A Pilot Scale Study on the Extraction & Optimization of Keratin from Human Hair – An Adapted Strategy for the Control of Environmental Menace

Journal of Environmental Treatment Techniques

2021, Volume 9, Issue 1, Pages: 342-348

J. Environ. Treat. Tech.

ISSN: 2309-1185

Journal web link: http://www.jett.dormaj.com

https://doi.org/10.47277/JETT/9(1)348

A Pilot-Scale Study on the Extraction &

Optimization of Keratin from Human Hair – An

Adapted Strategy for the Control of Environmental

Menace

Punam Sen , Arun. C. M , Divvyapriya. J

Associate Professor, Department of Microbiology, PSG College of Arts & Science, Coimbatore-641014

Department of Microbiology, PSG College of Arts & Science, Coimbatore-641014

Received: 15/09/2020

Accepted: 29/11/2020

Published: 20/03/2021

Abstract

Large aggregates of the keratin based pollutants are known to cause intense threat in contaminating the environment of contact

region than in the discrete region. Diversifying the application of keratin could be a promising phenomenon for reducing the keratin

pollution. Accordingly, the undertaken work was designed to optimize the parameters for the keratin extraction from human hair with

a potential source of application in the environment. Sequence of the effective extraction dealt with pre-treatment of human hair with a

surfactant (sodium dodecyl sulphate), followed by the digestion of hair with sodium sulfide (Na S) at pH 12 - 14 and then stirred with

magnetic stirrer at 50C. The aliquot was then centrifuged at pH of 3 - 4, following which the precipitated keratin was extracted, dried

and pulverized. The conformational study of the extracted keratin was done by performing Ninhydrin test and Fourier transform

infrared spectroscopy (FTIR) analysis. The extracted keratin can be exploited in several applications such as, active component in bone

replacement, hydro-gel preparation, cosmetics, scaffold preparation, bio degradable films etc. Hence, this work highlighted on the

optimal isolation of pure keratin from human hair, paving away the environmental pollutants and advent a healthy grid of societal

benefit.

Keywords: Keratin, SDS, Filtration, FTIR

preparation. The keratin extracted from human are found to be

Introduction

more biocompatible, less immune stimulating when used in

transplantation and are readily biodegradable [7]. Amidst the

substantial development that finds use of keratin into various

products like foods, catalysis, bone replacement, cosmetics

and fertilizers, still cumbersome keratin finds its way into

landfills only due to lack of efficient technology of

contaminant free extraction.

The Alkali reduction [8] method which was previously

found effective among the rest of available techniques of

extraction is employed in this experiment. Current work aims

to elevate the process in industrial scale view and

optimization aims the utmost productivity of the industry

which should be higher than 75% extraction ratio (maximum

achieved till date) [8]. The work here concentrated mainly on

to reduce the environmental impact caused by the keratin

based waste resources with effective extraction of pure

keratin, less labor and production cost and less resources

consumption. The commercial form of keratin finds its use as

additive in cosmetics, hair care products, sutures,

antimicrobial bio-films, and as active component of bone

replacement.

Solid waste management of urbanized society has become

a recent interest due to dreadful effects caused by the

untreated effluents and potential bio-compounds that can be

extracted. Keratin waste from various sources like poultry,

slaughter houses, leather industry and human hair are known

to cause deteriorating effect on human and environment [1].

Being recalcitrant to many simple proteases, keratin when

persists in the environment is anticipated to cause long term

effects like causing pollution in the environment and

imparting diseases like chlorosis and fowl cholera in human

2]. As no data over current keratin production is known, the

estimated potential to produce keratin from only 40×10 tons

of chicken feather [3] and 6.9×10 tons of human hair [4] is

.1×10 tons per year (in a rate of 80% extraction) excluding

the leather industry waste, wool waste and other slaughter

house waste. Recent trends of sustainable development and

wide attraction towards natural protein and its derived

materials lead to exploit keratin as reliable source for the day

to day application. This super coiled polypeptide with

extensive disulfide cross linking is classified into two types –

soft and hard Keratin, with 1% and 5% sulfur content

respectively.

[

Notable role of keratin is centered in cosmetics and

wound healing materials and with 50% share estimating to

about 3500 tons of keratin consumption annually, cosmetics

highly exploit the keratin resources. Keratin aids in wound

healing by directly activating the keratinocytes [9] – skin

cells, activating proliferation of the cells in the wounded

region. Alpha keratin builds outer skin; aids in hair care, nail

growth and beta keratin acts as a precursor for vitamin A,

Keratin today finds its uses in various fields like

cosmetics, Pharmacology, biomedical [5-6] and scaffold

Corresponding Author: Dr. Punam Sen, Associate

Professor, Department of Microbiology, PSG College of Arts

Science, Coimbatore-641014. Email:

punamsen@yahoo.co.in

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Journal of Environmental Treatment Techniques

2021, Volume 9, Issue 1, Pages: 342-348

hence promoting the eye sight. Amidst raising trends in

exploiting keratin into diverse application, the cost of keratin

acts as only constrains, so a proper protocol for keratin

extraction from available resources can abruptly increase the

utilization of the same. Reducing the need of synthetic protein

processing by promoting chemical extraction of natural

proteins will simultaneously decrease the environmental

contamination and increase the exploitation of natural

recourses in an economically feasible manner. Commercially,

keratin is used in the hair treatment medications, cosmetics,

medial application, and as coating in various medical

applications like sutures [10].

Keratin from human hair like other mammalian Keratin is

α-keratin. Previous approach for the extraction of the keratin

is found quite less feasible, with application of greater electric

and mechanical energy copulation. Works proposed earlier

demand the need for continuous digestion of the keratin over

the magnetic stirrer[11], which pulls the requirement of huge

amount of power consumption [12], [13].The proposed

method aims to reduce the contaminant and debris to the

maximum extent, ensuring the product stays extra pure. There

are very few literatures that are concentrated over the

extraction of keratin from human hair, due to unknown

notion, this area of research is still found to be least attracted

field of study. Moreover, the extraction of keratin from hair is

considered a tedious process compared to the extraction of

keratin from chicken feather [12], [13], but the current work

proposes that the extraction of keratin in much simpler,

economic and less time consuming processes comparatively,

while accounting the entire process from raw material

processing and to the keratin purification.

made over the working protocol, that aimed to demonstrate

and point out the necessary steps required and eliminate the

unwanted steps in order to optimize production. Parameters

for the comparison included effects of pretreatment of raw

material, need for effective pH maintenance of the hydro

lysate, effect temperature exposure. Keratin obtained was

analyzed for purity and quantified. It was subjected to

Ninhydrin Test and FTIR analysis, which revealed the amino

acid content in the keratin alongside confirming the presence

of keratin qualitatively and analytically. The dry weight of the

hair sample and the extracted keratin was measured for

calculating the efficiency of extraction. In Ninhydrin test [14],

copper sulphate solution and potassium hydroxide taken at 1%

each and equal volume of the corresponding solution were

mixed for about 5ml, and to the taken solution, 5 ml of

Keratin solution was added (1g keratin in 2ml of NaOH).

Results and Discussion

The keratin extracted with the optimized criteria, found to

be promising and efficient, with maximum extraction of the

protein in the given quantity of hair sample. The qualitative

and quantitative results of total percentage of keratin extracted

are detailed in following result.

3.1 Effect of pretreatment

The hair pretreated with SDS (detergent and surfactant) is

found to be contaminant free, while the other protein extracted

without pretreatment are mixed with contaminant and lots of

debris though previously washed with water. The color of

keratin is a good indicator of the purity of the keratin, a slight

milky grayish shade during recovery, which decolorized to

milky yellow after complete dehydration process. These

results are comparable with the existing research literatures

[3], [6], which supports that the color of keratin is milky

yellow. These results also correspond with keratin extracted

from various other raw materials like Chicken feather and pig

hair. Recording this result could potentially mark further

research works handy. The keratin extracted without

pretreatment are found to be blackish grey color, where

protein at this extract was found to be inseparable from debris.

The hydro lysate when treated to reduce the pH if untreated

then would result in foam formation upon reaction with acid,

resulting in the wastage of keratin.

Materials and methods

.1 Materials

Hair Samples (collected from nearby saloon), Sodium

Sulfide (Na S), Sodium dodecylsulphate (SDS), Over Head

Magnetic Stirrer, digestion tank or screw cap beaker, Stirrer,

Hot Air Oven.

.2 Methodology

Hair sample was collected from local saloon. The visible

debris from the sample were removed and cleaned from the

same. 70g of the sample taken is soaked in 2% SDS (1.5 L)

solution for a period of over 20 minutes. The SDS was

transferred to another container and can be reused for 4-5 time

and the treated hair was washed twice in distilled water. The

3.2 Optimum pH maintenance

Hair keratin dissolves and gets hydrolyzed only in the

alkaline condition and is non-reactive in neutral and acidic

pH. Hence, maintaining a pH range of 10.5 to 12 would bring

out the efficient hydrolysis of the keratin. Highly alkaline pH

causes the structural damage to the keratin and bring out the

altered conformational skeleton. pH maintenance during

recovery of the keratin from the hydro lysate was effective in

the extraction of the keratin without contamination. It was

found that keratin gets precipitated around the pH range of 2-3

(acidic).

sample with water was suspended in 1L of 1.5N Na S

solution, placed in dry region at room temperature for 5 hours

and the sample is mixed at 40ºCfor a period of 1-2 hour (in

industrial scale the need of magnetic stirrer can be replaced

with mechanical agitator which finely mix the hair and

produce a complete hydro lysate). The aliquot was centrifuged

at 5000 rpm for a period of 5 minutes at room temperature to

remove the unwanted debris (which if not removed may lead

to wastage of keratin by formation of foam on reaction with

the acid added).

The supernatant recovered and the pH of the solution was

brought to 2-3.5 to precipitate out the keratin from the hydro-

lysate, using 1N HCl. The solution was kept undisturbed for 2

hours in order to precipitate out the keratin in white

aggregates. Later, the Keratin was filtered from the precipitate

and dried at 45ºC, for 2 hours,following which flakes of

keratin was obtained. Then, keratin is recovered and

pulverized. The significance of work lies in optimizing the

parameters for potential extraction of keratin lies in the

optimization of parameters controlling the rate and quality of

reaction. For this purpose, there were certain comparisons

3.3 Influence of temperature over the keratin extraction

Temperature applied over the process of extraction of the

keratin played a significant role in the extraction procedure.

Initially during the digestion of sample, an ambient

temperature around 45C to 58 C were found to be effective,

since the temperature more than the optimum was known to

alter the side chain of the keratin sometimes leading to

degradation [7]. The observed temperature dependence

contradicts the previous literature observations which insisted

on input of temperature of at least 70C [15]. The temperature

of less than the given, if applied, known to increase the

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2021, Volume 9, Issue 1, Pages: 342-348

duration of extraction process, which can add up to the

excessive use of electrical and mechanical consumption. But

the undergone work revealed that 50C to 58C was ambient

for highest productivity, provided that the initial incubation

period is 3 – 4 hours (undisturbed). The other place where the

heat found its importance was in the drying of keratin during

the post extraction process, where exposure of keratin

extracted and washed were dried at 45 C for a period of 5

hours.

of the total dry weight of the human hair sample taken. The

color of the extracted keratin was found to be either pale

milky yellow or pale milky grey shade.

3.6 Ninhydrin Test

The keratin when added to Ninhydrin reagent, the solution

turned into deep blue to violet color indicating the presence of

the protein. This result corresponds to standard coloration of

protein extract [14]. The crude protein with contaminant will

not produce a significant coloration and thus promoting false

results. So Ninhydrin test being primary analysis

contamination in extraction would result in misinterpretation

of product obtained, thus a care must be taken and a

contamination free extraction must be ensured.Figure4

represents the observed result of Ninhydrin test.

Figure 2: Difference between centrifuged sample (left) and un-

centrifuged Hydro lysate sample (right)

(

a) Untreated Hair

(

a) Untreated Keratin

(

b) Treated Hair

Figure 1: Treated and untreated hair

.4 Post extraction Centrifugation

Comparable to pretreatment the post extraction

centrifugation of the hydro lysate for a period of 10 minutes at

0000 rpm in room temperature would result in effective

keratin extraction. Even these debris if not removed may

result in minimal foam formation and can add up to the

pigmentation of the final keratin extract [10]. Instead of

centrifugation, membrane filters too can be substituted for the

effective removal of debris allowing only the hydro lysate to

be separated.

b) Un-centrifuged pretreated sample (LEFT) and completely Treated

Keratin (RIGHT)

Figure 3: Extracted Keratin

.5 Keratin Extracted (Quantitative)

Out of 10g of the hair sample, a total of 7.28g dry weight

of the keratin was extracted which accounted for about 73%

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2021, Volume 9, Issue 1, Pages: 342-348

.7 FTIR Analysis of the Keratin

FTIR analysis aids in identification of types of

compounds present and especially the protein types based on

the functional group present in them [16], [17]. FTIR results

show that the peaks correspond to the keratin of the standard

chart and previous results of keratin [17].The Figure 5

represents the FTIR analysis of pure pale yellow keratin

sample effectively extracted from the experiment conducted.

-15

000

3600

3200

2800

2400

2000

1800

1600

1400

1200

1000

800

600

400

1/cm

Arun C.M(24-1-20)

Figure 5: FTIR Results obtained from final Keratin Powder

The peaks correspond to various functional group, the

peak at 3757 cm indicates the presence of H

O molecules,

-1

307.13 cm corresponds to O-H bonds in carboxylic acids

and derivatives, alcohols and phenols and 3062.96 indicates

the presence of C-H, CH2, C=C alkenes. The peak ranges

931.80, 2376 and 1668.78 represent C-H Systemic stretch of

CH and or at fatty acids, systemic stretching vibrations of

lipid acyl CH groups and NO bonding in nitro compounds,

Amide I band components of beta pleated structure of protein.

Figure 4: Ninhydrin Test Result (Positive)

) Centrifugation of the Keratin

) Membrane Filteration of the

Hydrolysate at 5000 rpm for 5

1) Pre treatment of Hair sample

Centrifuged sample

minutes

) Digestion of the Hydrolysate

in Magentic stirrerfor 1-2

hours at 40 -50 C

8) Recovery of the Keratin by

precipitatng it upon adding 1N

HCl

) Preparation of Stock Solution

of Sodium Sulfide

) Intermident mechanichal

) Addition of Hair to Soldium

stirring for 1min in every

9) Pulvarizing the sample and

subject for analysis

Sulfide Solution in ration of 70g

10mins and allow them to get

per 2 L of Na S

digested for 5 hours

Figure 6: Keratin Extraction technique - flow chart

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2021, Volume 9, Issue 1, Pages: 342-348

STEP 9

Figure 7: Methods involved in keratin extraction from human hair

The region at 1234.44 represents, Amide III band

components of proteins, C-N stretching vibrations from

amines, from free amino acids and P=O asymmetric stretching

of PO -, phosphodiesters. A slight peak at 1176 and distinctive

peak at 1041 indicates C-O, C-C, C-N, stretching, C-O-H, C-

O-C deformation of carbohydrates and carbohydrate glycoside

bonds, this infers the C=O glycol protein. 833.24 peak

milky yellow, but Pure keratin is in milky grey white and

milky yellow in color.

The extracted keratin may be highly pigmented due to the

presence of two major of keratin in demand. Less importance

is staged over such specific downstream processing of

extracting less pigmented keratin. In the future research upon

thawing light over the area could lead to customize pigments,

Eumelanin (Dark) and Phaeomelani [19] (Light) which can be

removed or retained according to the grade.

represents S-OR esters out of plane bending, NO -, NO - and

2 3

CO groups. The final peak at 624.95 infers C-H deformation,

, NH and NH groups. The peaks fluctuate and many

minor peaks seen near the major peeks are due to the fact that

the occurrence of hair from different age groups, since the

peaks of keratin of different age groups differs feebly

3.8 Quantity of Keratin extracted

Upon optimizing the above parameters and precisely

conducting the extraction process with no fluctuations from

the above mentioned protocol, the quantity of keratin obtained

was 7.28 g per 10 gram of human hair, where the maximum

possible theoretical keratin value is 9g of keratin per 10g of

[18].Keratin after extraction exhibits wide range of

pigmentation between a band of grey white, grey black or

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2021, Volume 9, Issue 1, Pages: 342-348

human hair sample (Estimating an average of 90% keratin

content). This is estimated to be about 79-80% of total keratin

in human hair, and accounts approximately 73% in dry weight

of dry hair sample. This method finds promising than the

previous results of 75 % [8]. Apart from this, the quantity of

keratin obtained from poorly treated and contaminated

substrates in its crude form weighed around 7.3g (treated hair

sample with poor centrifugation) and 7.9g (untreated hair

sample and improper centrifugation) where impurities

accounting for about 6 percent of total dry weight, with dark

pigmentation making the extract unfit for commercial

consumption. These parameters are not discussed in detail

among any parallel research papers, thus these results could

significantly influence the future research works.

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Acknowledgment

We are thankful to the host Institution PSG College of

Arts & Science, Coimbatore, for providing us with all the

facilities & support during the course of work.

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Competing interests

We declare that there is no conflict of interest that would

prejudice the impartiality of this scientific work.

Authors’ contribution

All authors of this study have a complete contribution for

data collection, data analysis and manuscript writing.

21. Lee, Hanna & Noh, Kwantae& Lee, Sang & Kwon, Il Keun&

Han, Dong-Wook& Lee, In-Seop& Hwang, Yu-Shik., Human

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