Transcriptomic analysis of the antibacterial mechanism of ε-polylysine-functionalized magnetic composites against Alicyclobacillus acidoterrestris and its application in apple juice.

BACKGROUND: Alicyclobacillus acidoterrestris is a common microorganism in fruit juice. It can produce off-odor metabolites and has been considered to be an important factor in juice contamination. Thus, the development of new strategy for the control of A. acidoterrestris has important practical significance. The primary objective of this work was to assess the antibacterial performance of ε-polylysine-functionalized magnetic composites (Fe3O4@MoS2@PAA-EPL) in apple juice and its effect on juice quality. Moreover, the molecular mechanism of Fe3O4@MoS2@PAA-EPL against A. acidoterrestris was explored by RNA sequencing (RNA-Seq). RESULTS: Experimental results indicated that the synthesized composites possessed the ability to inhibit the viability of A. acidoterrestris vegetative cells and spores. Besides, investigation on the quality of apple juice incubated with Fe3O4@MoS2@PAA-EPL implied that the fabricated composites displayed negligible adverse effects on juice quality. In addition, the results of RNA-Seq demonstrated that 833 differentially expressed genes (DEGs) were identified in Fe3O4@MoS2@PAA-EPL-treated A. acidoterrestris, which were associated with translation, energy metabolism, amino acid metabolism, membrane transport and cell integrity. CONCLUSION: These results suggested that the treatment of Fe3O4@MoS2@PAA-EPL disrupted energy metabolism, repressed cell wall synthesis and caused membrane transport disorder of bacterial cells. This work provides novel insights into the molecular antibacterial mechanism for ε-polylysine-functionalized magnetic composites against A. acidoterrestris. © 2024 Society of Chemical Industry.

Biosafety measures for Alicyclobacillus spp. strains across various levels of biohazard.

Alicyclobacillus bacteria are important contaminants in the beverage industry because their spores remain in the product after usual pasteurization. At the same time, their impact on human health has yet to be characterized, as it is generally assumed to be low or non-existent. However, these bacteria are causing quality concerns mainly due to odor and taste changes of the product. Since potential health effects are not precisely known, an experimental assessment was performed, including a biosafety assessment of six viable and non-viable vegetative and spore forms of Alicyclobacillus spp. strains using cell cultures and rodent study. The monolayer of Caco-2 (Cancer coli-2) cells was investigated for its adsorption effect on the epithelium of the small intestine of mice. Lactate dehydrogenase leakage (LDH) and transepithelial electrical resistance (TEER) tests were used to ensure the integrity of the cell membrane and tight junctions. The methylthiazole tetrazolium bromide (MTT) assay examined in vitro cytotoxicity in Caco-2 and HepG2 cell lines. The hemolysis of erythrocytes was spectrophotometrically measured. The results showed negligible cytotoxicity or non-toxic response in mice. In conclusion, Alicyclobacillus spp. exhibited biocompatibility with negligible cytotoxicity and minimal safety concerns.

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.

A novel endo-1,4-ß-xylanase from Alicyclobacillus mali FL18: Biochemical characterization and its synergistic action with ß-xylosidase in hemicellulose deconstruction.

A novel endo-1,4-ß-xylanase-encoding gene was identified in Alicyclobacillus mali FL18 and the recombinant protein, named AmXyn, was purified and biochemically characterized. The monomeric enzyme worked optimally at pH 6.6 and 80 °C on beechwood xylan with a specific activity of 440.00 ± 0.02 U/mg and a good catalytic efficiency (kcat/KM = 91.89 s-1mLmg-1). In addition, the enzyme did not display any activity on cellulose, suggesting a possible application in paper biobleaching processes. To develop an enzymatic mixture for xylan degradation, the association between AmXyn and the previously characterized ß-xylosidase AmßXyl, deriving from the same microorganism, was assessed. The two enzymes had similar temperature and pH optima and showed the highest degree of synergy when AmXyn and AmßXyl were added sequentially to beechwood xylan, making this mixture cost-competitive and suitable for industrial use. Therefore, this enzymatic cocktail was also employed for the hydrolysis of wheat bran residue. TLC and HPAEC-PAD analyses revealed a high conversion rate to xylose (91.56 %), placing AmXyn and AmßXyl among the most promising biocatalysts for the saccharification of agricultural waste.

Pyrococcus furiosus argonaute based Alicyclobacillus acidoterrestrsis detection in fruit juice.

Alicyclobacillus acidoterrestris is the major threat to fruit juice for its off-odor producing characteristic. In this study, Pyrococcus furiosus Argonaute (PfAgo), a novel endonuclease with precise DNA cleavage activity, was used for A. acidoterrestrisdetection, termed as PAD. The partially amplified 16 S rRNA gene of A. acidoterrestris can be cleaved by PfAgo activated by a short 5'-phosphorylated single strand DNA, producing a new guide DNA (gDNA). Then, PfAgo was activated by the new gDNA to cut a molecular beacon (MB) with fluorophore-quencher reporter, resulting in the recovery of fluorescence. The fluorescent intensity is positively related with the concentration of A. acidoterrestris. The PAD assay showed excellent specificity and sensitivity as low as 101 CFU/mL, which can be a powerful tool for on-site detection of A. acidoterrestris in fruit juice industry in the future, reducing the economic loss.

Antibacterial effects of various molecular weight chitosans against Alicyclobacillus acidoterrestris in orange juice.

Alicyclobacillus acidoterrestris has been gaining attention due to its unique thermo-acidophilic properties and being associated with the deterioration of pasteurized beverages. The objective of this study was to evaluate the antibacterial activity of chitosan with various molecular weights (MWs) (164, 85, 29.2, and 7.1 kDa) and concentrations (0-100 µg/mL) against A. acidoterrestris and its effect on guaiacol production. Various chitosan MWs were co-incubated for 7 days, and the bacterial growth, guaiacol, and vanillic acid contents during storage were determined. The chitosans performed antibacterial effects against A. acidoterrestris. Further, 164 kDa chitosan showed excellent results in controlling the growth and guaiacol formation in A. acidoterrestris. These findings demonstrated the efficacy of chitosan antibacterial activity against A. acidoterrestris and mitigating the guaiacol formation. Chitosan's antibacterial properties are attributed to the elimination of cells and suppression of guaiacol production. This study introduces a new approach for reducing A. acidoterrestris contamination in fruit juices, with potential product quality and safety advantages.

Enzyme-Assisted Endogenous Guide DNA Generation-Mediated Pyrococcus furiosus Argonaute for Alicyclobacillus acidoterrestris Detection.

Pyrococcus furiosusArgonaute (PfAgo) emerged as a novel endonuclease for the nucleic acid test recently. However, the input of exogenous guide DNA (gDNA) to activate PfAgo has reduced its flexibility. In this work, an enzyme-assisted endogenous gDNA generation-mediated PfAgo for the target detection strategy, termed EGG-PAD, was proposed. With the aid of EcoR Ι, the target double-strand DNA was cut, producing a phosphate group at the 5' end, functioning as gDNA to activate PfAgo for nucleic acid detection. The applicability of this assay was tested in the detection ofAlicyclobacillus acidoterrestris, a bacterium causing the spoilage of fruit juice, showing excellent sensitivity and specificity, ascribed to the "duplex amplification and triple insurance" mechanism. Moreover, EGG-PAD exhibited superior versatility in the identification of common foodborne pathogens. This powerful platform could also be an on-site test tool for detecting nucleic acid-containing organisms such as tumor cell, pathogen, and virus in the future.

Antibacterial mechanism of inosine against Alicyclobacillus acidoterrestris.

Inosine could potentially become a novel antibacterial agent against Alicyclobacillus acidoterrestris as low doses of inosine can prevent its contamination. However, until now the antibacterial mechanism of inosine targeting A. acidoterrestris is still unknown. In this study, to unravel the mechanism of inosine against A. acidoterrestris puzzle, the effects of inosine on bacterial surface hydrophobicity, intracellular protein content, cell membrane damage extent, and permeability of the A. acidoterrestris were investigated. The results showed that inosine can effectively inhibit the growth and reproduction of A. acidoterrestris by destroying the integrity of cell membrane and increasing its permeability, causing the leakage of intracellular nutrients. Furthermore, the interaction networks of inosine target proteins were analyzed. The interaction networks further revealed that damage to bacterial cell membranes might be relevant to inosine's effect on bacterial DNA replication and cell energy metabolism through regulating nucleotide synthesis and metabolism and the activity of translation initiation factors. Finally, the antibacterial mechanism of inosine against A. acidoterrestris was proposed.

Inactivation activity and mechanism of pulsed light against Alicyclobacillus acidoterrestris vegetative cells and spores in concentrated apple juice.

Alicyclobacillus acidoterrestris has received much attention due to its unique thermo-acidophilic property and implication in the spoilage of pasteurized juices. The objective of this study was to evaluate the sterilization characteristics and mechanisms of pulsed light (PL) against A. acidoterrestris vegetative cells and spores in apple juice. The results indicated that bacteria cells in apple juice (8-20°Brix) can be completely inactivated within the fluence range of 20.25-47.25 J/cm2, which mainly depended on the soluble solids content (SSC) of juice, and the spores in apple juice (12°Brix) can be completely inactivated by PL with the fluence of 54.00 J/cm2. The PL treatment can significantly increase the leakage of reactive oxygen species (ROS) and proteins from cells and spores. Fluorescence studies of bacterial adenosine triphosphate (ATP) indicated that the loss of ATP was evident. Scanning electron microscopy and confocal laser scanning microscope presented that PL-treated cells or spores had serious morphological damage, which reduced the integrity of cell membrane and led to intracellular electrolyte leakage. In addition, there were no significant negative effects on total sugars, total acids, total phenols, pH value, SSC and soluble sugars, and organic acid content decreased slightly during the PL treatment. The contents of esters and acids in aroma components had a certain loss, while that of alcohols, aldehydes and ketones were increased. These results demonstrated that PL treatment can effectively inactivate the bacteria cells and spores in apple juice with little effect on its quality. This study provides an efficient method for the inactivation of A. acidoterrestris in fruit juice.

Alicyclobacillus sp. SO9, a novel halophilic acidophilic iron-oxidizing bacterium isolated from a tailings-contaminated beach, and its effect on copper extraction from chalcopyrite in the presence of high chloride concentration.

Many acidophilic iron-oxidizing bacteria used in the mining industry for the bioleaching of sulfidic minerals are intolerant to high chloride concentrations, resulting in problems where chloride occurs in the deposit at high concentrations or only seawater is available. In search for strains tolerating such conditions a tetrathionate- and iron-oxidizing bacterium was isolated from a tailings-contaminated beach sample at Portman Bay, Cartagena-La Union mining district, Spain, in the presence of 20 g l-1 (0.34 M) sodium chloride. The isolate was able to form spores, did not grow in the absence of NaCl, and oxidized ferrous iron in the presence of up to 1.5 M (∼87 g l-1) NaCl. Genome sequencing based on a combination of Illumina and PacBio reads revealed two contigs, a circular bacterial chromosome of 5.2 Mbp and a plasmid of 90 kbp, respectively. The chromosome comprised seven different 16S rRNA genes. Submission of the chromosome to the Type (Strain) Genome Server (TYGS) without preselection of similar sequences revealed exclusively type strains of the genus Alicyclobacillus. In the TYGS analyses the respective most similar species were dependent on whether the final tree was derived from just 16S rRNA, from the genomes, or from the proteomes. Thus, TYGS analysis clearly showed that isolate SO9 represents a novel species of the genus Alicyclobacillus. In the presence of artificial seawater with almost 0.6 M chloride, the addition of Alicyclobacillus sp. SO9 improved copper dissolution from chalcopyrite (CuFeS2) compared to abiotic leaching without bacteria. The new isolate SO9, therefore, has potential for bioleaching at elevated chloride concentrations.

Inhibitory mechanism of quercetin on Alicyclobacillus acidoterrestris.

In this the antibacterial of quercetin against Alicyclobacillus acidoterrestris was evaluated by measuring the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). Subsequently, the effect of quercetin on A. acidoterrestris cell membrane was evaluated through scanning electron microscopy (SEM), surface hydrophobicity determination, diacetate fluorescein staining and propidium iodide (PI) staining. Additionally, the effects of quercetin on intracellular macromolecules and cell metabolism were explored by measuring the culture medium protein, bacterial protein and intracellular sodium and potassium adenosine triphosphate (ATP) enzyme activity. The results revealed that quercetin exhibited the MIC and MBC values of 100 ug/mL and 400 ug/mL, respectively, against A. acidoterrestris. The SEM results revealed that quercetin could induce irreversible damage to the cell membrane effectively. Moreover, quercetin could enhance the surface hydrophobicity of A. acidoterrestris. The results of flow cytometry and fluorescence microscopy analyses revealed that quercetin could promote cell damage by altering the cell membrane permeability of A. acidoterrestris, inducing the release of nucleic acid substances from the cells. Furthermore, the determination of protein content in the culture medium, bacterial protein content, and the Na(+)/K(+)-ATPase activity demonstrated that quercetin could reduce the intracellular protein content and impedes protein expression and ATPase synthesis effectively, leading to apoptosis.

Exploring the growth characteristics of Alicyclobacillus acidoterrestris for controlling juice spoilage with zero additives.

Fruit juice spoilage that caused by contaminated Alicyclobacillus has brought huge losses to beverage industry worldwide. Thus, it is very essential to understand the growth and metabolism processing of Alicyclobacillus acidoterrestris (A. acidoterrestris) in controlling juice spoilage caused by Alicyclobacillus. In this work, simulative models for the growth and metabolism of A. acidoterrestris were systematically conducted in the medium and fruit juice. The results showed that low temperature (4 ℃) and strong acidic environment (pH 3.0-2.0) of medium inhibited the growth and reproduction of A. acidoterrestris. In addition, with decreasing temperature, the color, smell and turbidity of commercially available juice supplemented with A. acidoterrestris significantly improved. This work provided a clear exploration of growth characteristics of A. acidoterrestris by applying theory (medium) to reality (fruit juices), and pave fundamental for exploring the zero additives of controlling juice spoilage.

Proposal of a novel subspecies: Alicyclobacillus hesperidum subspecies aegles.

Alicyclobacillus sp. DSM 11985T was isolated from geothermal soil but had not yet been classified at the species level. The strain produced guaiacol, which is of interest from the viewpoint of food spoilage in the food industry. 16S rRNA gene sequence analysis revealed that strain DSM 11985T was closely related (99.6 % similarity) to Alicyclobacillus hesperidum DSM 12489T. However, strains of A. hesperidum did not produce guaiacol; therefore, we performed the taxonomic characterization of strain DSM 11985T. The results showed that strain DSM 11985T and strains of A. hesperidum showed different phenotypic characteristics in biochemical/physiological tests including guaiacol production. Average nucleotide identity values between strain DSM 11985T and strain DSM 12489T were 95.4-95.9 %, and the in silico DNA-DNA hybridization value using the Genome-to-Genome Distance Calculator between strains DSM 11985T and DSM 12489T was 65.5 %. These values showed that strain DSM 11985T was genetically closely related but separated from strains of A. heparidum. From the above results, a novel subspecies of A. hesperidum, named Alicyclobacillus hesperidum subsp. aegles subsp. nov. is proposed. The type strain is DSM 11985T (=FR-12T=NBRC 113041T).

Genomic insight and physiological characterization of thermoacidophilic Alicyclobacillus isolated from Yellowstone National Park.

Introduction: Alicyclobacillus has been isolated from extreme environments such as hot springs, volcanoes, as well as pasteurized acidic beverages, because it can tolerate extreme temperatures and acidity. In our previous study, Alicyclobacillus was isolated during the enrichment of methane oxidizing bacteria from Yellowstone Hot Spring samples. Methods: Physiological characterization and genomic exploration of two new Alicyclobacillus isolates, AL01A and AL05G, are the main focus of this study to identify their potential relationships with a thermoacidophilic methanotroph (Methylacidiphilum) isolated from the same hot spring sediments. Results and discussion: In the present study, both Alicyclobacillus isolates showed optimal growth at pH 3.5 and 55°C, and contain ω-alicyclic fatty acids as a major lipid (ca. 60%) in the bacterial membrane. Genomic analysis of these strains revealed specific genes and pathways that the methanotroph genome does not have in the intermediary carbon metabolism pathway such as serC (phosphoserine aminotransferase), comA (phosphosulfolactate synthase), and DAK (glycerone kinase). Both Alicyclobacillus strains were also found to contain transporter systems for extracellular sulfate (ABC transporter), suggesting that they could play an important role in sulfur metabolism in this extreme environment. Genomic analysis of vitamin metabolism revealed Alicyclobacillus and Methylacidiphilum are able to complement each other's nutritional deficiencies, resulting in a mutually beneficial relationship, especially in vitamin B1(thiamin), B3 (niacin), and B7 (biotin) metabolism. These findings provide insights into the role of Alicyclobacillus isolates in geothermal environments and their unique metabolic adaptations to these environments.

Computational analysis of carboxylesterase genes and proteins in non-pathogenic food bacterium Alicyclobacillus acidocaldarius: insights from proteogenomics.

Over recent years, Alicyclobacillus acidocaldarius, a Gram-positive nonpathogenic rod-shaped thermo-acid-tolerant bacterium, has posed numerous challenges for the fruit juice industry. However, the bacterium's unique characteristics, particularly its nonpathogenic and thermophilic capabilities, offer significant opportunities for genetic exploration by biotechnologists. This study presents the computational proteogenomics report on the carboxylesterase (CE) enzyme in A. acidocaldarius, shedding light on structural and evolutional of CEs from this bacterium. Our analysis revealed that the average molecular weight of CEs in A. acidocaldarius was 41 kDa, with an isoelectric point around 5. The amino acid composition favored negative amino acids over positive ones. The aliphatic index and hydropathicity were approximately 88 and - 0.15, respectively. While the protein sequence showed no disulfide bonds in the CEs' structure, the presence of Cys amino acids was observed in the structure of CEs. Phylogenetic analysis presented more than 99% similarity between CEs, indicating their close evolutionary relationship. By applying homology modeling, the 3-dimensional structural models of the carboxylesterase were constructed, which with the help of structural conservation and solvent accessibility analysis highlighted key residues and regions responsible for enzyme stability and conformation. The specific patterns presented the total solvent accessibility of less than 25 (Å2) was in considerable position as well as Gly residues were noticeably have high accessibility to solvent in all structures. Ala was the more frequent amino acids in the conserved-SASA of carboxylesterases. Furthermore, unsupervised agglomerative hierarchical clustering based on solvent accessibility feature successfully clustered and even distinguished this enzyme from proteases from the same genome. These findings contribute to a deeper understanding of the nonpathogenic A. acidocaldarius carboxylesterase and its potential applications in biotechnology. Additionally, structural analysis of CEs would help to address potential solutions in fruit juice industry with utilization of computational structural biology.

Antibacterial Activity and Transcriptomic Analysis of Hesperetin against Alicyclobacillus acidoterrestris Vegetative Cells.

The aim of this research was to investigate the antimicrobial characteristics and mechanism of hesperetin against Alicyclobacillus acidoterrestris vegetative cells. The results presented show that hesperetin had effective antimicrobial activity on Alicyclobacillus acidoterrestris vegetative cells, minimum inhibition concentration (MIC) of 0.0625 g/L, and minimum bacterial concentration (MBC) greater than 2 g/L. Moreover, treatment of hesperetin caused significant damage to cell integrity, preventing the growth of Alicyclobacillus acidoterrestris vegetative cells, enhancing the leakage of nucleic acid and proteins, and destroying the vegetative cell morphology. To further investigate the mechanism, transcriptomic analysis was carried out, and 3056 differentially expressed genes (DEGs) were detected. Gene ontology (GO) enrichment analysis revealed that hesperetin inhibits Alicyclobacillus acidoterrestris by affecting the intracellular nitrogen metabolism and amino acid metabolism. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis explained that hesperetin was also able to prevent the growth of Alicyclobacillus acidoterrestris by affecting the processes of nutrient transport, energy metabolism, and flagella motility. These results provide new insights into the antimicrobial effects and mechanism of hesperetin against Alicyclobacillus acidoterrestris, which provides a new method for inactive Alicyclobacillus acidoterrestris in the juice industry.

Detection of Alicyclobacillus spp. and Identification of Guaiacol Production Using the GENE-UP® PRO ACB, IFU Method No. 12, and Cosmo Bio Assays.

Some species of Alicyclobacillus spoil beverages by producing guaiacol. Current culture-based methods detect the presence of Alicyclobacillus spp. and a subsequent peroxidase assay determines if the isolate can produce guaiacol. However, these methods are time-consuming and can yield false negatives due to differences in growth optima among species. The purpose of this study was to compare a RT-PCR-based method, the GENE-UP® PRO ACB assay, to the IFU Method No. 12 Enumeration and Enrichment methods. Ten species of Alicyclobacillus were detected using the tested RT-PCR assay, while A. dauci and A. kakegewensis were not detected using either IFU protocol. Low concentrations (1-10, 10-100, and 100-1,000 CFU/10 mL) of A. acidoterrestris, A. suci, and A. acidocaldarius were tested in five matrices. The proportion of positive samples identified using the tested RT-PCR assay (62/84) or the IFU Enrichment protocol (62/84) did not differ significantly from the proportion of inoculated samples (63/84). However, the IFU Enumeration method (32/84) detected statistically fewer positives. Additionally, methods identifying guaiacol production were compared. The proportion of correctly identified guaiacol producers using the tested RT-PCR assay (51/63) was not significantly different than those identified using the 3 h Cosmo Bio assay (54/63). Finally, four commercial samples of orange juice and sucrose solution were tested. Alicyclobacillus spp. were identified in all four samples using the IFU Enrichment method and in two samples using the tested RT-PCR assay. However, Alicyclobacillus was not detected in any sample using the IFU Enumeration method. Overall, this study showed consistent detection of Alicyclobacillus spp. using either the IFU Enrichment protocol or the tested RT-PCR assay, which both outperformed the IFU Enumeration protocol. Both the 3 h guaiacol bioassay and the tested RT-PCR assays consistently differentiated guaiacol-producing and nonproducing strains.

Characterization and Genome Study of a Newly Isolated Temperate Phage Belonging to a New Genus Targeting Alicyclobacillus acidoterrestris.

The spoilage of juices by Alicyclobacillus spp. remains a serious problem in industry and leads to economic losses. Compounds such as guaiacol and halophenols, which are produced by Alicyclobacillus, create undesirable flavors and odors and, thus, decrease the quality of juices. The inactivation of Alicyclobacillus spp. constitutes a challenge because it is resistant to environmental factors, such as high temperatures, and active acidity. However, the use of bacteriophages seems to be a promising approach. In this study, we aimed to isolate and comprehensively characterize a novel bacteriophage targeting Alicyclobacillus spp. The Alicyclobacillus phage strain KKP 3916 was isolated from orchard soil against the Alicyclobacillus acidoterrestris strain KKP 3133. The bacterial host's range and the effect of phage addition at different rates of multiplicity of infections (MOIs) on the host's growth kinetics were determined using a Bioscreen C Pro growth analyzer. The Alicyclobacillus phage strain KKP 3916, retained its activity in a wide range of temperatures (from 4 °C to 30 °C) and active acidity values (pH from 3 to 11). At 70 °C, the activity of the phage decreased by 99.9%. In turn, at 80 °C, no activity against the bacterial host was observed. Thirty minutes of exposure to UV reduced the activity of the phages by almost 99.99%. Based on transmission-electron microscopy (TEM) and whole-genome sequencing (WGS) analyses, the Alicyclobacillus phage strain KKP 3916 was classified as a tailed bacteriophage. The genomic sequencing revealed that the newly isolated phage had linear double-stranded DNA (dsDNA) with sizes of 120 bp and 131 bp and 40.3% G+C content. Of the 204 predicted proteins, 134 were of unknown function, while the remainder were annotated as structural, replication, and lysis proteins. No genes associated with antibiotic resistance were found in the genome of the newly isolated phage. However, several regions, including four associated with integration into the bacterial host genome and excisionase, were identified, which indicates the temperate (lysogenic) life cycle of the bacteriophage. Due to the risk of its potential involvement in horizontal gene transfer, this phage is not an appropriate candidate for further research on its use in food biocontrol. To the best of our knowledge, this is the first article on the isolation and whole-genome analysis of the Alicyclobacillus-specific phage.

Transcriptomic and Metabolomic Profiling Uncovers Response Mechanisms of Alicyclobacillus acidoterrestris DSM 3922T to Acid Stress.

Alicyclobacillus acidoterrestris, which has strong acidophilic and heat-resistant properties, can cause spoilage of pasteurized acidic juice. The current study determined the physiological performance of A. acidoterrestris under acidic stress (pH 3.0) for 1 h. Metabolomic analysis was carried out to investigate the metabolic responses of A. acidoterrestris to acid stress, and integrative analysis with transcriptome data was also performed. Acid stress inhibited the growth of A. acidoterrestris and altered its metabolic profiles. In total, 63 differential metabolites, mainly enriched in amino acid metabolism, nucleotide metabolism, and energy metabolism, were identified between acid-stressed cells and the control. Integrated transcriptomic and metabolomic analysis revealed that A. acidoterrestris maintains intracellular pH (pHi) homeostasis by enhancing amino acids decarboxylation, urea hydrolysis, and energy supply, which was verified using real-time quantitative PCR and pHi measurement. Additionally, two-component systems, ABC transporters, and unsaturated fatty acid synthesis also play crucial roles in resisting acid stress. Finally, a model of the responses of A. acidoterrestris to acid stress was proposed. IMPORTANCE Fruit juice spoilage caused by A. acidoterrestris contamination has become a major concern and challenge in the food industry, and this bacterium has been suggested as a target microbe in the design of the pasteurization process. However, the response mechanisms of A. acidoterrestris to acid stress still remain unknown. In this study, integrative transcriptomic, metabolomic, and physiological approaches were used to uncover the global responses of A. acidoterrestris to acid stress for the first time. The obtained results can provide new insights into the acid stress responses of A. acidoterrestris, which will point out future possible directions for the effective control and application of A. acidoterrestris.

Role and Mechanism of Cold Plasma in Inactivating Alicyclobacillus acidoterrestris in Apple Juice.

A. acidoterrestris has been identified as the target bacterium in fruit juice production due to its high resistance to standard heat treatment. Multiple studies have shown that cold plasma can effectively inactivate pathogenic and spoilage microorganisms in juices. However, we are aware of only a few studies that have used cold plasma to inactivate A. acidoterrestris. In this study, the inactivation efficacy of cold plasma was determined using the plate count method and described using a biphasic model. The effects of the food matrix, input power, gas flow rate, and treatment time on inactivation efficacy were also discovered. Scavenging experiments with reactive oxygen species (•OH, •O2-, and 1O2), scanning electron microscopy (SEM), Raman spectra, as well as an in vitro toxicology assay kit, were used to determine the inactivation mechanism. According to the plate count method, a maximum reduction of 4.14 log CFU/ mL could be achieved within 7 s, and complete inactivation could be achieved within 240 s. The scavenging experiments showed that directly cold plasma-produced singlet oxygen plays the most crucial role in inactivation, which was also confirmed by the fluorescence probe SOSG. The scanning electron microscopy (SEM) and Raman spectra showed that the cold plasma treatment damaged the membrane integrity, DNA, proteins, lipids, and carbohydrates of A. acidoterrestris. The plate count results and the apple juice quality evaluation showed that the cold plasma treatment (1.32 kV) could inactivate 99% of A. acidoterrestris within 60 s, with no significant changes happening in apple juice quality, except for slight changes in the polyphenol content and color value.