In recent decades, there has been massive growth in consumer demand for products containing live bacterial cultures, or "probiotics", driving a $75B market. Yet, even as market share has expanded, the relative effectiveness of different probiotic products is still not fully understood. Such products require further scientific substantiation before manufacturers can claim health benefits. Few studies have been conducted on how wide-ranging and adverse conditions in the gastro-intestinal tract can influence ingested "pro-biotic" culture function and viability. This research attempts to close this knowledge gap, providing a formal method of characterizing bacterial function under various gut conditions through the identification of biomarkers that are indicative of healthy “probiotic” cultures. L. Bulgaricus, L. Acidophilus and S. thermophilus cultures were evaluated after exposure to conditions simulating major components of the gastro-intestinal tract, their protein expression analyzed and correlated with growth. Simulated colonic conditions maximized bacterial growth, while simulated gastric conditions minimized it. The validity of the experimental model was thus reinforced, as it accurately reflected previous in vivo analysis of bacterial growth in different components of the GI tract. By linking growth and protein expression, the gene, oppa1, was identified as a possible biomarker of cell growth. This gene, activated in conditions that conferred sub-standard growth relative to a positive control, seems to present a key to understanding bacterial population health. This research presents a step forward in the evaluation of the quality of various “probiotic” products by understanding the influence of the human digestive system on live cultures.

Separation of proteins using metal ligand complexes is a well-established practice in the field of bioengineering and biochemistry.  electrospray ionization mass spectrometry (ESI-MS) can be used to identify bio and organic molecules. Previous studies have detected chelated metal ions using ESI-MS, this project focuses on the identification of a metal ligand complex comprised of a tridentate chelating agent Iminodiacetic acid (IDA) and a bidentate ligand, Histidine. By coordinating copper with IDA a binary complex is formed, this allows for the detection of copper by proxy of the IDA. By selecting a unique fragment related to IDA we can target in tandem mass spectrometry (MS/MS) for greater sensitivity; the complex can be selected for analysis out of solution. We will use this novel approach to build a parent-ion scanning technique to monitor metal-ligand complexes extracted from environmental matrices. first the ideal solution parameters are determined to maximize the complex formation and detection of the Cu-IDA complex. So far, a high ratio of copper to IDA coupled with a basic buffer have yielded the best data. Creating a ternary complex comprised of copper IDA and an imidazole ring containing compound, histidine. Selectively tuning to the peaks associated with the copper IDA complex, the ternary complexes can be selected for in depth analysis of its structure and bonding properties. Future work could focus on identification of metal ligand complexes from soil samples with other compounds containing imidazole rings such as the neonicotinoid imidacloprid which has been indicated in bee colony collapse. By chelating solid with IDA complex formed from pentacoordinate copper ions could be detected despite the low relative concentration.

Probiotics are living organisms that when ingested have been linked with health benefits to the gut and improvement of conditions such as irritable bowel syndrome (IBS). The gut contains a plethora of microorganism populations that make up the microbiota. To understand how these populations communicate with each other, and the tissues surrounding them, it is imperative to identify and characterize the method of communication. Extracellular vesicles are one such possible method. Extracellular vesicles (EVs) are lipid-bilayer delineated sacks of material secreted from cells. It has been established that EVs are used as a waste disposal system. However, new research revealed that EVs can be used by the cell for methods of communication. Furthermore, EVs are now being linked to cell-to-cell and cell-to-organism communication. If EVs have been linked to communication, then characterizing them is one step closer to understanding how probiotic bacteria function. Previous studies have mainly characterized EVs by their size and divided them into 3 main groups: exosomes (40-150 nm), microvesicles (100-1000 nm), and apoptic bodies (>2000 nm). However, an analysis of proteins found in these EVs has not been performed yet. Here we compare EV proteins to proteins in the cell, to determine which protein fractions are secreted by cells through vesiculation for signaling purposes. To separate the cellular fraction from the EVs fraction, cell suspensions were centrifuged. First, the cells were pelleted and collected at 300x g. The leftover supernatant was spun at 16,000x g to pellet the EVs. Then, proteins from cells and EVs were solubilized and digested with trypsin. The tryptic peptides will be analyzed using liquid chromatography-mass spectrometry. A comparison of proteins in cells and EVs, and their relative concentrations can help us learn more about how probiotic EVs function in the gut.

Probiotics are found in numerous fermented foods such as yogurt, sauerkraut, and kombucha. Research shows that eating live probiotics are beneficial for the gut because it helps supplement the plethora of native bacteria. Now, these microorganisms are gaining popularity throughout the world—people are ingesting them in the form of food, drink, and even pills. Most probiotics are anaerobic. In fact, excess oxygen can damage organelles, create ionic imbalance and eventually even kill the cells. However, in the process of manufacturing these fermented foods, the probiotics in them often get exposed to oxygen, for example, due to leaks in packaging. Even the FDA does not have a rule about standard manufacturing processes regarding anaerobic conditions and yogurt. This could mean that the probiotics people eat are highly compromised: damaged or dead. I hypothesize that probiotics exposed to aerobic environments for extended periods of time will express more DNA repair enzymes such as DNA pol. 1 and 2, p53 or photolyase. This is because oxygen will cause probiotic cells to change function, and therefore, they will start to express enzymes, such as these DNA repair enzymes, to protect them from oxidative damage. In order to test this, I left probiotics out to oxidize for various amounts of time—0 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, and 1 hour. After oxidizing, I cultured the cells for 16 hours. I then pelleted the cells by centrifugation at 300 x g and washed and lysed them. Then, I tryptically digested cell proteins and analyzed them by LCMS (Liquid Chromatography Mass Spectrometry) for identification. In the future, these proteins can provide insights to how these oxidized probiotics impact the gut. Are they really the miracle microorganisms that benefit the gut, or could their compromised condition end up being harmful?