Cora De Gol

Plant-based food products are rapidly gaining market share as consumers shift toward more sustainable and healthy diets. Compared to animal-based products, their production requires less land and water and generates a substantially lower carbon footprint. Among them, meat substitutes have become particularly popular for their ease of use. They are typically produced by extrusion, where plant-based protein isolate powders are hydrated and texturized into meat-like products. The functionality of the protein, primarily its gelling properties, is therefore crucial for the process. Conventional protein extraction from the raw material and drying steps involve high temperatures and chemical use, which not only consume large amounts of energy but also impair the gelling properties of the proteins. Additionally, most commercial meat substitutes are soy-based, raising concerns about deforestation, large-scale monoculture and global transport dependence.

This thesis aims to advance the development of plant-based food products by rethinking meat substitute manufacturing. It proposes replacing imported soy protein isolates by locally sourced protein ingredients and eliminating conventional energy-intensive protein extraction and drying steps. Instead, minimal processing of proteins was developed. Two protein ingredients, Chlorella vulgaris (Cv) suspension and yellow pea protein extract (PPE) were considered. PPE was obtained from air classified pea protein concentrate under mild, aqueous and room temperature conditions. A life cycle assessment (Chapter 6) revealed that substituting soy with pea reduces the protein’s carbon footprint by a factor of 5.5, and using PPE instead of pea protein isolate (PPI) powder lowers it by an additional factor of 1.8. Moreover, Chapter 2 demonstrated the feasibility of incorporating protein-rich plant-based liquids into extrusion without compromising the properties of the final product.

A major challenge with handling liquids is their microbial instability. While extrusion (high temperature, pressure and residence time) ensures the microbial safety of the final product, quality deterioration of the liquid during refrigerated storage and prior to extrusion limits processing flexibility. Key spoilage bacteria were identified in Cv (Pseudomonas guariconensis, Enterobacter soli and Lactococcus lactis) and PPE (Pseudomonas trivalis and Erwinia gerundensis) (Chapters 3 and 4). Pulsed electric field (PEF) treatment combined with cold storage was investigated as a mild preservation strategy. PEF treatment is based on the application of high-voltage short pulses to liquids, inactivating bacteria with minimal temperature increase and reduced damage to heat-sensitive compounds, predominantly present in liquid foods.

In Chapter 3, challenge tests using pilot-scale PEF treatment in continuous mode were performed on Cv. The optimized conditions (Eel = 28 kV/cm, f = 120 Hz, τ = 20 μs, t = 225 μs, Tin = 30 °C, Tout < 55 °C) allowed microbial reductions of ~ 4 logs for P. guariconensis, E. soli and L. lactis. These reductions enabled shelf-life extension of up to five days at 10 °C, which could be further increased through repetitive PEF cycles. Similarly, Chapter 4 demonstrated PEF-induced microbial reductions of ~ 4 logs for P. trivalis and E. gerundensis inoculated into PPE (Eel = 24 kV/cm, f = 50 Hz, τ = 20 μs, t = 126 μs, Tin = 40 °C, Tout < 60 °C). Shelf-life was extended by three days at 10 °C storage and six days at 5 °C. When applied to the PPE native microbiota, PEF caused a four-day lag phase extension at 5 °C storage, accompanied by a shelf-life extension of six days and a pH stability of ten days. Overall, PEF effectively preserved the quality of Cv and PPE with significant inactivation effects on both targeted spoilage bacteria and native microbiota. The implementation of PEF in the supply chain of plant-based foods was evaluated in Chapter 6. The growth rate of PEF-treated E. soli was significantly reduced, and the lag phase duration was extended when introducing one day storage (10 °C) prior to PEF. Chapter 6 further investigates the PEF-induced electroporation of the bacterial membrane, aiming for consistent lag phase extension to further enhance storage stability. In addition to lethal effects, PEF at high intensity (Eel = 28 kV/cm) caused the formation of sublethal injury in E. soli. Despite exhibiting delayed colony formation, detected as smaller colony size upon plating, this injury was not associated with a prolonged lag phase duration during shelf-life. The viability of PEF as a preservation method depends on its ability to maintain protein functionality. Chapter 5 examined the impact of PEF on pea proteins (albumins and globulins) structure and functionality and compared it with conventional heat pasteurization. Spectroscopic measurements revealed partial protein unfolding following PEF, promoting intermolecular interaction and enhanced (> 50%) gelling properties. In contrast, heat pasteurization caused protein aggregation and 60% reduction in gelling properties for equivalent microbial inactivation levels. Moreover, lab-scale extrusion trials (Chapter 6) confirmed that the incorporating (30%) of PEF-treated PPE in PPI did not affect the meat substitute texture, whereas the textural parameters were reduced with thermally treated PPE.

To conclude, the proposed approach – combining local sourcing, minimal processing, and mild preservation – significantly reduces carbon footprint and enhances protein functionality for meat substitute production. However, to validate its technological feasibility, further work is required to optimize processing for higher conductivity liquids (Chapter 6), to understand batch-to-batch variability (Chapter 2), and to assess potential adverse effects of increased protein concentration on inactivation level (Chapter 6). Overall, this thesis advanced the development of plant-based protein ingredients, bridging sustainability and functionality through innovative use of PEF technology.