Alicyclobacillus spp. in fruit-based products: Isolation, identification, quantitative assessment (SPME/GC-MS) of spoilage compounds and spore's resistance to thermal shocks.

Alicyclobacillus spp. is the cause of great concern for the food industry due to their spores' resistance (thermal and chemical) and the spoilage potential of some species. Despite this, not all Alicyclobacillus strains can spoil fruit juices. Thus, this study aimed to identify Alicyclobacillus spp. strains isolated from fruit-based products produced in Argentina, Brazil, and Italy by DNA sequencing. All Alicyclobacillus isolates were tested for guaiacol production by the peroxidase method. Positive strains for guaiacol production were individually inoculated at concentration of 103 CFU/mL in 10 mL of orange (pH 3.90) and apple (pH 3.50) juices adjusted to 11°Brix, following incubation at 45 °C for at least 5 days to induce the production of the following spoilage compounds: Guaiacol, 2,6-dichlorophenol (2,6-DCP) and 2,6-dibromophenol (2,6-DBP). The techniques of micro-solid phase extraction by headspace (HS-SPME) and gas-chromatography with mass spectrometry (GC-MS) were used to identify and quantify the spoilage compounds. All GC-MS data was analyzed by principal component analysis (PCA). The effects of different thermal shock conditions on the recovery of Alicyclobacillus spores inoculated in orange and apple juice (11°Brix) were also tested. A total of 484 strains were isolated from 48 brands, and the species A. acidocaldarius and A. acidoterrestris were the most found among all samples analyzed. In some samples from Argentina, the species A. vulcanalis and A. mali were also identified. The incidence of these two main species of Alicyclobacillus in this study was mainly in products from pear (n = 108; 22.3 %), peach (n = 99; 20.5 %), apple (n = 86; 17.8 %), and tomato (n = 63; 13 %). The results indicated that from the total isolates from Argentina (n = 414), Brazil (n = 54) and Italy (n = 16) were able to produce guaiacol: 107 (25.8 %), 33 (61.1 %) and 13 (81.2 %) isolates from each country, respectively. The PCA score plot indicated that the Argentina and Brazil isolates correlate with higher production of guaiacol and 2,6-DCP/2,6-DBP, respectively. Heatmaps of cell survival after heat shock demonstrated that strains with different levels of guaiacol production present different resistances according to spoilage ability. None of the Alicyclobacillus isolates survived heat shocks at 120 °C for 3 min. This work provides insights into the incidence, spoilage potential, and thermal shock resistance of Alicyclobacillus strains isolated from fruit-based products.

Isolation and identification of guaiacol producing Alicyclobacillus fastidiosus strains from orchards in Poland.

The genus Alicyclobacillus comprises a group of Gram-positive, thermo-acidophilic bacteria that are capable of producing highly resistant endospores during unfavorable environmental conditions. The members of this genus inhabit natural environments, including hot springs and soils. The main reason behind the spoilage of final commercial fruit products by Alicyclobacillus is the contamination of fruits with soil at the time of harvesting. Some of the Alicyclobacillus species, including Alicyclobacillus acidoterrestris, are categorized as spoilage bacteria due to their ability to produce off-flavor compounds (e.g., guaiacol and halophenols) that adversely affect the taste and aroma of beverages. In our study, Alicyclobacillus species were isolated from Polish orchard soils and fruits and were subjected to 16S rDNA sequencing. The results of the analysis showed that the isolated strains belonged to A. acidoterrestris and Alicyclobacillus fastidiosus species. All the three isolated strains of A. fastidiosus (f1, f2, f3) exhibited similar morphological and biochemical properties as the strain described in the literature. However, these isolated strains were able to produce guaiacol at temperatures of 20°C, 25°C, and 45°C. Thus, the strains of A. fastidiosus discovered in the present study can be included in the group of spoilage species as they possessed the gene responsible for the production of guaiacol.

DNA electrochemical biosensor for detection of Alicyclobacillus acidoterrestris utilizing Hoechst 33258 as indicator.

Alicyclobacillus acidoterrestris is an acidophilic and thermophilic bacterium present in the soil, often associated with the spoilage of acidic juices, such as orange juice. Their spores resist pasteurization and, when reactivated, modify the organoleptic properties of the juice, making it unsuitable for consumption, due mainly to production of guaiacol. Biosensors are detection devices that respond quickly and are easy to handle, with great potential for use in the juice production chain. In this context, this work reports an electrochemical genosensor for detection of A. acidoterrestris, based on a graphite electrode modified with electrochemically reduced graphene oxide, a polymer derived from 3-hydroxybenzoic acid and a specific DNA probe sequence complementary with the genomic DNA of A. acidoterrestris. Detection of the target was performed by monitoring the oxidation peak of the Hoechst 33258, a common DNA stainer. The genosensor detection limit was 12 ng mL-1 and it kept 77% of response after ten weeks, and a test showed that orange juice does not interfere with bacteria lysate detection. This biosensor is the first platform for electrochemical detection of the genomic DNA of A. acidoterrestris in the literature, and the first to use Hoechst 33258 as indicator with whole genomic DNA molecules.

Preparation of species-specific monoclonal antibody and development of fluorescence immunoassay based on fluorescence resonance energy transfer of carbon dots for accurate and sensitive detection of Alicyclobacillus acidoterrestris in apple juice.

The growth and metabolism of Alicyclobacillus acidoterrestris can lead to the spoilage of commercial fruit juice. Existing methods have some drawbacks such as complex sample pretreatment, skilled technician requirement, reduced sensitivity and specificity. Herein, a novel fluorescence immunoassay was developed using a monoclonal antibody (mAb) against A. acidoterrestris as the sensing element and carbon dots (CDs) as the signal response unit. The CDs can be quenched via fluorescence resonance energy transfer (FRET) by the oxidization product of p-phenylenediamine (PPD), a chromogenic substrate of horseradish peroxidase (HRP). This approach showed enhanced accuracy and sensitivity with relatively low limit of detection (LOD) of 6.16 × 102 CFU mL-1. Moreover, apple juice contaminated with 1 CFU mL-1 of A. acidoterrestris can be identified after 24 h enrichment. This fluorescence immunoassay could serve as a powerful tool for laboratory identification and on-site inspection of A. acidoterrestris, reducing the adverse effect on the quality of fruit juice.

Heat resistance and genomics of spoilage Alicyclobacillus spp. Isolated from fruit juice and fruit-based beverages.

Alicyclobacillus acidoterrestris is a spore-forming bacterium of importance to the fruit juice industry due to its remarkable heat resistance and production of guaiacol taint. Whole genome sequencing analysis reveals species demarcation corresponds to the two major genotypic groups to which A. acidoterrestris isolates belong. Heat resistance was significantly different between genotypic groups 1 and 2 with D90 values of 15.5 and 9.3 min, respectively (p < 0.01). Comparison of squalene-hopene cyclase (shc) encoding sequences reveals non-synonymous changes and the alteration of glutamine residues. Glutamine absence may link to the stability reinforcement of the enzyme structure against thermal denaturation. Genomic islands harbouring heavy metal resistance genes are found in the majority of genotypic group 1 genomes (63%) but occurs in only one genome (5%) of genotypic group 2. Distribution of the genomic islands in the genotypic groups 1 and 2 is also consistent with phylogenetic trees and ANI and dDDH values. Subsequently, we propose genotypic group 1 as a new species closely related to A. acidoterrestris that possesses enhanced heat resistance.

Development of the Saltatory Rolling Circle Amplification Assay for Rapid and Visual Detection of Alicyclobacillus acidoterrestris in Apple Juice.

A novel nucleic acid isothermal amplification method based on saltatory rolling circle amplification (SRCA) for rapid and visual detection of Alicyclobacillus acidoterrestris in apple juice was established. Fourteen A. acidoterrestris strains and 44 non-A. acidoterrestris strains were used to confirm the specificity. The sensitivity of SRCA was 4.5 × 101 CFU/mL by observing the white precipitate with the naked eye, while it was 4.5 × 100 CFU/mL by fluorescence visualization. The detection limit of SRCA in artificially inoculated apple juice was 7.1 × 101 and 7.1 × 100 CFU/mL via visualization of the white precipitate and fluorescence, respectively. Compared with the traditional PCR method, SRCA exhibited at least a 100-fold higher sensitivity and 100-fold lower detection limit. Seventy samples were investigated for A. acidoterrestris contamination, and the results showed 100% sensitivity, 97.01% specificity, and 97.14% accuracy compared with those by the conventional microbiological cultivation method. Overall, this method is a potentially useful tool for visual and rapid detection of A. acidoterrestris.

A novel thermal Cas12b from a hot spring bacterium with high target mismatch tolerance and robust DNA cleavage efficiency.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas), such as Cas9 and Cpf1, are RNA-guided endonucleases that target and degrade nucleic acids, providing powerful genomic editing and molecular diagnostic tools. Cas12b enzymes are distinct effectors; however, their features and catalytic boundaries require further characterization. We identified BrCas12b from the thermophile bacterium Brevibacillus sp. SYSU G02855 as a novel ortholog of cas12b. Biochemical analyses revealed that BrCas12b is a dual-RNA-guided endonuclease with higher optimum reaction temperature than that of other reported members of Cas12b. The seed sequence of BrCas12b is only 4 nt in length, indicating that it has greater target mismatch tolerance than that of previously reported Cas effectors; however, it contains a compensatory effect at the position of the cleavage site. Using fluorescence-based detection method to evaluate target cleavage efficiency, we showed that BrCas12b has robust enzymatic cleavage activity (Kcat/Km (s-1 M-1) = 8.80 × 1011), which is significantly higher than that of AacCas12b (Kcat/Km (s-1 M-1) = 7.56 × 108) from Alicyclobacillus acidoterrestris. The results increase our understanding of the catalytic mechanism of Cas12b family members and suggest that BrCas12b might be useful in the application of genomic editing and molecular diagnosis.

Diversity and guaiacol production of Alicyclobacillus spp. from fruit juice and fruit-based beverages.

Alicyclobacillus acidoterrestris is an acido-thermophilic, spore-forming bacterial species that can spoil acidic fruit juice and fruit-based beverages. The metabolism of taint compounds by this bacterial species has led to its status as a targeted microorganism in the fruit juice industry. This study aims to assess the genetic diversity of Alicyclobacillus spp. including A. acidoterrestris and its correlation to spoilage taint metabolism. Alicyclobacillus cultures, which were previously isolated from a wide range of domestic and international products including fruit juice, fruit drinks and fruit juice concentrates, were subjected to DNA fingerprint analysis by using randomly amplified polymorphic DNA (RAPD) - polymerase chain reaction. Isolates were classified on the basis of their RAPD profile and the results were used to select representative strains to undergo taint production assessment. The taint guaiacol produced by Alicyclobacillus spp. was measured by headspace gas chromatography and mass spectrometry. From produced RAPD profiles, two genotypic groups and two sub-groups were identified. The groups were independent of product types and geographical origins. A significant number of isolates were clustered in genotypic group I, including A. acidoterrestris ATCC 49025. These isolates produced significant levels of guaiacol, 8.7 mg/L on average. A smaller number of isolates was found in genotypic group II including A. acidocaldarius and they produced no guaiacol. Primer F-64 was useful to identify Alicyclobacillus at the species level, and permitted rapid identification of strains producing fruit juice taint compounds such as guaiacol.

Effect of ultraviolet (UV-C) radiation on spores and biofilms of Alicyclobacillus spp. in industrialized orange juice.

Bacteria of the genus Alicyclobacillus pose serious quality problems for the juice processing industries that have sought effective alternatives for its control. The present study evaluated the effect of UV-C radiation on the reduction of spores and biofilm formation of Alicyclobacillus spp. on stainless steel and rubber surfaces using industrialized orange juice as a culture medium. Four reference Alicyclobacillus spp. species and different UV-C dosages were investigated. After exposed for 20 min (16.8 kJ/m2) to UV-C, the spores of Alicyclobacillus acidoterrestris, Alicyclobacillus herbarius, and Alicyclobacillus cycloheptanicus decreased drastically more of 4 log CFU/mL, with counts below the detection limit of the method (<1.7 log CFU/mL), while the Alicyclobacillus acidocaldarius spores were more sensitive to UV-C, once this spore reduction was observed within 15 min (12.6 kJ/m2). Morphological changes in the Alicyclobacillus acidoterrestris spores were observed by scanning electron microscopy. A reduction of biofilm formation was observed for all UV-C treatments, and the higher reductions (approximately 2 log CFU/mL) were found for the Alicyclobacillus acidocaldarius species after 30 min (26.2 kJ/m2), on the stainless steel and rubber surfaces. The results suggest that UV-C can be used to reduce the biofilm formation and could be a promising alternative for controlling Alicyclobacillus spp. spores in industrialized orange juice.

Genetic Heterogeneity of Alicyclobacillus Strains Revealed by RFLP Analysis of vdc Region and rpoB Gene.

PCR-RFLP targeting of the 16S rDNA and rpoB genes, as well as the vdc region, was applied to identify and differentiate between the spoilage and non-spoilage Alicyclobacillus species. Eight reference strains and 75 strains isolated from spoiled juices, juice concentrates, drinks, its intermediates, and fresh apples were subject to study. Hin6I restriction patterns of the 16S rDNA gene enabled distinguishing between all the species analyzed, while the rpoB gene and vdc gene cluster analysis also revealed that there were two major types among the A. acidoterrestris isolates, one similar to the reference strain A. acidoterrestris DSM 2498, and the other similar to the reference strain A. acidoterrestris ATCC 49025. Heterogeneity was also observed among the A. acidocaldarius isolates. RFLP analysis of the 16S rDNA and rpoB genes, as well as vdc region, can be used successfully in the identification and research of intraspecies heterogeneity of the Alicyclobacillus species.

Synthesis of multifunctional fluorescent magnetic nanoparticles for the detection of Alicyclobacillus spp. in apple juice.

An approach based on multifunctional fluorescent magnetic nanoparticles was proposed for the enrichment and identification of Alicyclobacillus spp. in apple juice simultaneously. The prepared Fe3O4 magnetic particles (MNPs) were modified by the sol-gel process and a silica shell was formed to improve the reactivity, and then the obtained MNPs@SiO2-SH nanoparticles were conjugated with Thioglycolic acid functionalized CdTe/CdS QDs via thiols chemistry. The characteristic evaluation results indicated that the MNPs-QD nanocomposites exhibited good magnetic properties and optical characterization. The polyclonal anti-Alicyclobacillus IgG antibody was immobilized onto the surface of MNPs-QD materials via esterification reactions. The maximum antibody immobilization capacity was 119.62 µg/mg and the adsorption reaction could be accomplished in 60 min. The adsorption process could be represented by Langmuir model and pseudo-second order kinetics equation, respectively. Based on the high immunocapture efficiency and sensitive fluorescence characteristics, the obtained MNPs-QDs-antibody conjugates could be applied to recognize the contamination of Alicyclobacillus spp. and a quantitative analysis method was established for target cells detection. The minimum quantitative limit was 104 CFU (colony forming unit)/mL and the testing process could be completed in 90 min. The results indicated that the MNPs-QDs-antibody conjugates can be successfully applied for immunocapture and detection of Alicyclobacillus spp. in apple juice. That is to say, the developed MNPs-QDs-antibody conjugates have exhibit more attractive and great potential for the immunocapture and recognition of target bacteria, fully demonstrated a new method for enrichment and rapid detection of Alicyclobacillus spp. in fruit juices.

Description of Alicyclobacillus montanus sp. nov., a mixotrophic bacterium isolated from acidic hot springs.

Three morphologically similar thermo-acidophilic strains, USBA-GBX-501, USBA-GBX-502 and USBA-GBX-503T, were isolated from acidic thermal springs at the National Natural Park Los Nevados (Colombia). All isolates were spore-forming, Gram-stain-positive and motile, growing aerobically at 25-55 °C (optimum ~45 °C) and at pH 1.5-4.5 (optimum pH ~3.0). Phylogenetic analysis of the 16S rRNA gene sequences of these isolates showed an almost identical sequence (99.0 % similarity) and they formed a robust cluster with the closest relative Alicyclobacillus tolerans DSM 16297T with a sequence similarity of 99.0 %. Average similarity to other species of the genus Alicyclobacillus was 93.0 % and average similarity to species of the genus Effusibacillus was 90 %. In addition, the level of DNA-DNA hybridization between strain USBA-GBX-503T and Alicyclobacillus tolerans DSM 16297T was 31.7 %. The genomic DNA G+C content of strain USBA-GBX-503T was 44.6 mol%. The only menaquinone was MK-7 (100.0 %). No ω-alicyclic fatty acids were detected in strain USBA-GBX-503T, and the major cellular fatty acids were C18 : 1ω7c, anteiso-C17 : 0 and iso-C17 : 0. Based on phenotypic and chemotaxonomic characteristics, phylogenetic analysis and DNA-DNA relatedness values, along with low levels of identity at the whole genome level (ANIb and ANIm values of <67.0 and <91.0 %, respectively), it can be concluded that strain USBA-GBX-503T represents a novel species of the genus Alicyclobacillus, for which the name Alicyclobacillus montanus sp. nov. is proposed. The type strain is USBA-GBX-503T (=CMPUJ UGB U503T=CBMAI1927T).

Identification of Quorum-Sensing Signal Molecules and a Biosynthetic Gene in Alicycliphilus sp. Isolated from Activated Sludge.

Activated sludge is a complicated mixture of various microorganisms that is used to treat sewage and industrial wastewater. Many bacteria produce N-acylhomoserine lactone (AHL) as a quorum-sensing signal molecule to regulate the expression of the exoenzymes used for wastewater treatment. Here, we isolated an AHL-producing bacteria from an activated sludge sample collected from an electronic component factory, which we named Alicycliphilus sp. B1. Clone library analysis revealed that Alicycliphilus was a subdominant genus in this sample. When we screened the activated sludge sample for AHL-producing strains, 12 of 14 the AHL-producing isolates were assigned to the genus Alicycliphilus. A putative AHL-synthase gene, ALISP_0667, was cloned from the genome of B1 and transformed into Escherichia coli DH5α. The AHLs were extracted from the culture supernatants of the B1 strain and E. coli DH5α cells harboring the ALISP_0667 gene and were identified by liquid chromatography-mass spectrometry as N-(3-hydroxydecanoyl)-l-homoserine lactone and N-(3-hydroxydodecanoyl)-l-homoserine lactone. The results of comparative genomic analysis suggested that the quorum-sensing genes in the B1 strain might have been acquired by horizontal gene transfer within activated sludge.

Culture dependent and independent genomic identification of Alicyclobacillus species in contaminated commercial fruit juices.

Alicyclobacillus is a genus of thermo-acidophilic, endospore-forming, bacteria species which occasionally cause spoilage of heat-processed fruit juices by producing guaiacol taint. In this study, Alicyclobacillus contamination of commercial fruit juices in West Africa was investigated using culture-dependent and -independent approaches. Firstly, a total of 225 fruit juice products from Ghana (n = 39) and Nigeria (n = 186) were enriched with yeast-starch-glucose (YSG) broth (pH 3.7) following heat shock at 80 °C for 10 min. Alicyclobacillus was detected in 11.6% (26) of samples. Isolates were identified to the genus taxonomic level by genus-specific PCR which targeted the squalene-hopene-cyclase (shc) gene followed by analysis of the almost-complete 16S ribosomal RNA (rRNA) gene sequences that identified 16 Alicyclobacillus acidoterrestris, 7 Alicyclobacillus acidocaldarius and 3 Alicyclobacillus genomic species 1 (Alicyclobacillus sp. 1). Whole-genome fingerprinting using PCR-RAPD primers Ba-10, F-61 and F-64 grouped the 16 A. acidoterrestris isolates into two genetic clusters. Furthermore, high performance liquid chromatographic (HPLC) analyses revealed the activity of vanillic-acid decarboxylase (vdc) in all A. acidoterrestris isolates due to guaiacol production from vanillic-acid. Lastly, species-specific PCR-DGGE targeting the 16S rRNA gene clearly discriminated between the guaiacol-producing A. acidoterrestris and the non-spoilage A. acidocaldarius group. Information provided by this study is fundamental to the development of effective strategies for the improvement of quality and shelf-life of processed tropical fruit juices in W. Africa.

[Gene cloning, heterologous expression and enzyme characterizations of halo-tolerant endoglucanase from Alicyclobacillus D-1].

Objective: An endoglucanase gene (gluE1) was cloned from a thermalacidophilus (Alicyclobacillus tengchongensis CGMCC1504) isolated from a hot spring, and the sequence and biochemical characterization of enzyme were analyzed. Methods: The full-length gluE1 was obtained based on genome sequencing, analysis of amino acid sequence of GluE1. gluE1 was ligated into pEASY-E2 vector and expressed in Escherichia coli BL21 (DE3) cells. GluE1 was purified to electrophoretic homogeneity by Ni2+-NTA metal chelating affinity chromatography, and then the enzyme characterizations were determined. Results: The 1020 bp full-length gluE1 (50.5% GC content) encodes a 339 residues polypeptide (GluE1: 40.45 kDa). GluE1 showed the highest identity of 97% with endoglucanase in public databases, and <60% identities with other endoglucanase. GluE1 efficiently hydrolyzed CMC-Na, soluble starch and barley-ß-glucan, which showed apparent optimal at pH 6.5 and 55℃. GluE1 was stable and active (>60%) at pH 5.0-10.0, and had a high stability at 37℃; and it exhibited Km, Vmax and kcat values of 8.58 mg/mL, 416.67 U/mg and 280.90 s-1 respectively. GluE1 was strongly inhibited by Ag+, Hg2+ and SDS, partial promoted by ß-Mercaptoethanol, Pb2+, Mg2+, Ca2+ and Na+, 30% NaCl still retains more than 64% of the activity. The residual enzyme activity kept 93% after pre-incubation of the enzyme in 30% NaCl. Conclusion: Endoglucanase gene gluE1 from Alicyclobacillus was first reported, and GluE1 showed a good pH stability and strong halo-tolerant property. GluE1 might have greater potential applications.

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.

Alicyclobacillus fodiniaquatilis sp. nov., isolated from acid mine water.

Two novel, Gram-stain-variable, moderately thermophilic, acidophilic, rod-shaped, endospore-forming bacteria, G45-16T and G45-17, were isolated from acid mine water of Zijin copper mine in Fujian Province, China. Phylogenetic analysis of 16S rRNA gene sequences showed that they were closely related to Alicyclobacillus acidoterrestris ATCC 49025T with sequence similarities of 96.8 %. Cells grew aerobically at 20-45 °C (optimum, 40 °C), at pH 2.5-5.5(optimum, pH 3.5) and in the presence of 0-4.0 % (w/v) NaCl. Strains contained MK-7 as the major menaquinone and the major cellular fatty acids were ω-cyclohexane C19 : 0 and ω-cyclohexane C17 : 0. The DNA G+C content was 51.3 and 49.8 mol% (Tm) for G45-16T and G45-17, respectively. On the basis of phenotypic, chemotaxonomic and phylogenetic comparisons with their relatives and DNA-DNA relatedness values, it is concluded that strains G45-16T and G45-17 represent a novel species within the genus Alicyclobacillus, for which the name Alicyclobacillus fodiniaquatilis sp. nov. is proposed; the type strain is G45-16T(=CGMCC 1.15049T=NBRC 111483T).

Genotypic and phenotypic diversity of Alicyclobacillus acidocaldarius isolates.

UNLABELLED: The fruit juice industry recognizes Alicyclobacillus as a major quality control target micro-organism. In this study, we analysed 19 bacterial isolates to identify Alicyclobacillus species by polymerase chain reaction (PCR) and sequencing analyses. Phenotypic and genomic diversity among isolates were investigated by API 50CHB system and ERIC-PCR (enterobacterial repetitive intergenic consensus-PCR) respectively. All bacterial isolates were identified as Alicyclobacillus acidocaldarius, and almost all showed identical DNA sequences according to their 16S rRNA (rDNA) gene partial sequences. Only few carbohydrates were fermented by A. acidocaldarius isolates, and there was little variability in the biochemical profile. Genotypic fingerprinting of the A. acidocaldarius isolates showed high diversity, and clusters by ERIC-PCR were distinct to those obtained from the 16S rRNA gene phylogenetic tree. There was no correlation between phenotypic and genotypic variability in the A. acidocaldarius isolates analysed in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: Detection of Alicyclobacillus strains is imperative in fruit concentrates and juices due to the production of guaiacol. Identification of the genera originates rejection of the product by processing industry. However, not all the Alicyclobacillus species are deteriorative and hence the importance to differentiate among them. In this study, partial 16S ribosomal RNA sequence alignment allowed the differentiation of species. In addition, ERIC-PCR was introduced for the genotypic characterization of Alicyclobacillus, as an alternative for differentiation among isolates from the same species.

Fate of Alicyclobacillus spp. in enrichment broth and in juice concentrates.

In this study, the fate of Alicyclobacillus acidoterrestris spores in different types of juice concentrates stored under different conditions was investigated. In addition, the impact of dilution procedures during the enrichment step for the detection of Alicyclobacillus in lemon juice concentrates was studied. Pear, red grape, mango, tangerine, carrot and lemon juice concentrates (50-69.4°Brix, pH1.7-4.3) were inoculated with A. acidoterrestris spores (10(3) spore/mL) and stored at 4 °C and 20 °C, after which the spores were counted at 0, 2, 5, 9, 17, 21, 28, 36, 43, and 50 days. No significant differences in the number of Alicyclobacillus spores were observed at storage temperatures of 4 °C and 20 °C (p>0.05). The results also indicated that the number of spores of A. acidoterrestris remained stable in all types of juice concentrates during the storage period, except in lemon juice concentrate. In lemon juice concentrate, a decline in A. acidoterrestris spore populations of 0.3-0.8 log CFU/mL was observed within 5-10 days of storage. The decline in A. acidoterrestris spore populations was more pronounced in cloudy lemon juice concentrate, which contained higher concentrations of flavonoids (mainly eriocitrin and hesperidin) than clarified lemon juice concentrate. It was also found that dilution of lemon juice concentrate samples in the proportion of 1:19 allowed the germination of A. acidoterrestris spores and the growth of populations of up to 10(7) CFU/mL. In contrast, the proportion (1:9) recommended in internationally recognized methods led to a reduction in the population of this microorganism that would yield false negative results. Data presented in this study demonstrated that Alicyclobacillus spores remain stable in most juice concentrates during storage, but that natural antimicrobial compounds present in some of them may decrease spore counts and inhibit their recovery by detection procedures.

Tumebacillus algifaecis sp. nov., isolated from decomposing algal scum.

Bacterial strain THMBR28(T) was isolated from decomposing algal scum that was collected during an algal bloom in Taihu lake, China. Cells of strain THMBR28(T) were Gram-staining-positive, facultatively anaerobic and rod-shaped. Growth was observed at 20-45 °C (optimum, 30 °C), at pH 5.0-9.5 (optimum, pH 6.5-7.5), and in the presence of 0-1.0% (w/v) NaCl (optimum, 0.5%). Strain THMBR28(T) contained MK-7 as the major menaquinone and iso-C15 : 0 as the major cellular fatty acid. The polar lipid profile contained phosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidylethanolamine and six unidentified polar lipids. The diamino acid found in the cell-wall peptidoglycan was meso-diaminopimelic acid. The DNA G+C content was 57.6 mol% (Tm). Phylogenetic analysis of 16S rRNA gene sequences showed that strain THMBR28(T) belonged to the genus Tumebacillus, most closely related to Tumebacillus ginsengisoli DSM 18389(T) (95.0%) and Tumebacillus permanentifrigoris Eur1 9.5(T) (93.4%). Based on phylogenetic and phenotypic characterization, it is concluded that strain THMBR28(T) represents a novel species of the genus Tumebacillus, for which the name Tumebacillus algifaecis sp. nov. is proposed, with THMBR28(T) ( = CGMCC 1.10949(T) = NBRC 108765(T)) as the type strain.