Raw materials from renewable and agricultural sources have been proposed to produce plastics for food packaging and shopping bags for many years [1
]. With increasing environmental concerns surrounding non-biodegradable plastics, several options have been explored to replace such conventional plastics. Since some of the renewable materials, which are mainly plant-based, are first generation feedstocks, which compete with the food security, utilizing second-generation feedstocks (biomaterials which are by-products, residues, or wastes from other processes) is the preferable option. One such option is the production of plastics from whey proteins, which can be obtained from the by-product/residues of cheese-making processes and expired dairy products.
Cow and goat milk mainly consist of 80% casein proteins and 20% whey proteins [3
]. Casein is a major component of cheese [4
], i.e., in cheese production, casein is coagulated, while the residual whey is normally discarded. Whey proteins, also known as serum proteins [4
], consist of several globular proteins. The most abundant protein in whey is beta-lactoglobulin, making about 50% of the total whey protein, while the second-most abundant is the alpha-lactalbumin, making up about 20% of the whey protein [5
]. The remaining fractions are minor protein/peptide components (e.g., immunoglobulins, lactoferrin, lactoperoxidase, serum albumin, lysozyme, and growth factors) [5
Whey, a by-product of the cheese industry can be used for a wide variety of applications: for example, as a food supplement and in protein drinks [6
]. Generally, in order to make 1 kg of cheese, about 9 L of whey are generated [7
], of which approximately 0.55% of it is whey protein [8
]. The production of whey in the world is about 160 million tonnes in 2008, which has risen to 200 million tonnes in 2011, thereafter with an increase rate of 2% per year [5
]. These whey wastes were also discarded into waterways, causing environmental problems [10
]. Today, protein is extracted from the waste whey and valorized for applications in food and pharmaceutical industries, among others [6
]. Of the produced whey in the United States, 65% is utilized further for human consumption, and 35% for animal feed [13
]. In recent years it is not well documented how much product from the dairy industry is recycled and how much is discharged [13
]. In the UAE, large amount of fresh milk expires and is discharged daily. For example, in a Dubai dairy farm, 7 tonnes of expired milk is wasted daily [14
]. The whey protein from expired dairy products therefore have a high potential for producing protein-based plastics.
Different studies have investigated the use of proteins to form polymeric materials primarily for packaging purposes. Milk proteins have also been used to produce plastics, especially for food packaging. Films and coatings for food products, which are also edible, have been produced successfully from milk proteins [15
]. Ramos et al. [16
] studied the effect of whey protein purity and glycerol (plasticizer) content on the physical properties of edible films made from them. It was found that WPI films exhibited higher mechanical resistance, elasticity, and transparency values, but lower moisture content, film solubility, water vapor permeability, and color change values than their WPC counterparts, for the same content of glycerol. Plastic films from milk proteins were also created for specific application, such as for cheddar cheese packaging, from the study done by Wagh et al. [17
]. Casein and whey proteins were plasticized with glycerol and sorbitol, and the oxygen and water vapor barrier and tensile properties were determined. The tensile strengths and elongation of WPC plasticized with glycerol were generally greater than those with sorbitol, while the elastic moduli were somewhat equivalent [17
]. Jones et al. [18
] investigated the thermal, viscoelastic and antibacterial properties of albumin, soy, and whey bioplastics using three types of plasticizers: water, glycerol, and natural rubber latex (NRL). While the whey protein film plasticized with glycerol showed the highest tensile strength, the film plasticized with NRL has the highest modulus and the film plasticized with water the greatest elongation-at-break.
Besides milk proteins, proteins from different sources including plant (corn zein, wheat gluten, soy proteins) and animal proteins (collagen, gelatin, keratin and myofibrillar) have been explored for polymer production. Cuq et al. [1
] have extensively reviewed different types of plant and animal proteins that could be used to produce agricultural polymers which are used for packaging. A paper by Flieger et al. (2003) [2
] reviews the biodegradable plastics that are made from different renewable sources, including those from microbial fermentations such as polyesters and neutral polysaccharides, and those prepared from chemically-modified natural products such as starch, cellulose, chitin, or soy proteins. Soy protein is found to be one of the most common proteins that are used to produce biodegradable plastics [2
There are several methods in which proteins can be copolymerized with other monomers to produce plastic sheets or films. One such method is through free-radical polymerization, where the side chains and N-termini in the protein molecules can conjugate with other compounds to form polymer, after the protein structure has been modified by functionalizing it with a reactive group, e.g., methacrylating it with methacrylic anhydride, which is well-established in the field of synthesis of GelMA or gelatin methacrylate [21
]. The modified protein is then copolymerized with a monomer to form polymeric or plastic-like materials. Several factors are hypothesized to play an important role in formation of biomaterials and their final properties: protein formulation, polymerization stoichiometry, and the processing technique [15
]. Protein formulation represents the quantitative and qualitative properties of bulk protein fraction obtained from the dairy products. Protein purity, length of peptide chains (which can be controlled by hydrolysis), protein denaturation, and most certainly degree of methacrylation are some of the factors that can influence properties of the final materials. Polymerization stoichiometry refers to the mass ratio between the soft-segment building comonomer and hard-segment building proteins. The mechanical behavior of the elastomer is highly affected by this proportion. The third group, the processing parameters, is related to reaction conditions (heat or chemical catalysis), downstream processing (drying, molding, etc), and plasticizers used [15
In terms of economics, the purity of the protein, as well as its concentration in water can impact the cost of the final products. Commercial whey protein concentrates (WPC) are produced by fractionation with ultrafiltration (UF) and diafiltration (DF) methods, to remove lactose, minerals, and other low-molecular weight components, resulting in WPC with ≥75% protein [26
]. Whey protein isolates (WPI), on the other hand, are manufactured by stirred-bed ion exchange adsorption process, where the pH of the proteins is adjusted and the proteins are eluted from the ion exchanger, concentrated by UF and spray dried, resulting in WPI with ≥90% protein [26
]. The commercial value of the products is determined by the purity. The higher value intuitively requires more expensive modules, types of membrane, and even methods to control fouling [4
]. Therefore, the use of either WPC or WPI would definitely impact the cost of the process and the final products. Moreover, by using less protein (greater dilution in water), the cost may also be minimized. Hence, the purity and the concentration of proteins in the starting materials to produce polymers are also important factors, as far as the economy of the process is concerned.
The method that is followed in this paper is based on the research done by Chan et al. [24
]. This study goes beyond that work, optimizing different polymerization proportions with the copolymer poly(ethylene glycol) methyl ether methacrylate (PEGMA) and comparing the two different types of whey proteins: whey protein concentrate (WPC) and whey protein isolate (WPI). The objectives of the present manuscript are: (1) To compare the mechanical and thermal properties of the polymers obtained from WPI and WPC (2) to compare the mechanical properties of different protein-to-monomer proportions (namely 20:80 and 30:70), and (3) to analyze the mechanical properties of the different variations (11%–14%) of protein concentration in water, given a fixed dry weight concentration (20% and 30%).
2. Materials and Methods
Whey protein concentrate (WPC) that contains 77% protein, as per supplier information, was purchased from the supplier Natur-Drogeriet based in Hørning, Denmark, while whey protein isolate (WPI) that contains 91% protein, was purchased from the BiPro company (Eden Prairie, Minnesota, USA). These two protein sources were chosen as the model crude protein mixture as they are well-studied and widely available agricultural-based proteins, obtained mainly from the dairy industry. Poly(ethylene glycol) methyl ether methacrylate (PEGMA, Mn = 500 g·mol−1) was used as the co-polymerization monomer and was purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Methacrylic anhydride, tetra-methylethylenediamine (TEMED), and ammonium persulfate (APS) were also purchased from Sigma-Aldrich (St. Louis, Missouri, USA).
2.2. Methacrylation and Polymerization Reactions
Polymerizable whey protein was prepared through an amine-based reaction with methacrylic anhydride to introduce (meth)acryloyl moieties onto milk-derived proteins. It is important to make sure that the reactions are not carried out in acidic conditions since the proteins can precipitate at pH around 5. At higher pH conditions, the amine groups on lysine side chains and the protein N-termini can react with the anhydride in the methacrylation step.
Prior to the process of methacrylation, the pH of the protein solution is increased to above a neutral level of pH 7. This is mainly to prevent proteins precipitation when methacrylic acid forms during the reaction and the pH drops. Proteins precipitation occur at the isoelectric point (pH about 4–6), which must be avoided before polymerization.
Methacrylation was used to chemically activate the selected proteins before their further co-polymerization with PEGMA. Methacrylation is a reaction that occurs randomly on different amine groups along the polypeptide chain, more specifically on the ε-amines of lysines and the N-terminal amine of the protein chain [24
]. Figure 1
presents a diagram of the protein methacrylation reaction. Methacrylic anhydride was added, at a dosage of 50 µL/g_protein, to the WPC or WPI solutions, in a 20 mL reaction vessel, and incubated for 18 h at a temperature controlled Innova42 incubator shaker (New Brunswick Scientific, Göteborg, Sweden) at 25 °C and 100 rpm. The resulting solution contains methacrylic acid and the selected methacrylated proteins.
The resulting methacrylated protein was co-polymerized with PEGMA to form polyethylene-like plastics, at two protein/PEGMA mass ratios: 20:80 and 30:70. A 20% w
solution of ammonium persulfate (APS) was added as an initiator at a concentration of 4% v
w.r.t. PEGMA. TEMED was used as a catalyst at a concentration of 0.2% v
w.r.t. PEGMA. Figure 2
illustrates the reaction carried out to produce the polymer. The resulting mixture was then placed (by pipette) in between two parallel glass plates with a spacer of 1 mm and left at ambient conditions (25 °C) for 2 h to form gel-like sheets. The sheets were then peeled-off and dried at 60 °C for 46 h to form elastomers of thickness of about 0.5 mm. The resulting sheets were maintained at 55 ± 5% relative humidity for a minimum of 2 days before the tensile and thermal analyses. Additional to the two protein/PEGMA ratios (20:80 and 30:70), as mentioned above, 4 protein concentrations in water, 11–14% w
were tested, for both of the selected proteins WPI and WPC. This resulted in a total of 16 unique polymer sheets, which were tested for their tensile and thermal characteristics.
2.3. Mechanical and Thermal Analysis
The resulting sheets were cut into 50 × 10 mm rectangular strips with a gauge length of 30 mm. 3 strips were obtained from each sheet. Tests were carried out using a universal tensile testing machine (Zwick Roel Z005, Ulm, Germany) with 20 N load cell and at 1 mm/min strain rate. The thickness of the strips was measured with a digital Vernier caliper (detection limit = 0.01 mm). The thickness of the rectangular specimens used for tensile testing was about 0.45 ± 0.15 mm.
The thermal stability of the resulting polymer sheets was analyzed using a NETZSCH High Temperature TGA-STA449F3-Jupiter thermogravimetric analyzer (TGA, NETZSCH-Gerätebau GmbH, Selb, Germany), under inert atmosphere (nitrogen gas) at a heating rate of 10 °C/min from 30 °C to 800 °C using approximately 10 mg of samples.
2.4. Statistical Analysis
For each specimen, three representative samples data were collected, and average values were considered. Differences between samples were deliberated as significant by considering 95% confidence level. All results obtained are expressed as mean ± margin of error at a confidence level of 95%.