Rapid Proteomic Characterization of Bacteriocin-Producing Enterococcus faecium Strains from Foodstuffs

Enterococcus belongs to a group of microorganisms known as lactic acid bacteria (LAB), which constitute a broad heterogeneous group of generally food-grade microorganisms historically used in food preservation. Enterococci live as commensals of the gastrointestinal tract of warm-blooded animals, although they also are present in food of animal origin (milk, cheese, fermented sausages), vegetables, and plant materials because of their ability to survive heat treatments and adverse environmental conditions. The biotechnological traits of enterococci can be applied in the food industry; however, the emergence of enterococci as a cause of nosocomial infections makes their food status uncertain. Recent advances in high-throughput sequencing allow the subtyping of bacterial pathogens, but it cannot reflect the temporal dynamics and functional activities of microbiomes or bacterial isolates. Moreover, genetic analysis is based on sequence homologies, inferring functions from databases. Here, we used an end-to-end proteomic workflow to rapidly characterize two bacteriocin-producing Enterococcus faecium (Efm) strains. The proteome analysis was performed with liquid chromatography coupled to a trapped ion mobility spectrometry-time-of-flight mass spectrometry instrument (TimsTOF) for high-throughput and high-resolution characterization of bacterial proteins. Thus, we identified almost half of the proteins predicted in the bacterial genomes (>1100 unique proteins per isolate), including quantifying proteins conferring resistance to antibiotics, heavy metals, virulence factors, and bacteriocins. The obtained proteomes were annotated according to function, resulting in 22 complete KEGG metabolic pathway modules for both strains. The workflow used here successfully characterized these bacterial isolates and showed great promise for determining and optimizing the bioengineering and biotechnology properties of other LAB strains in the food industry.     

大曲中产阿魏酸酯酶菌株的筛选及其特性分析

目的 从浓香型白酒大曲中分离筛选出一株产阿魏酸酯酶的菌株。方法 将大曲样品中筛选出的11株菌进行划线纯化培养,进一步挑选出产阿魏酸酯酶酶活较强的菌株并鉴定,分析其安全性及生物学特性,最后对发酵产酶条件进行优化。结果 得到一株产阿魏酸酯酶酶活较强的菌株为L-7,经鉴定为屎肠球菌(Enterococcus faecium)。经毒力基因检测、有害代谢产物实验结果均为阴性;生物学特性研究实验结果为:在16 h时生物量达到最大,耐最低pH为4,最高为11, NaCl质量分数为8%的条件下仍能生长,耐受最高的乙醇体积分数为13%;具有较好的安全性且有耐乙醇、耐盐、广泛的pH耐受性等优良性能。通过发酵产酶条件优化,该菌株产阿魏酸酯酶最佳发酵条件为:接种量为6%体积分数、培养温度32℃、转速160r/min、培养时间96 h;此时该菌株产阿魏酸酯酶的酶活为(44.68±0.05) U/L,较优化前提高了57.05%。结论 本研究结果可为产阿魏酸酯酶的菌株筛选提供参考,且筛选所得屎肠球菌在提高浓香型白酒及其他发酵食品中阿魏酸含量及风味具有应用潜力。     

两株降胆固醇球菌生长条件的研究

本实验对粪肠球菌的基础培养基碳、氮源进行筛选,同时对其生长所需的温度、时间和接菌量进行研究,优化出适合粪肠球菌生长最佳培养基,以提高活菌数量。在优化后的培养基中菌数分别达5.6×109CFU/mL、8.0×109CFU/mL。     

自然发酵乳中粪肠球菌和屎肠球菌的4种鉴定方法的比较

采用传统生理生化鉴定方法,16S rRNA基因序列分析技术,16S-23S rRNA间区序列多态性分析技术,变性梯度凝胶电泳技术(DGGE),对分离于自然发酵乳中的9株粪肠球菌和6株屎肠球菌进行鉴定,并对4种鉴定方法进行比较和评价。结果表明,16S-23S rRNA间区序列多态性分析技术和DGGE技术不但可以快速、精确地区分粪肠球菌和屎肠球菌,而且能够将粪肠球菌和屎肠球菌种内的不同基因亚型区分开,而传统生理生化鉴定方法和16S rRNA基因序列分析技术较以上两种方法区分效果略差。     

Chemical composition,antimicrobial potential against cariogenic bacteria and cytotoxic activity of Tunisian Nigella sativa essential oil and thymoquinone

We investigate in this work the chemical composition by GC–EIMS, the antibacterial and the cytotoxic activities of Tunisian Nigella sativa essential oil and its bioactive compound, thymoquinone, were tested against various clinical cariogenic bacteria (n = 30). Eighty-four compounds were identified in the essential oil. The major one was p-cymene (49.48%) whereas thymoquinone represented only 0.79%. The essential oil (2.43 mg/disc) containing only 3.35 μg of thymoquinone showed pronounced dose dependant antibacterial activity against Streptococcus mitis, Streptococcus mutans, Streptococcus constellatus and Gemella haemolysans (15.5 ± 0.707 mm). However, pure thymoquinone compound (150 μg/disk) was active against all the studied strains especially S. mutans and S. mitis (24.5 ± 0.71 and 22 ± 1.41 mm inhibition zones, respectively).     

Water Diffusion from a Bacterial Cell in Low‐Moisture Foods

We used a Fick's unsteady state diffusion equation to estimate the time required for a single spherical shaped bacterium (assuming Enterococcus faecium as the target microorganism) in low‐moisture foods to equilibrate with the environment. We generated water sorption isotherms of freeze‐dried E. faecium. The water activity of bacterial cells at given water content increased considerably as temperature increased from 20 to 80 °C, as observed in the sorption isotherms of bacterial cells. When the water vapor diffusion coefficient was assumed as between 10^?12 and 10^?10 m²/s for bacterial cells, the predicted equilibration times (t_eq) ranged from 8.24×10^?4 to 8.24×10^?2 s. Considering a cell membrane barrier with a lower water diffusion coefficient (10^?15 m²/s) around the bacterial cell with a water diffusion coefficient of 10^?12 m²/s, the t_eq predicted using COMSOL Multiphysics program was 3.8×10^?1 s. This result suggests that a single bacterium equilibrates rapidly (within seconds) with change in environmental humidity and temperature.     

Application of bacteriocinogenic Enterococcus mundtii CRL35 and Enterococcus faecium ST88Ch in the control of Listeria monocytogenes in fresh Minas cheese

Several strains of Enterococcus spp. are capable of producing bacteriocins with antimicrobial activity against important bacterial pathogens in dairy products. In this study, the bacteriocins produced by two Enterococcus strains (Enterococcus mundtii CRL35 and Enterococcus faecium ST88Ch), isolated from cheeses, were characterized and tested for their capability to control growth of Listeria monocytogenes 426 in experimentally contaminated fresh Minas cheese during refrigerated storage. Both strains were active against a variety of pathogenic and non-pathogenic microorganisms and bacteriocin absorption to various L. monocytogenes, Enterococcus faecalis ATCC 19443 and Lactobacillus sakei ATCC 15521 varied according to the strain and the testing conditions (pH, temperature, presence of salts and surfactants). Growth of L. monocytogenes 426 was inhibited in cheeses containing E. mundtii CRL35 up to 12 days at 8 °C, evidencing a bacteriostatic effect. E. faecium ST88Ch was less effective, as the bacteriostatic affect occurred only after 6 days at 8 °C. In cheeses containing nisin (12.5 mg/kg), less than one log reduction was observed. This research underlines the potential application of E. mundtii CRL35 in the control of L. monocytogenes in Minas cheese.     

坚强肠球菌SQ-3-2基于Lux S群体感应系统信号分子AI-2的检测及方法优化

通过生物学方法检测坚强肠球菌SQ-3-2是否产生群体感应信号分子AI-2,并对检测条件进行优化。将坚强肠球菌SQ-3-2培养上清液加入到由哈维氏弧菌BB170构成的特异性报告系统中,使用多功能酶标仪化学发光模式检测荧光强度,通过与AB培养基空白对照进行荧光强度的比较得出坚强肠球菌SQ-3-2是否产生具有活性的AI-2信号分子,同时依据荧光强度的大小对加样比和酸碱度两个条件进行优化。研究结果表明,坚强肠球菌SQ-3-2培养上清液中含有AI-2信号分子,随着菌体密度的增加信号分子AI-2的浓度也随之增大,在对数期末期达到最大值。方法优化实验证明最佳检测条件为待测样品pH=7,加样比例为1∶100。实验为进一步研究坚强肠球菌SQ-3-2的Lux S系统打下基础。同时,方法优化实验为检测各类乳酸菌是否分泌AI-2信号分子提供了一定的实验基础和理论依据。     

Physicochemical and Theoretical Characterization of a New Small Non-Metal Schiff Base with a Differential Antimicrobial Effect against Gram-Positive Bacteria

Searching for adequate and effective compounds displaying antimicrobial activities, especially against Gram-positive bacteria, is an important research area due to the high hospitalization and mortality rates of these bacterial infections in both the human and veterinary fields. In this work, we explored (E)-4-amino-3-((3,5-di-tert-butyl-2-hydroxybenzylidene)amino) benzoic acid (SB-1, harboring an intramolecular hydrogen bond) and (E)-2-((4-nitrobenzilidene)amino)aniline (SB-2), two Schiff bases derivatives. Results demonstrated that SB-1 showed an antibacterial activity determined by the minimal inhibitory concentration (MIC) against Staphylococcus aureus, Enterococcus faecalis, and Bacillus cereus (Gram-positive bacteria involved in human and animal diseases such as skin infections, pneumonia, diarrheal syndrome, and urinary tract infections, among others), which was similar to that shown by the classical antibiotic chloramphenicol. By contrast, this compound showed no effect against Gram-negative bacteria (Klebsiella pneumoniae, Escherichia coli, and Salmonella enterica). Furthermore, we provide a comprehensive physicochemical and theoretical characterization of SB-1 (as well as several analyses for SB-2), including elemental analysis, ESMS, ¹H and ¹³C NMR (assigned by 1D and 2D techniques), DEPT, UV-Vis, FTIR, and cyclic voltammetry. We also performed a computational study through the DFT theory level, including geometry optimization, TD-DFT, NBO, and global and local reactivity analyses.     

Utilisation possibility of Enterococcus faecalis isolates from neonate's faeces for production of fermented sausages as starter cultures

This study aimed at assessing the utilisation possibility of Enterococcus faecalis as starter cultures for production of fermented sausages. The E. faecalis isolates isolated from faeces samples of 5‐day‐old healthy newborn babies, identified by 16SrDNA sequencing and free from virulence determinants as proven by genomic sequencing, were used for fermented sausages production as starter culture. Six treatments (four inoculated with four different E. faecalis isolates, one with commercial starter culture and one control) were prepared. The inoculation with E. faecalis showed a stronger acidification compared with the control. The treatments with E. faecalis showed significantly higher flavour and acceptability scores in comparison with the control or commercial starter culture. Both of commercial starter culture and E. faecalis were positive with tyramine. Apart from the amine production, the inoculation with E. faecalis generally improved the acidifying activity and flavour of the products, suggesting their application potentials in fermented sausages production as starter cultures.