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Fish Processing Waste: A Promising Source of Type-I Collagen
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Collagen is the sole most profuse protein in the animal kingdom. It has been subjected to various studies from time immemorial. Its applications are numerous and have been extracted from various sources such as land animals (mainly bovine and porcine) and birds. Although collagen sources are abundant the outbreak of varied diseases among land animals posed a threat to its utilization in our daily life. Thus a probe for an alternative source began which in turn revealed the immense untapped marine source. The present article deals with a brief description of collagen its characteristics,chemistry,common extraction procedure, application in various fields and sources. A lot of studies have been carried out on various land animals, birds and marine organisms and this review sums up the work performed to date in a concise manner.
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.
The passage of leukocytes through basement membranes involves proteolytic degradation of extracellular matrix (ECM) components executed by focalized proteolysis. We have investigated whether the migration of leukocytes through 3-dimensional collagenous tissue scaffolds requires similar ECM breakdown. Human T blasts and SupT1 lymphoma cells expressed mRNA of MMP-9, MT1-MMP, MT4-MMP, cathepsin L, uPA, and uPAR as well as ADAM-9, -10, -11, -15, and -17. Upon long-term migration within 3-dimensional collagen matrices, however, no in situ collagenolysis was obtained by sensitive fluorescein isothiocyanate-collagen fragmentation analysis and confocal fluorescence/backscatter microscopy. Consistent with nonproteolytic migration, T-cell crawling and path generation were not impaired by protease inhibitor cocktail targeting MMPs, serine proteases, cysteine proteases, and cathepsins. Dynamic imaging of cell-ECM interactions showed T-cell migration as an amoeba-like process driven by adaptive morphology, crawling along collagen fibrils (contact guidance) and squeezing through pre-existing matrix gaps by vigorous shape change. The concept of nonproteolytic amoeboid migration was confirmed for multicomponent collagen lattices containing hyaluronan and chondroitin sulfate and for other migrating leukocytes including CD8+ T blasts, monocyte-derived dendritic cells, and U937 monocytic cells. Together, amoeboid shape change and contact guidance provide constitutive protease-independent mechanisms for leukocyte trafficking through interstitial tissues that are insensitive toward pharmacologic protease inhibitors.
The body wall of sea cucumber, Bohadschia bivitatta, was studied with
respect to its collagen protein. About 70% of the total body wall protein was accounted
for by highly insoluble collagen fibers. Aim of this study was to isolate pepsinsolubilized
collagen (PSC) from crude collagen fibres, extracted from body wall of sea
cucumber Bohadschia bivitatta, and also to investigate 67 nm axial repeat D-banding of
collagen fibrils, which characterizes collagen. Body wall of Bohadshia bivitatta were cut
into small pieces followed by washing with distilled water and then replaced with 4 mM
ethylenediaminetetraacetic acid (EDTA), 0.1 M Tris–HCl, pH 8.0, and stirred for 3 days
to get precipitated crude collagen fibrils. Disaggregated insoluble crude collagen fibrils
were treated with 0.1 M NaOH and 0.5 M acetic acid containing porcine pepsin to get
PSC collagen. The ultra structures were analyzed by using Quanta™ Scanning Electron
Microscope, Netherlands, under 130 Pa pressure, at a working distance of 6-10 mm at
4.0 to 5.0 kV from magnification 5,000X to 100,000X. Pepsin-solubilized collagen was
successfully isolated from disaggregated crude collagen fibres, with 65% yield. SEM
analysis of crude collagen displayed a regularly repeated striated pattern, whereas, PSC
displayed non-banded sheet like fibers with twisted configuration. In conclusion high
yield of Pepsin-solubilized collagen was successfully isolated, and evidence of Dperiodic
bands confirms it as fibrillar collagen. This collagen could be useful as an
alternative to mammalian collagen in the nutraceutical and pharmaceutical industries.
The collagen of skin, scales and fins of Catla catla and Cirrhinus mrigala were isolated and characterised. Nine fishes of each fish species of three weight groups were collected from a commercial fish farm. Collagen characterisation using SDS-PAGE revealed the molecular weights (kDa) of the C. catla skin, scales, and fins which ranged from 120 to 210, 70 to 201, and 68 to 137 kDa, respectively. The size of the collagen of C. mrigala skin, scales and fins ranged from 114 to 201, 77 to 210, and 70 to 147 kDa, respectively. Glycine and alanine were the most abundant amino acid, whereas tryptophan was totally absent in all selected tissues. Thus, significant variation exists in type of collagen and amino acid profile within the weight groups of the two fish species. The imino acid (proline and hydroxyproline) contents estimated in C. catla and C. mrigala skin (161-165 and 160-168), scales (155-159 and 152-161) and fins (162-171 and (152-155) residues/1,000 residues, respectively. The proximate analysis was also performed for skin, scales and fins. The maximum protein content of the skin was determined as 26.10 % and 22.90 % in the C. catla and C. mrigala, respectively, from the W3 weight group. The scales of the W3 weight group exhibited maximum protein contents of 25.90 and 21.77 % for C. catla and C. mrigala, respectively. The maximum protein contents (19.04 % and 18.12 %) were recorded for C. catla and C. mrigala, respectively in the fins.
Acid-soluble collagen (ASC) and pepsin-solubilized collagen (PSC) were prepared from the waste freshwater carp fish scales. The results of SDS-PAGE showed that purified collagens were composed of at least two different chains which were in accordance with the type I collagen with α chain composition of (α 1) 2 α 2 . Compared with the carp fish ordinary muscle type I collagen, porcine dermis type I collagen and other seawater fish collagens, freshwater carp fish scales collagen contained relative high half-cystine (Cys-s), but lower denaturation temperature(Td) than the porcine dermis type I collagen. These collagens had evident absorption at 230 nm by UV-Vis spectra. The spectrum X-ray diffraction showed that the collagen remained single-helix and tri-helix configuration with the minimum values of the repeat spac-ings (d) of about 4.48 Å and 11.87 Å. Therefore, to make more effective use of limited-resources, carp fish scales can be a potential resource for the extraction of type I collagen or gelatin.
Tilapia are one of the most widely introduced fish globally that has clearly emerged as a promising group in aquaculture. Oreochromis niloticus was the first Tilapia species to be taken up for large Scale aquaculture. It is consumed widely due to its deliciousness and rich source of protein. During its processing, the scales, Fins, Skins etc are expelled out as waste Acid solubilized collagen (ASC) and Pepsin Solubilized collagen (PSC) were extracted from these processing wastes. Initial extraction by acid yielded 22% of collagen and subsequent digestion with pepsin yielded 56% on dry weight basis. The total protein of ASC and PSC was determined by Bradford method which contains 68.34mg/ml,23.24 mg/ml respectively. The FT-IR Spectrum showed that ASC and PSC are helpful in prediction and confirmation of Secondary structure of proteins. The denaturation temperature of ASC was 32°C while for PSC it is 29°C.SEM micrograph showed the fibrous nature of Collagen. This report indicates that Tilapia waste might be useful as a new source of collagen apart from usual bovine and pig skin.
Collagen was isolated from threadfin bream (Nemipterus japonicas) waste (mixture of scale and fin) by using 0.5 M citric acid or calamansi juice (Citrofortunella microcarpa) for 12 and 24 hrs at 4°C. The physico-chemical characteristics of the collagens were then compared with the commercial collagen. Shorter extraction time (12 hrs) and extraction using calamansi juice resulted in higher yield. The yield was 22% (12 hrs) and 20.37% (24 hrs) for collagen extracted using calamansi juice and 8.3% (12 hrs) and 6.9% (24 hrs) for collagen extracted using citric acid. Collagen extracted using calamansi juice were light yellow (L = 93.70, a = -1.84, b = 13.44) while citric acid collagens were white (L = 94.82, a = 0.31, b = 0.20). Sensory evaluation on odor recognition test showed that collagen extracted with calamansi juice has a pleasant natural fragrance which is sweet citrus. Electrophoresis profile indicated that the collagen were of type I comprising of a1 and a2 chains. Threadfin bream collagen contained higher amount of imino acids proline (254.72 to 275.50/1000 residues) and hydroxyproline (7.56 to 13.50/1000 residues) than commercial collagen which is 21.25 and 5.16/1000 residues, respectively. Maximum transition temperature (Tmax) falls within a close range for all the collagens ranging from 24.81 to 25.91°C. Calamansi juice collagens were more viscous compared to others. The extraction of threadfin bream collagen for 12 hrs using calamansi juice generally leads to collagen characterised by pleasant odor, reasonably high yield and more viscous. Therefore, natural source such as calamansi juice could be an alternative medium for collagen extraction.
Marine collagen has been attracting attention as a medical material in recent times due to the low risk of pathogen infection compared to animal collagen. Type I collagen extracted from the swim bladder of Bester sturgeon fish has excellent characteristics such as high denaturation temperature, high solubility, low viscosity and an extremely fast rate to form large bundle of fibers under certain conditions. These specific characteristics of swim bladder collagen (SBC) permit us to create stable, disk shaped hydrogels with concentric orientation of collagen fibers by the controlled diffusion of neutral buffer through collagen solution at room temperature. However, traditionally used animal collagens, e.g. calf skin collagen (CSC) and porcine skin collagen (PSC), could not form any stable and oriented structure by this method. The mechanism of the superstructure formation of SBC by a diffusion induced gelation process has been explored. The fast fibrillogenesis rate of SBC causes a quick squeezing out of the solvent from the gel phase to the sol phase during gelation, which builds an internal stress at the gel-sol interface. The tensile stress induces the collagen molecules of the gel phase to align along the gel-sol interface direction to give this concentric ring-shaped orientation pattern. On the other hand, the slow fibrillogenesis rate of animal collagens due to the high viscosity of the solution does not favor the ordered structure formation. The denaturation temperature of SBC increases significantly from 31 °C to 43 °C after gelation, whereas that of CSC and PSC were found to increase a little. Rheology experiment shows that the SBC gel has storage modulus larger than 15 kPa. The SBC hydrogels with thermal and mechanical stability have potential as bio-materials for tissue engineering applications. This journal is