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.

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.

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.

Analysis of phenotypic changes in high temperature and low pH extreme conditions of Alicyclobacillus sendaiensis PA2 related with the cell wall and sporulation genes.

The A. sendaiensis PA2 is a polyextremophile bacterium. In this study, we analyze the A. sendaiensis PA2 genome. The genome was assembled and annotated. The A. sendaiensis PA2 genome structure consists of a 2,956,928 bp long chromosome and 62.77% of G + C content. 3056 CDSs were predicted, and 2921 genes were assigned to a putative function. The ANIm and ANIb value resulted in 97.17% and 96.65%, the DDH value was 75.5%, and the value of TETRA (Z-score) was 0.98. Comparative genomic analyses indicated that three systems are enriched in A. sendaiensis PA2. This strain has phenotypic changes in cell wall during batch culture at 65 °C, pH 5.0 and without carbon and nitrogen source. The presence of unique genes of cell wall and sporulation subsystem could be related to the adaptation of A. sendaiensis PA2 to hostile conditions.

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.

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.

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).

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.

Effect of dual-frequency thermosonication, food matrix, and germinants on Alicyclobacillus acidoterrestris spore germination.

The off-odors associated with spoilage of acidic beverages are linked to the germination and growth of Alicyclobacillus acidoterrestris (AAT) spores. As a consequence, we determined the influence of nutrients, non-nutrient germinants, dual-frequency thermosonication (DFTS), and food matrix on spore germination. AAT spores in orange juice (OJ), supplemented by L-alanine (L-ala), had the highest germination rate and lowest DPA content at 10 h of incubation. The formation of microscopic pores in cell membranes during DFTS caused irreversible damage in AAT spores in citrate buffer solution (CBS); however, it stimulated AAT spore germination in CBS containing L-ala. Hence, the germination potential was established in the order: L-ala > Calcium dipicolinate > asparagine, glucose, fructose, and potassium ion mixture (AGFK) > L-valine. The conductivity analysis indicated that membrane damage could be a key factor contributing to the artificial germination in CBS. AFM images revealed that after 2 h of adding L-ala, the protein content increased with increased germinated cells. TEM showed that membrane poration and coat detachment were the main pre-germination morphological changes detected after DFTS treatment. This study provides evidence that germination stimulated with DFTS might be an effective strategy for reducing A. acidoterrestris spores in fruit juices.

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.

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.

Occurrence of Alicyclobacillus acidoterrestris in pasteurized and high hydrostatic pressure-treated fruit juices and isolates' characterization.

In recent years, acidophilic, heat-resistant, and spore-forming spoilage bacteria have been identified in pasteurized or treated by high hydrostatic pressure (HPP) fruit juices. Alicyclobacillus acidoterrestris is the bacteria more frequently linked to the spoilage of this type of product because its spores can survive conventional pasteurization and HPP treatments. Under favourable conditions, such as an acidic pH, its spores can germinate and multiply, with the consequent production of guaiacol. Guaiacol is a compound with an undesirable odour ("medicinal", "smoked" or "antiseptic"). In this context, our objective was to determine the prevalence of A. acidoterrestris in 150 Spanish pasteurized and HPP-treated fruit juices purchased from supermarkets or received from manufacturers. Then, the isolates and the reference strain (CECT 7094 T) were characterized to establish differences in terms of (i) growth capacity at different pH and temperatures, and in (ii) guaiacol production capacity. The results showed a high incidence of A. acidoterrestris (18.0 %) in the analysed juices. The 44.4 % of the isolates came from blends of fruit juices. Within juice blends, 9 juices contained apple juice among their ingredients. This represents a 18.8 % of incidence with respect to the total of blended juices with apple. A high incidence in monovarietal apple juices was also observed (3 out of 14 samples). Regarding the characterization of the isolates, EC1 (isolated from an apple concentrate) showed the highest growth capacity at pH 4.0 at temperatures from 20 to 55 °C. Besides, three strains (R42, EC10, and EZ13, isolated from clementine, plum and white grape juice, respectively) could grow at room temperatures (20 and 25 °C). For pH, only EZ13, isolated from white grape juice, was able to grow significantly at pH 2.5. Finally, the production of guaiacol ranged from 74.1 to 145.6 ppm, being the isolate EC1 the one that produced more guaiacol after 24 h of incubation at 45 °C (145.6 ppm). As we have observed, there is a high incidence of A. acidoterrestris in marketed juices and intermediate products despite the treatments received (pasteurization or HPP). Under favourable conditions for the development of this microorganism, it could produce enough guaiacol to spoil the juices before their consumption. Therefore, in order to improve the quality of fruit juices it is necessary to investigate in more detail the origin of this microorganism and to find strategies to reduce its presence in final products.

Contribution of amino acids to Alicyclobacillus acidoterrestris DSM 3922T resistance towards acid stress.

Spoilage of juice and beverages by a thermo-acidophilic bacterium, Alicyclobacillus acidoterrestris, has been considered to be a major and widespread concern for juice industry. Acid-resistant property of A. acidoterrestris supports its survival and multiplication in acidic juice and challenges the development of corresponding control measures. In this study, intracellular amino acid differences caused by acid stress (pH 3.0, 1 h) were determined by targeted metabolomics. The effect of exogenous amino acids on acid resistance of A. acidoterrestris and the related mechanisms were also investigated. The results showed that acid stress affected the amino acid metabolism of A. acidoterrestris, and the selected glutamate, arginine, and lysine contributed to its survival under acid stress. Exogenous glutamate, arginine, and lysine significantly increased the intracellular pH and ATP level, alleviated cell membrane damage, reduced surface roughness, and suppressed deformation caused by acid stress. Additionally, the up-regulated gadA and speA genes and the enhanced enzymatic activity confirmed that glutamate and arginine decarboxylase systems played a crucial role in maintaining pH homeostasis of A. acidoterrestris under acid stress. Our research reveals an important factor contributing to acid resistance of A. acidoterrestris, which provides an alternative target for effectively controlling this contaminant in fruit juices.

Inactivation of Alicyclobacillus acidoterrestris spores, single or composite Escherichia coli and native microbiota in isotonic fruit-flavoured sports drinks processed by UV-C light.

Pasteurized sports drinks and other fruit-based beverages are susceptible to deterioration due to thermal processing ineffectiveness to inactivate certain spoilage microorganisms, like Alicyclobacillus acidoterrestris. This represents a major challenge for the beverage industry. The goals of this study were to: i) investigate the UV-C inactivation (annular thin film unit, actinometrical delivered fluence: 795-1270 mJ/cm2, 10-15 min, 20 °C, 1.8 L/h, Reh = 391-1067, recirculation mode operation) and the evolution during refrigerated storage of A. acidoterrestris ATCC 49025 spores and single or composite Escherichia coli ATCC 25922 in isotonic sports drinks (ISDs) made from orange (orange-ISD, UVT% = 81) or orange-banana-mango-kiwi-strawberry-lemon juices (multi-fruit-ISD, UVT% = 91), compared to a turbid orange-tangerine juice (OT juice, UVT% = 40); ii) assess the effect of pH, °Brix, A254nm, turbidity, colour and particle size of the ISDs and juice on microbial inactivation, iii) evaluate the evolution of native microbiota during cold storage, iv) investigate the Coroller, biphasic, Weibull, and Weibull-plus-tail models' ability to describe microbial inactivation and v) measure 5-hydroxymethylfurfural (HMF) formation. The modified biodosimetry method was used to calculate the germicidal UV-C fluences. Heat pasteurization (T-coil, 80 °C/6 min) was evaluated as the control treatment. UV-C was highly effective at inactivating E. coli as 4.1-5.1 and 4.5-5.6 log reductions were determined in the multi-fruit-ISD and orange-ISD, respectively, barely impacted by the background microbiota. No significant differences were recorded for the inactivation of E. coli in the UV-C and T-coil systems. Whereas, a significantly higher inactivation of A. acidoterrestris spores was achieved by UV-C (3.7-4.0 log reductions), compared to the negligible one achieved by the thermal treatment. Even though E. coli inactivation curves were similar in shape, UV-C was less effective when a cocktail of other E. coli strains was present. In comparison to the OT juice, the ISDs' inactivation kinetics were markedly different in shape, with a rapid decrease in population during the first minutes of treatment. The germicidal fluence (Hd biod) corresponding to A. acidoterrestris (19.1 mJ/cm2) was selected as it was higher than the one obtained for E. coli (11.0 mJ/cm2). UV-C induced 2.8- or 1.3 and 2.3- or 0.8 log-reductions of total aerobes or moulds and yeasts in the multi-fruit-ISD and orange-ISD, respectively. Compared to the other models, the Coroller and biphasic models showed a better fit and more accurate parameter estimates. UV-C-induced HMF production was not significant in the ISDs. The current study found that the UV-C treatment was more effective than typical heat pasteurization for inactivating A. acidoterrestris spores in isotonic drinks, following a similar trend for E. coli and native microbiota.

Antimicrobial activity and mechanism of preservatives against Alicyclobacillus acidoterrestris and its application in apple juice.

Alicyclobacillus acidoterrestris has great influence on the quality of apple juice products. In this study, the antibacterial activity of five preservatives (ε-polylysine, propylparaben, monocaprin, octyl gallate and heptylparaben) against A. acidoterrestris and its underlying mechanism were investigated. Results showed that these five preservatives all exerted antibacterial activity through a multiple bactericidal mechanism, and monocaprin and octyl gallate had the highest antibacterial activity, with the minimum inhibitory concentration (MIC) values of 22.5 and 6.25 mg/L, respectively. Five preservatives all changed the permeability of the cell membrane and destroyed the complete cell morphology, with the leakages of the intracellular electrolytes. Moreover, the treatment of ε-polylysine, propylparaben and monocaprin increased the leakage of intracellular protein; propylparaben and octyl gallate reduced the levels of cellular adenosine triphosphate. Also, monocaprin and octyl gallate may stimulate bacteria to release a large amount of reactive oxygen species, so that certain oxidative damage can kill the bacteria. Furthermore, monocaprin and octyl gallate could effectively inactivate the contamination of A. acidoterrestris in apple juices, with the slightly decrease of soluble sugars and organic acids, without significant adverse effects on total sugars and titratable acids. This research highlights the great promise of using monocaprin and octyl gallate as the safe multi-functionalized food additives for food preservations.

Unveiling the complete genome sequence of Alicyclobacillus acidoterrestris DSM 3922T, a taint-producing strain.

Several species from the Alicyclobacillus genus have received much attention from the food and beverages industries. Their presence has been co-related with spoilage events of acidic food matrices, namely fruit juices and other fruit-based products, the majority attributed to Alicyclobacillus acidoterrestris. In this work, a combination of short and long reads enabled the assembly of the complete genome of A. acidoterrestris DSM 3922T, perfecting the draft genome already available (AURB00000000), and revealing the presence of one chromosome (4,222,202 bp; GC content 52.3%) as well as one plasmid (124,737 bp; GC content 46.6%). From the 4,288 genes identified, 4,004 sequences were attributed to coding sequences with proteins, with more than 80% being functionally annotated. This allowed the identification of metabolic pathways and networks and the interpretation of high-level functions with significant reliability. Furthermore, the additional genes of interest related to spore germination, off-flavor production, namely the vdc cluster, and CRISPR arrays, were identified. More importantly, this is the first complete and closed genome sequence for a taint-producing Alicyclobacillus species and thus represents a valuable reference for further comparative and functional genomic studies.

Pulsed multifrequency thermosonication induced sonoporation in Alicyclobacillus acidoterrestris spores and vegetative cells.

Alicyclobacillus acidoterrestris (AAT) was proposed as an index of pasteurization design for high-acid fruit products due to its spore resistance and repeated spoilage incidences in fruit juices. This study aimed to determine the effectiveness of pulsed multifrequency ultrasound to minimize AAT spores and vegetative cells in aqueous suspension. For this research, an investigation of the reactive oxygen species and antioxidant activity was performed to examine the effect of temperature and frequency on AAT spore inoculation. Total decreases in AAT bacteria were 5.99, 5.74 Log CFU/mL in vegetative cells for dual-frequency thermosonication (DFTS) and dual-frequency ultrasonication (DFUS), respectively, while 5.90 and 5.38 Log CFU/mL in spores for both DFUS and DFTS, respectively. The loss of the percentage of cells in ultrasound (US) and thermosonication (TS) treatments was inversely associated with the rate of O2-and H2O2 development. The fluorescence microscopy revealed a higher bactericidal efficacy of DFTS compared to the DFUS and control. Scanning Electron Microscopy (SEM) and Transmission electron microscopy (TEM) demonstrated ultra-structural modifications such as the interruption of cell walls by cavitation and pores in the membrane structure of the AAT bacteria induced by sonoporation. Several TS frequencies of 20/40/60, 20/40, and 20 kHz treated spores had a higher electrical conductivity than untreated ones, with an improvement of 7.94, 5.68, and 3.72 %, respectively. Fourier-transform infrared (FTIR) spectroscopy revealed major changes in the spectral region of membrane fatty acids and proteins of AAT. Simultaneously, AAT inactivation specific energy rate was significantly reduced using dual-frequency ultrasound, compared to mono-frequency thermosonication. The significant results of this work recommended pulsed DFUS as an alternative application to mono-frequency US in beverage industries.