Modulation of the bacterial population in commercial cucumber fermentations by brining salt type.

AIMS: Differences in the bacterial population of cucumber fermentations brined with no salt, 100 mmol l-1 (1·1%) calcium chloride (CaCl2 ) or 1·03 mol l-1 (6%) sodium chloride (NaCl) were studied. METHODS AND RESULTS: Changes in the microbiology and chemistry of commercial and laboratory scale cucumber fermentations occurring as a function of time were monitored using colony counts and metagenetic analysis, and a pH probe and high-performance liquid chromatography analysis respectively. Dissolved oxygen and carbon dioxide content were monitored in commercial fermentations. Fermentations brined with calcium chloride (CaCl2 ) or no salt sustained faster microbial growth and reduction in pH than those brined with 1·03 mol l-1 NaCl. Leuconostoc, Lactococcus and Weissella dominated in fermentations brined with no salt or 100 mmol l-1 CaCl2 on day 1 as compared to Weissella and enterobacteria in fermentations containing 1·03 mol l-1 NaCl. Lactobacilli dominated all fermentations by the third day, regardless of salt type, and was followed, in relative abundance by Pediococcus, Leuconostoc, Lactococcus and Weissella. From 84 to 96% of the population was composed of Lactobacillus by day 7 of the fermentations, except in the no salt fermentations in which a mixed population of LAB remained. The population of LAB found in commercial cucumber fermentations brined with 100 mmol l-1 CaCl2 (n = 18) or 1·03 mol l-1 NaCl (n = 9) mimicked that of laboratory fermentations. A declining population of aerobes was detected in commercial fermentations brined with CaCl2 on day 1. CONCLUSION: A reduced NaCl content in cucumber fermentation enhances microbial diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study fills a knowledge gap and aids in the design of improved reduced NaCl cucumber fermentations.

Evaluation of the use of malic acid decarboxylase-deficient starter culture in NaCl-free cucumber fermentations to reduce bloater incidence.

AIMS: Accumulation of carbon dioxide (CO2 ) in cucumber fermentations is known to cause hollow cavities inside whole fruits or bloaters, conducive to economic losses for the pickling industry. This study focused on evaluating the use of a malic acid decarboxylase (MDC)-deficient starter culture to minimize CO2 production and the resulting bloater index in sodium chloride-free cucumber fermentations brined with CaCl2 . METHODS AND RESULTS: Attempts to isolate autochthonous MDC-deficient starter cultures from commercial fermentations, using the MD medium for screening, were unsuccessful. The utilization of allochthonous MDC-deficient starter cultures resulted in incomplete utilization of sugars and delayed fermentations. Acidified fermentations were considered, to suppress the indigenous microbiota and favour proliferation of the allochthonous MDC-deficient Lactobacillus plantarum starter cultures. Inoculation of acidified fermentations with L. plantarum alone or in combination with Lactobacillus brevis minimally improved the conversion of sugars. However, inoculation of the pure allochthonous MDC-deficient starter culture to 107 CFU per ml in acidified fermentations resulted in a reduced bloater index as compared to wild fermentations and those inoculated with the mixed starter culture. CONCLUSIONS: Although use of an allochthonous MDC-deficient starter culture reduces bloater index in acidified cucumber fermentations brined with CaCl2 , an incomplete conversion of sugars is observed. SIGNIFICANCE AND IMPACT OF THE STUDY: Economical losses due to the incidence of bloaters in commercial cucumber fermentations brined with CaCl2 may be reduced utilizing a starter culture to high cell density.

Fermentation Cover Brine Reformulation for Cucumber Processing with Low Salt to Reduce Bloater Defect.

Reformulation of calcium chloride (CaCl2 ) cover brine for cucumber fermentation was explored as a mean to minimize the incidence of bloater defect. This study particularly focused on cover brine supplementation with calcium hydroxide (Ca[OH]2 ), sodium chloride (NaCl), and acids to enhance buffer capacity, inhibit the indigenous carbon dioxide (CO2 )- producing microbiota, and decrease the solubility of the gas. The influence of the cover brine formulations tested, on the cucumber fermentation microbiota, biochemistry, CO2 production, and bloating defect was studied using metagenetics, HPLC analysis, a portable gas analyzer and bloater index, respectively. Cover brine supplementation with Ca(OH)2 and acetic acid resulted in complete fermentations with final pH values 0.5 units higher than the un-supplemented control. Lactic acid production increased by approximately 22%, possibly inducing the observed reduction in the relative abundance of Enterobacteriaceae by 92%. Ca(OH)2 supplementation also resulted in an increased relative abundance of Leuconostocaceae by 7%, which likely contributed to the observed increment in CO2 levels by 25%. A 50% reduction on acetic acid formation was detected when cover brines were supplemented with Ca(OH)2 and 690 mM (4%) NaCl. No significant difference was observed in bloater index as the result of Ca(OH)2 or NaCl supplementation in cover brines, given that the CO2 levels remained at above the 20 mg/100 mL needed to induce the defect. It is concluded that the modified cover brine formulation containing Ca(OH)2 and NaCl enables the complete conversion of sugars, decreases production of CO2 and levels of Enterobacteriaceae, but insignificantly reduces bloater index. PRACTICAL APPLICATION: A cucumber fermentation cover brine containing Ca(OH)2 , 0.26% CaCl2 , 345 mM (2%) NaCl, and acetic acid to pH 4.7 has a functional combination of ingredients enabling a complete conversion of sugars to lactic acid with reduced production of acetic acid and CO2 . It represents a process ready cover brine formulation with the potential to allow the manufacture of cucumber pickles with low salt, enhanced food safety, and reduce environmental impact and water usage. Pilot commercial scale cucumber fermentations brined with such ingredients are to reveal the efficacy of this process ready formulation in the presence of oxygen from air in tanks, as opposed to 3.8 L (1-US gal) closed jars in the laboratory.

Reassessment of the succession of lactic acid bacteria in commercial cucumber fermentations and physiological and genomic features associated with their dominance.

A compositional re-assessment of the microbiota present in commercial cucumber fermentation using culture independent and dependent methods was conducted, with emphasis on lactic acid bacteria (LAB). Two commercial cucumber fermentation tanks were monitored by measuring pH, dissolved oxygen and temperature, and used as sources of samples for microbial plating, genomic DNA extraction and measurement of organic acids and carbohydrates by HPLC. Six additional commercial tanks were included to identify the dominant microorganisms using molecular methods. A comparative analysis of the publically available genome sequences corresponding to the LAB found in cucumber fermentations was completed to gain an understanding of genomic features possibly enabling dominance. Analyses of the microbiota suggest Lactobacillales prevail in cucumber fermentations, including in order of prevalence Lactobacillus pentosus, Lb. plantarum, Lb. brevis, Weissella spp., Pediococcus ethanolidurans, Leuconostoc spp. and Lactococcus spp. It was observed that Lb. pentosus and Lb. plantarum have comparatively larger genomes, higher gene counts, uniquely distribute the ribosomal clusters across the genome as opposed to close to the origin of replication, and possess more predicted amino acids prototrophies and selected biosynthesis related genes. It is theorized that Lb. pentosus and Lb. plantarum dominance in cucumber fermentations is the result of their genetic make-up.

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.

Characterization of the microbial diversity in yacon spontaneous fermentation at 20 °C.

The prebiotic fructooligosaccharide content of yacon makes this root an attractive alternative for the supplementation of a variety of food products. The preservation of yacon by fermentation has been proposed as an alternative to increase the probiotic content of the root concomitantly with its shelf life. Thus the fermented yacon could have significant functional content. The objective of this research was to characterize the biochemistry and microbiology of spontaneous yacon fermentation with 2% NaCl and define the viability of the proposed process. The biochemical analysis of spontaneous heterolactic fermentation of yacon showed a progressive drop in pH with increased lactic and acetic acids, and the production of mannitol during fermentation. The microbial ecology of yacon fermentation was investigated using culture-dependent and culture-independent methods. Bacterial cell counts revealed a dominance of lactic acid bacteria (LAB) over yeasts, which were also present during the first 2 days of the fermentation. Results showed that the heterofermentative LAB were primarily Leuconostoc species, thus it presents a viable method to achieve long term preservation of this root.

Microbial interactions associated with secondary cucumber fermentation.

AIMS: To evaluate the interaction between selected yeasts and bacteria and associate their metabolic activity with secondary cucumber fermentation. METHODS AND RESULTS: Selected yeast and bacteria, isolated from cucumber secondary fermentations, were inoculated as single and mixed cultures in a cucumber juice model system. Our results confirmed that during storage of fermented cucumbers and in the presence of oxygen, spoilage yeasts are able to grow and utilize the lactic and acetic acids present in the medium, which results in increased brine pH and the chemical reduction in the environment. These conditions favour opportunistic bacteria that continue the degradation of lactic acid. Lactobacillus buchneri, Clostridium bifermentans and Enterobacter cloacae were able to produce acetic, butyric and propionic acids, respectively, when inoculated in the experimental medium at pH 4.6. Yeast and bacteria interactions favoured the survival of Cl. bifermentans and E. cloacae at the acidic pH typical of fermented cucumbers (3.2), but only E. cloacae was able to produce a secondary product. CONCLUSIONS: The methodology used in this study confirmed that a complex microbiota is responsible for the changes observed during fermented cucumber secondary fermentation and that certain microbial interactions may be essential for the production of propionic and butyric acids. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the dynamics of the development of secondary cucumber fermentation aids in the identification of strategies to prevent its occurrence and economic losses for the pickling industry.

Bacteriophage ecology in a commercial cucumber fermentation.

To reduce high-salt waste from cucumber fermentations, low-salt fermentations are under development. These fermentations may require the use of starter cultures to ensure normal fermentations. Because potential phage infection can cause starter culture failure, it is important to understand phage ecology in the fermentations. This study investigated the phage ecology in a commercial cucumber fermentation. Brine samples taken from a fermentation tank over a 90-day period were plated onto deMan-Rogosa-Sharpe agar plates. A total of 576 lactic acid bacterial isolates were randomly selected to serve as potential hosts for phage isolation. Filtered brine served as a phage source. Fifty-seven independent phage isolates were obtained, indicating that 10% of the bacterial isolates were sensitive to phage attack. Phage hosts include Lactobacillus brevis (67% of all hosts), Lactobacillus plantarum (21%), Weissella paramesenteroides, Weissella cibaria, and Pediococcus ethanolidurans. Nearly 50% of phages were isolated on day 14, and the majority of them attacked L. brevis. Some phages had a broad host range and were capable of infecting multiple hosts in two genera. Other phages were species specific or strain specific. About 30% of phage isolates produced turbid pinpoint plaques or only caused reduced cell growth on the bacterial lawns. Six phages with distinct host ranges were characterized. The data from this study showed that abundant and diverse phages were present in the commercial cucumber fermentation, which could cause significant mortality to the lactic acid bacteria population. Therefore, a phage control strategy may be needed in low-salt cucumber fermentations.

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.

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.

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.