Enzymatic Effects of Pancreatic Amylase Inhibition by Kombucha Tea

Kombucha is a fermented tea beverage produced through use of a symbiotic culture of bacteria and yeast (SCOBY). The purported health benefits of this drink are being assessed through the use of an assay simulating breakdown of starches in the mouth.1 Analysis with spectrophotometry at 540 nm for porcine pancreas amylase using 0.100 mg/mL reported a Km of 0.440 mg/mL and Vmax of 0.00451 mg/min. Breakdown of starches was compared to a standard using maltose for comparison.2 Kombucha inhibition using IC50 inhibition analysis with a dose response curve of dilutions determined a value of 6.70x10-3 mL. Further IC50 analysis was performed using pH adjusted Kombucha and black tea samples determined values of 7.24x10-1 mL and 6.92x10-4 mL respectively. Using the Gallic Acid Equivalence method, black tea was prepared with similar phenolic concentration to that used in Kombucha during the fermentation process. In adjusting the pH of Kombucha and black tea samples, an acidified solution demonstrated the highest inhibitory function on pancreatic amylase. This data provides relevant information related to the absorption of starches in the mouth during intake of the beverage. The slowed breakdown of carbohydrates to monosaccharides could be pertinent in the overall health and digestive effects of Kombucha during consumption.

Production of Esters by Saccharomyces cervisiae Fermentation of Different Fusel Alcohols

A reaction between fusel alcohols and Acetyl-CoA and Acyl CoA in Saccharomyces cervisiae uses ester synthase enzymes, such as alcohol acetyltransferase (AATase), to produce esters. Fusel alcohols are a mixture of many alcohols that are produced during fermentation. Both the type of fusel alcohol and strain of yeast factor into which esters will be produced during fermentation. Esters are essential to the final flavor determination of the beer and with many factors affecting their production, problems can occur in a hurry. Low ester production could cause a poor flavor profile and cost brewers money and time if the beer is unusable and cannot be sold. This research has been dor:e in an attempt to compare yeast strains with similar types of produced esters, resulting in similar flavor profiles, and then factor in other characteristics such as price. This will allow the determination of which yeast, or combination of yeasts, would be most cost effective, resistant to diseases or temperature changes, and still give the same flavor profile. Several experimental setups were constructed in attempt to efficiently produce and characterize esters. S. cerevisiae was incubated with a fusel alcohol in aerobic conditions for 12-24 hours. The alcohols and esters were then extracted with pentane for gas chromatography mass spectroscopy (GC-MS) analysis. Data analysis has shown that we cannot jump into the pathway at this point, but instead must back up and try to influence fusel alcohol production at earlier points in the pathway. Most samples have shown little to no esters that can clearly be identified from their precursor alcohol. Research is currently being done, and will continue in the future, in order to find the best experimental method for ester production. Once that is accomplished, different strains of yeast will be tested to see what esters are produced by the different strains

Ethyl Acetate Extraction of Humulus Lupulus Compounds with Characterization by Gas Chromatography-Mass Spectrometry

Hops are an essential ingredient in flavoring and preserving beer. Hops contain two main types of compounds: α-acids and β-acids, which are the essential oils. I studied these volatile compounds such as humulone, an α-acid, and myrcene and humulene, which are β-acid. Humulone, like other α-acids, gives beer a bitter flavor via isomerization. Unlike α-acids, β-acids flavors are based on which chemical compounds are present in the hops. For example, myrcene produces a floral, citrus, and piney flavor, while humulene creates a spicy, and herbal aroma. Additionally I investigated the effect on my compounds of interest during the brewing process, more specifically when and how the hops were added. The relationship between bitter and aroma is crucial for obtaining the desired taste. Ethyl Acetate is used to extract the desired volatile compounds from my hops; this allowed me to use Gas Chromatography-Mass Spectroscopy to visualize and identify the desired compounds

Determination of Methods to Separate and Analyze Small and Large Chain Isoprenoids from Mycobacterium tuberculosis

The genes responsible for the isoprenoid carrier lipids involved in cell wall biosynthesis of Mycobacterium tuberculosis (M.tb) are believed to be Rv2361c, Rv1086, and Rv0989c. These genes encode prenyltransferases catalyzing production of isoprenoids of increasing length. Rv0989c can work in tandem with Rv1086 to create precursors to decaprenyl diphosphate, the final product of Rv2361c. Rv2361c can also work by itself to produce decaprenyl diphosphate. In addition to Rv2361c, Rv1086, and Rv0989c, M.tb encodes seven total different prenyltransferases, and in other biochemical studies it has been suggested that these genes may provide redundancy with the isoprenoids that are formed. These genes can be expressed in Escherichia coli and the isoprenoids extracted through lipid extraction. The samples are then dephosphorylated using alkaline phosphatase. Once dephosphorylated these samples can be analyzed using HPLC in order to separate and analyze the isoprenoid products. HPLC separation occurs with a mobile phase of water and an increasing gradient of 60:40 isopropanol:methanol at a rate of 1 mL/min. These samples are ran over a C18 column. The HPLC allows us to view the different products that may be formed during errant metabolism when alternate isoprenoid precursors are available. Continuing work with these products will allow us to see the influence of each gene with the other genes present in M.tb in the formation of decaprenyl diphosphate

Characterization of Alternative Grains Used in the Brewing Process and Analysis of Product Parameters

In recent months, an interest has arose in whether or not gluten free beers due in fact have gluten in them. During the first part of my research, a few gluten free beers were tested using an ELIZA (EZ Gluten) test to determine if there was any gluten in the beer (this test would be used later on the grains). The results showed that the gluten free beers did in fact not have gluten in them. After testing the beers, I will utilize alternate grains (sorghum, rice, corn, quinoa) to determine if any gluten is extracted during the fermentation process. During the process, standard tests used during the brewing process will be used on the alternative grain fermentation to analyze that the grains are a plausible alternative to standard grains used (barley, malt, etc.). The tests to be run include hydrometer readings to test specific gravity, a Brix test to determine sugar content and consumption, and a total protein assay to determine the proteins extracted by the fermentation process

Investigation of Evolutionary Events in Corynebacteriaceae Involving Prenyltransferases in Corynebacterium glutamicum

Terpenoids, a class of compounds with 30,000 unique structures, are known to include antitumor compounds, pigments, vitamins, antibiotics, and flavor molecules. Prenyltransferases are the primary enzyme responsible for assembling the carbon backbone of these compounds from five carbon isoprene units. Bioinformatic analysis has indicated that an evolutionary event has resulted in the acquisition of an additional prenyltransferase in Corynebacterium glutamicum (C. glutamicum) compared to the majority of the Corynebacteriaceae. This result suggests that C. glutamicum may produce a non-essential terpenoid compound, or secondary metabolite. Sequencing has confirmed the successful cloning of the aforementioned genes. Present work includes expressing these genes in Escherichia coli, isolating the protein product, and assessing the function in vitro. This data will be used to expand our understanding of the evolutionary path of C. glutamicum compared to other members of the genus. Continuing work will shed light on the function of the third gene for C. glutamicum, and understanding the role of terpenoids in the family Corynebacteriaceae and greater Actinobacteria

Gene Encoding of Corneybacterium Glutamicum

Antibiotics have been of great scientific and medical interest since the discovery of penicillin from a fungus. The research detailed below was done in order to determine similarities between genes of interest and to express them in order to determine their characteristics. Terpenoids are a class of compounds that have been shown in the past to exhibit antibiotic qualities such as is the case with terpentecin. First, bioinformatics was performed to determine similarities in genes that commonly encode for formation of terpenoids to those known to exist in the family Actinobacteriaceae. A BLAST analysis was done against the entire family with known proteins of interest. Corynebacteriaceae jumped out as a potential candidate with a vast number of hits as well as a diverse family. Further probing was done to determine what species within Corynebacteriaceae had sufficient genomic data as well as propensity to produce secondary metabolites of interest. Six species were found to be possible contenders, of which half were found to be pathogenic. Corneybacterium glutamicum(C. glutamicum) was found to be a worthy candidate due to its complete genome being sequenced as well as its production of terpenoids. The DNA of C. glutamicum was successfully extracted and purified. The genes of interest were then successfully amplified by PCR and confirmed by gel electrophoresis

Alzheimer\u27s-Like Behavior and Pathology in a Transgenic Mouse Model of Down Syndrome

Down syndrome (DS) is caused by trisomy of human chromosome 21 (Hsa21) and is correlated with various comorbidities, including Alzheimer’s disease in 50% of affected individuals. The Tc1 mouse model contains a copy of Hsa21, producing an animal model of DS. In an effort to understand the correlation between DS and Alzheimer’s disease, this study compared the learning and memory abilities with hippocampal and cortical brain pathology in the Tc1 mouse model. Genotyping of Tc1 mice indicated that Hsa21 was transmitted with a 25.0% frequency. Tc1 mice underwent open field, dark-light, and step down test of inhibitory avoidance tests at set timepoints. Acquisition of a hippocampal-dependent short-term memory task was intact while long-term memory trended towards impairment. Relative brain weight of Hsa21 positive subjects was significantly higher than control littermates with lateral ventricle area profoundly increased. Histological screening for Alzheimer’s-like neuropathology showed neurofibrillary tangles in the cortex of all Hsa21+ mice and a diffuse plaque in one Hsa21+ subject. Immunohistochemistry was used to analyze β-amyloid 1-42 levels in control and Hsa21+ mice. This heterogeneity in pathology is consistent with the spectrum of neuropathological deficits, including Alzheimer’s, seen in patients with DS

Characterization of Active Compounds Produced in the Biotransformation of Metabolites in Kombucha Tea

Kombucha is a fermented black tea that has been hypothesized to provide many health benefits. The exact origin of these benefits, however, is continually being investigated. This study aims to identify the various active compound produced in the biotransformation of the metabolites during the fermentation process of the tea beverage, as well as quantify functions such as antioxidant capacity. Hydrophilic and hydrophobic liquid-liquid extractions were performed on a filtered sample of GT’s Organic Raw Kombucha® using acetonitrile and ethyl acetate. Ferric reducing/antioxidant power (FRAP) assays were run on both the Kombucha tea and the extracts to determine their antioxidant capacity. The Kombucha tea, acetonitrile extract, and ethyl acetate extract were found to have FRAP values of 146.9, 102.7, and 71.04, respectively. The high retention of FRAP in the acetonitrile extract gives evidence that a polar hydrophobic molecule is functioning as an antioxidant in the Kombucha tea. Antioxidant capacity was further tracked using a variety of chromatographic techniques including the use of silica gel and C-18 functionalized silica flash chromatographies, as well as reverse-phase C-18 HPLC. These studies aim to further purify and characterize the compounds responsible for antioxidant capacity

Arkansas Symphony Orchestra

This is the program of the Arkansas Symphony Orchestra concert, directed by Philip Mann, featuring music by Dr. W. Francis McBeth, held on March 8, 2013, in the Jones Performing Arts Center