Lonicera caerulea Pomace Alleviates DSS-Induced Colitis via Intestinal Barrier Improvement and Gut Microbiota Modulation.

The objective of this investigation was to appraise the mitigative effects of Lonicera caerulea pomace (LCP)-supplemented diets on Dextran Sulfate Sodium (DSS)-induced colitis, and to discuss the potential mechanisms. LCP, a by-product of fruit juice processing, harbors a higher content of polyphenols and dietary fiber compared to the L. caerulea pulp. In a murine model of colitis, the LCP diet attenuated the symptoms of colitis, as evidenced by the reduction in the disease activity index (DAI), extension of colon length, and amelioration of histopathological damage. The anti-inflammatory attributes of LCP were substantiated by a decrease in myeloperoxidase (MPO) activity and suppression of inflammatory cytokine expressions within the colon. Meanwhile, LCP mediated the repair of the intestinal barrier, characterized by the upregulation of gene expressions of tight junction (TJ) proteins and Muc2. Furthermore, LCP altered the composition of the gut microbiota, manifested in increased alpha diversity, enhancement of the abundance of beneficial bacteria (Akkermansia, Coprococcus and Bifidobacterium), and diminishment in the abundance of pathogenic bacteria (Escherichia, Enterococcus, Mucispirillum and Clostridium). Dietary LCP also increased the concentrations of SCFAs within the intestinal luminal contents of colitis mice. Given the affirmative impact of LCP on colitis, LCP may possess great potential in promoting intestinal health.

Regulation of Cell Membrane Permeability Enhanced the Non-Classical Secretion of γ-Cyclodextrin Glycosyltransferase in Bacillus subtilis.

γ-Cyclodextrin glycosyltransferase (γ-CGTase, EC 2.4.1.19) is an essential enzyme required in the production of γ-cyclodextrin, which shows huge prospects in the food, medicine, materials, and chemical industries. In this study, γ-CGTase from Bacillus sp. G-825-6 STB17 was successfully cloned and expressed in Bacillus subtilis WB600. The final extracellular activity of γ-CGTase can reach 45.34 U/mL with the deletion of the signal peptide, which was about 11.3 times of the initial level of γ-CGTase secreted by the general pathway. By monitoring the whole cultivation process, secretion was divided into two stages, which were dominated by cell membrane changes and apoptosis. The measurement of lactate dehydrogenase and the results of fluorescence microscopy demonstrated that the cell membrane permeability changed significantly in the middle stage of fermentation, proving that it played a crucial role in the non-classical secretion of γ-CGTase. Furthermore, the addition of Triton X-100, a non-ionic surfactant, remarkably enhanced the secretion level of γ-CGTase by 21.5%, which was caused by the increase in cell membrane permeability. This work is the first to obtain the extracellular expression of CGTase via the non-classical secretion pathway in B. subtilis and provides a new strategy to enhance extracellular expression by regulating the cell membranes.

Effect of fermentation time and addition amount of rice sourdoughs with different microbial compositions on the physicochemical properties of three gluten-free rice breads.

This study aimed to better understand the impact of microbial growth and metabolism on rice sourdough and their relationship with quality characteristics of rice breads. Therefore, this research determined the microbial compositions and biochemical characteristics of indica rice (IR), japonica rice (JR) and waxy rice (WR) sourdough within 72 h fermentation. And the effects of different fermentation time and dosage of rice sourdough on rheology of rice doughs, quality characteristics of rice breads were studied. The results showed that the dominant bacteria Leuconostoc changed the processing properties of rice doughs through producing organic acids and reducing sugars. Adding 30 % rice sourdough had the best effect on improving specific volume of rice breads. And IR and JR breads with sourdough fermented 48 h had the highest chewiness, cohesiveness, resilience and the lowest hardness, while WR breads had high viscosity and poor overall structure, which was not suitable for making gluten-free bread.

Engineering the ß-Oxidation Pathway in Yarrowia lipolytica for the Production of trans-10, cis-12-Conjugated Linoleic Acid.

trans-10, cis-12-Conjugated linoleic acid (t10, c12-CLA) is an octadecadienoic acid with various biological benefits. Previously, linoleic acid isomerase from Propionibacterium acnes (PAI) was overexpressed in Yarrowia lipolytica (Y. lipolytica) to produce t10, c12-CLA. However, the t10, c12-CLA yield was restricted by the peroxisomal ß-oxidation pathway. In this study, to minimize the degradation of t10, c12-CLA, four genetically modified strains of Y. lipolytica (Δpox2-oPAI, Δpox3-oPAI, Δpox2Δpox3-oPAI, and Δpex10-oPAI) were constructed and compared in terms of production stability and yield of t10, c12-CLA using safflower seed oil as substrates. The Δpex10-oPAI strain exhibited the best results, as revealed by the reduction of the t10, c12-CLA degradation rate from 58.5 to 18.6 mg/L/h. Additionally, the YLUpex10mP recombinant strain bearing six copies of oPAI combined with PEX10 deletion enhanced t10, c12-CLA production to 7.4 g/L and exhibited a CLA degradation rate of 19.7 mg/L/h, a 78% decrease from that of the control strain. Finally, in a bioreactor containing low-cost volatile fatty acids as partial carbon sources, the t10, c12-CLA content in the YLUpex10mP strain increased to 9.7 g/L, 1.3 times higher than in flasks. To our knowledge, this is the highest t10, c12-CLA yield through microbial synthesis reported to date.

Beneficial Effects of Three Dietary Cyclodextrins on Preventing Fat Accumulation and Remodeling Gut Microbiota in Mice Fed a High-Fat Diet.

Globally, obesity and its metabolic complications, which are intimately linked to diet, are major public health problems. Cyclodextrins (CDs) are cyclic oligosaccharides consisting of (α-1,4)-linked D-glucopyranose units that can reduce fat bioavailability and affect metabolism by improving intestinal flora as prebiotics. We compared the effects of three CDs on preventing fat accumulation and remodeling gut microbiota in a high-fat diet-fed C57BL/6J mouse model. α-CD maximized energy expenditure by 12.53%, caused the RER value to drop from 0.814 to 0.788, and increased the proportion of fatty acid oxidation for energy supply. ß-CD supplementation resulted in a marked 24.53% reduction in weight gain and a decrease in epididymal-fat-relative weight from 3.76% to 2.09%. It also minimized ectopic fat deposition and improved blood lipid parameters. γ-CD maximized the concentration of SCFAs in the cecum from 6.29 to 15.31 µmol/g. All three CDs reduced the abundance ratio of Firmicutes and Bacteroidetes to a low-fat diet level, increased the abundance of Lactobacillus and Akkermansia, and reduced the abundance of Allobaculum and Ruminococcus. These findings imply that a combination of multiple CDs may exert superior effects as a potential strategy for obesity prevention.

Maltose binding site 2 mutations affect product inhibition of Bacillus circulans STB01 cyclodextrin glycosyltransferase.

The efficiency of enzymatic cyclodextrin production using cyclodextrin glycosyltransferases (CGTases) is limited by product inhibition. In this study, maltose binding site 2 (MBS2) of the ß-CGTase from Bacillus circulans STB01 was modified to decrease product inhibition. First, two point mutants were prepared at position 599 (A599V and A599N). Then, two double mutants incorporating alanine at position 633 (A599N/Y633A and A599V/Y633A) were prepared. Finally, the entire MBS2 region was replaced by that of the α-CGTase from Paenibacillus macerans JFB05-01 to form multipoint mutant MBS2 ߠ→ α. All five mutants exhibited mixed-type product inhibition, although both the competitive and uncompetitive components of this inhibition were decreased. The total cyclization activities of A599N, A599V and A599V/Y633A were 15.6%, 76.8% and 70.9% lower than that of the wild-type, respectively, while that of A599N/Y633A was 22.4% higher. Among the mutants, only MBS2 ߠ→ α showed catalytic efficiency (kcat/Km) comparable with that of the wild-type. Moreover, A599N, A599N/Y633A and MBS2 ߠ→ α produced cyclodextrin yields 13.1%, 15.8% and 19.7% greater than that of the wild-type, respectively. These results suggest that A599N, A599N/Y633A and MBS2 ߠ→ α may be more suitable than the wild-type for cyclodextrin production.

Rational Design of Disulfide Bonds for Enhancing the Thermostability of the 1,4-α-Glucan Branching Enzyme from Geobacillus thermoglucosidans STB02.

Disulfide bonds play crucial roles in thermostabilization, recognition, or activation of proteins. They are vital in maintaining the respective conformations of globular structures, thereby enhancing thermostability. Bioinformatic approaches provide practical strategies to build disulfide bonds based on structural information. We constructed nine mutants by rational analysis of the 1,4-α-glucan branching enzyme (EC 2.4.1.18) from Geobacillus thermoglucosidans STB02, which catalyzes the synthesis of α-1,6-glucosidic bonds by acting on α-(1,4) and/or α-(1,6) glucosidic linkages. Four of the mutations enhanced thermostability, and five of them had adverse or negligible effects on stability. Circular dichroism spectra and intrinsic fluorescence analysis showed that introducing disulfide bonds might only affect secondary structures. The results also demonstrated that the distances of Cα carbons and thiol groups, as well as the sequence between the two cysteines, need to be considered when designing disulfide bonds.

Preliminary safety assessment of a new Bacteroides fragilis isolate.

The novel commensal strain of Bacteroides fragilis HCK-B3 isolated from a healthy Chinese donor was discovered beneficial effects of attenuating lipopolysaccharides-induced inflammation. In order to contribute to the development of natural next-generation probiotic strains, the safety assessment was carried out with in vitro investigations of its morphology, potential virulence genes and antimicrobial resistance, and an in vivo acute toxicity study based on both healthy and immunosuppressed mice by cyclophosphamide injection. Consequently, the potential virulence genes in the genome of B. fragilis HCK-B3 have yet been identified as toxicity-associated. The absence of plasmids prevents the possibility of transferring antibiotic resistance features to other intestinal commensals. No intracorporal pathogenic properties were observed according to the body weight, hematological and liver parameters, cytokine secretions and tissue integrity. In addition, B. fragilis HCK-B3 performed alleviations on part of the side effects caused by the cyclophosphamide treatment. Thus, the novel strain of B. fragilis HCK-B3 was confirmed to be non-toxigenic and did not display adverse effects in both healthy and immune-deficient mice at a routinely applicable dose.

Complexity and variability of gut commensal microbiota in polyphagous lepidopteran larvae.

BACKGROUND: The gut of most insects harbours nonpathogenic microorganisms. Recent work suggests that gut microbiota not only provide nutrients, but also involve in the development and maintenance of the host immune system. However, the complexity, dynamics and types of interactions between the insect hosts and their gut microbiota are far from being well understood. METHODS/PRINCIPAL FINDINGS: To determine the composition of the gut microbiota of two lepidopteran pests, Spodoptera littoralis and Helicoverpa armigera, we applied cultivation-independent techniques based on 16S rRNA gene sequencing and microarray. The two insect species were very similar regarding high abundant bacterial families. Different bacteria colonize different niches within the gut. A core community, consisting of Enterococci, Lactobacilli, Clostridia, etc. was revealed in the insect larvae. These bacteria are constantly present in the digestion tract at relatively high frequency despite that developmental stage and diet had a great impact on shaping the bacterial communities. Some low-abundant species might become dominant upon loading external disturbances; the core community, however, did not change significantly. Clearly the insect gut selects for particular bacterial phylotypes. CONCLUSIONS: Because of their importance as agricultural pests, phytophagous Lepidopterans are widely used as experimental models in ecological and physiological studies. Our results demonstrated that a core microbial community exists in the insect gut, which may contribute to the host physiology. Host physiology and food, nevertheless, significantly influence some fringe bacterial species in the gut. The gut microbiota might also serve as a reservoir of microorganisms for ever-changing environments. Understanding these interactions might pave the way for developing novel pest control strategies.

Gender-specific bacterial composition of black flies (Diptera: Simuliidae).

Although hematophagous black flies are well-known socioeconomic pests and vectors of disease agents, their associated bacteria are poorly known. A systematic analysis of the bacterial community associated with freshly emerged adult black flies of four North American species, using cultivation-independent molecular techniques, revealed 75 nonsingleton bacterial phylotypes. Although 17 cosmopolitan phylotypes were shared among host species, each fly species had a distinct bacterial profile. The bacterial composition, however, did not correlate strongly with the host phylogeny but differed between male and female flies of the same species from the same habitat, demonstrating that a group of insects have a gender-dependent bacterial community. In general, female flies harbor a less diverse bacterial community than do males. The anatomical locations of selected bacteria were revealed using fluorescence in situ hybridization. Understanding the physiological function of the associated bacterial community could provide clues for developing novel pest-management strategies.

Wood identification with PCR targeting noncoding chloroplast DNA.

Wood identification is extremely important in the modern forest industry. It also has significant applications in forensics, as well as in archeology and ecological research. In this study, five universal primer pairs amplifying chloroplast noncoding sequences of 300-1,200 bp were designed. Sequencing these amplicons in combination can lead to reliable identification of logs and wood products to cultivar, ecotype, or even the falling population. These primer pairs work on both gymnosperms and angiosperm trees. They also are potentially applicable to accurately identify shrubs and herbaceous species. In addition, a wood DNA purification method is proposed in which N-phenacylthiazolium bromide (PTB) is used to increase the quality and quantity of extracted DNA. This method was first validated using air-dried timber disks from three different tree species that were felled 4 years ago. The sapwood and outer heartwood provided the best locations for DNA extraction. The method was also successfully applied to extract DNA from the recalcitrant processed white oak wood, randomly selected staves of wine barrels. The single nucleotide polymorphism detected on the oak DNA sequences showed correlation to their geographical origins.

Crystallization of α- and ß-carotene in the foregut of Spodoptera larvae feeding on a toxic food plant.

In the animal kingdom, carotenoids are usually absorbed from dietary sources and transported to target tissues. Despite their general importance, the uptake mechanism is still poorly understood. Here we report the "red crop" phenomenon, an accumulation of α- and ß-carotene in crystalline inclusions in the enlarged foregut of the polyphagous Spodoptera larvae feeding on some potentially toxic plant leaves. The carotene crystals give the insect foregut a distinctive orange-red color. The crystals are embedded in a homogenous lawn of the bacterium Enterococcus casseliflavus, but the carotene seems to be selectively taken from the food plant. Caterpillars which fail to develop these carotene crystals exhibit a high mortality or fail to develop to adulthood. The crystallization of carotene and the enlargement of the foregut thus appears to manifest a multiple-step physiological adaptation of the insects to toxic food plants.

[Community constitute and phylogenetic analysis on soil uncultured microorganism].

Many evidences show bachteria that can be cultured in lab are only the minority of germs existing in nature. Most of bacteria, containing rich valuable gene resource, are unknown for they are uncultured. To research these uncultured bacteria, Directly extract crude DNA from farm soil, separate 16S rDNA mixture amplified by PCR from the crude DNA, sequence it and compare the sequences with the records of NCBI to classify the germs. The significant microbe community difference from respective soil, which have possibility in developing a new tool for researching soil bacteria diversity were finded.