Birendra B. Adhikari, Vadim Kislitsin, Pooran Appadu, Michael Chae, Phillip Choi and David C. Bressler* Specified risk materials (SRM) constitute the proteinaceous waste of slaughterhouses and are currently being disposed off either by incineration or by land filling. Over the last few years, our efforts have focused on developing technology platforms for deployment of this renewable resource for various value-added industrial applications. This report describes a technology for utilization of SRM for the development of an environmentally friendly plywood adhesive with an improved water resistance property. The feedstock (SRM) was first thermally hydrolysed according to a standard protocol, and the hydrolysed protein fragments (peptides) were recovered from the hydrolysate. The recovered peptides were chemically modified through esterification reaction using ethanol, and then chemically crosslinked with polyamideamine-epichlorohydrin (PAE) resin to develop a wood adhesive system. Plywood specimens were then developed using the peptides –PAE resin-based adhesive. The effects of crosslinking time, solid content of the adhesive formulation, ratio of peptides and crosslinking agent in the formulation, and curing conditions of specimen preparation on lap shear strength of resulting plywood specimens were systematically evaluated. Despite the hydrophilic nature of hydrolysed protein fragments, the peptides –PAE resin formulations exhibited remarkable water resistance property after curing. Capping of polar carboxyl groups of peptides by converting them to esters further improved the water resistance property of this adhesive system. Under the optimum conditions of adhesive preparation and curing, the ethyl ester derivative of peptides and PAE resin-based formulations resulted in plywood specimens having comparable dry as well as soaked shear strengths to those of commercial phenol formaldehyde resin.
Industrial processing of agricultural and animal products generates a significant amount of agro-industrial residues, which are composed of compounds of great industrial interest such as sugars, fibres, and proteins. Over the years, the interest in chemical conversion of waste biomass to valuable products has risen sharply as this approach has shown tremendous potential for recovering a wide range of value-added products from bio-waste. Development of sustainable technology platforms for efficient utilization of such bio-wastes as renewable resources of energy and materials helps address the pressing issues related to depleting resources, energy, and the environment. Consequently, the concept of discovering environmentally benign and innovative ways for processing and converting biomass to value-added products is of great importance, and has emerged as an integral part of the holistic approach of sustainable development. Of the various agro-industrial sectors producing substantial amount of bio-waste, the meat industry produces considerable amounts of proteinaceous waste from animal slaughtering and processing of meat. This proteinaceous waste consists of animal products and by-products that are not considered edible and cannot be sold as dressed meat. 1–3 One way to utilize the slaughterhouse waste is processing of the waste through renderers into sellable by-products such as proteinaceous meals. 4,5 However, specified risk materials (SRM) are a type of slaughterhouse waste that cannot enter the feed and food chain, and require segregation, removal, and disposal. These are the tissues of bovine, ovine, and caprine animals where an abnormal form of protein (called prion protein) are likely to concentrate, and these prion proteins are believed to cause a neurodegenerative disease called Bovine Spongiform Encephalopathy (BSE) or "mad cow disease" in cattle, and equivalent diseases in ovine and caprine animals, as well as in humans. 6–9 Due to the fear of potential BSE infectivity, the Canadian Food Inspection Agency (CFIA) has imposed an enhanced feed ban that precludes SRM and any tissues that come in contact with SRM from all animal feed, pet food and fertilizer applications. 6 Similarly, the United States Food and Drug Administration (FDA) prohibits the use of SRM tissues in human food, dietary supplements, and cosmetics. 10 Legislation from the European Food Safety Authority (EFSA) requires the segregation and removal of SRM from cattle, sheep, and goats so that these tissues do not enter the feed and food chain. 9
These enhanced feed bans have led to the generation of significant amount of proteinaceous waste around the world that is being disposed off either by incineration or landfilling with considerable economic burden to meat industry as well as environmental impact. 11
The CFIA and FDA recommended methods of SRM disposal include landfilling, incineration, composting, burial, and thermal or alkaline hydrolysis. 7,11 Of these, thermal or alkaline hydrolysis is attractive as this method allows recovery of hydrolysed protein fragments from the hydrolysate, which then can be utilized in various value-added applications. For instance, protein recovered from SRM hydrolysates has shown great promise for development of bio-composites, 12 bio-plastics, 13 and as an adhesive for oriented strand boards 14 and plywood. 15
Since the international agency for research on cancer (IARC) has identified formaldehyde as a carcinogen, 16 there are several health and environmental concerns with the use of formaldehyde-based resins for production of engineered wood products. Nevertheless, a recent market research report revealed that urea –formaldehyde and melamine –urea –formaldehyde resins accounted for over 70% of total volume (1.96 million tons) of wood adhesive consumed in 2015. 17 As a potential alternative to such formaldehyde-based adhesives, there has been growing interest in development of protein-based wood adhesives. 17,18
Even though the protein-based adhesives offer environmental benefits over petrochemical-based products, one of the major issues concerning the use of protein-based adhesives in engineered wood products is poor moisture resistance, which makes these renewable resource-based products less attractive for real time applications. The adhesives developed from proteinaceous material recovered from SRM also exhibited promising adhesive properties under dry conditions, but the resulting wood composites had limited moisture resistance. 14,15
Review of literature on current trends of protein-based adhesive formulations from various protein sources indicates that the approaches of chemical modification of protein or chemical crosslinking of protein with suitable crosslinking agent and/or combination thereof have resulted in protein-based wood adhesive formulations demonstrating enhanced adhesive strength as well as moisture resistance, with some formulations satisfying the performance requirements of the American Society for Testing and Materials (ASTM) and International Organisation for Standardisation (ISO). 19 The multi-functional compounds possessing reactive sites that are prone to react with the functional groups such as amine, hydroxyl, and carboxyl of protein and/or peptides are effective protein cross-linking reagents, and have shown great promise as crosslinking agents for formulation of protein-based adhesive systems for wood bonding applications. 14,19 –22
The polyamideamine epichlorohydrin (PAE) resins are such polymeric resins that contain reactive sites susceptible to react with nucleophilic groups such as amine, hydroxyl, and carboxyl groups. 23 Because of the potentiality of these resins to react with such functional groups, the PAE resins are widely used as commercial wet strength additive in manufacturing of wet strength papers. 24,25 Kymene ™ 557H, a representative PAE resin, has also shown promise as a crosslinking agent for adhesive development from soy protein isolate. 20,22 Recently, we reported on wood adhesive potential of SRM hydrolysates after crosslinking with PAE resin. 26 In a continuation of our research aimed to develop an environmentally sound plywood adhesive that incorporates protein sourced from SRM and meets the industry requirements, we report on the effect of chemical modification of SRM hydrolysates on the water resistance property of the formulated adhesive.
Thermal hydrolysis of SRM was performed using a 5.5 L high temperature and high pressure stainless steel Parr reactor vessel (Parr 4582) equipped with Parr reactor controller (Parr 4848, Parr Instrument Company, Moline, IL, USA). Size exclusion high performance liquid chromatography (SEC-HPLC) analysis of hydrolysed protein was conducted using an Agilent Technologies 1200 series HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with an autosampler, variable wavelength UV detector, and two size exclusion columns – Superdex ™ 200 and Superdex ™ peptide (GE Healthcare Biosciences AB, Uppsala, Sweden). Viscosity of adhesive formulations was measured using an OFITE model 900 viscometer (OFI Testing Equipment, Houston, TX, USA). Hot pressing of plywood specimens was performed using a Carver hot press (Model 3890 Auto 'M', Carver Inc., Wabash, IN, USA). A CMP 6050 plant growth chamber (Conviron, Winnipeg, MB, Canada) was used for conditioning of plywood specimens prior to testing. The plywood specimens were tested using mechanical test system (MTS 810, MTS Systems Corporation, Eden Prairie, MN, USA) equipped with a 10 kN load cell. The scanning electron microscope (SEM) images of wood specimens were acquired using Zeiss Sigma 300 VP Field Emission SEM (Carl Zeiss AG, Oberkochen, Germany) operating at 20 kV.
The feedstock (SRM) was supplied in dried, ground form, and was handled following the CFIA recommended protocol for safe handling and disinfection of SRM. 27 Kymene ™ 557H resin from Solenis (Wilmington, DE, USA) was used as a representative PAE resin. The phenol formaldehyde (PF) resin was generously provided by a commercial supplier located in Edmonton, AB, Canada. According to the information provided by respective suppliers, the PAE and PF resins contained 12.5% and 43% solids, respectively. The poplar veneers used in this study were of 1.6 mm thickness, and were purchased from a local supplier. Solvents and all other chemicals used in this study were of ACS grade, and were acquired from Fisher Scientific (Fair Lawn, NJ, USA).
The Canadian Food Inspection Agency (CFIA) approved protocol for thermal hydrolysis of SRM requires a temperature of $180 °C and pressure of $1200 kPa for 40 min per cycle. 7 Following these CFIA guidelines, we have developed and optimized the conditions for thermal hydrolysis of SRM, 27 and the previously optimized conditions (180 °C, $174 psi, 40 minutes) were used for conducting thermal hydrolysis of SRM in this study. Before hydrolysis, the SRM sample was handled as a potential biohazard and all the safety measures were practiced as described in previous reports. 26,27 Recovery of hydrolysed protein from the post hydrolysis material was also achieved by following our standard protocol published elsewhere. 15,26 The hydrolysed protein fragments (referred to as peptides hereafter) were recovered on average of 35 ± 1% of the feedstock. In order to ensure batch-to-batch reproducibility, thermal hydrolysis was done in at least three replicates, and the recovered peptides were characterised by following the standard characterization methods being practiced in our lab that consists of elemental analysis, analysis of end functional groups (carboxyl and amines) and analysis of molecular size distribution by SEC-HPLC. 26,27 Analysed in at least three replicates, all these studies ensured batch-to-batch reproducibility of the starting material (peptides).
The hydrolyzed peptides were chemically modified through conversion of terminal carboxyl groups to esters via esterification reaction using ethanol in presence of catalytic amount of concentrated HCl. The esterification reactions were conducted by adopting the conditions previously reported for esterification of a number of proteins as well as polyglutamic acid, 28 which was also reported to be very effective for esterification of soy protein. 29 In a typical esterification reaction, a slurry of 20 g peptides and 400 mL ethanolic HCl (0.1 M HCl in ethanol) was stirred at room temperature for 48 hours (this condition was found to be the best suited condition as determined by the evaluation of extent of esterification and percentage yield of the product; details in Table 3). Afterwards, the pH was adjusted to 5.5 with NH 4 OH and the chemically modified peptides were recovered after vacuum filtration at average yields of 65 ± 1%. The percentage yield reported here is of the solid residue obtained after removal of ethanol by vacuum filtration at the end of the reaction. The chemically modified peptides are named
