The extraction, characterization, modification, and texturization of novel pennycress (Thlaspi arvense) protein for food applications

Abstract

The food industry is actively seeking functional, nutritious, and sustainably produced crops as novel sources of plant protein ingredients to aid in feeding the growing population and address consumer demands. Pennycress (Thlaspi arvense) protein is an attractive alternative to market leading proteins, soy and wheat, as it is currently non-allergenic, non-GMO, and has vast environmental benefits. As a winter cover crop, pennycress provides soil stabilization, nutrient sequestration, and reduced nitrate leaching. Furthermore, pennycress oilseeds are high in protein and oil contents, which are attractive to valorize as two potential food ingredients. The oil from pennycress is currently utilized for industrial biofuels, leaving behind a proteinaceous meal as a by-product. Extracting protein from the meal will increase crop value, creating incentive for farmers to grow this sustainable crop, and will aid in addressing the growing consumer demands for alternative sources of plant proteins. Research on the utilization of pennycress oilseeds for food applications is limited due to antinutritional compounds, namely erucic acid and glucosinolates. Recent agricultural advancements have identified accessions of pennycress with no erucic acid, which are suitable for human consumption. In addition, glucosinolates, which are typically abundant in pennycress meal, are lost during protein isolation steps. Determining optimal, scalable protein extraction conditions that have a high yield of functional, nutritious protein isolates is crucial when evaluating novel crops for food applications. Furthermore, identifying differences in protein structural and functional characteristics among genetically diverse lines is an instrumental knowledge in the advancement of breeding efforts for pennycress. Many plant proteins are known to have inferior functionality compared to whey and soy protein, limiting their use in a variety of applications. Accordingly, this research was divided into two studies to evaluate protein extraction conditions and their impact on structural, functional, and nutritional characteristics of pennycress protein, and then to enhance inferior functional properties through targeted structural modification techniques. Therefore, the objectives of the first study were to 1) optimize protein extraction conditions to maximize yield and purity following two extraction methods, alkaline solubilization coupled with isoelectric precipitation and salt solubilization coupled with ultrafiltration and 2) characterize structural, functional, and nutritional properties of pennycress protein isolates as impacted by the extraction method, scaling up, and difference in genetic variety. Wild-type (W), and zero erucic acid (0EA) pennycress seeds harvested in 2017 were screw-pressed to expel the oil, milled to 60-mesh, and then residual oil was extracted using hexane to produce defatted pennycress meal (DPM). W-DPM was utilized for protein extraction following alkaline solubilization coupled with isoelectric precipitation and salt solubilization coupled with ultrafiltration. Pennycress protein isolate (PcPI) from alkaline extraction (W-PcPI-pH) had a dark, undesirable color, therefore, sodium sulfite was utilized during alkaline solubilization as a reducing agent to mitigate browning. Salt extracted pennycress protein isolate (W-PcPI-Salt) had superior color and functionality. Therefore, salt extraction was used for pilot plant scale up production of PcPI and for protein extraction from 0EA-DPM. Structural and functional characterization was performed on PcPI produced following selected alkaline (with and without sodium sulfite, W-PcPI-pH and W-PcPI-pH-S, respectively) and salt extraction conditions, scaled up salt extraction, and from 0EA seeds. Structural and functional properties of the PcPI samples were compared to native (nSPI) and commercial (cSPI) soy protein isolates. Furthermore, PcPI-salt and W-DPM were evaluated for in-vitro and in-vivo protein digestibility corrected amino acid score (PDCAAS). PcPI-pH, produced with and without the use of sodium sulfite, had relatively poor functionality overall as a consequence of excessive protein denaturation and aggregation and high surface hydrophobicity. On the other hand, W-PcPI-Salt had similar gel strength, three times higher solubility under acidic conditions, and 1.5 times higher emulsification capacity compared to cSPI. 0EA-PcPI-Salt had comparable functionality to that of W-PcPI-Salt. The scaling up process of W-PcPI-Salt resulted in partial denaturation and mild polymerization that contributed to enhanced surface hydrophilic/hydrophobic balance, water holding capacity (WHC), and gel strength compared to its bench scale counterpart. Protein profiling showed that PcPI contains primarily small molecular weight proteins compared to nSPI, contributing to inferior gelation and WHC. Finally, the in-vitro (0.87) and in-vivo (0.72) PDCAAS of PcPI-Salt was superior or comparable to other commercially available plant protein sources. Protein crosslinking and formation of soluble aggregates are required for the development of a strong 3-dimensional gel network that entraps water. Proteins of relatively large molecular weight are correlated with a high potential to form cohesive, strong gel networks. Crosslinking proteins in PcPI will increase gel strength and WHC for enhanced texturization potential, and incorporation into high-value meat analogue applications. Protein crosslinking can be induced by either transglutaminase (TG) or physical treatment with cold atmospheric plasma (CAP). Therefore, the objectives of the second study were to 1) evaluate the effect of CAP and TG modifications on the structural and functional characteristics of PcPI, and 2) to determine the texturization potential of the modified PcPI. CAP treatment with dielectric barrier discharge (DBD) was utilized to polymerize PcPI (PcPI-CP). The production of TG modified PcPI (PcPI-TG) was optimized for enzyme dose, the use of pre-treatment denaturation, and time based on lysine crosslinking and protein profile. PcPI-CP and PcPI-TG were evaluated for structural and functional properties compared to unmodified PcPI. Micro-compounding was utilized for bench scale texturization of unmodified, PcPI-CP, and PcPI-TG at 50% water content. The texturization potential was assessed through mechanical responses during micro-compounding, structural properties, and texture profile analysis. CAP treatment induced polymerization primarily through intermolecular disulfide interchange, whereas TG resulted in a relatively higher extent of polymerization that was induced through a combination of inter- and intramolecular disulfide linkages and other covalent interactions involving acidic subunits of cruciferin. Compared to unmodified PcPI, PcPI-CP and PcPI-TG had double and triple the gel strength, respectively. Furthermore, PcPI-TG had the highest WHC (almost 100%). Upon micro-compounding, unmodified PcPI did not form fibrous structures and instead was a soft mass with low resilience and cohesiveness. Micro-compounding of PcPI-CP resulted in hard, dense fibrous structures due to the low WHC. However, the high gel strength and WHC of PcPI-TG resulted in fibrous structures with more air incorporation upon micro-compounding. Results confirmed that polymerization, especially with TG, can enhance gelation properties and texturization potential of PcPI. This work was the first to optimize protein extraction conditions from pennycress and provide a comprehensive structural, functional, and nutritional comparison among the resulting isolates. Overall, this work demonstrated that PcPI can be successfully extracted from DPM with high protein purity and yield, and acceptable color. Furthermore, the characterization of PcPI from genetically diverse lines provided a benchmark of knowledge to progress pennycress breeding efforts. Results confirmed that salt extraction is scalable and can result in PcPI with favorable functional properties that are comparable, or in some cases superior to cSPI. The low gel strength of PcPI was overcome by inducing polymerization through CAP and TG treatment, ultimately enhancing texturization potential. In particular, TG modification increased the WHC of PcPI, which resulted in textural properties that are desirable for meat analogue applications. This research provided foundational knowledge for the processing, modification, and utilization of PcPI. The introduction of PcPI into the protein ingredients market provides a sustainable, nutritious, and highly functional protein source for use in a wide range of food applications.