Effects of In-Situ Produced Exopolysaccharides on Techno-Functional Properties of Fermented Lupin Flour
Creators: name: Kurukulasooriya Senanayakage, Dhananga
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 300607
subject: Food technology

description: The increasing global demand for alternative protein sources is driven by concerns over resource scarcity, sustainability, and cost-effectiveness. Legumes have emerged as a promising option, but their techno-functional, sensory, and nutritional limitations pose challenges for their use in the food industry. Microbial fermentation is one of the food processing technologies with the potential to overcome these challenges. The integration of microbial fermentation with exopolysaccharide (EPS) producing lactic acid bacterial (LAB) strains greatly enhances the ability to address these limitations effectively. 

Among legumes, lupin (Lupinus spp.) stands out for its high protein content (34-44%), which is comparable to soybeans. Additionally, lupin is rich in dietary fibre and contains various bioactive compounds, including antioxidants, which contribute to its health-promoting properties. Despite these advantages, lupin remains underutilised as a human food due to its techno-functional limitations such as poor solubility, inferior foaming, emulsification, and rheological properties. As a result, the applications of lupin in the food industry so far are limited. 

In the realm of legume fermentation, significant attention has been devoted to crops such as soybeans, chickpeas, and peas. Extensive research has been conducted on these legumes, including studies on EPS-producing LAB and their role in overcoming limitations associated with legumes, including techno-functional constraints. However, there is a critical lack of research related to lupin fermentation in general. Fermentation of lupin with EPS-producing LAB strains is exceedingly rare to our knowledge. Therefore, it is crucial to investigate this underexplored domain to harness the potential of lupin and enhance its industrial applications, with a specific focus on addressing techno-functional limitations. 

With this objective, this research sought to enhance the techno-functional properties of lupin through the application of EPS-producing LAB. Initially, 26 novel LAB strains, including Lactiplantibacillus plantarum and Leuconostoc mesenteroides, were assessed for their genetic potential to produce EPS using a customised bioinformatics pipeline, which incorporates a specialised database for bacterial EPS, various bioinformatics tools and steps. The strains L. mesenteroides C13, C18, and C20 exhibited the highest genetic potential for EPS production, containing 50, 51, and 39 EPS production-related genes, respectively. This genetic potential was confirmed experimentally through EPS quantification using MRS broth as a model medium. These three strains were the highest EPS producers among all the strains tested, with EPS production levels of 1.67 mg/mL, 1.57 mg/mL, and 0.82 mg/mL. Furthermore, it was found that these strains predominantly possessed genes for the extracellular EPS biosynthesis pathway, suggesting the production of dextransucrase and levansucrase by these strains. They did not possess genes related to the synthase-dependent EPS producing pathway but did have certain genes related to the ABC-transporter dependent pathway and the wzx/wzy-dependent pathway. 

Based on previous research indicating that the application of abiotic stress on LAB can enhance EPS production, the second experimental chapter investigated the effects of pre-stressing L. mesenteroides C13 and C18 at various pH (3.0, 4.5, and 6.0) and temperatures (10 °C, 25 °C, and 40 °C) levels on their EPS production, proteomics, and transcriptomics profiles. Pre-stressing at pH 3.0 resulted in a notable reduction in colony-forming units but increased EPS production per cell. Proteomics identified key proteins related to EPS production and stress response, including dextransucrase, levansucrase, sucrose phosphorylase, biofilm regulatory proteins, cold shock-like proteins, and universal stress proteins, which were upregulated on per cell basis in response to acid stress (pH 3.0). 

Based on these findings, L. mesenteroides C18 was selected for use as a starter culture in experimental trials to assess the feasibility of fermentation using EPS producing LAB for improving the techno-functional properties of lupin. The experimental design included pre-stressing the starter cultures at pH 3.0 (25 °C for 2 h in MRS broth), followed by fermentation for 24 h at 25 °C. Lupin suspension (20 %) was used as fermentation substrate. Fermentation using unstressed culture (pH 6.0, 25 °C for 2 h in MRS broth) was conducted under the same condition for comparison. Unfermented and acidified unfermented samples were used as controls. While pre-stressing did not significantly increase the overall EPS production compared to unstressed fermentation, it did enhance EPS production per cell. Fermentation, particularly using non-stressed cultures significantly improved the viscosity, and increased foaming, water and oil absorption, emulsification, and protein solubility of lupin suspensions compared to the unfermented treatments. This enhancement indicates a correlation between EPS production and improvement in these techno-functional attributes. Therefore, the future research should focus on optimising fermentation conditions to maximise EPS yield under unstressed pH conditions and temperature (pH 6.0, 25 °C) and exploring other stress types such as osmotic, oxidative, and nutrient stress to further enhance lupin's techno-functional properties. 

The work produced from this thesis will extend the understanding of lupin's techno-functional properties and how they can be enhanced under different pH and temperature conditions. Additionally, the bioinformatics pipeline developed in this study can be used with other microorganisms to identify the presence of various genes of interest. This research also provides valuable insights into the EPS-producing pathways in novel strains tested.