Stabilisation of dried Lactobacillus rhamnosus against temperature-related storage stresses : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Manawatū, New Zealand

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In the past few years, research has established a link between gut health and overall health and wellbeing. A diverse microbiome is a major step towards a healthy gut. Probiotics could help by improving the gut microbiome diversity and thus, are being added to a wide range of food products. However, maintaining them in a viable state within these food products is a considerable challenge. In order to increase the shelf-life of probiotics, numerous encapsulation systems have been developed to help protect them. Techniques such as emulsification, coacervation, or drying methods have all been employed with varying levels of success. While the final encapsulated bacteria may have enhanced protection and stability, a range of stresses are imposed on the bacterial cells during the actual encapsulation process, including mechanical, physical and chemical. Drying is the technique that confers the most protection to the probiotics, potentially stabilising them for up to several years. However, water plays a structural role and upon its removal, forces appear between cell components leading to the denaturation of proteins or the phase transition of the phospholipids membrane. Thus, bacterial cells need to be dried in the presence protectants that can prevent detrimental events from occurring and damaging the cells. It is thought that there are three main mechanisms by which protectants will confer superior stability. Firstly, the protective matrix can form a glassy system preventing further chemical reactions from happening, and thus protecting the bacteria. Secondly, if protectants are introduced for a period prior to drying, they can interact with the cellular biomolecules, replacing the structural role of the water, and maintaining the biomolecules in their native state when the water is removed from the system. Finally, the protectants can increase the free energy of water, maintaining it in the vicinity of the biomolecules, so that when the water is removed, the biomolecules are still hydrated and in their native state. Therefore, it is obvious that the role of protectants during the drying step is critical. The question that has remained largely unanswered, however, is how long and under what conditions should the protectants be introduced, and what type of protectants work best? Once the probiotics are successfully dehydrated, storage stresses may impair their stability on the shelf. Among these stresses, high temperatures of the surrounding environment is one that has been well documented to be detrimental to the cells and generally leads to a rapid drop in shelf stability. These temperatures can be experienced not only during the life of the product on the supermarket shelves, but also during transport of these consumables around the globe. The effect of changes in temperature on bacterial cell viability is an area which has not been explored in great depth, and the impact that encapsulation may have on the viability under these conditions even less so. Once again, like in the case of the protectants, the materials used to encapsulate the bacteria will be critical to final stability. Materials such as ‘phase change materials’ (PCM), which can absorb and release heat over different temperature ranges could be the key to protecting bacteria under extreme conditions. The aim of this thesis was thus to stabilise a model probiotic: Lactobacillus rhamnosus HN001 to high temperatures occurring during storage and transport. In order to do so, the study was separated into four principal research questions. Firstly, can a pre-drying step (for example the uptake of protectants) help the stability/viability of the bacteria during storage? Secondly, what are the best protectants for long-term storage of Lb. rhamnosus HN001, and why? Thirdly, is it possible that combinations of the most suitable protectants act in synergy, bringing increased storage stability compared to either protectant on its own? Finally, can the inclusion of PCM in the encapsulation matrix give extra protection to the cells during storage? This question would be of particular significance when examining the effect of the fluctuating temperatures experienced during the transport of the probiotics. The first study, therefore, consisted of establishing a protocol to prepare the cells for drying, by finding the early stationary phase where cells are known to be most stable to stress, and then optimising the exposure of the cells to potentially protective solutions of glucose and sucrose at 4 and 20°C. The uptake of the solutes was explored using HPLC, before drying the cells and evaluating the effect that their uptake had on the shelf-life stability of freeze-dried cells. In order to try and understand any interactions between the intracellular biomolecules and the protectants, the Nano DSC was used. Results showed that when cells were exposed to glucose at 20°C, metabolisation took place, and the longer the exposure, the lower the stability of the cells after drying and over storage. Overall, the study revealed that cells exposed to sucrose at 20°C for 4 hours presented best stability indicating that both the type of protectant, and exposure settings are critical to a successful outcome. The results from the Nano DSC showed that sucrose interacted with some of the cell biomolecules, rendering them more stable. The exposure temperature for the rest of the experiments was thus set at 4°C to avoid metabolisation, and the time was set at one hour so that exposure settings would be adapted for both sugars. In the second part of the study, a range of nine protectants (glucose, fructose, galactose, sucrose, lactose, trehalose, betaine, monosodium glutamate (MSG) and sorbitol) were compared for their ability to stabilise freeze-dried Lb. rhamnosus at 30°C for 6 months. Inulin was used as a carrier. The impact of galactose, sucrose, betaine, MSG and sorbitol was studied using a Nano DSC to again try and establish links between biomolecule interaction and stability during storage. Interestingly, MSG led to the best stability overall with a cell loss of 0.19 /month, even though it had the highest water activity of all the samples following freeze-drying. This is contradictory to general thought on how water activity affects bacterial cell stability, with higher water activity generally resulting in increased cell death over time. It was shown, using the Nano DSC, that MSG interacted with most of the cell biomolecules rendering them more stable. MSG was thus selected for further study. Three additional protectants were selected (galactose, sucrose and sorbitol) to look for potential synergistic effects with MSG in terms of protecting the bacteria during storage. The study followed a mixture design of experiment (DoE) in order to obtain an optimal protective matrix. The powder structure was also studied at this point by microscopy along with analysis using the DSC to try and comprehend the importance of the powder structure on the stability of the dried cells. Multivariate analysis was used to link all factors and their relative impact on the cell death rate together. Interestingly, it was found that neither a high glass transition temperature (Tg) nor a low water activity helped to stabilise the bacteria. Instead, the amount of MSG was clearly shown to improve the shelf-life, and a synergy was found between sorbitol and MSG. Microscopy showed that this powder led to a unique structure that most likely collapsed during drying resulting in the shrinkage of the cake and the loss of the porous structure, thus lowering the exposure of the bacteria to oxygen. In addition, a small amount of the sorbitol present in the matrix seemed to help in stabilising additional biomolecules as shown by the Nano DSC. The slowest death rate results obtained were 0.04 /month when MSG alone was mixed with inulin, but the model predicted an even lower death rate due to the synergy occurring between MSG and sorbitol. Finally, this optimised stabilisation matrix was used to study the impact of further protection, in the form of an encapsulate containing a PCM, on the stability of the bacteria. Powders with two different structures were compared using freeze-drying and spray drying techniques. The viability of the resulting powders was assessed during two separate storage studies designed to test the cells against fluctuating temperatures (20 to 50°C) and at constant temperature (35°C). The results showed that PCM appeared to have little impact on the overall stability of the powder. However, it was confirmed that a dense and smooth powder structure helped to maintain the bacteria in a viable state for a longer time than a more porous structure. This was most likely due to the lower surface-area ratio decreasing the exposure with the environment and preventing detrimental reaction such as oxidation. The bacteria in the optimised stabilisation matrix had the best stability, with a death rate of 0.07 /month at 35°C and 0.18 /month under fluctuating temperature from 20 to 50°C. In conclusion, it was found that the interaction of the protectants with cells is of paramount importance in maintaining the cells in a dried, viable state for longer periods at elevated temperatures. In addition, the structure of the powder should also be considered as one of the main mechanisms for protecting the bacteria, as it has a substantial impact on the shelf-life of the powder. Conversely, in this body of work it was shown that a high glass temperature did not enhance, or indeed help to maintain cell viability as has been suggested by many previous studies. A dense structure is, however, believed to protect the bacteria through preventing exchanges with the environment, especially with oxygen. If future work is to be done, it should follow the oxidation of the cells during storage and link it with measures of the powder porosity to gain further insight into the impact of the structure on oxidation stress.
Figure A.5 is re-used under a Creative Commons Attribution 4.0 International (CC BY 4.0) license
Lactobacillus, Probiotics, Drying, Storage, Microencapsulation