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A Novel Enzymatic Method for Preparation and Characterization of Collagen Film from Swim Bladder of Fish Rohu ( Labeo rohita )


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A novel enzymatic method for extraction and preparation of fish collagen from swim bladder revealed the occurrence of α, β and γ bands with approximately 12.1 g/100g collagen corresponding to 89% of collagen and thus confirmed the nativity and purity of the fish collagen. FT-IR studies confirmed the retention of all three amide bands of I, II and III, and triple helixcity. UN-crosslinked and UV-crosslinked fish collagen membrane records a very high temperature of helix denatura-tion at 197˚C and 215˚C, shrinkage temperature at 50˚C ± 3.2˚C and 62˚C ± 2.7˚C and tensile strength at 16.89 ± 2.5 and 120.02 ± 1.0 Kg/cm 2 respectively. Fish collagen matrix promoted NIH 3T3 and L6 cellular growth and proliferation. The study indicates that availability of pure fish col-lagen could replace bovine collagen in tissue engineering applications.
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Food and Nutrition Sciences, 2015, 6, 1468-1478
Published Online November 2015 in SciRes.
How to cite this paper: Sripriya, R. and Kumar, R. (2015) A Novel Enzymatic Method for Preparation and Characterization of
Collagen Film from Swim Bladder of Fish Rohu (Labeo rohita). Food and Nutrition Sciences, 6, 1468-1478.
A Novel Enzymatic Method for Preparation
and Characterization of Collagen Film from
Swim Bladder of Fish Rohu (Labeo rohita)
Ramasamy Sripriya1, Ramadhar Kumar2
1Sree Balaji Medical College & Hospital, Bharath University, Chennai, India
2Department of Life Sciences (R&D), Datt Mediproducts Ltd., Gurgaon, India
Received 27 October 2015; accepted 21 November 2015; published 24 November 2015
Copyright © 2015 by authors and Scientific Research Publishing Inc.
This work is licensed under the Creative Commons Attribution International License (CC BY).
A novel enzymatic method for extraction and preparation of fish collagen from swim bladder re-
vealed the occurrence of α, β and γ bands with approximately 12.1 g/100g collagen corresponding
to 89% of collagen and thus confirmed the nativity and purity of the fish collagen. FT-IR studies
confirmed the retention of all three amide bands of I, II and III, and triple helixcity. UN-crosslinked
and UV-crosslinked fish collagen membrane records a very high temperature of helix denatura-
tion at 197˚C and 215˚C, shrinkage temperature at 50˚C ± 3.2˚C and 62˚C ± 2.7˚C and tensile
strength at 16.89 ± 2.5 and 120.02 ± 1.0 Kg/cm2 respectively. Fish collagen matrix promoted NIH
3T3 and L6 cellular growth and proliferation. The study indicates that availability of pure fish col-
lagen could replace bovine collagen in tissue engineering applications.
Fish, Swim Bladder Collagen, FT-IR, DSC, Cytocompatibility
1. Introduction
Current work deals with a novel enzymatic method for extraction and preparation of fish collagen (FC) from
discarded swim bladder of Rohu (Labeo rohita). Rohu is a species of the carp family, found in rivers in South
Asia. It is an omnivore. It reaches a maximum length of 2 m (6.6 ft) and a weight of about 110 kg (240 lb). It
may reach even greater weights in the northern part of Nepal. It is an important aquacultured freshwater species
in South Asia. Rohu is very commonly eaten in India, Bangladesh, Nepal, and Pakistan. It is also a very popular
food fish in Iraq.
R. Sripriya, R. Kumar
Collagen is the major protein of connective tissues and the most abundant protein of animal origin [1]-[3]. For
industrial purposes, collagen is extracted mainly from skins and bones of cattle and pigs skins and has been
widely used in the pharmaceutical, food, healthcare, and cosmetic industries [4] [5]. However, the incidence of
diseases such as bovine spongiform encephalopathy (BSE or the popular “mad cow”), and foot and mouth dis-
ease has raised concerns about safe use of animal proteins. In addition, the collagen extracted from pigs cannot
be used due to religious barriers. For this reason, interest in alternative sources of collagens, which present no
health and social risks, has expanded [6]. The other source found for collagen extraction includes skin, bone, fin
and scales of fresh water and marine fish, chicken skin, bull frog skin, squid skin, octopus arms and marine
sponge [7]-[9]. Among collagen alternatives, fish provided the best source of raw material because of its high
availability, no risk of disease transmission, and no religious barriers [10]. Collagens from fish skin or swim
bladders which are the waste products in fish processing, may be good substitutes, because of their safety and
solubility in neutral salt solutions and dilute acids. Fish collagen is absorbed up to 1.5 times more efficiently into
the body, meaning that it has superior bioavailability over bovine or porcine types. Because it is absorbed more
efficiently and enters the bloodstream more quickly, it is considered to be the best source of collagen for phar-
maceutical applications. Moreover, 75% of the total weight of fish is discarded as wastes in the form of skins,
bones, fins, heads, guts, scales and bladder during processing [11]. If we can reutilize fish processing wastes for
the production of collagen-like biomaterials, it may increase the economic value of the fish and fish processing
industry. The parts of collagen that are attributed to its immunogenicity namely telopeptides, are eliminated in
the process of atelocollagen production. Therefore atelocollagen possesses little immunogenicity [12]. Chemical
treatments confer remarkably high strength and stability to collagen matrix but may result in potential cytotoxic-
ity or poor biocompatibility [13], while physical treatment has no cytotoxicity and can provide sufficient stabili-
ty [14] [15]. The properties of biocompatibility and biodegradation of fish atelocollagen are some of the features
that enable the fish collagen to be considered as suitable for the scaffold in tissue engineering, though these
phenomena strongly depend on the procedures for crosslinking [16]. If substantial amounts of collagen could be
obtained from fish wastes (scale, skin and bone), they would provide an alternative source to bovine collagen in
food, cosmetics and biomedical materials.
There are studies that have focused on extraction procedures and characterization of fish-based collagens [5]
[9]. In general, the collagen from fish skins and bladder was extracted by soaking them in NaOH for a minimum
duration of 36 hrs with stirring followed by defatting using alcohol solution. After defatting the tissue shall be
suspended in acid and enzyme treatment shall be given for 3 days. Finally the fish collagen shall be collected by
salt precipitation followed by centrifugation. Therefore the extraction procedures so far reported for collagen
extraction was time-consuming taking a minimum of three days for homogenization, two more days for swelling
in NaOH and further a minimum of 2 - 3 days for enzyme treatment which is followed by salt precipitation and
centrifugation. In contrast, the extraction method reported in the current study is a simple and novel method de-
veloped to extract pure fish collagen from the swim bladder. The pure fish collagen thus obtained was reconsti-
tuted in the form of a film and crosslinked by UV irradiation to enhance its thermo-stability. The fish collagen
from the swim bladder was further characterized for its physico-chemical and biological properties. Its biologi-
cal properties are reported for the first time to validate the use of swim bladder extracted collagen application as
a biocompatible polymeric material with potential applications as injectables in coating cardiovascular prosthe-
ses, support for cell growth and in systems for wound dressing with controlled drug delivery.
2. Materials and Methods
2.1. Extraction of Fish Collagen
The swim bladders were collected from the fresh water fish Rohu (Labeo rohita) from the local fish market,
Chennai, India and they were cut into very fine pieces of random sizes with surgical blade and were suspended
overnight in antibiotic-antimycotic solution (Invitrogen). The fish collagen was thus extracted from the fish
swim bladder tissues by following a procedure reported earlier with slight modification [17] (Figure 1). In brief,
the pieces of the swim bladder were washed by using 0.3% w/v of Triton X-100, a non-ionic surfactant and were
suspended in 0.3% w/w of sodium peroxide solution for 3 hrs followed by washing with Milli Q water. The re-
sultant tissue was further treated with enzyme pepsin (0.3% w/w in distilled water, pH 2.5 - 3.0) for 10 - 12 hrs.
Pepsin solubilized fish collagen was further purified by differential salt precipitation (NaCl up to 2.5 molL1),
R. Sripriya, R. Kumar
Figure 1. Flow chart of the procedure adopted for extraction of collagen from swim blader.
followed by centrifugation, washing, and lyophilization. The obtained fish collagen (without any further purifi-
cation) was used for further studies viz. biological, physico-chemical characterization and membrane preparation.
All the processes were performed at 4˚C and used ice cold water wherever required.
2.2. Preparation of Fish Collagen Film
The lyophilized fish collagen was dissolved in Millipore water (0.06 µs purity) acidified to pH 3.5 using HCl to
get a pure collagen solution. The fish collagen solution was degassed and the clear solution was allowed to dry
in Teflon troughs kept in a dust-free chamber and maintained at a temperature of 20˚C ± 5˚C. The fish collagen
membranes were crosslinked by UV (NUV) radiation (wavelength, 365 nm) for 3 hrs.
2.3. Estimation of Hydroxyproline
Hydroxyproline of collagen from the swim bladder of the fish collagen was estimated to quantify the collagen
purity by using the method of Neuman & Logan [18] which gives a correlation between collagen content and
hydroxyproline content.
2.4. Gel Electrophoresis
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the fish collagen samples was
done by using Laemmli method [19]. The lyophilized fish collagen samples were dissolved in sample buffer and
incubated for 30 minutes at 65˚C. The supernatant sample without residues was loaded onto 8% separating gel.
R. Sripriya, R. Kumar
The SDS-PAGE gel was stained with 0.25% Coomassie brilliant blue R-250 containing 25% ethanol and 10%
acetic acid and destained with 5% methanol and 7.5% acetic acid.
2.5. Differential Scanning Calorimetric (DSC) Studies
DSC studies on the fish collagen film were performed by using universal V4.4A TA instruments at 24˚C and
65% R. H. Fish collagen membranes (un-crosslinked & crosslinked) were shielded in aluminium containers and
heated at a rate of 10˚C per minute from 0˚C to 300˚C in N2 atmosphere and the thermograms were recorded.
2.6. Fourier Transform Infrared Spectroscopy (FT-IR) Studies
Collagen-potassium bromide pellets was prepared by powdering lyophilized fish collagen with potassium bro-
mide and further subjected to FTIR studies by using a Thermo Nicolet avatar 320 FTIR spectrometer (Nicolet
Instrument Con., Madison, WI). The spectra of the fish collagen were recorded in the range of 400 to 4000 cm1
at 25˚C.
2.7. Studies of Shrinkage Temperature
The shrinkage temperature of the fish collagen membranes was measured in micro shrinkage meter fitted with a
field microscope. Fish collagen membrane cut into the size of 1 sq·cm, in distilled water was placed on the wet
surface (distilled water) of a micro slide and the temperature of the slide was raised slowly by 1˚C/min and the
shrinkage of the fish collagen membrane was determined through the microscope. The temperature at which the
fish collagen membrane shrinks approximately half to its original size was taken it as the shrinkage temperature.
2.8. Studies of Tensile Strength
The fish collagen membrane was cut into 16 mm dumbbells with 5.15 mm inner width and immersed in distilled
water for 30 min and the tensile force were applied at an extension rate of 10 mm/min. The ends of the fish col-
lagen membrane were held by pneumatic grips (40 psi grip pressure) and the tensile strengths of the collagen
membranes were tested by Instron series II Automated Materials Testing System.
2.9. Biological Characterization and Culture of Cell Lines
Biological characterization of the fish collagen was performed by evaluating the degree of cell proliferation,
growth and morphological examinations of mouse embryonic fibroblasts (NIH 3T3) and L6 myocytes cell lines.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay was performed to evaluate the degree of cell
proliferation [20] as viable cells with active metabolism convert MTT into a purple colored formazan product.
The quantity of formazan (presumably directly proportional to the number of viable cells) is measured by re-
cording changes in absorbance at 630 nm using a plate reading spectrophotometer [21]. All cell lines (NIH 3T3
& L6 myocytes) were obtained from National Centre for Cell Science (NCCS), Pune, India and the cell cultures
were maintained in DMEM with 10% fetal calf serum (FCS) supplemented with penicillin (120 units/ml), strep-
tomycin (75 mg/ml), gentamycin (160 mg/ml) and amphotericin B (3 mg/ml) at 37˚C with 5% CO2.
2.10. Experimental Design for Cytoproliferative Effects of Collagen Matrix
The cytoproliferative effects of the fish collagen matrix and polystyrene were determined. Polystyrene cell cul-
ture plates were coated with 0.5% fish collagen solution and dried under a laminar air flow hood followed by
UV sterilization. The uncoated wells were used as control. The cells were seeded at the density of 2 × 104 per
well and were incubated at 37˚C in a humidified atmosphere containing 5% CO2. In all cell culture conditions,
the fresh cell culture medium was renewed every day. After 24 and 48 hrs of incubation, the supernatant of each
well was replaced with MTT diluted in serum-free medium and the cell culture plates were incubated at 37˚C for
3 hrs. After aspirating the MTT solution, acid isopropanol (0.04 N HCl in isopropanol) was added to each well
and pipetted up and down to dissolve all of the dark blue crystals and then left at room temperature for a few
minutes to ensure all crystals are dissolved. Finally, absorbance (optical density) was measured at 630 nm by
using a UV spectrophotometer. Each experiment was repeated at least three times. The sets of three wells for the
MTT assay were used for each experimental variant.
R. Sripriya, R. Kumar
2.11. Statistical Analysis
The results of the current study are expressed as mean ± standard error of the mean (SEM). Data obtained from
the current study were analysed by student’s t-test and differences at the 95% level were considered to be statis-
tically significant.
3. Results
3.1. Hydroxyproline Content of Fish Collagen
The hydroxyproline content of the extracted fish collagen protein mass was found to be approximately 12.1
g/100g collagen which corresponds to more than 89% purity of collagen in the extracted mass.
3.2. Gel Electrophoresis (SDS-PAGE) of Fish Collagen
The extracted pure fish collagen from swim bladder was characterised by SDS-PAGE (8%) gel. The electro-
phoretic pattern of the protein sample of the fish swim bladder (Labeo rohita) is shown in Figure 2. The fish
collagen constituted largely of α with a considerable amount of β (dimer) and γ (trimer) band. Two α bands cor-
responding to α1 and α2, which were the unfolding polypeptide chains of the triple helix of the fish collagen.
3.3. Differential Scanning Calorimetric (DSC) Studies
Both un-crosslinked and UV crosslinked fish collagen membrane obtained in the current study were analyzed
for their thermal stability by DSC for which the samples were heated from 0˚C to 300˚C and the thermograms
recorded and illustrated (Figure 3 and Figure 4). In the case of un-crosslinked fish collagen, the endothermic
peak observed at 84˚C could be attributed to water loss/evaporation and the occurrence of another peak around
197˚C reflected the melting temperature of the fish collagen due to helix denaturation (Figure 3) whereas in UV
crosslinked fish collagen membrane, the water loss was observed near 110˚C and helix denaturation occurred
around 215˚C (Figure 4).
3.4. Fourier Transform Infrared Spectroscopy (FT-IR) Studies of Fish Collagen Matrix
The FT-IR spectrum (Figure 5) of the fish collagen obtained in the present study showed the presence and re-
tention of all major three amide band viz. I, II, and III at the region of 1636 - 1661 cm1, 1549 - 1558 cm1 and
Figure 2. SDS-PAGE electrophoretogram of the fish colla-
gen sample shows the occurrence of band pattern of α, β and
γ isomers. No other major protein bands were observed.
R. Sripriya, R. Kumar
Figure 3. Differential scanning calorimetric scan of fish collagen recorded using calorimeter
(DSC 204) with collagen membrane shielded in aluminium containers in the temperature
range of 5˚C and 200˚C in the N2 atmosphere is showed in the figure. The DSC scan shows
an endothermic peak at 84˚C attributed to water loss/evaporation. The occurrence of another
peak around 197˚C reflected the melting temperature of the fish collagen due to helix dena-
Figure 4. Differential scanning calorimetric Scan of UV-crosslinked fish collagen recorded
using calorimeter (DSC 204) with collagen membrane shielded in aluminium containers in
the temperature range of 5˚C and 200˚C in the N2 atmosphere is shown in the figure. The
DSC scan records endothermic peak at 110˚C attributed to water loss and helix denaturation
around 215˚C.
R. Sripriya, R. Kumar
Figure 5. Fourier transform infrared spectrum of fish collagen recorded using collagen films through Nicolet 2DDKB FTIR
spectrometer at 25˚C using OMNIC (Version 6.0) software is shown in the figure. The FT-IR spectra show the secondary
structure of the fish collagen proteins as amide I, amide II and amide III band. The ratio of IR absorption intensity between
1237 cm1 (amide III) and 1450 cm1 (amide II) band was approximately equal to 1.0, which confirms the triple helical
structure of collagen.
1200 - 1300 cm1 which in turn validated the integrity of the proteins in the conformation of the collagen mole-
cules. While the peak situated in the range of 670 - 640 cm1 in the fish collagen can be attributed to the C-S
stretching vibrations. The protein band occurring close to 1450 cm1 is probably associated with C-H bending
modes and the amide-A band (NH stretching), was observed at 3400 cm1.
3.5. Shrinkage Temperature Analysis
The shrinkage temperature of the un-crosslinked fish collagen membrane records at 50˚C ± 3.2˚C whereas the
UV crosslinked collagen showed shrinkage temperature at 62˚C ± 2.7˚C.
3.6. Tensile Strength Analysis
The mean tensile strength of un-crosslinked fish collagen membrane records at 16.89 ± 2.5 Kg/cm2 whereas, the
UV crosslinked fish collagen membrane shows 120.02 ± 1.0 Kg/cm2 executing the increased strength of the
biomaterial fish collagen membrane after crosslinking.
3.7. Cytoproliferative Effects of Fish Collagen Matrix
In the present study, we used the mouse embryonic fibroblasts (NIH 3T3) and rat myoblasts (L6) to observe the
degree of cellular growth and proliferation on fish collagen matrices and polystyrene surface. Interestingly, we
found that the rate of proliferation of both L6 and NIH3T3 was relatively more in comparison to control (Figure
6) with good morphology of cells in the cell culture as observed under a phase contrast inverted microscope
(Figure 7). Moreover, L6 myoblasts proliferation and differentiation into myotubes was more pronounced on
collagen matrix (data not shown).
R. Sripriya, R. Kumar
Figure 6. Degree of cellular proliferation of both L6 and NIH3T3 evaluated
by MTT assay and results were evaluated after 24 and 48 hrs of incubation as
shown in the figure. The rate of proliferation of both L6 and NIH3T3 was
relatively more in comparison to control. Bars represent mean ± SEM of five
wells (p < 0.05).
Figure 7. Microscopic images (×200) of NIH 3T3 fibroblasts (A) on polys-
tyrene (control) substratum, (B) on fish collagen substratum; L6 myoblasts
(C) on polystyrene substratum, (D) on fish collagen substratum. Cellular pro-
liferation with good morphology of cells in the cell culture as observed under
a phase contrast inverted microscope. The scale bar used is 30 µm.
4. Discussion
On the basis of the amino acid analysis of the fish collagen, the quantity of hydroxyproline is present to the ex-
tent of 12.8 g/100g of collagen [22] or approximately 13% of its weight [23]. The current study showed hy-
droxyproline value which is comparable with reported values indicating that the collagen extracted from the fish
swim bladder is of high purity. The occurrence of the pattern of α, β and γ isomers of the fish collagen and no
other major protein bands were observed indicating the nativity of the extracted fish collagen. The pattern of α, β
and γ isomers of the fish collagen from the current study compares favourably with the electrophoretic pattern
reported earlier [16], for the fish collagen from swim bladder of catfish (Tachysurus maculatus). DSC values
obtained in the present study are relatively higher than the DSC values of tropical fishes: i.e. fish collagen from
fish such as Arius parkeri (Gurijuba), Cynoscion acoupa (Pescada Amarela) and Cynoscion leiarchus (Pescada
R. Sripriya, R. Kumar
Branca) showed difference in their denaturation temperature ranging from 65.9˚C to 74.8˚C [6]. Thus the in-
creased temperature for water loss in UV crosslinked fish collagen could have been due to tightly bound water
molecules with collagen as a result of crosslinking. Swim bladder collagen-chitosan of cat fish records thermal
decomposition by thermo gravimetric analysis at 209˚C for un-crosslinked and 240˚C for glutraldehyde cros-
slinked collagen indicating the crosslinking lead to thermo stability [16]. Moreover thermostability of fish col-
lagen from the internal tissues (swim bladders and bones) was slightly higher than that of pepsin solubilized
collagen from the external tissues (fins, scales and skins) [24]. Collagen film prepared from bovine serosa regis-
tered a denaturation temperature range from 55.23˚C to 80.55˚C [25] [26]. Moreover the thermal stability of the
fish collagen depends on several factors like physiological temperature of the fish, species to which it belongs
and has direct correlation with imino acid (proline and hydroxyproline) content. Triple helical structure of the
collagen molecule is more stable with higher imino acid content as these facilitate intra and intermolecular cros-
slinking [27]. The denaturation of the fish collagen membrane also depends on the factors like solvent used, io-
nic strength, pH and humidity. The results of the current study indicated that the extracted fish collagen to exhi-
bit good thermal stability which increased significantly after crosslinking. There are also interesting conse-
quences of variation in denaturation temperature with variation in temperature of their living environment. The
deep sea fishes have lower denaturation temperature in comparison to fresh water fish therefore the isolated type
I collagen from fish swim bladder may find wide biomedical application due to its high denaturation tempera-
ture. FT-IR has been shown to be useful tool for testing unknown biological materials such as fish collagen that
might have been altered, and moreover it relies on objective criteria such as changes of the IR absorption fre-
quency and intensity in various functional groups of biological molecules. The usefulness of FT-IR spectroscopy
has also been demonstrated by determining the secondary structure of the fish collagen proteins as amide I,
amide II and amide III band. The exact frequencies of these stretching vibrations can be noted for amide I which
depends upon the strength of the hydrogen bond to the carbonyl oxygen and the environment dictated by the lo-
cal peptide conformation. Amide A band is almost symmetric, suggesting that the amount of water present in the
fish collagen must be low. The ratio of IR absorption intensity between 1237 cm1 (amide III) and 1450 cm1
(amide II) band was approximately equal to 1.0, which confirms the triple helical structure of collagen. The
measurement of fish collagen shrinkage by a micro-shrinkage apparatus has been shown to be a useful research
tool, relating to gross tissue changes to events occurring at the molecular structural level, and the temperature of
isotonic contraction (shrinkage temperature) could be used to investigate these molecular changes. Shrinkage of
the fish collagen occurs as a result of hydrothermal denaturation of the collagen protein molecules and is moni-
tored with a micro-shrinkage apparatus. Thus the present study has shown that the shrinkage temperature of the
fish collagen membrane is also known to be influenced by different factors, most of which appear to be influ-
enced by the number and nature of crosslinking interactions between adjacent polypeptide chains of the protein
molecules. The increased strength of the crosslinked fish collagen membrane is due to the UV irradiation that in
turn could have promoted bonds formation between free radicals generated on aromatic amino-acid residues as
reported by Cooper & Davidson [28] and Fujimori [29]. The current study has thus confirmed the usefulness of
collagen in promoting the proliferation and differentiation of the cells in culture. The use of collagen-based
biomaterials has been reported on several occasions in the literature to aid in biomedical implant or as a coating
material, wound healing and in reconstructive surgery [30] [31] due to which it occupies prominent position in
the field of tissue engineering. Collagen substrates have been shown to influence proliferation, migration, and
differentiation of a number of cell types in vitro [32]. The cell proliferation and viability of live cells were
measured by the MTT assay which determines the active mitochondrial enzymes present in the viable cell which
in turn utilizes the MTT as substrate, converting it to a coloured (Formazan) product. In contrast dead cells does
not have the ability to convert MTT into formazan, thus colour formation serves as a useful and convenient
marker of only the viable cells. It is a colorimetric assay in which the degree of absorbance of Formazon directly
reflects the number of viable cells. Cell attachment, proliferation and differentiation depend on the nature of the
matrix and cell type [33] [34].
5. Conclusion
The current study has shown the novelty of the preparation method by its retention of nativity, purity, triple he-
lixcity, biocompatibility and very high thermal stability of fish collagen extracted from the swim bladder of
fishes which are normally left as waste materials as an alternate source of collagen for industrial purposes. The
R. Sripriya, R. Kumar
fish collagen extracted by the current novel method resembles in many ways with the more widely studied col-
lagen of mammals in its physico-chemical properties and thus shows potential as a cell substrate, suggesting the
possibility of use as a source collagen for pharmaceutical and biomedical application.
The authors Dr. R. Sripriya and Dr. Ramadhar Kumar dedicate this research article to Late Dr. P. K. Sehgal,
(Chief Scientist, Central Leather Research Institute), for his valuable guidance, inspiration, coaching and enthu-
siasm and they gratefully acknowledge the Council of Scientific Research (CSIR) for financial support at the
Central Leather Research Institute (CLRI), Chennai 600020, India. We acknowledge Sree Balaji Medical Col-
lege & Hospital (SBMCH), Bharath Institute of Higher Education and Research for extending the research facil-
ities to complete the work.
[1] Muyonga, J.H., Cole, C.G.B. and Duodu, K.G. (2004) Characterisation of Acid Soluble Collagen from Skins of Young
and Adult Nile Perch (Lates niloticus). Food Chemistry, 85, 81-89.
[2] Usha, R., Maheshwari, R., Dhathathreyan, A. and Ramasami, T. (2006) Structural Influence of Mono and Polyhydric
Alcohols on the Stabilization of Collagen. Colloid Surface B, 48, 101-105.
[3] Pinazo, A. (1996) Effect of Surfactant Structure on Diffusion through a Collagen Membrane. Colloid Surface B, 8,
[4] Reid, G., Lam, D., Policova, Z. and Neumann, A.W. (1993) Adhesion of Two Uropathogens to Silicone and Lubricious
Catheters: Influence of pH, Urea and Creatinine. Journal of Materials Science: Materials in Medicine, 4, 17-22.
[5] Ogawa, M., et al. (2004) Biochemical Properties of Bone and Scale Collagens Isolated from the Subtropical Fish Black
Drum (Pogonis cromis) and Sheepshead Seabream (Archosargus probatocephalus). Food Chemistry, 88, 495-501.
[6] Fernandes, R.M., Couto Neto, R.G., Paschoal, C.W., Rohling, J.H. and Bezerra, C.W. (2008) Collagen Films from
Swim Bladders: Preparation Method and Properties. Colloid Surface B, 62, 17-21.
[7] Sadowska, M., Kolodziejska, I. and Niecikowska, C. (2003) Isolation of Collagen from the Skins of Baltic Cod (Gadus
morhua). Food Chemistry, 81, 257-262.
[8] Nagai, T. and Suzuki, N. (2000) Isolation of Collagen from Fish Waste MaterialSkin, Bone and Fins. Food Chemi-
stry, 68, 277-281.
[9] Nagai, T. and Suzuki, N. (2002) Preparation and Partial Characterization of Collagen from Paper Nautilus (Argonauta
argo, Linnaeus) Outer Skin. Food Chemistry, 76, 149-153.
[10] Senaratne, L.S., Park, P.J. and Kim, S.K. (2006) Isolation and Characterization of Collagen from Brown Backed Toad-
fish (Lagocephalus gloveri) Skin. Bioresource Technology, 97, 191-197.
[11] Shahidi, F. (1994) Seafoods: Chemistry, Processing, Technology and Quality. Blackie Academic and Professional,
[12] Sano, A., Maeda, M., Nagahara, S., Ochiya, T., Honma, K., Itoh, H., et al. (2003) Atelocollagen for Protein and Gene
Delivery. Advanced Drug Delivery Reviews, 55, 1651-1677.
[13] Huang-Lee, L., Cheung, D.T. and Nimni, M.E. (1990) Biochemical Changes and Cytotoxicity Associated with the
Degradation of Polymeric Glutaraldehyde Derived Crosslinks. Journal of Biomedical Materials Research, 24, 1185-
[14] Koide, M., Osaki, K., Konishi, J., Oyamada, K., Katakura, T., Takahashi, A. and Yoshizato, K. (1993) A New Type of
Biomaterial for Artificial Skin: Dehydrothermally Cross-Linked Composites of Fibrillar and Denatured Collagen.
Journal of Biomedical Materials Research, 27, 79-84.
[15] Safandowska, M. and Pietrucha, K. (2013) A New Method of Determination of Collagen Conjugated with Keratin.
Autex Research Journal, 13, 37-39.
[16] Bama, P., Vijayalakshimi, M., Jayasimman, R., Kalaichelvan, P.T., Deccaraman, M. and Sankaranarayanan, S. (2010)
Extraction of Collagen from Cat Fish (Tachysurus maculatus) by Pepsin Digestion and Preparation and Characteriza-
tion of Collagen Chitosan Sheet. International Journal of Pharmacy and Pharmaceutical Sciences, 2, 133-137.
R. Sripriya, R. Kumar
[17] Sripriya, R., Ahmed, M.R., Sehgal, P.K. and Jayakumar, R. (2003) Influence of Laboratory Ware Related Changes in
Conformational and Mechanical Properties of Collagen. Journal of Applied Polymer Science, 87, 2186-2192.
[18] Neuman, R.E. and Logan, M.A. (1950) Determination of Collagen and Elastin in Tissues. The Journal of Biological
Chemistry, 186, 549-556.
[19] Leammli, U.K. (1970) Discontinuous Buffer System for Slab Gel. Nature, 227, 680-681.
[20] Mosmann, T. (1983) Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cy-
totoxicity Assays. Journal of Immunological Methods, 65, 55-63.
[21] Marshall, N.J., Goodwin, C.J. and Holt, S.J. (1995) A Critical Assessment of the Use of Microculture Tetrazolium As-
says to Measure Cell Growth and Function. Growth Regulation, 5, 69-84.
[22] Spackman, D.H., Stein, W.H. and Moore, S. (1958) Automatic Recording Apparatus for Use in the Chromatography of
Amino Acids. Analytical Chemistry, 30, 1190-1205.
[23] Oliver, R.F., Barker, H., Cooke, A. and Grant, R.A. (1982) Dermal Collagen Implants. Biomaterials, 3, 38-40.
[24] Liu, D., Liang, L., Joe, M. and Zhou, R.P. (2012) Extraction and Characterisation of Pepsin-Solubilised Collagen from
Fins, Scales, Skins, Bones and Swim Bladders of Bighead Carp (Hypophthalmichthys nobilis). Food Chemistry, 133,
[25] Goes, J.D., Figueiro, S.D., de Paiva, J.A.C., de Vasconcelos, I.F. and Sombra, A.S.B. (2002) On the Piezoelectricity of
Anionic Collagen Films. Journal of Physics and Chemistry of Solids, 63, 465-470.
[26] Silva, C.C., Pinheiro, A.G., Thomazini, D., Góes, J.C., Figueiró, S.D., de Paiva, J.A.C. and Sombra, A.S.B. (2001) Ef-
fect of the pH on the Piezoelectric Properties of Collagen Films. Materials Science and Engineering: B, 83, 165-172.
[27] Wong, D.W.S. (1989) Mechanism and Theory in Food Chemistry. Van Nostrand Reinhold Company, New York.
[28] Cooper, D.R. and Davidson, R.J. (1965) The Effect of Ultraviolet Irradiation on Soluble Collagen. Biochemical Jour-
nal, 97, 139-147.
[29] Fujimori, E. (1965) UV Light-Induced Change in Collagen Macromolecules. Biopolymers, 3, 115-119.
[30] Singer, A.J. and Clark, R.A. (1999) Cutaneous Wound Healing. The New England Journal of Medicine, 341, 738-746.
[31] Sefton, M.V. and Woodhouse, K.A. (1998) Tissue Engineering. Journal of Cutaneous Medicine and Surgery, 3, 18-23.
[32] Schor, S.L. (1980) Cell Proliferation and Migration on Collagen Substrata in Vitro. Journal of Cell Science, 41, 159-
[33] Chen, F., Yoo, J.J. and Atala, A. (1999) Acellular Collagen Matrix as a Possible “Off the Shelf” Biomaterial for
Urethral Repair. Urology, 54, 407-410.
[34] El-Kassaby, A.W., Retik, A.B., Yoo, J.J. and Atala, A. (2003) Urethral Structure Repair with an Off-the-Shelf Colla-
gen Matrix. The Journal of Urology, 169, 170-173.
... It can be said that, collagen from snakehead fish skin (Channa striata) is safe to use. and below 135 kDa, respectively. Based on previous studies, it was reported that α chain of collagen from snakehead fish skin is around 130 kDa [19]. ...
... There are also β (dimers) and ɣ (trimers) subunits indicating crosslinking of collagen [19]. There were two protein bands in collagen samples of 5 and 10 mg/ml (A1-B3) around 245 kDa. ...
... This indicates that there are β (dimers) and ɣ (trimers) subunits in samples with molecular weights below and above 245 kDa, respectively. Based on previous studies, it was reported that β subunit of collagen sourced from fish was found at 200-250 kDa [19]. ...
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Objective: Liquid crystals are special state of matters which have regularity of solid arrangement but had a liquid-like flow characteristics. Collagen is a biopolymer that qualified for requirements as the system of a liquid crystal because it is mesogenic and rigid in a triple-helix section. There are various sources of collagen that have been used; one of them is snakehead fish skin (Channa striata). Methods: The stages of research were collagen isolation, collagen identification, liquid crystals formation, and characterization. Collagen liquid crystals were formed by lyotropic method using 0.5 M acetic acid and treated with and without sonication at 30, 60, and 80 mg/ml concentrations. The formation of Liquid crystal phase characterized by using Polarization Ligh microscopy. Results: Mesophase analysis using polarized light microscope showed the presence of cholesteric phase (fingerprint pattern) which seen from the lowest concentration used in this study (30 mg/ml). The increasing of collagen concentration and sonication treatment can trigger the formation of clearly liquid crystal cholesteric phase under polarized light microscope. Infrared spectra of collagen liquid crystals both sonicated or not, showed no change in triple-helix. Conclusion: The formation of lyotropic liquid crystal of collagen from snakehead fish skin showed the cholesteric pattern without changing the triple-helix collagen structure.
... Fish collagen exhibited an endothermic peak at 89.58 C owing to evaporation of water. Moreover, another peak occurred around 200 C, reflecting fish collagen's melting temperature and was attributed to the denaturation of the double helix (Sripriya & Kumar, 2015). The thermogram of a-arbutin revealed one sharp characteristic endothermic peak at 205 C, which is denoted to its melting point and reveals a-arbutin crystalline state (Ayumi et al., 2019). ...
... The protein double bands occurring at 1450.93-1400.46 cm À1 were probably accompanied with C-H bending vibration modes (Sripriya & Kumar, 2015). The FT-IR spectrum of plain CSNPs (Supplementary 4(f)) showed peak shift from 3367.92-3441.01 ...
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The increase in the production of melanin level inside the skin prompts a patient-inconvenient skin color disorder namely; melasma. This arouses the need to develop efficacious treatment modalities, among which are topical nano-delivery systems. This study aimed to formulate functionalized chitosan nanoparticles (CSNPs) in gel form for enhanced topical delivery of alpha-arbutin as a skin whitening agent to treat melasma. Ionic gelation method was employed to prepare α-arbutin-CSNPs utilizing a 2⁴ full factorial design followed by In vitro, Ex vivo and clinical evaluation of the nano-dispersions and their gel forms. Results revealed that the obtained CSNPs were in the nanometer range with positive zeta potential, high entrapment efficiency, good stability characteristics and exhibited sustained release of α-arbutin over 24 h. Ex vivo deposition of CSNPs proved their superiority in accumulating the drug in deep skin layers with no transdermal delivery. DSC and FTIR studies revealed the successful amorphization of α-arbutin into the nanoparticulate system with no interaction between the drug and the carrier system. The comparative split-face clinical study revealed that α-arbutin loaded CSNPs hydrogels showed better therapeutic efficacy compared to the free drug hydrogel in melasma patients, as displayed by the decrease in: modified melasma area and severity index (mMASI) scores, epidermal melanin particle size surface area (MPSA) and the number of epidermal monoclonal mouse anti–melanoma antigen recognized by T cells-1 (MART-1) positive cells which proved that the aforementioned system is a promising modality for melasma treatment.
... Collagen has two endothermic transitions in the range of 0 -300 °C, Figure 1B. The thermogram has the characteristics of other collagen thermograms found in the literature, with subtle differences in parameters depending on the type and origin of the collagen [45][46][47][48][49][50].The first transition occurs in temperatures from 30 to150 °C showing the endothermic peak (Td') of maximum between 80 °C and 104 °C. This thermal effect has been assigned in the literature to the dehydration process and overlaps the denaturation endotherm of fibrillar collagen [49,50]. ...
... This thermal effect has been assigned in the literature to the dehydration process and overlaps the denaturation endotherm of fibrillar collagen [49,50]. According to the literature, the transition is related to the thermal denaturation of collagen [45][46][47][48][49][50][51]. This broad endothermic peak is associated with the helixcoil transition induced by thermal disruption of amino acid-derived hydrogen bonds stabilizing the triple-helical collagen structure. ...
Collagen is a biocompatible and bioresorbable material, an excellent active component for the formulation of medical dressings and scaffolds. Its current popularity and the developing methods of producing such medical materials imply the need for methods of controlling and processing collagen used in medicine. One of the basic methods used to sterilize medical devices is radiation sterilization. In this study, we undertook the determination of the influence of humidity, radiation sterilization, and the influence of these factors on the structure of collagen. Spectroscopic tests (EPR, DRS UV-VIS, FTIR), gas chromatography, and thermal methods (TGA, DSC) were used. Humidity appears to affect the amount, and especially the rate, of oxidative damage and the reorganization of oxygen addition products. Despite radiation damage observed by spectral methods, from the point of view of its thermal properties, collagen I remains stable upon irradiation within the dose range up to 50 kGy, probably due to its unique macromolecular and intermolecular structure, containing numerous crosslinks. The high thermal stability of collagen I in response to radiation gives good indications for radiation sterilization of collagen I based medical products. This is in agreement with the observed structural stability of radiation-sterilized collagen scaffolds used in skin wound healing. Nevertheless, oxidative modification was observed, which ultimately introduces new oxygen-containing functional groups in collagen. The result should be considered when designing and sterilizing collagen-based dressing materials and scaffolds.
... Previous authors have reported a wide range of collagen yields varying from 19% from the skin of O. niloticus (35) to 51.9% from the skin of Probarbus Jullieni (36). Swim bladders tend to have higher yields when compared to skin and bones, as their structure is primarily made up of pure collagen (37). The extraction efficiency of rohu swim bladder collagen in the present study was, on average, higher than previous reports, which ranged from 46.5% (38) to 54.5% (39). ...
This study attempts to identify the significant role played by the secondary and tertiary structure of collagen-derived peptides that are involved in lipid peroxide quenching in food products. Fish collagen hydrolysate (CH) was extracted with an efficiency of 70%. The constituent peptides of CH (8.2-9.7kDa) existed in a polyproline-II (PP-II) conformation and at a minimum concentration of 1mg/ml and pH range 7-8, assembled into a stable, hierarchical, quasi-fibrillar (QF) network. The peroxide quenching activity of this QF-CH increased with increasing ionic stability of the assembly and decreased upon proteolytic dismantling. Upon being used as an additive, the QF-CH reduced peroxide formation by 84.5-98.9% in both plant and fish-based oil and increased the shelf life of soya oil by a factor of 5 after 6 months of storage. The addition of QF-CH to cultured cells quenched peroxide ions generated in situ and decreased stressor activity by a factor of 12. 16 abundant peptides were identified from the CH. The reason behind the high efficacy displayed by CH was attributed to its unique charge distribution, prevalence of proton-donating amino acid residues and proximal charge delocalization by the QF network, making fish derived CH a suitable substitute for antiperoxide agents in lipid-rich food.
... The bands at 1549 and 1532 cm −1 suggested that the N-O and C-H groups corresponded. A new band that formed at 1337 cm −1 after biosorption was probably dye to C-H bending [15]. ...
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The presence of eriochrome black T (EBT) dye in waste water causes a significant hazard to human health and ecology. In the current study, biosorption was employed to eliminate EBT from water. Thus, we utilized endophytic fungi strain Exserohilum rostratum NMS1.5 mycelia biomass as biosorbent agent. The process was carried out at room temperature by magnetic stirring. The results indicated that an increase in pH would decrease adsorption capacity and removal percentage. In addition, an increased EBT concentration would decrease the removal percentage and increase bio-sorption capacity. The equilibrium time indicated that after 300 min of mixing, the percentage removal and biosorption capacity were 80.5% and 100.61 mg/g, respectively. The biosorption iso-therms and kinetics were compatible with the Freundlich model and the pseudo-second-order. This research indicates that E. rostratum NMS1.5 may be utilized as an environmentally friendly and affordable alternative biosorbent material for EBT removal.
... Figure 2e shows the FT-IR spectrum of fish scale before and after dye adsorption. The band at 2924.0 cm −1 for both fish scale and dye-adsorbed fish scale is owing to the asymmetric −CH 3 stretching vibration [25]. The bands at 1122.4 cm −1 and 1192.2 cm −1 are characteristics of the −C-N bond, which confirms the presence of an azo group in the dye. ...
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Methyl orange, an anionic dye, is injurious to health and the environment which must be treated before discharging. The processed fish (Labeo rohita) scales were characterized by scanning electron microscope (SEM), the attenuated total reflectance (ATR) Fourier transform infrared spectroscopy (FT-IR), and BET surface area analyzer. The BET surface area and pore diameter were observed to be 192 m² g⁻¹ and 44 nm, respectively. Influences of parameters such as pH, temperature, and concentration of adsorbent were studied by response surface methodology and analysis of variance (ANOVA) to optimize methyl orange dye uptake in adsorption process by fish scale. The influences of factors on adsorption capacity followed the order (initial concentration ˃ temperature ˃ pH). The fish scale attained a high sorption capacity (Langmuir capacity of 520 mg g⁻¹ at pH 5.3, 283 K) towards methyl orange. The thermodynamics analyses implied that the physisorption was an exothermic and spontaneous process (ΔG⁰ = negative, ΔH⁰ = −9.17 kJ mol⁻¹ and ΔS⁰ = + 0.03 J mol⁻¹ K⁻¹). The interaction of fish scale biosorbent with methyl orange dye was also explored with the assistance of a mechanistic pathway. The results indicate that the fish scale could be employed as an effective biosorbent for the removal of methyl orange dye from aqueous solution.
Collagen is an active macromolecule, but direct comparisons between different sources were scarce. The purpose of this study was to compare collagen from three fish species to find a collagen with higher bioactivity. Collagen from the swim bladder of Ctenopharyngodon idella, Nibea coibor and Protonibea diacanthus were isolated and named CASC, NASC and PASC, respectively. SDS-PAGE and spectroscopic analysis indicated that ASCs contained triple-helix type I collagen. The microstructure of collagen after lyophilization was uniform and porous. In addition, DSC analysis of the ASCs showed that the denaturation temperature (Td) and melting temperature (Tm) were 79 °C–93 °C and ∼216 °C, respectively. Moreover, the antioxidant activity of ASCs and its effect on fibroblast viability and collagen synthesis were investigated. Current studies have shown that ASCs had antioxidant capacity and were not toxic to fibroblasts. NASC had a higher antioxidant capacity (Hydroxyl, DPPH and ABTS radical scavenging rates were 33.73%, 33.49% and 89.81%, respectively) and significantly promoted fibroblast viability compared to the other two ASCs. It was further found that NASC can promote collagen synthesis in fibroblasts. Collagen extracted from swim bladder may be a promising functional product for the food, cosmetics and biomedical fields.
Collagen, a natural extracellular matrix protein, plays a key role in tissue architecture and regeneration during wound healing process. The concept of providing exogenous sources to improve wound healing facilitated the use of collagen in the biomedical industries. The availability of raw material, biocompatibility, biodegradability, and the ability of reconstitution in various forms allowed to develop several collagen-based biomaterials for wound healing and tissue engineering applications. A wide number of studies have been reported on the efficacy of collagen in tissue regeneration. Here we describe elaborately about the different sources for collagen, extraction protocol followed, and the method of reconstitution for biomedical applications. We also provide the strategies developed to use the collagen for wound healing and tissue engineering applications.
Faster wound healing is critical to restore homeostasis. Chronic wounds that fail to heal pose a challenge for health care professionals and may result in socioeconomic and psychological imbalances. Wound dressing materials could enhance wound healing dramatically. The choice of suitable material for dressing depends on type of wound and cause. Natural polymers boast the attributes that can aid in rapid wound healing. This chapter discusses properties and applications of various natural polymers used in wound dressings and skin substitutes. A concise list of commercially available wound healing products that were made using natural polymers or their derivatives is also provided.
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This study was conducted to synthesize and characterize silver nanoparticles (AgNPs) using a rapid green synthesis approach. Antibacterial properties of AgNPs were evaluated. Extract of Melia Dubia (Neem) and collagen produced from the fish scales were used as reducing and stabilizer agents respectively. Uv-Vis Spectroscopy, Scanning Electron Microscopy (SEM), Energy-Dispersive X-Ray Spectroscopy (EDX), Fourier-Transform Infrared Spectroscopy (FTIR), and Dynamic Light Scattering (DLS) were used to characterize the synthesized AgNPs. Evaluation of the synthesized AgNP's antibacterial activity was conducted, with Staphylococcus Aureus (S. aureus) as Gram-positive and Escherichia Coli (E. coli) as the negative bacteria. The peak of absorbance for the synthesized AgNPs was at 454 nm, indicating conformed AgNPs. SEM image showed semi-evenly distributed rod shapes. The EDX data revealed presence of the metallic silver. Meanwhile, FTIR analysis indicated presence of C2H2, C=O, N-H groups. DLS showed an average size of 437.6 nm. XRD showed calculation for particle size using d = Kλ/βcosθ. Average size of AgNPs was 141.81 ± 5 nm. AgNPs also displayed tangible antibacterial activity towards the S. aureus and pathogenic E. coli. AgNPS were successfully synthesized and evaluated for their antibacterial properties. The outcomes being the multifaceted biological activities alongside application of biocompatible green nanoparticles, is discoverable in the field of Nanomedicine.
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Collagen was extracted by pepsin digestion from the swim bladder of catfish (Tachysurus maculatus) processing wastes. The total collagen yield extracted was 40% on the basis of lyophilized dry weight. According to the electrophoretic pattern, the swim bladder of the fish consisted of comparable amounts of two α chain-sized components designated as ά 1, ά 2 and the β. Collagen was cross linked with chitosan. The formed collagen- chitosan sheet was characterized and showed that there is a possibility use in medical field.
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The paper describes the possibility of using Sirius red dye for the determination of collagen conjugated with keratin of wool. Sirius red assay was shown to be feasible for collagen detection, which was enzymatically coupled onto wool fibers and woven fabric. The effectiveness of combination of keratin protein with collagen was evaluated.
For the first time in over twenty-five years, this unique and popular textbook on food chemistry mechanism and theory has received a full update. Emphasizing the underlying chemical reactions and interactions that occur in foods during processing and storage, this book unifies the themes of "what", "how" and "why" in the language of equations, reactions and mechanisms. This book is the only work which provides in-depth focus on aspects of reaction mechanisms and theories in the chemistry of food and food systems. With more than 500 chemical equations and figures, this book provides unusual clarity and relevance, and fills a significant gap in food chemistry literature. It is a definitive source to consult regarding the important mechanisms that make food components and reactions tick. Mechanism and Theory in Food Chemistry has been a popular resource for students and researchers alike since its publication in 1989. This important new edition contains updates on the original text encompassing a quarter century of advances in food chemistry. Many parts of the original chapters are revised to make for smoother navigation through the subjects, to better explain the underlying chemistry concepts and to fulfill the need of adding topics of emerging importance. New sections on fatty acids, lipid oxidation, meat, milk, soybean and wheat proteins, starch and many more have been incorporated throughout the revision. This updated edition provides an excellent source of all the important chemical mechanisms and theories involved with food science.
Seafoods are important sources of nutrients for humans. Proteins and non­ protein nitrogenous compounds play an important role in the nutritional value and sensory quality of seafoods. Consumption of fish and marine oils is also actively encouraged for the prevention and treatment of cardio­ vascular diseases and rheumatoid arthritis. Highly unsaturated long-chain omega-3 fatty acids are regarded as the active components of marine oils and seafood lipids. The basic chemical and biochemical properties of seafood proteins and lipids, in addition to flavour-active components, their microbiological safety and freshness quality, are important factors to be considered. A presentation of the state-of-the-art research results on seafoods with respect to their chemistry, processing technology and quality in one volume was made possible by cooperative efforts ofan international group of experts. Following a brief overview, the book is divided into three sections. In Part 1 (chapters 2 to 8) the chemistry of seafood components such as proteins, lipids, flavorants (together with their properties and nutritional significance) is discussed. Part 2 (chapters 9 to 13) describes the quality of seafoods with respect to their freshness, preservation, micro­ biological safety and sensory attributes. The final section of the book (chapters 14 to 16) summarizes further processing of raw material, underutilized species and processing discards for production of value­ added products.
Seafood processing discards account for approximately three-quarters of the total weight of catch. Despite the presence of valuable components in discards, these have not been used in North America. Although some composting of processing discards has taken place, discards are generally dumped in-land or hauled into the ocean. Nonetheless, meal and silage production has also been used as a possible means of waste utilization.
Recent progress in recombinant gene technology and cell culture technology has made it possible to use protein and polynucleotides as effective drugs. However, because of their short half-lives in the body and the necessity of delivering to target site, those substances do not always exhibit good potency as expected. Therefore, delivery systems of such drugs are important research subjects in the field of pharmacology, and to prolong the effect of these drugs, many studies are being conducted to control the release of proteins and polynucleotides from various carrier materials. Collagen is one of the most useful carrier materials for this purpose. In this article, we report on the controlled release of protein drugs using collagen, focusing on a new drug delivery system (DDS), the Minipellet, as our basic technology. Then we introduce our recent work about gene therapy using collagen-based DDS. Basic formulation study showed that collagen DDS protects DNA degradation from both chemical cleavage and enzymatic digestion. A single injection of collagen DDS containing plasmid DNA produced physiologically significant levels of gene-encoding proteins in the local site and systemic circulation of animals and resulted in prolonged biological effects. These results suggest that collagen DDS containing plasmid DNA may enhance the clinical potency of plasmid-based gene transfer, facilitating a more effective and long-term use of naked plasmid vectors for gene therapy. Also, variety kinds of application of collagen DDS for gene therapy using adenovirus vector, antisense DNA and DNA vaccine, will be discussed.
The objective of this study was to extract and characterise pepsin-solubilised collagens (PSC) from the fins, scales, skins, bones and swim bladders of bighead carp and to provide a simultaneous comparison of five different sources from one species. The PSC were mainly characterised as type I collagen, containing two α-chains, and each maintained their triple helical structure well. The thermostability of PSC from the internal tissues (swim bladders and bones) was slightly higher than that of PSC from the external tissues (fins, scales and skins). The peptide hydrolysis patterns of all PSC digests using the V8 protease were similar. All PSC were soluble at acidic pH (1–6) and lost their solubility at NaCl concentrations above 30 g/l. The resulting PSC from the five tissues would all be potentially useful commercially.