The Pennsylvania State University The Graduate School College of Agricultural Sciences A STUDY ON THE EFFECT OF DELIVERY VEHICLES ON THE EFFICACY OF BIFIDOBACTERIUM ANIMALIS SUBSP. LACTIS BB-12 IN HUMANS A Dissertation in Food Science by Zhaoyong Ba © 2016 Zhaoyong Ba Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2016 The dissertation of Zhaoyong Ba was reviewed and approved* by the following: Robert F. Roberts Department Head of Food Science Professor of Food Science Dissertation Advisor Chair of Committee Ryan J. Elias Frederik Sr. and Faith E. Rasmussen Professor of Food Science Associate Professor of Food Science Rodolphe Barrangou Adjunct Professor of Food Science Associate Professor of Food, Bioprocessing and Nutrition Sciences North Carolina State University Penny M. Kris-Etherton Distinguished Professor of Nutrition James L. Rosenberger Director of SCC and Online Programs Professor of Statistics *Signatures are on file in the Graduate School Abstract Bifidobacterium animalis subsp. lactis BB-12 (BB-12) is a probiotic strain that has been used as a food ingredient and food supplement worldwide since 1985 with well- documented health benefits. It has been commonly delivered in fermented dairy products for perceived health benefits including facilitating bowel transit, increasing gut short chain fatty acid (SCFA) production, and modifying gut microbiota (increase the ratio of potentially beneficial to harmful microbes). In addition to traditional fermented dairy- based probiotic-containing products, many new types of probiotic-containing products such as juice-based, chocolate-based, and capsules have been developed. While these provide more options for people to access probiotics, little research has been done on the effect of delivery matrix (dairy vs. non-dairy) on the efficacy of BB-12 in humans. In addition, it was not clear how yogurt fermentation might impact the survival of BB-12 in the product or on its performance in vivo. In order to address these questions, a randomized clinical trial was conducted using BB-12 delivered by capsule (CAP) or yogurt smoothies. The first step in the project was to identify a selective medium for the enumeration of BB-12 in yogurt product where high concentration of live cultures Lactobacillus delbrueckii subsp. bulgaricus (LB) and Streptococcus thermophilus (ST) are present. After evaluation of different selective media, the MRS-NNLP agar was found to effectively inhibit growth of ST and LB, and was subsequently chosen for BB-12 enumeration. Then three yogurt smoothies: 1) a control yogurt smoothie without BB-12 (YS), 2) a yogurt smoothie with BB-12 added pre-fermentation (PRE), or 3) post- iii fermentation (POST), were developed. Compositions (fat and total solids) of the yogurt smoothies were measured following each production. The concentration of BB-12, ST, LB, and pH of the products were measured weekly during shelf life. Data from 27 batches indicated the BB-12 population in the yogurt smoothies was stable during shelf life and comparable within and across batches. The population of BB-12 declined in both PRE and POST throughout shelf life and was found to decrease faster in the POST drink as compared to the PRE drink. The BB-12 remained at the specified dose level (log10 10 ± 0.5 CFU per/serving) throughout shelf life of the products. The second objective was to evaluate the effect of BB-12 interventions on the gut transit time and fecal SCFA production in healthy adults. A total of 36 healthy adults aged 18-40 years with a BMI of 20-35 kg/m2 were recruited in this controlled, 4-period crossover, free-living study. Participants received each of the 4 treatments in a random order. When consuming yogurt smoothies, participants consumed 240 g of yogurt smoothie per day or on the capsule treatment, they consumed a probiotic capsule per day, receiving a dose of log 10 ± 0.5 CFU of BB-12/day. Dietary intake of total calories and various nutrients were assessed via self-reported 3-day dietary recall. Gut transit times were measured at baseline using the blue dye and a telemetry device known as a SmartPill®, and after each treatment using SmartPill® only. Stool and blood samples were collected at baseline and after each treatment. Fecal SCFAs were extracted using ethyl acetate and analyzed by gas chromatography (GC). A significant positive correlation (Spearman rho = 0.67, P < 0.0001) was observed between blue dye and SmartPill® transit times, suggesting that the blue dye method remained a reliable cost-efficient approach for iv baseline screening for whole gut transit time (WGTT). No significant treatment effect was observed on either GTT or fecal SCFAs. However, this study is the first to demonstrate the possible relationships among regional gut transit times and fecal SCFAs in healthy adults, and the results from this study confirmed a number of correlations that have been reported previously. Notably, the predominant SCFAs negatively correlated with WGTT, colonic transit time (CTT), and gastric emptying time (GET), but had little to do with small bowel transit time (SBTT). The third objective was to survey the gut microbiota of participants before and after different BB-12 interventions. Bacterial genomic DNA was isolated from fecal samples using the MOBIO PowerSoil DNA isolation kit according to the manufacturer’s protocol with modifications. The DNA samples were amplified for the V4 region of the 16S rRNA gene. Amplicons were then sequenced at the DNA Technologies Core Facility of the University of California, Davis on an Illumina® Genome Analyzer II sequencing platform. After removing samples with low quality and poor compliances, about 2.4 million sequences from 147 stool samples were analyzed using QIIME. Overall, 10 phyla and 109 genera were identified in the participants. Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria accounted for > 98% of the sequences at the phylum level. No significant treatment effect on the gut microbiota was detected due to the large interpersonal and intrapersonal variations observed except that yogurt interventions resulted in a higher relative abundance of Streptococcus than the capsule treatment. A significant gender effect was observed when comparing the gut microbiota of the present study cohort. v In an effort to understand the relationships between gut microbiota and host immune and metabolic responses, the beta-diversity results were grouped by parameters including sex, body mass index (BMI), blood pressure, glucose, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides (TGs), tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), and etc., a number of statistically significant differences were found. However, further studies are needed to validate these relationships, because either only a small percentage of difference could be explained by the grouping, or only a few data points were in one of the two arms. To further explore the effect of the interventions on the relative abundance of members of Bifidobacterium genus, DNA samples were analyzed using bifidobacteria specific terminal restriction fragment length polymorphism (Bif-TRFLP). Interestingly, the two BB-12-containing yogurt smoothies (PRE and POST) resulted in significantly higher percentage of B. animalis when compared to baseline, to the BB-12-free yogurt smoothie (YS), to the BB-12 containing capsule (CAP), and after final washout. In conclusion, the BB-12 survived well in yogurt smoothies throughout shelf life (< 1 log decrease) and survived better in the pre-added treatment than in the post-added product perhaps due to BB-12’s adaptation to the acidic environment during fermentation. Little treatment effect was observed on either GTT or fecal SCFAs. The predominant SCFAs negatively correlated with regional transit times except for SBTT. Small treatment effect on the gut microbiota was detected while gender effect was found to be significant in the present study cohort. BB-12 containing yogurt smoothies resulted in higher relative abundance of B. animalis than BB-12 containing capsule. vi Table of Contents List of Figures ................................................................................................................... xi List of Tables .................................................................................................................. xiii List of Abbreviations ..................................................................................................... xiv Acknowledgements ........................................................................................................ xix Chapter 1 - Literature Review ......................................................................................... 1 1.1. Probiotics ................................................................................................................. 1 1.2. The Genus Bifidobacterium ................................................................................... 2 1.3. Bifdobacteriam animalis subsp. lactis .................................................................... 4 1.4. Bifidobacterium animalis ssp. lactis BB-12 ........................................................... 5 1.5. Probiotic Effects of BB-12 ..................................................................................... 6 1.5.1. BB-12 and GI Health ......................................................................................... 7 1.5.2. BB-12 and Immune Health .............................................................................