Modification of azo dyes by lactic acid bacteria.

AIM: The ability of Lactobacillus casei and Lactobacillus paracasei to modify the azo dye, tartrazine, was recently documented as the result of the investigation on red coloured spoilage in acidified cucumbers. Fourteen other lactic acid bacteria (LAB) were screened for their capability to modify the food colouring tartrazine and other azo dyes of relevance for the textile industry. METHODS AND RESULTS: Most LAB modified tartrazine under anaerobic conditions, but not under aerobic conditions in modified chemically defined media. Microbial growth was not affected by the presence of the azo dyes in the culture medium. The product of the tartrazine modification by LAB was identified as a molecule 111 daltons larger than its precursor by liquid chromatography-mass spectrometry. This product had a purple colour under aerobic conditions and was colourless under anaerobic conditions. It absorbed light at 361 and 553 nm. CONCLUSION: LAB are capable of anabolizing azo dyes only under anaerobic conditions. IMPACT AND SIGNIFICANCE OF THE STUDY: Although micro-organisms capable of reducing the azo bond on multiple dyes have been known for decades, this is the first report of anabolism of azo dyes by food related micro-organisms, such as LAB.

Determination of 5-log pathogen reduction times for heat-processed, acidified vegetable brines.

Recent outbreaks of acid-resistant food pathogens in acid foods, including apple cider and orange juice, have raised concerns about the safety of acidified vegetable products. We determined pasteurization times and temperatures needed to assure a 5-log reduction in the numbers of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella strains in acidified cucumber pickle brines. Cocktails of five strains of each pathogen were (separately) used for heat-inactivation studies between 50 and 60 degrees C in brines that had an equilibrated pH value of 4.1. Salmonella strains were found to be less heat resistant than E. coli O157:H7 or L. monocytogenes strains. The nonlinear killing curves generated during these studies were modeled using a Weibull function. We found no significant difference in the heat-killing data for E. coli O157:H7 and L. monocytogenes (P = 0.9709). The predicted 5-log reduction times for E. coli O157:H7 and L. monocytogenes were found to fit an exponential decay function. These data were used to estimate minimum pasteurization times and temperatures needed to ensure safe processing of acidified pickle products and show that current industry pasteurization practices offer a significant margin of safety.

Commercial Scale Cucumber Fermentations Brined with Calcium Chloride Instead of Sodium Chloride.

Development of low salt cucumber fermentation processes present opportunities to reduce the amount of sodium chloride (NaCl) that reaches fresh water streams from industrial activities. The objective of this research was to translate cucumber fermentation brined with calcium chloride (CaCl2 ) instead of NaCl to commercial scale production. Although CaCl2 brined cucumber fermentations were stable in laboratory experiments, commercial scale trials using 6440 L open-top tanks rapidly underwent secondary cucumber fermentation. It was understood that a limited air purging routine, use of a starter culture and addition of preservatives to the cover brine aids in achieving the desired complete cucumber fermentation. The modified process was used for subsequent commercial trials using 12490 and 28400 L open-top tanks packed with variable size cucumbers and from multiple lots, and cover brines containing CaCl2 and potassium sorbate to equilibrated concentrations of 100 and 6 mM, respectively. Lactobacillus plantarum LA0045 was inoculated to 10(6) CFU/mL, and air purging was applied for two 2-3 h periods per day for the first 10 d of fermentation and one 2-3 h period per day between days 11 and 14. All fermentations were completed, as evidenced by the full conversion of sugars to lactic acid, decrease in pH to 3.0, and presented microbiological stability for a minimum of 21 d. This CaCl2 process may be used to produce fermented cucumbers intended to be stored short term in a manner that reduces pollution and waste removal costs.

Sulfite analysis of fruits and vegetables by high-performance liquid chromatography (HPLC) with ultraviolet spectrophotometric detection.

Free and total sulfite were analyzed in acidified vegetable products, instant mashed potatoes, and dried apples. Sulfite was separated by HPLC and quantified with a UV-vis detector. Resolution from components of food samples was achieved by varying the acid concentration of the eluant solution and by appropriate choice of the analytical wavelength. The minimum detectable levels for sulfite were 0.5 mg/L for a 10-cm analytical column and 1.5 mg/L for a 30-cm column. For analyses done with a 30-cm column, the coefficient of variation was <2% for analysis of free sulfite and total sulfite in acidified vegetables. For dried apples and instant potatoes, it ranged from 1 to 6.5%. The corresponding analytical errors were <4% and 1.2-5.6%, respectively, for the 10-cm column.

Development of oxidized odor and volatile aldehydes in fermented cucumber tissue exposed to oxygen.

Changes in volatile compounds in fermented cucumber tissue during exposure to oxygen were investigated by purge and trap sampling, followed by GC-MS. Hexanal and a series of trans unsaturated aldehydes, (E)-2-pentenal, (E)-2-hexenal, (E)-2-heptenal, and (E)-2-octenal, increased in fermented cucumber slurries exposed to oxygen. Sensory evaluation of oxidized odor was correlated with the increase in aldehyde concentrations. Other identified volatile components present after fermentation did not show major changes during exposure to oxygen. There was no decrease in the formation of aldehydes in fermented cucumber samples that were heated to inactivate enzymes before exposure to oxygen. These results indicated that the formation of aldehydes in oxygen was due to nonenzymatic reactions.

Inhibition of formation of oxidative volatile components in fermented cucumbers by ascorbic acid and turmeric.

Two naturally occurring antioxidants, ascorbic acid and turmeric, were effective in inhibiting formation of hexanal, (E)-2-penenal, (E)-2-hexenal, (E)-2-heptenal, and (E)-2-octenal when slurries of fermented cucumber tissue were exposed to oxygen. Added ascorbic acid prevented formation of most of these oxidative aldehydes at 175 ppm or greater. Turmeric, which is used commercially as a yellow coloring in cucumber pickle products, was found to almost completely prevent aldehyde formation at 40 ppm.

Solid-phase microextraction (SPME) technique for measurement of generation of fresh cucumber flavor compounds.

Investigations were carried out to determine whether flavor compounds characteristic for fresh cucumbers could be rapidly determined using a solid-phase microextraction (SPME) dynamic headspace sampling method combined with gas chromatography and flame ionization detection. Cucumbers were sampled, during blending, for fresh cucumber flavor compounds (E,Z)-2,6-nonadienal and (E)-2-nonenal. The GC was such that the two target compounds were separated and baseline-resolved. Relative standard deviations for analysis of both (E,Z)-2,6-nonadienal and (E)-2-nonenal using this SPME sampling method were +/-10%. Utility of the analytical method was demonstrated by determining the effect of heat treatments on the ability of cucumbers to produce these flavor impact compounds.

Investigations into genotypic variations of peanut carbohydrates.

Carbohydrates are known to be important precursors in the development of roasted peanut quality. However, little is known about their genotypic variation. A better understanding of the role of carbohydrates in roasted peanut quality requires first an understanding of the genotypic variation in the soluble carbohydrate components. Ion exchange chromatography was used to isolate 20 different carbohydrate components in 52 genotypes grown in replicated trials at two locations. Inositol, glucose, fructose, sucrose, raffinose, and stachyose were quantitated, and 12 unknown peaks were evaluated on the basis of the peak height of the unknown relative to the cellobiose internal standard peak height. Peaks tentatively identified as verbascose and ajugose could not be properly integrated because of tailing. Of the 18 carbohydrates that were estimated, 9 exhibited significant variation between test environments, 5 among market types, 14 among genotypes within market types, and 11 exhibited some significant form of genotype x environment interaction. Genotypes accounted for 38-78% of the total variation for the known components, suggesting that broad-sense heritability for these components is high. The observed high genotypic variation in carbohydrate components is similar to the high genotypic variation observed for the sweetness attribute in roasted peanuts, which raises the question regarding possible interrelationships. The establishment of such interrelationships could be most beneficial to peanut breeding programs to ensure the maintenance of flavor quality in future peanut varieties.

Relationships of sweet, bitter, and roasted peanut sensory attributes with carbohydrate components in peanuts.

Certain roasted peanut quality sensory attributes have been shown to be heritable. Currently the only means of measuring these traits is the use of a trained sensory panel. This is a costly and time-consuming process. It is desirable, from a cost, time, and sample size perspective, to find other methodologies for estimating these traits. Because sweetness is the most heritable trait and it has a significant positive relationship to the roasted peanut trait, the possible relationships between heritable sensory traits and 18 carbohydrate components (inositol, glucose, fructose, sucrose, raffinose, stachyose, and 12 unknown peaks) in raw peanuts from 52 genotypes have been investigated. Previously reported correlations among sweet, bitter, and roasted peanut attributes were evident in this study as well. Where there was positive correlation of total sugars with sweetness, there also was positive correlation of total sugars with roasted peanut attribute and negative correlation of total sugars with bitterness and astringency. The expected generalized relationship of total sugars or sucrose to sweetness could not be established because the relationship was not the same across all market-types. Further work is needed to determine the nature of the chemical components related to the bitter principle, which appear to modify the sweet response and interfere with the sensory perception of sweetness, particularly in the Virginia market-type. Also, certain carbohydrate components showed significant relationships with sensory attributes in one market-type and not another. These differential associations demonstrate the complexity of the interrelationships among sweet, bitter, and roasted peanut sensory attributes. Within two market-types it is possible to improve the efficiency of selection for sweetness and roasted peanut quality by assaying for total carbohydrates. On the basis of the regression values the greatest efficiency would occur in the fastigiate market-type and then the runner.

Use and removal of sulfite by conversion to sulfate in the preservation of salt-free cucumbers.

Cucumbers (Cucumis sativus) were microbiologically stable in cover liquid containing 300 ppm of added sodium metabisulfite (calculated as SO2), 20 mM calcium chloride, and HCl to give an equilibrated pH of 3.5. The sulfur(IV) oxoanions could be easily removed to nondetectable levels (< 3 ppm) by addition of an equimolar amount of hydrogen peroxide, which rapidly converted S(IV) primarily to sulfate ions. When sulfur(IV) oxoanions were removed from stored fresh cucumbers, 85% of the added metabisulfite could be accounted for by formation of sulfate ions. If cucumbers were heated before addition and removal of sulfur(IV) oxoanions, 96% of that added was converted to sulfate by hydrogen peroxide. Preservation of cucumbers in this way does not require fermentation, so addition of salt is not needed to select for lactic acid bacteria.

Independent effects of acetic acid and pH on survival of Escherichia coli in simulated acidified pickle products.

Our objective was to determine the effects of organic acids and pH on the rate at which selected strains of Escherichia coli O157:H7 die in acid solutions representative of acidified pickle products (pH < 4.6). We used gluconic acid/sodium gluconate (pKa = 3.7) as a noninhibitory buffer to maintain pH at selected values in the absence of other organic acids. This was possible because we found that the inhibitory effects of this acid on E. coli strains at pH 3.1 were independent of acid concentration over a range of 2 to 200 mM. By this method, the lethal effects of acetic acid solutions (100 to 400 mM) at selected pH values between 3.1 and 4.1 were compared with the effects of pH alone (as determined using gluconate buffer). We found D-values were two- to fourfold lower with acetic acid compared with the effect of pH alone for simulated pickle brines in this pH range. Glutamic acid, an amino acid that is known to enhance acid resistance in E. coli and is a component of pickle brines, protected the E. coli strains from the specific effects of acetic acid.

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.

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

UNLABELLED: 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.

Preservation of acidified cucumbers with a natural preservative combination of fumaric acid and allyl isothiocyanate that target lactic acid bacteria and yeasts.

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 degrees 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.

Effects of pH adjustment and sodium ions on sour taste intensity of organic acids.

Protonated organic acid species have been shown to be the primary stimuli responsible for sour taste of organic acids. However, we have observed that sour taste may be modulated when the pH of acid solutions is raised using sodium hydroxide. Objectives were to evaluate the effect of pH adjustment on sour taste of equimolar protonated organic acid solutions and to investigate the potential roles of organic anions and sodium ions on sour taste perception. Despite equal concentrations of protonated acid species, sour taste intensity decreased significantly with increased pH for acetic, lactic, malic, and citric acids (P < 0.05). Total organic anion concentration did not explain the suppression of sour taste in solutions containing a blend of 3 organic acids with constant concentration of protonated organic acid species and hydrogen ions and variable organic anion concentrations (R(2)= 0.480, P = 0.12). Sour taste suppression in these solutions seemed to be more closely related to sodium ions added in the form of NaOH (R(2)= 0.861, P = 0.007). Addition of 20 mM NaCl to acid solutions resulted in significant suppression of sour taste (P = 0.016). However, sour taste did not decrease with further addition of NaCl up to 80 mM. Presence of sodium ions was clearly shown to decrease sour taste of organic acid solutions. Nonetheless, suppression of sour taste in pH adjusted single acid solutions was greater than what would be expected based on the sodium ion concentration alone, indicating an additional suppression mechanism may be involved.

Microbiological preservation of cucumbers for bulk storage using acetic acid and food preservatives.

Microbial growth did not occur when cucumbers were preserved without a thermal process by storage in solutions containing acetic acid, sodium benzoate, and calcium chloride to maintain tissue firmness. The concentrations of acetic acid and sodium benzoate required to ensure preservation were low enough so that stored cucumbers could be converted to the finished product without the need to wash out and discard excess acid or preservative. Since no thermal process was required, this method of preservation would be applicable for storing cucumbers in bulk containers. Acid tolerant pathogens died off in less than 24 h with the pH, acetic acid, and sodium benzoate concentrations required to assure the microbial stability of cucumbers stored at 30 degrees C. Potassium sorbate as a preservative in this application was not effective. Yeast growth was observed when sulfite was used as a preservative.

Lactobacilli and tartrazine as causative agents of red-color spoilage in cucumber pickle products.

The cucumber pickling industry has sporadically experienced spoilage outbreaks in pickled cucumber products characterized by development of red color on the surface of the fruits. Lactobacillus casei and Lactobacillus paracasei were isolated from 2 outbreaks of this spoilage that occurred about 15 y apart during the last 3 decades. Both organisms were shown to produce this spoilage when inoculated into pickled cucumbers while concomitantly degrading the azo dye tartrazine (FD&C yellow nr 5). This food dye is used as a yellow coloring in the brine cover solutions of commercial pickled cucumber products. The red color does not occur in the absence of tartrazine, nor when turmeric is used as a yellow coloring in the pickles. Addition of sodium benzoate to the brine cover solutions of a pickled cucumber product, more specifically hamburger dill pickles, prevented growth of these lactic acid bacteria and the development of the red spoilage.

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

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.

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

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.

Protective effects of organic acids on survival of Escherichia coli O157:H7 in acidic environments.

Outbreaks of disease due to acid-tolerant bacterial pathogens in apple cider and orange juice have raised questions about the safety of acidified foods. Using gluconic acid as a noninhibitory low-pH buffer, we investigated the killing of Escherichia coli O157:H7 strains in the presence or absence of selected organic acids (pH of 3.2), with ionic strength adjusted to 0.60 to 0.68. During a 6-h exposure period in buffered solution (pH 3.2), we found that a population of acid-adapted E. coli O157:H7 strains was reduced by 4 log cycles in the absence of added organic acids. Surprisingly, reduced lethality for E. coli O157:H7 was observed when low concentrations (5 mM) of fully protonated acetic, malic, or l-lactic acid were added. Only a 2- to 3-log reduction in cell counts was observed, instead of the 4-log reduction attributed to pH effects in the buffered solution. Higher concentrations of these acids at the same pH aided in the killing of the E. coli cells, resulting in a 6-log or greater reduction in cell numbers. No protective effect was observed when citric acid was added to the E. coli cells. d-Lactic acid had a greater protective effect than other acids at concentrations of 1 to 20 mM. Less than a 1-log decrease in cell numbers occurred during the 6-h exposure to pH 3.2. To our knowledge, this is the first report of the protective effect of organic acids on the survival of E. coli O15:H7 under low-pH conditions.