1. A method for extracting keratin from waste nails, comprising (i) Washing and cleaning the waste nails with fresh water and cutting into pieces, (ii) Dissolving the clean nails in a lysis buffer under pre-determined conditions to obtain lysed nails, (iii) Placing the strands of the lysed nails into a fresh tube containing 400µl of the lysis buffer to obtain protein solution, (iv) Stirring the protein solution and keeping for 30 min. at RT, (v) Centrifuging the stirred protein solution to take out nail strand fraction from the protein solution as sediment, (vi) Pulverizing the nail strand fraction to obtain pulverized nail extract, (vii) Centrifuging the pulverized nail extract and taking out the supernatant containing keratin protein (referred as Extract 1) in a fresh tube and kept at 5o C, (viii) Pulverizing the sediment, after centrifugation in step (vii), containing undissolved nail fraction, followed by centrifugation and collecting supernatant containing keratin protein (referred as Extract 2) in the fresh tube kept at 5o C, (ix) Pulverizing the sediment, after centrifugation in step (viii), containing undissolved nail fraction, followed by centrifugation and collecting supernatant containing keratin protein (referred as Extract 3) in the fresh tube kept at 5o C, (x) Dialysing the supernatant collected in the fresh tube to obtain dry keratin powder, (xi) Precipitating the dry keratin powder using acetone to obtain protein pellets, and (xii) Suspending the pellets in a suitable buffer, wherein said suitable buffer comprises 7M urea, 2M thiourea, 4% 3-[3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 2% ampholytes and 5% glycerol.

2. The method as claimed in claim 1, wherein the lysis buffer comprises 0.1 M sodium hydroxide (NaOH), 1% sodium dodecyl sulfate (SDS), 2% mercaptoethanol (ß ME), and 0.02 M ethylenediamine tetraacetic acid (EDTA).

3. The method as claimed in claim 1, wherein pre-determined conditions of dissolving the clean nails in lysis buffer is duration of 30 min at 900 C.

4. The method as claimed in claim 1, wherein yield of the extracted keratin is in the range of 70% to 78%.

5. The method as claimed in claim 1, wherein the average yield of the extracted protein is 70.3%.

6. The method as claimed in claim 1, wherein the NaOH concentration in the lysis buffer is in the range of 6g/L to 7g/L.

7. The method as claimed in claim 1, wherein the total processing time is 2.5 h to 3 h. Dated this the 27th day of November, 2024.

Description:FIELD OF THE INVENTION
The present invention relates to the field of sustainable chemistry, and waste management. More particularly, the present invention relates to a method of extracting keratin from waste nail.

BACKGROUND OF THE INVENTION
The background information herein below relates to the present disclosure but is not necessarily prior art.

Hair, wool, nails, feathers, and reptile skin all include keratin, a biological macromolecule with exceptional mechanical capabilities. The use of keratin, a type of functional material, in biology, materials, and other disciplines has significantly increased in recent years.

Recent years have seen a large amount of research on human KAPs. There are numerous established methods for removing proteins from nail samples. One of the most popular techniques, the Shindai method, was developed based on the approach utilized by Fujii and associates to extract protein from hair. In the Shindai approach, the KAPs and keratin fractions were recovered from the samples to approximately 10% and 50% of their initial quantities, respectively.

Proteome analysis of the human nail is still challenging due to the low solubility of nail proteins and the challenges associated with their extraction.

The Lee et al. technique and the previously published Shindai approach. The alkaline lysis approach lowered the time of extraction from five days to just 90 minutes, in addition to being straightforward and repeatable. These methods were used to extract the keratein protein, but they are not very effective. The method we are using for the extraction of keratein from the nails is the first time reported and that is a two-hour protocol and the spectrophotometer is reflected in the fluorescence behaviour of the nail-extracted protein. The drawbacks of this approach are the lengthy extraction of keratein protein from nails and the challenge of detecting fluorescent nail behaviour.

The protein keratein may be extracted in an environmentally benign and economical manner, making it a great material for use in a variety of applications.

OBJECT OF THE INVENTION
Some of the objects of the present disclosure, which at least one embodiment herein satisfy are as follows:

The main object of the present invention is to provide a method of extracting keratin protein from waste nails.

Another object of the present invention is to provide a method of extracting keratin protein from waste human nails.

Yet another object of the present invention is to provide a method of extracting keratin protein from waste human nails using alkaline lysis.

Other objects and advantages of the present disclosure will be more apparent from the following description when read in conjunction with the accompanying figures, which are not intended to limit the scope of the present disclosure.

SUMMARY OF THE INVENTION
Accordingly, there is provided a method of alkaline lysis for the extraction of proteins from hair shafts. The method of the present disclosure is a rapid extraction method for nail protein using an alkaline-based buffer. Characterization of the extracted protein was carried out using SDS-polyacrylamide gel electrophoresis, and FTIR, XRD, SEM-EDX, ICP analysis, TGA, and spectrophotometer analysis were employed to characterize the extracted protein from the nail. Identification of these extracted keratin proteins and the detection of nails' fluorescence behaviour are essential for improving the understanding of the molecular basis of nails for possible applications in the future.

To extract the keratin from discarded human nails, present invention employs sodium hydroxide as a stratum corneum disruptor, and sodium dodecyl sulphate as a keratin stabilizing agent. The yield of the keratin extracted was measured along with the reaction duration and the impacts of concentration of sodium hydroxide. The extracted keratin was also subjected to a fourier transform infrared spectroscopy analysis to confirm its structure.

According to the findings, the yield of the extracted keratin is in the range of 70% to 78%. The average yield of the extracted protein is 70.3%. Additionally, FTIR, TGA and XRD demonstrate that during extraction, keratin's molecular structure changed from a a-helix to a ß-sheet.

The method of the present disclosure provides a rapid protocol for high yield protein extraction from human nails by comparing its efficacy and percent recovery to previously reported procedures. The development of an improved extraction process for nail proteins could lead to investigations into their roles in biochemical pathways and potential applications as markers via spectrophotometer, which is reflected in the protein's fluorescence behaviour.

These and other aspects herein will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following descriptions are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the invention herein without departing from the spirit thereof.
BRIEF DESCRIPTION OF DRAWINGS
Fig. 1 shows flow diagram of the process steps of the present invention.
Fig. 2A shows SEM images of nails: Surface morphology of non-extracted nail (a) 10 µm (b) 20µm.
Fig. 2B shows SEM photos of retrieved keratin from nails show the surface morphology of the nail. (a) 10 µm (b) 20µm.
Fig. 3 shows EDAX Spectrum of nail after keratin extraction.
Fig. 4 shows effect of NaOH concentration on keratin yield.
Fig. 5 shows effect of reaction duration on keratin yield.
Fig. 6 shows FTIR spectra of (a) nail (b) extracted keratin.
Fig. 7 shows crystalline nature of the isolated proteins is demonstrated by XRD.
Fig. 8 shows fluorescent nature of the isolated proteins demonstrated by Fluorescence spectroscopy.
Fig 9 shows thermogravimetric analysis of extracted keratin protein.

DETAILED DESCRIPTION OF THE INVENTION WITH NON-LIMITING EMBODIMENTS AND EXAMPLES
In the following detailed description of the invention, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be obvious to a person skilled in the art that the invention may be practiced with or without these specific details. In other instances, well known methods, procedures and components have not been described in details so as not to unnecessarily obscure aspects of the invention.

Furthermore, it will be clear that the invention is not limited to these alternatives only. Numerous modifications, changes, variations, substitutions and equivalents will be apparent to those skilled in the art, without parting from the scope of the invention.
In one of the embodiments of the present disclosure, there is provided a method for extraction of keratin protein from waste nails.
In one of the embodiments of the present disclosure, there is provided a method for extraction of keratin protein from waste human nails.

The method of the present invention is based on alkaline lysis. The waste nails are washed and cleaned with fresh water and cut into pieces. The cleaned nail are dissolved the clean nails in a lysis buffer under pre-determined conditions to obtain lysed nails. The lysed nails have nail strands. The strands of the lysed nails are placed into a fresh tube containing 400µl of the lysis buffer to obtain protein solution. The protein solution is stirred and kept for 30 min. at RT, followed by centrifugation of the stirred protein solution to take out nail strand fraction from the protein solution as sediment. The sediment is pulverized to obtain pulverized nail extract, which is centrifuged to obtain a supernatant and a sediment. The supernatant (referred as Extract 1) is taken in a fresh tube and kept at 5o C. The sediment is pulverized and centrifuged to collect the supernatant (referred as Extract 2) in the fresh tube containing Extract 1 and kept at 5o C. The subsequent sediment is again pulverized, centrifuged and supernatant (referred as Extract 3) is collected in the fresh tube containing Extract 1 and Extract 2) kept at 5o C. The supernatant collected in the fresh tube contains extracted keratin protein. The supernatant collected in the fresh tube is dialyzed to obtain dry keratin powder. The dry keratin powder is precipitated with acetone to obtain protein pellets. The protein pellets are suspended in a suitable buffer. The suitable buffer comprises 7M urea, 2M thiourea, 4% 3-[3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 2% ampholytes and 5% glycerol.

In an embodiment of the present disclosure, the pre-determined conditions of dissolving the clean nails in lysis buffer is duration of 30 min at 90 0C.

In an embodiment of the present disclosure, the yield of the extracted keratin is in the range of 70% to 78%.

In an embodiment of the present disclosure, the NaOH concentration in the lysis buffer is in the range of 6g/L to 7g/L.

In an embodiment of the present disclosure, the total processing time is 2.5 h to 3 h.

As illustrated in Fig. 1, in an embodiment of the present disclosure, human waste nails have been collected from local persons. Collected nails have been thoroughly washed and cleaned with fresh water and cut into small pieces. Cleaned nail samples have been dissolved in lysis buffer for 30 min at 900 C to obtain lysed nails. The lysis buffer comprises 0.1 M sodium hydroxide (NaOH), 1% sodium dodecyl sulfate (SDS), 2% mercaptoethanol (ß ME), and 0.02 M ethylenediamine tetraacetic acid (EDTA). The strands of lysed nails have been carefully placed into a fresh tube containing 400µl of the lysis buffer using a pair of sterile forceps to obtain protein solution. The protein solution has been centrifuged to filter out nail strand fraction from the protein solution. The nail strand fraction is stirred to pulverize the nail strands in the nail strand fraction to obtain pulverized nail extract. The pulverized nail extract has been kept for 30 min at room temperature (RT), followed by centrifugation, and supernatant containing keratin protein (referred as Extract 1) is collected in a fresh tube and kept at 5o C. The undissolved nail fraction left after centrifugation has been pulverized again, followed by centrifugation and collecting supernatant containing keratin protein (referred as Extract 2) in the same fresh tube kept at 5o C. Again, the undissolved nail fraction has been pulverized, followed by centrifugation and collecting supernatant containing keratin protein (referred as Extract 3) in the same fresh tube. Thus, the maximum protein has been extracted by repeating the process three times. The supernatant containing keratin protein is dialysed to produce keratin powders. The keratin powder obtained by the method of present disclosure is precipitated using acetone to obtain protein pellets. The pellets are suspended in a suitable buffer.

When compared to the Shindai approach that had previously been described and the Lee et al. procedure, present method extracts much greater % of protein. The extraction time is also been reduced from five days to just 90 minutes using the alkaline lysis approach, in addition to being simple and reproducible.

Characterization of extracted protein from nails

Surface morphology
Using SEM (TM3000, Japan), the surface morphology of the non-extracted nails and keratin extracted from nail has been investigated. (Fig. 2A and 2B).

EDAX Spectrum
Fig. 3 shows EDAX Spectrum of nail after keratin extraction.

NaOH concentration's impact on keratin synthesis
Cuticle layer is destroyed by NaOH. The keratin extraction is significantly increased when the NaOH concentration increased from 0 to 6 g/L (Fig. 4). 6g/L to 7g/L of NaOH is the optimal concentration.

Keratin yield and the impact of response time
The yield of keratin is significantly influenced by lysis time. Fig. 5 observes that the keratin extraction grew noticeably over time from 1 to 2.5 hours and peaked at 70.3% at 3 hours. The output of keratin dropped after three hours. However, human nail keratin's peptide bond scission took longer.

Fourier transform infrared spectroscopy
The isolated keratin and nail's molecular structure and chemical bonds have been analyzed using FTIR. The wave number range for the FTIR has been 600 cm-1 to 4000 cm-1. (Fig. 6)
The nail and extracted keratin FTIR spectra depicted bands of absorption in the spectra assigned to peptide bonds (>CONH-). FTIR was used to characterize the protein extracts' structural composition.
The >N-H stretching vibrations responsible for the typical medium absorption peak for amide vibrations were found at 3276 cm-1. A strong amide II peak that appeared at 1517 cm-1 has been attributed to >N-H bending vibrations and >C-N stretching vibrations. In contrast, the strong amide I band at 1640 cm-1 is connected to the >C=O stretching vibrations. A weak band at 1234 cm-1, known as amide III, appeared due to the stretching vibrations of >C-N and RC-OR and the bending vibrations of >N-H.

X-ray diffraction (XRD)
An X-ray diffractometer has been used to conduct the X-ray diffraction research on the regenerated proteins (Fig. 7).
The keratin extracted by various extraction techniques was examined using XRD. The XRD pattern revealed two different peaks at approximately 9° that are connected to both a-helix and ß-sheet structures, as well as a more prominent rise at around 20°, which is associated with the sheet structure, demonstrating the crystalline nature of the regenerated keratins. Lower crystallinity values imply more significant structural damage and poor spinnability, whereas higher values suggest less deterioration.

Fluorescence spectroscopy
Fig. 8 shows fluorescent nature of the extracted proteins demonstrated by Fluorescence spectroscopy.

Thermogravimetric Analysis (TGA)
The TGA measures the change in sample weight caused by controlled temperature changes.
TGA The thermal stability and degradation of the extracted keratin has been studied by using TGA. As shown in (Fig. 9). The extracted keratin shows two stages of decomposition started to degrade between 61°C and 267°C and produced a significant amount of residue. There was occurrence of sharp weight loss from 200°C to 400°C, which is associated with helix structure denaturation and skeletal degradation and destruction of chain linkage, peptide bridges. There are several chemical reactions occur which decompose keratin into lighter products and volatile compounds such as CO2, H2S, HCN and H2O. Thermal degradation started at 61°C and become faster above 158°C.
FTIR, XRD study demonstrates that keratin's supramolecular structure changed from an a-helix to a ß-sheet. The XRD pattern revealed two different peaks at approximately 9° that are connected to both a-helix and ß-sheet structures, as well as a more significant height at around 20°, which is associated with the sheet structure, demonstrating the crystalline nature of the extracted keratins.
From the features as applied to various alternatives, it can be understood that various omissions, substitutions, and changes in the form and details of the present disclosure can be made without departing from the scope of the disclosure. As can be recognized, certain alternatives described herein can be embodied within a form that does not provide all of the features and benefits set forth herein, as some features can be used or practiced separately from others.
, Claims:I/We claim:
1. A method for extracting keratin from waste nails, comprising
(i) Washing and cleaning the waste nails with fresh water and cutting into pieces,
(ii) Dissolving the clean nails in a lysis buffer under pre-determined conditions to obtain lysed nails,
(iii) Placing the strands of the lysed nails into a fresh tube containing 400µl of the lysis buffer to obtain protein solution,
(iv) Stirring the protein solution and keeping for 30 min. at RT,
(v) Centrifuging the stirred protein solution to take out nail strand fraction from the protein solution as sediment,
(vi) Pulverizing the nail strand fraction to obtain pulverized nail extract,
(vii) Centrifuging the pulverized nail extract and taking out the supernatant containing keratin protein (referred as Extract 1) in a fresh tube and kept at 5o C,
(viii) Pulverizing the sediment, after centrifugation in step (vii), containing undissolved nail fraction, followed by centrifugation and collecting supernatant containing keratin protein (referred as Extract 2) in the fresh tube kept at 5o C,
(ix) Pulverizing the sediment, after centrifugation in step (viii), containing undissolved nail fraction, followed by centrifugation and collecting supernatant containing keratin protein (referred as Extract 3) in the fresh tube kept at 5o C,
(x) Dialysing the supernatant collected in the fresh tube to obtain dry keratin powder,
(xi) Precipitating the dry keratin powder using acetone to obtain protein pellets, and
(xii) Suspending the pellets in a suitable buffer, wherein said suitable buffer comprises 7M urea, 2M thiourea, 4% 3-[3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 2% ampholytes and 5% glycerol.
2. The method as claimed in claim 1, wherein the lysis buffer comprises 0.1 M sodium hydroxide (NaOH), 1% sodium dodecyl sulfate (SDS), 2% mercaptoethanol (ß ME), and 0.02 M ethylenediamine tetraacetic acid (EDTA).
3. The method as claimed in claim 1, wherein pre-determined conditions of dissolving the clean nails in lysis buffer is duration of 30 min at 900 C.
4. The method as claimed in claim 1, wherein yield of the extracted keratin is in the range of 70% to 78%.
5. The method as claimed in claim 1, wherein the average yield of the extracted protein is 70.3%.
6. The method as claimed in claim 1, wherein the NaOH concentration in the lysis buffer is in the range of 6g/L to 7g/L.
7. The method as claimed in claim 1, wherein the total processing time is 2.5 h to 3 h.

Dated this the 27th day of November, 2024.