The impact of storage buffer and storage conditions on fecal samples for bacteriophage infectivity and metavirome analyses.

BACKGROUND: There is an increasing interest in investigating the human gut virome for its influence on the gut bacterial community and its putative influence on the trajectory towards health or disease. Most gut virome studies are based on sequencing of stored fecal samples. However, relatively little is known about how conventional storage buffers and storage conditions affect the infectivity of bacteriophages and influence the downstream metavirome sequencing. RESULTS: We demonstrate that the infectivity and genome recovery rate of different spiked bacteriophages (T4, c2 and Phi X174) are variable and highly dependent on storage buffers. Regardless of the storage temperature and timespan, all tested phages immediately lost 100% (DNA/RNA Shield) or more than 90% (StayRNA and RNAlater) of their infectivity. Generally, in SM buffer at 4 °C phage infectivity was preserved for up to 30 days and phage DNA integrity was maintained for up to 100 days. While in CANVAX, the most effective buffer, all spiked phage genomes were preserved for at least 100 days. Prolonged storage time (500 days) at - 80 °C impacted viral diversity differently in the different buffers. Samples stored in CANVAX or DNA/RNA Shield buffer had the least shifts in metavirome composition, after prolonged storage, but they yielded more contigs classified as "uncharacterised". Moreover, in contrast to the SM buffer, these storage buffers yielded a higher fraction of bacterial DNA in metavirome-sequencing libraries. We demonstrated that the latter was due to inactivation of the DNases employed to remove extra-cellular DNA during virome extraction. The latter could be partly avoided by employing additional washing steps prior to virome extraction. CONCLUSION: Fecal sample storage buffers and storage conditions (time and temperature) strongly influence bacteriophage infectivity and viral composition as determined by plaque assay and metavirome sequencing. The choice of buffer had a larger effect than storage temperature and storage time on the quality of the viral sequences and analyses. Based on these results, we recommend storage of fecal virome samples at in SM buffer at 4 °C for the isolation of viruses and at - 80 °C for metagenomic applications if practically feasible (i.e., access to cold storage). For fecal samples stored in other buffers, samples should be cleared of these buffers before viral extraction and sequencing. Video Abstract.

Replication Protein Rep Provides Selective Advantage to Viruses in the Presence of CRISPR-Cas Immunity.

Anti-Clustered regularly interspaced small palindromic repeat (CRISPR) (Acr) phages cooperate to establish a successful infection in CRISPR-containing host. We report here the selective advantage provided by a replication initiator, Rep, toward cooperative host immunosuppression by viruses encoding Acrs. A rep knockout mutant (Δgp16) of Sulfolobus islandicus rod-shaped virus 2 produced around fourfold less virus in a CRISPR-null host, suggesting that Rep is the major replication initiator. In addition to Rep-dependent replication initiation from the viral genomic termini, we detected Rep-independent replication initiation from nonterminal sites. Intriguingly, despite the presence of Acrs, lack of Rep showed a profound effect on virus propagation in a host carrying CRISPR-Cas immunity. Accordingly, the co-infecting parental virus (rep-containing) outcompeted the Δgp16 mutant much more quickly in the CRISPR-containing host than in CRISPR-null host. Despite the nonessentiality, rep is carried by all known members of Rudiviridae, which is likely an evolutionary outcome driven by the ubiquitous presence of CRISPR-Cas in Sulfolobales.

Komagataeibacter hansenii CGMCC 3917 alleviates alcohol-induced liver injury by regulating fatty acid metabolism and intestinal microbiota diversity in mice.

The potential effects of Komagataeibacter hansenii CGMCC 3917 cells on alcohol-induced liver injury and their probable mechanisms were investigated. Male Kunming mice were orally administered with alcohol (10 mL per kg BW) alone or in combination with administration of K. hansenii CGMCC 3917 cells at 2 × 108 and 2 × 106 CFUs for 10 weeks. Administration of strain CGMCC 3917 cells, especially high dose administration, decreased the liver weights, fat gain, and fatty-acid metabolism-related enzyme SCD-1, ACC and FAS expressions and endotoxin release, which were elevated by alcohol treatment. Furthermore, the total contents of long chain fatty acids of the liver and serum in alcohol-treated mice supplemented with a high dose of strain CGMCC 3917 cells were decreased to 5.44 ± 0.19 µg mL-1 and 3.66 ± 0.15 µg mL-1 from 6.65 ± 0.31 µg mL-1 and 4.52 ± 0.21 µg mL-1, respectively. Conversely, the SCFAs decreased by ethanol treatment, particularly the acetic acid, propionic acid and butyric acid, were obviously enhanced in the faeces, colon and cecum of the mice supplemented with strain CGMCC 3917 cells. Moreover, strain CGMCC 3917 cells could regulate gut microbiome by significantly decreasing the abundance of Actinobacteria, Proteobacteria and Firmicutes, and dramatically increasing the abundance of Bacteroidetes in alcohol-treated mice. These findings suggest that K. hansenii CGMCC 3917 cells alleviate alcohol-induced liver damage via regulating fatty acid metabolism and intestinal microbiota diversity in mice.

Improved characterization of nanofibers from bacterial cellulose and its potential application in fresh-cut apples.

The present research aimed to study the nanofibers from bacterial cellulose (BC) by HCl hydrolysis and explore its new potential application in fresh-cut apples. Bacterial cellulose nanofibers (BCNs) showed low and more homogeneity particle size, as well as higher zeta potential and transparency in comparison with BC, which was confirmed by morphological analysis. Physical properties analysis showed that BCNs was more excellent semi-crystalline polymer with higher thermal stability as compared with BC. Rheological results displayed that BCNs suspensions presented a shear thinning behaviour with higher apparent viscosity, storage (G') and loss (G'') moduli at the same concentration in comparison with BC. Furthermore, BCNs suspensions were more stable than BC suspensions under storage condition of 4 °C. Additionally, 2% (wt%) of BCNs suspensions were coated on fresh-cut apples. Results showed that the samples coated with BCNs suspension displayed more excellent properties of keeping fresh-cut apples as compared with that coated with BC suspensions, including delaying weight loss, improving firmness and soluble solids content, reducing browning index and titratable acidity. Therefore, the low cost and high biocompatibility of BCNs can be used as new coatings for fresh-cut apples and have great potential to coat fresh-cut fruits and vegetables in food industry.

Artemisia sphaerocephala Krasch polysaccharide prevents hepatic steatosis in high fructose-fed mice associated with changes in the gut microbiota.

High fructose (HF) diet-induced liver steatosis is associated with intestinal microbiota dysbiosis. The aim of this study was to assess the modulatory effects of Artemisia sphaerocephala Krash seed polysaccharide (ASKP) on fatty acid metabolism and intestinal microbiota in mice fed with HF water. Administration of HF-fed mice with ASKP prevented fat accumulation and blunted metabolic inflammation and endotoxaemia. ASKP-treated mice displayed improved glucose tolerance and fully exhibited protection against hepatic steatosis. Besides, ASKP was effective in improving the changes in the composition of liver fatty acids via modulating hepatic SREBP-1c, SCD-1, ACC and FAS expressions. 16S rRNA gene sequencing showed that ASKP treatment modified the gut microbial species at the phylum level with a decrease of Firmicutes and a slight increase of Bacteroidetes (p > 0.05). Interestingly, ASKP markedly increased the proportion of the mucin-degrading bacterium Akkermansia at the genus level in HF-fed mice. These findings support the beneficial metabolic effects of ASKP through amelioration of the HF-induced features of liver steatosis, which is associated with health maintenance of the intestinal microecosystem.

Antioxidant, antimicrobial, and antiproliferative activity-based comparative study of peel and flesh polyphenols from Actinidia chinensis.

BACKGROUND: Kiwifruit (Actinidia chinensis) peel has been always considered as useless because of the harsh taste. To promote the full utilization of kiwifruit resources it is essential to explore the nutritional benefits of kiwifruit peel. OBJECTIVE: Our studies explored the difference in polyphenolic composition and biological activity including antioxidant, antimicrobial, and antiproliferative activity of the flesh and peel of kiwifruit. DESIGN: Antioxidant activity of the extracted polyphenols of the peel and flesh of A. chinensis was checked by 2,2-diphenyl-1-picrylhydrazyl, 2,2'-azino-bis3-ethylbenzothiazoline-6-sulphonic acid (ABTS), hydroxyl ion reduction, and ion chelating ability. Antibacterial activity against Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus and antiproliferative activity against HepG2 was tested in a dose- and time-dependent manner. Liquid chromatography/mass spectrometry (LC/MS) chromatogram of the peel and flesh further differentiated the phenolic acid profile. RESULTS: The pericarp of kiwifruit was found to be more abundant in polyphenols and flavonoids than the flesh, with contents of 12.8 mg/g and 2.7 mg/g, respectively. LC/MS analysis revealed that the catachin, quercetin and epigallocatechin content (the main polyphenols in kiwifruit) in the peel was significantly higher than in the flesh (P < 0.05). The antioxidant and antibacterial activity of the peel was significantly higher when compared to the flesh. Moreover, the proliferation of HepG2 cells was time- and dose-dependently inhibited by kiwifruit polyphenols, with IC50 values of 170 µg/mL and 291 µg/mL for peel and flesh polyphenols after 72 h of treatment time, respectively. CONCLUSION: Kiwifruit peel, with higher content of phenolics and flavonoids, exerts more potent antioxidant, antibacterial, and anticancer activity than the flesh. Our study provides scientific evidence for the development of kiwifruit, especially peel-based, novel natural products with excellent bioactivity.

Effects of Dietary Fiber Supplementation on Fatty Acid Metabolism and Intestinal Microbiota Diversity in C57BL/6J Mice Fed with a High-Fat Diet.

This work was to assess possible impacts of novel insoluble fiber 8% bacterial cellulose (BC), soluble fiber 8% konjac glucomannan (KGM), and their mixture (4% BC/4% KGM) on fatty acid metabolism and intestinal microbiota of C57BL/6J mice fed with a high-fat diet (HFD). HFD-fed mice receiving the dietary fibers (DFs) for 16 weeks exhibited an improvement in lipid-associated cytokines and a decrease in inflammation factors, which was associated with the improved hepatic and serum fatty acid composition. The DFs, notably the mixed BC/KGM, elevated the HFD-caused decrease in the contents of acetic acid (from 23.9 ± 0.85 to 32.2 ± 0.84 mM/g; p < 0.05), propionic acid (from 6.53 ± 0.28 to 12.8 ± 0.58 mM/g; p < 0.05), and butyric acid (from 7.73 ± 0.43 to 13.5 ± 0.47 mM/g; p < 0.05). Furthermore, the mixed BC/KGM significantly decreased the abundance of Firmicutes (from 90.4 to 67.6%) and Mucispirillum (from 4.77 to 1.58%) and dramatically increased the abundance of Bacteroidetes (from 7.83 to 25.0%) and Akkermansia (from 0.69 to 2.80%) in the gut of HFD-fed mice at the genus level. Moreover, correlation analysis revealed that the multiplicity of gut microbiota was useful in sustaining colonic integrity through producing short-chain fatty acids to some extent. This finding suggests that a mixture of insoluble BC and soluble KGM has positive effects on modulation of the intestinal microecosystem in mice.

Chemical characteristics, antioxidant capacities and hepatoprotection of polysaccharides from pomegranate peel.

This proposed work aimed to investigate the chemical characteristic, antioxidant capacities and hepatoprotection effect of pomegranate peel polysaccharides (PPP) on CCl4-induced oxidative damage in mice. PPP was identified as the acidic heteropolysaccharides by HPLC methods. In vitro test showed that PPP had excellent reducing power and scavenging effects against free radicals. Administration of PPP (50, 100 and 200 mg/kg·bw) in mice before the injection of CCl4 could observably antagonize the increased serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and hepatic malonaldehyde level in CCl4-induced mice, especially administrated with 200 mg/kg·bw of PPP. Hepatic enzymatic activities of total superoxide dismutase, glutathione peroxidase, catalase and non-enzymatic activity of glutathione were markedly increased at high dosage of PPP, respectively. In addition, histopathological observation of liver further proved these biochemical characteristics. Therefore, it can be concluded that PPP exhibits strong protective effects against CCl4-induced liver injury in mice.

Enhanced anti-obesity effects of bacterial cellulose combined with konjac glucomannan in high-fat diet-fed C57BL/6J mice.

This study aimed to investigate the effects of supplementation with bacterial cellulose (BC), konjac glucomannan (KGM) and combined BC/KGM fiber on high-fat (HF)-diet-induced obesity in C57BL/6J mice. The results showed that combined supplementation with BC/KGM in HF-fed mice was more efficient in reducing body weight, lowing serum lipid profiles and suppressing insulin resistance than single supplementation with BC or KGM. Moreover, supplementation with combined BC/KGM fiber more efficiently alleviated HF-diet-induced liver injury by decreasing hepatic steatosis in comparison with supplementation with BC or KGM alone. Furthermore, supplementation with combined BC/KGM fiber in HF-fed mice had a more positive effect on obesity-associated hepatic inflammation by reducing levels of TNF-α and IL-6 and suppressing the protein expression of Nrf-2/ARE in comparison with supplementation with BC or KGM alone. Consumption of these dietary fibers, especially mixed BC/KGM, resulted in an improved antioxidant defense system and reduced lipid peroxidation in the liver by increasing the activity of antioxidant enzymes and reducing the formation of MDA in the liver. Moreover, supplementation with these fibers regulated the levels of leptin and adiponectin and inhibited the protein expression of PPARγ by reducing the size of cells in the adipose tissue of HF diet-fed mice. Therefore, fiber supplementation (especially with combined BC/KGM) efficiently inhibited HF-induced obesity in mice by reducing insulin resistance, liver injury and inflammation, enhancing the antioxidant defense system and regulating the secretion of adipocytokines and adipogenesis-associated proteins.

Composition characterization, antioxidant capacities and anti-proliferative effects of the polysaccharides isolated from Trametes lactinea (Berk.) Pat.

This study was designed to investigate the chemical characterization and bioactivity of the Trametes lactinea (Berk.) Pat polysaccharides (TLP). The crude TLP was fractionated into two fractions, namely TLP-1 and TLP-2 with Cellulose DEAE-52 and Sephadex G-150. HPLC and FT-IR analysis showed that TLP-1 and TLP-2 were heteropolysaccharides mainly composed of glucose with the average molecular weights of 443.19kDa and 388.83kDa, respectively. TLP-1 from water elution possessed of higher reducing power and scavenging activities against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, superoxide radical and hydroxyl radical than TLP-2 eluted by 0.1M of NaCl. In comparison with TLP-2, TLP-1 showed stronger growth inhibition against human hepatoblastoma HepG-2 cells and caused higher LDH leakage. However, TLP-1 showed lower growth inhibition against normal hepatocyte L-02 cells and lower LDH leakage than TLP-2. Flow cytometric analysis showed that TLP-1 had a stimulatory effect on apoptosis of HepG-2 cells. These findings suggested that the polysaccharides, especially TLP-1 could contribute to the potential anticancer effects of T. lactinea (Berk.) Pat, which might be valuable as a natural antioxidant source applied in both healthy medicine and food industry for health benefits.

Bacterial Cellulose Relieves Diphenoxylate-Induced Constipation in Rats.

This study was to probe the effects of bacterial cellulose (BC) on diphenoxylate-induced constipation in rats. Administration with BC at 500 mg/kg of body weight in diphenoxylate-induced constipation rats distinctly improved the carmine propulsion rate (83.5 ± 5.2%), shortened the defecating time of the first red feces (249.0 ± 23.3 min), and increased the weight of carmine red feces within 5 h (2.7 ± 1.3 g). The levels of aquaporins (AQP-2, AQP-3, and AQP-4) and inhibitory neurotransmitters (nitric oxide, nitric oxide synthetase, vasoactive intestinal peptide, and arginine vasopressin) in the BC-treated groups reduced by 31.9-40.0% ( p < 0.01) and 21.1-67.7% ( p < 0.01) compared to those in the constipation group, respectively. However, the secretion of excitability neurotransmitters (substance P and motilin) in the BC-treated groups was increased by 20.0-39.9% ( p < 0.01). The activities of ATPases in the colon of constipation rats were significantly weakened by BC administration ( p < 0.01). Histological morphology of the colon showed that BC supplementation could effectively increase the length of villus cells and the thickness of colonic mucosa and muscle ( p < 0.01). Moreover, BC supplementation could protect colonic smooth muscle cells against apoptosis. All of the findings suggest that BC supplementation effectively relieves constipation in rats and BC would be used as a great promising dietary fiber for alleviating constipation.

Emulsions stabilized by nanofibers from bacterial cellulose: New potential food-grade Pickering emulsions.

In the present work, we investigated the formation and stability of Pickering emulsions stabilized by nanoparticles generated from bacterial cellulose (BC) by hydrochloric acid hydrolysis. The resulting particles, called nanofibers, presented a ribbonlike shape with diameters of 30-80nm and range in length from 100nm to several micrometers. The obtained nanofibers showed good hydrophilic and lipophilic properties and had significant ability to reduce the surface tension of oil/water droplets from 48.55±0.03 to 34.52±0.05mN/m. The oil-in-water Pickering emulsions with a peanut oil concentration of 15% (v/v) were stabilized by only 0.05% (w/v) nanofibers and displayed a narrow droplet size distribution and high intensity with an average droplet size of 15.00±0.82nm. The morphological studies confirmed the nano-scaled droplets of emulsions. The effects of pH values and temperatures on the creaming ability and physical stability were also evaluated by zeta-potential and droplet sizes. Results showed that emulsions displayed relatively lower creaming ability at pH<7, while displayed optimal physical stability and dispersibility at pH≥7. The temperature (20-100°C) and time-dependent test (0-4weeks) indicated that the Pickering emulsions stabilized by only 0.05% (w/v) nanofibers displayed excellent stability. Due to the sustainability and good bio-compatibility of nanofibers from BC, the developed emulsions stabilized by low concentration of nanofibers can be used as new food-grade Pickering emulsions and have great potential to deliver lipophilic bioactive substances in food industry.

Optimization for pectinase-assisted extraction of polysaccharides from pomegranate peel with chemical composition and antioxidant activity.

In this work, based on response surface methodology (RSM), an optimized method of extracting water-soluble polysaccharide from pomegranate peel (PPP) with pectinase was proposed. Additionally, the chemical composition and the antioxidant activities of the extracted PPP were studied. The effects of three factors of enzymolysis time, ratio of liquid to solid and dosage of enzyme on the PPP yield were investigated. The experimental results showed that the optimal conditions were as following: enzymolysis time 19.70 min, ratio of liquid to solid 20.5:1(mL/g), dosage of enzyme 0.68%. Under the best conditions, the maximum PPP yield was 27.3 ±â€¯0.08%. FT-IR and GC-MS revealed that PPP was the typical acidic heteropolysaccharides and mainly consisted of d-mannose (19.30%), d-galactose (47.93%) and l-arabinose (23.08%). Moreover, the antioxidant activity assay in vitro showed that PPP exhibited strong reducing power and good scavenging activities on superoxide anion, hydroxyl, and DPPH radicals. The PPP extracted with pectinase had excellent antioxidant properties and could be developed into a new type of dietary supplement or functional food.

Chemical characteristics of an Ilex Kuding tea polysaccharide and its protective effects against high fructose-induced liver injury and vascular endothelial dysfunction in mice.

The present study was designed to investigate the protective effects of Ilex Kuding tea polysaccharides (IKTP) on high fructose (HF)-induced liver injury and vascular endothelial dysfunction in mice. IKTP were identified as acidic heteropolysaccharides by FT-IR and HPLC. Healthy male Kunming mice fed 20% fructose in drinking water for 8 consecutive weeks significantly displayed dyslipidemia, hepatic steatosis, oxidative stress and vascular endothelial dysfunction. However, continuous administration of IKTP at 200, 400 and 800 mg per kg bw in HF-fed mice could prevent the damage caused by HF-diets, especially at dosages of 400 and 800 mg per kg bw (p < 0.01). IKTP significantly reduced the HF-induced elevation of the serum TC, TG, LDL-C, TXA2 and ET-1 levels, as well as AST and ALT activities, while markedly increased the HF-induced decline of HDL-C, PGI2 and eNOS levels in the serum compared to HF-fed mice. Meanwhile, the hepatic MDA level was lowered while SOD and GSH-Px activities were increased in IKTP-treated mice, compared to HF-fed mice. Histopathology of the liver and cardiovascular aortic by H&E or oil red O staining confirmed the liver steatosis and the vascular injury induced by HF-diets and the protective effects of IKTP. These findings suggest that HF causes oxidative damage, and IKTP alleviates liver injury and vascular endothelial dysfunction.

Response surface optimization of ultrasound-assisted polysaccharides extraction from pomegranate peel.

Ultrasonic technique was employed to extract polysaccharides from pomegranate peel. The optimal conditions for ultrasonic extraction of pomegranate peel polysaccharide (PPP) were determined by response surface methodology. Box-Behnken design was applied to evaluate the effects of four independent variables (ratio of water to raw material, extraction time, extraction temperature, ultrasonic power) on the yield of PPP. The correlation analysis of mathematical-regression models indicated that quadratic polynomial model could be employed to optimize the ultrasonic extraction of PPP. The optimum extraction parameters were as follows: ratio of water to raw material, 24 ml/g; extraction time, 63 min; extraction temperature, 55°C; and ultrasonic power, 148 W. Under these conditions, the polysaccharide yield was 13.658 ± 0.133% for the pomegranate peel, which well matches with the predicted value.