in dilute
organic acids and is highly biocompatible and biodegradable as well as having good water
adsorption ability. The DD of chitin and chitosan samples can be determined using various
methods such as FTIR, NMR and DSC [18, 19]. There are more than 200 applications reported
for chitin and its derivatives (Figure 1). Chitin and chitosan have been used in agriculture,
veterinary, environmental and food applications [20, 21]. Furthermore, chitosan is widely used
for medical and pharmaceutical implants, and has be used in products that are either ingested or
injected without presentation of any inflammatory issues [22]. Due to its N-acetyl-glucosamine
residues which are found in the extracellular matrix of most living tissues, chitosan has wide
applications in tissue engineering [19]. The highly cationic nature of chitosan has enabled its use
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it many biomaterial applications as a result of the properties of bioadhesion, increased
absorption, transfection efficiency, anti-hypercholesterolemic, anti-microbial, anti-inflammatory
and anti-tumour characteristics (56,57). Chitosan has been shown to exhibit significant
antimicrobial properties that are thought to be the result of its cationic nature disrupting cell wall
synthesis, structure and cellular transport (56). Chitosan has accelerated healing and
biocompatibility properties (56). Chitosan is biodegraded by chitosanases, but as these are absent
in mammals, the majority of degradation occurs by lysozyme. Wound healing properties are due
to macrophage activation and the production of cytokines. The activation of macrophages also
protects against infection (56). Chitosan has excellent biocompatibility with body tissue and
hence no pathological inflammatory response (58). The chitosan material can be moulded into
various structures depending on usage, including microspheres, paste, membranes, sponges,
fibres, and porous scaffolds (12,56).
Chitosan also has wide application in the food industry. Chitosan based films are very effective
in food preservation and chitosan polymer can be synthesized in different shapes such as films,
fibers and beads for packaging of various food products. Additionally, chitosan has antimicrobial
properties against a range of different microorganisms which make it a suitable candidate for
extending the shelf life of food products including fruits and vegetables [23, 24], in addition,
chitin and chitosan based materials have wide application in immobilization of enzymes in
various food industry including wine, sugar and fish industry [25].
Extraction methods
Waste from sea-food contains chitin (20-30%), protein (30-40%), calcium carbonate (30-50%)
and pigments [20, 26] and typically represents more than 50% of the weight of the raw material.
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There is considerable literature on the production of chitin/chitosan from shrimp, crab, crayfish
and scampi shell and from squid pen (see Table 1). Chitin-containing waste from the sea-food
industry is the main source for commercial production of chitin and chitosan. Landfill,
composting and sea dumping are common disposal methods for fish industry wastes; however,
these disposal methods are often environmentally incompatible. Stricter waste management
procedures have been implemented in major production sites which add to the cost of
commercial operation. Several studies have investigated the optimal extraction conditions of
chitin and chitosan from shell waste and examined the functional and nutritional properties of the
extracts. Chitin is located in the cuticles of crustacean shells, where it is complexed with calcium
carbonate, proteins, pigments and lipids [26]. The extraction process can be carried out using
chemicals, enzymes, or by microbial fermentation (Table 1). The conventional method for
production of chitin is a chemical process that uses strong alkali solution (50-55% w/v) for
deproteinization and strong acids such as HCl for demineralization (Figure 2) [27-29]. During
this process proteins are lost and chitin structure is affected [30]. Decolourization is normally
achieved using chemicals such as acetone, chloroform, ethyl acetate, sodium hypochlorite, and
hydrogen peroxide to remove the pigments. Chemical methods have strong environmental
impacts due to the heavy alkali polluted waste water and high energy input [10]. Chitin obtained
by chemical processing has a low quality due to non-homogeneous depolymerisation and
deacetylation. Furthermore, the use of these harsh chemicals results in the protein component
being unusable, that otherwise could be used in other applications [31-33].
Microbial fermentation is an alternative to the chemical process for demineralization and
deproteinization of shell waste and has been used widely over the years for extraction of chitin
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from various shell wastes (Table 1) including crab, prawn and shrimp shells [26, 34, 35].
Fermentation affords the recovery of other valuable compounds in addition to chitin. For
example, several studies have demonstrated the potential for producing high value products such
as proteins and amino acids [36, 37], pigments [38], chitinases [39] and antioxidant molecules
[40] from shell waste, in addition to chitin. A combined microbial fermentation process includes
application of a protease-producing bacterial species such as Serratia or Aspergillus and an

organic acid producing bacterial species such as Pseudomonas or lactobacillus to depolymerize
chitin in shell waste. However, chitin recovery by fermentation suffers from several
disadvantages due to low efficacy, long processing time and relatively high processing cost that
hinders uptake of the technology at an industrial scale [26]. However, the ability to recover
protein and the perception of the method as a green process, because it avoids the use of harsh
chemicals and does not generate significant environment pollution problems, makes
fermentation a promising method worthy of further research to make it more attractive for
industrial applications [26]. The fermentation process is highly dependent on microbial inoculum
concentration, carbon source concentration, pH and reaction time duration [35, 41]. Recent
studies have employed various experimental designs to optimize the extraction yield. Bellaaj et
al, (2011) found that glucose concentration, incubation time and inoculum size were very
significant factors in extracting chitin from shrimp shells using Pseudomonas aeruginosa A2.
The authors found the highest deproteinization and demineralization (89% and 96% respectively)
was achieved using the following conditions; shrimp shell concentration 50 g/l, glucose 50 g/l, 5
days of treatment and an inoculum of 0.05 OD. The same group carried out a similar study in
2013 using Bacillus pumilus A1 and found that shrimp shell concentration could be increased to
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70 g/l under the same conditions as above. This led to 88% demineralization and 94%
deproteinization [42, 43]. Perhaps a combined microbial fermentation using Bacillus pumilus A1
and Pseudomonas aeruginosa A2 could improve the process further. Beaney et al., [44]
compared a biological method involving fermentation using Lactobacillus salvarius,
Enteroccus facium and Pediococcus acidilactici for 5 days reaction at 30°C to a chemical
method involving 1 M hydrochloric acid in a 1:15 solid-to-liquid ratio and a 2h reaction at the
ambient temperature for the extraction of chitin from prawn shell waste. They reported that the
biological method was not as effective as the chemical method and produced a chitin preparation
of a lower quality. Biological methods could therefore be useful for preparing a partially purified
chitin preparation that could be suitable for some use applications.
Fermentation with Lactobacillus plantarum PTTC1058 was found to be very effective for the
recovery of chitin [45]. The authors found that the use of three strains of lactic acid bacteria was
more effective for the extraction of chitin compared to a chemical method. The advantages of
this fermentation method include the following: the organic acid produced by the bacteria is low
cost, the process is environmental friendly, the natural properties of the resultant chitin
preparation are preserved and the organic salts produced during the demineralization process
could be used as anti-icing or preservative agents. Similarly, Das and Ganesh 2010 [46],
compared use of the lactic acid producer strain L. plantarum to the use of HCl at different
concentrations, in a procedure similar to that used by Khanfari et al. [45] and found that L.
plantarium was effective in demineralization process to produce chitin preparations.
The order of deproteinization (DP) and demineralization (DM) steps can play an important role
in the recovery yield and purity of chitin. Wahyutari et al 2011 used Bacillus licheniformis F11.1
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and Lactobacillus acidophilus FNCC116 for DP and DM processes, respectively. The authors
concluded that DM followed by DP resulted in higher chitin recovery and with decreases to
47.37% protein and 50.23% ash compared to the starting material, whereas a DP followed by
DM process decreased to 79.61% protein and 88.65% ash compared to the starting material.
Microbial strains such as Lactobacillus acidophilus SW01 have been isolated from shrimp waste
[47] . Jung et al. (2006; 2007) compared single and co-fermentation of red crab shells using
Lactobacillus paracasei subsp., tolerans KCTC-3074 and Serratia marcescens FS-3. They
showed that co-fermentation was very effective for DM and DP compare to fermentation using a
single microbe; however, protein removal was not still complete. Bhaskar et al. 2007 [48]
investigated the ability of five lactic acid bacterial strains to extract chitin from shrimp waste.
Pediococcus acidolactici CFR2182 was found to be the best strain generating the highest DP and
DM. As the cost of commercially available purified proteases such as alkaline protease, trypsin,
papain, and pepsin is high, the use of protease producing bacteria has advantages for chitin
extraction.
Extraction of calcium compounds from biogenic resources
Shells provide the soft body tissues of host animals with protection against the external
environment. Shells consist mainly of calcium carbonate and small amounts of organic materials
(Table 2). Marine shells use amorphous calcium carbonate to build a crystalline phase [49].
Marine shells and corals have evolved specific structures to provide a suitable habitat for the host
animal tissues to proliferate and differentiate. Calcium carbonate has been used since earliest
civilisation and has various food (food preservative and color retainer) and industrial applications
(building materials, limestone or precipitated calcium carbonate) that are still recognised in
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modern times. Calcium carbonate is nontoxic, has high porosity, and high surface area to volume
ratio that makes it beneficial for many industries. Calcium carbonate exists in four main
polymorphs: calcite, vaterite, aragonite and amorphous calcium carbonate. Aragonite is the main
polymorph in marine shells and exists in a needle-like arrayed structure. In the following section,
a summary of major applications of shell waste includes; applications in tissue engineering;
antibacterial and antifungal agents; catalyst oil and fat; and treatment of wastewater will be
discussed with emphasis on the properties required for each application.
Use of shell waste as potential calcium supplement
Sea shells are an important source of by-products that can be used in the production of inorganic
calcium with application in food industries (e.g. milk fortification, supplements). Sea shell is a
proper source of calcium carbonate for feed application. It has been reported that calcium
obtained from oyster shell [50, 51] and mussel shell [52] has been widely used as a supplement
in the poultry industry. Muir et al 1976, evaluated granular limestone, aragonite, oyster shell,
clam shell and egg shell as feed supplement for laying hens and found clam shells to be a
suitable calcium source for the hens [53]. In another study, Finkelstein et al. found clam shells as
effective Ca supplements for lactating dairy cows [54]. It has been reported that shrimp waste is
a prominent source of calcium (3000mg/100g) and phosphorous [55], however, few studies have
dealt with the possible recovery and its application. Shono et al., fed rats with nacre powder
(mother of pearl, from Pinctada maxima) and evaluated its effect on reducing fat accumulation
and high blood triglycerides. The authors reported that body weight was reduced as well as the
amount of fat and the level of triglycerides without detrimental effect on body size, food intake
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or muscle tissue [56]. Furthermore, hydroxyapatite obtained from sea shells and fish bones also
can be used as supplement, and its bioavailability has been suggested previously [57].
The effectiveness of hydroxpatite in controlling and preventing bone loss has been documented
in several studies. HA has been compared with calcium carbonate in prevention of bone mass
loss and it has been suggested that HA was more effective in controlling bone loss in
postmenopausal women, and generally HA had a greater anabolic effect on bone than calcium
carbonate [58, 59]. Additionally, in studies by Castelo-Branco et al. [60, 61], a decrease in bone
mass was observed in subjects treated with calcium carbonate, while the bone mass of a patient
treated with HA did not decrease after 24 months of treatment, and HA was more effective than
calcium supplements in maintaining bone mass. Hydroxyapatite has also been suggested to be
more effective than tricalcium phosphate in the prevention of bone loss [62]. Moreover the
hydroxyapatite crystals can be modified and substituted with different elements such as
magnesium [63], citrate [64], zinc [65] and silicate [66] to enhance its biocompatibility and
biological response.
Shells comprise 83 percent of the waste produced by shellfish processing industries and mainly
consist of calcium carbonate, and its current applications are limited to low value products for
roads, mining industry etc. Although calcium enriched food can potentially help consumers to
achieve required calcium intake, it is necessary to evaluate the adsorption of Ca derived from
different sources (such as sea shell and fish bone) in comparison with available commercial
products for human consumption [67]. Shells contain about 5% organic materials, which need to
be considered when shell extracts are planned to be used as a food supplement [68]. The
elemental concentration of black oyster (Pinctada margaritifera) shell was evaluated by Chang
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et al., and they reported that the measured concentrations are lower that the safety limit
recommended for human consumption [69]. The bioavailability of calcium salts can influence
how effective they are for treating osteoporosis. In a study by Fujita et al., oyster shell
electrolysate (OSE) was used as a supplement (900 mg/day) for elderly osteoporotic females and
reported that radial bone mineral density significantly increased after 12 and 24 months in
subjects given OSE compared to a control group [70]. In a separate study [71], compared the
effects of heated oyster shell-seaweed calcium (AAA Ca), calcium carbonate, and a placebo in
58 elderly females, and reported AAA Ca to be an effective supplement for increasing bone
mineral density in elderly subjects. Despite this, Ross et al. [72] reported that calcium
supplements sourced naturally such as from oyster shell contain a high amount of lead, and in a
related report, Palaniappan et al. [73], observed that oyster shell calcium supplement might
induce parotid swelling .
Tissue engineering applications
The calcium carbonate component of shells can be converted to calcium-based compounds
suitable for biomedical applications. The major mineral component of bone is calcium phosphate
(CaP) [74, 75]. Bone is a complex matrix composed of extracellular matrix (ECM) (90%) and
water (10%). The ECM consists of organic and inorganic materials, in which the inorganic
component confers strength and stiffness while the organic component makes it flexible against
stress and pressure. The inorganic component contributes about 60-70% of the ECM. The
remaining 30-40% organic component consists of 90 % type Ӏ collagen and 10%
non-collagenous proteins such as glycosaminoglycans. Hydroxyapatite (HA) and beta tricalcium
phosphate (β-TCP) have similarity to the mineral phase of the bone. Therefore, synthetic apatites
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for use in bone graft repairs have been of interest for decades [76]. HA is a crystalline form of
calcium phosphate with the chemical formula Ca10[(PO4)6(OH2)] (29). Naturally produced HA
makes up the majority of the mineral phase of bone (37). As synthetic HA has a similar chemical
makeup to human hard tissue it has a favourable chemical and biological affinity to bone tissue
(37,38). Due to these similar chemical properties with mineralized bone matrices, synthetic HA
therefore provides excellent osteoconductive, bioactive and biocompatible properties (29,36,38).

HA can be obtained directly or can be chemically synthesised from marine shell waste (Table 3).
This research area has been an interesting field due to some special characteristics of the material
produced from some of these sources, as well as due to economic and environmental benefits
obtained from adding-value to waste and its reduction [77-79]. Apart from the economic benefits
of converting low cost waste to a value-added product, HA produced from biogenic resources
contains valuable trace ions and is suggested to have better biocompatibility than synthetic HA.
Although HA has good cytocompability, it has some disadvantages, including low solubility at
physiological pH and different mechanical properties from bone [36]. Despite such
disadvantages, HA is widely accepted as a biomaterial for bone regeneration (37).
Nano-hydroxyapatite (nHA) is stable at physiological temperature and pH, and is poorly soluble
in water. Additionally, nHA has a high hardness, is bioactive, is not cytotoxic and is also
biocompatible with skin and muscle. The use of nHA in bone grafting results in rapid bone
regeneration and a solid biological fixation to bone due to its osteoconductive and bioactive
properties (33). nHA has a similar structure to the minerals found in bone and is thought to
enhance the mechanical and osteoconductive properties of implants. Compared to
micro-hydroxyapatite (µHA), nHA has a high surface area to volume ratio that could control
protein interactions (adsorption, configuration and bioactivity) and therefore enhance osteoblast
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adhesion (37). Tri-calcium phosphate (TCP) is also a biocompatible crystalline structure like HA
with the formula Ca3[(PO4])2. Its structure is similar to the mineral phase of bone with higher
degradation rates compared to HA, which occurs by dissolution and resorption by osteoclasts.
Osteoconduction is improved by the small particle size of TCP and an interconnected
sponge-like matrix is formed that allows fibrovascular invasion and bone production (29,36).
Calcium phosphate cements are also available that offer the advantage of bonding the bone graft
to the natural bone surface. The disadvantages of cements include the requirement for close
contact with bone for osteoconduction and a lack of a porous matrix for tissue invasion (36). In
the following section, various methods for conversion of shells to calcium-based materials for
biomedical application are discussed.
Methods for the conversion of shell material to HA
The aim of bone tissue engineering is to produce materials that mimic natural bone, and so the
developed biomaterials need to have a similar chemical composition and structural requirement
to that of natural bone. The major component of hard tissue in all animals is composed of
calcium phosphate arranged in a micron to nano size pore structure. Some marine shell structures
have excellent micro structure which is porous and interconnected, with the required elemental
composition.
The application of natural coral as potential bone grafts dates back to 1970s. Coral natural
structures have a similar structure to cancellous bone and are also osteoconductive and
biodegradable [80]. Furthermore, they are good carriers for growth factors and provide a suitable
surface for cell growth and proliferation [80]. However, the declining resources of coral in the
world make this material less attractive for use in the future. In addition to calcium and
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phosphate, the presence of minerals such as Mg, Sr and Si found in sea shells also has been
reported to improve the biocompatibility of the produced implant and its acceptance by the host
bone structure.
Calcium carbonate originating from marine shells usually exhibits characteristic porosity and
interconnectivity similar to that found in human bone [81, 82]. Sea shells are composed mainly
of aragonite. This calcium carbonate form can be converted hydrothermally (140–260°C) to
hydroxyapatite (HA) using a phosphate solution by a solid-state topotactic ion-exchange reaction
(equation 1) in which the HA retains the orientation of the original aragonite structure [83, 84].
The following equation summarizes the process
10CaCO3 + 6(NH4)2HPO4 + 2H2O Ca10(PO4)6(OH)2 +6(NH4)2CO3 + 4H3CO3 (1)
Aragonite from marine sources has tailored architectures with inherently high mechanical
properties and with potentially desirable porous structure. For instance, seashells have dense
lamellar structures, while coral, cuttlebone and sea urchin spines have porous structures.
Preservation of these unique properties during processing can be important for end-use
applications. Some marine shells, such as red algae [85], sea urchin spines [86], conch and clam
shells [87] have structures that have a specific interconnected pore morphology and mechanical
properties that make them suitable for biomedical applications. Considering the successful
clinical application of coral structures [88, 89], several studies have explored other marine
sourced materials for tissue engineering applications, including marine sponges, sea shells, and
cuttlefish pens. In these studies, researchers were able to preserve the natural structure, porosity
and pore size of the materials that were used for cells to proliferate and grow. A range of
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different techniques have been used for production of calcium phosphate materials from marine
structures (Table 4). Hydrothermal processing methods are the most used techniques that
maintain the shell structure during formation of HA from aragonite. Marine shell structure can
generally be used in biomedical applications in two distinct ways. The first is a direct method
where the natural marine structures can be used directly as a template for cells to proliferate and
generate a new tissue. In this direct method, the microstructure of the shells is preserved using a
hydrothermal processing method. This method is used when the marine shell has a natural
porous, interconnected microstructure, with a relatively high surface area. In the majority of
conversion methods, and depending on the type of shells, the organic content of shell needs to be
removed, however, when the morphology of the shell needs to be preserved, special attention is
required during the removal of organic materials in order not to damage the structure. Therefore,
In this method, first the organic materials are removed using either heat treatment [85, 87] or
chemicals (e.g. NaClO) or in the case of conch and clam shell no organic removal steps are
deemed necessary [87]. With heat treatment the time and temperature required for the removal of
organic materials depends on the shell structure and particle size. Use of temperatures of up to ~
750°C have been reported [85, 90]. Higher temperatures may weaken the structure of the shell,
as at 700 to 800°C, decomposition of CaCO3 to CaO occurs. After removal of organics,
depending on the proposed conversion process, HA can be formed in the shell in either
crystalline or amorphous form. If the aim is to produce a crystalline structure Vecchio et al. [87],
for example, converted conch and calm shell to H using an autoclave with a temperature range
of 180-240 for up to 20 days. They did not use any sintering step and found that clam shell
material was completely converted after 10 days at 200ºC. The original structure of the material
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was preserved and the converted shell samples had good mechanical strength, and the average
fracture strength of the product was 71 MPa, close to that of compact human bone. The
conversion process was enhanced at higher temperatures up to 240 , and the thickness of the
HA layer was increased with increasing conversion time [91]. In a similar way calcitic sea urchin
spine was converted to apatite at 180 °C in 24 h [92]. However, if the aim is to convert shell
material partially to HA without the requirement of a crystalline structure, then lower

temperatures and pressures for a shorter time can be used.
Other materials, such as mineralised red algae have also been used to prepare HA. Walsh et al.
[85] converted red algae to hydroxyapatite under atmospheric pressure using a heating step. The
authors removed organic matter by heating at 700°C for 12 hours, followed by a stirring step in
phosphate solution ((NH4)2H2PO4) and a hot plate at a temperature of 100°C for 12 hrs. pH
adjusted to 10-11 using NH4OH to enhance the crystal growth.
The authors’ successfully converted mineralised red alga to hydroxyapatite while maintaining
the natural structure intact [85, 90]. Additionally, some studies have performed a post reaction
sintering step at up to 1000°C in a furnace to complete the conversion to HA [93]. In another
study, heating on a hot plate stirrer at 80-100°C for 2-8h was used [93, 94] and various calcium
phosphates with different purities were produced over the duration of the thermal treatment, such
as tricalcium phosphate, brushite, calcite and HA. Using this method, pure hydroxyapatite is
normally obtained after sintering at temperatures above 1000°C. The pH of the phosphate
solution plays an important role in the conversion process. Having the starting solution of the
material at high pH around 11 has the benefit of preventing the carbonate form of HA being
generated [95]. At high pH the increased hydroxyl ions in the solution enhance HA formation
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through formation of an octacalcium phosphate phase [96, 97]. In a study by Vecchio et al. [51],
spines from the sea urchins Heterocentrotus trigonarius and Heterocentrotus mammillatus were
converted to bioresorbable Mg-substituted tricalcium phosphate (β-TCMP) by a hydrothermal
reaction at 180°C. The three dimensional interconnected porous structure of the original spine
was preserved and an average fracture strength of 23 MPa was recorded for converted spines
(β-TCMP) in compression tests [86]. This technique however, is not universally suitable to all
marine shells. Zaremba et al [83] found that bivalve and cephalopod shell was converted into
HA powder using the above processing conditions while gastropod shell was able to retain its
structure during the HA conversion process [83]. It is documented that the conversion process is
accelerated at higher temperatures, and the thickness of the HA layer increases with increasing
conversion time [83, 98]. Vecchio et al., in another study, converted conch and clam shell to
hydroxyapatite (HA) by hydrothermal processing. The converted conch and clam shell had very
high fracture strength, of 218 and 150 MPa respectively [87]. HA produced from sea urchin,
conch and clam shell had good biological compatibility and osteoconductivity [86, 87]. The
physical properties and bone regeneration ability of HA from shell (sHA) and eggshell (eHA)
were studied recently by Lee et al. [99]. The authors found that the morphology and
characteristics of both HA were similar to pure HA, however higher levels of sodium and
strontium were found in sHA, and eHA had higher magnesium. After 4 weeks of implantation,
sHA had higher mineral density but over the same time eHA caused less inflammation. The
authors concluded that eHA was better than sHA for bone formation than in their study [99]. The
second method is indirect, in that it is based on the conversion of the shell structure to calcium
compounds and restructuring the compounds into a bioscaffold using different organic materials
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such as collagen and chitosan. This method has been used when the shell is non-porous and has
no porous structure as with mussel shells, and the shell structure does not play an important role
in the final properties of the HA product. In this method, shell material is milled and crushed
either before or after pyrolysis, and the organic components are removed either by heating [93]
or with chemicals such as acetic or hydrochloric acid [100]. In this method shell material is
heated to ≥ 900° to remove all organic materials. Then the aragonite is partially or completely
converted to calcium hydroxide which is then reacted with a phosphate source under defined
temperature and pressure conditions to produce HA crystals. In some cases, the heating step can
be replaced by chemical treatment, such as reaction with HCl or HNO 3 to produce calcium
nitrate or calcium chloride [101]. With regard to the phosphate source, Boonyang et al.
compared three different phosphate precursors (NH4)2HPO4, Ca3(PO4)2, and H3PO4, in a high
sintering temperature method and found that only (NH4)2HPO4 and Ca3(PO4)2 were suitable for
the production of monophasic HA [102, 103]. The HA obtained using this method had low
mechanical properties, since the structure was not preserved due to the effect of high sintering
temperature and consequently it was not suitable for load bearing applications. In a recent study,
sea coral was dried, heated at 900°C for 2h, then soaked in phosphate solution and autoclaved at
150°C for 8 days followed by heat treatment at 1250°C [104]. The processing conditions used
resulted in a hydroxyapatite material with a high degree of crystallinity (87%), however, the high
temperature that was used for removal of organic materials weakened the structure and the
compression strength of the final product was only 2 ± 0.5 MPa. In addition to the hydrothermal
HA conversion process, a number of other methods are available, such as microwave and sol gel
processing for production of HA from shell materials without preserving their structures.
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Microwave irradiation can be used to accelerate the reaction. Microwave energy provides a
stable and uniform heating of the mixture that prevents the formation of a temperature gradient
with the material. This uniform heating process enhances the homogeneity of the material and
increases the thermodynamics and kinetics of the reaction and therefore reduces the time (usually
< 30 minutes) required for crystal formation, and produces nano-sized crystals. Microwave
heating is a more energy efficient method compared to conventional heating [105]. Microwave
energy can be used to accelerate HA production in three different modes, continuous refluxing,
hydrothermal-microwave assisted processing and conventional microwave processing.
Furthermore, there have been some attempts to improve hydrolysis and combustion with
microwave heating. In this regard, some recent studies applied microwave treatment under reflux
conditions using EDTA as the chelating agent for production of HA [106, 107]. In all instances,
either HA nano powder or nano strips were obtained with particle sizes of less than 100 nm with
different morphologies (rod shape, flower like, elliptical, bowknot) depending on the treatment
conditions (time, temperature and pressure). EDTA plays an important role in the formation of
crystal shape and morphology as a result of the formation of EDTA-Ca complexes [108-110].
The pH of the treated material has a significant effect on the stability of the EDTA-Ca complex
and the final form of the HA structure. The high concentration of hydroxyl ions available at a
strong basic pH of 12-13 creates a high specific surface energy that enhances the adsorption of
hydroxyl ions on the crystal planes [111]. Due to its high ionic interaction, the surface hydroxyl
groups act as the active sites for adsorption of Ca-EDTA complexes, and the Ca-EDTA becomes
a part of the obtained crystallite. During further microwave heat treatment Ca-complexes are
decomposed and HA crystals are formed [77]. Additionally, EDTA plays another role as a
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combustion agent. The combustion synthesis method is based on a self-sustained heating
generated from an exothermic reaction where once the reaction is started at the ignition
temperature, the reaction itself becomes a heating source that enhances the temperature of the
reaction [112]. During microwave heating, the authors reported the occurrence of sparks at the
end of process, and these intermittent sparks were taken as evidence of combustion caused by
EDTA superheating [113]. Microwave irradiation has the advantage of enabling this combustion
process that does not occur with conventional heating. Considering that nucleation and particle
growth depends on heat distribution, the inhomogeneous heat distribution of conventional
heating results in HA particles of various sizes unlike that achieved with microwave heating.
Microwave irradiation can be carried out under normal atmospheric pressure until complete
drying of the HA product is achieved [114-116]. A nano crystalline HA with uniform shape and
size has been reported to be produced in a short time [117]. The hydrothermal assisted
microwave method is the same as the conventional hydrothermal method [118] except that the
reaction vessel was a polymeric reactor rather than stainless steel. Gypsum powder was reacted
with (NH4)2HPO4 solution under hydrothermal microwave reaction conditions and nano rods of
5–50 nm in diameter and 30–300 nm in length of crystalline HA were obtained after 5 min
reaction [119]. Isolated mature apatite crystals have been studied with transmission electron
microscopy and have been clearly shown to be platelet-like, with an average length range of 50–
100 and width range of 25–50 nm, and the deposited crystals were 4–6 nm in thickness [120].
Therefore, microwave assisted production of HA is a promising heating method to achieve HA
crystals with desired shape and size that would suit biomedical applications. It has been reported
that the microwave power used and the ratio of calcium to phosphate are important parameters in
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defining the final properties of the HA produced. Low microwave power of less than 450 W
resulted in a low purity HA that contained Ca(OH)2, CaHPO4, while at 550 W a single phase HA
was obtained [77]. Sol-gel is another process which has been used for more than a century for
ceramic production. In this process, the 'Sol' component is a dispersion of colloidal particles, and
the 'gel' component is a polymer with interconnected structure. This method does not need an
elevated pH or a high temperature and it can be used to produce different forms of calcium
phosphate-containing powders and platelets by changing the chemistry and other parameters of a
given solution. The high reactivity of the sol gel system removes the need for high temperature
or high pH [121]. Additionally, a high purity, homogeneous nano-crystalline HA with a different
crystalline morphology can be obtained by slight modification of the reaction chemistry or
process conditions [121]. In this method, precursors are mixed at a molecular level, which
improves the homogeneity of the final product. However, the high cost of production and
inconsistency of the products are drawbacks of this method [122]. Briefly, this process starts
with making of the sol and after molecules are cross-linked during an aging step, the cross
linking is developed and increases. Finally, by drying and sintering the excess solvent and
residual organics is removed, and crystal structure develops. The crystallinity of the product can
be adjusted by variation of the calcium to phosphate ratio [123]. It has been reported that
addition of sucrose makes the solution more viscous and prevents precipitation of the ions and
retains the homogeneity of the solution [124].
Marine shell waste as an adsorbent of phosphate, metal ions and dye
The use of shell material in waste management to remove phosphorous, toxic metalic ions, and
adsorption of toxic dyes from waste water can contribute to preserving marine ecosystems and
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assist with waste discharge problems [6-8]. The use of alkali sorbents such as calcium carbonate,
calcium oxide and calcium hydroxide for removal of acidic gases including sulphur dioxide has
been well documented [125, 126]. Considering that hygroscopicity and cost of sorbent are very
important factors, calcium based materials with high hygroscopicity and low price are highly
promising commercial adsorbents [4]. The use of shell material for the absorption of metallic
ions, acids and gases is economical and also is important for waste recycling [4]. Onoda et al.
[84, 85] evaluated the Corbicula, a Japanese littleneck shell, and oyster shell for the production
of calcium phosphate with the objective of recovering lanthanum cations from waste water. The
authors concluded that calcium phosphate samples prepared from Corbicula shell had the same
binding capacity of rare earth elements comared to that of calcium phosphate prepared from
commercial calcium carbonate. Generally, oyster shell produced lower yields of calcium
phosphate than corbicula shell [127, 128]. Oyster shell is an industrial waste generated by the
shellfish processing industry, that has attracted very limited use compared to limestone. Oyster
shell would therefore be attractive for use in the above application, and. furthermore, oyster
shell, even without calcination, is reported to have a high desulfurization efficiency [4]. The
efficiency of element removal is dependent on the shell particle size. Jeon and Yeom [86] used
crab shell for removal of phosphate and the authors concluded that with a shell particle size of
less than 1000 µm, more than 85% of phosphate can be removed in 24 hours from a 500 mg/l
phosphate solution, whereas larger shell particles of 3350 µm removed only 50% of the
phosphate. The process is more effective under acidic conditions at pH 2, and temperatures
between 15-45°C had negligible effect on the removal efficiency. Under optimum process
conditions, crab shell can remove 108.9 mg of phosphate per 1 gram of shell, which is around 5
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times higher than scallop shells. This better performance of crab shell compared to scallop shells
is due to its higher organic compound content containing functional ions that play a role in
binding phosphate. The possibility of using shell waste to remove dyes from waste water was
investigated by a number of researchers (Table 5). Haddad et al. [129, 130] used calcined mussel
shell to remove Rhodamine B, Alizarin Red S and Orange II textile dyes and safranin from waste
water. Calcined mussel shell exhibited higher adsorption capacity (196.67 mg/g) compared to
sugar pulp (147), rice husk (178.09) and calcined bone (107.76). Furthermore, bivalve shell
powder was reported to have a higher Basic Green 4 dye adsorption capacity (42.3 mg/g) than
many other adsorbents such as bentonite clay (7.72 mg/g) [131], marine algae (26.7 mg/g) [132]
and activated charcoal (0.179 mg/g) [133] [134]. In addition to calcium-based shell material that
has been used for pollution removal, chitosan obtained from shell waste also has demonstrated
good adsorption capacities. In a recent study by Shaheen et al. [135] the authors examined the
Cd, Cu, Pb and Zn metallic ion removal capability of chitosan (CH), egg shell (ES), potassium
humate (KH), and sugar beet factory lime (SBFL). Chitosan demonstrated the highest removal
efficiency. Due to the high electronegativity and lower pK values of Pb and Cu, these minerals
can be adsorbed better on chitosan compared to Cd and Zn. The dye sorption capacity of
chitosan has also been reported by Daneshvar et al. [135]. The authors reported that the shrimp
Penaeus indicus shell is a good biosorbent with high adsorption capacity of 645.2 mg/g for
removal of Acid Blue 25. Considering that Acid Blue 25 is an anioinic dye, it is not clear
whether chitosan can be also a useful sorbent for cationic dyes, as most industrial effluent is
normally a mixture of various pollutants. Most of the experimental studies have used a single or
a combination of minerals in their studies without consideration of the impact of organic
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components in normal waste water. It would be of interest to examine the effects of the
interaction of various pollutants such as metallic ions, dyes and organic components on the
removal efficacy of bio-sorbents within the same study. In most of the studies investigating the
sorption of pollutants onto marine shell material, sorption capacity had a direct relationship with
the waste water pH, adsorbent dosage, temperature and particle size, and the adsorption followed
a pseudo-second order kinetics. Despite that many studies have indicated that waste natural
materials such as shell material are a good source of adsorbent for the removal of pullutants such
as metallic ions and dyes, but most of these studies have usually been conducted in batch mode
which is normally expensive to operate and would not be a viable option for industries requiring
flow-through pollutant removal. In addition, the desorption capability to regenerate the adsorbent
is an important consideration in order to reuse the adsorbent. Furthermore, the pH adjustment
employed in many small scale studies may not be practical for industry due to use of expensive
chemicals. Moreover, the effect of adsorbent particle size and problem related to adsorbent
sedimentation also has to be considered.
Use of shell waste as catalyst agent
Oils and fats that are not suitable for human consumption (vegetable oil from inedible plants,
animal fat drippings and waste cooking oil) can be used for biodiesel production. Due to their
high viscosity, vegetable oils and animal fats usually cannot be used directly as fuel as they are
not suitable for combustion, have poor atomization and can lead to functional problems in an
engine. In order to meet the required standards such as ASTM D 6751 for Biodiesel [136] and to
overcome the problems mentioned above, the molecular weight of the oil needs to be reduced
through conversion of triacyglycerols to long chain [C16-18] fatty acid monoalkyl esters that
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have reduced viscosity and can be used as a clean fuel in diesel engines [137]. In general, four
different methods have been suggested for processing the feedstock, involving
transesterification, pyrolysis, micro-emulsion and dilution with petroleum diesel, of which
transesterification is the most wildly used and investigated method. During the esterification
process, oil is reacted with alcohol in presence of a catalyst (e.g. NaOH, KOH) and glycerine is
separated from the product methyl esters which can be used as biodiesel (Error! Reference
source not found.). The biodiesel fuel produced from this process can be blended with
conventional diesel and used in diesel engines without further modification [136]. Waste shell
material can be converted by heat treatment to calcium oxide, a metal oxide that can be used as a
catalyst for production of biodiesel. For this purpose, different temperatures have been used and
reported (Table ). The conversion of calcium carbonate to calcium oxide normally occurs at
temperatures > 700°C and the generated calcium oxide can be used without any further
modifications. The catalyst concentration, reaction time and reaction temperature are important
factors in the esterification process. Boey et al [138, 139] investigated the optimization of the
esterification process of palm olein using response surface methodology. The authors suggested
the catalyst (calcium oxide from waste Scylla serrata shell) has a positive influence on purity of
the methyl esters but had a negative effect on the yield. The waste catalyst performed similar to
laboratory grade CaO and the catalyst could be reused up to 11 times. Recently Boro et al. [140]
examined the effect of modification of catalyst. In this study barium doped CaO derived from
waste Turbonilla striatula shell was used as a heterogeneous base catalyst in the
transesterification of waste cooking oil (WCO) at 1% and achieved a 98% conversion yield in
the process. Several studies have applied a higher ratio of diverse catalysts to improve the yield
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(Table ). The inclusion of barium increased the alkalinity of the CaO derived from the waste T.
striatula shell and an increase in transesterification conversion with the barium loading was
attributed to the presence of both CaO and BaO, as the catalytic species during the reaction
[140]. In another study by Xie et al. [106] Biont shell was incompletely carbonized and
impregnated with potassium floride. The transesterification yield of rapeseed oil to make
biodiesel reached 97.5% within 3 hours of reaction time with 3% catalyst dosage. It was
suggested that reaction of incomplete carbonized Biont shells with KF produced a catalysis site
for transesterification [141].
Use of shell waste as antibacterial and antifungal agents
Calcium carbonate is the main component of shell waste. Upon heat treatment of calcium
carbonate at ≥ 700° it is converted to calcium oxide that has antibacterial activity (Table 7)
[142, 143]. The mechanism of antibacterial action is related to the alkalinity of the
environment and production of reactive oxygen species that cause bacterial inhibition. Li et al.
[95] investigated the antibacterial activity of mussel shell powder doped with silver particles.
The authors concluded that the antibacterial mechanism was mainly due to Ag+ and the high pH
value exerted by the mussel shell powder. Particle size is an important factor for the antibacterial
properties of heat-treated shell powder [143, 144]. Watanabe et al. found that nanoparticles had a
much higher antibacterial effect compared to microparticles [143] and they decreased the number
of E.coli after a 60 second treatment at all of the concentrations (0.1, 0.25 and 0.5 mg/ml) tested,
and to a greater extent (1 more log10 CFU/ml) than the micro particles. Sawai et al [145, 146]
evaluated antimicrobial (against coliforms), antifungal (Trichophyton mentagrophytes
NBRC5466) and sporicidal action (Bacillus subtilis spores) activities of heated scallop shell
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powder. The antimicrobial activity of scallop shell powder increased as the concentration of the
powder increased up to the solubility limit of Ca(OH)2. Scallop shell heat-treated powder
(1000°C for 1 h) was able to reduce the total count of bacteria in shredded cabbage with the
antimicrobial activity increasing with increasing powder concentration and treatment
temperature over the range of 0.1 to 1.0 g/dm3 and 10 - 40°C, respectively. Complete inhibition
of viable coliforms was obtained within 5 min using Ca(OH)2 at 0.1 g/dm3. Heat-treated scallop
powder resulted in a 20-30% reduction in the L-ascorbic content of cabbage which was similar to
that obtained by the use of a 200 µg/dm3 sodium hypochlorite treatment. In addition to the
antimicrobial properties of the heat-treated scallop shells, antimicrobial activity has been
reported for a polyhydroxylated naphthoquinone pigment extract obtained from the shells of
Salmacis virgulata ( MI = 100 μg/ml) [147] and from shell powder extracts of the mollusc
Donax faba [148].
Pigments
Echinochrome and spinochrome in sea urchins
Sea urchin species are coloured differently ranging from purple to brown and red, which shows
that the shell and spines contain various pigments. The pigments present in the shell (test) and
spines of sea urchins are mainly naphthoquinones, principally echinochrome and spinochrome
depending on their original sources [149]. The sea urchin quinones are derivatives of either
juglone (5-hydroxy-1,4-naphthoquinone) (Figure 3, structure 1) or naphthazarin
(5,8-dihydroxy-1,4-naphthoquinone) (Figure 3, structure 2) with additional hydroxy, ethyl,
acetyl, methoxy and amino substituents [150]. The first identification of naphthoquinone
pigments was made by MacMunn in 1883 [149]. Since then, more than twenty naphthoquinone
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pigments have been identified from sea urchin [149, 151-153]. Spinochromes are classified by
letters (Spinochromes A–E) [149] (Figure 3, structures 3-8).
The majority of species examined so far contain up to six spinochrome pigments which are
predominantly echinochrome and the spinochromes A, B, C, D and E. [149], as well as other
minor components identified more recently. The structures of all known spinochromes are shown
in Figure 3 (structures 3-30). Anderson [149] summarized 19 different naphthoquinone pigments
which were identified across fifty-four species of echinoids. Some novel pigments have also
been found. The UV–Vis absorption spectra and structural elucidation of all identified
naphthoquinone pigment structures are shown in Table 8.
Mathieson et al. [154] found that the spines of Diademaantillarurn were mainly pigmented with
6-ethyl-2,3,7-trihydroxynaphthazarin (Figure 3, structure 3) and its 2- and 3-mono-methylethers.
Temnopleurustoreumaticus contains 3-acetyl-2,6,7-trihydroxy juglone (Fig.1, structure 13) and
six other known spinochromes. Spatangus purpureus has two common naphthazarins and two
novel binaphthaquinones, ethylidene-3,3'-bis(2,6,7-trihydroxynaphthazarin) (Figure 3, structure
26) and its anhydro-derivative (Figure 3, structure 25). In 2005, two new spinochromes,
echinamine A (Figure 3, structure 22) and echinamine B (Figure 3, structure 23) were isolated
from Scaphechinus mirabilis [151]. Echinamine A was extracted and obtained as a dark brown
powder, while echinamine B was extracted and obtained as dark brown needles. The 13C NMR
and IR spectra of echinamine A and B showed that they are isomers of
5,8-dihydroxy-1,4-naphthoquinone having an ethyl radical, an amino, and two α-hydroxyl
groups located in different positions [155]. Shikov et al. [156] used HPLC-DAD-MS analysis to
show the presence of six pigments in Strongylocentrotus droebachiensis including spinochrome
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B and D, and three spinochrome dimers: ethylidene-6,6’-bis (2,3,7-trihydroxynaphthazarin)
(Figure 3, structure 26) anhydroethylidene-6,6’-bis (2,3,7- trihydroxynaphthazarin) and its
isomer (Figure 3, structure 25). One additional pigment was preliminarily identified as a
spinochrome dimer with the structural formula C22H16O16. Zhou et al. [157] reported two new
compounds that were tentatively identified as aminopentahydroxynaphthoquinone and
acetylaminotrihydroxynaphthoquinone in Strongylocentrotus nudus. The molecular formulas for

these two compounds were deduced based on their molecular mass. The compounds were
identified to be aminated PHNQ, but the molecular mass and molecular formula of these two
compounds did not match with any known compounds reported previously [155]. Therefore,
these two compounds possibly were new PHNQ. Very recently Powell et al. [153] using LC-MS
identified several pigments in Psammechinus miliaris that had been reported earlier except for a
new sulphated spinochrome E sulphate that was identified.
Extraction, purification and identification method
PHNQ pigments are present in the calcified skeleton consisting of shell and spines of sea
urchins. To facilitate the extraction of the pigments, the shells and spines are usually dissolved in
an aqueous solution of HCl or H2SO4 for decalcification and then the pigments are extracted
using organic solvents such as diethyl ether [156], chloroform [155], ethyl acetate [153] or
butanol [158]. The solvent extract of sea urchin pigments is then washed with an aqueous
solution of NaCl to remove acid and then dried with anhydrous sodium sulphate followed by
freeze drying. [156]. Powell et al. [153] used solid phase extraction (SPE) as a clean-up step for
the pigments extracted from Psammechinus miliaris. The LC-MS profiles of the SPE extraction
and the original acid extracts were similar but the SPE step generated more reproducible data.
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Some alternative methods have been investigated. Zhou et al.[157] obtained a crude pigment
extract from ground spine powder of the purple sea urchin using an ethanol-HCl-aqueous
solution, followed by use of a macroporous resin to further purify this PHNQ pigment. This
method achieved a higher yield [159] compared to using only an organic solvent extraction
process. Silica gel column chromatography and acid-treated thin-layer chromatography (TLC)
have been used to analytically separate pigments from sea urchin shells [149]. Chromatography
with a Sephadex LH-20 column was used for separation and purification of the pigments from
Strongylocentrotus franciscanus [160]. However, methods such as these have low resolution and
efficiency. The utilization of HPLC and UPLC allowed for faster analysis with better resolution
and hence gave a more detailed identification of pigments. HPLC-MS or LC-MS are stable and
reliable methods to identify the structures of the PHNQ pigments. A summary of the HPLC
conditions reported in the literature is shown in Table 9. Shikov et al. [156] successfully used
TLC for the fractionation of sea urchin pigments and further separated and identified the
pigments with HPLC-DAD-MS. Compared to HPLC, UPLC has better resolution, sensitivity,
and speed. PHNQ pigments from S. nudus spines were extracted by macroporous adsorption
resin and were identified tentatively by using UPLC/Q-TOF MS [156].
Characteristics of polyhydroxylated naphthoquinones
Autoxidation of chemical compounds is a relatively slow, radical-catalysed process that proceeds
via a chain reaction [161]. Using 1H- and 13C-NMR and IR spectroscopy it was concluded that
there was only one final product of echinochrome A autooxidation, a monohydrate of
naphthotetraketone, which was the same product generated during the reaction of echinochrome
A with O2 [161].
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The reaction scheme for the autoxidation of echinochrome A was proposed:
H5Nq+O2 H4Nq • +O2 •- +H+ (1)
H4Nq•+O2 H3Nq +O2 •- +H+ (2)
H5Nq+O2 • +H+ H4Nq • +H2O2 (3)
H4Nq•+ O2 • +H+ H3Nq+ H2O2 (4)

2H4Nq• H5Nq+ H3Nq (5)
As PHNQs have several acid dissociation constants, PHNQ will be present in either the
non-ionised or various anion forms in aqueous solution at different pH. The state of ionisation of
PHNQ can affect their colour and stability [162, 163]. When the pH was changed from 2.0 to
10.0, the colour of the pigment changed from orange-red to deep-earth-yellow, corresponding to
a change from non-ionised to various ionised forms as a result of the dissociation of β-hydroxyl
groups [162-164]. In addition, the pigment in aqueous solution was most stable at pH 2.0–3.0,
moderately stable at pH 4.0–8.0 and was the least stabile at pH 9.0–10.0 [164].
The pigment was prone to degradation upon exposure to light. Only 40.11% of the initial
pigment absorbance remained after storage under natural light for 14 days, while 65.14%
remained after storage under the same time in the dark. The rate of degradation of the pigment
increased with increasing temperature. After 24 h at 20℃, 90.49% of the initial absorbance of the
pigment was retained, however, this decreased to 66.61% at 80℃. Vitamin C was shown to
protect and enhance the colour of PHNQ pigment [159, 164]. The stability of echinochrome A
solutions is summarized in Table 10, the ultraviolet-visible absorption spectra of spinochromes is
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summarised in Table 8 [152], and the distribution of spinochromes in echinoids is summarized in
Table 11.
Bioactivities of spinochrome
In vitro antioxidant activity
Since the structure of PHNQ pigments has several phenolic OH groups, it was hypothesized that
they would act as antioxidants, scavenging free radicals by donating hydrogen atoms, as has been
observed with other well-known polyphenolic antioxidants such as the catechins, quercetin and
Gallic acid [165]. Free radicals are potentially capable of abstracting hydrogen atoms from
hydroxyl groups of PHNQ compounds to become stable diamagnetic structures. The antioxidant
ability of PHNQ is thought to be the result of a combination of iron chelation, reducing power
and free-radical scavenging activity. The pigments from sea urchin exhibited low antioxidant
potential at low concentration but at high concentration the antioxidant capacity was found to be
comparable to reference positive controls such as vitamin C and BHT [157]. The antioxidant
activity and mechanism of PHNQ is summarized in Table 12.
DPPH
One method to evaluate the antioxidative activity of specific compounds or extracts is to react
them with a stable radical such as 2, 2-diphenyl-picrylhydrazyl (DPPH). The reduction of
DPPH° is followed by monitoring the decrease in absorbance at a characteristic wavelength
during the reaction. In its radical form, DPPH absorbs at 515 nm, but upon reduction by an
antioxidant (AH), the absorption disappears [161]. Pigment extracts prepared from Glyptocidaris
crenularis and Streptococcus intermedius were found to have DPPH radical scavenging ability
that was dose-dependent. Compared with vitamin C as a positive control, the scavenging ability
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of the pigment extracts was found to be weak at low concentrations, but similar to that of vitamin
C at high concentrations [159]. Pozharitskaya et al. [166] showed that the DPPH
radical-scavenging ability of the pigments was better than that of α-tocopherol. Lebedev et al.
(2008) have shown that the antiradical activity of echinochrome A and spinochromes C and D
toward the DPPH radical were almost identical. PHNQs were found to be stronger DPPH radical
scavengers than either trimethoxyechinochrome (TMEch), α-tocopherol or quercetin [165]. Free

radicals are potentially capable of abstracting hydrogen atoms from hydroxyl groups of PHNQ
compounds to generate a stable diamagnetic structure. In losing hydrogen atoms, PHNQ may
become naphthosemiquinone as the intermediate product and naphthotetraketone as the final
reaction product [157, 162].
Super anion-radical
The mechanism of scavenging super anion radicals by echinochrome A includes a two-stage
reaction scheme, involving naphthosemiquinone as an intermediate product and H2O2 and
naphthotetraketone as the final reaction products [162, 165]. Lebedev et al. [167] studied the
superoxide anion radical scavenging activity of several polyhydroxylated naphthoquinones,
echinochrome A and spinochromes. The authors suggested that compounds containing hydroxyl
groups at the C-2 and C-3 positions in the molecules may act as powerful superoxide anion
radical scavengers. They suggested that 1, 2, 3, 4-tetraketones are formed from echinochrome A
and spinochromes D and E via O2 . -induced oxidation of their OH-groups in the C-2 and C-3
positions. While hydroxyl groups at the C-5 and C-8 positions interact with the p-quinone group
at the C-1 and C-4 positions, they are thought not to directly participate in the radical scavenging
activity [168, 169].
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Reducing power
In the anion form, PHNQ may transmit two electrons consecutively to a ferric ion and become
naphthotetraketone as the final reaction product as summarized in Figure 4. [157].
The electron-donating potential of a given compound, termed as reducing capacity, may serve as
a significant indicator of its antioxidant potential. The reducing power of the pigment extract
exhibited a dose-dependent effect over a concentration range of 16 to 500 μg/ml, over which the
reducing power of the pigment extract was lower than that of vitamin C, but was comparable to
that of vitamin at concentrations above 500μg/ml [156].
Hydroxyl radical scavenging activity
Hydroxyl radicals are known to be one of the most reactive oxygen containing species. The
hydroxyl radical scavenging ability of pigments was evaluated by measuring the competition
between deoxyribose and pigments for hydroxyl radicals generated by FeSO4 and hydrogen
peroxide, and was found to be negligible [169]. Nevertheless, because the pigments were found
to have significant scavenging activity toward hydrogen peroxide and superoxide anion radicals
[167, 168] that could be precursors of hydroxyl radicals in vivo, it is considered that sea urchin
pigments would be able to depress the generation of reactive oxygen radical species [170].
Metal ion chelation
Free ferrous ion is quite sensitive to oxygen and gives rise to ferric ion and superoxide, thereby
generating hydrogen peroxide. Reaction of ferrous ion with hydrogen peroxide generates
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hydroxyl radicals, the most reactive and detrimental reactive oxygen species (ROS) in biological
systems. In this process, known as the Fenton reaction, hydroxyl radical production is directly
related to the concentration of ferrous ion [171]. Chelators can form complexes with metal ions
and inhibit the Fenton-induced oxidation.[157] The presence of orthohydroxyl groups and the
ketol structure of the sea-urchin pigments, echinochrome and the spinochromes, making them
suitable molecules for metal ion chelation [165]. Studies have indicated that PHNQ has metal ion
chelating capacity, but it was found to be weaker than that of EDTA [159, 165]. Free hydroxyl
groups at positions 2, 3 and 7 of PHNQ compounds are thought to be important for binding metal
ions. This is based on echinochrome a not being able to form adducts with calcium ions when the
hydroxyl groups at the C-2, C-3 and C-7 positions were methoxylated [162, 163]. The main
forms of PHNQ containing 2, 3,7-hydroxyl groups form di- and polyvalent anions at neutral and
weakly alkaline pH, due to dissociation of the hydroxyl groups. In the di- and polyvalent anion
forms, PHNQs with 2,3-hydroxyl groups may form complexes with ferrous ions [159, 163].
Liposome Oxidation
Echinochrome, spinochrome C and spinochrome E, like EDTA, were effective inhibitors of
ferrous/ascorbate-induced lipid peroxidation, whereas butylated hydroxytoluene and
α-tocopherol were less effective. Spinochrome D had a weak effect at low concentration.
Trimethoxy echinochrome did not inhibit liposome peroxidation at any tested concentration
[165]. Zhou et al. [157] investigated the potential of the pigment extract to inhibit lipid
peroxidation in a rat liver homogenate as a result of induced oxidation by a Fe2+-vitamin C
system. The pigment extract showed significant inhibitory activity. Although the activity was
weaker than that of BHT at low concentrations, it was higher than that of BHT at concentrations
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above 250 μg/ml. It was postulated that the inhibitory ability of PHNQ is the result of a
combination of iron chelation and free-radical scavenging activity [165]. In contrast, BHT can
inhibit lipid peroxidation by scavenging radicals only.
Protection against oxidative stress
The antioxidant activity of the pigment extract was also evaluated in a cellular system. Tert-butyl
hydroperoxide (t-BOOH) is an organic hydroperoxide and is widely used to induce oxidative

stress in a variety of cells [172]. Exposure of rat peritoneal macrophages to t-BOOH can generate
considerable oxidative stress, resulting in damage that can cause cell death which can either be
necrotic or apoptotic [157]. Zhou et al. [157] found that incubation with the pigment extract
before t-BOOH challenge significantly improved the recovery of macrophages from the
oxidative stress caused by t-BOOH. Polyphenols such as flavonoids can inhibit t-BOOH-induced
cytotoxicity by scavenging ROS and preventing lipid peroxidation [173], which could explain
the protection mechanism of PHNQ as these compounds have similar polyphenolic structures.
Other bioactivities
In addition to antioxidant activity, PHNQ may possess significant pharmaceutical activities
because of its quinine structure and functional groups. Echinochrome and spinochromes from sea
urchins have been shown to possess antimicrobial, anti-inflammatory and anti-allergenic
activities (Table 13) [174-179]. Echinochrome A is the active substance in the drug Histochrome
[178]. One form of Histochrome is used in the treatment of ocular diseases [179] and another
form of Histochrome is used for preventing reperfusion damage during myocardial infarction
[177]. In order to understand the cardioprotective mechanisms of echinochrome A, Jeong et al.
[180] found that cardiotoxic agents caused mitochondrial dysfunction as a result of increased
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ROS level and decreased mitochondrial membrane potential were inhibited with PHNQ.
Co-treatment with echinochrome A could prevent these cardiotoxic agents affecting
mitochondrial oxidative phosphorylation and adenosine triphosphate levels. Lennikov et al.
[174] reported that echinochrome ameliorated intraocular inflammation caused by
endotoxin-induced uveitis, and decreased the expression of NFκB and TNF-α. The authors
suggested that echinochrome may be a potential candidate for reducing intraocular inflammation.
Pozharitskaya et al. [175] found that sea urchin extract could significantly reduce
histamine-induced contractions of isolated guinea pig ileum, had an inhibitory effect on ocular
allergenic inflammation, surpassing that of the model reference drug olopatadine, and did not
show any irritating effect in rabbits. The sea urchin extract predominantly contained
polyhydroxy-1,4-naphthoquinone, as well as spinochrome D, spinochrome B, an unidentified
spinochrome dimer, anhydroethylidene-6,6-bis(2,3,7-trihydroxynaphthazarin),
ethylidene-6,6-bis(2,3,7-trihydroxynaphthazarin)andanhydroethylidene-6,6-bis(2,3,7-trihydroxyn
aphthazarin) isomer, which would be responsible for the pharmacological activity. Shankarlal et
al. [147] showed that a methanolic extract of Salmacis virgulata possessed both antibacterial and
antifungal activities due to the presence of polyhydroxylated naphthoquinone pigment in the
crude extract. Natural PHNQ, such as echinochrome A and spinochrome A appeared to be
general inhibitors of hydroxylases such as tyrosine hydroxylase and dopamine-B-hydroxylase,
which are targets for treatment of hypotension and other conditions [181, 182], however the
inhibition mechanism is still not well understood. This may be due to complex formation through
an enzyme-bound metal, or due to the non-enzymatic oxidation of the reducing cofactor and the
resultant consumption of oxygen in the reaction mixture [182]. Taking into account the free
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radical scavenging activity, iron-chelating ability and other known bioactivities, PHNQs could
be considered to be suitable for pharmaceutical usage.
Astaxanthin
Structure
Astaxanthin (3,3′-dihydroxy-β, β-carotene-4,4′-dione) is a xanthophyll carotenoid which can be

found in various microorganisms and marine animals. It is the most abundant carotenoid found
in crustacean wastes such as shrimp and crab shells [183]. The structure of astaxanthin, consists
of two terminal rings joined by a polyene chain. The stereoisomers (3S, 3′S) and (3R 3′R) are the
most abundant in nature [184].
Extraction methods
Astaxanthin is a lipophilic compound, which can be dissolved in organic solvents and oils.
Extraction with organic solvents, edible oils and supercritical CO2, and microbial fermentation
and enzymatic methods has been used to obtain astaxanthin from natural sources (Table 14). For
wet tissues, non-polar solvents are not recommended because of their penetration through the
hydrophobic mass surrounding the pigment is limited. It was postulated that efficient extraction
of carotenoids could be achieved with samples of low moisture content by use of slightly polar
plus non-polar solvents [185]. Some studies have been carried out to assess the extractability of
carotenoids in shrimp waste with different organic solvents and solvent mixtures and to optimize
the extraction conditions for maximum yield [186, 187]. It was shown that a 50:50 mixture of
isopropyl alcohol and hexane resulted in the highest (43.9 μg/g waste) carotenoid extraction
yield compared to using acetone, methanol, ethanol, isopropyl alcohol, ethyl acetate, ethyl
methyl ketone, petroleum ether, and hexane individually, as well as a mixture of acetone and
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hexane [188]. The Soxhlet method achieved a higher yield compared to maceration of the
starting material, using the same extraction solvent [189]. It may be that the high temperature
used and the solvent/solute interactions in the Soxhlet method contributed to the highest
solubilisation of components from the starting material. Also, the use of solvent at its boiling
temperature reduces its viscosity and surface tension, allowing the solvent to solvate soluble
substances inside the solid matrix. Pressurized liquid extraction is similar in principle to Soxhlet
extraction, except that elevated pressure, in addition to temperature are used, which enables
extractions using small amounts of solvents to be completed in a relatively short time. Quan et al.
[190] examined the effects of changing different pressurized liquid extraction parameters on the
extraction yield of astaxanthin from shrimp waste. The authors found that temperature and
extraction time had a significant influence on the astaxanthin recovery, while pressure only had a
minor effect. In another study [191], a new method using high pressure processing (HPP) to
extract astaxanthin from shrimp (Litopenaeusvannamei) shell and head at ambient temperature
was studied. Compared to the conventional extraction methods, HPP showed significant
advantages with higher yield and shorter extraction time. The use of HPP to extract astaxanthin
from shrimp discards is a potential alternative to conventional methods. Various organic solvents
have been used for the extraction and recovery of carotenoids from shellfish waste and the
efficiency of extraction were correlated to the elevated temperatures used and to the solvent–
solute interactions. However, the limitations of organic solvent extraction procedures are high
energy costs due to elevated extraction temperature and the effect of temperature on thermolabile
compounds, use of organic solvents and the need for their recovery and the time duration of the
process [188, 189].
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Vegetable oils have also been successfully used as solvents for the extraction of carotenoid
components from crustaceans [189, 192]. The advantage of using vegetable oils is that they are
considered as a good barrier against oxygen, which reduces oxidation processes. Edible
vegetable oils are compatible and can also contribute as an energy source if the extracted
carotenoid product is subsequently applied in food formulations. Mezzomo et al. [189] found
cold oil extraction can be a good alternative to selectively extract astaxanthin and protect the
extract, although process optimization is required to enhance the extraction yield. Extraction of
carotenoids from shrimp waste using refined sunflower oil achieved good carotenoid yield after
optimizing the oil to waste ratio, temperature and extraction time [192]. An extract obtained from
shrimp waste silage produced a better yield of astaxanthin than that obtained from raw shrimp
waste [193]. In another study, the total carotenoid recovery from ensilaged shrimp waste was
higher than that from non-ensilaged waste as assessed by HPLC analysis, achieving up to 115
μg/g of waste material [194]. Ensilaging in organic/ inorganic acids or by fermentation is used to
preserve shrimp waste. Acid ensilaging of shrimp waste resulted in a gradual conversion of
astaxanthin diester to monoesters during storage of the silage. As carotenoids are prone to
degradation by acids, mild treatments such as fermentation may have beneficial effects on the
stability of carotenoids. Two lactobacillus species L. plantarum and L. acidophilus have been
used to ferment shrimp waste [45]. It was shown that the microbial method of extraction of
astaxanthin was more effective than chemical methods based on solvent extraction of carotenoids
from natural matrices. Sachinadra et al. [195] found that fermentation was superior to acid
ensilaging with respect to stability and extractability of carotenoids from shrimp waste. In
crustaceans, carotenoids occur as a complex with protein which imparts stability to the pigment.
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Attempts have been made to recover crustacean carotenoids as caroteno-protein complexes
[196-199]. Although fermentation of shrimp waste was found to induce protein hydrolysis it
resulted in an increased yield of carotenoids [195].
Extraction with supercritical carbon dioxide has become an important separation technique in the
area of nutraceutical supplements and functional foods and is a promising alternative to

traditional processing methods. This extraction technique is advantageous when thermo-labile
compounds are present, in addition to avoiding the use of toxic solvents, since CO2 is a cheap
and easy to separate from the extract [200] but has environmental issues. The advantages of
supercritical fluid extraction relative to traditional organic solvents allow carotenoid losses
through heat-induced degradation to be reduced. Some reports have used supercritical fluid
extraction successfully in the extraction of lipids and carotenoids from shrimp or crab shell
[201-204]. In addition, because it avoids the use of organic solvents, the extracted compounds
can be employed more readily as nutritional additives and in pharmacological applications.
Characteristics of pigments
Pigment compounds can be extracted from shellfish waste such as crab or shrimp shells. The
extraction process can be achieved by several methods, including use of solvents or oils [188,
189], and by supercritical fluid extraction [201-204]. Astaxanthin stability is enhanced in nature
due to various mechanisms, including association with other biomolecules [38], such as
esterification with fatty acids [205], binding to chitin through ester bonds [196] and association
with proteins to form caroteno-protein complexes [38]. However, once extracted the pigment
becomes unstable. Purified or semi-purified carotenoids are highly sensitive to environmental
factors such as light, oxygen and temperature, among others. Franco et al. [183] studied the
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stability of astaxanthin in two model systems, a protein solution and an edible oil. If astaxanthin
is incorporated into a protein system, the pigment undergoes oxidation to astacene after only 17
days, even when stored in air-free, refrigerated conditions, and in the dark, indicating that a
protein system alone is not really effective at preventing pigment degradation, and a lipid
environment has a more efficient protective effect. Sachindra et al. [206] reported that the
stability of carotenoids recovered from shrimp waste using organic solvents and vegetable oils
was affected by antioxidants and pigment carriers. Solvent extracted carotenoids can be stored by
mixing the extract with carriers such as sodium alginate or corn starch. As starch or alginate is
used as one of the main ingredients in minced fish products, the pigmented carrier could be used
directly as a source of colouring agent in these products. Addition of antioxidants such as TBHQ
or α-tocopherol and storing them in metalized polyester pouches reduced the carotenoid
degradation during storage. The oil-extracted carotenoid was found to be protected from
degradation by addition of antioxidants and storage in amber coloured bottles. Armenta and
Isabbl [207] assessed the effect of oxidation factors on the stability of free astaxanthin contained
in caroteno-protein powder extracted from shrimp by-products. It was found that natural
astaxanthin from fermented shrimp by-product had moderate stability levels. Although natural
astaxanthin oxidized faster than the synthetic pigment, its stability may be improved by
antioxidant and polymer addition.
Bioactivities
Astaxanthin, the red-orange carotenoid has been referred to as a powerful biological antioxidant
with beneficial health effects in both experimental animal and clinical studies. Its promising
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healthy benefits include antioxidant activities [208-210], including enhancement of the immune
system [211, 212] and protection of the cardiovascular system [213]. Astaxanthin has important
food applications as it can be used as a colorant additive in foods because of its colour and
antioxidant capacity.
Antioxidant activities
One of the most important characteristics of astaxanthin is its ability to act as an antioxidant
[210]. Very few reports are available on the antioxidant activity of astaxanthin-containing
crustacean waste extracts. Sowmya et al. [208] reported that the crude extract and fractions rich
in astaxanthin had strong antioxidant activity, indicated by radical scavenging activity, reducing
activity and metal chelating activity, comparable to that of the known antioxidants such as
α-tocopherol and TBHQ. The higher antioxidant activity of the astaxanthin-rich fraction was
indicated by a higher protection factor compared to α-tocopherol against singlet
oxygen-mediated oxidation of liposomes. Santos et al. [214] examined the ability of shrimp
astaxanthin to modulate the production of superoxide, nitric oxide and TNF-α in rat alveolar
macrophages. The shrimp extract showed a suppressive effect on the generation of free radicals
such as O−2 and NO, while astaxanthin isolated from shrimps had a specific effect on NO only.
Only the crude shrimp extract completely inhibited TNF-α, compared with superoxide
dismutase, butylated hydroxytoluene and commercial astaxanthin. The antioxidant action
demonstrated in these studies indicates that the shrimp extract is a promising source of
antioxidant bioactivity, which could be used in food and biomedical applications.
Anti-inflammatory activities
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It is well known that reactive oxygen species (ROS) can up-regulate proinflammatory cytokines
such as TNF-α, IL-1, and IL-6 whereas astaxanthin has been found to reduce the levels of
markers of inflammation such as TNF-α [214]. Astaxanthin is a strong antioxidant with the
ability to reduce markers of inflammation. Screening studies of anti-inflammatory natural
compounds showed that astaxanthin significantly inhibits NF-κB activity, an effect that is
associated with its major antioxidant activity [215]. Furthermore, maternal ethanol-induced
up-regulation of toll-like receptor 4 (TLR4), and the down-stream myeloid differentiation factor
88 (MyD88), NF-kB, TNF-a, and IL-1 in embryos, can be inhibited by astaxanthin pre-treatment,
which was demonstrated to produce a protective effect for fetal alcohol spectrum disorder, and
suggested that oxidative stress and inflammatory reaction are involved in this process [216].
Immune enhancement
The immune system involves both innate and acquired immunity. Acquired immunity involves
lymphocytes, which are highly active cells that constantly generate reactive oxidative products
(ROS) as a part of their normal cellular activity [217]. The antioxidant capability of astaxanthin
can potentially reduce the toxic effects of reactive oxygen species (ROS). Recent studies on the
role of carotenoids in gene regulation, apoptosis and angiogenesis have advanced our knowledge
on the possible mechanism by which carotenoids regulate immune function. Using a rodent
model, Chew et al. [218] demonstrated that astaxanthin stimulated an immune response in mice.
Mice supplemented with astaxanthin had increased in vitro splenocyte antibody response to
T-dependent antigens, lymphoblastogenic response and cytotoxic activity. Some research [212]
demonstrated an enhanced immune response in humans. It was shown that dietary astaxanthin
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decreased a DNA damage biomarker and acute phase protein, and enhanced immune response in
young healthy females.
Anti-UV activities
UV-induced skin damage is considered to be partly caused by reactive oxygen species (ROS),
such as singlet oxygen, generated in the skin by UV irradiation [219]. Thus natural antioxidative
agents such as astaxanthin could be expected to prevent UV-induced skin damage. Hama et
al.,[220] demonstrated that astaxanthin had potent antioxidative activity against singlet oxygen
generated in an in vitro system. Application of astaxanthin-egg yolk
phosphatidylcholine-liposomes (Asx-EPC-lipo) inhibited skin damage due to a decrease in
collagen that was induced by UV irradiation. Moreover, a combination of cationic
Asx-DOTAP-lipo and iontophoresis prevented melanin production in deep regions of the skin
[221]. Supplementation with dietary astaxanthin combined with a collagen hydrolysate improved
facial elasticity and decreased matrix metalloproteinase-1 and -12 expression [221]. Induction of
procollagen type I mRNA was observed after 12 weeks of treatment, along with significant
decreases in the expression of the collagen-degrading enzyme MMP-1 mRNA and the
elastin-degrading enzyme MMP-12 mRNA after UV irradiation. It was thought that
improvements in facial skin elasticity might be related to these molecular changes.
Cardiovascular disease prevention
It is known that an excess of reactive oxygen species (ROS) is associated with inflammation,
growth and vasoconstriction contributing to vascular injury in many cardiovascular diseases,
such as hypertension, hyperlipidemia, and diabetes [222]. Diabetes mellitus (DM) is a health
problem that affects millions of individuals all over the world. Hyperglycemia has been found to
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play a key role in reactive oxygen species (ROS)-generated damage [223]. The antioxidant
property of astaxanthin may have a role in the treatment of diabetes. Sila et al. [224] showed that
astaxanthin extracted from shrimp shell waste had protective effects on diabetes and kidney
function complications. It was also shown that supplementation with astaxanthin was able to
suppress oxidative stress. The administration of astaxanthin to diabetic rats led to hypoglycemic
and antihyperglycemic effects by reducing the fasting blood glucose level, which may be
explained by stimulation of residual pancreatic β-cell activity. The anti-diabetic activity of
astaxanthin may be due to its protective role in quenching free radicals and inhibition of lipid
peroxidation to ensure a beneficial role in the survival of pancreatic β-cells. Results from other
research indicated that astaxanthin could be a good adjuvant for recovery from lymphocyte
dysfunctions associated with diabetic patients, especially when focusing on the re-establishment
of redox balance and achieving a hypothetical anti-apoptotic effect in lymphocytes [225].
Monroy et al. [213] reported that supplementing the diet with astaxanthin had beneficial effects
on hypertension, by decreasing blood pressure, improving cardiovascular remodeling and
oxidative stress. The results suggested that systolic blood pressure was lower in
astaxanthin-treated groups than the control group from the first week of treatment, and the left
ventricular hypertrophy index (LVH) was significantly reduced. Administration of astaxanthin
improved endothelial function in resistance arteries, but had no effect on aorta.
Scallop shell matrix protein
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Scallop shells are made up mainly of two layers of calcium carbonate, comprising an outer layer
of calcite and an inner layer of aragonite. The lustrous inner layer, called the nacre, is mainly
composed of aragonite tablets oriented in multiple layers, each surrounded by an organic matrix
[226], which makes up less than 5% of the nacre composition and is composed mainly of
polysaccharides and protein [227]. Over the past five decades, many studies of the shell material
have been carried out, mainly via electron microscopy and examination of the amino acid
composition of the organic shell matrix fraction [228, 229]. More recently the matrix protein of
the shell has been characterised (Table 15). Shell matrix proteins have drawn increasing attention
due to their unique physical and biological properties. Matrix proteins are often classified as
either soluble or insoluble based on their solubility after decalcification with a weak acid or a
calcium-chelating agent such as EDTA.
Bioactivities of matrix protein
Several matrix proteins have been identified in the nacre and are known to play an important role
in mineralization of the shell [230, 231]. In addition to their role in regulating the mineralization
process of nacre, soluble matrix proteins are known to contribute to biological activity
[232-234].
Osteoblast Differentiation
The biological activity associated with matrix proteins in the nacre has made them attractive for
use in traditional pharmaceutical preparations used for stimulating bone growth and enhancing
bone density. Natural products in nacre are thought to be able to promote biomineralization and
to stimulate bone formation by osteoblasts [235]. Several in vitro and in vivo studies have shown
that nacre has excellent biocompatibility and osteogenic properties, which suggests that nacre
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contains signalling molecules in the soluble matrix of the nacre that are osteoinductive. Lopez et
al. [236] found that human osteoblasts cultured in the presence of nacre produced bone structure.
Liao et al. [237] determined that the components involved in bone tissue generation were
localised in the outer prismatic and the inner nacreous layers of the shell of the fresh water
dwelling Margaritifera. The authors showed that Margaritifera shell is biocompatible,
biodegradable and osteoconductive. Bonding between the natural aragonite and bone seems to
occur via a phosphorous-rich intermediate layer. Atlan and Lamgjari [238, 239] suggested that
there is a dynamic continuity at the bone-nacre interface, giving rise to the hypothesis that the
organic matrix of nacre contains diffusible soluble factors that have osteogenic activity. Almeida
et al. [240] reported the extraction of four water-soluble matrix (WSM) fractions (SE1–SE4)
from nacre. The WSM and SE4 when tested in a human foetus lung tissue fibroblast culture
increased alkaline phosphatase (ALP) activity, while the SE1 fraction caused a decrease in ALP
activity. It was also found that WSM greatly increased the level of B-cell lymphoma-2（Bcl-2) in
the cytoplasm and nucleus of osteoblasts. [232]. However, as nacre proteins tend to aggregate
and form various biopolymer-associations it remained unclear as to the bioactivity of specific
nacre molecules [241]. Kim et al. [235] found that water soluble nacreous factors (WSNF) which
can prevent osteoporotic bone loss through both osteoblast activation and osteoclast inactivation,
contained calcium, phosphate and unknown proteinaceous components. However, treatment with
calcium at the same concentration of calcium contained in WSNF, did not affect
biomineralization in vitro. The acid soluble matrix of the sea snail, Aliotisdiversicolor reeve
was shown to contain a factor, thought to be the mantle protein somesuke, which may be
involved in osteoblastic cell differentiation [242]. Four proteins have been identified in the oyster
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Crassostrea gigas nacre WSM [243] that were investigated for osteogenic and osteoinductive
properties. Furthermore, three proteins, namely P60, P10, and PFMG3 have been identified from
Pinctada fucata, which induced osteoblast differentiation in murine pre-osteoblast cells
[244-246]. However, it remains to be seen whether these bioactive molecules are represented in
WSM, and if so, then whether they act individually or in harmony with other active molecules in
order to induce bone differentiation.

Ability of shell matrix extracts to protect skin
Skin, which has a highly differentiated and complex structure, is vulnerable to free radical
damage because of its contact with oxygen in the atmosphere. Free radical damage to skin causes
alteration to collagen and elastin in the extracellular matrix of connective tissue, resulting in loss
of skin tone and formation of wrinkles. An increase in elastase activity in skin destroys elastic
fibers, resulting in reduced skin elasticity [221]. Water soluble matrix components from shells of
Patinopecteny essoensis exhibited radical scavenging activity which inhibited generation of the
superoxide anion and also the fibroblast cell growth rate was increased by this extract. Scallop
shell extract was shown to have strong inhibitory activities against elastase [233] and protected
keratinocyte cells against UV-B-induced damage in vitro by acting as an antioxidant and a
growth promoter [247]. To establish a mechanism for the skin-protection activity, scallop shell
extract was investigated in vivo by Liu et al. [248], who showed that wound healing of erythema
and eschar and recovery of the epidermal layer was promoted. These results suggested that
scallop shell extract activates rat keratinocyte cells and promotes the turnover of skin stratum
corneum [248]. Scallop shell extract was also found to inhibit squalene mono
hydroperoxide-induced skin erythema and wrinkle formation in rat [249]. However, as it is
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unclear whether bioactive substances in the extract are proteins or other non-protein components,
there is a need to isolate and identify these components [249]. Torita et al. [250, 251]
investigated the bio functionality of organic components extracted from Patinopecteny essoensis
shells and found that, a reduction in collagen content is associated with the loss of tensile
strength and the wrinkling of skin. These changes occur not only as a result of aging but also as a
result of UV exposure from the Sun. Collagen turnover is controlled by collagen degrading
matrix metalloproteinase (MMP) and their tissue inhibitors (TIMP). Treatment of human skin
fibroblast cells with scallop shell extract increased the mRNA expression levels of type I
collagen, MMP-1 and TIMP-1, suggesting that the scallop shell extract may activate collagen
metabolism in skin fibroblasts. These results suggest that the scallop shell extract may be
effective for the treatment of photo aged and aging skin, which have undergone collagen loss
[250]. The authors also investigated the effect of scallop shell extract on the expression of
keratinocyte growth factor (KGF) from human skin fibroblasts. Scallop shell extract increased
the mRNA expression level of KGF in skin fibroblasts and the secretion of KGF from the
fibroblasts. These results suggested that the scallop shell extract may promote recovery from
UVB-induced injury to skin through an increase in the secretion of KGF from the fibroblasts. As
scallop shell extract contains many proteins, sugars, and other organic compounds, the biological
activity of scallop shell extract may be attributed to more than one component [251]. When a
nacre extract of Patinopecteny essoensis was implanted in the dermis of rats, it resulted in
enhanced extracellular matrix synthesis and the production of components implicated in cell to
cell adhesion and communication and tissue regeneration. It was shown that the effects of the
implant were not due to calcium carbonate, and the authors suggested the possibility that here the
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nacre organic matrix may cause a release of biologically active factors that influence the main
phenomena of tissue regeneration [252]. The comparative effects of nacre water-soluble matrix
and dexamethasone on the alkaline phosphatase activity of MRC-5 fibroblasts have been
evaluated [253]. The effect of WSM on alkaline phosphatase activity of bone marrow stromal
cells was similar to that obtained by dexamethasone. These results imply that human diploid
MRC-5 fibroblasts respond to differentiating factors that promote an osteoblastic phenotype in

bone-derived cell cultures. The WSM and MR14 fractions that were separated from the pearl
powder of Hyriopsiscumingii lea was able to significantly promote fibroblast proliferation and
promote collagen accumulation [254]. The MR14 fraction inhibited MMP-2 activity significantly
and all of the fractions promoted TIMP-1 production. This study elucidated the mechanism by
which pearl powder promotes wound healing through an ability to stimulate fibroblast mitosis,
collagen deposition, and TIMP-1 production, and the major active fraction may be MR14 [254].
When water soluble nacre（WSN）extract was applied to a skin burn area, the burn-induced
granulation sites were rapidly filled with collagen, and the damaged dermis and epidermis were
restored to the appearance of normal skin. WSN enhanced the wound healing recovery properties
for burn-induced apoptotic and necrotic cellular damage and spurred angiogenesis. Additionally,
WSN-treated murine fibroblast NIH3T3 cells showed increased proliferation and collagen
synthesis [255].
Ability of shell extracts to control weight
Liu et al. [256] investigated feeding scallop shell powder to rats and the authors reported a
decrease in body weight including a decrease of white adipose tissue. This result provided initial
evidence that the scallop shell powder was useful for lowering fat mass and raised the possibility
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of a new application of the powder as a health supplement. The organic components in scallop
shell were found to promote lipolysis in differentiated 3T3-L1 and C3H10T1/2 adipocytes [257].
Scallop shell extract, which contains unidentified proteins and saccharides, may contribute to the
decrease in amount of white adipose tissue of rats fed scallop shell powder through an increase in
the expression of UCP1 and acyl CoA dehydrogenase [234, 258]. Feeding powdered nacre
resulted in reduced body weight, with a decrease in visceral fat [259], but the specific nature of
the substance causing the fat reduction was not reported. In 2012, in order to clarify the
mechanism of action, Takahashi et al. [260] investigated the effects of scallop shell extract on
the differentiation of 3T3-L1 pre-adipocyte cells and found that scallop shell extract reduced
lipid accumulation in 3T3-L1 cells during differentiation and inhibited adipocyte differentiation.
In addition, scallop shell extract suppressed the expression of adipogenic transcription factors
and inhibited differentiation-associated mitotic clonal expansion. It was found that glycoproteins
isolated from scallop shell extract via affinity chromatography could inhibit
differentiation-associated mitotic clonal expansion and suppressed the expression of an
adipocyte-specific protein, fatty acid binding protein [260]. In order to find out which substance
could cause a decrease in white adipose tissue in rat, Fukuda et al. [261] treated the extract with
pepsin and pancreatin and identified a protein with a molecular weight of 90 kDa that was
resistant to digestion, and was found to have a free radical-scavenging capability and had the
ability to bind bile acids and potentially inhibit the absorption of bile acid.
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Other bioactivities of shell extracts
Nacre has potential as a traditional Chinese medicine for treatment of eye diseases. Xu et al.
[262] reported a form-sense-deprived model (FDM) for chicks that was developed to examine
the effect of nacre on the elongation of axis oculi. It was shown that nacre can prevent and treat
myopia, however, the specific substances involved in the nacre powder that have this activity
require identification. Oyster shell extract significantly decreased intracellular ROS levels and
increased antioxidant enzyme activities, blocked NO production and expression of TNF-α,
IL-1β, IL-6, NF-κB, iNOS and OX-2 in LPS-stimulated Raw 264.7 cells. Together, these
results suggest oyster shell extract could be useful as a natural anti-oxidant and
anti-inflammatory resource [263]. It was also shown that a water soluble matrix extract of nacre
had ability of vitro scavenging of ABTS and DPPH free radicals and inhibition of lipid
peroxidation [264]. Mitsuhashi et al. [265] reported a radical scavenging glycoprotein with a
molecular weight of approximately 90 kDa m the scallop shell
Conclusions
Worldwide the seafood harvesting and processing industry generates considerable waste
co-products. A significant proportion of the waste is in the form of shell material that is currently
under-utilised. With the expansion of particularly shellfish aquaculture, this waste stream will be
perpetuated. Much of the shell waste stream is disposed of into landfill with the potential to
create increasing environmental problems in the future. An increasing volume of literature
indicates considerable potential for utilisation of the chemical mineral major component of shells
in applications ranging from food-grade antibacterial protection to bone implants with
demonstrated functional and health-promoting advantages. The lower abundant protein and
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pigment contents present in many shell types are also a significant resource that are recognised to
have a variety of applications including fortification of consumed food products and use in
topically applied pharmaceutical products. These materials have their own unique structures and
can have potential applications in various industries. With the increasing trend in preservation
and central processing of seafood (e.g. production of individually quick-frozen mussels and
half-shell products) that ensure production efficiency and product diversity, there is no shortage
of a large supply of marine shells for production of the above mentioned products. A challenge
for utilisation of these materials is the development of industrial scale processes for
interconversion of shell material and for the extraction of protein and pigments using processes
that do not require the need for use of harsh chemicals that may in turn create additional
environmental waste problems. Several reports have demonstrated potential ways forward by use
of processes such as fermentation of shell material and extraction of lipophilic molecules such as
pigments with edible oils and other green technologies. Development of scalable processes to
utilise the health-promoting and functional material resources available in shell waste material is
underway and holds great promise for the future.
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[349] Hatate H, Murata H, Hama Y, Tanaka R, Suzuki N. Antioxidative activity of spinochromes
extracted from shells of sea urchins. Fish Sci. 2002;68:1641-2.
[350] Ma C, Zhang C, Nie Y, Xie L, Zhang R. Extraction and purification of matrix protein from
the nacre of pearl oyster pinctada fucata. Tsinghua Sci & Techno. 2005;10:499-503.
100
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Table 1 Extraction of chitin/chitosan from sea shell waste
Shell Method/Microorga optimized DM DP Referen

source/meth nisms conditions/prop (%) (%) ces
od erties
Microbial
Shrimp Solid state 34.6°C 58.19% Chitinase [266]
(Parapeneopsi fermentation v/w moisture activity:
s hardwickii)
Citrobacter content. 143.15 U/gds
freundii str. nov. 2.36 % w/w
haritD11 yeast
Shrimp Biological 70 g/l shrimp, 88 94 [42]
(Metapeneaus treatment, using 35°C, 6 days
monoceros)
the Bacillus glucose
pumilus A1 concentration
of 50 g/l, pH
5.0
Bacillus cereus 40 g/l shrimp protease 88 [267]
Shrimp SV1 60°C, pH activity: 5,900
(Metapeneaus
Monoceros)
6-9.5, and U/mL)
<55°C, pH 8.0
Shrimp Treatment with 6 h, room 88.8 [268]
Crude Protease temperature,
from Bacillus DA 89%
cereus SV1 remarkable
antioxidant
activities
Fresh Pseudomonas 80 g/l SWP and protease 85 [269]
shrimp aeruginosa A2 135 g/L activities:
waste FSW,60°C, pH 17,000 and
(FSW) 8.0 12,000 U/mL
Shrimp
waste
powder
(SWP)
Shrimp Pseudomonas 50 g/l shrimp, protease 96 89 [43]
shells waste aeruginosa A2 glucose 50 g/l, activities:
(Metapenea 25°C. 18,000 U/ml
us 5 days.
monoceros) inoculum of
0.05 OD
Shrimp shells Serratia sp. chitinase and Chitinase: [270]
waste TKU016 protease were 22 mU/ml
101
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(pH 7, 50° C, protease:

pH 6-7, <50°C) 160 mU/ml,
and (pH 8-10,
40°C, pH 5-10,
<50°C),
DPPH
scavenging
ability was
about 76%
Shrimp shell Lactobacillus 5days [45]
waste plantarum (PTTC
(Penaeus 1058)
semisulcatus Lactobacillus
) acidophilus
(PTTC 1643)
Lactobacillus
rhamnosus (PTCC
1637)
Crangon 56.82°C for 83 [271]
2.55h
Shrimp shell Pediococcus sp. 50 g/l, 37°C for 83.03 [272]
waste L1/2. 24h
pH 7.00
Shrimp shell Lactobacillus 10% (w/v) of 44 91 [9]
waste helveticus shrimp shells
(Parapenaeus
longirostris)
inoculated with
a L. helveticus
strain milano
(10% v/v).
The use of date
juice, as an
alternative to
the use of a
primary carbon
source such as
glucose
80 g L−1 sugar
content and
30°C
Shrimp shell Bacillus subtilis - 72 84 [32]
waste
Shrimp shell Bacillus cereus 14 day, 10% 95 97.1 [273]
waste and shrimp shell
Exiguobacterium waste broth,
102
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acetylicum 37°C
Shrimp shell Pediococcus 72h, 15% 72.5± 97.9± [48]

waste acidolactici (w/w) glucose, .5 0.3
(Penaeus
monodon)
CFR2182 37°C
Prawn waste Lactococcus lactis1% v/v 78.8 66.5 [274]
and inoculant, shell 23. 77.8
Teredinobacter waste (10% 3
turnirae w/v) pH
8.8 ± 0.5, 15%
glucose, 4 day
Shrimp shells Bacillus 96h 229.7 ± 10.0 93.5 87 [275]
waste licheniformis and U/mL
Litopenaeus
vannamei
Gluconobacter
oxydans
Shrimp shells Lactobacillus 48h 99.02 91.54 [47]
waste acidophilus SW01 15% glucose,
Penaeus
vannamei
37°C
Shrimp shells Fermentation shrimp shell 88% 94% [41]
waste using Bacillus concentration
pumilus A1 of 70 g/l,
glucose
concentration
of 50 g/l, pH of
5.0 incubated
with 0.225 OD
of B. pumilus
A1 inoculum,
35°C and
150 rpm for 6
days.
Red crab Lactobacillus Supplementatio 94.3 68.9 [34]
shell paracasei n of N-source
(Chionoecet KCTC-3074 and in culture
es Serratia media
japonicas) marcescens FS-3 improved the
extraction
efficiency of
chitin from
crustacean
shell. 7 days
Crab shell Pseudomonas 30°C, 7day 92 63 [35]
aeruginosa F722 10% glucose
103
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supplement,
10%
inoculation
Snow crabs Serratia marcescens 7day, 30°C 60.7 U/ml 84 47 [276]
(Chionoecetes FS-3
opilio)
Chemical
Shrimp, Chemical method Chitin from [277]
squid pen, using NaOH and squid pens did
crab, lobster autoclave not require
steeping in
sodium
hydroxide
solution/ after
15 min of
heating a
degree of
deacetylation of
90% was
achieved.
Pink Shrimp 75°C, 2.5 N [278]
(Solenocera NaOH and 1.7
melantho) M HCl
solution to solid
ratio of 5 mL/g
from NaOH (3.5%) [279]
exoskeleton with a solvent
of shrimp to solid ratio of
(Metapenae 10:1 (v/w) for 2
us h at 65°C
monoceros) 1 N HCl for 0.5
h at ambient
temperature
with constant
stirring and a
solvent to solid
ratio of 15:1
(v/w).
chitin from Lactobacillus lactic acid [46]
trash crab plantarum concentration
(Podophthal of 69.5 g/L at
mus vigil) pH 6.9, acid to
shells ratio
1:25, 40°C
104
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Shrimp shell shells soaked in [280]

waste 0.5 M NaOH at
room
temperature for
24 h
Demineralized
by 0.25 M HCl
for 45 min at
room
temperature.
deproteinizatio
n by three
repeated
six-hour
treatments with
2.5 M NaOH at
80°C
Shrimp shell 24 h & 45 min [28]
waste NaOH 1 M
(Parapenae within 24 h at
opsis 70°C
stylifera) solid-to-liquid
ratio of about
1/40 (w/v)
Shrimp, Chemical and MW Microwave [281]
lobster, heating reduced
crab, cuttle the time of
fish heating from 6–
10 h to 10–
15 min, 0.25 M
HCl solution at
ambient
temperature
with a
solution-to-soli
d ratio of
40 mL/g,
Deprotenization
: 1.0 M
NaOH
(20 mL/g) at
70°C
Shrimp 2.5h >98 >98 % [282]
shells waste Response surface NaOH solution %
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(Penaeus methodology 3.6 % (w/v),

sp) reaction time
2.5 h, treatment
temperature
69.0 ± 1°C,
solid liquid
ratio of NaOH
solution 1:7.4
(w/v)
106
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Table 2 Chemical composition of some of sea shells [283, 284]
Component Oyster Mussel Clam Crab

shell shell shell crust
SiO2 0.40 0.20 0.46 -
Al2O3 0.22 0.13 0.20 -
Fe2O3 0.04 0.03 0.04 0.02
CaO 53.81 53.70 53.92 22.34
MgO 1.70 0.33 0.22 1.07

CaCO3 - - - -
K2O - - - 0.01
Na2O - - - 0.07
P2O5 - - - 3.08
107
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Table 3 Synthesis of HA powder from natural resources. Research published in the period of
2010-2014.
Time Powder Phosphate Method Source Ref

properties source
10 Flower shape, Na2HPO4 Microwave Eggshell [109]
min 78 nm, Mg ,
EDTA used,
b-tcp,
Globular like, H3PO4 Precipitation Eggshell [285]

30 nm, stable to
900 C
Spheroidal, Ca2P2O7 Solid state Eggshell [286]
pure β-TCP Na,
Mg and Sr.
72 h Flower like NH4H2PO4 Hydrothermal Eggshell [287]
smooth prisms
or rough
particles HA
phases.
Whitlockites
24 needle-like or H3PO4 Hydrothermal Eggshell [288]
-72 h rod-like, Na,
Mg, and Sr.
24 h H3PO4 Precipitation Eggshell [289]
24 h Flower like NH4H2PO4 Eggshell [290]
sphere, 15-35
µm
48 h whisker, 0.06 CaHPO4 Hydrothermal Eggshell [291]
µm
1-7 Amorphous, 15 K2HPO4 Hydrothermal Eggshell [292]
days nm circular
3-12 flower like K2HPO4 Egg shell [293]
days
24 h needle-like (NH4)2HPO4 Eggshell [294]
morphology, 5
nm in diameter
and 50–100 nm
length
Up Spherical, HA, Ca2P2O7 Mechanical Oyster [295]
to 8 B-TCP, solid milling
108
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h state reaction,
12 h, Microspheres, Na2HPO4·12H2O Hydrothermal Nacre [296]
~ 5 μm
5h irregular shape, KH2PO4 Precipitation Mussel shell [297]
24 h rod like, Mg, Na Hydrothermal [298]
Sea Urchin
4 the starting (NH4)2HPO4 Hydrothermal Sea Urchin [86]
days (NH4)2HPO4 Heterocentrotus
solution was trigonarius and
−1
0.2 g ml and Heterocentrotus
temperature was mammillatus
180°C
∼200°C (NH4)2HPO4 Hydrothermal [87]
Strombus gigas
(conch) shells and
Tridacna gigas
(Giant clam) shells
20 180°C, 137–218 (NH4)2HPO4 Hydrothermal [98]
days MPa, conch and clam
seashells
β-MgTCP, 80 C hot [93]
monetit and HA plate 2 hour , Sea urchin and sea
sintering up snail
to 850 C
coralline 250 ±C and 3.8 Hydrothermal [299]
hydroxyapatite MPa pressure Coral
retains thewith excess
interconnectivity (NH4)2HPO4.
of coral dried at 70 ±C.
150°C forfired at 900°C Hydrothermal
8 days followed for 2 h Coral
by sintering at KH2PO4 Coralin
1250°C hydroxyapatite
~ 87%
crystallinity, 70–
75% porosity
and 2 ± 0.5 MPa
compressive
strength
250°C and 3.8 (NH4)2HPO4. Hydrothermal
MPa pressure . Coral
200°C(~ 15 KH2PO4 Hydrothermal

atm), 4 hours Cuttlefish
109
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9h 200°C, after of Hydrothermal

HT Cuttlefish bones
pore size (∼80
μm in width and
∼100 μm in
height)
110
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Table 4 Advantages and disadvantages of different HA production methods
Method Advantages Disadvantages Ref

Heterogeneity of phase
Good for mass production of HA composition, owing to
powder, small diffusion
Solid state Stoichiometric, structure a large coefficients of ions [300-302]
surface area within the solid phase
small cluster size High sintering time and
temperature
Poorly crystallized.
without any regular
shape. Non
stoichiometric ratio,
Simple, low temperature,
Precipitation high pH and sintering [303-305]
Nano HA particles, large scale possible,
temperature, sensitive
to pH and rate of
stirring, drying
temperature
Molecular-level mixing of reactants, Generation of secondary
Improving the chemical homogeneity phase.
of the resulting powder, High cost of some of the
Low temperature formation starting materials,
[65,
Sol-gel Easy to perform; Inexpensive especially
306-311]
Stochimetric ratio of HA alkoxide-based
Nano HA particles, phase purity and precursors, Expensive
homogeneous mixing of molecules, chemicals, difficult to
Low temperature and pressure. hydrolyse phosphate,
Relatively stoichiometric and highly Elevated temperature
crystalline. Third most popular method and pressure. Need
after the conventional precipitation expensive equipment,
Hydrothermal and combination methods. Capability high pressure [312-315]
to induce 1D growth, Well crystallised, equipment,
homogenous powder, nano HA agglomeration of the
powder. product
111
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Table 5 Literature relating to application of shell material for treatment of waste water
Adsorbent pollution Adsorption capacities and Optimum condition Ref

comments
Corbicula Phosphoric Precipitate calcium hydrogen phosphate, Batch stirring [128]
shells and acid (0.1 for up to 6 hours

Japanese mol/l)
littleneck
shells
Oyster Phosphoric 0.1 and 0.05 mol/l of phosphoric acid [127]
Shells acid(0.1
mol/l)Calcium
Phosphate
Mussel, Removal Clam shells showed highest value of SO2 (>1.0 mmol/g) [4]
clam, oyster SO2/NOx and NOx (0.1 mmol/g) adsorption
shell
Crab shells Phosphate 108.9 mg/g; Particle <1000 µm more than 85% of the [283]
500mg/l of in 24 hours at pH 2,
Crab shells Copper(II) Copper 243.9 and cobalt 322.6 mg/g, 2 h, particle size [316]
and cobalt(II) 0.767 mm, (pH 6); biosorbent dosage (5 g/l). Eluting
agents, EDTA (pH 3.5, in HCl) performed well and also
caused low biosorbent damage. five cycles
Pretreated Lead and Crab: lead 19.83 ± 0.29 copper 38.62 ± 1.27 mg/g ; Arca [317]
crab and copper shell: 18.33 ± 0.44 mg/g and 17.64 ± 0.31 mg/g
arca shell
Scallop Phosphate 23.0 mg-phosphate/g-shell; 45 μm, 100 mg/L of [318]
phosphate in 3 h; removal was not much affected by the
pH in the range of 2.0–7.5 but it was scarcely removed
beyond pH 8.0.constant removal over 35°C.
HCl-treatment increased the adsorption capacity of the
shell removal efficiency was over 85%
Crab shell Chelating ability of chitosan makes it an excellent [319]
Nickel adsorbent for removing pollutants.The sorption occurred
primarily within 5 min.pH was also found not to be
prominent
Oyster shell Cupper and Cu2+ and Ni2+ were 49.26–103.1 and 48.75– [320]
nickle 94.3 mg g−1,BET surface area of 15.20 m2 g−1, an average
112
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pore size of 0.46 nm, and an average pore volume of

0.04 cm3 g−1. 60°C, contact time of 240 min
Crushed Hydrogen 12 mg-S g−1.specific surface area 0.25 m2 g−1 [321]
oyster shell sulfide
Oyster shell 1 year of operation under the overall hydraulic retention [322]
time of 3.5 days, the integrated system was found to be
highly effective in removing BOD5 (92.3%), N (85.7%),
P (98.3%) and total suspended solids (TSS) (94.4%)
compounds
Recover [323]
Oyster and Boron from 122 mg-B/g-treated shell, 95% within 10 min of reaction time by
clam shell wastewater adding 0.3 g. microwave hydrothermal treatment

Phosphate [324]
Hydrothermally removal 800°C for 1 h; hydrothermal annealing at 150°C for 12 h. 74% or 92%
modified phosphate removal after 2 or 4 h, respectively.
fumed silica
and pulverized
oyster shell
Brilliant Red −1
[325]
Waste shell of HE-3B dye, pH 1.2, shells dose of 40 g L , dye initial concentration of 50–
−1
Rapana batch 50 mg L ; the equilibrium time decreases with increasing of dye
gastropod
conditions concentration
Remove Basic pH 8.0. –OH, –CO3, and –PO4 functional groups were [134]
Bivalve type Green 4 (BG mainly responsible for the adsorption process.
sea shell 4) cationic42.33 mg g−1 at 303 K at 50 mg L−1 initial BG 4
dye concentration, temperature has strong influence on the
adsorption process equilibrium at 120 min.
Textile dyes [129]
Calcined Rhodamine B, pH 9 for biosorption of Rhodamine B and between 4 and 6 for
mussel shell Alizarin Red Alizarin Red S and Orange II. The equilibrium for all dyes in 60 min
S and Orange 45.67 mg/g for Rhodamine B, 39.65 mg/g for Alizarin Red S and
II
41.75 mg/g for Orange II.
Safranin dye [130]
Calcined 87.56% using 200 mg of biosorbent and 150 mg/L as safranin
mussel shell concentration and for a pH above 9.2
113
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Table 6 waste sea shells as catalyst
Catalyst Feedsto Calcination Reaction Methanol/ Catalys Yield and Ref

ck oil ratio t comment
amount
Tim Temp Tim Temp
e (°C) e (°C)
(h)
(h)
Combuste soybean 3 700 C 5h 25wt.% 70% [326
d oyster oil . ]
shell
Waste Palm 2 700 C 65°C 0.5:1 5 wt.% Re-employ [138
mud crab olein ed up to 11 ]
(Scylla times,
serrata) 500 rpm
shell
Waste palm 800 2–4h 2h 90% [327
shells of olein oil ]
egg,
golden
apple
snail, and
meretrix
venus
Waste palm 900 2h 0.54:1 4.9 [139
cockle olein wt.% ]
shell
(Anadara
granosa)
Waste mustard 900 65 ± 5° 9:1 3.0 wt. 93.3% [328
shell of oil C % ]
Turbonill
a striatula
Ba doped Ba 3h 65°C 6:1 1.0 wt >98% [140
CaO waste between % ]
shells of 0.5 to
Turbonilla
striatula 1.5 wt%
Biont and 25 500 3 3 wt% 97.5% [141
shell wt% KF ]
impregnat solution
ed with
KF
114
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Waste Palm oil 900 2 h. 6 60°C 8:1 3 wt % 93 ± 2.2%, [329

capiz 170 700 rpm ]
(Amusium mesh
cristatum) screens
shell
Snail shell Waste 7h 2.0 wt. 87.28%. [330
frying % ]
oil
Calcium Waste 3.5 1173 3h 333 K 6.03:1 3 wt % (>89%) [331
oxide frying K conversion ]
derived oil (>97%)
from .
Mereterix
mereterix
calcined
clamshell
Waste 1050 8 60°C 24:1 12 wt. 5 times [332
mussel % was ]
shell studied
94.1%,
Calcined 800 3 65°C 9:1 3.0% 98 % [333
river-snail ]
shell
Waste 1.5 70°C 12:1 5% 90% [334
freshwater h 7 cycles ]
mussel shell surface
area
(23.2 m2 g−
1)
115
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Table 7 Waste seashells as antimicrobial agent
Waste shell Antibacterial application Antibacterial characteristic

Scallop shell, oyster Physalospora piricola Non-treated oyster shell [335]
shell and their Nose (P. piricola) and powder antifungal activity at
pyrolyzed products Rhizoctonia solani Kühn 25,000 ppm; Heat treated at
(R. solany) 1050 C: antifungal activity at
500 ppm, 100% inhibition of
R. solany was observed;
oyster shell affect the

membrane permeability of the
fungus
Scallop shell powder Listeria monocytogenes, SSP (0.5%) exposure cleaned [336]
Staphylococcus aureus pathogens by 3–5 logs with a
and Escherichia coli 1 min exposure. Escherichia
O157:H7 coli O157:H7 was most
sensitive bacteria
Scallop shell heated Coliform 1000°C for 1 hour, 0.1 g [146]
powder dm-3 in 5 min removed
bacteria at 20°C
Oyster shell BOD, COD, TP and TSS Average removal efficiency [337]
removal of COD, BOD, NH3-N, TP
and TSS was 80.05%,
85.02%, 86.59%, 50.58% and
85.32%,
Oyster shell as Phosphorus Removal At pH 10, 90.6% of total [338]
biological aerated phosphorus was removed
filter medium
Oyster shell Extension of shelf life of 0.05% and 0.1% addition had [339]
Tofu a shelf life of above 2 days
longer than that prepared with
a single use of MgCl2; good
sensory evaluation and the
extension of shelf life.
Oyster shell powder Extension of shelf-life of 0.5% of powder enhanced the [340]
Kimchi shelf-life and the quality of
Kimchi for preservation and
consumption
Mussel shell waste E. coli and S. aureus, 400 and S. aureus was much [341]
doped with silver 600 ppm thicker, which played a
defense system and
protected the cell from
116
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penetration of silver ions

into the cytoplasm.
Oysters, calcined Total aerobic counts and E. Clams had the highest [342]
scallops, clams and coli activity; antibacterial activity
roll shells was due to the strong
alkalinity of aqueous
solutions of this calcined
calcium preparation.
CaO scallop-shell Escherichia coli, Listeria 0.05% CaO solution for 10 [343]
powder monocytogenes, and min cleaned pathogens 1-3
Salmonella typhimurium. log; the bactericidal action
was maintained for at least

24 h of storage, E.coli was
most sensitive bacteria
Heated scallop-shell Antibacterial activity of Antibacterial activity of the [143]
nano-particles nano-particles (20 nm) nano-particles was much
micro-particles (30 μm) higher than that of
micro-particles, >3 log
reduction for B. subtilis
spores was confirmed
following a 30 min treatment
at 5 mg/ml and 60°C.
Antibacterial 50–900 nm [144]
nanoparticles from nanoparticles,1200-1300°C
scallop for 3-4 hour
117
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Table 8 UV–Vis absorption spectral data and structural elucidation of naphthoquinone pigments
structures
Num Structure elucidation Molecu λM X Molar Refere

ber in lar mass nce
Fig.1 formula
1 5-hydroxy-1,4-naphthoquinone C10H6O 420nm(met 174.1 [344]
3 hanol ) 5
2 5,8-dihydroxy-1,4-naphthoquinone C10H6O / 190.1 [344]
4 5
3 6-ethyl-2,3,5,7,8-pentahydroxy- C12H9O 507, 312, 265.0 [153]
naphthoquinone 7 250 35
4 2-acetyl-3,5,6,8-tetrahydoxy-1,4- C12H7O 263.0, 263.0 [153]
naphthoquinone, 7 235.0 194
5 2,3,5,7-tetrahydroxy-1,4- C10H6O 269, 321, 221.0 [159]
naphthoquinone, 6 390, 489 068
6 2-acetyl-3,5,6,7,8-pentahydroxy-1,4- C12H7O 467, 350, 279.0 [153]
7 2,3,5,7,8-pentahydroxy-1,4- C10H6O 265, 325, 237.0 [159]
8 2,3,5,6,7,8-hexahydroxy-1,4- C10H6O 266, 345, 252.9 [159]
20 2,3,5,8-Tetrahydroxy-1,4-naphthoquion C10H6O 230,246,46 221 [151]
6 0,489
21 6-ethyl-2,3,5,8-tetrahydroxy-1,4-naphthoqui C12H11 217, 275, 266 [345]
none NO6 343,
480
22 Echinamine A C12H11 217, 233, 266 [345]
NO6 274,
345, 479
23 Echinamine B C12H11 217, 275, 266 [345]
NO6 343,
480
24 7,5’-anhydroethylidene-6,6’bis(2,3,7-trihydr C12H11 263,323,45 [150]
oxynaphthazarin) O13 1 483.0
2
26 Ethylidene-6,6’-bis(2,3,7-trihydroxynaphtha C12H11 266,342,48 264 [150,
zarin) O13 2,528 156,
346]
[154]
25 7,7’-anhydroethylidene-6,6’-bis(2,3,7 C12H11 340,480 484.0 [150,
trihydroxynaphthazarin) O13 2 154,
118
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156,
346]
27 2,3,5,6,8- pentahydroxy-1,4- 238 [154]
naphthoquinone
3-acetyl-2,7-dihydroxy-6-methylnaphthazari 261,324,53 278 [347]
n 5
Spinochrome E sulphate C10H5O 475,330,26 332.8 [153]
derivative 11S 0
Spinochrome B sulphate C10H5O 420, 300, 300.8 [153]
derivative 9S 262
Aminopentahydroxy naphthoquinone C10H6O 471, 369, 252.1 [153,
7N 273 157]
119
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Table 9 Method of extraction, purification and identification of spinochrome and HPLC
conditions
Source Extraction Purification Mobile grad Flo wavel Colu Refe

method and phase ient w ength mn renc
detection rat e
method e
Psammechinus Solid LC-MS A: 20 0-30 0.2 280/3 C18 [153
miliaris phase mmol/L min ml/ 65/52 ]
extraction formic acid 5%- mi 0 nm

B:20 40% n
mmol/L B
formic acid
acetonitrile
Glyptocidariscr Ethanol-H Macroporous A: 0-20 0.2 475 100m [159
enularis Cl-aqueou resin acetonitrile min ml/ nm m×2. ]
Strongylocentro s solution UPLC B: 0.1% 10-5 mi 1 mm
tusintermedius Q-TOFMS formic acid 0% n C18
A 1.7
μm
Strongylocentro Ethanol-H Macroporous [157
tusnudus Cl-aqueou resin ]
s solution UPLC
Q-TOFMS
Strongylocentro Solvent TLC-HPLC– A:0.2% 0-3 0.3 340 100m [156
tusdroebachiens extraction DAD–MS formic acid min ml/ nm m×2. ]
is B:acetonitril 5%B mi 1 mm
e 0.2% 0-40 n C18,
formic acid min 3.5
5%- μm
40%
B
40-4
5
min
40%
B
Purple sea Solvent RP-HPLCwit A:0.1% 50% 0.5 340 [152
urchin extraction hDAD,LC-M formic acid A ml/ nm ]
S B:MeOH:ac and mi 520
etonitrile(5: 50% n nm
9) B
120
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with
isocr
atic
eluti
on
Scaphechinus Solvent HPLC [155
mirabilis extraction ]
Strongylocentro Solvent SephadexLH [160
tus extraction -20 column ]
franciscanus chromatogra
phy
121
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Table 10 Echinochrome A in solution stability
Condition Effect on autoxidation

Aerobic conditions pH <= 7 Stable
pHvalues above 8.5-9.0 Rate of autoxidation increased
EDTA and SOD and Vitamin C Rate of autoxidation decreased
Anaerobic with the glucose/glucose Stable
oxidase system(any pH)
Calcium ions Rate of autoxidation sharply increased
EDTA added in excess of CaCl2 Stopped the autoxidation
Mg2+, Ba2+, Sr2+, Co2+, Cd2+, Mn2+, No effect on the echinochrome A

Pb2+,Fe2+ autoxidation in the pH range from 7 to 9
Cu2+ and Hg2+ ions Oxidized echinochrome A directly in
redox reactions
122
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Table 11 Distribution of spinochromes in echinoids
Source Structure in Fig 1 Reference

Strongylocentrotus 3.5.8 [160]
franciscanus
Scaphechinus mirabilis 3.5.7 [155]
Toxopneustespileolus 4.5.6 [158]
Strongylocentrotus 4.5.6.7.8 [156, 346]
droebachiensis 25.20.21
Strongylocentrotus nudus 3.4.5.6.7.8.18.23 [157, 347, 348]
Aminopentahydroxynaphthoquinone
Acetylaminotrihydroxynaphthoquinone3-acety
l-2,7-dihydroxy-6-methylnaphthazarin
Glyptocidaris crenularis 5.7.8 [159]
Strongylocentrotus 4.5.6.7 [159]
intermedius
Psammechinus miliaris Spinochrome E sulphate derivative [153]
3.4.5.6.8
Aminopentahydroxynaphthoquinone
Diademaantillarum 3 [154]
Temnopleurus toreumaticus 3.4.5.6.7.8 13 [154]
Spatangus purpureus Ethylidene-3,3’-bis(2,6,7-trihydroxynaphthaza [154]
rin) and its anhydro-derivative
Scaphechinus 20,21 [151]
mirabilis
123
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Table 12 Antioxidant activity and mechanism of PHNQ
Source Par Assay Pigments Mechanism Refer

t ence
Glyptocid Spi DPPH / As losing hydrogen atoms, [159,
aris ne PHNQ may become 165,
crenularis Sh naphthosemiquinone 166,
Strongylo ells As the intermediate product 169]
centrotus and and naphthotetraketone as the
intermedi spi final reaction product
us nes
Anthocida
ris
crassispin
a
Strongylo
centrotus
intermedi
us
Strongylo
centrotus
droebachi
ensis
Anthocida Sh O2- Echinochrome A Echinochrome-naphthosemiqu [162,
ris ells inone-naphthotetraketone 169]
crassispin and
a spi
nes
Anthocida Echinochrome A 1,2,3,4-tetraketones are [167,
ris >spinochrome D formed from echinochrome A 168]
crassispin >spinochrome C < NBT and spinochromes D and E via
a <trimethoxyechinochrome O2.-induced oxidation of their
A OH-groups in the 2nd and 3rd
positions.
Strongylo Spi Reduci / Anion form transmits two [157]
centrotus nes ng electrons consecutively to
nudus power ferric ion and becomes
naphthotetraketon as the final
reaction product
Anthocida Hydrox / Could not be competitively [169]
ris yl inhibited by the pigments
crassispin radical
a scaven
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ging
activity
Strongylo Iron Echinochrome A Formation of iron(III)-PHNQ [165]
centrotus Bindin and iron(II)-PHNQ complexes
intermedi g
us
Glyptocid Spi Fe2+ch / PHNQ had metal ion chelating [159]
aris ne elating capacity, but weaker than
crenularis Capacit EDTA
Strongylo y
centrotus
intermedi
us
Strongylo Iron-In Ech> >E>BHT>α-Tocoph / [165]
centrotus duced erol>D>EDTA>TEech
intermedi Liposo
us me
Oxidati
on
Lipid / / [349]
peroxid
ation
Strongylo spi Inhibiti / Result of a combination of [157]
centrotus nes on of iron chelation and free-radical
nudus lipid scavenging activity
peroxid
ation in
rat liver
homog
enate
Strongylo spi Protecti / Inhibit t-BOOH-induced [157]
centrotus nes on cytotoxicity by scavenging
nudus against ROS and preventing lipid
oxidati peroxidation
ve
stress
125
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Table 13 Bioactivities of PHQN pigments
Source Pigments Function Reference

Histochrome Echinochrome Anti-inflammatory [174]
Histochrome Echinochrome Cardioprotective activity [176-178]
Histochrome Echinochrome Retinoprotective activity [179]
Green Sea Antiallergic activity [175]

Urchin
Salmacisvirgulata Shells Antimicrobial activity [147]
Spinochrome A Inhibitory activity on [182]

Echinochrome dopamine-β-hydroxylase
A
Spinochrome A Inhibitory activity on [181]
Echinochrome bovine adrenal tyrosine
A hydroxylase.
126
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Table 14 Astaxanthine extraction method
Extraction Solvent Optimized condition Carotenoid Reference

method extraction yield
Solvent Acetone (Ac), methanol 60% hexane in solvent 43.9 μg/g waste [188]
extraction (Meth), ethanol mixture, solvent mixture
(EtOH), isopropyl to waste ratio of 5:1 in
alcohol (Isop), ethyl each extraction and
acetate (EA), ethyl three extractions

methyl ketone (EMK),
petroleum ether (PE),
and hexane (Hx)
individually and to a
mixture of acetone and
hexane
Soxhlet Hx; EtOH; Ac; (5 g) was packed inside HxIPA： [189]
Isop and Isop:Hx a cartridge and 198μg/g extract
(50:50, v/v) transferred
to a 250mL extractor
device and submitted to
8-h recycling
extractionwith
150mLsolvent at boiling
temperature
Maceration Hx,EtOH,Ac, Isop, and 25 g of the sample Hx：188μg/g [189]
the binary Hx:Isop into 100mL of selected extract
(50:50, v/v) organic solvent for five
days at room
temperature,
light protection and one
daily manual agitation.
Hot and Sunflower oil and Mixing 10 g of raw OilC-Sunflower [189]
cold oil soybean oil material ： 5.18μg/g
extraction with 40mL of vegetable extract
oil in a 250mL flask
(light protected),
submitted to hot plates
with 2-h agitation period
at room temperature
(OilC) or at 70 ◦C(OilH
127
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Vegetable refined sunflower in sunflower oil were Sunflower oil: [192]

oil oil,groundnut oil, determined to be oil 27.56μg/g
extraction gingelly oil, mustard level to waste of 2, waste
oil, soy oil, coconut oil temperature of 70 C and
and rice bran oil heating time of 150min
128
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Table 15 Matrix Proteins extracted from shells of shellfish
Protein name Species MW (kDa) Reference

AP7 Haliotis 7.565 Michenfelder et al.,
2003
Aspein Prisms Pinctadafucata 41 Tsukamoto et al.,
2004
Prismalin-14 Pinctadafucata 11.89 Suzuki et al., 2004
Asprich family Atrinarigida 20-30 Gotliv et al., 2005
Calprismin Pinna nobilis 38 Marin et al., 2005
MSI7 Pinctadafucata 7 Zhang et al., 2003

Casparti Pinna nobilis 38 Marin et al., 2001,
2005
MSP-SC Patinopectenyessoensis 14 [231]
P14 Pinctadafucata 14.5 [350]
MSP-1 Patinopectenyessoensis 74.5 [230]
129
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Figure 1 Potential products from marine shell waste and current direction of research
Crustacean shell
Deproteinization
Demineralization
α-chitin
Deacetylation
α-chitsoan
130
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Figure 2 Chitin and chitosan production by chemical and microbiological method
131
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Figure 3 Structures of spinochrome pigments in echinoids
Cpd R1 R2 R3 R4 R5 Name
No.
1 H H H H H Juglone (5-hydroxy-1, 4-naphthoquinone)
2 H H H H OH Naphthazarin (5, 8-dihydroxy-1, 4-naphthoquinone
3 OH OH Eth OH OH Echinochrome A (6-ethyl-2, 3, 5, 7, 8-pentahydroxy-
naphthoquinone )
4 OH Ac H OH OH Spinochrome A（2-acetyl-3,5,6,8-tetrahydoxy-1,4-
naphthoquinone）
5 OH OH H OH H Spinochrome B (2, 3, 5, 7-tetrahydroxy-1, 4-
naphthoquinone)
6 Ac OH OH OH OH Spinochrome C (2-acetyl-3, 5, 6, 7, 8-pentahydroxy-1,
4-naphthoquinone)
7 OH OH OH H OH Spinochrome D (2, 3, 5, 7, 8-pentahydroxy-1,
4-naphthoquinone)
8 OH OH OH OH OH Spinochrome E (2, 3, 5, 6, 7, 8-hexahydroxy-1,
4-naphthoquinone)
9 H H Eth H H 6-Ethyl-5-hydroxynaphthalene-1,4-dione
10 OH H Ac OH H 6-Acetyl-2,5,7-trihydroxynaphthalene-1,4-dione
11 OH OH Eth OH H 6-Ethyl-2,3,5,7-tetrahydroxynaphthalene-1,4-dione
12 OH OH Ac OH H 6-Acetyl-2,3,5,7-tetrahydroxynaphthalene-1,4-dione
132
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13 OH Ac OH OH H 3-Acetyl-2,5,6,7-tetrahydroxynaphthalene-1,4-dione
14 OH H H H OH 2,5,8-Trihydroxynaphthalene-1,4-dione
15 OH H Eth H OH 6-Ethyl-2,5,8-trihydroxynaphthalene-1,4-dione
16 OH H Ac H OH 6-Acetyl-2,5,8-trihydroxynaphthalene-1,4-dione
17 OH Ac H OH OH 2-Acetyl-3,5,8-trihydroxynaphthalene-1,4-dione
18 OH H H OH OH 2,5,7,8-Tetrahydroxynaphthalene-1,4-dione
19 OH Ac H OH OH 2-Acetyl-3,5,6,8-tetrahydroxynaphthalene-1,4-dione
20 OH OH OH OH OH 2,3,5,6,7,8-Hexahydroxynaphthalene-1,4-dione
21 OH OH Eth H OH 6-Ethyl-2,3,5,8-tetrahydroxynaphthalene-1,4-dione
22 NH2 OH Eth OH OH 2-Amino-6-ethyl-3,5,7,8-tetrahydroxynaphthalene-1,4-dione
23 NH2 OH OH Eth OH 2-Amino-7-ethyl-3,5,6,8-tetrahydroxynaphthalene-1,4-dione
24
7,5’-Anhydroethylidene-6,6’bis(2,3,7-trihydroxynaphthazarin)
25
7,7’-a\Anhydroethylidene-6,6’-bis(2,3,7 trihydroxynaphthazarin)
133
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26
Ethylidene-6,6’-bis(2,3,7-trihydroxynaphthazarin)
27
2,3,5,6,8-Pentahydroxynaphthalene-1,4-dione
134
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Echinochrome A Naphthosemiquinone Napthotetraketone

Figure 4 A) Mechanisms of DPPH scavenging activities of PHNQ (with spinochrome E as an
example), B) Super anion-radical scavenging activity of PHNQ (with Echinochrome A as an
example), C) reducing power activity of PHNQ (with spinochrome E as an example), D) metal ion
chelation activity of PHNQ (with spinochrome E as an example)
135
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Critical Reviews in Environmental Science and

ISSN: 1064-3389 (Print) 1547-6537 (Online) Journal homepage: http://www.tandfonline.com/loi/best20

Marine shells: potential opportunities for

To link to this article: http://dx.doi.org/10.1080/10643389.2016.1202669

Accepted author version posted online: 20

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Marine shells: potential opportunities for extraction of functional and health-promoting

NGd, RANDY CHI FAI CHEUNGd, ALAA EL-DIN A. BEKHITa*

Hong Kong, China

amin.shavandi@postgrad.otago.ac.nz; Ph: ++64 3 479 5463,

aladin.bekhit@otago.ac.nz; Ph: ++64 3 479 4994

Waste shells, Chitin, Hydroxyapatite, Pigment

production is utilized in further processing. The large quantity of processed aqua-cultured

the sustainability of shellfish production.

processing according to animal by-product regulations such as EC Regulation 1069/2009. After

for scaling up the processes.

Chitin, chitosan from waste shells

oligomer and is composed of α-(1-4)-linked 2-acetamido-2-deoxy-D-glucose [6-9]. Chitin is

lipid, meat offcuts, calcium carbonate and pigments.

Properties and applications of chitin

it many biomaterial applications as a result of the properties of bioadhesion, increased

absorption, transfection efficiency, anti-hypercholesterolemic, anti-microbial, anti-inflammatory

various structures depending on usage, including microspheres, paste, membranes, sponges,

fibres, and porous scaffolds (12,56).

by chemical processing has a low quality due to non-homogeneous depolymerisation and

being unusable, that otherwise could be used in other applications [31-33].

Microbial fermentation is an alternative to the chemical process for demineralization and

application of a protease-producing bacterial species such as Serratia or Aspergillus and an

organic acid producing bacterial species such as Pseudomonas or lactobacillus to depolymerize

compared a biological method involving fermentation using Lactobacillus salvarius,

chitin preparation that could be suitable for some use applications.

plantarium was effective in demineralization process to produce chitin preparations.

Extraction of calcium compounds from biogenic resources

a summary of major applications of shell waste includes; applications in tissue engineering;

discussed with emphasis on the properties required for each application.

Use of shell waste as potential calcium supplement

induce parotid swelling .

Tissue engineering applications

The calcium carbonate component of shells can be converted to calcium-based compounds

remaining 30-40% organic component consists of 90 % type Ӏ collagen and 10%

non-collagenous proteins such as glycosaminoglycans. Hydroxyapatite (HA) and beta tricalcium

therefore provides excellent osteoconductive, bioactive and biocompatible properties (29,36,38).

disadvantages, HA is widely accepted as a biomaterial for bone regeneration (37).

enhance the mechanical and osteoconductive properties of implants. Compared to

Osteoconduction is improved by the small particle size of TCP and an interconnected

biomedical application are discussed.

Methods for the conversion of shell material to HA

of aragonite. This calcium carbonate form can be converted hydrothermally (140–260°C) to

hydroxyapatite (HA) using a phosphate solution by a solid-state topotactic ion-exchange reaction

The following equation summarizes the process

10CaCO3 + 6(NH4)2HPO4 + 2H2O Ca10(PO4)6(OH)2 +6(NH4)2CO3 + 4H3CO3 (1)

as at 700 to 800°C, decomposition of CaCO3 to CaO occurs. After removal of organics,

material partially to HA without the requirement of a crystalline structure, then lower

temperatures and pressures for a shorter time can be used.

adjusted to 10-11 using NH4OH to enhance the crystal growth.

sintering step at up to 1000°C in a furnace to complete the conversion to HA [93]. In another

converted to bioresorbable Mg-substituted tricalcium phosphate (β-TCMP) by a hydrothermal

hydrothermal-microwave assisted processing and conventional microwave processing.

combustion agent. The combustion synthesis method is based on a self-sustained heating

growth depends on heat distribution, the inhomogeneous heat distribution of conventional

Therefore, microwave assisted production of HA is a promising heating method to achieve HA