**9. Collagen extraction research and its applications**

The popularity of collagen extraction continues to increase due to many reasons. It is a high strength protein, bio-derived, has excellent biocompatibility, biodegradability, and has weak antigenicity. Another main reason that relates to waste valorisation and sustainability is the fact that collagen can be extracted from almost any mammalian skin, bones, cartilage, fish skin and even chicken feet. Most often, the meat industry results in these by-products that can end up in landfill. These advantageous characteristics have made collagen one of the most useful biomaterials.

Research to this day is being carried out to improve extraction methods in terms of efficiency and economics. In addition to improving extraction methodologies, research is being carried out on collagen to enhance its use in several industries (**Table 9**).



**Period Collagen extraction research**

*DOI: http://dx.doi.org/10.5772/intechopen.94266*

*Collagen: From Waste to Gold*

recommended.

for food purposes.

types were identified.

pepsin-solubilised collagen.

FTIR, and solubility analysis.

be 24.6°C.

efficiency.

PAGE).

was carried out with 0.4 M NaCl.

**1990– 1999**

**2000– 2009**

**225**

collagen content and acetic acid or neutral salts were used to extract collagen. Using SDS-

• Kurita et al. [110] analysed the changes in collagen types during the healing of rabbit tooth extraction wounds. Collagen type was identified by use of SDS-PAGE analysis and

Collagen quantification and understanding types of collagen present in diseased tissue vs.

• Montero et al. [111] extracted collagen from Plaice skin and analysed its functional properties. Acetic acid was the main solubilising agent in this study and homogenisation

• Ambrose et al. [112] extracted and characterised collagen from bone and teeth for isotopic analysis. Carbon to nitrogen ratios of bone and teeth collagen was analysed the use of purification procedures that removed acid and alkaline-soluble contaminants were

• Ciarlo et al. [114] extracted collagen from hake skin. Acetic acid was used for solubilisation and collagen was characterised for its viscosity and collagen type (SDS-

the study of collagen in human tissue was still predominant.

solubilised collagen having a higher Td (30.87°C).

type II obtained from Sigma Aldrich.

Muslim religions not accepting certain meat products).

• Bishop et al. [115] extracted and characterised collagen types II and IX from bovine vitreous. Centrifugation and precipitation with 4.5 M NaCl were applied and the collagen

In this era, extraction of collagen from waste materials such as fish skin began, however, the methodologies were mainly focused on salt extractions, which is not very efficient, and

• In 2007, Nalinanon et al. [59] extracted collagen from the skin of bigeye snapper using pepsin. Acid-extracted collagen resulted in lower collagen yields in comparison to

• In 2003, Sadowska et al. [73] isolated collagen from the skin of Baltic cod. The aim of this investigation was also to determine optimum conditions for the extraction of collagen from cod skin. Acetic acid and citric acid were used as the main collagen solvents and within the two solvent extractions, time of treatment and digestion were varied. • Jongjareonrak et al. [117] extracted and characterised collagen from bigeye snapper. Acid and pepsin solubilised collagen were isolated and characterised for their properties. The Td of the acid-solubilised and pepsin solubilised collagen varied slightly with, pepsin

• Zhang et al. [69] extracted and characterised collagen from the skin of grass carp via the method of pepsin-solubilisation. A collagen yield of 46.6% (dry-basis) was obtained and SDS-PAGE showed that the extracted collagen was type I and collagen Td was found to

• Nalinanon et al. [118] used pepsin from the stomach of tuna fish to use it for collagen extraction of threadfin bream skin. Pepsin from different tuna species were obtained to use for collagen extraction and to determine the differences in collagen extraction

• Cao et al. [119] extracted and characterised type II collagen from chick sternal cartilage. Pepsin was the main solubilising agent in this study and NaCl was used for collagen precipitation. SDS-PAGE confirmed the presence of collagen type II and the amino acid composition of the type II collagen extracted was very close to the reference collagen

In this period, a vast number of different fish species were analysed for their collagen extractability. The main reason for this was due to adding value to waste fish skin and due to a large acceptance of fish collagen by a diverse group of people (Jewish, Hindu and

• Woo et al. [116] extracted collagen from yellowfin tuna skin. Methodology was optimised by varying NaOH concentration, treatment time and pepsin concentration. The objective of this study was to determine the optimum conditions for extracting collagen from yellowfin tuna skin and characterisation was carried out by SDS-PAGE,

• Nomura et al. [113] extracted and analysed properties of type I collagen from fish scales. Collagen was extracted with 0.5 M acetic acid and it was concluded that a large portion (80% of collagen remained insoluble which was further denatured to gelatine to be used

PAGE analysis, 42.6% of extracted collagen to be type III.

hydroxyproline analysis was applied to observe collagen content.

normal human tissue was the focus of collagen research in this period.

**Period Collagen extraction research**

*Biotechnological Applications of Biomass*

8.7 at 3–90°C.

hexosamine.

methodologies.

images.

ageing.

feasible.

ethanol.

chains.

SDS-PAGE analysis.

dried acetone powder.

solubilisation.

**1970– 1979**

**1980– 1989**

**224**

in the range 5.5–6°C.

intermolecular cross-linking.

• In 1968 Rigby [92] analysed the amino acid composition and thermal stability of ice-fish skin. The fish skin was swollen in 0.1 M HCl for extraction purposes. Td was found to be

• Young et al. [93] extracted cod skin collagen with mild solvents in the pH range of 3.4–

• Grant et al. [94] studied the carbohydrate content of bovine collagen. It was shown that crude preparations of collagen were contaminated with mannose, fucose and

• Bronstein et al. [95] studied human collagen and the relation between intra and

• Miller et al. [96] extracted and characterised chick bone collagen with acetic acid. Specific methodology is not given, and the focus of this study was to understand collagen

During this period, research was mainly carried out to understand collagen as a protein. There was no research on method optimization or investigation of different extraction

• Anderson et al. [97] extracted bovine nasal collagen with 4 M guanidinium chloride or 1.9 M CaCl2 and examined the structure by studying their scanning electron microscopy

• Pierson et al. [98] studied the effect of post-mortem ageing, time and temperature on pH, tenderness and soluble collagen fractions in bovine Longissimus muscle. Salt and acid-soluble collagen were not affected by temperature nor length of post-mortem

• Maekawa et al. [99] extracted collagen from the skin of mice. Extraction was carried out

• Francis et al. [100] extracted collagen from biopsies of human skin. The study concluded to show that polymeric collagen of normal and diseased human skin from biopsies was

• Uitto et al. [101] analysed the solubility of skin collagen in normal human subjects and in patients with generalised scleroderma. Extraction was carried out with 0.14 M NaCl and

• In 1976, Trelstad et al. [102] applied differential separation to separate native collagen types I, II, and III. The main precipitants were ammonium sulfate, sodium chloride and

The focus of collagen extraction in this period remained to be for medical purposes. The

• Merkel et al. [104] studied the content of type I and III collagen of healing wounds in fatal and adult rats. Collagen was extracted with 0.5 M acetic acid with pepsin. Collagen content ratio was estimated from densitometer scans of electrophoretically separated α-

• Graham et al. [105] extracted and quantified types of collagen both in control intestine and as well as in both in inflamed and structured intestine resected from patients with Crohn's disease. This study mainly focused on differences in collagen types between the

• Murata et al. [106] studied the changes in collagen types in various layers of the human aorta and their changes with the atherosclerotic process. Collagen from human aortas was extracted by repeated pepsin digestion and the collagen types were identified by

• Elstow et al. [107] extracted and characterised type V foetal calf skin collagen. Neutral salt solutions (pH 9.2) with phosphate-buffered saline (PBS) were used to extract collagen and SDS-PAGE analysis was used to characterise and identify type V collagen. • Laurent et al. [108] showed a simplified method for quantification of the relative amounts of type I and type III collagen in rabbit lung samples. This extracted involved repetitive homogenisation of the collagenous tissue in 2% sodium dodecyl sulfate and

• Van Amerongen et al. [109] analysed the concentration and extractability of collagen in human dental pulp. Premolar and third molar dental pulps were studied for their

• Riemschneider et al. [103] extracted collagen from cow placenta via pepsin

compositional changes with ageing via chromatography.

with 0.5 M acetic acid at 40°C for different times.

main sources of collagen were of human skin and rat skin.

controlled and both inflamed and structured intestines.

number of extractions were varied.


Muslim religions not accepting certain meat products).


electron microscopy (SEM) and transmission electron microscopy (TEM); collagen

*10.1.1 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)*

The results from these analyses can be compared to standard collagen found in the market to compare yields and quality. The investigation of physiochemical properties of collagen through these characterisation methods is a way of optimising

SDS-PAGE analysis can be used to differentiate between the different collagen types and their individual chains. SDS-PAGE patterns of the extracted collagen can be obtained through any electrophoresis device such as the Mini-Protean or a PhastGel system. The collagen sample is boiled in SDS, resulting in collagen to break down into its polypeptide chains so that the α and β components of the collagen

Though SDS electrophoresis has been utilised for preparative separation of collagen [125], it has been mainly used to compare collagen from different tissue types and to identify collagen types and polypeptide chains. Wu et al. [126] extracted bovine collagen and applied SDS-PAGE to identify the different collagen types

In order to assess the collagen for abnormal formation and organisation and changes in its secondary structure, Fourier Transform Infrared Spectroscopy (FTIR) can be applied to reveal the collagen bio-distribution. FTIR has been used to study collagen denaturation [127], collagen cross-linking [128], and thermal self-

The vibrational bands characteristic of peptide groups and side chains provide information on protein structures. Spectral changes in amide A, amide I (1636–

are indicative of changes in collagen secondary structure [127]. An increase in the intensity of amide III and broadening of amide I are related with increased intermolecular interactions via hydrogen bonding in collagen. Among these, the amide I band (peptide bond C=O stretch) is especially sensitive to secondary structures. A reduction in the intensity of amide A, I, II and III peaks and narrowing of

An FT-IR spectrophotometer can be used to obtain a spectrum for collagen. Approximately 2–4 mg of collagen in 100 mg potassium bromide (kBr) can be used

.

Hydroxyproline is an amino acid found in collagen, comprising about 13% of the collagen molecule, this amino acid is not found in any other proteins apart from elastin. Thus, determining the hydroxyproline content in a specified tissue enables the calculation of the total amount of collagen present. Experimentally, the amount of hydroxyproline content in a sample for mammals [130] is multiplied by 7.46 to

Many studies on collagen extraction have applied hydroxyproline analysis to calculate collagen content [81, 87, 131, 132]. Researchers have developed methods to effectively measure hydroxyproline concentration of collagen using calorimetric

amide I band are associated with collagen denaturation (Td) [127].

), and amide III (1200–1300 cm<sup>1</sup>

) regions

moisture content, and hydroxyproline analysis.

*DOI: http://dx.doi.org/10.5772/intechopen.94266*

*10.1.2 Fourier transform infrared spectroscopy (FTIR)*

), amide II (1549–1558 cm<sup>1</sup>

to obtain spectra from 4000 to 1000 cm<sup>1</sup>

give the amount of collagen in the sample.

**10.2 Collagen content: hydroxyproline analysis**

future collagen extraction methods.

*Collagen: From Waste to Gold*

**10.1 Collagen molecular stability**

molecule can be analysed.

present.

assembly [129].

1661 cm<sup>1</sup>

**227**

#### **Table 9.**

*Timeline of advancements in collagen extraction (1960s – 2015).*

## **10. Methods used to investigate the physiochemical properties of collagen and collagen-based films**

There are at least 27 collagen types with 42 distinct polypeptide chains identified. Types I to XXVII collagen are fibril-forming collagens, containing triple-helix structures that can bundle into fibrils. Some collagen types are only present in certain tissues, for example, collagen types II, IX and XI are mostly found in cartilage tissues. Collagen types I to III are the ones mostly present in all collagencontaining tissues, type I being mainly present in skin tissue. Collagen characterisation is carried out to acquire information on structure, denaturation temperature, quantity, quality, thermal stability and fibril arrangement. Understanding the properties of each type of collagen will result in a better picture of what applications it can further be applied in.

The properties of the extracted collagen can be characterised by Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), Fourier transform infrared spectroscopy (FTIR), thermal stability (thermogravimetric analysis (TGA), differential scanning calorimetry (DSC)), morphology analysis, such as scanning

#### *Collagen: From Waste to Gold DOI: http://dx.doi.org/10.5772/intechopen.94266*

electron microscopy (SEM) and transmission electron microscopy (TEM); collagen moisture content, and hydroxyproline analysis.

The results from these analyses can be compared to standard collagen found in the market to compare yields and quality. The investigation of physiochemical properties of collagen through these characterisation methods is a way of optimising future collagen extraction methods.

### **10.1 Collagen molecular stability**

## *10.1.1 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)*

SDS-PAGE analysis can be used to differentiate between the different collagen types and their individual chains. SDS-PAGE patterns of the extracted collagen can be obtained through any electrophoresis device such as the Mini-Protean or a PhastGel system. The collagen sample is boiled in SDS, resulting in collagen to break down into its polypeptide chains so that the α and β components of the collagen molecule can be analysed.

Though SDS electrophoresis has been utilised for preparative separation of collagen [125], it has been mainly used to compare collagen from different tissue types and to identify collagen types and polypeptide chains. Wu et al. [126] extracted bovine collagen and applied SDS-PAGE to identify the different collagen types present.

### *10.1.2 Fourier transform infrared spectroscopy (FTIR)*

In order to assess the collagen for abnormal formation and organisation and changes in its secondary structure, Fourier Transform Infrared Spectroscopy (FTIR) can be applied to reveal the collagen bio-distribution. FTIR has been used to study collagen denaturation [127], collagen cross-linking [128], and thermal selfassembly [129].

The vibrational bands characteristic of peptide groups and side chains provide information on protein structures. Spectral changes in amide A, amide I (1636– 1661 cm<sup>1</sup> ), amide II (1549–1558 cm<sup>1</sup> ), and amide III (1200–1300 cm<sup>1</sup> ) regions are indicative of changes in collagen secondary structure [127]. An increase in the intensity of amide III and broadening of amide I are related with increased intermolecular interactions via hydrogen bonding in collagen. Among these, the amide I band (peptide bond C=O stretch) is especially sensitive to secondary structures. A reduction in the intensity of amide A, I, II and III peaks and narrowing of amide I band are associated with collagen denaturation (Td) [127].

An FT-IR spectrophotometer can be used to obtain a spectrum for collagen. Approximately 2–4 mg of collagen in 100 mg potassium bromide (kBr) can be used to obtain spectra from 4000 to 1000 cm<sup>1</sup> .

#### **10.2 Collagen content: hydroxyproline analysis**

Hydroxyproline is an amino acid found in collagen, comprising about 13% of the collagen molecule, this amino acid is not found in any other proteins apart from elastin. Thus, determining the hydroxyproline content in a specified tissue enables the calculation of the total amount of collagen present. Experimentally, the amount of hydroxyproline content in a sample for mammals [130] is multiplied by 7.46 to give the amount of collagen in the sample.

Many studies on collagen extraction have applied hydroxyproline analysis to calculate collagen content [81, 87, 131, 132]. Researchers have developed methods to effectively measure hydroxyproline concentration of collagen using calorimetric

**10. Methods used to investigate the physiochemical properties of**

There are at least 27 collagen types with 42 distinct polypeptide chains identified. Types I to XXVII collagen are fibril-forming collagens, containing triple-helix structures that can bundle into fibrils. Some collagen types are only present in certain tissues, for example, collagen types II, IX and XI are mostly found in cartilage tissues. Collagen types I to III are the ones mostly present in all collagencontaining tissues, type I being mainly present in skin tissue. Collagen characterisation is carried out to acquire information on structure, denaturation temperature, quantity, quality, thermal stability and fibril arrangement. Understanding the properties of each type of collagen will result in a better picture of what applications

Method optimization and comparison of different acids and enzymes for extraction efficiency had started in this period, due to a high demand for collagen from various

• In 2010, Uriarte-Montoya et al. [120] extracted and characterised collagen from Jumbo squid and analysed its potential to be formed into a composite film with chitosan. Acidsolubilised collagen was extracted, and film blends of chitosan-collagen were prepared by casting. The films were characterised for their thermal and mechanical properties. The purpose of these films was to be used as bio-friendly packaging materials. • Muralidharan et al. [71] extracted collagen from skin, bone and muscle of both trash fish and leather jacket fish. The collagen was characterised for their properties. Three methods of extraction were applied, and each method resulted in different collagen yields with the highest collagen yield being 71%. It was concluded that collagen from both trash fish and leather jacket fish could be used to extract collagen use it for potential

• In 2012, Liu et al. [121] extracted collagen from fins, scales, skin, bones, and swim bladders of bighead carp. The aim of this study was to characterise pepsin-solubilised collagen from the five sources for simultaneous comparison purposes. It was concluded that all five tissues could be used as a potential substitute for mammalian collagen. • Matmaroh et al. [122] extracted collagen from the scale of spotted golden goatfish via acid and pepsin solubilisation. SDS-PAGE showed both methods had revealed type I collagen and FTIR confirmed the presence of collagen triple helical structure. The main purpose of this study was to study collagen from the scale of spotted golden goatfish. • Kittiphattanabawon et al. [123] extracted and characterised collagen from the skin of brown-banded bamboo shark. Both acid soluble and pepsin soluble collagen were extracted and the collagen yield with pepsin solubilisation was slightly lower than with acid solubilisation. It was concluded that collagen from skin of brownbanded bamboo

• Singh et al. [124] isolated collagen from skin of striped catfish. Once again, both methods of acid and pepsin solubilisation were applied to extract collagen. Collagen yield with pepsin-solubilisation was slightly higher (7.7%) in comparison to acid solubilised

In this period and currently, most research being carried out on collagen extraction is based on method improvement and investigating novel tissues for possibility of collagen extraction. Sources that were not investigated and sources that potentially be

environmentally friendly are being analysed for collagen extraction in order to find cheaper

The properties of the extracted collagen can be characterised by Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), Fourier transform infrared spectroscopy (FTIR), thermal stability (thermogravimetric analysis (TGA), differential scanning calorimetry (DSC)), morphology analysis, such as scanning

**collagen and collagen-based films**

and efficient means of extraction.

*Timeline of advancements in collagen extraction (1960s – 2015).*

collagen (5.1%).

**Period Collagen extraction research**

*Biotechnological Applications of Biomass*

pharmaceutical and biomedical applications.

shark could serve as an alternative source of collagen.

markets.

**2010– 2015**

**Table 9.**

**226**

it can further be applied in.

assays [19, 133], high-performance liquid chromatography, and enzymatic methods [133]. Calorimetric methods usually require complete hydrolysis of collagen to its individual amino acids, oxidising hydroxyproline present to a pyrrole, and then reacting the pyrrole with a colour forming agent. This colour change is measured using a UV/Vis spectrophotometer and compared against calibration data to determine hydroxyproline concentration [19]. To obtain the amount of collagen in a sample for mammals, the amount of hydroxyproline in the sample (mg) is multiplied by a factor of 7.46 [67].

recorded while the sample is heated from room temperature up to 800°C at a rate of 10°C per minute. The first derivative of percentage mass change versus temperature can also be calculated to investigate temperature regions where mass loss was

Ramanathan et al. [140] used TGA to assess the thermal stability of fish skin collagen which was extracted via acid-solubilisation. They report using samples of approximately 5 mg and heating samples at 10°C/min in the temperature range of 0–800°C. The acid-solubilised collagen showed two weight loss steps on the TGA thermogram, relating the first stage to the loss of structural and bound water and stage two to thermal degradation of the polypeptide chain. The study concluded to show that the two peaks observed on the TGA differential curve were of collagen

The protein morphology of the extracted collagen can be studied using SEM. The

morphology of the extracted collagen can be compared to the standard bovine

*SEM images of extracted collagen, with 1) acid-soluble collagen of Catla fish (a), pepsin-soluble collagen of Catla fish (B), acid-soluble collagen of Rohu fish (C) and pepsin-soluble collagen of Rohu fish (D) [139], (2) being from buffalo skin [14] and 3) being SEM image of porcine skin collagen [142] (used with permission).*

denaturation and collagen degradation respectively.

**10.4 Collagen morphology**

*Collagen: From Waste to Gold*

*DOI: http://dx.doi.org/10.5772/intechopen.94266*

*10.4.1 Scanning electron microscopy (SEM)*

occurring.

**Figure 8.**

**229**

#### **10.3 Collagen denaturation temperature (td) and thermal stability**
