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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. [ABSTRACT FROM AUTHOR]

In this study, sweet potato β-amylase (SPA) was modified by methoxy polyethylene glycol maleimide (molecular weight 5000, Mal-mPEG5000) to obtain the Mal-mPEG5000-SPA modified β-amylase and the interaction mechanism between SPA and Mal-mPEG5000 was investigated. the changes in the functional groups of different amide bands and modifications in the secondary structure of enzyme protein were analyzed using infrared spectroscopy and circular dichroism spectroscopy. The addition of Mal-mPEG5000 transformed the random curl in the SPA secondary structure into a helix structure, forming a folded structure. The Mal-mPEG5000 improved the thermal stability of SPA and protected the structure of the protein from breaking by the surrounding. The thermodynamic analysis further implied that the intermolecular forces between SPA and Mal-mPEG5000 were hydrophobic interactions and hydrogen bonds due to the positive values of ΔHθ and ΔSθ. Furthermore, the calorie titration data showed that the binding stoichiometry for the complexation of Mal-mPEG5000 to SPA was 1.26, and the binding constant was 1.256 × 107 mol/L. The binding reaction resulted from negative enthalpy, indicating that the interaction of SPA and Mal-mPEG5000 was induced by the van der Waals force and hydrogen bonding. The UV results showed the formation of non-luminescent material during the interaction, the Fluorescence results confirmed that the mechanism between SPA and Mal-mPEG5000 was static quenching. According to the fluorescence quenching measurement, the binding constant (KA) values were 4.65 × 104 L·mol−1 (298K), 5.56 × 104 L·mol−1 (308K), and 6.91 × 104 L·mol−1 (318K), respectively. [ABSTRACT FROM AUTHOR]

Alicyclobacillus acidoterrestris is a major concern in fruit juice industry due to its spoilage potential of acidic fruit juice. Quantifying the expression levels of functional genes by real-time quantitative polymerase chain reaction (RT-qPCR) is necessary to elucidate the response mechanisms of A. acidoterrestris to acid stress. However, appropriate reference genes (RGs) for data normalization are required to obtain reliable RT-qPCR results. In this study, eight novel candidate RGs were screened based on transcriptome datasets of A. acidoterrestris under acid stress. The expression stability of eight new RGs and commonly used RG 16s rRNA was assessed using geNorm, NormFinder, and BestKeeper algorithms. Moreover, the comprehensive analysis using the RefFinder program and the validation using target gene ctsR showed that dnaG and dnaN were the optimal multiple RGs for normalization at pH 4.0; ytvI , dnaG , and 16s rRNA at pH 3.5; icd and dnaG at pH 3.0; and ytv I, dnaG , and spoVE at pH 2.5. This study revealed for the first time that A. acidoterrestris had different suitable RGs under different acid conditions, with implications for further deciphering the acid response mechanisms of this spoilage-causing bacterium. [ABSTRACT FROM AUTHOR]

BACKGROUND Fusarium head blight (FHB) is one of the disasters that seriously harm wheat and other small grain crops. It causes spoilage and mildew of the grain leading to a significant decline in the yield and quality of the grain. This research aimed to isolate antagonistic bacteria to purify antifungal proteins. A strain was isolated from the rhizosphere of healthy wheat in a wheat field affected by a severe FHB epidemic. This isolated strain was tentatively identified as Paenibacillus polymyxa 7F1, which displayed a strong inhibitory effect against several other pathogens. One novel antifungal protein was purified from the P. polymyxa 7F1 and successfully expressed. RESULTS: A crude culture of P. polymyxa 7F1 demonstrated antifungal activity that was stable at a temperature range of 60–90 °C and a pH range of 2.6–9.0. However, the antifungal activity of the P. polymyxa 7F1 was inhibited with proteinase K, trypsin, and neutral protease treatment. A 36 kDa protein with broad‐spectrum antifungal activity was purified from the P. polymyxa 7F1. A glycosyl hydrolase domain was identified from this protein through liquid chromatography–mass spectrometry (LC–MS) analysis. A recombinant plasmid pET32a(+)/36kd for prokaryotic expression was constructed, and the renatured p36kd protein demonstrated similar antifungal activity to the 36 kDa protein purified from the P. polymyxa 7F1. CONCLUSION: A novel antifungal protein produced by P. polymyxa 7F1 was purified and expressed. The recombinant protein showed good antifungal activity as the novel purified protein. The novel antifungal protein provides an effective way to control the Fusarium head blight. © 2020 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

DnaK gene coding for the 70-kDa heat shock protein (Hsp70) plays an essential role in protecting microorganisms from a hostile environmental and food preservation conditions such as heating, acidity, alkalinity and chilling and is believed to be associated with the thermo-acidophilic resistance of Alicyclobacillus acidoterrestris ( A . acidoterrestris ). In this study, the genome walking technique was used to clone DnaK gene and the bioinformatics characteristics of DnaK was predicted using online software. The growth and sporulation of A. acidoterrestris were explored and the expression levels of DnaK gene in A. acidoterrestris were determined by quantitative real time PCR (qRT-PCR) under heat, acid and cold stresses. DnaK gene (GenBank Accession No. HQ893543) from A. acidoterrestris DSM 3922 T contains an open reading frame of 1854 bp coding for 617 amino acids and the deduced amino acid sequence is homologous to the DnaK chaperone protein of Alicyclobacillus acidocaldarius . The protein translated from DnaK gene of A. acidoterrestris was characterized as a thermostable protein based on the primary and secondary structure analyses which predict the functions of amino acid sequence, and folding and coiling polypeptides, respectively. Three environmental stresses including heat stress at 70 °C, acid stress at the pH of 1.0, and cold stress at 0 °C were imposed on A. acidoterrestris to investigate the expression of DnaK . The transcriptional level of DnaK gene under heat stress increased quickly to peak level in 5 min and then dropped to the minimum level in 15 min; the transcriptional levels of DnaK gene under acid stress increased markedly in 0.5 h to peak level in 1 h and then decreased to the minimum level in 3 h, and the peak mRNA expression levels differed between heat and acid stress. DnaK expression levels of 5 min heat stress and 1 h acid stress were 1.4 times and 4.3 times higher than those of the control under non-stress condition at 45 °C, respectively. In response to cold stress at 0 °C, the DnaK expression level decreased drastically to 0.48 times of the control after 1 h cold treatment. Spores of A. acidoterrestris were produced after the bacteria received 40 min heat stress, 5 h acid stress or 6 h cold stress, which suggested that DnaK gene regulates the stress resistance of A. acidoterrestris against abrupt abiotic stimuli differently from sporulation. However, the expression of DnaK under stress conditions demonstrated the different pattern of changes as the induced stresses continued, especially in the case of acid stress, which implied that a more complicated mechanism of DnaK regulation is involved in the stress response of A. acidoterrestris . [ABSTRACT FROM AUTHOR]

This article describes the cloning, sequence analysis and expression of the DnaJ gene from Alicyclobacillus acidoterrestris. The genome walking technique was used to clone the full-length sequence of DnaJ and quantitative real-time PCR was used to analyze DnaJ expression under stress conditions. AadnaJ (GenBank accession nr: HQ893544) containing an open reading frame of 1137 bp encoding 378 amino acid residues was cloned from A. acidoterrestris DSM 3922T. The nucleotide sequence of AadnaJ shows 77% homology with the DnaJ of A. acidocaldarius LAA1. The DnaJ expression level was upgraded rapidly under heat or acid stress. Its mRNA expression level reached a peak value at 25 min after the onset of heat stress (70 °C) and at 1 h after the onset of acid stress (pH = 1). Acid stress at pH 1 for 25 and 60 min led to the DnaJ expression levels 2.1 times and 35.7 times above that of the control, respectively. In response to cold stress at 0 °C, the DnaJ expression level decreased drastically to 0.04 times that of the control level after 1 h. The expression patterns of DnaJ in response to the stress conditions shown here explained the heat and acidity endurance of A. acidoterrestris. Practical Application: This study directly addresses the role of the DnaJ gene in temperature and acid endurance in A. acidoterrestris. This provides a basis for the development of genetic and molecular techniques that may minimize the adverse effects of A. acidoterrestris in fruit juice production. This study also sheds light on the design of heat- and acid-tolerant recombinases and the understanding of the molecular mechanisms underlying heat and acid resistance in A. acidoterrestris. [ABSTRACT FROM AUTHOR]

The article presents a study on the molecular cloning, overexpression, and biochemical properties of PsBg16A, which is part of a family 16 endo-β-glucanase from Paecilomyces species. The study uses the genomic DNA of Paecilomyces FLH30 as template for subsequent polymerase chain reaction (PCR) amplification. According to multiple sequence alignment, phylogenetic analysis, and substrate specificity, GH family 16 proteins were categorized into five subgroups.

[Display omitted] • Integrated transcriptome and proteome revealed heat responses of A. acidoterrestris. • Peptidoglycan and fatty acid composition were modified to obtain intact cell surface. • Terminal oxidases, cytochrome bd and aa 3 , in respiratory chain were upregulated. • Heat induced ribosome hibernation and higher ratio of initiation factor to ribosome. • Heat caused lower dissolved oxygen solubility and imbalance in cellular redox state. Alicyclobacillus acidoterrestris can survive pasteurization and is implicated in pasteurized fruit juice spoilage. However, the mechanisms underlying heat responses remain largely unknown. Herein, gene transcription changes of A. acidoterrestris under heat stress were detected by transcriptome, and an integrated analysis with proteomic and physiological data was conducted. A total of 911 differentially expressed genes (DEGs) was observed. The majority of DEGs and differentially expressed proteins (DEPs) were exclusively regulated at the mRNA and protein level, respectively, whereas only 59 genes were regulated at both levels and had the same change trends. Comparative analysis of the functions of the specifically or commonly regulated DEGs and DEPs revealed that the heat resistance of A. acidoterrestris was primarily based on modulating peptidoglycan and fatty acid composition to maintain cell envelope integrity. Low energy consumption strategies were established with attenuated glycolysis, decreased ribosome de novo synthesis, and activated ribosome hibernation. Terminal oxidases, cytochrome bd and aa 3 , in aerobic respiratory chain were upregulated. Meanwhile, the MarR family transcriptional regulator was upregulated, reactive oxygen species (ROS) was discovered, and the concentration of superoxide dismutase (SOD) increased, indicating that the accompanied oxidative stress was induced by high temperature. Additionally, DNA and protein damage repair systems were activated. This study provided a global perspective on the response mechanisms of A. acidoterrestris to heat stress, with implications for better detection and control of its contamination in fruit juice. [ABSTRACT FROM AUTHOR]

The sweet potato β-amylase (SPA) was modified by 6 types of methoxy polyethylene glycol to enhance its specific activity and thermal stability. The aims of the study were to select the optimum modifier, optimize the modification parameters, and further investigate the characterization of the modified SPA. The results showed that methoxy polyethylene glycol maleimide (molecular weight 5000, Mal-mPEG5000) was the optimum modifier of SPA; Under the optimal modification conditions, the specific activity of Mal-mPEG5000-SPA was 24.06% higher than that of the untreated SPA. Mal-mPEG5000-SPA was monomeric with a molecular weight of about 67 kDa by SDS-PAGE. The characteristics of Mal-mPEG5000-SPA were significantly improved. The Km value, Vmax and Ea in Mal-mPEG5000-SPA for sweet potato starch showed that Mal-mPEG5000-SPA had greater affinity for sweet potato starch and higher speed of hydrolysis than SPA. There was no significant difference of the metal ions' effect on Mal-mPEG5000-SPA and SPA. [ABSTRACT FROM AUTHOR]

Abstract For the purpose of investigating the heat resistance mechanism of Alicyclobacillus acidoterrestris , label-free quantification was used to reveal some cellular changes in A. acidoterrestris during heat stress. Totally, 545 differential expression proteins were respectively identified at heat stress of 65 °C for 5 min, of which 258 proteins were up-regulated and 287 proteins were down-regulated. These significantly changed proteins were mapped to 100 pathways and some of them were mostly related to protection or repair of macromolecules such as proteins and DNA, cell wall formation, which indicated that these proteins might play crucial roles in response to heat stress. The KEGG pathway analysis combined with protein functional analysis and further validation at mRNA level suggested that A. acidoterrestris sensed the temperature rise in environment through alterations in the secondary structure of DNA and RNA molecules. The biosynthesis of antibiotics pathway and the ribosomes might be involved in signal transduction in heat stress and further trigger a large number of proteins playing a critical role in the regulation of heat stress in A. acidoterrestris. The study firstly demonstrated the global physiological response to heat stress and the results provided a better understanding of thermal adaption mechanism of A. acidoterrestris. Graphical abstract Image 1 Highlights • At 65 °C heat stress for 5 min, 545 differential expression proteins were identified. • The proteins were related to repair of macromolecules and transcriptional regulators. • The results indicated A. acidoterrestris sensed temperature through DNA and proteins. • Biosynthesis of antibiotics pathway and ribosomes related to heat stress regulation. • The study firstly demonstrated the global heat stress response in A. acidoterrestris. [ABSTRACT FROM AUTHOR]

Abstract Inhibition of spore germination or sterilization after induction of spore germination would effectively control low pH food spoilage caused by Alicyclobacillus acidoterrestris spores. However, the characteristics and mechanisms of A. acidoterrestris spore germination in low ambient pH remains poorly understood. In this study, the germination rate of A. acidoterrestris spores at different ambient pH conditions was determined, and subsequently the proteomic profiles of A. acidoterrestris in spore germination were analysed by label-free quantification, in which the specific metabolic pathways involved were identified and key functional proteins were screened and validated using RT-qPCR (real time quantitative PCR). The suitable ambient pH value for the spore germination of A. acidoterrestris ranged from 3.0 to 5.0 with the optimum pH of 4.0. According to the LC-ESI-MS/MS (liquid chromatography electrospray ionization tandem mass spectrometry) analysis, 98 proteins of geminated spores of A. acidoterrestris incubated for 2 h at pH 3.0 were changed significantly in comparison to non-germinated spores, the expression of 20 proteins were up-regulated and that of 78 proteins down-regulated respectively. Those differential expressed proteins were mainly involved in cell wall hydrolysis, cell morphological changes, protein synthesis and folding, perception of external stimuli and signal transduction etc., and we observed that germination receptor D (GerD), cell wall hydrolase, transpeptidase, peptidase S1 and two-component regulatory system phoR were significantly up-regulated, but hydrolase NlpC/P60, peptidoglycan glycosyltransferase, spore coat proteins CotX, CotJB and the Lrp/AsnC (leucine-responsive regulatory protein/asparagine synthase C products) protein were significantly down-regulated in the experiment, which implied the important roles of identified proteins during the spore germination. Furthermore, the pathway analysis showed the possible involvement of differentially expressed proteins in the β-lactam resistance, ribosome, biosynthesis of secondary metabolites, pyruvate metabolism, two-component system and other metabolic pathways, which indicated that synthesis and hydrolysis of cell wall, intracellular substance synthesis, energy generation and signal transduction were likely associated with the initiation of spore germination and restoration of vegetative growth. In conclusion, the quantitative proteomic landscape of A. acidoterrestris spores could provide the theoretic and experimental evidences for the hazard control of A. acidoterrestris spores in the thermal pasteurization process of acidic beverages industry. Graphical abstract Unlabelled Image Highlights • When A. acidoterrestris spores were incubated for 2 h at pH 3.0, 98 differential expression proteins were identified. • The proteins were involved in cell wall hydrolysis, cell morphological changes, feelings of stimuli and signal transduction. • Cell wall hydrolysis, intracellular substance synthesis and signal transduction were related to spore germination. [ABSTRACT FROM AUTHOR]

Changes in physicochemical properties, oxidative properties, and protein denaturation of porcine longissimus muscles, which were frozen by different methods and then stored at −18 °C for various times, were investigated. As the freezing rate was increased, the deterioration was reduced. In addition, over a period of 168 d, the quality reduction was proportional to the levels of oxidation and protein denaturation. The obtained results showed that a slower freezing rate and long-term duration had a negative influence on the porcine quality. At 168 d, although the thawing loss, total volatile base nitrogen, and electrical conductivity of the −40 °C frozen samples were higher than those of the −80 °C and liquid nitrogen frozen samples, they remained within the acceptable limits. However, compared with freezing at – 18 °C, freezing at −40 °C reduced oxidation, and maintained the sample thermal stability and viscoelasticity. Therefore, in terms of energy conservation, freezing at −40 °C could preserve porcine quality over 168 d, thereby confirming its suitability for application in the meat industry. • Changes in the properties of porcine muscles upon freezing were investigated. • As the freezing rate was increased, the quality deterioration was reduced. • The quality reduction was proportional to the oxidation and protein denaturation. • Slower freezing rate and long-term duration had a negative effect on quality. • To save energy, freezing at −40 °C was selected to maintain quality for 168 d. [ABSTRACT FROM AUTHOR]

3-Phenoxybenzoic acid (3-PBA) is a general metabolite of synthetic pyrethroids. It could be used as a generic biomarker for multiple pyrethroids exposure for human or pyrethroid residues in the environment. In this study, monoclonal antibodies (mAbs) against 3-PBA were developed by using PBA–bovine serum albumin (BSA) as an immunogen. In the competitive enzyme-linked immunosorbent assay (ELISA) format, the I 50 and I 10 values of purified mAbs were 0.63 and 0.13 μg/ml, respectively, with a dynamic range between 0.19 and 2.04 μg/ml. Then, the colloidal gold (CG)-based lateral flow immunoassay was established based on the mAbs. The working concentration of coating antigen and CG-labeled antibodies and the blocking effects were investigated to get optimal assay performance. The cutoff value for the assay was 1 μg/ml 3-PBA, and the detection time was within 10 min. A total of 40 river water samples were spiked with 3-PBA at different levels and determined by the lateral flow immunoassay without any sample pretreatments. The negative false rate was 2.5%, and no positive false results were observed at these levels. This lateral flow immunoassay has the potential to be an on-site screening method for monitoring 3-PBA or pyrethroid residues in environmental samples. [ABSTRACT FROM AUTHOR]

The aim of the research was to obtain a high healthcare honeysuckle beverage with strong antioxidant activity. Honeysuckle ( Lonicera japonica Thunb ) was used as the raw material in this experiment. The effects of fermentation temperature, fermentation time, lactic acid bacteria inoculation amount, and sugar addition amount on the sensory quality of honeysuckle beverage were investigated by single factor test and orthogonal test, and the best process was obtained. The physicochemical indexes and antioxidant activity of honeysuckle beverages fermented with lactic acid bacteria were studied. The results showed that the fermentation temperature of the beverage was 37 °C, the fermentation time was 24 h, the inoculation amount of Lactiplantibacillus plantarum and Lactobacillus acidophilus mixed starter (1:1) was 3%, and 8% white granulated sugar was added. The highest sensory score was 87.30 ± 0.17, which was the optimal process. The honeysuckle liquid mixed inoculation with Lactiplantibacillus plantarum and Lactobacillus acidophilus was fermented for 24 h. The number of viable bacteria reached 9.84 ± 0.02 lg cfu/mL, the pH value was 3.10 ± 0.01, and the total polyphenol content was 7.53 ± 0.03 mg GAE/g. The number of lactic acid bacteria, pH, total polyphenol content, and free radical scavenging rate were significantly increased ( p  < 0.05) compared with the non-inoculated and single-inoculated lactic acid bacteria. To sum up, it was concluded that a better quality beverage could be obtained by fermenting a solution of honeysuckle with Lactiplantibacillus plantarum and Lactobacillus acidophilus mixed fermentation agent, providing a new approach and new ideas for the development of deep processing and fermented beverages using honeysuckle., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ran, Tang, Mao, Meng, Jiao, Li, Zhao and Zhou.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Liang, Tu, Li, Sun, Zhao, Ran, Jiao, Huang and Li.)