Small Molecule Binding,Docking, and Characterization of the Interaction between Pth1 and Peptidyl-tRNA

Bacterial Pth1 is essential for viability. Pth1 cleaves the ester bond between the peptide and nucleotide of peptidyl-tRNA generated from aborted translation, expression of mini-genes, and short ORFs. We have determined the shape of the Pth1:peptidyl-tRNA complex using small angle neutron scattering. Binding of piperonylpiperazine, a small molecule constituent of a combinatorial synthetic library common to most compounds with inhibitory activity, was mapped to Pth1 via NMR spectroscopy. We also report computational docking results, modeling piperonylpiperazine binding based on chemical shift perturbation mapping. Overall these studies promote Pth1 as a novel antibiotic target, contribute to understanding how Pth1 interacts with its substrate, advance the current model for cleavage, and demonstrate feasibility of small molecule inhibition.     

Fermentation of Cucumbers Brined with Calcium Chloride Instead of Sodium Chloride

ABSTRACT: Waste water containing high levels of NaCl from cucumber fermentation tank yards is a continuing problem for the pickled vegetable industry. A major reduction in waste salt could be achieved if NaCl were eliminated from the cucumber fermentation process. The objectives of this project were to ferment cucumbers in brine containing CaCl₂ as the only salt, to determine the course of fermentation metabolism in the absence of NaCl, and to compare firmness retention of cucumbers fermented in CaCl₂ brine during subsequent storage compared to cucumbers fermented in brines containing both NaCl and CaCl₂ at concentrations typically used in commercial fermentations. The major metabolite changes during fermentation without NaCl were conversion of sugars in the fresh cucumbers primarily to lactic acid which caused pH to decrease to less than 3.5. This is the same pattern that occurs when cucumbers are fermented with NaCl as the major brining salt. Lactic acid concentration and pH were stable during storage and there was no detectable production of propionic acid or butyric acid that would indicate growth of spoilage bacteria. Firmness retention in cucumbers fermented with 100 to 300 mM CaCl₂ during storage at a high temperature (45 °C) was not significantly different from that obtained in fermented cucumbers with 1.03 M NaCl and 40 mM CaCl₂. In closed jars, cucumber fermentations with and without NaCl in the fermentation brine were similar both in the chemical changes caused by the fermentative microorganisms and in the retention of firmness in the fermented cucumbers.     

Novel quantitative assays for estimating the antimicrobial activity of fresh garlic juice

Novel agar diffusion and broth dilution assays were developed for quantitatively estimating the antimicrobial activity of fresh garlic juice. Bacteria found to be inhibited by garlic juice in agar diffusion assay included two gram-positive and five gram-negative species. Leuconostoc mesenteroides was not inhibited. Escherichia coli B-103 (HB101, with pJH101, ampicillin resistant, 100 microg ml(-1)) was inhibited and chosen as the standard culture for quantitative assays. The agar diffusion assay was based on the slope ratio method, where the slope of dose response for garlic juice was divided by the slope of dose response for methylmethane thiosulfonate (MMTSO2). Juice from fresh garlic varied in activity between 1.76 and 2.31 microg of MMTSO2 per mg of garlic juice. The activity of juice decreased during 11 months of storage of garlic cloves at 5 degrees C from 2.31 to less than 0.1 microg of MMTSO2 per mg of juice. The broth dilution assay also used the E. coli B-103 culture, which permitted selective enumeration of this bacterium when 100 microg ml(-1) of ampicillin was incorporated into the enumerating agar. Selective enumeration was essential since the garlic juice was not sterile and, thus, contained natural flora. Growth of E. coli was unaffected by 0.1%, delayed by 0.25%, and completely inhibited at 0.5 and 2% garlic juice in broth during 24 h of incubation at 37 micro C. The minimum inhibition concentration of garlic juice by broth dilution assay was, thus, estimated to be 0.5%, which is equivalent to 3.46 microg of MMTSO2 per mg of garlic juice by the agar diffusion assay.     

Phenolic acid content and composition in leaves and roots of common commercial sweetpotato (Ipomea batatas L.) cultivars in the United States

ABSTRACT:  Phenolic acids in commercially important sweet potato cultivars grown in the United States were analyzed using reversed-phase high-performance liquid chromatography (HPLC). Caffeic acid, chlorogenic acid, 4,5-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, and 3,4-di-O-caffeoylquinic acid were well separated with an isocratic elution in less than 25 min compared to about 120 min for analyzing and re-equilibrating the column with a gradient method. The isocratic elution order of these caffeoylquinic acid derivatives was confirmed by LC-MS/MS. Chlorogenic acid was the highest in root tissues, while 3,5-di-O-caffeoylquinic acid and/or 4,5-di-O-caffeoylquinic acid were predominant in the leaves. Steam cooking resulted in statistically nonsignificant increases in the concentration of total phenolics and all the individual phenolic acids identified. Sweetpotato leaves had the highest phenolic acid content followed by the peel, whole root, and flesh tissues. However, there was no significant difference in the total phenolic content and antioxidant activity between purees made from the whole and peeled sweet potatoes.     

Determination of 5-log reduction times for food pathogens in acidified cucumbers during storage at 10 and 25 degrees C

Outbreaks of acid-resistant foodborne pathogens in acid foods with pH values below 4.0, including apple cider and orange juice, have raised concerns about the safety of acidified vegetable products. For acidified vegetable products with pH values between 3.3 and 4.6, previous research has demonstrated that thermal treatments are needed to achieve a 5-log reduction in the numbers of Escherichia coli O157:H7, Listeria monocytogenes, or Salmonella enterica. For some acidified vegetable products with a pH of 3.3 or below, heat processing can result in unacceptable product quality. The purpose of this study was to determine the holding times needed to achieve a 5-log reduction in E. coli O157:H7, L. monocytogenes, and S. enterica strains in acidified vegetable products with acetic acid as the primary acidulant, a pH of 3.3 or below, and a minimum equilibrated temperature of 10 degrees C. We found E. coli O157:H7 to be the most acid-resistant microorganism for the conditions tested, with a predicted time to achieve a 5-log reduction in cell numbers at 10 degrees C of 5.7 days, compared with 2.1 days (51 h) for Salmonella or 0.5 days (11.2 h) for Listeria. At 25 degrees C, the E. coli O157:H7 population achieved a 5-log reduction in 1.4 days (34.3 h).     

Preservation of Acidified Cucumbers with a Natural Preservative Combination of Fumaric Acid and Allyl Isothiocyanate that Target Lactic Acid Bacteria and Yeasts

ABSTRACT: Without the addition of preservative compounds cucumbers acidified with 150 mM acetic acid with pH adjusted to 3.5 typically undergo fermentation by lactic acid bacteria. Fumaric acid (20 mM) inhibited growth of Lactobacillus plantarum and the lactic acid bacteria present on fresh cucumbers, but spoilage then occurred due to growth of fermentative yeasts, which produced ethanol in the cucumbers. Allyl isothiocyanate (2 mM) prevented growth of Zygosaccharomyces globiformis, which has been responsible for commercial pickle spoilage, as well as the yeasts that were present on fresh cucumbers. However, allyl isothiocyanate did not prevent growth of Lactobacillus plantarum. When these compounds were added in combination to acidified cucumbers, the cucumbers were successfully preserved as indicated by the fact that neither yeasts or lactic acid bacteria increased in numbers nor were lactic acid or ethanol produced by microorganisms when cucumbers were stored at 30 °C for at least 2 mo. This combination of 2 naturally occurring preservative compounds may serve as an alternative approach to the use of sodium benzoate or sodium metabisulfite for preservation of acidified vegetables without a thermal process.     

Influence of sodium chloride, pH, and lactic acid bacteria on anaerobic lactic acid utilization during fermented cucumber spoilage

Cucumbers are preserved commercially by natural fermentations in 5% to 8% sodium chloride (NaCl) brines. Occasionally, fermented cucumbers spoil after the primary fermentation is complete. This spoilage has been characterized by decreases in lactic acid and a rise in brine pH caused by microbial instability. Objectives of this study were to determine the combined effects of NaCl and pH on fermented cucumber spoilage and to determine the ability of lactic acid bacteria (LAB) spoilage isolates to initiate lactic acid degradation in fermented cucumbers. Cucumbers fermented with 0%, 2%, 4%, and 6% NaCl were blended into slurries (FCS) and adjusted to pH 3.2, 3.8, 4.3, and 5.0 prior to centrifugation, sterile-filtration, and inoculation with spoilage organisms. Organic acids and pH were measured initially and after 3 wk, 2, 6, 12, and 18 mo anaerobic incubation at 25 °C. Anaerobic lactic acid degradation occurred in FCS at pH 3.8, 4.3, and 5.0 regardless of NaCl concentration. At pH 3.2, reduced NaCl concentrations resulted in increased susceptibility to spoilage, indicating that the pH limit for lactic acid utilization in reduced NaCl fermented cucumbers is 3.2 or lower. Over 18 mo incubation, only cucumbers fermented with 6% NaCl to pH 3.2 prevented anaerobic lactic acid degradation by spoilage bacteria. Among several LAB species isolated from fermented cucumber spoilage, Lactobacillus buchneri was unique in its ability to metabolize lactic acid in FCS with concurrent increases in acetic acid and 1,2-propanediol. Therefore, L. buchneri may be one of multiple organisms that contribute to development of fermented cucumber spoilage. PRACTICAL APPLICATION: Microbial spoilage of fermented cucumbers during bulk storage causes economic losses for producers. Current knowledge is insufficient to predict or control these losses. This study demonstrated that in the absence of oxygen, cucumbers fermented with 6% sodium chloride to pH 3.2 were not subject to spoilage. However, lactic acid was degraded by spoilage microorganisms in reduced salt, even with pH as low as 3.2. Efforts to reduce salt in commercial brining operations will need to include control measures for this increased susceptibility to spoilage. Lactobacillus buchneri was identified as a potential causative agent and could be used as a target in development of such control measures.     

Preparation of a Lactobacillus plantarum starter culture for cucumber fermentations that can meet kosher guidelines

A method is described for growth of a Lactobacillus plantarum starter culture in jars of commercially available pasteurized fresh-pack kosher dill cucumbers so that jars can be used to inoculate commercial scale cucumber fermentation tanks. A procedure is also described to transfer lactic acid bacteria from frozen storage in MRS broth into cucumber juice and commercial jars of kosher dill cucumbers so that a selected strain of lactic acid bacteria can be kosher certified for commercial fermentations in processing plants that operate under kosher certification. The strain of L. plantarum used in these experiments grew to maximum cell numbers in 4 d at 20 to 25 °C and then maintained viable cell numbers for 2 wk at >10(8) CFU/mL so the culture was suitable for inoculation of fermentation tanks. Refrigeration of jars of culture after they grow to maximum numbers minimizes die-off of cells sufficiently so that a pure culture can be maintained by aseptically transferring brine containing viable bacteria to a new pH-adjusted jar only once every 4 mo. PRACTICAL APPLICATION: This report describes a method to prepare a lactic acid bacteria starter culture suitable for kosher vegetable fermentations.     

A chemical basis for sour taste perception of acid solutions and fresh-pack dill pickles

ABSTRACT:  Sour taste is influenced by pH and acids present in foods. It is not currently possible, however, to accurately predict and modify sour taste intensity in foods containing organic acids. The objective of this study was to investigate the roles of protonated (undissociated) organic acid species and hydrogen ions in evoking sour taste. Sour taste intensity increased linearly with hydrogen ion concentration ( R ²= 0.995), and with the concentration of protonated organic acid species at pH 3.5 ( R ²= 0.884), 4.0 ( R ²= 0.929), and 4.5 ( R ²= 0.975). The structures of organic acid molecules did not affect sour taste after adjusting for the effects of protonated organic acid species and hydrogen ions. Sour taste intensity was also linearly related to the total concentration of protonated organic acid species in fresh-pack dill pickles ( R ²= 0.957). This study showed that the sour taste of organic acids was directly related to the number of molecules with at least 1 protonated carboxyl group plus the hydrogen ions in solution. Conclusions from these results are that all protonated organic acids are equally sour on a molar basis, that all protonated species of a given organic acid are equally sour, and that hydrogen ions and protonated organic acids are approximately equal in sour taste on a molar basis. This study provides a new understanding of the chemical species that are able to elicit sour taste and reveals a basis for predicting sour taste intensity in the formulation of acidified foods.