Studies on the stability of probiotic bacteria during long term storage : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand

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According to the Food and Agriculture Organization (FAO) and the World Health Organization (WHO), probiotics are defined as ‘‘live microorganisms which when administered in adequate amounts confer a health benefit for the host’’ (FAO/WHO, 2001). Lactobacilli and bifidobacteria are two major group of organisms considered to have probiotic properties. The primary objective of this project was to develop a novel stabilization technology for probiotic bacteria, through which a range of probiotic bacterial strains could potentially be delivered to the host through shelf stable dry and intermediate moisture foods. For preliminary experiments (reported in Chapter 4.0), Lactobacillus casei 431, a commercial strain from Chr Hansen, Denmark, was chosen as the experimental strain and milk powders (both skimmed and full-fat) were chosen as the principal supporting agent while stabilizing the bacterial cells. Stabilization efficiency in terms of long term ambient temperature storage viability was compared using freeze and fluidized bed drying techniques. Fluidized bed drying was able to retain 2.5 log cfu/g higher viability after 52 weeks of storage at 25 °C. A combination of fluidized bed drying and osmotic stress adaptation to the probiotic cells yielded further improvement of 0.83 log cfu/g higher viability compared to the unstressed cells. The findings were validated with other two lactobacilli and two bifidobacterium strains with probiotic characteristics and significant improvements in storage stability over freeze-dried samples were observed. Fortification of vitamin E in the stabilization matrix as an antioxidant improved the stability by 0.18 log cfu/g during 20 weeks storage period at 25 °C, whereas any similar benefit of fortifying inulin as a prebiotic was not observed. Incubation in simulated gastric fluid and intestinal fluid (in vitro) revealed that the L. casei 431 cells were better protected within the stabilized matrix than in the free form. The survival of the stabilized cells were 5.0 and 2.1 log cycles higher than free cells in gastric juice and bile salt solution respectively. Physical characterization of the probiotic ingredient showed very good flow-ability and solubility, with 470 Kg/m3 bulk density, water activity of 0.27 and agglomerated particles of 125.6 μm mean diameter. Thereafter, the project aimed to understand the underlying mechanism of the processes responsible for gradual decay in cell viability of another probiotic strain (Lactobacillus reuteri LR6) during long term storage at 37 °C (Chapter 5.0 onwards). Vacuum drying of sorbitol- or xylitol-coated Lactobacillus reuteri LR6 cells and fluidized bed drying of the same coated cells with different excipients were compared for the cell viability post drying. LR6 cells coated with xylitol and desiccated in unsupported form or together with skim milk powder as an excipient were found to be better protected when exposed to moderate as well as high drying temperatures. In Chapter 6.0, a closer examination of the protein and polypeptide components of the cell envelopes (amide regions) via Fourier transform infrared spectroscopy revealed different degrees of structural deformation in individual samples, which correlated well with the residual cell viability. It was also important to understand the underlying mechanisms responsible for the loss of viability of stabilized probiotic cells when stored at non-refrigerated temperatures. In Chapter 7.0, the stabilized Lactobacillus reuteri LR6 cells were stored at 37 °C and at two water activity (aw) levels. Superior storage stability was recorded in a lower aw environment, supported by a stronger glassy matrix when skim milk powder was used as the excipient. Fourier transform infrared spectroscopic examination of the cell envelopes revealed substantial dissimilarities between samples at the beginning and at the end of the storage period. In milk powder-based matrices, adjusting the aw to 0.30 resulted in a weaker or no glassy state whereas the same matrices had a high glass transition temperature at aw 0.11. This strong glassy matrix and low aw combination was found to enhance the bacterial stability at the storage temperature of 37 °C. During storage of the stabilized cells for 121 days at 37 °C, the measured Tg for all the samples was slightly lower than what was recorded at the beginning. Scanning electron microscopy revealed the formation of corrugated surfaces and blister-type deformations on the cell envelopes during the stabilization process whereas the freshly harvested cells were found to be with a smooth surface and undamaged membrane. Inspection of the cell bodies via transmission electron microscopy showed freshly harvested cells with normal shapes with no damage in the inner membrane structure. An almost intact but slightly waved outer membrane structure was observed. The findings emphasize the importance of protecting the integrity of the membrane of probiotic cells by using suitable protecting agents to enhance their stability during long term storage. The stabilized cell matrix samples were segregated into 4 groups based on the average particle diameter by passing through sieves of different mesh sizes. The degree of agglomeration had a very important role in offering physical protections to the LR6 cells during the desiccation process. The viable cell populations in the higher particle size groups (above 500μm and 1000μm) were between 9.5 to 9.9 log cfu/g whereas the same for the lower particle size (below 500μm but above 250μm) group was only 7.8 log cfu/g. The minimum viable cell concentration was recorded (7.3 log cfu/g) in the finer particles having less than 250μm diameter but having the maximum mass fraction. In case of stored samples, it was found that the bacterial cells adhered to the finest particles suffered the maximum loss in viability (41.4%) whereas the minimum loss (14.9%) was within the particles with average diameter above 500μm. In order to assess the effect of stabilization and storage (12 weeks, 37 °C) on the common probiotic attributes of the LR6 cells, an in vitro study on acid, bile salts tolerance and surface hydrophobicity was conducted. The results showed considerable reductions in cell viability for the desiccated as well as stored cells when incubated in simulated gastric (acid tolerance) and intestinal (bile salts tolerance) environments. A coating of xylitol over the cell bodies during desiccation was found to be marginally protective against these stresses. High aw storage was found to be more detrimental to the cells in terms of their ability to survive in the acid or bile environments. The cell surface hydrophobicity towards various hydrocarbons was also found to be adversely affected due to desiccation and non-refrigerated storage. Considerable degradation in hydrophobicity was found to be occurring in the cells stored at aw 0.30, a trend similar to the acid and bile resistance properties.
Listed in 2019 Dean's List of Exceptional Theses
The following Figures and Tables were removed for copyright reasons but they may be accessed via their sources: Figures 2.1. (=Holtzapfel et al., 1998, Fig 2); 2.4 (=Garcia, 2011, Fig 2); 2.5 (=Santivarangkna et al., 2007, Fig 1); 2.6 (=Gardiner et al., 2000, Fig 2); 2.7 (=Mugnier & Jung, 1985, Fig 5). Tables 2.1 (Williams, 2010, Table 1); 2.3 (Linders et al., 1997, Table 1).
Lactobacillus, Dried milk, Microbiology, Probiotics, Dean's List of Exceptional Theses