An investigation into the use of stress to improve the survivability and robustness of Lactobacillus plantarum B21
Creators: name: Parlindungan, Elvina
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 300604
subject: Food packaging, preservation and processing
classificationCode: 310799
subject: Microbiology not elsewhere classified

description: Food waste is a global challenge with significant environmental, economic and social impacts. Around one billion tonnes of food are wasted each year worldwide. This costs the global economy around USD 940 billion (US$20 billion in Australia), consumes nearly a quarter of the water used in agriculture, and produces 8% of global greenhouse gas emissions. In a world where many people don¿t have enough food this amount of waste is unsustainable. Spoilage due to microbial contamination is a major cause of food waste and can also contribute to gastrointestinal diseases and associated health costs. The use of chemical preservatives has become increasingly unpopular with the public due to their perceived health risks so there is an increasing need to develop natural, biopreservatives. A group of peptides, collectively known as bacteriocins, have significant potential in this regard as some can control a wide range of pathogenic and spoilage bacteria. Elucidating the conditions needed to grow the bacteria that produce bacteriocin would have a significant impact on reducing food waste and improving food safety and quality and is the main aim of this PhD.<br><br>At present, nisin is the only bacteriocin licenced for use as a food preservative. This compound took ~30 years to be commercialized since its discovery but the global nisin market is expected to reach USD 545.5 million by 2020. However, nisin has some significant limitations, in terms of its low solubility in water and the fact that it is not pH or temperature stable. This makes it unsuitable for use in the many food products that have a high pH and/or are heated during production. Lactobacillus plantarum B21, originally isolated from a Vietnamese fermented meat product (and classified as safe by the US Food and Drug Agency) produces another antimicrobial bacteriocin known as plantacyclin B21AG. This peptide is pH and temperature stable and effective against many species, including lactic acid bacteria (commonly associated with spoilage), and known pathogens such as Listeria and Clostridium. This thesis proposed that plantacyclin B21AG had high potential for future industrial applications as a natural biopreservative but that more research was needed on the biology of both the bacteriocin and the L. plantarum B21 that produces it. <br><br>For effective functional food technology applications bacteria need to survive the harsh conditions encountered during production and manufacturing. These can include heat, lack of water and changes in pH levels. A significant drop in a bacteria¿s viability and ability to produce useful compounds may occur due to their sensitivity and intolerance to stress and this is an important factor to consider for industrial processing of any bacterial species. The aims of my project were therefore to:<br>i. discover the growth conditions that promote the secretion of plantacyclin B21AG,<br>ii. investigate the conditions which retain or enhance the robustness and stability of the B21 strain while retaining its ability to secrete bacteriocin,<br>iii. characterize the structure of the B21 bacteriocin and fully assess its antibacterial properties. <br><br>To achieve these aims a multi-disciplinary approach was utilised, as outlined below.<br><br>a. Multiple culturing experiments were undertaken to find the optimum conditions for bacterial growth and bacteriocin production.<br>b. Well diffusion assays were used to assess the effectiveness of the B21 bacteriocin as per industry standard assessment.<br>c. Scanning and transmission electron microscopy were used to observe changes in the ultrastructure of L. plantarum B21 under different conditions, and to investigate the mechanism of action of plantacyclin B21AG against target bacteria.<br>d. Spray drying was utilized to mimic industrial processing conditions and allow microencapsulation of the L. plantarum B21 into a dry, powdered form. This was used to test the bacteria¿s robustness and stability at different temperatures for extended periods of time (8 weeks) in a form suitable for use in food.<br>e. Biochemical analysis using nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS) in conjunction with advanced multivariate analysis was utilized to understand the biochemical changes occurring at the cellular level during all experiments.<br><br>Important findings from my PhD are outlined below.<br>a. Nutrient stress, particularly the lack of a major carbohydrate source was found to promote both bacteriocin production and long-term robustness in L. plantarum B21 cultures.<br>b. Well diffusion assays showed B21 bacteriocin (plantacyclin B21AG) had high antimicrobial efficacy while electron microscopy revealed it worked by damaging the cell walls of target organisms, allowing their cell contents to leak out.<br>c. L. plantarum B21 survivability remained high and stable when stored for 8 weeks under ambient temperatures after spray drying, which is not only the industrially preferred method of drying, but has the added benefit of being cheaper, time-efficient and more environmentally friendly than the other commonly used method of freeze drying.<br>d. Biochemical analysis unravelled the metabolic changes that contributed to the B21 strain's resistance and tolerance to stress and also generated possible useful general biomarkers of bacterial robustness, including elevated levels of alanine, glutamic acid, aspartic acid, valine, proline, and norleucine. In addition, the modulation of metabolic responses through stress was identified as a viable strategy to reroute bacterial metabolic pathways to produce a range of desirable compounds (e.g. lactate) for high-scale industrial production.<br><br>Developing new antimicrobial agents that are stable during processing and storage is crucial if we are to reduce the billions of dollars lost annually each year to food waste. The findings from this research will be highly useful in this regard and this highly translatable to industry. Given its high efficacy, plantacyclin B21AG may also have a role to play as in the fight against the increasing resistance to antibiotics and other antimicrobial agents currently being seen in clinical settings. In addition, it was recently discovered that bacteriocins could be used as an adjuvant or even as a therapeutic agent to fight against various diseases, including irritable bowel syndrome, and chronic infections. This project therefore has a very high potential for impact by improving food safety, reducing food spoilage, (thus saving billions of dollars) as well as, potentially treating diseases and saving lives.