Available at: https://digitalcommons.calpoly.edu/theses/473
Date of Award
MS in Agriculture - Dairy Products Technology
Probiotic bacteria are increasingly prevalent in food and nutritional products today. These remarkable microorganisms are capable of imparting exceptional health benefits on their host, including prevention of infection by pathogens and stimulation of immune system function. Their most common mode of delivery is through dairy products (e.g. yogurt), which are also one of their preferred habitats. The interactions between probiotic bacteria and dairy systems have been studied, but are still not well discerned. There is a need for better understanding of these associations, as well as those surrounding the mode of bacterial transfer from the food product to the human gastrointestinal tract. Discoveries into the optimal means of probiotic transport to the body may lead to great advancements in both the design of probiotic foods and their exploitation in the support of human health.
Much of the previous research on probiotic bacteria has explored their possible means of adherence in the intestine, as well their strengths in the promotion of human health. Studies relating to their interaction with dairy products are lacking, however, thus this work aims to elucidate some of these aspects. The primary endeavor of this thesis was to develop a technique to quantify the binding affinity of probiotic lactic acid bacteria for milk phospholipids. An additional objective was to exploit these bacteria, as well as dairy ingredients rich in bioactive molecules, in the creation of a highly nutritious food product.
In these experiments, a collection of methods were used in progression in order to arrive at a novel protocol to assess binding with excellent reproducibility and simplicity. These included various membrane blotting techniques, as well as thin-layer chromatography. Essentially, phospholipids from both animal-derived standards and milk extracts were applied to a surface (e.g. PVDF membrane), and bacteria were incubated with them to allow binding reactions. The lactic acid bacteria selected for the final assays consisted of four strains of Lactobacillus, including L. reuteri (SD2112 and T-1), L. acidophilus, and L. casei (LC-10). Their adhesion to phospholipids was detected by either colorimetric or fluorescent labeling systems. To illustrate this, the final method developed was a procedure in which bacteria fluorescently stained with acridine orange were allowed to bind to dots of PVDF membrane coated with phospholipids. The results of this study showed that lactic acid bacteria undeniably exhibit selective binding affinity for phospholipids as opposed to other lipids such as triglycerides. The bacteria demonstrated significantly greater binding for a phospholipid extract from milk as opposed to individual phospholipid standards from other sources (p<0.05). Nonetheless, adhesion to all phospholipids was substantially greater than that to triglycerides. These findings, as well as the development of this method, should prove valuable in future research regarding the associations of probiotics with dairy systems.
An additional purpose of this thesis was to design a dairy-based food product containing ingredient sources rich in milk bioactives. A gel-type product was created using primarily colostrum, buttermilk powder, and whey protein isolate, as well as selected strains of Lactobacillus. With the inclusion of immunoglobulin-rich colostrum, the product was analyzed alongside fluid milk and colostrum in order to quantify and compare these bioactive molecules. An enzyme-linked immunosorbent assay (ELISA) was used to complete this, and the results revealed concentrations that would be expected by the literature. Specifically, immunoglobulin G (IgG) was quantified by interpolation from a bovine IgG standard regression curve. The results showed that the concentration of IgG in the gel was nearly twice that of colostrum, and almost eight-fold higher than that of milk. This indicates that use of bioactive-rich substances, such as colostrum, in a food product may serve as a means of delivering more concentrated doses of bioactives than their respective ingredients.
The research completed in this thesis is significant in that it contributes a valuable method to the elucidation of bacterial binding interactions with milk components, and also demonstrates the successful application of dairy ingredients to an innovative food product high in beneficial compounds. The insight provided by these studies could encourage further work in improving the understanding of probiotic delivery and advancing the development of bioactive-rich food products.