Novel Eco-Friendly Method to Extract Keratin from Hair - American

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A NOVEL ECO-FRIENDLY METHOD TO EXTRACT KERATIN FROM HAIR Ana Carolina Cassoni, Ricardo Freixo, Ana Pintado, Manuela Amorim, Carlos Dias Pereira, Ana Raquel Madureira, and Manuela E. Pintado ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b02680 • Publication Date (Web): 06 Aug 2018 Downloaded from http://pubs.acs.org on August 6, 2018

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ACS Sustainable Chemistry & Engineering

A NOVEL ECO-FRIENDLY METHOD TO EXTRACT KERATIN FROM HAIR Ana C. Cassonia’, Ricardo Freixoa’, Ana I. E. Pintadoa, Manuela Amorima, Carlos D. Pereirab, Ana Raquel Madureiraa, Manuela M. E. Pintadoa*.

a

CBQF – Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola

Superior de Biotecnologia, Universidade Católica Portuguesa/Porto, Rua Arquiteto Lobão Vital, Apartado 2511, 4202-Porto, Portugal b

Escola Superior Agrária, Instituto Politécnico de Coimbra, Bencanta, 3045-601

Coimbra, Portugal

‘Equal contribution *Corresponding author Manuela M. E. Pintado Address: Rua Arquiteto Lobão Vital, 172, 4200-374 Porto Tel.: 22 558 0001 Fax: 22 509 0351 E-mail address: [email protected]

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Pig slaughtering for meat production is a major worldwide industry that generates many by-products that can cause environmental problems. Nonetheless, it is possible to have a management of these residues towards the re-use and valorization of these by-products. One opportunity focusses on the extraction of value added compounds such as keratin since pig hair is composed by 80% of this protein. There are some methods for keratin extraction from human hair as other sources such as wool and feathers, but they bring many problems as pollution, time-consumption and high costs. This work uses a commercial detergent belonging to the category of degreasers, capable of fast and efficient dissolution of pig hair, followed by a simple filtration to remove residues from skin trimmings with fat and lard. The resulting solution is submitted to an ultrafiltration process to obtain a solution with higher protein content and purity, both at laboratory scale and pilot scale-up. Use of this keratin green extraction method allows to obtain a keratin product with protein purity up to 70% with a yield extraction of ca. 50%, which although lower than the previous methods allow protein integrity and cleaner technology. This method allows a relevant valorization of pig hair, using for the first time a simple, cost-effective and environmental friendly approach.

KEY-WORDS Keratin; Ultrafiltration; Extraction; Valorization; Residues.


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INTRODUCTION Worldwide millions of pigs are slaughtered, comprising one of the most efficient animals for meat production [1]. Only in 2016, ca. 330 million pigs were slaughtered [2] and this industry generates different kinds of by-products, e.g. blood, hooves, fat and bones, that can be lost and cause environmental problems [3]. Therefore, management and re-use of these by-products has great importance for a sustainable meat production and is highly suggested. One example of by-product from this industry is pig hair. The dehairing of pigs consumes a lot of water and generates wastewaters with high levels of organic matter, fat and dirt [3]. Hence, the hair is a by-product worthy of management towards its reuse and valorization – nowadays the hair is used for brushes, felt, rugs, upholstery, plaster binding and insulation and glue [3]. One of the major components of hair is keratin, also present in wool, feathers and other keratinous substances, consisting in ca. 80% keratin [4]. There are two major protein groups: α-keratin, found in hair, and β-keratin found in e.g. epidermis and feathers. The α-keratins molecular sizes are described with values of ca. 40-65 kDa [5] and have a high content of cysteine and stable cross-linked disulfide bridges between different amino acids [4,6–8]. They can be used especially in biomedical applications such as for regenerative medicine materials, tissue engineering, in the production of keratin based films (coatings) and fibers as polymer reinforcements [8]. Also, keratin is a challenging protein to digest and extract [6] since the disulfide bonds provide a compact structure with resistance to chemical or enzymatic reactions [8] and protective functions to the external environment [5]. Several studies have reported the extraction of keratin from human hair, however research on the extraction of keratin from pig hair has not been reported. According to a recent review from Holkar et al. [4], two of the existing methods for keratin extraction from hair are “Shindai method” [9] and “Reduction method” [10]. The first uses ethanol, a mixture of chloroform/methane, Tris–HCl, thiourea, urea and 2mercaptoethanol with a recovery yield of 75% protein. “Reduction method” [10] uses using urea, SDS, and 2-mercaptoethanol with recovery of 78,1 % amino acids. Nevertheless, there are other methods available to extract keratin from similar sources such as feathers. Gupta et al. [11] uses sodium sulfide to promote digestion and ammonium sulfate to recover keratin from feathers with ca. 53% of yield. Shavandi et al. [8] analyzed several methods reported for keratin extraction and enumerated the



various problems are associated with great part of the existing methods, such as hydrolyzed keratin products, use of non-environmental friendly approaches, high-cost, time-consumption and limited industrial implementation. The use of membrane technology in a continuous process, such as ultrafiltration, can help overcome some problems associated with the extraction of keratin since it is a greener option with reduced energy and carbon footprint as well improvement of safety [12,13]. Hence, the aim of this work was to develop an easier, sustainable and environmental friendly method of extracting keratin from pig's hair. The proposed method uses a detergent treatment with membrane purification, in order to valorize this by-product and obtain a relevant high quality ingredient with reduced hydrolysis degree.

MATERIAL AND METHODS The pig hair was provided by ICM Pork, Primor Group (Famalicão, Portugal) as raw material, which brought together residues such as skin with fat, lard, some hooves and blood. The raw pig hair was rinsed with tap water to remove bigger residues and weighed.

Chemicals used For the extraction of keratin from pig hair the following reagents were used: Ethyl ether (99.5%) from LabChem (LaborSpirit, Loures, Portugal), Sodium sulfide (>98%) and Ammonium sulfate (>99%) from Sigma-Aldrich, Sodium hydroxide (pellets; >99%) from AkzoNobel (Bohus, Sweden) and Mistolin® HTG 50 (MSTN Group, Vagos, Portugal).

Keratin extraction via traditional method In order to compare a traditional extraction method with our novel method, in parallel, the same pig hair batch, in the same quantities, was submitted to the method described by Gupta et al. [11], which was developed for keratin extraction from chicken feathers, further referred as “precipitation method”. Briefly, this method started with a pre-treatment, soaking the hair in ether for 24 h to clean it, followed by dissolution with 0.5 M sodium sulfide at 30 oC with continuous stirring for 6 h. The resultant solution is filtered and centrifuged at 8000 rpm for 5 min. The supernatant was then precipitated with ammonium sulfate 70% (m/v) at a ratio of


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1:1 and the precipitated was recovered by centrifugation. Finally, the solid particles were washed with deionized water, centrifuged and dissolved in NaOH 2 M.

New Keratin extraction method - Laboratorial scale The extraction of keratin was performed initially at laboratory scale combining dissolution with ultrafiltration method as shown in Fig. 1. A commercial degreaser, Mistolin® HTG 50, highly alkali (pH=13-14) constituted by nonionic surfactants (0.05). However, the same method (referred in this work as precipitation method) for extraction of keratin from pig hair had lower protein yield (35.2%) than the obtained for chicken feathers (53%), which suggests that this method could not be the best one for the extraction of keratin from pig hair and eventually could be optimized for this by-product.


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Table 1 – Means of the total protein yield (± standard deviation) obtained with different processes applied (ultrafiltration, laboratory and scale-up and traditional precipitation method).

Process applied Protein yield (%) Ultrafiltration – Laboratory 48.1 (± 8.7)a Ultrafiltration – Pilot scale-up 28.5b* Precipitation method 35.2 (± 6.5)a a,b

Means of protein yields values with the same superscript are not statistically different at a

significance level (P>0.05); *single test.

In addition, performing a pilot scale-up ultrafiltration generated a lower protein yield when comparing with the other two processes (P90% biodegradability) and used by food industry. Furthermore, the safety data sheet provided by the company states that the product does not cause harm to the environment, although it has to be treated as residue accordingly to legislation. The use of this reagent makes the final product obtained a brown liquid with a pH of 12.5 due to the NaOH, nonetheless according to EPA, products containing sodium hydroxide are not likely to cause adverse effects on the environment [24].


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Economically, the purification of the protein is achieved by ultrafiltration, a membrane separation process used to retain macromolecules, that has high throughput of product, easy to scale-up for industrial production and with potential lower costs [25-27]. Estimated costs associated with the different methods tested are depicted in Table 3. The ultrafiltration membrane used is reusable, hence the cost of production of keratin extract is much lower. Nowadays, this process is widely used in the food and dairy industry, but it is completely new to the field of extraction of keratin. Using this process reduces the execution time and simplifies the method, so this method takes about 9 h to get the final product, which is much lower than the Shindai and Gupta methods that take about 2 days. Also, the recovery of the solvent is also possible and the optimization of the cleaning process of this solvent can be made in order to remove all particles and dust resulting from digestion process. The reverse osmosis is a process that may involve high energetic costs but, in this method, this step is optional since the concentration of the final product will be dependent on the purpose and use of the keratin extract.

Table 3. Estimated cost per Kg of keratin produced by precipitation method, ultrafiltration method and commercial keratin. The values per kg or L are based on Sigma-Aldrich reagents. The quantities were estimated regarding the obtained yields in this work.

Ether Precipitation Sodium Sulfide method Ammonium sulfide Sodium Hydroxide Ultrafiltration Mistolin ® method Ultrafiltration Commercial Keratin a

Quantity Volume Weight (L) (Kg) 11 11 0.7 5 3.5 4 0.35 5 -

Sum of energetic cost and membrane at pilot scale;

Cost per Kg or L (in €) 8.61 131.34 127.92 6.83 2.83

Total cost (€) 94.71 91.94 447.7 2.391 14.15 36a

636,8

50.15 75b

b

Cost of a keratin powder (Hefei TNJ

Chemical Industry Co. Ltd., Hefei, China)

CONCLUSIONS Pig hair is a by-product of the pig farming industry that has a high percentage of keratin and since the dehairing can potentially cause environmental problems, turns its re-use of highly importance. In this research a method to extract keratin from pig hair that uses ACS Paragon Plus Environment


only one biodegradable reagent combined with a physical ultrafiltration method was developed. The yield of protein is similar to the obtained by other studies, but when compared, it is possible to significantly simplify the extraction of keratin process with the elimination of harmful reagents and amount of time spent in the process. Hence, the costs are lower and it is possible to obtain keratin with low environmental impact, which is of great importance in the current global framework. However, more tests need to be done in the scale-up process to be suitable at an industrial level. The final product consists in a fairly pure extract of keratin, as shown in FPLC and DSC. Also, the method uses a commercial degreaser, which we believe can be used for other keratinous by-products. This new keratin extraction method is innovative, simpler, cost-effective and environmental friendly.

ACKNOWLEDGMENTS This work was supported by Compete 2020 through project “R4Textiles - Development of sustainable textiles based on the valorization of textile and agrofood waste”, and National Funds from FCT through project UID/Multi/50016/2013. Also, special thanks are extended to the project partners Riopele, CeNTI and Citeve.


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ABSTRACT GRAPHIC

SYNOPSIS The valorization of residues from industry with a more sustainable method for extracting keratin contributes for the circular economy and sustainability.


Novel Eco-Friendly Method to Extract Keratin from Hair - American

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