U.S. patent application number 13/158954 was filed with the patent office on 2012-05-10 for collagen extraction from aquatic animals.
This patent application is currently assigned to Universiti Putra Malaysia. Invention is credited to Badlishah Sham Baharin, Jamilah Bakar, Kaur Harvinder, Harvinder Kaur, Dzulkifly Mat Hashim, Umi Hartina Mohamad Razali, Awis Qurni Sazili, Russly Abdul Rahman.
Application Number | 20120114570 13/158954 |
Document ID | / |
Family ID | 41278503 |
Filed Date | 2012-05-10 |
United States Patent
Application |
20120114570 |
Kind Code |
A1 |
Bakar; Jamilah ; et
al. |
May 10, 2012 |
COLLAGEN EXTRACTION FROM AQUATIC ANIMALS
Abstract
The present invention relates to the use of fish skin as novel
industrial source of collagen. Advantageously, said skin is
obtained after the filleting or cutting of the fresh fish and
frozen immediately after filleting/cutting, thus guaranteeing a
very good quality of the base material, both from the
bacteriological standpoint and from the standpoint of the native
property of the protein.
Inventors: |
Bakar; Jamilah; (Selangor
Darul Ehsan, MY) ; Mohamad Razali; Umi Hartina;
(US) ; Mat Hashim; Dzulkifly; (US) ; Qurni
Sazili; Awis; (US) ; Kaur; Harvinder; (US)
; Rahman; Russly Abdul; (US) ; Baharin; Badlishah
Sham; (US) ; Harvinder; Kaur; (US) |
Assignee: |
Universiti Putra Malaysia
|
Family ID: |
41278503 |
Appl. No.: |
13/158954 |
Filed: |
June 13, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/MY2009/000122 |
Aug 19, 2009 |
|
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13158954 |
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Current U.S.
Class: |
424/59 ; 426/580;
426/590; 426/631; 426/657; 435/273; 514/17.2; 73/61.52 |
Current CPC
Class: |
A61P 29/00 20180101;
A61P 43/00 20180101; C07K 14/78 20130101; G01N 33/6887 20130101;
C07K 14/461 20130101 |
Class at
Publication: |
424/59 ; 435/273;
514/17.2; 426/657; 426/590; 426/580; 426/631; 73/61.52 |
International
Class: |
A61K 8/65 20060101
A61K008/65; A61K 38/39 20060101 A61K038/39; A61P 29/00 20060101
A61P029/00; A61Q 17/04 20060101 A61Q017/04; G01N 30/00 20060101
G01N030/00; A23L 1/305 20060101 A23L001/305; A23L 2/66 20060101
A23L002/66; A23C 9/152 20060101 A23C009/152; A23G 1/44 20060101
A23G001/44; C12S 3/16 20060101 C12S003/16; A61P 43/00 20060101
A61P043/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 23, 2008 |
MY |
PI20085247 |
Claims
1. A process of extracting collagen, the collagen is obtained from
skins of aquatic animals, wherein the aquatic animals include Lates
calcarifer and Oreochromis nilotica.
2. The process of extracting collagen as claimed in claim 1,
wherein the collagen is extracted from fish skin using an
enzyme.
3. The process of extracting collagen as claimed in claim 1,
wherein the enzyme is papain.
4. The process of extracting collagen as claimed in claim 1,
wherein the process comprising of a starting material consisting
essentially of fish skin, extracting collagen from said the fish
skin, and recovering the collagen.
5. The process of extracting collagen as claimed in claim 2, fish
skin is a flat fish skin.
6. The process of extracting collagen as claimed in claim 2,
wherein said skin is obtained by cutting the skin from fresh or
frozen fish.
7. The process as claimed in claim 1, wherein the process of
extracting collagen from skins of Lates calcarifer (barramundi)
comprising the steps of: a) mixing the skin with alkaline solution
for at least 6 hours; b) washing the skin with water to eliminate
residual the alkaline solution; c) soaking the skin with alcohol
solution for at least 18 hours; d) washing the skin with a neutral
solution; e) treating the skin from step (d) with an acidic
solution; f) hydrolyzing the skin from step (e) with papain; g)
obtaining a mixture from step (f) and stirring the mixture at a
working temperature of 4.degree. C. for at least 24 hours; h)
centrifuging the mixture from step (g) at 4-5.degree. C.; i)
precipitation of collagen by introducing the collagen into a sodium
chloride solution to precipitate collagen fibers. j) collecting the
collagen fibers from step (i) and centrifuging the collagen for at
least 60 minutes; k) obtaining collagen pellet; l) dissolving the
collagen pellet into acetic acid solution m) freeze-drying the
dialysed suspension.
8. The process as claimed in claim 1, wherein the process of
extracting collagen from skins of Oreochromis nilotica (red
tilapia) comprising the steps of: a) homogenizing the fish skin
with sodium; b) obtaining a suspension from step (a) c) stirring
the suspension for at least 24 hours; d) centrifuging the
suspension from step (c) for at least 20 minutes; e) obtaining a
precipitate from step (d); f) homogenizing the precipitate from
step (e) with alkaline solution; g) stirring the precipitate from
step (f) for at least 24 hours and for at least 3 repeats; h)
washing the precipitate with water and acetic acid solution; i)
stirring the precipitate from step (h) with papain at a working
temperature of 5.degree. C. for at least 24 hours; j) obtaining a
mixture from step (i) k) centrifuging the mixture from step (j) for
at least 20 minutes; l) obtaining collagen fibers from step (k); m)
introducing the collagen into a sodium chloride solution n)
collecting the collagen fibers from step (m) and centrifuging the
collagen for at least 20 minutes; o) obtaining collagen pellet; p)
freeze-drying the dialysed suspension.
9. The process as claimed in claim 7, wherein the collagen obtained
from the skin having a percentage of working yield between 14 and
40% by weight.
10. The process as claimed in claim 8, wherein the collagen
obtained from the skin having a percentage of working yield between
14 and 40% by weight.
11. A process of analyzing the collagen as claimed in claim 7,
wherein the process includes amino acid analysis, peptide in the
collagen and type of collagen.
12. The process as claimed in claim 11, wherein the amino acid
includes glycine, proline, alanine and arginine.
13. The process as claimed in claim 11, wherein the apparent
molecular weights of the peptide in the collagen is between 37 and
205 kilodalton (KDa).
14. The process as claimed in claim 11, wherein the type of
collagen is type 1 collagen.
15. A process of analyzing the collagen as claimed in claim 8,
wherein the process includes amino acid analysis, peptide in the
collagen and type of collagen.
16. The process as claimed in claim 15, wherein the amino acid
includes glycine, proline, alanine and arginine.
17. The process as claimed in claim 15, wherein the apparent
molecular weights of the peptide in the collagen is between 37 and
205 kilodalton (KDa).
18. The process as claimed in claim 15, wherein the type of
collagen is type 1 collagen.
19. A pharmaceutical composition comprising collagen of claim 1 and
a cosmetic or topical preparation.
20. The pharmaceutical as claimed in 19, wherein the cosmetic
includes cream, lotion eye cream, ointment or gel, sun-screen, oral
administration, face mask cream, anti-inflammatory medicine, and/or
anti-irritant medicine.
21. A food composition comprising the collagen of claim 1 and a
food product.
22. The food as claimed in 21, wherein the food product/s include
beverages, dairy products, confectionaries, chocolates, and any
application in food formulation/s as an ingredient or for any
functional properties.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of PCT Application No.
PCT/MY2009/000122, filed on Aug. 19, 2009, which claims priority to
Malaysian Patent Application No. PI 20085247, filed on Dec. 23,
2008, the entirety of which is incorporated herein by
reference.
FIELD OF INVENTION
[0002] The present invention relates to a collagen obtained from
fishes and a method of producing such fish collagen. In particular,
the present invention is directed to a collagen obtainable from the
skin of fishes and a method of extracting and producing the
same.
BACKGROUND OF INVENTION
[0003] A great amount of waste portions of fish, which are
generally considered useless or untapped, had been discarded. This
has been one of the major problems that we should address in the
modern society to find various ways to use that seemingly
unserviceable portion of fishes in many applicable fields. In view
of collagen being obtained from mammals and widely available as an
edible material, researches are now being made about fish collagen
and in particular about salmon and trout skins, in which a collagen
is a main ingredient of the tissues. Recent years, thus, have seen
some methods proposed to extract and produce a collagen from fish
skins, including the skins of salmon and trout in question. The
fish skin collagen, however, differs in characteristics from
mammalian collagen and requires relatively less harse treatment due
to the more delicate matrix of the skin. Collagen products have a
number of applications in various industries. In one such
application, collagen powders are used in clarification or
precipitation processes, for example for clarifying potable liquors
such as beer and wine. During the fermentation of liquors various
particulate materials such as yeasts and proteins become suspended
in the liquor and need to be removed. Collagen finings are added to
the liquor to clarify it by aiding the precipitation of the
suspended materials. Collagen and gelatin can also be used in juice
clarification processes.
[0004] Collagen is generally prepared from fish isinglass, which
constitutes a very pure source of collagen prepared from the dried
swim bladders of fish. Many investigations have been made into the
extraction of collagen from animal and fish skins including cold
water fish skins (U.S. Pat. No. 4,295,894, U.S. Pat. No. 5,698,228,
U.S. Pat. No. 5,162,506, U.S. Pat. No. 5,420,248, JP 4037679, JP
9-278639, JP 2-291814, PL 312122, RU 2139937). The collagen
extraction processes known involve a wide range of chemical and
mechanical extractions, or combinations thereof. The properties of
the collagen products obtained by these processes vary widely. Many
of the extraction processes applied to fish skins are adaptations
and modifications of mammalian collagen extraction techniques. The
applicants have identified that many of the processing steps
applied to mammalian collagen extraction are not directly
applicable for fish skin collagen extraction since the treatment
may be quite harsh or too strong for the fish skin matrix. Such
steps include chemical washes and extractions with strong acids or
alkali, excessive filtering and decantation steps amongst others. A
simplified extraction process which eliminates many of these steps
would be desirable to increase yield and to reduce denaturation of
the extracted collagen.
[0005] Collagen is recognized as a difficult and expensive protein
to quantify because of the insoluble nature of most collagens. Yet,
solubility is a key functional property important in a variety of
applications such as healthcare products. The applicants have also
determined that the conformation of the native collagen molecule
determines molecular functionality, with transition to the random
coiled confirmation of gelatin upon denaturing resulting in a
significant loss in fining ability. Collagen has also been reported
to be extracted from several fish species such as hake (Merluccius
merluccius L.), yellow sea-bream (Dentex tumiforms), tiger puffer
(Takifugu rubripes), carp (Cyprimus carpio); squids (Illex
argentinus) (Ilona Kolodziejska, 1999); and also jellyfish
(Rhopilema asamushi) (Takeshi Nagai et. al, 2000). All procedures
reported were very similar where non-enzymatic extractions were
employed and, if enzymatic reactions were used, then pepsin was the
most common enzyme.
[0006] The use of by-products from fish processing for collagen and
gelatine production, as an alternative introduces some questions,
the diversity of aquatic species and also the higher susceptibility
of this collagen to deterioration when compared to those from
mammals, which is more stable and facilitates its preservation
(Fernandez-Diaz et al., 2003). Moreover, after degutting and
filleting of fish, skins are kept with the rest of the discard and
they are subjected to rapid enzymatic and microbial damage, which
are natural and this may lead to quality variation of the extracted
collagen and the gelatins. Enzymatic activities in aquatic animals
are known to be higher than land animals.
[0007] Collagen exists in several polymorphic forms, the common
ones are Type I, III and V; type II and IV which are uncommon and
can only be found in certain collagens, which have also been
reported (Foegeding et al., 2001). Collagens and their denatured
forms, gelatines, are composed of long chains of amino acids,
connected by peptide bonds (Ockerman and Hansen, 1988; Ward and
Courts, 1977). The number and type of chemical covalent bonds
between the chains are altered as the animal ages, fewer numbers in
younger animals. This influences the molecular properties of the
resultant gelatine and glue (Ockerman and Hansen, 1988). Fish
collagens, in general, have lower amino acids contents than
mammalian collagens and this may be the reason for the lower
denaturation temperature (Grossman and Bergman, 1992, Jamilah and
Harvinder, 2002). This in turn appears to be related to the body
temperature of the species (Johns, 1977). There are many properties
of collagen that make it an attractive substance for various
medical applications, such as for implants, transplants, organ
replacement, tissue equivalents, vitreous replacements, plastic and
cosmetic surgery, surgical suture, surgical dressings for wounds,
burns, etc. (See e.g., U.S. Pat. Nos. 5,106,949, 5,104,660,
5,081,106, 5,383,930, 4,485,095, 4,485,097, 4,539,716, 4,546,500,
4,409,332, 4,604,346, 4,835,102, 4,837,379, 3,800,792, 3,491,760,
3,113,568, 3,471,598, 2,202,566, and 3,157,524, all of which are
incorporated herein by reference; Prudden, Arch. Surg. 89:1046-1059
[1964]; and Peacock et al. Ann. Surg., 161:238-247 [1965]). For
example, by itself, collagen is a relatively weak immunogen, at
least partially due to the masking of potential antigenic
determinants within the collagen structure. Also, it is resistant
to proteolysis due to its helical structure. In addition, it is a
natural substance for cell adhesion and the major tensile
load-bearing component of the musculoskeletal system. Thus,
extensive efforts have been devoted to the production of collagen
fibers and membranes suitable for use in medical, as well as
veterinary applications. Collagens have been actively incorporated
in beverage formulations (both instant and traditional), of
late.
SUMMARY OF INVENTION
[0008] The present invention relates to a process of extracting
collagen (type 1 collagen), the collagen is obtained from skins of
aquatic animals (preferably Lates calcarifer and Oreochromis
nilotica). The collagen is extracted from fish skin using an
enzyme, whereby the enzyme is papain. Furthermore, the process
comprises a starting material consisting essentially of fish skin,
extracting collagen from said the fish skin, and recovering the
collagen. The skin is obtained by removing the skin from fresh or
frozen fish. Moreover, the process further includes: extracting
collagen from skins of Lates calcarifer (barramundi) which includes
mixing the skin with alkaline solution (such as sodium) for at
least 6 hours; washing the skin with water to eliminate residual of
alkaline; soaking the skin with an alcohol (such as butyl alcohol)
solution for at least 18 hours; washing the skin with a neutral
solution; treating the skin with an acidic solution; hydrolyzing
the skin with papain; obtaining a mixture and stirring the mixture
at a working temperature of 4.degree. C. for at least 24 hours;
centrifuging the mixture at 4.degree. C.; precipitation of collagen
by introducing the collagen into a sodium chloride solution to
precipitate collagen fibers; collecting the collagen fibers and
centrifuging the collagen for at least 60 minutes thus obtaining
collagen pellet; dissolving the collagen pellet into acetic acid
solution and/or freeze-drying the dialysed suspension.
[0009] In addition, the process of extracting collagen from skins
of Oreochromis nilotica (red tilapia) further includes;
homogenizing the fish skin with alkaline solution such as sodium;
obtaining a suspension and stirring the suspension for at least 24
hours; centrifuging the suspension for at least 20 minutes;
obtaining a precipitate; homogenizing the precipitate with alkaline
solution; stirring the precipitate for at least 24 hours and for at
least 3 repeats; washing the precipitate with water and acetic acid
solution; stirring the precipitate with papain at a working
temperature of 4-5.degree. C. for at least 24 hours; obtaining a
mixture; centrifuging the mixture for at least 20 minutes;
obtaining collagen fibers; introducing the collagen into a sodium
chloride solution; collecting the collagen fibers and centrifuging
the collagen for at least 20 minutes; as a result collagen pellet
is obtained; and freeze-drying the dialysed suspension. The
collagen obtained from the skin/s of Lates calcarifer and
Oreochromis nilotica having a percentage of working yield between
14 and 40% by wet weight. The characterization of the properties of
the extracted collagen) analysis of the followings: amino acid
analysis, peptide in the collagen and type of collagen. The amino
acid which was obtained includes glycine, proline, alanine and
arginine. The peptide obtained had an apparent molecular weight
distribution of 37 and 205 kilodalton (kDa)
[0010] In addition, the present invention also relates to the use
of the collagen for the manufacture of a pharmaceutical composition
cosmetic or topical preparation or food product/s. The cosmetic
includes cream, eye cream, lotion, ointment or gel, sun-screen,
oral administration, face mask cream, anti-inflammatory medicine,
and/or anti-irritant medicine. The food product/s include(s)
beverages, dairy products, confectionaries, chocolates, and any
application in food formulation/s as an ingredient or for any
functional properties.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows SDS-PAGE Patterns of Collagen from Skins of Red
Tilapia.
[0012] FIG. 2 shows SDS-PAGE Patterns of Collagen from Skins of Red
Tilapia as Affected by Different Enzyme Extraction and Storage
Study.
[0013] FIG. 3 shows an electrophoretic pattern of collagen samples
from barramundi skin with comparison to type 1 collagen from calf
skin.
[0014] FIG. 4 shows Energy Dispersive X-ray (EDX) Chromatogram of
Collagen Sample from Red Tilapia Skins.
DETAILED DESCRIPTION OF THE INVENTION
[0015] The present invention relates to methods for preparing
collagen from aquatic animals. In particular, the present invention
provides methods for the preparation of collagen suitable as the
raw material for biomedical, and pharmaceutical applications; and
for food application.
DEFINITIONS
[0016] Creams, as is a well known arts of pharmaceutical and
cosmeceutical composition, are viscous liquids and semisolids
emulsions, either oil-in-water or water-in-oil. Cream bases are
water-washable, and contain an oil phase, and emulsifier, and an
aqueous phase. The aqueous phase usually exceeds the oil phase in
volume, and generally contains substance. The emulsifier is a cream
composition generally nonionic, anionic, cationic or amphoteric
surfactant.
[0017] The creams, lotions, gels, emulsions and paste or the like
may be spread on the affected surface and gently rubbed in. A
solution may be applied with a measuring device, swab, or the like,
and carefully applied to the affected areas.
[0018] Unless stated otherwise, the amounts are given in % by
weight, based on the total weight of the respective
preparations.
[0019] The present invention relates to the use of fish skin as
novel industrial source of collagen. Advantageously, said skin is
obtained after the filleting or cutting of the fresh fish and
frozen immediately after filleting/cutting, thus guaranteeing a
very good quality of the base material, both from the standpoint of
bacteriological and the native properties of the protein.
EXAMPLES
Materials
[0020] Red tilapia (Oreochromis nilotica) is a cultured freshwater
fish obtained from a local fish farm in Ulu Langat, Selangor. The
red tilapia weighed between 500 g to 600 g. Upon arrival at the
laboratory, the fish were killed, filleted and the skin was
manually removed. The skins were stored at -20.degree. C. until to
be used.
Chemicals
[0021] Pepsin (crystallized and lyophilized, EC 3.4.23.1, from
porcine stomach mucosa) with a declared activity of 3460 units/mg
protein was obtained from Sigma Chemical. Trypsin (crystallized and
lyophilized, EC 3.4.21.4 from porcine pancreas) with a declared
activity of 40 U/mg protein and Papain (dry powder, EC 3.4.22.2,
from Carica papaya) with a declared activity of 30000 USP-U/mg were
obtained from Merck (USA). All other chemicals used were of
analytical grade.
BEST MODE TO CARRY OUT THE INVENTION
Extraction
[0022] Storage study of the red tilapia skins was also carried out
to determine the effects of frozen storage on collagen
characteristics. Skins were stored frozen up to 8 weeks and
subjected to treatments at 2 weeks interval. The fish skins were
cut into small pieces with scissors and thoroughly rinsed in excess
water to remove superfluous material. They were then homogenized
with 10 volumes of 0.1M NaOH to remove non-collagenous proteins and
to prevent the effect of endogenous proteases on collagen (Sato and
others, 1987). The suspension was stirred overnight and centrifuged
at 10000.times.g for 20 min. The resultant precipitate was
rehomogenized with 20 volumes of 0.1M NaOH and stirred slowly
overnight. This procedure was repeated 3 or 4 times. The residue
after alkali extraction was carefully and gently washed with
distilled water and then suspended in 0.5M acetic acid.
[0023] The suspension was then stirred with enzyme at an
enzyme/substrate ratio of 1/1000 (w/w) for 24 h at 5.degree. C.
Enzymes used were pepsin, trypsin and papain. The suspension was
then centrifuged at 10000.times.g for 20 min. The collagen in the
resultant supernatant after centrifugation was salted out by adding
NaCl to give a final concentration of 2.0M. After centrifugation at
10000.times.g for 20 min, the resultant precipitate was
freeze-dried.
Analyses
[0024] Yield of collagen (% w/w)
[0025] Yield is determined by weight of collagen extracted as to
weight of wet skins used. Yield is calculated as follows.
Yield ( % w / w ) = collagen ( dry weight ) Fish skins ( Wet weight
) .times. 100 % ##EQU00001##
[0026] Visual Observations and Instrumental Colour
[0027] Visual observations for colour and texture were also noted.
Colour measurements were made using Hunterlab Ultrascan Sphere
Spectrocolorimeter (model Minolta Cr-300 Series). The samples were
filled in a clear plastic and readings were then taken. Readings
were carried out in triplicates.
[0028] Yields of dried collagens obtained from red tilapia skins
(Oreochromis nilotica) are shown in Table 1. Yield is calculated
based on the dry weight of collagen over the wet weight skins.
These yields are in the range as those reported by Takeshi and
Nobutaka (2000) for Japanese-sea-bass (55.4%), chub mackerel
(49.8%) and bullhead shark (50.1%). Nagai et al., (2000) recorded a
yield of 35.2% on a dry weight basis of collagen extracted from
rhizostomous jellyfish. Pepsin-digested extraction 0-week storage
collagen recorded the highest yield of 74.77.+-.11.36% followed by
pepsin-digested extraction 4-week storage of 63.62.+-.6.05% and
pepsin-digested extraction 8-week storage of 62.98.+-.2.37%. The
lowest yield was recorded by trypsin-digested extraction 0-week
storage i.e. 31.59.+-.5.95%.
[0029] In the storage study, 0-week storage gave the highest yield
of 74.77% followed by 4-weeks storage (63.62%) and 8-weeks storage
62.98%. Lowest yield is recorded by 2-weeks storage (37.66%). This
lower yield could be due to the loss of extracted collagen through
leaching during the series of washings steps. Thus, lower
concentrations of collagen were extracted. Another possible reason
for the lower yield could be due to the incomplete hydrolysis of
the collagen. The extraction time and temperature combination may
not be sufficient enough to allow complete hydrolysis of the
collagen. Protein composition of the tilapia skins may also
influence the yield of collagen obtained.
TABLE-US-00001 TABLE 1 Yield (%) and Protein Content (%) of
Collagen Samples from Red Tilapia Skins Yield And Protein Content
of Collagen Samples Collagen Sample Yield (%) Protein Content (%)
Papain-digested 40.15 .+-. 4.55.sup.CD 25.83 .+-. 1.01.sup.B
Trypsin-digested 31.59 .+-. 8.41.sup.D 30.15 .+-. 0.83.sup.A
Pepsin-0-week 74.77 .+-. 16.07.sup.A 14.24 .+-. 1.26.sup.E
Pepsin-2-week 37.66 .+-. 7.58.sup.CD 26.29 .+-. 0.40.sup.B
Pepsin-4-week 63.62 .+-. 6.05.sup.AB 15.65 .+-. 2.96.sup.E
Pepsin-6-week 51.51 .+-. 7.81.sup.BCD 20.09 .+-. 0.59.sup.D
Pepsin-8-week 62.98 .+-. 2.37.sup.AB 24.68 .+-. 0.40.sup.B
[0030] Values are the means.+-.standard deviations of 3 replicates.
Means with the same superscripts within each column are not
significantly different (p<0.05).
Protein Content
[0031] Protein content was determined by the micro-Kjedhal method
(AOAC, 1995) and a nitrogen conversion factor of 5.3 was used.
Protein digestion was done with concentrated sulfuric acid to
ensure complete hydrolysis of collagen. Analyses were carried out
in triplicates. Protein content of trypsin-digested collagen was
twice the protein content of pepsin-digested collagen; 30.15% and
14.24% respectively. Papain-digested collagen has a protein content
of 25.83% which is lower than trypsin-digested collagen. In the
storage study, 0-week storage and 4-weeks storage have the lowest
protein content (<15%) whereas 2-weeks storage had the highest
protein content of 26.29%.
Visual Observation and Instrumental Color
[0032] The visual observation is as shown in Table 2. All samples
except papain digested collagen were of snowy white and
light-textured. Papain digested collagen however, has light yellow
and was light-textured in appearance. Plate 1 and 2 shows the
collagen samples extracted by different enzymes and storage study
respectively.
TABLE-US-00002 TABLE 2 Visual Observation of Red Tilapia Skin
Collagen as Extracted By Different Enzymes and Storage Study.
Collagen Samples Appearance Trypsin digested Snowy white and
light-textured Papain digested Light yellow and light-textured
Pepsin 0-week Snowy white and light-textured Pepsin 2-week Snowy
white and light-textured Pepsin 4-week Snowy white and
light-textured Pepsin 6-week Snowy white and light-textured Pepsin
8-week Snowy white and light-textured
[0033] Table 3 shows the Hunter values of collagen extracted by
different enzymes and storage study. The L*, a* and b* values were
quite similar for all collagen samples. L* value which indicates
the lightness value was >93 for all samples. This shows that all
samples extracted by different enzymes and subjected to storage
study were white in colour and this corresponds with the visual
observation made. The negative a* values (-a) also shows a slight
red hue in all samples whereas the higher b* value indicates more
yellowish hue. However, papain digested collagen sample has the
highest b* value, thus more yellowish in appearance. Furthermore,
Table 3 illustrates all samples L* value of >95 indicating
whiter samples. However, the b* value of pepsin 6-week collagen
sample was the lowest compared to other samples. Nevertheless, the
L*, a* and b* values for all samples in the storage study were
almost similar and not much difference were observed which
corresponds with the visual appearance of the thus samples. The
colour of the collagen depends on the raw material. However, it
does not influence other functional properties (Ockerman and
Hansen, 1988).
TABLE-US-00003 TABLE 3 Instrumental Color of Collagen Samples As
Affected by Different Enzyme Extractions and Storage Period
Instrumental Colour Sample L a b Trypsin 95.62 .+-. 0.07.sup.BC
-1.10 .+-. 0.04.sup.EF 6.38 .+-. 0.49.sup.BC digested Papain 94.63
.+-. 0.30.sup.D -0.65 .+-. 0.04.sup.B 7.24 .+-. 0.37.sup.A digested
Pepsin 0-week 95.70 .+-. 0.67.sup.BC -0.49 .+-. 0.09.sup.A 6.77
.+-. 0.82.sup.AB Pepsin 2-week 95.85 .+-. 0.10.sup.BC -1.01 .+-.
0.02.sup.E 5.06 .+-. 0.25.sup.DE Pepsin 4-week 97.23 .+-.
0.10.sup.A -0.80 .+-. 0.04.sup.C 5.15 .+-. 0.15.sup.DE Pepsin
6-week 96.89 .+-. 0.16.sup.A -0.62 .+-. 0.06.sup.B 4.47 .+-.
0.08.sup.E Pepsin 8-week 95.94 .+-. 0.27.sup.B -0.90 .+-.
0.07.sup.D .sup. 5.73 .+-. 0.40.sup.CD
[0034] Values are the means.+-.standard deviations of 3
replicates.
[0035] Means with the same superscripts within each row are not
significantly different (p<0.05)
TABLE-US-00004 Properties Control Pepsin-extracted Papain-extracted
Yield (%) 2.8 14.0 14.1 Protein 68.72 .+-. 0.95 92.82 .+-. 2.68
111.16 .+-. 1.05 content (%) Moisture 18.46 .+-. 1.23 18.55 .+-.
0.67 16.05 .+-. 0.31 content (%) Hunter color values `L` 65.41 .+-.
0.08 61.33 .+-. 0.04 44.76 .+-. 0.02 `a` 0.14 .+-. 0.01 2.59 .+-.
0.02 0.74 .+-. 0.02 `b` 3.16 .+-. 0.03 5.35 .+-. 0.04 2.14 .+-.
0.04
Amino Acids Composition
[0036] The amino acids composition in collagen was determined using
Waters-Pico Tag Amino Acid Analyzer High Performance Liquid
Chromatography, Model: Waters 501 Millipore Corporation, USA with
column size 3.9.times.150 mm. Each sample was hydrolyzed with 6N
HCl at 110.degree. C. for 24 hrs. The hydrolysis was analyzed for
their free amino acid content on a Waters auto analyzer, as
recommended in the Waters-501 Instruments Manual (1991). Table 5
shows the amino acid composition of the collagen from red tilapia
skins due to different enzyme extractions and storage periods. The
amino acid profile obtained was from an acid hydrolysate. The amino
acid content of trypsin and papain-digested collagen are higher
than that of pepsin-digested collagen. The amino acid profile of
papain and trypsin-digested collagen are not significantly
different (p<0.05).
TABLE-US-00005 TABLE 5 Amino Acids Composition in Various Species
of Fish Collagen Amino Amino Acid Content (mg/g sample) Acids Red
Tilapia.sup.a C H Mackerel.sup.b Y S Bream.sup.b Tiger Puffer.sup.b
Asp 4.5 .+-. 0.2 42.9 .+-. 1.4 40.7 .+-. 0.9 44.6 .+-. 0.4 Glu 11.0
.+-. 0.1 72.5 .+-. 0.6 72.6 .+-. 1.2 68.0 .+-. 0.3 Ser 11.9 .+-.
0.4 35.8 .+-. 0.2 41.2 .+-. 0.6 42.9 .+-. 0.4 Gly 71.9 .+-. 0.3
361.5 .+-. 0.3 351.3 .+-. 2.0 349.9 .+-. 2.1 His ND 4.4 .+-. 0.0
3.8 .+-. 0.2 3.6 .+-. 0.1 Arg 35.6 .+-. 0.8 53.0 .+-. 0.4 52.0 .+-.
1.3 52.7 .+-. 0.1 Thr 10.8 .+-. 0.1 30.1 .+-. 0.4 29.8 .+-. 0.5
29.1 .+-. 0.5 Ala 37.3 .+-. 0.4 120.6 .+-. 0.7 124.7 .+-. 1.2 117.5
.+-. 1.6 Pro 55.8 .+-. 0.9 115.2 .+-. 2.2 110.7 .+-. 0.4 112.5 .+-.
0.3 Tyr 3.2 .+-. 0.4 1.5 .+-. 0.1 2.0 .+-. 0.0 1.6 .+-. 0.1 Val 8.0
.+-. 0.0 16.1 .+-. 0.4 16.3 .+-. 0.3 21.7 .+-. 0.0 Met 6.0 .+-. 1.1
10.1 .+-. 0.9 11.4 .+-. 2.0 14.0 .+-. 0.2 Cys ND ND ND ND Ile 5.1
.+-. 0.1 8.2 .+-. 0.2 6.9 .+-. 0.1 7.4 .+-. 0.0 Leu 11.4 .+-. 0.2
18.7 .+-. 0.1 17.0 .+-. 0.7 15.4 .+-. 0.2 Phe 6.5 .+-. 4.9 11.2
.+-. 0.0 12.2 .+-. 0.5 12.3 .+-. 0.1 Lys 10.9 .+-. 0.7 23.7 .+-.
0.9 25.6 .+-. 1.3 29.0 .+-. 1.8 Total 289.9 925.5 918.2 922.2
ND--Not Detected .sup.aTrypsin digested collagen results
.sup.bSourced from Yata et al. (2001)
[0037] The total amino acids content increases gradually from
4.sup.th to 8.sup.th week storage. Glycine and proline are the
major amino acids, constituting a quarter and a fifth of total
amino acid content of the collagen samples respectively. This
characteristic distinguishes collagen from other proteins. Proline
content in both trypsin (55.81) and papain-digested (51.54)
collagens are double than in pepsin-digested 0-week storage
(25.95). However, in this present invention, hydroxyproline content
could not be determined. These compositions are different from
which was reported from horse mackerel, yellow seabream and tiger
puffer.
TABLE-US-00006 TABLE 6 Amino acid (g/100 g) Control
Pepsin-extracted Papain-extracted Aspartic acid 3.797 .+-. 0.14
4.075 .+-. 0.19 4.038 .+-. 0.05 Serine 1.927 .+-. 0.07 1.989 .+-.
0.09 2.30 .+-. 0.03 Glutamic acid 6.847 .+-. 0.25 7.061 .+-. 0.35
7.256 .+-. 0.10 Glycine 14.992 .+-. 0.56 15.539 .+-. 0.76 19.208
.+-. 0.31 Histidine 1.449 .+-. 0.04 1.298 .+-. 0.02 Not detected
Arginine 5.943 .+-. 0.07 6.38 .+-. 0.06 8.316 .+-. 0.08 Threonine
2.066 .+-. 0.05 2.171 .+-. 0.02 2.485 .+-. 0.02 Alanine 6.996 .+-.
0.27 6.865 .+-. 0.20 8.890 .+-. 0.12 Proline 7.887 .+-. 0.21 7.543
.+-. 0.18 12.772 .+-. 0.07 Cysteine Not detected Not detected Not
detected Tyrosine 0.258 .+-. 0.01 0.4925 .+-. 0.03 0.298 .+-. 0.01
Valine 1.739 .+-. 0.07 1.8715 .+-. 0.09 2.080 .+-. 0.03 Methionine
1.299 .+-. 0.06 1.345 .+-. 0.06 1.676 .+-. 0.48 Lysine 2.561 .+-.
0.09 2.872 .+-. 0.14 2.338 .+-. 0.01 Isoleucine 0.903 .+-. 0.04
1.063 .+-. 0.05 1.135 .+-. 0.02 Leucine 1.664 .+-. 0.07 1.931 .+-.
0.09 2.229 .+-. 0.03 Phenylalanine 1.436 .+-. 0.09 1.369 .+-. 0.15
1.898 .+-. 0.02 Total 61.40 63.862 76.919
Molecular Weight Determination
[0038] Molecular weight was determined by SDS-PAGE (Laemmli, 1970)
and the run was made in a 5% T gel containing 0.1% SDS. Molecular
weight marker SDS-6H (Sigma) was used as the standard. Samples
(4-50 .mu.g/well) were applied to the gel and the gel was stained
for protein with Coomassie Brilliant Blue R-250. FIGS. 1 and 2
shows the SDS-PAGE patterns of collagen from skins of red tilapia
extracted with different enzymes and storage period respectively.
Visually 16, 15 and 7 bands were observed for trypsin, papain and
pepsin-digested extraction respectively. Apparent molecular weights
of peptide detected were in the range of 20,300 Da to 221,900 Da
for papain-digested extraction whereas 31,500 Da to 200,000 Da for
trypsin-digested extraction. In pepsin-digested extractions
(storage study), no peptide bands above 144,200 Da were observed.
The molecular weight patterns for the storage study samples are
also almost similar. Identification of the molecular bands
associated with the types of collagens is the common results report
for the SDS-PAGE electrophoresis.
[0039] FIG. 3 represents an electrophoretic pattern of collagen
samples from barramundi skin with comparison to type 1 collagen
from calf skin. Apparent molecular weights of peptide detected were
in the range of .about.30,000 to 250,000 kDa. Collagen from
barramundi skin contained two different .alpha. chain
(.alpha..sub.1 and .alpha..sub.2) and .beta.-component. These
electrophoretic patterns were similar to calf skin collagen and the
kind of dimmer was also observed in collagen samples from other
aquatic sources. The acid-soluble collagen and pepsin-soluble
collagen had similar electrophoretic pattern with type 1 collagen
from calf skin. However, papain-extracted collagen shows slightly
different pattern, the .beta. dimmer was not observed. Collagen
with papain treatment also contained products of enzymatic
hydrolysis below 100 kDa. This result suggested that different type
of proteases have different cleavage properties of the collagen
samples, as shown in the SDS-PAGE chromatogram.
Mineral Content
[0040] Mineral analysis was determined using Energy Dispersive
X-ray (EDX). The collagen sample was mounted onto the stub and
viewed by EDX, using QBSD signal. Table 7 shows the mineral
analysis of collagen from tilapia skins by different enzyme
extractions and different storage period. Mineral analysis by EDX
was carried out to determine the mineral elements of collagen
obtained. Four elements were detected in all samples, namely
carbon, oxygen, sodium and chlorine as shown by the EDX
chromatogram (FIG. 4). Collagen is not a metalloprotein, therefore
the absence of heavy metals indicates that these collagens are safe
(GRAS). The presence of sodium and chlorine in all collagens are
probably due to the effect of salting process (NaCl) to precipitate
out collagen after extraction step. Trypsin-digested extraction
showed the highest carbon and oxygen content of 48.64% and 7.69%
respectively. Chlorine is the major element in all samples except
for trypsin-digested extraction (33.61%). Oxygen is the minor
element detected in all samples ranging from 1-7%. The carbon
content indicates that the collagens are of organic matter. To this
date, there are no reported findings of mineral analysis of
collagens.
TABLE-US-00007 TABLE 7 Mineral Analysis (in Weight %) of Collagen
from Red Tilapia Skins by Different Enzyme Extraction and Storage
Period Sample Carbon Oxygen Sodium Chlorine Trypsin 48.64 .+-. 4.03
7.69 .+-. 0.41 10.05 .+-. 1.11 33.61 .+-. 3.15 0-wk Papain 21.54
.+-. 12.15 2.03 .+-. 1.49 18.72 .+-. 2.65 57.79 .+-. 11.15 0-wk
Pepsin 13.56 .+-. 2.89 1.02 .+-. 0.36 22.47 .+-. 0.36 65.95 .+-.
2.58 0-wk Pepsin 30.04 .+-. 3.48 4.00 .+-. 0.99 11.45 .+-. 4.12
54.50 .+-. 7.56 2-wk Pepsin 33.30 .+-. 0.94 4.32 .+-. 0.15 15.45
.+-. 0.40 46.92 .+-. 0.78 4-wk Pepsin 12.07 .+-. 1.81 1.56 .+-.
0.35 20.49 .+-. 2.10 65.88 .+-. 3.73 6-wk Pepsin 23.25 .+-. 3.15
2.33 .+-. 0.47 16.33 .+-. 1.68 58.09 .+-. 4.41 8-wk
[0041] Values are the means.+-.standard deviations of 3
replicates.
[0042] Means with the same superscripts within each row are not
significantly different (p<0.05)
Storage Study
[0043] Skins were stored frozen (-20.degree. C.) for up to 2
months. Every week, the skins were thawed and subjected to
treatment to extract collagen. The collagen obtained was then
analyzed as before to determine the effects of frozen storage of
skins on the collagen characteristics.
Statistical Analysis
[0044] All data collected were analysed using the analysis of
variance (ANOVA) and Duncan's Multiple Range Test to determine the
significant differences between means (SAS, 1987). Extraction
processes of collagen and the following treatments reflect the
different quality of gelatine obtained. Collagen from skins of red
tilapia was extracted by a series of washings with 0.1 M NaOH
followed by enzyme-aided extraction for 24 hrs at 5.degree. C. The
colloidal suspension was salted out with NaCl and freeze-dried.
Enzymes used were pepsin, trypsin and papain. Storage study of red
tilapia skins was also carried out to determine the effects of
frozen storage on collagen characteristics. Visually, all collagen
samples were appeared to be similar and the differences could only
be detected by chemical analyses such as protein content, amino
acid composition, molecular weight profile and mineral content. The
collagens from different enzymes extraction and storage period were
snowy white and light-textured in appearance. The collagen yield
was in the range 32.54% to 74.77% with pepsin digested collagen
showing the highest yield. However, the protein content of pepsin
digested collagens was much lower compared to trypsin and papain
digested collagens. Protein content recorded was in the range 15%
to 30%. Amino acid content of trypsin and papain digested collagen
are higher than pepsin digested collagen. Apparent molecular
weights of peptide detected were in the range 20,000 Da to 222,000
Da. In the mineral analysis, four elements were detected in all
collagen samples namely carbon, oxygen, sodium and chlorine. SEM
observations of all collagen samples show similar network
structure. The collagen fibres are long cylindrical protein
embedded in the protein matrix. Based on the results, trypsin
digested collagen showed the best results in terms of protein
content and amino acid profile but gave a low yield. Pepsin
digested collagen on the other hand showed high yields but lower
protein and amino acid content. Therefore, papain which showed
reasonably high yield, protein and amino acid content will be
chosen for the study of effects of enzyme on gelatin extraction and
characteristics in the next chapter. Papain, being a plant based
enzyme will also overcome the religious issues associated with
certain communities such as Islam and Judaism. In the storage
study, frozen storage of tilapia skins does not have significant
effects on the collagen characteristics. Therefore, skins may be
stored frozen for weeks without much affecting the characteristics
of the gelatin extracted.
* * * * *