Author’s Accepted Manuscript

Isolation, characterization and valorizable

applications of fish scale collagen in food and
agriculture industries

Prashant K. Bhagwat, Padma B. Dandge

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PII: S1878-8181(16)30092-5
DOI: http://dx.doi.org/10.1016/j.bcab.2016.06.010
Reference: BCAB415
To appear in: Biocatalysis and Agricultural Biotechnology
Received date: 5 May 2016
Accepted date: 16 June 2016
Cite this article as: Prashant K. Bhagwat and Padma B. Dandge, Isolation,
characterization and valorizable applications of fish scale collagen in food and
agriculture industries, Biocatalysis and Agricultural Biotechnology,
http://dx.doi.org/10.1016/j.bcab.2016.06.010
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 Isolation, characterization and valorizable applications of fish scale collagen in

food and agriculture industries.

Prashant K. Bhagwata, Padma B. Dandgeb*

a
Department of Microbiology, Shivaji University, Kolhapur, 416004 India
b
Department of Biochemistry, Shivaji University, Kolhapur, 416004 India
*
Corresponding author: Dr. (Mrs.) Padma B. Dandge, Department of Biochemistry, Shivaji

University, Kolhapur 416004, India, Co. No.: +919921111722. E-mail:

pbd_biochem@unishivaji.ac.in

Abstract

Collagen has enormous applications in food, biomedical and pharmaceutical industries;

but its high cost severely limits its use. Fish processing waste is a promising and cost efficacious

source of collagen, which otherwise stated as an earnest environmental pollutant. Carp (Cyprinus

carpio) is one of the main species of freshwater fish consumed in India. Acid-soluble collagen

(ASC) was isolated from scales of Cyprinus carpio. Scales were demineralized by EDTA

treatment and ASC was extracted from the demineralized scales. The yield of scale ASC was

9.79% (on the wet weight basis). SDS–PAGE and FTIR corroborated the isolated protein as

collagen. Denaturation temperature (Td) of isolated collagen was found to be 37ºC. Considering

bioactive properties of collagen, a milk based food product - paneer was developed by

incorporation of the extracted collagen. Composed paneer was found to be acceptable with good

sensorial and textural attributes. Same scales were further treated enzymatically and the released

metabolites were tested for their competency to promote the plant growth. Released metabolites

1
showed excellent plant growth promotion and hence could be successfully employed as an

economic source of nitrogen fertilizers for plants.

Keywords: Collagen, Fish processing waste, Cyprinus carpio, Scales, Paneer, nitrogen fertilizers.

1. Introduction

Fishing industry is one of the established food sectors which supply ample amount of food

to deliberately growing population. Fish continues to be one of the most-traded food

commodities worldwide. India’s worldwide share in fishery industry is increasing day by day.

India stands at 7th position worldwide in case of fish captures from marine sources and it is at 2 nd

position in fish captures from inland sources. Interestingly inland fish production has been

growing with a great speed showing necessity of fish as a food source in noncoastal regions of

India (FAO, 2014).

A huge amount of fish production also tends to produce nearly equal amount of waste as

that of final product. Processing of fish involves stunning, grading, slime removal, deheading,

washing, scaling, gutting, cutting of fins, meat bone separation and steaks and fillets. During

these steps significant amount of waste (20-80% depending upon the level of processing and type

of fish) is generated (Ghaly et al., 2013). Very large quantity of waste produced should be

properly disposed, but a good care cannot be taken for all the waste produced. Currently waste is

disposed in a number of ways, polluting our environment in a very rigid manner. Landfilling and

incineration are some of the methods which are used many times, but they are not fruitful as they

are costly as well as require a good maintenance (Kim and Venkatesan, 2014). Moreover using

these techniques waste having important biomolecules are not utilized properly and world’s

current scenario does not allow us to do so, hence we should try to use and isolate the important

constituent present in those wastes in a technological way.

2
 Collagen is very important biomolecule and is most abundant protein in mammals

representing nearly 30% of total proteins in the animal body. (Pati et al., 2010) Enormous health

benefits of collagen have led to the establishment of collagen supplements industry. Food and

pharmaceutical industries all over the world are witnessing an increasing demand for collagen.

The collagen may advance the function of skin dermis and epidermis by increasing the water

absorption ability of the outermost skin layer. Hydration of skin tissue is directly allied to

smoothness and reduces wrinkling (King’ori, 2011). Collagen supplement can boost up lean

muscle gain, decrease recovery time, reconstruct damaged joint structure and improve

cardiovascular performances. Therefore, collagen is in demand within the sports nutrition field

(Hashim et al., 2015). Among various types, type I collagen has been extensively used as

biomaterial for the development of tissue engineering constructs and wound dressing systems

due to its low antigenic and high direct cell adhesion properties (Falguni et al., 2010).

Mammalian collagens (porcine and bovine) are the most popular and widely used but are

facing problems due to their allergic reactions as well risk of transmissible diseases like bovine

spongiform encephalopathy (mad cow disease), ovine and caprine scrapie, and other zoonoses

(Lynn et al., 2004; Pati et al., 2012). The major protein constituent of seafood processing waste

like skin, bone, swim bladder, and scales resembles, in many ways, the more widely studied

collagen of mammals (Eastoe, 1957; Giraud-Guille et al., 2000). As fish collagen reportedly

possesses similar characteristics to porcine collagen, and may, thus, be considered as an

alternative to mammalian collagen (Kim and Venkatesan, 2014, Pati et al., 2012).

So current study aims for i) Isolation and characterization of collagen from the scales of

Cyprinus carpio. ii) Use of extracted collagen in paneer production. iii) Further recovery of

3
collagen from the same scales by enzymatic treatment. iv) Use of enzymatically solubilized

collagen extracts for plant growth promotion studies.

2. Materials and methods

2.1. Collagen extraction from fish scales

Extraction of collagen from fish scales was carried out in two steps. First step was used

for demineralization of calcium from fish scales using EDTA and subsequently acid soluble

collagen (ASC) was isolated by dilute acetic acid treatment. Both processes were performed at

the temperature no higher than 4°C to reduce temperature induced defragmentation of collagen.

2.1.1. Demineralization of fish scales

Fish scales of Cyprinus carpio were collected from local market of Kolhapur, (MS) India.

These scales were thoroughly washed five times with distilled water. Then for removal of

unwanted surface proteins, scales were treated in a system containing 1.0 M NaCl, 0.05 M Tris

HCl, 20.0 mM EDTA at pH 7.5 for 48 hours. It was followed by the demineralization process, in

which scales were treated with 0.5 M EDTA solution at pH 7.4 for 48 hours. The demineralized

fish scales were washed thrice with distilled water and used further.

2.1.2. Isolation of collagen

Demineralized fish scales were treated with 0.5 M acetic acid solution at pH 2.5 for 48

hours and the insoluble part of the scales were filtered out. Filtrate having soluble collagen is

then recovered by salting out technique using NaCl to a final concentration of 0.9 M and kept

undisturbed for 24 h. After incubation it was centrifuged at 8000 rpm for 20 min and precipitate

was again solubilized in 0.5 M acetic acid. Both the processes of salting out and centrifugation

4
were repeated twice for better purification of collagen. This purified collagen was then dialyzed

against 0.1 M acetic acid and distilled water for 24 h each and freeze-dried. (Pati et al., 2010)

2.2. Characterization of the extracted collagen

2.2.1. FTIR analysis

Sample of collagen was mixed with approximately 5 times of vaccum dried KBr and

pressed into pellets by hydraulic press. Infrared spectra were obtained in the range between 4000

and 650 cm-1 using an infrared spectrophotometer (Model – Jasco FTIR-410).

2.2.2. SDS–PAGE

SDS–PAGE was performed by following the method of Laemmli (Laemmli, 1970) with

8% separating gel and 4% stacking gel. Extracted collagen sample was dissolved to 4 mg/ml in

sample buffer, type I collagen from calf skin was parallely run for comparison. About 50 µl of

sample was loaded per well. The gels were stained using 0.1% (w/v) Coomassie brilliant blue

R250 dissolved in methanol-water-acetic acid (5:4:1, v/v/v, respectively) and destained with

distilled water-methanol-acetic acid (20:3:2).

2.2.3. Temperature induced change in viscosity

The denaturation of collagen was determined from temperature induced viscosity change

using an Ostwald’s viscometer. Solutions of 0.1% (w/v) collagen were prepared in 0.1 M acetic

acid at 10°C. The solutions were loaded to viscometer and incubated at 10°C for 30 min through

submerging in water bath. The temperature was raised stepwise to 50°C and incubated for 10

min at each temperature. Fractional viscosity was calculated for each temperature as follows:

Fractional viscosity =

(measured viscosity - minimum viscosity) / (maximum viscosity - minimum viscosity);

5
The denaturation temperature (Td) is the temperature at which fractional viscosity is 0.5 and was

estimated from graph (Pati et al., 2010).

2.3.1. Use of extracted collagen in paneer production

Buffalo milk paneer was prepared by following the procedure of Sunil Kumar et al.

(Kumar et al., 2008) with slight modifications. The standardized milk (6% fat) sample was

divided into two groups. Milk samples were held at 82°C for 5 minutes and then cooled to 70°C

one group was added with 2.5% collagen (wet weight by volume) and labeled as test while other

group without addition of collagen was labeled as control. Milk samples were coagulated by 2%

of citric acid with continuous agitation till clear whey separated out. The curd was left to settle

down for 10 minutes without agitation; whey was removed and curd was pressed in hoops lined

with muslin cloth. Curd blocks were pressed for 15 minutes by applying pressure with a weight

of 5 kg, then immersed in chilled water and placed on wooden planks to allow loose water to

drip down. Physico-chemical properties of the paneer samples were determined by the standard

methods of Association of Analytical Chemists (AOAC, 1980). Sensory evaluation of the

products was done by a panel of 5 experienced judges using 9 point hedonic scale (1 = disliked

extremely, 9 = liked extremely).

2.3.2. Texture profile analysis of paneer

The samples were analyzed with the help of TA-XT2 Texture Analyzer, Stable Micro

System Ltd., London. Texture profile analysis was carried out using probe P/75 (75 mm

Compression platen), in compression mode at pretest speed 1.00 mm/s, test speed 5.00 mm/s,

post-test speed 5.00 mm/s. Target mode was kept at 40.00 % strain and trigger force 5.0 g. Data

acquisition rate was kept at 200.00 pps (Gatade and Sahoo, 2015).

6
2.4.1. Enzyme aided recovery and degradation of collagen from scales

Acid solubilization treatment does not completely extract collagen from the scales.

Therefore after extraction of collagen by acid hydrolysis treatment from scales, same scales were

again treated with partially purified collagenolytic protease of Pseudomonas strain SUK which

was previously isolated in our lab (Bhagwat et al., 2015). 10% scales were autoclaved and strain

SUK enzyme (50 U) was added in the test. Similarly in control flask, heat denatured enzyme was

added. It was kept shaking on 120 rpm at room temperature, samples were periodically

withdrawn and were tested for its amino acid and protein content up to 48 hours. The amount of

released free amino acid was measured by the ninhydrin method using standard leucine curves

(Moore and Stein, 1954) and the protein content was determined by Lowry method using bovine

serum albumin (BSA) as a standard protein (Lowry et al., 1951).

2.4.2. SDS-PAGE analysis

SDS–PAGE analysis was carried out in order to confirm the action of enzyme on

collagen. Samples from both the control and enzyme treated flasks were analyzed with the

method of Laemmli (Laemmli, 1970) using 8% separating gel and 4% stacking gel. Both the

samples were concentrated up to 4 mg/ml and mixed with sampling buffer. About 50 µl of

sample was loaded per well. The gels were stained using Coomassie brilliant blue R250

dissolved in methanol-water-acetic acid (5:4:1, v/v/v, respectively) and destained with distilled

water-methanol-acetic acid (20:3:2).

2.4.3. Plant growth promoting ability of enzymatically treated collagenous waste

Enzyme treated scales were filtered and its aqueous soluble extract (ASE) was autoclaved

and checked for its efficiency to promote the seed germination and root growth of Vigna radiata.

Seeds of V. radiata were surface-sterilized with 75% (v/v) ethanol for 10 min and washed

7
thoroughly several times with distilled water. For germination and root growth experiment, seeds

were placed in petri dishes containing the ASE solutions at the following concentrations: 25%,

50%, 75% and 100%. The dishes for each treatment were scored for radicle emergence and root

length at 12, 24 and 36 hours after the start of imbibition, and both factors were calculated as a

percent of the control (deionized water) treatment. All germination and growth experiments were

maintained in darkness, at room temperature. All assays were replicated at least three times; each

replicate was carried out on 100 seeds with 10 ml ASE solutions for germination and root growth

measurements.

2.5. Statistical analysis

Results obtained were mean of three or more determinants. One way ANOVA was done

using Microsoft Office data analysis tool pack. Difference of p ≤ 0.05 was considered as

significant.

3. Results and discussion

3.1. Collagen extraction from fish scales:

Fish namely Cyprinus carpio was used in this study. Generally scales of fish were having

abundant amount of collagen protein which is packed firmly in calcium hydroxyapatite crystals

(Olson and Watabe, 1980). Collagen from the scales can be extracted by the acid hydrolysis but

before that scales has to be de-calcified, decalcification of respective scales exposes collagen

fibers which can be easily extracted. EDTA treatment was used for the decalcification of the

mineralized scales, where hydroxyapatite crystals were solubilized by chelating Ca2+ ions.

Further collagen was solubilized in 0.5 M acetic acid and was recovered by salt precipitation.

Cyprinus carpio scales yielded around 9.79% yield of collagen on wet weight basis which was

greater than carp (1.35%), catla (3.9%), mrigala (3.2%) (Gaurav and Suresh, 2016).

8
3.2. Characterization of the extracted collagen

3.2.1. FTIR analysis

FTIR spectra with peak locations was assigned for collagen from Cyprinus carpio as

shown in fig. 1. The absorption intensity between 1241 cm-1 (amide III) and 1411 cm-1 (amide II)

band ratio was approximately equal to 1.0, which confirms the triple helicle structure of

collagen. (Plepis et al., 1996) Normal absorption range of the amide II bands position is 1550-

1600 cm-1 which was found to be shifted to lower frequency, 1411 cm -1 which indicates the

existence of hydrogen bonding in collagen (Pati et al., 2010) and COO- symmetrical stretching

(Jackson et al., 1995). Whereas, amide III band with NH bending coupled with CN stretching

was found at 1241 cm-1 (Payne and Veis, 1988).

Amide I bond associated with streching vibrations of carbonyl groups (C=O) along the

polypeptide backbone, with characteristic frequencies in the range from 1600-1700 cm-1, which

is a marker of peptide secondary structure was found to be at 1596 cm-1 in this study (Payne and

Veis, 1988; Surewicz and Mantsch, 1988). The amide A band is associated with the N-H

streching frequency which occurs in the range of 3400-3440 cm-1, it is shifted to lower

frequencies when NH group of peptide is involved in hydrogen bonding (Pati et al., 2010). In

this case amide A band of Cyprinus carpio was at 3312 cm-1 (Veeruraj et al., 2013), whereas

amide B band with CH2 asymmetrical stretching was observed at 2920 cm-1 (Abe and Krimm,

1972).

3.2.2. SDS–PAGE

ASC extracted from scales of Cyprinus carpio and type I collagen from calf skin (sigma

aldrich) were analyzed by polyacrylamide gel electrophoresis in presence of SDS, using 8%

resolving gel (Fig. 2). Electrophoretic pattern showed that ASC from Cyprinus carpio was

9
composed of one β chain and at least two different α chains, α1 and α2; and density wise α1 was

far intense than α2 chain. On the basis of subunit composition of SDS-PAGE compared to the

type I collagen derived from calf skin, it could be inferred that ASC was mainly composed of

type I collagen. All these results were in agreement of the previous reports (Duan et al., 2009;

Pati et al., 2010; Thuy et al., 2014).

3.2.3. Temperature induced change in viscosity

The denaturation temperature (Td) of acid soluble collagen of Cyprinus carpio was

calculated by using temperature induced change in viscosity (Fig. 3). The Td of fish scale

collagen from Cyprinus carpio was found to be 37°C which was similar to the Td of porcine skin

collagen (37°C) and very near to that of fish scale collagen of Labeo rohita and catla catla

(36.5°C) which are fresh water fishes (Pati et al., 2010). Whereas, collagen extracted from

marine fishes such as eel (30.2°C), common mackerel (26.9°C), saury (24.0°C), chum salmon

(20.6°C) (Kimura et al., 1988) have Td less as compared to fresh water fishes. Cyprinus carpio is

a fresh water fish of tropical countries, habituated to temperature variation as low as 5°C in the

winter to about 45°C in the summer. So scales of Cyprinus carpio may act as thermal cushions.

(Pati et al., 2010).

The utilization of collagen as a biomaterial is currently undergoing a renaissance in the

tissue engineering field (Friess, 1998). Collagen from fish scales reported to have a good

biocompatibility with potential applications in tissue engineering (Pati et al., 2012). Owing to

fair propinquity in Td with mammalian collagen, the isolated collagen may accommodate as an

alluring alternative to mammalian collagen for biomedical and pharmaceutical applications.

3.3.1. Use of extracted collagen in paneer production

10
 Collagen is one of the important proteins in our body; it is foundation of connective

tissues that makes our skin elastic and flexible. Aging is a natural process where body’s collagen

production reduces which increases the chance of wrinkles. Collagen supplements are intended

to uphold skin, hair, nails and body tissues of the users. The metabolites of collagen assemble

bone, skin and ligaments by attracting fibroblasts that generate the synthesis of new collagen.

Therefore, the thickness, suppleness and resilience, as well as hydration of the tissues will be

enhanced (King’ori, 2011).

Owing to this fact paneer incorporated with collagen was produced using buffalo milk.

Physico-chemical analysis of formed paneer showed that concentration of moisture and protein

was more in test samples as compared to control samples indicating supplemented collagen is

well retained in paneer samples (Table 1A). Sensory analysis revealed that both the test and

control samples have good acceptability. Supplementation of collagen in to paneer improved the

protein concentration of the final product fulfilling the objective of the study. This is the first

report on use of collagen in paneer as a supplement however, use of seafood-derived collagen

previously has been reported in preparation functional food, beverage, dietary supplements and

confectionery (Antoniewski & Barringer, 2010; Bilek & Bayram, 2015; Hashim, Ridzwan,

Bakar, & Hashim, 2015; Regenstein & Zhou, 2007).

3.3.2. Texture profile analysis of paneer

Both the test and control samples were then analyzed of with the help of TA-XT2 texture

analyzer (Table 1B). Hardness, adhesiveness and gumminess of test samples were moderately

altered as compared to control samples whereas, springiness, cohesiveness, chewiness, and

resilience were slightly altered. Similar results were observed in case of chicken frankfurters

11
made with added collagen (Meullenet et al., 1994). These results clearly indicate that

incorporation of collagen in paneer does not alter its textural attributes extremely.

3.4.1. Enzyme aided solubilization and degradation of collagen from scales

Acid hydrolysis mediated collagen recovery extracts maximum concentration of collagen

from scales, although minute quantities of collagen remain in the treated scales. So further these

scales were treated enzymatically to recover the collagen. Collagenolytic protease of

Pseudomonas strain SUK (50 U) was used to treat 10% autoclaved scales for the period of 48

hours. Concentrations of soluble amino acids as well as proteins were found to be increasing in

the test as compared to control suggesting enzymatic solubilization of collagen (Fig. 4).

3.4.2. SDS-PAGE analysis

SDS-PAGE analysis of the test and control samples corroborates the above results (Fig.

5). In control samples one β chain and two different α chains, α1 and α2; of collagen can be

clearly seen whereas in test samples only β chain can be seen and most of the components were

found to be solubilized due to action of enzyme.

3.4.3. Plant growth promoting ability of enzymatically treated collagenous waste

Aqueous soluble extract (ASE) of enzyme treated scales were used for seed germination

and root growth of V. radiata (Fig. 6 and 7). ASE which is rich in soluble amino acid as well as

protein concentrations act as a natural stimulant and enhance the growth of V. radiata. Soluble

amino acids stimulate protein synthesis and hence seed germination index as well as root length

of ASE treated seeds were found to be increased. The results suggest that enzymatically

solubilized end products of scales have a beneficial effect on seed germination and root length of

V. radiata. Some of the studies showed similar results where soluble fractions of chicken

12
feathers degraded by Bacillus subtilis PF1 and Paenibacillus woosongensis TKB2 promoted the

plant growth (Bhange et al., 2016; Paul et al., 2013).

4. Conclusion

This study gives an innovative idea of use of fish waste in a technological way which

could be efficiently utilized in fish waste management. Seafood processing waste like scales

which otherwise cause earnest environmental pollution are opulent sources of novel and valuable

biomolecule “collagen”. Up till now study has been oriented on recovery of collagen from fish

waste, but the remaining waste also has potential and can be used for agricultural purposes.

This is the first report on the use of fish scales in a diverse way with lucrative outcomes.

Collagen from the scales of Cyprinus carpio was extracted and its biochemical characteristics

were analyzed. Extracted collagen may serve as a captivating alternative to mammalian collagen

and can be exploited in food, biomedical as well as in pharmaceutical fields. Further, paneer was

prepared supplementing extracted collagen; which showed to have good acceptability.

Subsequently remaining waste was again treated enzymatically and the released metabolites

were successfully utilized for growth studies of V. radiata.

Acknowledgement

Prashant K. Bhagwat is thankful to UGC for awarding BSR meritorious Fellowship for

doctoral research. Corresponding author wish to thank UGC-MRP with sanction grant No

F.No.41-1282/2012 (SR).

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Fig. 1 FTIR analysis of fish scale collagen of Cyprinus carpio

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Fig. 2 SDS-PAGE pattern of acid soluble collagen from scales of Cyprinus carpio (ASC).

Lane 1: ASC; lane 2: Type I collagen from calf skin.

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Fig. 3. Change in fractional viscosity with temperature of fish scale collagen of Cyprinus carpio

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Fig. 4. Amino acid content (A) and protein content (B) released from scales treated with

collagenolytic protease of Pseudomonas strain SUK, Control (●) and Test (♦).

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Fig. 5. SDS-PAGE pattern of collagen from scales of Cyprinus carpio treated with collagenolytic

protease (ASC). Lane 1: Control; lane 2: Test.

Fig. 6. Germination (A) and root length (B) of Vigna Radiata seeds at various concentrations of

ASE after 12 (♦), 24 (■), and 36 (▲) h. For each treatment 100 seeds were used and the

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 germination percentage and root length represent the mean number of germinated seeds from

three independent experiments.

Fig. 7. ASE promotes the growth of Vigna radiata: A - Control, B - 25%, C - 50%, D - 75%, E -

100%

Table 1A: Physico-chemical analysis of paneer enriched with collagen from Cyprinus carpio

Sensory
Moisture (%) Protein (%) Ash (%) Lactose (%) Fat (%)
scores
Test 47.64±0.43 21.44±0.86 3.26±0.1 2.58±0.03 24.49±0.49 7.3±0.7
Control 46.50±0.41 19.44±0.57 3.60±0.2 2.73±0.05 25.74±0.31 7.9±0.4

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Table 1B: Texture profile analysis of paneer enriched with collagen from Cyprinus carpio

Hardnes Adhesivene Springines Cohesivene Gummine Chewines Resilienc

s ss s ss ss s e
4469 ± -8.571 ± 0.592 ± 0.365 ±
Test 1631 ± 23 965 ± 99 0.1 ± 0.0
83 -3.8 0.069 0.002
Contro 3998 ± -1.002 ± 0.626 ± 0.39 ± 0.12 ±
1559 ± 36 977 ± 60
l 41 -0.8 0.024 0.013 0.01

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Graphical abstract