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.

Characterization of polysaccharide from Lonicera japonica Thunb leaves and its application in nano-emulsion.

The polysaccharides in honeysuckle leaves (PHL) were separated and characterized for the first time. The nano-emulsion stabilized by PHL and whey protein isolate (WPI) were also fabricated based on the ultrasonic method. The results indicated that PHL was mainly composed of glucose (47.40 mol%), galactose (19.21 mol%) and arabinose (20.21 mol%) with the weight-average molecular weight of 137.97 ± 4.31 kDa. The emulsifier concentration, WPI-to-PHL ratio, ultrasound power and ultrasound time had significant influence on the droplet size of PHL-WPI nano-emulsion. The optimal preparation conditions were determined as following: emulsifier concentration, 1.7%; WPI/PHL ratio, 3:1; ultrasonic power, 700 W; ultrasonic time, 7 min. Under the above conditions, the median diameter of the obtained nano-emulsion was 317.70 ± 5.26 nm, close to the predicted value of 320.20 nm. The protective effect of PHL-WPI emulsion on ß-carotene against UV irradiation was superior to that of WPI emulsion. Our results can provide reference for the development of honeysuckle leaves.

Apple polyphenol biotransformation using probiotics in vitro and dynamic simulated digestion by bionic rats.

BACKGROUND: The present study investigated the effects of fermentation by Lactobacillus rhamnosus zrx01 (LR-zrx01), Lactobacillus acidophilus zrx02 (LA-zrx02), and Lactobacillus plantarum zrx03 (LP-zrx03), as well as dynamic simulated digestion by bionic rats, on the biotransformation and antioxidant potential of apple polyphenols. Polyphenols were determined by ultra-high-performance liquid chromatography-mass spectrometry, the dynamic simulated digestion of fermented apple pulp was determined by bionic rats, and the antibacterial and antioxidant activities were analyzed. RESULTS: The polyphenol content of apple pulp fermented using the three strains was respectively 1.41, 1.38, and 1.36 times that of non-fermented pulp. The antibacterial activity of apple pulp improved dramatically after fermentation. Moreover, the antioxidant potential of apple pulp increased after fermentation and digestion. After dynamic simulated digestion by bionic rats, the polyphenol content in unfermented and the three fermented groups increased significantly by 1.19, 1.23, 1.20, and 1.19 times compared to that before digestion, respectively. The major polyphenols in each group with obvious changes were epicatechin, rutin, kaempferol, quercetin-3 galactoside, p-coumaric acid, and two unknown substances, 1 and 2. CONCLUSION: Fermented and digested apple polyphenols showed better biotransformation effects and mostly existed in the form of small molecules, which was conducive to the improvement of polyphenol bioavailability and beneficial to the absorption of active substances by the human body. These findings build a foundation for the development of functional food beverages. © 2023 Society of Chemical Industry.

Pseudomonas Acts as a Reservoir of Novel Tigecycline Resistance Efflux Pump tmexC6D6-toprJ1b and tmexCD-toprJ Variants.

Several variants of the plasmid-carried tigecycline resistance gene cluster, tmexCD-toprJ, have been identified. This study characterized another novel variant, tmexC6D6-toprJ1b, located on the chromosome of environmental-origin Pseudomonas mendocina. TMexC6D6-TOprJ1 mediates resistance to multiple drugs, including tigecycline. The promoter activity of tmexC6D6-toprJ1b and negative transcriptional repression by the upstream regulator tnfxB6 are crucial for the expression of tmexC6D6-toprJ1b. tmexC6D6-toprJ1b was found in the plasmids or chromosomes of different Pseudomonas species from six countries. Two genetic backgrounds, class 1 integrons and int-carrying integrase units, were found adjacent to the tmexC6D6-toprJ1b gene cluster and might mediate the transfer of this novel efflux pump gene cluster in Pseudomonas. Further phylogenetic analysis revealed Pseudomonas as the major reservoir of tmexCD-toprJ variants, warranting closer monitoring in the future. IMPORTANCE Tigecycline is one of the treatment options for serious infections caused by multidrug-resistant bacteria, and tigecycline resistance has gained extensive attention. The emergence of a transferable tigecycline resistance efflux pump gene cluster, tmexCD-toprJ, severely challenged the efficiency of tigecycline. In this study, we identified another novel tmexCD-toprJ variant, tmexC6D6-toprJ1b, which could confer resistance to multiple classes of antibiotics, including tigecycline. Although tmexC6D6-toprJ1b was found only in Pseudomonas species, tmexC6D6-toprJ1b might spread to Enterobacteriaceae hosts via mobile genetic elements resembling those of other tmexCD-toprJ variants, compromising the therapeutic strategies. Meanwhile, novel transferable tmexCD-toprJ variants are constantly emerging and mostly exist in Pseudomonas spp., indicating Pseudomonas as the important hidden reservoir and origin of tmexCD-toprJ variants. Continuous monitoring and investigations of tmexCD-toprJ are urgent to control its spread.

Study on the Interaction Mechanism of Methoxy Polyethylene Glycol Maleimide with Sweet Potato ß-Amylase.

In this study, sweet potato ß-amylase (SPA) was modified by methoxy polyethylene glycol maleimide (molecular weight 5000, Mal-mPEG5000) to obtain the Mal-mPEG5000-SPA modified ß-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.

Paenibacillus polymyxa Antagonism towards Fusarium: Identification and Optimisation of Antibiotic Production.

An antibiotic produced by Paenibacillus polymyxa 7F1 was studied. The 7F1 strain was isolated from the rhizosphere of a wheat field. Response surface methodology was used to optimize the physicochemical parameters. The strain showed broad-spectrum activity against several plant pathogens. Identification of the strain was realized based on 16s rRNA gene and gyrB gene sequencing. The antibiotic was optimized by one-factor-at-a-time (OFAT) and response surface methodology (RSM) approaches. The suitable antibiotic production conditions were optimized using the one-factor-at-a-time method. The individual and interaction effects of three independent variables: culture temperature, initial pH, and culture time, were optimized by Box-Behnken design. The 16SrRNA gene sequence (1239 nucleotides) and gyrB gene (1111 nucleotides) were determined for strain 7F1 and shared the highest identities to those of Paenibacillus polymyxa. The results showed the optimal fermentation conditions for antibiotics produced by Paenibacillus polymyxa 7F1 were a culture temperature of 38 °C, initial pH of 8.0, and culture time of 8 h. The antibiotics produced by Paenibacillus polymyxa 7F1 include lipopeptides such as iturin A and surfactin. The results provide a theoretical basis for the development of bacteriostatic biological agents and the control of mycotoxins.

Early prediction of pathological complete response to neoadjuvant chemotherapy combining DCE-MRI and apparent diffusion coefficient values in breast Cancer.

INTRODUCTION: Improving the early prediction of neoadjuvant chemotherapy (NAC) efficacy in breast cancer can lead to an improved prediction of the final prognosis of patients, which would be useful for promoting individualized treatment. This study aimed to explore the value of the combination of dynamic contrast-enhanced (DCE)-MRI parameters and apparent diffusion coefficient (ADC) values in the early prediction of pathological complete response (pCR) to NAC for breast cancer. METHODS: A total of 119 (range, 28-69 years) patients with biopsy-proven breast cancer who received two cycles of NAC before breast surgery were retrospectively enrolled from our hospital database. Patients were divided into pCR and non pCR groups according to their pathological responses; a total of 24 patients achieved pCR, while 95 did not. The quantitative (Ktrans; Kep; Ve; IAUC) and semiquantitative parameters (W-in; W-out; TTP) of DCE-MRI that were significantly different between groups were combined with ADC values to explore their value in the early prediction of pCR to NAC for breast cancer. The independent T test was performed to compare the differences in DCE-MRI parameters and ADC values between the two groups. Receiver operating characteristic (ROC) curves were plotted, and the area under the ROC curve (AUC), sensitivity and specificity were calculated to evaluate the performance of the prediction. RESULTS: The Ktrans, Kep, IAUC, ADC, W-in and TTP values were significantly different between the pCR and non pCR groups after NAC. The AUC (0.845) and specificity (95.79%) of the combined Ktrans, Kep, IAUC and ADC values were both higher than those of the individual parameters. The combination of W-in, TTP and ADC values had the highest AUC value (0.886) in predicting pCR, with a sensitivity and specificity of 87.5% and 82.11%, respectively. CONCLUSIONS: The results suggested that the combination of ADC values and quantitative and semiquantitative DCE-MRI parameters, especially the combination of W-in, TTP, and ADC values, may improve the early prediction of pCR in breast cancer.

Germline variants profiling of BRCA1 and BRCA2 in Chinese Hakka breast and ovarian cancer patients.

OBJECTIVE: To investigate the prevalence and spectrum of BRCA1 and BRCA2 mutations in Chinese Hakka patients with breast and ovarian cancer. METHODS: A total of 1,664 breast or ovarian cancer patients were enrolled for genetic testing at our hospital. Germline mutations of the BRCA gene were analysed by next-generation sequencing, including the coding regions and exon intron boundary regions. RESULTS: The 1,664 patients included 1,415 (85.04%) breast cancer patients and 245 (14.72%) ovarian cancer patients, while four (0.24%) patients had both the breast and ovarian cancers. A total of 151 variants, including 71 BRCA1 variants and 80 BRCA2 variants, were detected in the 234 (14.06%) patients. The 151 variants included 58 pathogenic variants, 8 likely pathogenic variants, and 85 variants of unknown significance (VUS). A total of 56.25% (18/32) and 65.38% (17/26) of pathogenic variants (likely pathogenic variants are not included) were distributed in exon 14 of BRCA1 and exon 11 of BRCA2, respectively. The most common pathogenic variants among this Hakka population are c.2635G > T (p.Glu879*) (n = 7) in the BRCA1 gene and c.5164_5165del (p.Ser1722Tyrfs*4) (n = 7) in the BRCA2 gene among the Hakka population. A hotspot mutation in the Chinese population, the BRCA1 c.5470_5477del variant was not found in this Hakka population. The prevalence and spectrum of variants in the BRCA genes in the Hakka patients are different from that in other ethnic groups. CONCLUSIONS: The most common pathogenic variant in this population is c.2635G > T in the BRCA1 gene, and c.5164_5165delAG in the BRCA2 gene in this population. The prevalence and spectrum of variants in the BRCA1 and BRCA2 genes in the Hakka patients from southern China are different from those in other ethnic groups.

Influence of adding steam-exploded apple pomace on wheat flour characteristics and biscuit quality.

Apple pomace treated by steam explosion (SE-AP) was mixed with wheat flour, the wheat dough characteristics and biscuit quality are deserved to investigate. In this paper, the characteristics of wheat dough blended with SE-AP, including sedimentation values, pasting properties, and farinographic features were measured; the textural properties and sensory evaluation of the blended biscuits were analyzed. The results showed that the sedimentation values of wheat dough gradually decreased when SE-AP was less than 10%, which was almost no influence on the biscuit quality. The more SE-AP was added, the less values of peak viscosity, trough viscosity and final viscosity, which was disadvantage to the processing quality of wheat flour; however, the values of breakdown and setback increased with the addition of SE-AP, which improved the processing quality. Dough development time, stability time, and farinograph quality number decreased with the addition of SE-AP, which was unfavourable to the quality of wheat flour. When the addition of SE-AP was less than 10%, the hardness of biscuits decreased, springiness and resilience increased, and the chewability improved. According to the texture properties and organoleptic evaluation, the sensor score of the biscuits made from weak-gluten wheat with 10% (m/m) SE-AP added was the highest.

Structures, fabrication mechanisms, and emulsifying properties of self-assembled and spray-dried ternary complexes based on lactoferrin, oat ß-glucan and curcumin: A comparison study.

Protein-polyphenol-polysaccharide non-covalent ternary complexes possess many unique structural and functional properties. However, rare work is available to fabricate the neutral polysaccharide-based ternary complexes. Herein, the ternary complexes composed of lactoferrin (LF), oat ß-glucan (OG), and curcumin (Cur) with three binding sequences were successfully developed through self-assembly technique and spray drying technique, respectively. Spray drying could enhance the extent of the intermolecular associations among LF, OG, and Cur, leading to the formation of ternary complexes with smaller particle sizes and lower turbidities. Cur can be loaded in LF-OG complexes to form an amorphous complex through the intermolecular interactions (mainly hydrophobic interactions and hydrogen bonding). The ternary complexes can be used as potential emulsifiers to stabilize oil-in-water Pickering emulsions. The emulsifying capacity (to enhance physical stability) of the complexes was in the order as follows: the spray-dried ternary complexes > the spray-dried LF-OG complexes > the self-assembled ternary complexes > the self-assembled LF-OG complexes. The structural and functional properties (e.g., emulsifying property) of OG-based ternary complexes can be controlled by adjusting the binding sequences. These results will broaden our current understanding of protein-polyphenol-polysaccharide ternary complexes and provide more applications of OG in food, cosmetics, and pharmaceutical industries.

Analysis of differential expression proteins reveals the key pathway in response to heat stress in Alicyclobacillus acidoterrestris DSM 3922T.

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.

Label free quantitative analysis of Alicyclobacillus acidoterrestris spore germination subjected to low ambient pH.

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 ß-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.

Optimization of DHEA Extraction from Sweet Potato Pomace by Ultrasonic-Microwave Synergistic Employing Response Surface Methodology.

Background: A lot of sweet potato residues (SPR) were discarded and wasted. Objective: To make full use of the SPR. Methods: Ultrasonic microwave synergistic (UMS) extraction method was used to extract dehydroepiandrosterone (DHEA) in SPR. The extraction conditions were optimized by response surface methodology based on single factors. Results: The optimum extraction conditions were 1:25 (solid-liquid ratio), 300 W (microwave power), 30 min (extraction time), and 30°C (extraction temperature). The extraction yield of DHEA from SPR reached 117.25 µg/100 g. Conclusions: The advantage of UMS extraction technology is to make full use of the synergistic effect of ultrasound and microwave to improve extraction efficiency. Highlights: The technology provides an effective way to improve the DHEA extraction yield from the SPR in industrial production.

Chemical Modification of Sweet Potato ß-amylase by Mal-mPEG to Improve Its Enzymatic Characteristics.

The sweet potato ß-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.

Heterogeneous expression of DnaK gene from Alicyclobacillus acidoterrestris improves the resistance of Escherichia coli against heat and acid stress.

Alicyclobacillus acidoterrestris, an acidophilic and thermophilic bacteria, is an important microbial resource for stress resistance genes screening. In this study, DnaK gene from A. acidoterrestris was subcloned to construct the recombinant plasmid pET28a-DnaK. The successful construction of the plasmid was verified by double-enzyme digestion and sequencing analysis. The recombinant plasmid was transformed into Escherichia coli BL21 and isopropy-ß-D-thiogalactoside (IPTG) was used to induce recombinant E. coli to express DnaK gene. A 70 kD fusion protein was identified by SDS-PAGE, which suggested that DnaK gene from A. acidoterrestris was successfully expressed. The recombinant and wild BL21 were treated with high temperatures of 54, 56 and 58 °C at pH values of 5.0-7.0 to compare the effects of heterogeneous expression of the DnaK gene from A. acidoterrestris on the stress resistance. The experimental results showed that survival rate of recombinant BL21-DnaK has been improved considerably under heat and acid stresses in contrast with the wild BL21, and D-values of recombinant BL21 were 14.7-72% higher than that of wild BL21, which demonstrated that heterogeneous expression of DnaK gene from A. acidoterrestris could significantly enhance the resistance of host bacteria E. coli against heat and acid stresses.

Optimization of glutamine peptide production from soybean meal and analysis of molecular weight distribution of hydrolysates.

The process parameters of enzymatic hydrolysis and molecular weight distribution of glutamine (Gln) peptides from soybean meal were investigated. The Protamex(®) hydrolysis pH of 6.10, temperature of 56.78 °C, enzyme to substrate ratio (E/S) of 1.90 and hydrolysis time of 10.72 h were found to be the optimal conditions by response surface methodology (RSM) for a maximal degree of hydrolysis (DH) value of 16.63% and Gln peptides content at 5.95 mmol/L. The soybean meal was hydrolyzed by a combination of Protamex(®) and trypsinase so that DH and Gln peptides would reach 22.02% and 6.05 mmol/mL, respectively. The results of size exclusion chromatography indicated that the relative proportion of the molecular weight <1000 Da fraction increased with DH values from 6.76%, 11.13%, 17.89% to 22.02%, most notably the 132-500 Da fractions of hydrolysates were 42.14%, 46.57%, 58.44% and 69.65%. High DH values did not lead to high Gln peptides content of the hydrolysate but to the low molecular weight Gln peptides.

[Arginine metabolism in wine malolactic bacteria].

Arginine metabolism in wine malolactic bacteria may increase the concentration of ethyl carbamate (EC, a known carcinogen with the potential toxicological effects) in wine, which makes a profound impact on wine hygienic quality. In fact, recent advanced studies have proved that arginine metabolism in wine malolactic bacteria is via arginine deiminase pathway (ADI pathway), and this pathway is composed of three enzymes: arginine deiminase (ADI), ornithine transcarbamylase (OTC), carbamate kinase (CK). The possible mechanisms of arginine transport and the regulation of major enzymes for arginine metabolism as well as the mechanism of ADI pathway and its molecular characteristics are reviewed. The study on arginine metabolism in wine malolactic bacteria has practical and theoretical significance for the safety and high quality of wine.