Yeast heterogeneity during spontaneous fermentation of black Conservolea olives in different brine solutions

Aims: To assess the yeast community structure and dynamics during Greek‐style processing of natural black Conservolea olives in different brine solutions. Methods and Results: Black olives were subjected to spontaneous fermentation in 6% (w/v) NaCl brine solution or brine supplemented with (i) 0·5% (w/v) glucose, (ii) 0·2% (v/v) lactic acid and (iii) both glucose and lactic acid. Yeast species diversity was evaluated at the early (2 days), middle (17 days) and final (35 days) stages of fermentation by restriction fragment length polymorphism and sequence analyses of the 5·8S internal transcribed spacer and the D1/D2 ribosomal DNA (rDNA) regions of isolates. Analysis revealed a relatively broad range of biodiversity composed of 10 genera and 17 species. In all treatments, yeasts were the main micro‐organisms involved in fermentation together with lactic acid bacteria that coexisted throughout the processes. Metschnikowia pulcherrima was the dominant yeast species at the onset of fermentation, followed by Debaryomyces hansenii and Aureobasidium pullulans. Species heterogeneity changed as fermentations proceeded and Pichia membranifaciens along with Pichia anomala evolved as the main yeasts of olive elaboration, prevailing at 17 and 35 days of the process. Molecular techniques allowed for the identification of five yeast species, namely A. pullulans, Candida sp., Candida silvae, Cystofilobasidium capitatum and M. pulcherrima, which have not been reported previously in black olive fermentation. Conclusions: By using molecular techniques, a rich yeast community was identified from Conservolea black olive fermentations. Metschnikowia pulcherrima was reported for the first time to dominate in different brines at the onset of fermentation, whereas Pichia anomala and P. membranifaciens evolved during the course. The addition of glucose and/or lactic acid perturbed yeast succession and dominance during fermentation. Significance and Impact of the Study: Yeasts have an important role in black olive fermentation and contribute to the development of the organoleptic characteristics of the final product. At the same time, certain species can cause significant spoilage. The present study adds to a better knowledge of yeast communities residing in olive fermentations towards a well‐controlled process with minimization of product’s losses.     

Lactic fermentation during the storage of 'Alorena' cultivar untreated green table olives

The development of lactic fermentation processes for the storage of directly brined olives (Aloreña cultivar) was investigated by three procedures: (1) a modification of the traditional method with an initial brine containing 9% (w/v) NaCl and 0.2% (w/v) acetic acid; (2) induced lactic fermentation with 6% NaCl and 0.2% acetic acid; and (3) conservation in acidified brine containing 6% NaCl and 0.6% acetic acid. In all cases, strains of Lactobacillus plantarum and Pediococcus spp. were present in each, indicating the great tolerance of these micro-organisms to high levels of lactic and acetic acids. They also appeared in an altered sequence. Counts of Pediococcus remained moderate (higher than Lact. plantarum ) throughout the last part of the preservation period. A commercial starter improved colonization by Lact. plantarum. Yeasts coexisted with the lactic bacteria throughout the preservation period although their importance in the fermentation process was very limited. The brine characteristics obtained after fermentation were suitable for assured product preservation. There was no spoilage. These results encourage research on the mechanism of lactic acid bacteria inhibition in brines and the development of lactic fermentation processes for directly brined olives from other olive cultivars.     

Separate and combined biochemical activities of the subunits of a naturally split reverse gyrase

Reverse gyrase reanneals denatured DNA and induces positive supercoils in DNA, an activity that is critical for life at very high temperatures. Positive supercoiling occurs by a poorly understood mechanism involving the coordination of a topoisomerase domain and a helicase-like domain. In the parasitic archaeon Nanoarchaeum equitans, these domains occur as separate subunits. We express the subunits, and characterize them both in isolation and as a heterodimer. Each subunit tightly associates and interacts with the other. The topoisomerase subunit enhances the catalytic specificity of the DNA-dependent ATPase activity of the helicase-like subunit, and the helicase-like subunit inhibits the relaxation activity of the topoisomerase subunit while promoting positive supercoiling. DNA binding preference for both single- and double-stranded DNA is partitioned between the subunits. Based on a sensitive topological shift assay, the binding preference of helicase-like subunit for underwound DNA is modulated by its binding with ATP cofactor. These results provide new insight into the mechanism of positive supercoil induction by reverse gyrase.     

Induced lactic acid fermentation during the preservation stage of ripe olives from Hojiblanca cultivar

The influence of initial sodium chloride concentration (6 and 0%, w/v), acetic acid concentration (0.6, 0.3 and 0.0%, v/v), type of process (natural and inoculated), and storage system (anaerobic and aerobic) on the inducement of a lactic fermentation for the preservation stage of Hojiblanca cultivar ripe olives was investigated. The addition of 6% NaCl prevented colonization by lactic acid bacteria in all cases. A high level of acetic acid (0.6%) was effective in preserving olives for 2 months, although yeast growth was not inhibited for longer periods of storage. Natural growth of Lactobacillus plantarum did not occur. Inoculation with this micro-organism was effective only in the two treatments with tap water (with no NaCl) as the initial covering solution, although survival was reduced to a half of the added organisms when the initial pH was corrected with 0.3% acetic acid. In these two treatments pH quickly reached appropriate values (<4.0) for olive stabilization. Aerobic conditions led to low concentrations of carbon dioxide, without disturbing growth of lactic acid bacteria. Thus, the aerobic lactic acid fermentation, with tap water initially, was the most adequate preservation procedure for the storage of ripe olives prior to their oxidation treatment. Results of trials conducted on an industrial scale showed the same pattern and confirmed the viability of the new procedure.     

The fate of aflatoxin in naturally contaminated corn during the ethanol fermentation

Corn naturally contaminated with aflatoxin was used as a substrate in the ethanol fermentation. Distribution of toxin in several process and recovery fractions was identified. Although little degradation of the mycotoxin occurred during fermentation, no toxin appeared in the distilled alcohol. As accumulation of toxin in spent grains represents a potential problem in use of the material as animal feed, several decontamination procedures were tested. Sodium hydroxide, ammonium hydroxide, sodium hypochlorite, and hydrogen peroxide were identified as efficient agents of toxin degradation.     

Microbial population and biochemical changes during production of protein-enriched fufu

Summary Candida utilis strain BKT4 and Saccharomyces cerevisiae strain BKT7 isolated from burukutu (a local wine brewed from sorghum) were used to enrich fufu. During the fermentation process, there were changes in the microbiological and biochemical characteristics of the cassava. The total viable counts increased with increasing fermentation time while the counts of the lactics and fungi increased at the later stages of the fermentation due to the acidity of the medium. Various bacteria (Bacillus, Staphylococcus, Klebsiella, Escherichia, Streptococcus, Lactobacillus, Leuconostoc, Corynebacterium), moulds (Penicillium, Aspergillus, Fusarium, Mucor, Rhizopus) and yeasts (Candida, Saccharomyces, Hansenula, Rhodotorula) were found to be associated with the fermentation process. The pH of the fermenting cassava increased from 4.2 to 5.7 after 72 h while the cyanide level decreased from 2.2 mg/kg to 0.7 mg/kg over the same period of fermentation. Fufu (prepared by crushing and sieving fermenting cassava roots) enriched with 0.5 g of C. utilis strain BKT4, S. cerevisiae BKT7 and a mixed culture of the two organisms revealed a crude protein of 7.90, 6.34 and 10.0% respectively as compared to 2% protein content of the enriched fufu. There was a corresponding increase in protein content of the product as the quantity of the enrichment yeast was increased from 0.5 to 3.0 g. The aroma of the enriched fufu was preferred to that of the commercial fufu. Generally, good acceptability and organoleptic qualities (colour, taste, texture and aroma) of the protein enriched fufu was best achieved within 48 h of enrichment. The results of this study suggest that fufu can be made more nutritious with yeasts particularly Candida utilis strain BKT4 and Saccharomyces cerevisiae strain BKT7.     

Microbial colonization and controls in dryland systems

Drylands constitute the most extensive terrestrial biome, covering more than one-third of the Earth's continental surface. In these environments, stress limits animal and plant life, so life forms that can survive desiccation and then resume growth following subsequent wetting assume the foremost role in ecosystem processes. In this Review, we describe how these organisms assemble in unique soil- and rock-surface communities to form a thin veneer of mostly microbial biomass across hot and cold deserts. These communities mediate inputs and outputs of gases, nutrients and water from desert surfaces, as well as regulating weathering, soil stability, and hydrological and nutrient cycles. The magnitude of regional and global desert-related environmental impacts is affected by these surface communities; here, we also discuss the challenges for incorporating the consideration of these communities and their effects into the management of dryland resources.     

Microbial characterization of organic carrier colonization during a model biofiltration experiment

AIMS: Dynamic microbial characterization of the colonization of organic carrier during a model biofiltration experiment using methanol as air pollutant. METHODS AND RESULTS: A model biofilter was used in order to characterize the micro-organisms involved in the colonization of a model organic carrier. The model system consisted of closed vial as biofilter, peanut shells as lignocellulosic carrier and methanol as air pollutant. The micro-organisms involved in biofiltration were identified and characterized for their lignocellulolytic and methylotrophic activities. Fungi presented a higher lignocellulolytic activity than bacteria. A steady-state was reached after 15 to 20 days. CONCLUSIONS: The consortium naturally associated to peanut shells is limited to few aerobic bacteria and lignocellulolytic fungi. This consortium was able to degrade methanol without external nutrient supply. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first paper that focuses on carrier degradation processes and the micro-organisms involved during the start-up period of a biofiltration process.     

Microbial population dynamics and temperature changes during fermentation of kimjang kimchi

A distinct subset of lactic acid bacteria that are greatly influenced by temperature play an important role during kimchi fermentation. However, microbial population dynamics and temperature control during kimjang kimchi fermentation, which is traditionally fermented underground, are not known. Here we show that Lactobacillus sakei predominates in kimjang kimchi, perhaps due to suitable fermentation (5∼9°C) and storage (−2°C) temperatures. The temperature of this kimchi gradually decreased to 3.2°C during the first 20 days of fermentation (−0.3°C/day) and then was stably maintained around 1.6°C, indicating that this simple approach is very efficient both for fermentation and storage. These findings provide important information towards the development of temperature controlling systems for kimchi fermentation.     

Microbial communities and chemical changes during fermentation of sugary Brazilian kefir

The microorganisms associated with sugary Brazilian kefir beverage were investigated using a combination of culture-dependent and -independent methods. A total of 289 bacteria and 129 yeasts were identified via phenotypic and genotypic methods. Lb. paracasei (23.8%) was the major bacterial isolate identified, followed by Acetobacter lovaniensis (16.31%), Lactobacillus parabuchneri (11.71%), Lactobacillus kefir (10.03%) and Lactococcus lactis (10.03%). Saccharomyces cerevisiae (54.26%) and Kluyveromyces lactis (20.15%) were the most common yeast species isolated. Scanning electron microscopy showed that the microbiota was dominated by lemon-shaped yeast cells growing in close association with Lactobacillus (long and curved). Some lactic acid bacteria detected by sequence analysis of DGGE (denaturing gradient gel electrophoresis) bands were not recovered at any time through fermentation by plating. Conversely, DGGE fingerprints did not reveal bands corresponding to some of the species isolated by culturing methods. The bacteria Acetobacter lovaniensis and the yeast Kazachstania aerobia are described for the first time in sugary kefir. During the 24 h of fermentation, the concentration of lactic acid ranged from 0.2 to 1.80 mg/ml, and that of acetic acid increased from 0.08 to 1.12 mg/ml. The production of ethanol was limited, reaching a final mean value of 1.24 mg/ml.     

Influence of pasteurization and lye treatment on the fermentation of spanish-style manzanilla olives

The effect of lye concentration and pasteurization on the fermentation of Spanish-style Manzanilla green olives was investigated. Results showed that a rapid, normal fermentation always occurred when the proper lye was used, and only in the case of an inappropriate alkaline treatment could pasteurization help the growth of lactobacilli. Although pasteurization does not seem to affect color of processed olives, texture tests gave lower values for pasteurized samples than for those obtained from nonpasteurized olives.     

Measurement of α-amylase and glucoamylase activities produced during fermentation

A method for the automatic measurement of α-amylase and glucoamylase activities during fermentation has been developed. Soluble starch dyed with Remazol Brilliant Orange was used as the substrate for α-amylase and 4-nitrophenyl α-d-glucopyranoside for glucoamylase. The same automatic analysis system could be used for both of these enzymes because the reaction products were measured at the same wavelength. Simultaneous pick-up of enzyme and the respective substrate was enabled by using two samplers. The presence of α-amylase did not interfere with the glucoamylase determination. Absolute values for α-amylase activity were obtained using a mathematical correction. Monitoring of these enzymes was accomplished during microbial fermentation.     

Microbial activities during composting of pulp and paper-mill primary solids

In this study, bench-scale aerobic reactors were used to monitor microbial activities and determine the degradability of pulp and paper-mill primary solids. Over the 43-day composting period, the level of microorganisms detected increased four-fold. However, based on amounts of CO2 produced, the increase expected was 19-fold, suggesting lysis of some cells in the population. The composition of the microbial population, determined through species identification by the BIOLOG® method, and by spot plating, changed throughout the composting process with Cellulomonas spp. not appearing until day 22. Chemical analyses suggested a fall of 33% in the cellulose content, but no other statistically significant chemical changes were observed.     

Microbial populations and activities in the rhizoplane of rock-weathering desert plants. I. Root colonization and weathering of igneous rocks

Dense layers of bacteria and fungi in the rhizoplane of three species of cactus (Pachycereus pringlei, Stenocereus thurberi, Opuntia cholla) and a wild fig tree (Ficus palmeri) growing in rocks devoid of soil were revealed by bright-field and fluorescence microscopy and field emission scanning electron microscopy. These desert plants are responsible for rock weathering in an ancient lava flow at La Purisima-San Isidro and in sedimentary rock in the Sierra de La Paz, both in Baja California Sur, Mexico. The dominant bacterial groups colonizing the rhizoplane were fluorescent pseudomonads and bacilli. Seven of these bacterial species were identified by the 16S rRNA molecular method. Unidentified fungal and actimomycete species were also present. Some of the root-colonizing microorganisms fixed in vitro N(2), produced volatile and non-volatile organic acids that subsequently reduced the pH of the rock medium in which the bacteria grew, and significantly dissolved insoluble phosphates, extrusive igneous rock, marble, and limestone. The bacteria were able to release significant amounts of useful minerals, such as P, K, Mg, Mn, Fe, Cu, and Zn from the rocks and were thermo-tolerant, halo-tolerant, and drought-tolerant. The microbial community survived in the rhizoplane of cacti during the annual 10-month dry season. This study indicates that rhizoplane bacteria on cacti roots in rock may be involved in chemical weathering in hot, subtropical deserts.     

Correlation between antioxidative activities and metabolite changes during Cheonggukjang fermentation

Liquid chromatography mass spectrometry and multivariate analysis were employed to investigate the correlation between fermentation time-dependent metabolite changes in cheonggukjang, a traditional fermented soybean product, and changes in its antioxidant activity over 72 h. The metabolite patterns were clearly distinguished not by strains but by fermentation time, into patterns I (0-12 h), II (12-24 h), and III (24-72 h), which appeared as distinct clusters on principal component analysis. The compounds that significantly contributed to patterns I, II, and III were soyasaponins, isoflavonoid derivatives, and isoflavonoid aglycons respectively. Partial least square analysis for metabolite to antioxidant effects showed correlations between the ferric reducing/antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay during 24-36 h, and 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) test and total phenol content (TPC) during 36-72 h. Compared with the strong negative correlations of glucosylated-isoflavonoids with DPPH, ABTS and TPC during fermentation, the isoflavonoid aglycon displayed strong positive correlations with these compounds during fermentation.