Structural and functional properties of probiotic strains as affected by multi-stress adaptation process and subsequent freezing

Abstract

Consumers have become more aware of the importance of consuming foods and products that boost health. This is one of the reasons why the probiotic industry has been booming for the past several decades. Clinical studies have shown that when consumed in sufficient numbers, probiotics are capable of exerting certain beneficial characteristics on the host. However, their shortfall is their sensitivity to environmental stress factors, which alter their physiological state and thereby hinder their viability and functionality. The reality is, probiotics must endure various technological and gastrointestinal (GIT) stress factors before they arrive at their active site in the intestines. This therefore means that robust strains capable of withstanding threats to viability and functionality during production and after ingestion are a must. Moreover, understanding which food matrices facilitate viability during storage and in the conditions of the GIT can help in developing probiotic products that deliver on their claims. Much research has been conducted focused on producing stress tolerant probiotic strains. For example, by exposing cells to a sub-lethal dose of stress, this produces a stress response that later allows them to survive a more lethal dose of the same stress, a process known as stress adaptation. Adaptation to one stress is known to provide protection against other stresses, this is called cross protection. Research has also shown that adapting cells to multiple stress factors is better than adaptation to just one. Strains that undergo this type of manipulation must be assessed once more for retention of their probiotic properties. Taking that into consideration, the current study aimed to determine whether long term storage of multi-stress adapted (acid, bile and temperature) strains altered their functional and structural properties when compared to non-adapted and freshly adapted cells. In the first experimental chapter, five probiotic strains (Bifidobacterium bifidum LMG 11041, Bifidobacterium. longum LMG 13197, Bifidobacterium longum Bb46, Lactobacillus acidophilus LA14 150B and Lactobacillus plantarum) were sequentially adapted to acid, bile and temperature. The results show that after exposure to each stress factor, the strains not only survived better, but were capable of proliferating. Investigation into acid and bile tolerance showed insignificant differences in the strains’ ability to withstand acidic conditions p>0.05 However, bile resistance was better for the non-adapted cells compared to their adapted counterparts. Results from bile salt hydrolase (BSH) assay showed that only freshly-adapted cells and non-adapted L. acidophilus could hydrolyse bile salts. However, this did not enable these cells to survive bile exposure better than the cells that tested negative for BSH. The bile resistance was therefore attributed to other stress response genes. The freshly-adapted cells could, however, be beneficial for reducing serum blood cholesterol, which has been linked to BSH activity. The tests for antimicrobial activity showed that inhibition by old-adapted L. plantarum was significantly lower than non- and freshly adapted counterparts (p<0.05). The antibiotic sensitivity profile of the cells remained largely unchanged except for B. longum Bb46 and L. plantarum, which developed sensitivity following fresh adaptation and long-term storage. Additionally, the auto-aggregation percentages were reduced by the fresh stress adaptation as well as long term storage. Moreover, scanning electron microscopy (SEM) revealed that cold storage following stress adaptation changed the morphology of strains and there were significant changes that occurred in cell surface hydrophobicity (CSH) of the strains as a result of fresh adaptation and long term storage (p<0.05). Auto-aggregation, cell morphology and CSH are properties linked to adhesion to epithelial cells. It is likely therefore that adherence would also be affected by stress adaptation and subsequent cold storage. Since the alteration of probiotic properties by long-term storage was unique for each strain studies that look at each strain individually were necessary. Furthermore, in many cases foods are the vehicles used to deliver probiotic products to the body. So, investigating how different food matrices affect survival of probiotics in the product and in GIT conditions is an important aspect to consider. Multi-stress adapted L. plantarum was used for the second experimental chapter of the study. Its survival during storage in yoghurt, carrot and cranberry juice was determined, followed by assessing its GIT survival within the same foods. A decline in viability was observed for all cells in all three foods by the end of storage. This was attributed to be as a result of the low pH maintained by these foods throughout storage. Both adapted cultures survived better in the foods compared to non-adapted cells. However, in simulated GIT, the survival of L. plantarum was better for freshly adapted, followed by old-adapted and lowest for non-adapted cells. It was evident that process of adaptation improved stability of the cells in the foods during storage but the results also show that the long-term cold storage negatively affected viability in simulated GIT conditions. In terms of the matrices themselves carrot juice had the highest number of surviving cells, followed by yoghurt and cranberry juice had the least numbers. To our knowledge there has been no work done looking at how stress-adaptation of probiotics is affected by storage over extended periods of time. The results demonstrate how particular probiotic properties are changed in certain strains following long term storage. This had further implications for L. plantarum in simulated GIT conditions in which survival was lowered in the old-adapted cells. This study is relevant for both the probiotic food industry and consumers alike. The study showed how storage can negatively affect the properties of certain strains. Therefore, industries that use pre-stress treatment as a means to boost viability should explore different methods of storage that do not disrupt functionality and viability. Furthermore, these industries should incorporate the probiotic strains in food matrices that facilitate the survival of probiotics. As this study showed, carrot juice resulted in the highest number of surviving cells after storage. This information would also be useful to consumers as they would be able to make informed decisions about which probiotic foods will deliver the highest number of cells. This also benefits the probiotic manufacturers because satisfied customers will drive the continued growth of the industry.