Identification of thermo-acidophilic bacteria isolated from the soil of several Japanese fruit orchards

Aims: To investigate the occurrence and distribution of thermo‐acidophilic bacteria (TAB) associated with various commercial fruit crop soils in Japan and to assess their ability to produce the odorous phenolic compound, guaiacol. Methods and Results: Phylogenetic analysis based on the 5′ end of the 16S rRNA gene (approximately 500 bp), was performed on 62 TAB isolated from the soil of several Japanese fruit orchards. The results suggested that 60 of the bacterial strains analysed belonged to the genus Alicyclobacillus, while the remaining two belonged to the genus Bacillus. The majority of strains (58%) were identified as Alicyclobacillus acidoterrestris. This group partitioned into three phylogenetically distinct subgroups (A–C). Isolates identified as A. acidiphilus (two strains), A. acidoterrestris (36 strains), and A. hesperidum subsp. aigle (one strain), produced guaiacol from vanillic acid. Levels of guaiacol production varied significantly among strains. The guaiacol producing phenotype was conserved among certain species, however no correlation was observed between levels of guaiacol production and 16S rRNA gene‐based phylogenetic relatedness. Conclusions: Alicyclobacillus acidoterrestris and Alicyclobacillus contaminans were widely distributed among various fruit orchards in Japan. Guaiacol production was common at the species/subspecies level; however the amount of guaiacol produced by each strain varied significantly. Significance and Impact of the Study: This study provides a comprehensive phylogenetic survey of Alicyclobacillus species in Japanese fruit orchards. Quality control standards for guaiacol producing Alicyclobacillus have also been described.     

Genotypic and Phenotypic Heterogeneity in Alicyclobacillus acidoterrestris: A Contribution to Species Characterization

Alicyclobacillus acidoterrestris is the main cause of most spoilage problems in fruit juices and acidic products. Since soil borne species often contaminate fruit juices and do not need strict extreme requirements for survival, it is a great concern to investigate whether and how soil species could evolve from their ecological niches in microbial community to new environments as fruit juices. In this study, 23 isolates of thermo-acidophilic, spore-forming bacteria from soil were characterized by cultural and molecular methods. In addition, 2 strains isolated from a spoilage incident in pear juice were typed. Strains phenotyping showed that they could be grouped into 3 different clusters, and some isolates showed identical or quite similar patterns. Analyzing pH and temperature ranges for growth, the majority of strains were able to grow at values described for many species of Alicyclobacillus. Qualitative utilization of lysine, arginine and indole production from tryptophan revealed, for the first time, deamination of lysine and decarboxylation of arginine. Resistance to 5% NaCl as well as the ability to hydrolyze starch and gelatin, nitrate reduction, catalase and oxidase activities confirmed literature evidences. Examining of 16S rRNA, showed that isolates were divided into three blocks represented by effectively soil species and strains that are moving from soil to other possible growing source characterized by parameters that could strongly influence bacterial survival. RAPD PCR technique evidenced a great variability in banding patterns and, although it was not possible to obtain genotypically well-distinguished groups, it was feasible to appreciate genetic similarity between some strains. In conclusion, the investigation of a microbial community entails a combination of metagenomic and classic culture-dependent approaches to expand our knowledge about Alicyclobacillus and to look for new subspecies.     

Isolation and identification of species of Alicyclobacillus from orchard soil in the Western Cape, South Africa

Alicyclobacilli were isolated from orchard soil collected from an apple and pear farm in Elgin, Western Cape, South Africa. Morphological, biochemical and physiological characteristics of the isolates were used to presumptively classify them as belonging to the genus Alicyclobacillus. Strains were identified to species level by polymerase chain reaction (PCR) with genus-specific primers, and 16S ribosomal RNA (rRNA) gene sequencing. To our knowledge this is the first report on the isolation of Alicyclobacillus acidoterrestris and Alicyclobacillus acidocaldarius from orchard soil. The presence of these organisms in the soil suggests a possible source of contamination for the final fruit juice, concentrate or pulp.     

Detection of Alicyclobacillus spp. in Fruit Juice by Combination of Immunomagnetic Separation and a SYBR Green I Real-Time PCR Assay

An approach based on immunomagnetic separation (IMS) and SYBR Green I real-time PCR (real-time PCR) with species-specific primers and melting curve analysis was proposed as a rapid and effective method for detecting Alicyclobacillus spp. in fruit juices. Specific primers targeting the 16S rDNA sequences of Alicyclobacillus spp. were designed and then confirmed by the amplification of DNA extracted from standard strains and isolates. Spiked samples containing known amounts of target bacteria were used to obtain standard curves; the correlation coefficient was greater than 0.986 and the real-time PCR amplification efficiencies were 98.9%- 101.8%. The detection limit of the testing system was 2.8×10¹ CFU/mL. The coefficient of variation for intra-assay and inter-assay variability were all within the acceptable limit of 5%. Besides, the performance of the IMS-real-time PCR assay was further investigated by detecting naturally contaminated kiwi fruit juice; the sensitivity, specificity and accuracy were 91.7%, 95.9% and 95.3%, respectively. The established IMS-real-time PCR procedure provides a new method for identification and quantitative detection of Alicyclobacillus spp. in fruit juice.     

Influence of Different Filling,Cooling, and Storage Conditions on the Growth of Alicyclobacillus acidoterrestris CRA7152 in Orange Juice

The prevention of spoilage by Alicyclobacillus acidoterrestris is a current challenge for fruit juice and beverage industries worldwide due to the bacterium''s acidothermophilic growth capability, heat resistance, and spoilage potential. This study examined the effect of storage temperature on A. acidoterrestris growth in hot-filled orange juice. The evolution of the A. acidoterrestris population was monitored under six different storage conditions after pasteurization (at 92°C for 10 s), maintenance at 85°C for 150 s, and cooling with water spray to 35°C in about 30 min and using two inoculum levels: <10¹ and 10¹ spores/ml. Final cooling and storage conditions were as follows: treatment 1, 30°C for the bottle cold point and storage at 35°C; treatment 2, 30°C for 48 h and storage at 35°C; treatment 3, 25°C for the bottle cold point and storage at 35°C; treatment 4, 25°C for 48 h and storage at 35°C; treatment 5, storage at 20°C (control); and treatment 6, filling and storage at 25°C. It was found that only in treatment 5 did the population remain inhibited during the 6 months of orange juice shelf life. By examining treatments 1 to 4, it was observed that A. acidoterrestris predicted growth parameters were significantly influenced (P < 0.05) either by inoculum level or cooling and storage conditions. The time required to reach a 10⁴ CFU/ml population of A. acidoterrestris was considered to be an adequate parameter to indicate orange juice spoilage by A. acidoterrestris. Therefore, hot-filled orange juice should be stored at or below 20°C to avoid spoilage by this microorganism. This procedure can be considered a safe and inexpensive alternative to other treatments proposed earlier.The first Alicyclobacillus sp. discovered was isolated in 1982 from spoiled apple juice aseptically packed in Germany and was considered at that time strictly limited to thermophilic and acidic environments (5). The spoilage of fruit juices by Alicyclobacillus is characterized by “off” flavors (medicinal or phenolic) due to guaiacol, 2,6-dibromophenol, and 2,6-dichlorophenol (20, 32, 25). As the spoilage does not show any evident signs like swelling of the container or any overt changes in the fruit juice (e.g., pH or turbidity), it is often not recognized until the packages are opened, the product is tasted, and consumer complaints are received by the manufacturer (6).Alicyclobacillus acidoterrestris, Alicyclobacillus cycloheptanicus and Alicyclobacillus acidocaldarius were the first three species described when the Alicyclobacillus genus was created in 1992 (31). Although there are currently more than 15 species described (27), only four Alicyclobacillus species (A. acidoterrestris, A. pomorum, A. herbarius and A. acidophilus) have shown the ability to produce off flavors in fruit juices or beverages (5, 13, 9, 1). Of these, A. acidoterrestris is considered the most important spoilage species within the Alicyclobacillus genus either by its frequency of occurrence or by its linkage to the spoilage problems of fruit juices and beverages. The broad temperature range for A. acidoterrestris growth (25 to 60°C) (33, 21, 20, 30, 11), its ability to grow under acidic environments (pH 2.5 to 6.0) (20, 30, 19, 22, 6), and its high heat resistance in orange juice (D at 95°C of 2.7 min) (8) together provide adequate conditions for both survival through pasteurization and growth during juice storage.A. acidoterrestris growth and the consequent orange juice spoilage can lead to enormous economic losses; therefore, this microorganism is currently considered a major challenge for the fruit juice industries. It is known to be difficult, if not impossible, to guarantee the absence of Alicyclobacillus spores on the surface of fruits used to make juices since the soil is the primary niche of Alicyclobacillus spp. (7). Subsequently, control measures such as avoiding fruit contact with soil and the use of sanitizers during the fruit washing step before crushing have been studied (16, 12). In addition, fruit juice producers need to better control the pasteurization conditions and to redesign their thermal processes for targeting Alicyclobacillus spp. (28, 24). However, the limited effectiveness of sanitizers against Alicyclobacillus spores and the sensory and nutritional problems that may arise from increased time and temperature regimes in pasteurization are recognized. Since the complete inactivation of Alicyclobacillus spores from raw materials may not be feasible and since juice spoilage by this microorganism depends on the germination of spores and outgrowth, studies reporting conditions that avoid spoilage by controlling Alicyclobacillus spore germination are necessary. The best option to manage the challenge that Alicyclobacillus presents to the fruit juice industries will be one that results in the fewest alterations in processing and storage conditions, preserves the nutritional and sensory aspects of the final products, does not impact production costs and commercial practices, and at the same time ensures the control of the microorganism.In the fruit juice industry, two main types of thermal processes are commonly applied: pasteurization followed by a hot-fill process or pasteurization followed by a cold-fill process (6). In the former, after the product is heated to >90 to 95°C, it is held hot for 15 to 20 s. As the temperature decreases to 82 to 84°C, the product is filled into the package. Next, the product is held for approximately 2 min before the packages are cooled to room temperature. Hot filling has been extensively used in the manufacturing processes of fruit-based drinks and beverages, but problems due to spoilage caused by Alicyclobacillus may arise during fruit juice shelf life. This is due to the extended time that the product is maintained at temperature conditions adequate for the germination and outgrowth of acidothermophilic spore-forming microbes. Despite several studies regarding the factors that affect A. acidoterrestris growth and heat resistance (12, 18, 2, 29), there is a lack of research on the effects of hot-filled fruit juice storage conditions on A. acidoterrestris growth during juice storage. Thus, this study aimed at estimating and comparing the growth parameters (maximum population ratio, κ; lag time, λ; and maximum growth rate, μ) of A. acidoterrestris survival in hot-filled orange juice that was cooled and stored under several conditions that simulate industrial and commercial practices. Primary growth parameters were estimated by using the Baranyi predictive model (3). Additionally, orange juice cooling and storage conditions that avoided germination, growth, and guaiacol production by A. acidoterrestris CRA 7152 were determined.     

Alicyclobacillus tengchongensis sp. nov., a thermo-acidophilic bacterium isolated from hot spring soil

A thermo-acidophilic bacterium, designated strain ACK006^T, was isolated from the soil of a hot spring at Tengchong in China. Cells were Gram-staining-positive, motile, catalase-positive and oxidase-negative, spore-forming rods. The isolate grew aerobically at 30–50°C (optimum at 45°C), pH 2.0–6.0 (optimum pH 3.2) and 0–5.0% (w/v) NaCl (optimum 1% NaCl). Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain ACK006^T belongs to the genus Alicyclobacillus with the sequence similarity of 92.3, 92.4, 92.5, and 92.8% to Alicyclobacillus cycloheptanicus SCH^T, Alicyclobacillus ferrooxydans TC-34^T, Alicyclobacillus contaminans 3-A191^T and Alicyclobacillus disulfidooxidans SD-11^T, respectively. Similarity to other species of the genus Alicyclobacillus was 90.3–92.8% and similarity to species of the genus Tumebacillus was 85.9–87.8%. The genomic DNA G+C content was 53.7 mol%. The predominant menaquinone was MK-7. Major fatty acids were ω-cycloheptane C_18:0, iso-C_17:0 and anteiso-C_17:0. The cell-wall peptidoglycan was the A1γ type; containing meso-diaminopimelic acid as the diagnostic diamino acid. On the basis of polyphasic analysis from this study, strain ACK006^T represents a novel species of the genus Alicyclobacillus for which the name Alicyclobacillus tengchongensis sp. nov. is proposed. The type strain is ACK006^T (=KCTC 33022^T =DSM 25924^T).     

Characterization of bacteriocin bificin C6165: a novel bacteriocin

Aims

To purify and primarily characterize an anti‐Alicyclobacillus bacteriocin produced by Bifidobacterium animalis subsp. animalis CICC 6165, suggested to be named bificin C6165.

Methods and Results

During purification of the bificin C6165, optimal recovery was achieved with ammonium sulfate precipitation followed by two chromatographic steps. Mass spectrometry analyses revealed a distinctive peak corresponding to a molecular mass of 3395·1 Da. This bacteriocin was heat stable, effective after refrigerated storage and freeze–thaw cycles. The primary mode of action of bificin C6165 is most probably due to pore formation, as indicated by the efflux of K⁺ from metabolically active cells of Alicyclobacillus acidoterrestris. In the presence of 10 mmol l^?1 gadolinium, bificin C6165 did not affect cells of Alicyclobacillus acidoterrestris. This suggests that the mode of action of bificin C6165 relies on a net negatively charged cell surface.

Conclusions

Bificin C6165 is indeed a novel bacteriocin and it exhibited remarkable potency for Alicyclobacillus control.

Significance and Impact of the Study

Application of bacteriocins in preservation of fruit juices has seldom been studied. Bificin C6165 may be an alternative method to control juice spoilage by this Alicyclobacillus acidoterrestris and meet increasing consumer demand for nature and artificial chemical additive‐free food products.     

Prevention of the accumulation of Alicyclobacillus in apple concentrate by restricting the continuous process running time

Aims: To study the accumulation of vegetative cells and endospores of Alicyclobacillus, as well as viable aerobic counts during the continuous production of apple juice concentrate. Methods and Results: Apples were processed for a continuous process running time of 108 h (processing rate 1·8–2·0 t h^?1) without clean‐in‐place (CIP) procedures in‐between different batches. Samples from single‐strength apple juice, concentrate after evaporation (±30°Brix), the final product (concentrate pasteurized at 102–104°C for 90 s) and condensate water (by‐product of the juice concentration process) were collected every 12 h. From 12 to 84 h of processing, vegetative Alicyclobacillus counts in single‐strength apple juice increased significantly (P < 0·05) from 1 to 3·15 log₁₀ CFU ml^?1. Accumulation patterns of vegetative cells in apple concentrate and the final product were similar from 24 to 84 h of processing, with the respective counts increasing from 0·13 to 1·63 and 0·01 to 1·69 log₁₀ CFU ml^?1. The highest Alicyclobacillus endospore counts in single‐strength juice, concentrate and the final product was at 84 h of processing with 1·32, 1·59 and 1·64 log₁₀ CFU ml^?1, respectively. Conclusions: Alicyclobacillus vegetative cells and endospores accumulate in fruit concentrates during a continuous process running time of 108 h. Significance and Impact of the Study: In conjunction with good manufacturing practices, fruit concentrate manufactures can minimize Alicyclobacillus accumulation in fruit concentrates by limiting the continuous process running time between clean‐ups to under 84 h.     

Effect of Yeast Cell Morphology,Cell Wall Physical Structure and Chemical Composition on Patulin Adsorption

The capability of yeast to adsorb patulin in fruit juice can aid in substantially reducing the patulin toxic effect on human health. This study aimed to investigate the capability of yeast cell morphology and cell wall internal structure and composition to adsorb patulin. To compare different yeast cell morphologies, cell wall internal structure and composition, scanning electron microscope, transmission electron microscope and ion chromatography were used. The results indicated that patulin adsorption capability of yeast was influenced by cell surface areas, volume, and cell wall thickness, as well as 1,3-β-glucan content. Among these factors, cell wall thickness and 1,3-β-glucan content serve significant functions. The investigation revealed that patulin adsorption capability was mainly affected by the three-dimensional network structure of the cell wall composed of 1,3-β-glucan. Finally, patulin adsorption in commercial kiwi fruit juice was investigated, and the results indicated that yeast cells could adsorb patulin from commercial kiwi fruit juice efficiently. This study can potentially simulate in vitro cell walls to enhance patulin adsorption capability and successfully apply to fruit juice industry.     

Comparison of Enterococcus faecium and Enterococcus faecalis Strains Isolated from Water and Clinical Samples: Antimicrobial Susceptibility and Genetic Relationships

Enterococci are part of the normal intestinal flora in a large number of mammals, and these microbes are currently used as indicators of fecal contamination in water and food for human consumption. These organisms are considered one of the primary causes of nosocomial and environmental infections due to their ability to survive in the environment and to their intrinsic resistance to antimicrobials. The aims of this study were to determine the biochemical patterns and antimicrobial susceptibilities of Enterococcus faecalis and E. faecium isolates from clinical samples and from water (groundwater, water from the Xochimilco wetland, and treated water from the Mexico City Metropolitan Area) and to determine the genetic relationships among these isolates. A total of 121 enterococcus strains were studied; 31 and 90 strains were isolated from clinical samples and water (groundwater, water from the Xochimilco wetland, and water for agricultural irrigation), respectively. Identification to the species level was performed using a multiplex PCR assay, and antimicrobial profiles were obtained using a commercial kit. Twenty-eight strains were analyzed by pulsed-field gel electrophoresis (PFGE). E. faecium strains isolated from water showed an atypical biochemical pattern. The clinical isolates showed higher resistance to antibiotics than those from water. Both the enterococci isolated from humans, and those isolated from water showed high genetic diversity according to the PFGE analysis, although some strains seemed to be closely related. In conclusion, enterococci isolated from humans and water are genetically different. However, water represents a potential route of transmission to the community and a source of antimicrobial resistance genes that may be readily transmitted to other, different bacterial species.     

Inhibitory Effects of High Pressure and Heat on Alicyclobacillus acidoterrestris Spores in Apple Juice

The effectiveness of combined high pressure and heat treatment for reducing spore levels of Alicyclobacillus acidoterrestris, a thermoacidophilic spore-forming bacterium, in commercial pasteurized apple juice was investigated. Spores suspended in apple juice were successfully destroyed by combining high pressure with a mild or high temperature (45, 71, or 90°C).     

Identification of medicinal off-flavours generated by Alicyclobacillus species in orange juice using GC-olfactometry and GC-MS

AIMS: The objective of this study was to identify compounds responsible for medicinal off-flavours produced by different species and strains of Alicyclobacillus in orange juice using a combination of chromatographic-coupled olfactometric techniques and gas chromatography-mass spectrometry (GC-MS). METHODS AND RESULTS: Each of five Alicyclobacillus strains was inoculated into separate juice samples and incubated up to 28 days at 45 degrees C. Aroma compounds in the juice were analysed by GC-olfactometry (GC-O) and confirmed using GC-MS. GC-O identified three components that were described as medicinal/antiseptic. Microbial populations were enumerated at timed intervals by spiral plating onto Alicyclobacillus agar. Within 28 days incubation, all five strains produced medicinal off-aromas from guaiacol and at least one halogenated phenol. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to evaluate individual juice aroma components produced by Alicyclobacillus species using olfactometry and to demonstrate that at least three medicinal off-flavour compounds are associated with the growth of alicyclobacilli in orange juice.     

A real-time polymerase chain reaction-based method for rapid and specific detection of spoilage Alicyclobacillus spp. in apple juice

AIMS: To develop a real-time PCR-based rapid detection method for spoilage Alicyclobacillus spp. in juice products. METHODS AND RESULTS: The squalene-hopene cyclase-encoding gene was targeted for primer-and-probe development. Gene fragments from representative strains were cloned, and PCR primers and probe were designed by DNA sequence comparison. Selected bacteria were examined for cross-reactivity by the new method. Cells were serially diluted in apple juice and saline, and examined by the new method to establish detection sensitivity. Using the newly developed Taqman real-time PCR-based method, strains of Alicyclobacillus acidocaldarius and A. acidoterrestris were detected without cross reactivity with other common food-borne micro-organisms. Detection of <10 cells per PCR reaction from juice samples was accomplished within 3-5 h. CONCLUSION: This is the first reported real-time PCR-based detection method for Alicyclobacillus spp. and its application in juice products is demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: As a favourable alternative for the laborious and time-consuming culture- or biochemical characterization-based techniques, the system has great potential for industrial applications from raw material screening to final product quality control.     

Bacteriophage Ecology in Commercial Sauerkraut Fermentations

Knowledge of bacteriophage ecology in vegetable fermentations is essential for developing phage control strategies for consistent and high quality of fermented vegetable products. The ecology of phages infecting lactic acid bacteria (LAB) in commercial sauerkraut fermentations was investigated. Brine samples were taken from four commercial sauerkraut fermentation tanks over a 60- or 100-day period in 2000 and 2001. A total of 171 phage isolates, including at least 26 distinct phages, were obtained. In addition, 28 distinct host strains were isolated and identified as LAB by restriction analysis of the intergenic transcribed spacer region and 16S rRNA sequence analysis. These host strains included Leuconostoc, Weissella, and Lactobacillus species. It was found that there were two phage-host systems in the fermentations corresponding to the population shift from heterofermentative to homofermentative LAB between 3 and 7 days after the start of the fermentations. The data suggested that phages may play an important role in the microbial ecology and succession of LAB species in vegetable fermentations. Eight phage isolates, which were independently obtained two or more times, were further characterized. They belonged to the family Myoviridae or Siphoviridae and showed distinct host ranges and DNA fingerprints. Two of the phage isolates were found to be capable of infecting two Lactobacillus species. The results from this study demonstrated for the first time the complex phage ecology present in commercial sauerkraut fermentations, providing new insights into the bioprocess of vegetable fermentations.     

Control of Alicyclobacillus acidoterrestris in fruit juices by a newly discovered bacteriocin

Alicyclobacillus acidoterrestris is one of the most spoilage-causing bacteria in fruit juices. Control of A. acidoterrestris in fruit juices by bificin C6165 (Pei et al. in J Appl Microbiol 114(5):1273–1284, 2013), a bacteriocin produced by Bifidobacterium animalis subsp. animalis CICC 6165, was described in this study. Activity spectrum of bificin C6165 was investigated and sixteen strains of A. acidoterrestris were sensitive to bificin C6165 in diluted Apple Juices. In the commercial fruit juices, vegetative cells of A. acidoterrestris were inactivated by bificin C6165 at 40 μg/ml. The inhibitory effect of bificin C6165 was better at lower pH (pH 3.5) and at a higher temperature of 45 °C. Furthermore, electron microscopy examination of the vegetative cells treated with bacteriocin revealed substantial cell damage and bacterial lysis. The result suggested that primary mode of action of bificin C6165 was most probably due to pore formation. Although no significantly activity of bificin C6165 was observed against the endospores of A. acidoterrestris in commercial apple juice, the addition of bacteriocin contributed to the reduction of the thermal resistance of A. acidoterrestris spores. Additionally, encapsulation of bificin C6165 with Ca-alginate gel was investigated. Encapsulation of bificin C6165 provided a promising method to control A. acidoterrestris in food juice industry.     

Identification and Characterization of Leuconostoc fallax Strains Isolated from an Industrial Sauerkraut Fermentation

Lactic acid bacterial strains were isolated from brines sampled after 7 days of an industrial sauerkraut fermentation, and six strains were selected on the basis of susceptibility to bacteriophages. Bacterial growth in cabbage juice was monitored, and the fermentation end products were identified, quantified, and compared to those of Leuconostoc mesenteroides. Identification by biochemical fingerprinting, endonuclease digestion of the 16S-23S intergenic transcribed spacer region, and sequencing of variable regions V1 and V2 of the 16S rRNA gene indicated that the six selected sauerkraut isolates were Leuconostoc fallax strains. Random amplification of polymorphic DNA fingerprints indicated that the strains were distinct from one another. The growth and fermentation patterns of the L. fallax isolates were highly similar to those of L. mesenteroides. The final pH of cabbage juice fermentation was 3.6, and the main fermentation end products were lactic acid, acetic acid, and mannitol for both species. However, none of the L. fallax strains exhibited the malolactic reaction, which is characteristic of most L. mesenteroides strains. These results indicated that in addition to L. mesenteroides, a variety of L. fallax strains may be present in the heterofermentative stage of sauerkraut fermentation. The microbial ecology of sauerkraut fermentation appears to be more complex than previously indicated, and the prevalence and roles of L. fallax require further investigation.     

Yeast identification in grape juice concentrates from Argentina

Aims: The purpose of this study was to identify yeast species present in spoiled and unspoiled grape juice concentrates from Argentine industries. Methods and Results: Osmophilic and osmotolerant yeasts were isolated from spoiled – visually effervescent – and unspoiled – without any visible damage – grape juice concentrates by the spread‐plate technique in two culture media. Yeast identification was done by classical and molecular methods. Zygosaccharomyces rouxii was the only species isolated from spoiled grape juice concentrates. In unspoiled samples, five different species were identified: Z. rouxii was isolated at a higher frequency, followed in decreasing order by Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia anomala and Kluyveromyces delphensis. Conclusions: Yeasts isolated from grape juice concentrates were characterized by a limited taxonomic diversity, where Z. rouxii was the main species isolated. Significance and Impact of the Study: Grape production in Argentina is mainly devoted to the industry where wine and grape juice concentrates represent major types of commercial products. Little information on common yeast contaminants is available for grape juice concentrates. This study constitutes the first report of osmophilic yeast species present in spoiled and unspoiled grape juice concentrates elaborated in Argentina.     

Abiotic and Microbiotic Factors Controlling Biofilm Formation by Thermophilic Sporeformers

One of the major concerns in the production of dairy concentrates is the risk of contamination by heat-resistant spores from thermophilic bacteria. In order to acquire more insight in the composition of microbial communities occurring in the dairy concentrate industry, a bar-coded 16S amplicon sequencing analysis was carried out on milk, final products, and fouling samples taken from dairy concentrate production lines. The analysis of these samples revealed the presence of DNA from a broad range of bacterial taxa, including a majority of mesophiles and a minority of (thermophilic) spore-forming bacteria. Enrichments of fouling samples at 55°C showed the accumulation of predominantly Brevibacillus and Bacillus, whereas enrichments at 65°C led to the accumulation of Anoxybacillus and Geobacillus species. Bacterial population analysis of biofilms grown using fouling samples as an inoculum indicated that both Anoxybacillus and Geobacillus preferentially form biofilms on surfaces at air-liquid interfaces rather than on submerged surfaces. Three of the most potent biofilm-forming strains isolated from the dairy factory industrial samples, including Geobacillus thermoglucosidans, Geobacillus stearothermophilus, and Anoxybacillus flavithermus, have been characterized in detail with respect to their growth conditions and spore resistance. Strikingly, Geobacillus thermoglucosidans, which forms the most thermostable spores of these three species, is not able to grow in dairy intermediates as a pure culture but appears to be dependent for growth on other spoilage organisms present, probably as a result of their proteolytic activity. These results underscore the importance of abiotic and microbiotic factors in niche colonization in dairy factories, where the presence of thermophilic sporeformers can affect the quality of end products.     

Molecular application for identification of polycyclic aromatic hydrocarbons degrading bacteria (PAHD) species isolated from oil polluted soil in Dammam,Saud Arabia

Soil contamination with petroleum hydrocarbon products such as diesel and engine oil is becoming one of the major environmental problems. This study describes hydrocarbons degrading bacteria (PHAD) isolated from long-standing petrol polluted soil from the eastern region, Dammam, Saudi Arabia. The isolated strains were firstly categorized by accessible shape detection, physiological and biochemistry tests. Thereafter, a technique established on the sequence analysis of a 16S rDNA gene was used. Isolation of DNA from the bacterial strains was performed, on which the PCR reaction was carried out. Strains were identified based on 16S rDNA sequence analysis, As follows amplified samples were spontaneously sequenced automatically and the attained results were matched to open databases. Among the isolated bacterial strains, S1 was identified as Staphylococcus aureus and strain S1 as Corynebacterium amycolatum.     

Alicyclobacilli from an unexplored geothermal soil in Antarctica: Mount Rittmann

The isolation of three species from soil samples from an unexplored site in Antarctica, belonging to the Alicyclobacillus genus, is reported. The isolates, named MR1, MR2 and MR4, were from 30 samples of geothermal soil from Mount Rittmann. All the isolates had lipids based mainly on fatty acids possessing a terminal cyclohexane; hopanoid and quinones of MK-7 were also present. All isolates were characterized by phenotypic features and the isolate MR1 was subjected to genetic analyses. The isolates were thermoacidophilic, showing an optimum temperature of 63°C and an optimum pH of 3.5–4.0. They were able to grow in the temperature range 45–70°C. The percentage C+G of DNA of MR1 was 64.9% and showed a 16S rDNA similarity of 99.3% with Alicyclobacillus acidocaldarius ATCC 43034 and a DNA-DNA homology of 69.7% with A. acidocaldarius DSM 446. The strain MR1 is unable to produce amylolytic activity under different growth conditions. It is proposed to accommodate MR1 in the genus Alicyclobacillus as subspecies rittmannii of A. acidocaldarius. Received: 29 May 1997 / Accepted: 26 September 1997