Alicyclobacillus Acidoterrestris Research Articles

Identification of guaiacol producing <i>Alicyclobacillus</i> recovered from commercial orange juices distributed in Greek markets

The objective of this work was the isolation and characterization of Alicyclobacillus acidoterrestris strains from orange juice. In all, 72 isolates were recovered from commercial orange juice bought from the Greek market. The isolates were subjected to polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) to discriminate them at strain level and A. acidoterrestris types I and II. The restriction patterns of 16S rRNA gene revealed that isolates were categorized in different groups depending on the restriction endonucleases. Moreover, the digestion of the part of vdc operon gene with HphI restriction enzyme successfully differentiated between A. acidoterrestris types I and II. In conclusion, RFLP succeeded in differentiating A. acidoterrestris isolates at strain and type levels.

Heat resistance of five spoilage microorganisms in a carbonated broth

Despite their acidic pH, carbonated beverages can be contaminated by spoilage microorganisms. Thermal treatments, before and/or after carbonation, are usually applied to prevent the growth of these microorganisms. However, the impact of CO2 on the heat resistance of spoilage microorganisms has never been studied. A better understanding of the combined impact of CO2 and pH on the heat resistance of spoilage microorganisms commonly found in carbonated beverages might allow to optimize thermal treatment.Five microorganisms were selected for this study: Alicyclobacillus acidoterrestris (spores), Aspergillus niger (spores), Byssochlamys fulva (spores), Saccharomyces cerevisiae (vegetative cells), and Zygosaccharomyces parabailii (vegetative cells). A method was developed to assess the impact of heat treatments in carbonated media on microbial resistance.The heat resistances of the five studied species are coherent with the literature, when data were available. However, neither the dissolved CO2 concentration (from 0 to 7 g/L), nor the pH (from 2.8 to 4.1) have an impact on the heat resistance of the selected microorganisms, except for As. niger, for which the presence of dissolved CO2 reduced the heat resistance. This study improved our knowledge about the heat resistance of some spoilage microorganisms in presence of CO2.

Inactivation of Alicyclobacillus acidoterrestris in apple juice using pulsed light

We aimed to investigate the effect of pulsed light (PL) on inactivating Alicyclobacillus acidoterrestris in apple juice, the mechanism behind the inactivation, and the resultant effects on the quality of the juice. Our results showed that PL had a good inactivation effect on both spores and vegetative cells of A. acidoterrestris in apple juice, with a reduction of 3.5 ± 0.1 and 2.1 ± 0.1 log CFU/mL, respectively, following 3 s of treatment. This effect was better when lower concentrations of apple juice and lower liquid layer depths were used. Transmission electron microscopy (TEM) showed that the morphology and structure of A. acidoterrestris spores did not change significantly before and after treatment. When we compared the dipicolinic acid (DPA) release rate and death rate of the spores, we hypothesized that PL might have damaged the cortical proteins on A. acidoterrestris that are associated with the resistance of the spores, thus leading to their death. However, TEM revealed that the cell membranes of vegetative cells of A. acidoterrestris were intact and smooth before the treatment, but significantly wrinkled afterward. Raman spectroscopy revealed that the main targets of PL on A. acidoterrestris vegetative cells were: amide I on the proteins, nucleic acids [adenine(A) and guanine (G), in particular] and DNA (O–P–O‾ stretching) structures, and lipids (C–H2 deformation). The PL treatment increased the sugar content significantly, while having little to no effect on the other basic physicochemical parameters of apple juice.

Synergic effect of multi-frequency thermosonication and germinant on Alicyclobacillus acidoterrestris spore inactivation and underlying mechanisms

Alicyclobacillus acidoterrestris spores have strong stress resistance, and their spores cannot be eliminated by pasteurization, which is considered a major threat to the beverage industry. In this study, based on the spore control strategy of “germinate to eradicate”, the spores were germinated by multi-frequency thermosonication (MFTS) combined with germinant. The process parameters were optimized, and the underlying mechanism was explored. The results showed that when A. acidoterrestris spores were treated with MFTS (60 °C, 20/40/50 kHz) and 300 mM AGFK for 30 min, incubated at 45 °C for 2 h, and heated to 80 °C for 20 min, a maximum 1.4 ± 0.04 log reduction can be achieved. The spores showed the darkest phase and the best germination effect after MFTS treatment, but leakage of proteins and electrolytes inside the spores were also caused. The surface of the vegetative cells formed by spore after MFTS treatment shrank extensively, and the shape was irregular. Spore resistance was reduced, making them more likely to die after pasteurization. This study could provide theoretical evidence for the technological innovation and practical application of A. acidoterrestris spores hazard control in the beverage industry.

Resistance of Alicyclobacillus acidoterrestris spores to atmospheric cold plasma: Insights from sporulation temperature and mechanism analysis

Environmental conditions during sporulation, such as temperature, pH value, and mineral content, can significantly influence the formation production and resistance of spores. In this study, we investigated the sporulation capacity and resistance of Alicyclobacillus acidoterrestris (AAT) spores obtained at different temperatures (25 °C, 35 °C, 45 °C) to Dielectric Barrier Discharge Atmospheric cold plasma (DBD-ACP, 20 and 30 kV for 2–8 min). The results indicated that the efficiency of sporulation was highest at the optimum temperature (45 °C), deviation from optimal temperature would have a negative effect on sporulation. Otherwise, sporicidal efficiency of DBD-ACP increased with longer treatment time and lower sporulation temperatures, suggesting that AAT spores exhibit weaker resistance to DBD-ACP at lower sporulation temperature. Results from the release of DPA, and electron microscopic observations confirmed that DBD-ACP caused disruption of the cortex and membrane structure of AAT spores, leading to the release of intracellular contents and subsequent spore's death. Further analysis through proteomics revealed intriguing insights into the differences between spores formed at 25 °C and those at 45 °C. Specifically, spores formed at 25 °C exhibited lower expression levels of proteins related to sporulation, intracellular energy metabolism, membrane transport, and significant down-regulation of proteins involved in peptidoglycan and spore coat formation compared to those at 45 °C. These alterations likely contribute to the development of a thinner and looser spore structure, rendering it less effective in defending, i.e. less resistant to DBD-ACP treatment. Industrial relevanceAAT spores' resistance to pasteurization and persistence in NFC juices impact juice quality and safety. This study investigates the resistance of AAT spore to DBD-ACP treatment under different sporulation temperatures. Insights into sporicidal behavior of DBD-ACP and the effect of sporulation temperature on resistance provide essential knowledge for optimizing juice processing and enhancing microbial safety. The findings offer practical guidance for the juice industry to develop improved preservation strategies, ensuring longer shelf life and consumer satisfaction.

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.

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.

Molecular mechanism of acid stress response of Alicyclobacillus acidoterrestris DSM 3922T under sublethal pH

Alicyclobacillus acidoterrestris (A. acidoterrestris) causes the spoilage of pasteurized acidic fruit juice due to its unique thermoacidophilic properties, which caused economic losses to fruit juice industry. To reveal the molecular regulatory mechanism of how A. acidoterrestris responded to acid stress is the key to hazard control of this microbe. Herein, acid-responsive proteome expression profiles of A. acidoterrestris were analyzed using label-free quantitative mass spectrometry to investigate its acid resistance mechanism at sublethal pH. Totally, 325 differential expression proteins were identified during acid stress (pH 2.5, 15 min duration), of which the expressions of 83 proteins were up-regulated and the other 242 protein expressions were down-regulated. The identified differentially expressed proteins were mainly involved in small molecule metabolism, organic nitrogen compounds metabolism, organic acid metabolism, and signal transduction. Overall, they were mapped into 97 metabolic pathways. Combination of KEGG pathway analysis and protein functional analysis suggested that the pH homeostasis system variation and relevant changes in metabolic pathways, cell membrane permeability and DNA repair are the main acid resistance mechanisms of A. acidoterrestris. Our finding implied that A. acidoterrestris might sense and transmit pH signals from the external environment through NhaB protein which holds histidine-dependent acid resistance system, initiating a series of acid tolerance reactions. Taken together, our study demonstrated global physiological response of A. acidoterrestris to sublethal pH, which provided a better understanding of acid adaption mechanism of A. acidoterrestris.

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 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.

Rapid and sensitive detection of nucleic acids using an RAA-CRISPR/Cas12b one-pot detection assay (Rcod)

Rapid, sensitive and specific methods are crucial for nucleic acid detection. CRISPR/Cas12b has recently been widely used in nucleic acid detection. However, due to its thermophagic property, DNA isothermal recombinase-aided amplification (RAA) and subsequent CRISPR/Cas12b detection require two separate reactions, which is cumbersome and inconvenient and may cause aerosol pollution. In this study, we propose an RAA-CRISPR/Cas12b one-pot detection assay (Rcod) for Bordetella pertussis detection without additional amplification product transfer steps. The time from sample processing to response time was less than 30 min using nucleic acid extraction-free method, and the sensitivity reached 0.2 copies/μL. In this system, Alicyclobacillus acidoterrestris Cas12b protein (AacCas12b) exhibited strong and specific trans-cleavage activity at a constant temperature of 37 °C, while the cis-cleavage activity was weak. This characteristic reduces the interference of AacCas12b with nucleic acids in the system. Compared with real-time PCR, our Rcod system detected B. pertussis in 221 clinical samples with a sensitivity and specificity of 97.96 % and 99.19 %, respectively, with nucleic acid extraction-free method. The rapid, sensitive and specific Rcod system provides ideas for the establishment of CRISPR-based one-step nucleic acid detection and may aid the development of reliable point-of-care nucleic acid tests. ImportancePertussis is an acute respiratory infection caused by B. pertussis that is highly contagious and potentially fatal, and early diagnosis is essential for the treatment of whooping cough. In this study, we found that AacCas12b has high and strongly specific trans-cleavage activity at lower temperatures. A RAA-CRISPR/Cas12b one-step detection platform (Rcod) without interference with amplification was developed. In addition, the combination of Rcod and nucleic acid extraction-free method can quickly and accurately detect the qualitative detection of B. pertussis, and the detection results are visualized, which makes the pathogen nucleic acid detection and analysis process simpler, and provides a new method for the rapid clinical diagnosis of B. pertussis.

Impact of plasma activated water (PAW) at different temperatures on Alicyclobacillus acidoterrestris spores

This study aimed to determine the impact of plasma activated water (PAW) combined with heating on the Alicyclobacillus acidoterrestris spores’ inactivation. For this, viable spore counts were determined after contact with PAW at 20–90 °C/5–150 min and inactivation kinetic parameters were determined at 90 °C by fitting Bigelow and Weibull models. Results showed that A. acidoterrestris spores were resistant to PAW at room temperature (reduction <0.8 log CFU/mL after 2h of exposure), while PAW at 80 and 90 °C for 30 min inactivated 2.8 and 3.6 log CFU/mL, respectively, showing a synergistic effect (p < 0.05). Moreover, Weibull model fitted the data better (R2 ≥ 0.95) than Bigelow (R2 ≥ 0.90), due to the tail behavior observed. Although PAW sanitation is insufficient to guarantee high levels of inactivation of A. acidoterrestris spores, its association with mild temperatures reduces 1–2 log CFU/mL, potentially improving the shelf life of fruit products.

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.

Stereochemistry of aminoacylated cardiolipins and phosphatidylglycerols from bacteria.

Hydrophilic interaction liquid chromatography (HILIC) connected with electrospray high-resolution tandem mass spectrometry (MS) was used for the analysis of unusual amino acid (AA) substituted phosphatidylglycerols (PG) and cardiolipins (CL) in mesophilic and thermophilic bacteria. Individual peaks from the lipid class separation by HILIC were isolated and hydrolyzed to determine the absolute configuration of the aminoacyl side chain. The configuration of the aminoacyl side chain was assigned by indirect liquid chromatography (LC) enantiomer separation after the hydrolysis of the aminoacylated (aminoacyl) lipids using N-(4-nitrophenoxycarbonyl)-l-phenylalanine 2-methoxyethyl ester as chiral derivatizing agent and reversed phase LC-MS for analysis. When two chromatographic methods were combined, less common AAs, such as d-allo-Ile and d-allo-Thr, were identified. The taxonomic classification of bacteria showed that bacteria of the family Bacillaceae (Bacillus and Geobacillus) produce branched-chain AAs, that is, d-allo-Ile, d-Ile, and d-Leu. These AAs were present only in the genera Bacillus and Geobacillus and not in Alicyclobacillus acidoterrestris (family Alicyclobacillaceae). On the contrary, hydroxy AAs, that is, l- and d-Thr, and l- and d-allo-Thr, were identified as aminoacyl-PG and aminoacyl-CL in A. acidoterrestris and were not present in the genera Bacillus and Geobacillus. Therefore, the complete analysis made it possible to identify the stereochemistry of AAs in aminoacyl PGs and CLs and use this fact for chemotaxonomy.

Proteomics analysis reveals the inactivation mechanisms of Alicyclobacillus acidoterrestris spores by atmospheric cold plasma

SummaryAlicyclobacillus acidoterrestris is a typical endospore‐forming microorganism, whose spores are able to germinate in Not From Concentrate juice causing its spoilage. In this work, dielectric barrier discharge atmospheric cold plasma (DBD‐ACP) at the voltage of 30 kV was used to inactivate Alicyclobacillus acidoterrestris spores in NFC juice (2–8 min). The results showed spores reduced nearly 3‐log CFU mL−1 after 8 min treatment, and the morphology of spores were severely disrupted, causing the release of their contents. Furthermore, proteomic analysis showed totals of 122 and 170 different expression proteins were detected in group C (control)/Ct1 (30 kV, 4 min) and C/Ct2 (30 kV, 8 min), respectively, indicating that metabolic activities of spores including amino acid and energy metabolic, protein biosynthesis, DNA replication and spore germination are inhibited. This study confirms the inactivation of Alicyclobacillus acidoterrestris spores by DBD‐ACP and provides some new perspectives on the inactivation mechanisms of spores by DBD‐ACP.

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.

Effect of weak acids, combined with pH and temperature, on the growth or inactivation of Alicyclobacillusacidoterrestris

Alicyclobacillus acidoterrestris is a thermophilic, acidophilic, and spore-forming microorganism, able to spoil fruit juices. Few data are available on the effects of different acids of juices; thus, the goal of this research was to study the effect of 5 weak acids (malic, citric, ascorbic, tartaric, and lactic acids), compared to hydrochloric acid (strong acid, control) on two isolates of A. acidoterrestris from soil (C1 and C13). Acids were combined through a full randomized design with pH (2, 3, and 4), temperature (5 or 45 °C) and duration of storage (2, 7 and 14 days) as categorical predictors. Total population (spores + cells) and spores were assessed. The results of this paper suggest that acid-resistance of A. acidoterrestris could be strongly affected by the kind of acid; on total population, malic and ascorbic acids exerted the highest antimicrobial action, probably depending on pKa and acid concentration, but also on other factors (for example the size and the ability of acids to enter the cells). Results also confirm that weak acids could mainly act on activated spores/cells, rather than on spores. To some extent, acids could play an antimicrobial effect also on spores mainly at pH 2. In conclusion, this paper offers a first insight on potentialities and limits of weak acids on A. acidoterrestris and could contribute to understand the growth/inactivation of this spoiling microorganism, as a preliminary step to design and/or optimize new approaches for controlling it.

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.

Inactivation of Alicyclobacillus acidoterrestris vegetative cells and spores induced by atmospheric cold plasma

This study aimed to inactivate Alicyclobacillus acidoterrestris vegetative cells and spores (107–108 CFU/mL) using atmospheric cold plasma (ACP). Alicyclobacillus acidoterrestris vegetative cells decreased by 6.63 and 6.47 log under only ACP treatment of 30 kV for 6 min in saline solution and apple juice, respectively. ACP was less effective against spores, resulting in 2.64 and 2.43 log reductions at the same treatment parameter. However, better inactivation (4.03 log and 3.95 log) was achieved in apple juice and saline solution using ACP at 30 kV combined with mild heat (85 °C) for 6 min. Morphological observation and intracellular substance release revealed severe structural damage to spores induced by ACP. The application of ACP resulted in improved color, reduced pH, and increased levels of total phenolic content and total soluble solids in apple juice. This study offers an effective method for inactivating Alicyclobacillus acidoterrestris in apple juice. Industrial relevanceThe application of atmospheric cold plasma in combination with mild heat to effectively inactivate Alicyclobacillus acidoterrestris in apple juice offers a promising method for ensuring juice product quality. The enhancement of color, reduction in pH, and increase in total phenolic content and total soluble solids further underscore the potential benefits of this technique for enhancing the sensory attributes of apple juice. This study offers a potential approach that may directly contribute to enhancing production practices and product value.

Electron coupling effect-triggered monatomic copper laccase-mimicking nanozyme for the degradation and detection of guaiacol produced by Alicyclobacillus acidoterrestris

The generation of guaiacol by Alicyclobacillus acidoterrestris (A. acidoterrestris) in fruit juices negatively affects public health and causes severe environmental pollution. Therefore, the sensitive detection and efficient degradation of guaiacol in real samples are crucial. Here, we develop an electrochemical sensor utilizing a copper single-atom nanozyme (CuN4-G) to detect and degrade guaiacol at the picomolar level. Density functional theory (DFT) calculations verify that the bonding electron coupling effect in the CuN4-G facilitates rapid electron transfer, enhances electrical conductivity, and provides abundant active sites, thereby leading to exceptional catalytic performance. Moreover, CuN4-G demonstrates a Km value similar to that of natural laccase but a higher Vmax, highlighting its potential as a highly efficient biocatalyst. The CuN4-G-based electrochemical sensor achieves a detection from 5 to 50,000 pM for guaiacol, with a 1.2 pM (S/N = 3) detection limit. Additionally, CuN4-G-modified electrodes display high selectivity and excellent stability. CuN4-G nanozyme can keep its activity in conditions of pH (3–9), temperature (30–90 °C), ionic strength (0–400 mM), and organic solvent (0–50% (v/v)), overcoming the deficiencies of natural enzymes. Furthermore, our electrochemical sensor can not only accurately detect guaiacol, but also degrade it in actual fruit juice samples infected by A. acidoterrestris, demonstrating its potential applications in food and environmental monitoring.