Butyrogenic effect of galactosyl and mannosyl carbohydrates and their regulation on piglet intestinal microbiota.

Diarrhea is a global problem that causes economic losses in the pig industry. There is a growing attention on finding new alternatives to antibiotics to solve this problem. Hence, this study aimed to compare the prebiotic activity of low-molecular-weight hydrolyzed guar gum (GMPS) with commercial manno-oligosaccharide (MOS) and galacto-oligosaccharide (GOS). We further identified their combined effects along with probiotic Clostridium butyricum on regulating the intestinal microbiota of diarrheal piglet by in vitro fermentation. All the tested non-digestible carbohydrates (NDCs) showed favorable short-chain fatty acid-producing activity, and GOS and GMPS showed the highest production of lactate and butyrate, respectively. After 48 h of fermentation, the greatest enhancement in the abundance of Clostridium sensu stricto 1 was observed with the combination of GMPS and C. butyricum. Notably, all the selected NDCs significantly decreased the abundances of pathogenic bacteria genera Escherichia-Shigella and Fusobacterium and reduced the production of potentially toxic metabolites, including ammonia nitrogen, indole, and skatole. These findings demonstrated that by associating with the chemical structure, GMPS exhibited butyrogenic effects in stimulating the proliferation of C. butyricum. Thus, our results provided a theoretical foundation for further application of galactosyl and mannosyl NDCs in the livestock industry. KEY POINTS: • Galactosyl and mannosyl NDCs showed selective prebiotic effects. • GMPS, GOS, and MOS reduced pathogenic bacteria and toxic metabolites production. • GMPS specifically enhanced the Clostridium sensu stricto 1 and butyrate production.

Complete-genome sequence and in vitro probiotic characteristics analysis of Bifidobacterium pseudolongum YY-26.

AIMS: The aim was to isolate a neotype bifidobacteria strain and evaluate its in vitro probiotic potential. METHODS AND RESULTS: Bifidobacterium pseudolongum YY-26 (CGMCC 24310) was isolated from faeces of mice treated with low-molecular-weight hydrolyzed guar gum (GMPS) and identified based on 16S rRNA sequence and genome sequence. Whole-genome sequencing obtained using PacBio's single-molecular and Illumina's paired-end sequencing technology. A genome of 2.1 Mb in length, with 1877 predicted protein-coding sequences was obtained. Carbohydrate-Activity enZyme analysis revealed that YY-26 encodes 66 enzymes related to carbohydrate metabolism. Whole genome sequence analysis revealed the typical probiotic characteristics of YY-26, including safety in genetic level and ability to produce beneficial metabolites and extracellular polysaccharides. Ability of extensive carbon source utilization and short-chain fatty acid production was observed with single YY-26 cultivation. Considerable acetic acids and lactic acids were determined in GMPS utilization. YY-26 showed tolerance to simulated gastrointestinal tract and displayed appreciable antioxidant activity of free radical scavenging. CONCLUSIONS: B. pseudolongum YY-26 was identified with numerous probiotic-associated genes and its probiotic characteristics were verified in vitro. SIGNIFICANCE AND IMPACT OF STUDY: This study supplemented with limited publicly information regarding the genomes of B. pseudolongum strains and revealed the probiotic potential of YY-26.

Characterization of Recombinant Antimicrobial Peptide BMGlv2 Heterologously Expressed in Trichoderma reesei.

Antimicrobial peptides (AMPs) serve as alternative candidates for antibiotics and have attracted the attention of a wide range of industries for various purposes, including the prevention and treatment of piglet diarrhea in the swine industry. Escherichia coli, Salmonella, and Clostridium perfringens are the most common pathogens causing piglet diarrhea. In this study, the antimicrobial peptide gloverin2 (BMGlv2), derived from Bombyx mandarina, was explored to determine the efficient prevention effect on bacterial piglet diarrhea. BMGlv2 was heterologously expressed in Trichoderma reesei Tu6, and its antimicrobial properties against the three bacteria were characterized. The results showed that the minimum inhibitory concentrations of the peptide against E. coli ATCC 25922, S. derby ATCC 13076, and C. perfringens CVCC 2032 were 43.75, 43.75, and 21.86 µg/mL, respectively. The antimicrobial activity of BMGlv2 was not severely affected by high temperature, salt ions, and digestive enzymes. It had low hemolytic activity against rabbit red blood cells, indicating its safety for use as a feed additive. Furthermore, the measurements of the leakage of bacterial cell contents and scanning electron microscopy of C. perfringens CVCC 2032 indicated that BMGlv2 exerted antimicrobial activity by destroying the cell membrane. Overall, this study showed the heterologous expression of the antimicrobial peptide BMGlv2 in T. reesei and verified its antimicrobial properties against three common pathogenic bacteria associated with piglet diarrhea, which can provide a reference for the applications of AMPs as an alternative product in industrial agriculture.

A multi-functional genetic manipulation system and its use in high-level expression of a ß-mannanase mutant with high specific activity in Pichia pastoris.

To further extend the practical application of a thermostable and acidic resistance ß-mannanase (ManAK) in animal feed additives, an effective strategy that combined directed evolution and metabolic engineering was developed. Four positive mutants (P191M, P194E, S199G and S268Q) with enhanced specific activity (25.5%-60.9%) were obtained. The S199G mutant exhibited 56.7% enhancement of specific activity at 37°C and good thermostability, and this was selected for high-level expression in P. pastoris X33. A multi-functional and scarless genetic manipulation system was proposed and functionally verified (gene deletion, substitution/insertion and point mutation). This was then subjected to Rox1p (an oxygen related transcription regulator) deletion and Vitreoscilla haemoglobin (VHb) co-expression for high enzyme productivity in P. pastoris X33VIIManAKS199G . An excellent strain, named X33VIIManAKS199G ∆rox1::VHb, was achieved by combining these two factors, and then the maximum enzymatic activity was further increased to 3753 U ml-1 , which was nearly twice as much as the maximum production of ManAK in P. pastoris. This work provides a systematic and effective method to improve the enzymatic yield of ß-mannanase, promotes the application of ManAK in feed additives, and also demonstrated that a scarless genetic manipulation tool is useful in P. pastoris.

Improvement of the Catalytic Ability of a Thermostable and Acidophilic ß-Mannanase Using a Consensus Sequence Design Strategy.

In order to improve the catalytic efficiency of a thermostable and acidophilic ß-mannanase (ManAK; derived from marine Aspergillus kawachii IFO 4308), three mutants were designed by amino acid sequence consensus analysis with a second ß-mannanase (ManCbs), which also belongs to the glycoside hydrolase family 5 (GH5) and has excellent catalytic efficiency. Three mutants were constructed and their biochemical characteristics were measured after heterologous expression in Pichia pastoris. The results revealed that the kcat/Km values of the three recombinant mannanases ManAKC292V, ManAKL293V, and ManAKL294H were enhanced by 303.0, 280.4, and 210.1%, respectively. Furthermore, ManAKL293V showed greater thermostability than ManAK, retaining 36.5% of the initial enzyme activity after incubation at 80°C for 5min. This study therefore provides a rational design strategy based on consensus sequence analysis to develop industrially valuable ß-mannanase for future applications in marine aquafeed.

Functionalizing metal nanostructured film with graphene oxide for ultrasensitive detection of aromatic molecules by surface-enhanced Raman spectroscopy.

Surface-enhanced Raman spectroscopy (SERS) as a powerful analytical tool has gained extensive attention. Despite of many efforts in the design of SERS substrates, it remains a grand challenge for creating a general substrate that can detect diverse target analytes. Herein, we report our attempt to address this issue by constructing a novel metal-graphene oxide nanostructured film as SERS substrate. Taking advantages of the high affinity of graphene oxide (GO) toward aromatic molecules and the SERS property of nanostructured metal, this structure exhibits great potential for diverse aromatic molecules sensing, which is demonstrated by using crystal violet (CV) with positive charge, amaranth with negative charge, and neutral phosphorus triphenyl (PPh(3)) as model molecules.

In situ controllable growth of Prussian blue nanocubes on reduced graphene oxide: facile synthesis and their application as enhanced nanoelectrocatalyst for H2O2 reduction.

As a single-atom-thick carbon material with high surface area and conductivity, graphene provides an ideal platform for designing composite nanomaterials for high-performance electrocatalytic or electrochemical devices. Herein, we demonstrated a facile strategy for controllably growing high-quality Prussian blue nanocubes on the surface of reduced graphene oxide (PBNCs/rGO), which represents a new type of graphene/transition metal complex heterostructure. The merit of this method is that the composite nanomaterials could be produced directly from GO in an in situ wet-chemical reaction, where the reduction of GO and the deposition of PBNCs occurred simultaneously. The obtained composite nanomaterials were characterized by transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), thermogravimetric analysis (TGA), Raman spectroscopy, and electrochemical techniques. It was found that uniform PBNCs with controlled size and good dispersion were directly grown on the surface of graphene nanosheets. Moreover, we also investigated the performance of PBNCs/rGO nanocomposites as amperometric sensor toward reduction of H(2)O(2). Such a sensor showed a rapid and highly sensitive response to H(2)O(2) with a low detection limit (45 nM), which might find promising applications in developing a new type of enzymeless biosensor.

Fluorescent magnetic nanoprobes: design and application for cell imaging.

Multifunctional nanoprobes combining magnetic nanoparticles with organic dyes have attracted tremendous interest due to their promising applications in biomedical field. Here we demonstrate a facile and general strategy for the fabrication of robust fluorescent magnetic nanoprobes with high payloads of dye molecules and their use as multimodal nanoprobes for cell imaging. These nanoprobes not only effectively keep photochemical stability of dyes, but also provide a platform for grafting other functional or targeted moieties into silica surface via primary amines. Moreover, the nanoprobes are uniformly spherical morphology and can be dispersed well in aqueous solution, which are very desirable for biomedical applications. Importantly, this method can be extended to synthesize other bifunctional nanoprobes by using the dyes with isothiocyanate group.

Platinum-coated gold nanoporous film surface: electrodeposition and enhanced electrocatalytic activity for methanol oxidation.

This report describes the preparation of Pt-nanoparticle-coated gold-nanoporous film (PGNF) on a gold substrate via a simple "green" approach. The gold electrode that has been anodized under a high potential of 5 V is reduced by freshly prepared ascorbic acid (AA) solution to obtain gold nanoporous film electrode. Then the Pt nanoparticle is grown on the electrode by cyclic voltammetry (CV). The resulting PGNF electrode has highly ordered arrangement and large surface area, as verified by scanning electron microscopy (SEM) and CV, suggesting that the nanoporous gold film electrode provides a good matrix for obtaining PGNF with high surface area. Furthermore, the as-prepared PGNF electrode exhibited high electrocatalytic activity toward methanol oxidation in a 0.5 M H 2SO 4 solution containing 1.5 M methanol. The present novel strategy is expected to reduce the cost of the Pt catalyst remarkably.