Performing Department
(N/A)
Non Technical Summary
More than 8 million metric tons of plastics pollute our world's waterways each year, with more than half of that coming from food-related sources. Sustainable, biodegradable food packaging alternatives are urgently needed. Recently, protein films have become a promising plastic alternative for food packaging. An integral part of their manufacturing is the addition of plasticizers, to both enhance films' flexibility and ensure cohesive, coherent film structures. Glycerol, as the most widely used plasticizer, is highly effective; however, it reduces film tensile strength and readily migrates out of the protein matrix since it is not tightly bound to proteins. Phenolic compounds are potential alternative bioplasticizers that can modify and plasticize film structure. That said, most research into phenolic compounds in films has focused on their ability to create active packaging, i.e., for their antimicrobial, antibacterial, or antioxidative properties. Further, methods to solubilize proteins and facilitate their interactions with phenolic compounds are needed. Our central hypothesis is that coupling the plasticizing effects of phenolic compounds with the protein solubilizing and intermolecular bond breaking of formic acid will enhance protein film properties and produce edible, compostable films. We will test this hypothesis in two main objectives: (1) Characterize effects of naturally occurring bioplasticizers on protein film properties and their interaction mechanisms, and (2) Identify effects of solvents and thermal processing on protein and bioplasticizer stability and resultant film properties. Ultimately, we aim to develop methods that facilitate interactions between proteins and phenolic compounds and produce edible, compostable films as sustainable plastic alternatives for food packaging.
Animal Health Component
40%
Research Effort Categories
Basic
50%
Applied
40%
Developmental
10%
Goals / Objectives
Overall, we aim to identify effective bioplasticizers and processing conditions to produce edible, compostable protein films as sustainable plastic alternatives. Producing breakthroughs in the scientific knowledge of forming biopolymer films with desired structural integrity (low permeability and high tensile strength) will enable biopolymers to replace plastic food packaging and to improve novel foods through restructuring proteins. Our central hypothesis is that coupling the plasticizing effects of phenolic compounds with the protein solubilizing and intermolecular bond breaking of formic acid will enhance protein film properties and produce edible, compostable films.Objective 1: Characterize effects of naturally occurring bioplasticizers on protein film properties and their interaction mechanisms. Hypothesis 1: The proposed phenolic-based bioplasticizers will be more effective than glycerol in plasticizing protein films.Hypothesis 2: The proposed phenolic-based bioplasticizers will be more resistant to migration out of the protein films than glycerol, thus resulting in films that are more robust.Objective 2: Identify effects of solvents and thermal processing on protein and bioplasticizer stability and resultant film properties.Hypothesis 1: Solvents that denature the proteins will enhance the ability of bioplasticizers to plasticize films.Hypothesis 2: Prolonged thermal processing (>50 C, >24 h) will produce undesired hydrolysis reactions in the film-forming solutions, thus reduce film quality.Hypothesis 3: Bioplasticizers will most effectively improve protein film integrity when conjugated to the proteins.
Project Methods
Objective #1 - Characterize effects of naturally occurring bioplasticizers on protein film properties and their interaction mechanisms. We seek to demonstrate that phenolic compounds can outperform glycerol as bioplasticizers in protein films. Our working hypotheses are that the proposed phenolic-based plasticizers will be (1) more effective than glycerol in plasticizing protein films; and (2) more resistant to migration out of the protein films than glycerol, thus resulting in films that are more robust. Our preliminary data suggests that formic acid is an excellent solvent to solubilize proteins and that small MW phenolic compounds effectively plasticize films. We will test our hypotheses by combining these two findings: forming solution-cast protein films using formic acid to solubilize the proteins and assessing phenolic compounds to plasticize the films. When the proposed objective is completed, we expect the overall outcome to demonstrate an initial method to plasticize protein films with phenolic compounds. Though the goal is to eventually valorize proteins from low value and waste streams for these films, our work will start with protein isolates to identify effects of bioplasticizers on protein films.Methodology:Materials: Proteins will include plant-based protein isolates with different structural and functional attributes: soy protein isolate, which is one of the most used plant protein isolates thus has a well-established supply chain; and three pulses that are increasing popular in plant-based foods including pea, fava bean, and lentil protein isolate, which showed promising results in our preliminary study. To plasticize these proteins, we will test select phenolic acids and flavan-3-ols (gallic acid, ferulic acid, caffeic acid, rosmarinic acid, chlorogenic acid, ECG, EGCG) vs glycerol as a control.Methods: Proteins will be solubilized in neat formic acid solutions, the selected bioplasticizer will be added while shearing, and the solutions will be stirred for 60 min at room temperature to ensure full hydration and interaction. The glycerol (control) and phenolic compound bioplasticizers will be tested at two different levels: 5 and 20 w/w% of the protein. Based on method development work done in our lab, the protein-film solutions will be aliquoted into petri dishes and dried overnight under a fume hood to form films. After drying, the films will be equilibrated in desiccators controlled to 25, 50, and 75% relative humidity for 24 h before further testing.Protein-phenolic compound interactions: Prior to drying, we will aliquot protein-film solutions. To determine the amount of phenolic compound bound to proteins, we will precipitate proteins and quantify free phenolics with Folin assay. We will also assess effects of treatments on protein surface hydrophobicity and free/total -SH content. To detect and measure levels of formylation and modification by phenolic compounds, we will use mass spectrometric analysis. Further investigation into protein interactions will occur with electrophoresis and FTIR, detailed below in the Advanced Film Analyses.Basic film analyses: Film physical attributes (thickness, visible defects, and color with Hunter colorimeter) will be determined. Tensile strength of the films will be assessed using a texture analyzer (TA-XT2)1 at 22°C. Aqueous stability and water holding capacity of the films will be tested by placing the films in buffered solutions (pH 2-9) at 22 or 37°C and assessing films over 24 h1. These films will also be dried and assessed for tensile strength to indicate overall stability of the film, including stability of plasticizer, to aqueous environments.Advanced film analyses: We will investigate the properties of the films made with the most promising bioplasticizers further. Dynamic mechanical analysis (DMA) and thermal degradation analysis (TGA) will be determined to identify film glass transition and degradation temperatures, respectively26. Film barrier properties will be characterized using standard methods from ASTM International for water vapor permeability (ASTM E96-00) and oxygen gas transmission rate (ASTM D3985-17). Images of the film surface will be obtained with standard photography (visible) and scanning electron microscope (SEM, micron scale)1. FTIR will be used to identify changes in protein secondary structures. Covalent aggregation and crosslinking will be tested with SDS-PAGE analysis and electroblotting with staining as appropriate. Compostability will be tested by standard methods (ASTM D6400-21).Experimental Design All experiments in Obj. 1-2 will be evaluated as a completely randomized block design with three replications. Specifically, each replication containing the control and treatments will constitute a block. Controls will be protein films plasticized with glycerol. ANOVA will be used to detect treatment effects. Dunnett's test will be used to identify differences within each protein from its respective control with significance determined at P < 0.05.Objective #2 - Identify effects of solvents and thermal processing on protein and bioplasticizer stability and resultant film properties We seek to demonstrate that processing conditions can be modulated to produce desired functional attributes in protein films. The goals are to identify effects of alcohols, acids, and pH as well as thermal processing on protein films and the ability of phenolic compounds to plasticize them. Our working hypotheses are that: (1) solvents that denature the proteins will enhance the ability of bioplasticizers to plasticize films; (2) prolonged thermal processing will produce hydrolysis reactions in the film-forming solutions, thus reduce film quality; and (3) bioplasticizers will most effectively improve protein film integrity when conjugated to the proteins. We will test our hypotheses by varying the solvent used to make solution-cast protein films as well as the thermal processing conditions. When the proposed objective is completed, we expect the overall outcome to be optimized conditions for protein film quality. This result is expected to have a significant impact on our understanding of novel ways to plasticize protein films that can lead to their viability in replacing plastic food packaging.Methodology:Materials: The proteins used in Obj. 1 (soy, pea, lentil, and fava bean protein isolate) will be made into solution-cast films using either glycerol (control) or one of three selected phenolic compounds for the plasticizer.Methods: To understand the effects of solvents in conjunction with bioplasticizers on protein film properties, we will investigate several solvent systems: alcohol (ethanol), acid (formic acid), and pH controlled. Ethanol and formic acid will be assessed neat and in aqueous solution (25-75 v/v%). A solvent consisting of 50 v/v% aqueous ethanol will be used to assess the effects of pH (2-11, controlled with HCl or NaOH) on film properties33. We will also vary the thermal treatment used on the film forming solution. The control will be Obj. 1 conditions (60 min, room temperature), with treatments consisting of varied time (15 min - 48 h) and temperature (-20 - 75 °C). Methods to study protein-phenolic compound interactions and analyze films will be the same as listed above in Obj. 1.