Effect of Additives on the Tensile Performance and Protein Solubility

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Effect of Additives on the Tensile Performance and Protein Solubility of Industrial Oilseed Residual Based Plastics William R. Newson,*,† Ramune Kuktaite,† Mikael S. Hedenqvist,§ Mikael Gal̈ lstedt,# and Eva Johansson† †

Department of Plant Breeding, The Swedish University of Agricultural Sciences, SE-230 53 Alnarp, Sweden KTH, School of Chemical Science and Engineering, Fibre and Polymer Technology, SE-100 44 Stockholm, Sweden # Innventia, Box 5604, SE-114 86 Stockholm, Sweden §

S Supporting Information *

ABSTRACT: Ten chemical additives were selected from the literature for their proposed modifying activity in protein−protein interactions. These consisted of acids, bases, reducing agents, and denaturants and were added to residual deoiled meals of Crambe abyssinica (crambe) and Brassica carinata (carinata) to modify the properties of plastics produced through hot compression molding at 130 °C. The films produced were examined for tensile properties, protein solubility, molecular weight distribution, and water absorption. Of the additives tested, NaOH had the greatest positive effect on tensile properties, with increases of 105% in maximum stress and 200% in strain at maximum stress for crambe and a 70% increase in strain at maximum stress for carinata. Stiffness was not increased by any of the applied additives. Changes in tensile strength and elongation for crambe and elongation for carinata were related to changes in protein solubility. Increased pH was the most successful in improving the protein aggregation and mechanical properties within the complex chemistry of residual oilseed meals. KEYWORDS: oilseed meal, biobased plastics, Crambe abyssinica, Brassica carinata, compression molding

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behavior of plant protein based materials,15,16 but environmental and occupational safety issues make them unattractive.17 More benign approaches have been developed to improve the behavior of seed proteins such as modification of the pH environment,16,18−21 carboxylic acid cross-linking,22,23 chemical protein denaturation,24−29 and enzymatic cross-linking.30 Many of these approaches require solution processing during product formation (film casting) or after forming (solution soaking), which increases cost and environmental impact through the production of liquid waste. To reduce cost and environmental impact, approaches that can be integrated into solid-state process steps such as compression molding or extrusion6,24 are preferred. In this work the residual meals of crambe and carinata oilseeds were treated with additives and simultaneously converted into plastic materials using hot compression molding without extra processing steps. The materials were characterized for tensile properties and water absorption related to their possible use as plastic materials and combined with an examination of protein solubility and molecular weight profile changes via size exclusion high-performance liquid chromatography (SE-HPLC). Our intention is to survey the effect of additives that have shown activity in other protein systems with a view to improving the performance of oilseed meal based plastics in an environmentally sound way.

INTRODUCTION Global concerns regarding sustainability, petroleum availability, and climate change have fueled interest in biobased sources to replace petroleum products. Among the promising sustainable oil plants to replace petroleum products are the industrial oilseeds crambe (Crambe abyssinica Hochst.) and carinata (Brassica carinata A. Braun).1,2 Crambe and carinata are of interest as they have good agronomic characteristics in temperate zones, crambe showing relatively good drought tolerance and carinata excellent drought and heat tolerance3,4 with adequate yields. Crambe is already considered an industrial oil crop with high levels of erucic acid, whereas both crambe and carinata are amenable to genetic manipulation while being unlikely to cross with the important commercial crop oilseed rape.1,2,5 To improve the economic proposition of industrial oilseed meals, value-added applications such as biobased plastics are needed. Biobased plastics from hot-pressed oilseed meals are not thermoplastic, have poor water resistance, and lack cohesion, limiting them as plastic materials in industrial applications.6−8 To enhance their plasticity, polyols such as glycerol have been added to plant protein materials to modify their behavior by disrupting protein−protein interactions and acting as chemical chaperones in protein rearrangement.6,9,10 Water resistance and cohesion are increased by heating, with the accepted mechanism of protein denaturation and increased protein− protein interaction.6 Oilseed meals and oilseed protein isolates have been explored in adhesives,11 cast films,12,13 hot compression molding,14 and injection molding.8 Chemical means have been explored to improve protein− protein interaction in various plant protein systems. Crosslinking with aldehydes has been successful in modifying the © XXXX American Chemical Society

Received: April 4, 2014 Revised: June 25, 2014 Accepted: June 27, 2014

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Table 1. Selected Additives, Their Acronyms, Proposed Actions, and Literature Sources type base acid/base acid

reactive denaturant

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reductant

additive

abbrev

proposed action

ref

NaOH NH4OH citric acid/NaOH salicylic acid citric acid ascorbic acid Jeffamine EDR 176 benzoyl peroxide sodium dodecyl sulfate urea sodium bisulfite

NaOH AH CA/N SA CA AA JF BP SDS U SB

promote protein−protein interactions promote protein−protein interactions cross-linking retard protein−protein interactions cross-linking improve protein behavior provide reactive amine sites thermally decomposing oxidant allow protein rearrangement allow protein rearrangement allow protein rearrangement

19−21 16 18 22, 23 21, 32 33, 34 35 36 30, 35, 37 26, 27 24, 28, 29 38, 39

Hot Compression Molding. The oilseed meal was mixed with the additive/glycerol/water blend by hand using a mortar and pestle until evenly mixed (3−5 min). The mixture was then formed in a heated hydraulic press (Polystat 400s, Servitech, Germany) between aluminum plates with polyethylene terephthalate release film using a 0.5 mm thick aluminum frame with a 100 mm × 100 mm central opening to control the size and thickness of the film. A molding temperature of 130 °C, a pressure of 10 MPa on the sample area, and a pressing time of 10 min were used on all samples on the basis of previous work.6 After removal from the press, the sheets were cooled in the frame between room temperature aluminum sheets. The cooled oilseed meal sheets were removed from the frame using a scalpel. This process produced a wide range of macroscopic morphologies, from well-formed free-standing films to sticky incoherent films that were impossible to remove from the release films whole. All samples were processed via HPLC and water immersion; only well-formed films proceeded to tensile testing. SE-HPLC. The amount and size distribution of soluble protein in the films were determined by a three-step extraction procedure.40 Samples of each film were hand cut to a maximum particle size of approximately 0.2 mm, and triplicate samples of 16.5 (±0.05) mg each were weighed into 1.5 mL centrifuge tubes. Protein extractions were carried out in 1.4 mL of extraction buffer, 0.5% (w/v) SDS, 0.05 M NaH2PO4, pH 6.9. Three extractions were carried out serially on each sample. The extractions were as follows; extraction 1, vortexing for 10 s followed by shaking 5 min at 2000 rpm; extraction 2, 30 s of ultrasonication at an amplitude of 5 μm (Sanyo Soniprep, Tamro, Sweden); extraction 3, 30 + 60 s of ultrasonication at 5 μm. After each extraction, the supernatant was recovered for further analysis, and the residuals were resuspended in extraction buffer for the next extraction. Analysis was performed with a Waters 2690 separations module and a Waters 996 photodiode array detector at a flow of 0.2 mL/min (50% ACN, 0.1% TFA; 50% H2O, 0.1% TFA). A 20 μL sample of supernatant was injected onto a column, Phenomenex Biosep-SEC-S 4000 (300 mm × 4.5 mm, Torrance, CA, USA) with a prefilter (Phenomenex SecurityGuard GFC 4000). Absorption spectra were collected in 3D for 30 min and analyzed with Empower Pro software (Waters, Empower 2). Chromatograms were extracted at 210 nm and integrated into two arbitrary fractions: high molecular weights (HMw) from 8 to 18.3 min and low molecular weights (LMw) from 18.3 to 30 min. The solubility is defined as the area of the elution intervals normalized to the total protein extracted from the raw oilseed meals and corrected for individual sample meal content (eq 1). Error bars reported in HPLC data indicate one standard deviation. Interference due to the absorption of salicylic acid at 210 nm was removed from the data by manual peak selection.

MATERIALS AND METHODS

Materials. Oilseed meals of crambe (11.1 ± 0.02% db water) and carinata (10.1 ± 0.06% db water) were produced by the petroleum distillate method of Appelqvist31 performed at room temperature to avoid protein thermal denaturation. Carinata seeds were graciously provided by Agriculture Canada (Saskatoon, Canada) and crambe seeds (with pod) supplied by the Plant Research Institute (Wageningen, The Netherlands). Monosodium phosphate (NaH2PO4), as monohydrate, was provided by Baker (Deventer, The Netherlands), and glycerol (99.5%) was graciously supplied by Karlshamns Tefac AB (Karlshamn, Sweden). Trifluoroacetic acid (TFA) and acetonitrile (ACN) (Merck) were of spectroscopy grade and isocratic chromatography grade, respectively. All water was purified on a Milli-Q system (Millipore Corp., Bedford, MA, USA). Additives Used. Ten different chemical additives across five different types were selected on the basis of previous literature. The selected chemical additives, the reason for their selection, and reference(s) related to their previous use in protein based plastics are summarized in Table 1. Addition levels were selected from the references. Methods. Protein Content. Nitrogen content of the deoiled meal was performed in duplicate with the Dumas method (Flash 2000 NC Analyzer, Thermo Scientific, USA), nitrogen to protein conversion factor N × 6.25, and protein content together with an example of other constituents are given in Table 2.

Table 2. Selected Industrial Oil Meal Components (Percent, Dry Basis)

a

component

carinata

crambe

proteina ash glucosinolates phytic acid fiber soluble sugars polyphenols

43.7 ± 1.9 5.747 5.647 3.447 34.947 6.247 0.347

20.5 ± 1.2 7.462 4.5−762 NRb 22.162 8.348 NR

Current study. bNR, not reported.

Mixing Procedure. All samples contained the same amount of water, 5.25 parts per 70 parts oilseed meal, to match the water contained in the highest level of ammonium hydroxide (25% solution). Where the additive was hydrated, this was accounted for in the water addition. Additives, water (if necessary), and glycerol (30 parts per 70 parts oilseed meal by weight) were premixed before addition to the oilseed meals. In cases when the additives were not soluble under these conditions (NaOH 4.5 parts, SDS 4.5 parts, SA, BP), they were added as a dry powder to the meal before mixing. All additive doses are listed as parts additive per hundred parts combined oilseed meal and glycerol (70 parts and 30 parts, respectively).

% solubility integrated fraction area total sample mass = × × 100 total integrated raw meal area sample meal mass

(1) Tensile Testing. Tensile specimens were cut with an ISO 37, type 3, sample cutter (Elastocon AB, Sweden) and conditioned for a B

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Figure 1. Tensile properties of compression-molded crambe and carinata oilseed meals. Error bars represent one standard deviation. minimum of 48 h at 23 °C and 50% relative humidity prior to testing. The thickness was calculated as an average of five measurements, and the specimens were tested at 23 °C and 50% relative humidity using a crosshead speed of 10 mm/min and a 30 mm clamp separation (Instron 5566, Instron AB, Sweden).

The stress was calculated from the force divided by the cross-section area and the strain using the length of the reduced width section.41 Strain at maximum stress was considered as the measurement for ductility as the failure mode was not consistent with slow tearing, making the determination of the strain at break inconsistent. Young’s C

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modulus (E modulus) was calculated from the maximum slope after the initial toe correction.41 A minimum of nine replicates were used; error bars in reported data indicate one standard deviation. Films excluded from tensile testing due to poor quality on pressing are listed as n/a in the Supporting Information. Immersion Testing. Five replicate samples were removed from the oilseed meal films with a 5 mm diameter punch and weighed. Samples were lyophilized (Modulo, Edwards, UK) for a minimum of 48 h, weighed, and immersed in water for 24 h at 4 °C to prevent microbial growth. Following immersion, disks were removed from the water and held vertically for 10 s; the pendant drop was removed on fresh filter paper, and the disk was blotted between two dry pieces of filter paper (Munktell 1701, Falun, Sweden) under a 25 g weight, resulting in a pressure of 64.5 Pa for 10 s, then weighed. The disks were again lyophilized for a minimum of 48 h and weighed. The water absorption and mass loss during immersion were calculated on a dry basis. Error bars in reported data indicate one standard deviation. Films that lost coherence during immersion are listed as n/a in the Supporting Information.

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RESULTS AND DISCUSSION The present study on the effect of various additive types on tensile properties, protein solubility, protein molecular weight (Mw) distribution, and water uptake upon immersion produced a wide range of positive and negative results compared to a control with no additives. Here we have selected to present the results of those films with additives that

Figure 3. Protein solubility of compression-molded crambe and carinata oilseed meals divided into HMw and LMw fractions. Solubility is normalized to the total solubility of raw meal. Error bars represent one standard deviation.

Figure 4. Water absorption of compression-molded crambe and carinata oilseed meals. Error bars represent one standard deviation. Figure 2. Total protein solubility of compression-molded crambe and carinata oilseed meals. Solubility is normalized to the total solubility of raw meal. Error bars represent one standard deviation.

contributed to interesting tensile properties. Those films with additives either having little effect or showing a negative D

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such as lanthionine and isopeptide cross-links43,45,46 and, as sugars are present in both meals,47,48 Maillard type crosslinks.49 Thus, building on previous results of increased protein polymerization by creating a basic environment in protein systems, in this study we propose that increases in protein aggregation have occurred in the present oil crop residue based plastics in a similar way, despite the low protein content. Increasing the NaOH dose above 1.5 resulted in decreased mechanical properties (Figure 1a), whereas at these doses solubility increased markedly (Figure 2). Increased levels of AH did not induce further strength increases in crambe (Figure 1a), nor did the solubility decrease for either meal (Figure 2). These tensile-solubility relationships at higher levels of NaOH and AH may be the result of exhaustion of the available mechanisms for solubility reduction already occurring at the lowest additive level. A pH environment far from the isoelectric point is known to improve the properties of protein-based films.19 Although the isoelectric points for the individual proteins in our meals are not specifically known, combined carinata proteins demonstrated solubility minima at pH 3.5 and 5,47 whereas the minimum for crambe is approximately 5.5,50 both from aqueous alkaline extracted seed meal. The most common storage proteins in brassica, cruciferin and napin, have isoelectric points of approximately 7.2 and ≤10, respectively, as measured in rapeseed.51 An alternative explanation of the effect of NaOH and AH is that the properties vary related to the distance from the isoelectric points of the proteins. The pH is also known to affect the interaction between proteins and other constituents of brassica oilseed meals. Although the bulk of the evidence in this area is from aqueous solutions, increased pH enhances many processes including interactions between proteins and phenolic compounds,52 Maillard reactions with sugars,53 and phytic acid as a protein complexing agent that depends on pH.51 Indeed, phenolic acids have been added to purified plant proteins as cross-linking agents.54 E modulus was not positively affected by NaOH and AH in any case examined, despite changes in protein solubility. E modulus is a composite property resulting from deforming of all components, whereas maximum stress and strain at maximum stress are local phenomena. If the protein fraction is considered under the polymer entropic elasticity theory,55 secondary structure such as α-helix and β-sheet have been shown to have an effect on the response of protein materials that is not accounted for in their increased molecular weight due to aggregation. In our case the proteins exhibit increased aggregation, but do not contribute to an increased E modulus. The analysis of VanKleef55 suggests that changes in secondary structure, which do not contribute to the SE-HPLC response, may be responsible for a lowered stiffness of the protein fraction despite increased aggregation. Water uptake varied greatly between NaOH and AH, with NaOH showing markedly higher water uptake (Figure 4). It has been shown that in protein-based materials made with AH there is no residual pH effect as the free ammonia evaporates.16 For NaOH it is expected that there will be a residual pH effect that can continue to modify the protein environment during the water uptake test, possibly allowing more complete protein unfolding in water. Lower protein aggregation at higher NaOH levels coupled with the residual pH effect could result in a very loose and easily expanded protein network, allowing high water uptake levels.

Figure 5. Selected tensile properties versus protein solubility of compression-molded crambe (A, B) and carinata (C) oilseed meals. Solubility is normalized to the total solubility of raw meal. Error bars represent one standard deviation.

influence on properties are treated to only a limited extent, although all results are included in the Supporting Information. Effect of Additives with a Basic pH. Addition of NaOH (1.5) and AH (2.5) resulted in increased strain at maximum stress for both crambe and carinata (Figure 1a) as well as increased maximum stress in crambe (Figure 1a). Protein solubility decreased with additions of NaOH (1.5) and AH (all levels) (Figure 2). In particular, the extractability of the HMw fraction decreased compared to controls at the NaOH doses that resulted in increased tensile properties (Figure 3). Previous work on protein-based plastic materials has shown increased strength and decreased protein solubility, especially of HMw proteins, when AH was used as an additive. The increased strength and decreased protein solubility at increased pH have been attributed to reorganization and polymerization of the proteins.42−44 Thermal processing of proteins under basic conditions has been found to favor the formation of residue−residue reactions E

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Citric Acid−NaOH Blends. Addition of CA(3)/N(x) blends resulted in increased strain at maximum stress for both crambe and carinata while also increasing the maximum stress in crambe in a dose-dependent manner (Figure 1b). Protein solubility showed a slight decrease with increased NaOH, although the lowest NaOH level (3/0.5) in crambe demonstrated a pronounced increase in solubility due to the high acidity (Figure 2). This indicates low protein polymerization, similar to additions of CA alone (Supporting Information). CA is known to suppress lysinoalanine crosslinking in food protein systems,45 one possible mechanism for the increased solubility in this case. Solution pH is also known to be a determining factor in the interaction of secondary plant metabolites in oilseed meal with proteins. For example, maximum precipitation of phytates occurs at low pH values for both napin and cruciferin in rapeseed,56 whereas phenolic compounds (sinapic acid) have been found to bind tightly to canola cruciferin at pH 4 and adversely affect heat-set gels. It should be noted that the cited works on phenolic and phytate interaction with brassica proteins are in solution or gels at