In this study, we aimed to examine the inhibitory effect of PA003, a Pediococcus acidilactici that produces lactic acid and antimicrobial peptides pediocin, on pathogenic biofilm formation on abiotic surfaces. PA003 and pathogens (Escherichia coli, Salmonella enterica serovar Typhimurium, Staphylococcus aureus and Listeria monocytogenes) were used to evaluate auto-aggregation, hydrophobicity, biofilm formation and biofilm formation inhibition on stainless steel, polyvinyl chloride and glass slides in terms of exclusion, displacement and competition. The results showed the highest auto-aggregation abilities were observed for one of the E. coli strains EAggEC (E58595) and the highest hydrophobic strain was observed with EPEC (E2348/69) (51.9%). The numbers of biofilm cells of E. coli, S. Typhimurium, S. aureus and L. monocytogenes on stainless steel, polyvinyl chloride and glass slide coupons were effectively reduced by approximately 4 log CFU/coupon. These results demonstrate that lactic acid bacteria can be used as an alternative to effectively control the formation of biofilms by food-borne pathogens.

The objective of this study was to investigate pig fed by Bacillus coagulans-fermented distillers' dried grains with solubles (DDGS) on the faecal microbial composition and diversity using 454 pyrosequencing. Healthy crossbred (Duroc×Yorkshire×Landrace) growing and fattening pigs (n=48), with an average initial body weight of 65 kg, were divided into two groups (24 replicates per group; four pens per group; six pigs per pen), and given either DDGS feed as the control, or B. coagulans-fermented DDGS feed as the treatment. Faecal samples were collected on day 0, 7, 14, 21, and 28. DNA was extracted, and the V3–V6 region of the 16S rRNA gene was amplified. The fermented DDGS feed affected the relative abundance of bacteria populations at the phylum, genus, and species levels. At the genus level, the consumption of fermented DDGS feed led to higher relative abundances of faecal Prevotella, Lactobacillus, Clostridium, Bifidobacterium, Roseburia, and Bacillus, and lower relative abundances of faecal Escherichia, Ruminococcus, Dialister, unclassified Lachnospiraceae, unclassified Ruminococcaceae, and unclassified Enterobacteriaceae than in the control. At the species level, the consumption of fermented DDGS feed led to higher relative abundances of faecal Prevotella sp., Lactobacillus johnsonii, Lactobacillus fermentum, Lactobacillus mucosae, Lactobacillus reuteri, Clostridium butyricum, Bifidobacterium sp., and Roseburia sp., and lower relative abundances of faecal Prevotella copri, Escherichia coli, Ruminococcus gnavus, Ruminococcus flavefaciens, and Dialister sp. than in the control. Principal coordinates analysis indicated a distinct separation in the faecal microbial communities of pigs that were fed the fermented and unfermented DDGS feed. Fermented DDGS feed significantly increased the average daily gain (ADG) of pigs, and significantly decreased the average daily feed intake (ADFI) of feed and feed/gain (F/G). Thus, our results demonstrate a beneficial shift in the faecal microbiota of pigs consuming fermented DDGS feed, with potential applications in livestock production.

•By introducing microscale thermophoresis technology, we provided a simple and institutional analysis for molecular binding system.•A relatively complete and convictive discussion about the Hg2+ detecting principle of molecular binding system was presented.•Based on T-Hg2+-T coordination chemistry and molecular beacon, a sensitive and convenient detection method for Hg2+ has been established.A novel evaluation assay for molecular beacon (MB)-binding system was developed by microscale thermophoresis (MST) and thymine–mercuric–thymine coordination chemistry which was applied for the detection of mercury (II) ion (Hg2+) upon opening the hairpin and facilitating fluorescence recovery through binding with suitable mismatched target. The affinities between the loop of MBs and the complementary ssDNAs in the binding system were varied, determined and optimized by MST in the presence or absence of Hg2+. It suggested that the relative low background could be obtained when the number of mismatches of ssDNA was four and in the present of Hg2+ displayed strong recovery of fluorescence. The lowest detection limit of Hg2+ was 3 nM which was below the maximum contaminant level defined by U.S. Environmental Protection Agency (10 nM). The working range was 5–320 nM with negligible possible interference from the different background composition of water samples by optimization of the binding conditions. Through evaluating by MST, the molecular interaction mechanism was elaborated and the rapid, sensitive and selective detection of Hg2+ was achieved by the designed MB-binding system. It herein has great potential for appraising and analyzing molecular interaction among the interactants.Download high-res image (163KB)Download full-size image

A great amount of foodborne pathogens were Gram-positive (G+) bacteria, a threat to public health. In this study, considering the binding ability of nisin towards G+ bacteria and the stable fluorescent ability of EGFP protein, a fluorescent nisin–EGFP protein probe was constructed by a gene engineering method. Nisin and EGFP were used as the receptor and fluorophore, respectively, to detect G+ bacteria. The nisin and egfp gene were amplified separately according to the sequence published in GenBank using unique primers. The two genes were cloned into a pET-28b(+) vector resulting in a pET-28b(+)–nisin–egfp vector. The vector was transferred into Escherichia coli (E. coli) BL21 (DE3) for expression. The expressed protein was extracted, purified by a Ni–NTA column, and then tested by the SDS-PAGE method to confirm its molecular weight. Listeria monocytogenes (L. monocytogenes), Staphylococcus aureus (S. aureus), and Micrococcus luteus (M. luteus) were used as the representations of G+ bacteria. E. coli O157, representing the gram-negative (G−) bacteria, was used as a negative control. The binding specificity of the recombinant protein was performed on two types of bacteria and then detected through fluorescent microscopy. The results indicated that the nisin–EGFP probe could detect G+ bacteria at 108 CFU/mL.

The objective of this study was to describe the fecal microbiota succession of piglets from birth to post-weaning by pyrosequencing. The dominant bacterial phyla were Bacteroidetes, Firmicutes, Proteobacteria, and Fusobacteria, although the composition of the fecal microbiota changed as the piglets grew. Bacteroides, Parabacteroides, Prevotella, Lactobacillus, unclassified Lachnospiraceae, Ruminococcus, Oscillospira, Phascolarctobacterium, and Desulfovibrio were the core genera in the feces of piglets and existed from birth to post-weaning. Bacteroides was the most abundant at birth; however, the proportion of Bacteroides decreased as the piglets aged, while the proportion of Prevotella increased until it became the most abundant genus at the post-weaning stage. Principal coordinate analysis indicated that the microbial communities of piglet fecal samples from birth to post-weaning could be separated into four groups according to the similarity of community composition. These corresponded to time points on day 0 (birth); day 7; days 14, 21, and 28 (pre-weaning); and day 35 (post-weaning). This study suggests that the development of the intestinal microbiota in the piglet is a gradual and sequential process. This methodology establishes a basis for broader studies to investigate the microbiota of the piglet intestinal tract.

•The Fe3O4-COOH@MIL-101 composites combined the excellent properties of MIL-101 and Fe3O4-COOH particles.•Fe3O4-COOH@MIL-101 composites exhibited the efficient protein enrichment properties, size-exclusion performance and magnetic responsiveness.•Fe3O4-COOH@MIL-101 composites were successfully applied in the selective enrichment of the protein biomarkers from bacterial cell lysates and the discrimination of Escherichia coli at the strain level.Rapid and efficient characterization and identification of pathogens at the strain level is of key importance for epidemiologic investigations, which still remains a challenge. In this work, solvothermically Fe3O4-COOH@MIL-101 composites were fabricated by in situ crystallization approach. The composites combine the excellent properties of both chromium (III) terephthalate (MIL-101) and carboxylic-functionalized magnetite (Fe3O4-COOH) particles and possess the efficient peptides/proteins enrichment properties and magnetic responsiveness. Fe3O4-COOH@MIL-101 composites as magnetic solid phase extraction materials were used to increase the discriminatory power of MALDI-TOF MS profiles. BSA tryptic peptides at a low concentration of 0.25 fmol μL−1 could be detected by MALDI-TOF MS. In addition, Fe3O4-COOH@MIL-101 composites were successfully applied in the selective enrichment of the protein biomarkers from bacterial cell lysates and discrimination of Escherichia coli at the strain level. This work provides the possibility for wide applications of magnetic MOFs to discriminate pathogens below the species level.

A novel aptamer-based suspension array detection platform was designed for the sensitive, specific and rapid detection of human α-thrombin as a model. Thrombin was first recognized by a 29-mer biotinylated thrombin-binding aptamer (TBA) in solution. Then 15-mer TBA modified magnetic beads (MBs) captured the former TBA–thrombin to form an aptamer–thrombin–aptamer sandwich complex. The median fluorescence intensity obtained via suspension array technology was positively correlated with the thrombin concentration. The interactions between TBAs and thrombin were analyzed using microscale thermophoresis (MST). The dissociation constants could be respectively achieved to be 44.2 ± 1.36 nM (TBA1–thrombin) and 15.5 ± 0.637 nM (TBA2–thrombin), which demonstrated the high affinities of TBA–thrombin and greatly coincided with previous reports. Interaction conditions such as temperature, reaction time, and coupling protocol were optimized. The dynamic quantitative working range of the aptamer-based suspension array was 18.37–554.31 nM, and the coefficients of determination R2 were greater than 0.9975. The lowest detection limit of thrombin was 5.4 nM. This method was highly specific for thrombin without being affected by other analogs and interfering proteins. The recoveries of thrombin spiked in diluted human serum were in the range 82.6–114.2%. This innovative aptamer-based suspension array detection platform not only exhibits good sensitivity based on MBs facilitating highly efficient separation and amplification, but also suggests high specificity by the selective aptamer binding, thereby suggesting the expansive application prospects in research and clinical fields.

The putrefaction of alkaline silica sol was investigated in this paper. The total colony numbers in three alkaline silica sol samples were 1.47×105, 1.25×104, and 9.45×104 cfu·mL−1, respectively. The salt- and alkali-tolerant strains were isolated and selected using nutrient agar medium at 2.5% salinity and pH 9.5. Basic morphological, physiological and biochemical tests were conducted to confirm the preliminary characterizations of the strains. Based on API 50 CH test and 16S rDNA gene sequence analysis, the isolated strains were finally identified as Exiguobacterium aurantiacum, Cyclobacteriaceae bacterium, Microbacterium sp., Acinetobacter sp., Stenotrophomonas maltophilia and Bacillus thuringiensis. The survivability of the strains under different conditions such as salinities, acidities and temperatures was also studied. Some suitable methods for degerming, such as product pipe steam sterilization and regular canister cleaning, were proposed. To explore the possibility of isolates in industrial application, their alkaline protease and amylase production abilities were preliminarily studied. Five strains produced alkaline protease, whereas two strains produced alkaline amylase. Thus, understanding of the putrefaction on alkaline silica sol would be beneficial for improving industrial production.

Antifungal lipopeptide produced by Bacillus sp. BH072 was extracted from fermentation liquor and determined as iturin A by liquid chromatography-mass spectrometry (LC-MS). For industrial-scale production, the yield of iturin A was improved by optimizing medium components and fermentation conditions. A one-factor test was conducted; fermentation conditions were then optimized by response surface methodology (RSM) to obtain the following: temperature, 29.5°C; pH 6.45; inoculation quantity, 6.7%; loading volume, 100 ml (in 500 ml flasks); and rotary speed, 150 rpm. Under these conditions, the mass concentration of iturin A was increased from 45.30 mg/ml to 47.87 mg/ml. The following components of the medium were determined: carbon sources (glucose, fructose, sucrose, xylose, rhamnose, and soluble starch); nitrogen sources (peptone, soybean meal, NH4Cl, urea, and ammonium citrate); and metal ions (Zn2+, Fe3+, Mg2+, Mn2+, Ca2+, and K+). The effects of these components on iturin A production were observed in LB medium. We selected sucrose, soybean meal, and Mg2+ for RSM to optimize the conditions because of several advantages, including maximum iturin A production, high antifungal activity, and low cost. The optimum concentrations of these components were 0.98% sucrose, 0.94% soybean meal, and 0.93% Mg2+. After iturin A production was optimized by RSM, the mass concentration reached 52.21 mg/ml. The antifungal specific activity was enhanced from 350.11 AU/mg to 513.92 AU/mg, which was 46.8% higher than the previous result. The present study provides an important experimental basis for the industrial-scale production of iturin A and the agricultural applications of Bacillus sp. BH072.

A new erythritol-producing yeast (strain BH010) was isolated in this study. Analysis of the D1/D2 domain of the 26S rDNA sequence, the ITS/5.8S rDNA sequence, and the 18S rDNA sequence allowed the taxonomic position of strain BH010 to be discussed and it was identified and named Moniliella sp. BH010. Physiological characteristics were described. Scanning electron micrography clearly indicated that the cells were cylindrical to elliptical with an average size of 5 × 10 μm when growing in liquid medium, and that pseudohyphae and blastoconidia were observed when cultivated in agar plates. The erythritol reductase genes were cloned, sequenced, and analyzed. BLAST analysis and multiple sequence alignment demonstrated that erythritol reductase genes of Moniliella sp. BH010 shared very high homology with that of Trichosporonoides megachiliensis SNG-42 except for the presence of introns. The deduced amino acid sequences showed high homology to the aldo–keto reductase superfamily.

Research shows some potential applications for ionic liquids (ILs) with biomaterials. In this work, the ILs 1-butyl-3-methylimidazolium tetrafluoroborate ([C4mim]BF4), 1-butyl-3-methylimidazolium chloride ([C4mim]Cl), 1-butyl-3-methylimidazolium bromide ([C4mim]Br), and 1,3-dimethylimidazolium iodine ([dmim]I) were employed to investigate their effects on the solubility of lysozyme in aqueous solutions at pH 4.5. The results demonstrate that lysozyme solubility increases with the addition concentration of [C4mim]BF4 and [C4mimCl] and is nearly invariable with the increase of the [C4mim]Br concentration. Interestingly, the solubility slowly decreases with the increase of [dmim]I concentration. At constant added IL concentration, the solubility increases with increasing temperature. The effect of ILs on solubility can be attributed to changing interaction among lysozyme molecules. The effect of ILs employed in this work may provide crucial insight into the preparation of high-quality crystals and the development of new crystal forms as well as the design optimization of protein crystallization.

Two heterologous expression systems using thioredoxin (trxA) as a gene fusion part in Escherichia coli were developed to produce recombinant pediocin PA-1. Pediocin PA-1 structural gene pedA was isolated from Pediococcus acidilactici PA003 by the method of polymerase chain reaction (PCR), then cloned into vector pET32a(+), and expressed as thioredoxin-PedA fusion protein in the host strain E. coli BL21 (DE3). The fusion protein was in the form of inclusion body and was refolded before purification by nickel-iminodiacetic acid (Ni-IDA) agarose resin column. Biological activity of recombinant pediocin PA-1 was analyzed after cleavage of the fusion protein by enterokinase. Agar diffusion test revealed that 512-arbitrary unit (AU) recombinant pediocin PA-1 was obtained from 1 ml culture medium of E. coli (pPA003PED1) using Listeria monocytogenes as the indicator strain. Thioredoxin-PedA fusion gene was further cloned into pET20b(+). Thioredoxin-PedA fusion protein was detected in both the periplasmic and cytoplasmic spaces. The recombinant pediocin PA-1 from the soluble fraction attained 384 AU from 1 ml culture medium of E. coli (pPA003PED2). Therefore, biologically active pediocin PA-1 could be obtained by these two hybrid gene expression methods.