Effects of mulberry leaf powder water extract supplementation on the growth performance, immunity, antioxidant, meat quality and intestinal microbiota of yellow feather broilers.

A 50-day feeding trial was conducted to evaluate the effects of mulberry leaf powder water extract (MLE) on the growth performance, immunity, antioxidant, meat quality and intestinal microbiota of yellow feather broilers. A total of 720 birds (initial body weight 40.07 ± 0.05 g) were randomly distributed into four groups with six replicates per group and 30 birds per replicate. Four diets were formulated with 0% (CON), 200 mg/kg MLE (MLE200), 400 mg/kg MLE (MLE400) and 600 mg/kg MLE (MLE600) supplementation. Results showed that the addition of 200-600 mg/kg MLE to the diet significantly increased the body weight (BW) and average daily weight gain (ADG), but feed to gain ratio (F/G) were linearly decreased (p = 0.045) as dietary MLE increased. Birds fed MLE400 had higher (p < 0.05) total antioxidant capacity (T-AOC), interleukin-10 (Il-10), secretory immunoglobulin A (SIgA) and complement 3 (C3) contents than those fed CON, whereas MLE400 had lower malondialdehyde (MDA) content than CON (p < 0.05). Analysis of 16 S rDNA indicated that supplementation with 200 mg/kg MLE increased the Shannon indices in the caecum (p < 0.05). Supplementation with MLE decreased the abundance of the phylum Proteobacteria and genus Helicobacter, and increased the abundance of the phylum Bacteroidetes in the caecum in broiler chickens (p < 0.05). The drip loss rate in the MLE600 was significantly diminished (p < 0.05), whereas the shear force was significantly elevated (p < 0.05). In summary, dietary supplementation with MLE can effectively improve growth performance, intestinal immunity, serum antioxidant capacity, meat quality and intestinal microbiota of yellow feather broilers. The most appropriate MLE supplementation level was 400 mg/kg. This study provides a practical strategy for the dietary application of MLE in yellow feather broilers.

The oligosaccharides of Xiasangju alleviates dextran sulfate sodium-induced colitis in mice by inhibiting inflammation.

Xiasangju (XSJ) is a traditional Chinese herbal formula consisted of Prunella spica, Mulberry leaf and Chrysanthemi indici flos, which can be used to treat fever, headache and ulcer. To explore the effects of oligosaccharides from XSJ (OX) on colitis, we used dextran sulfate sodium (DSS) to establish colitis mouse models. After administration of OX with different doses on the control and colitis mice, we measured their body weights, disease activity indexes (DAI), lengths and histopathologic changes of colons, spleen indexes. The inflammatory cytokines and oxidative stress-related factors in serum, and the intestinal microbial community in feces were also detected. We found that colitis mice with oral administration of OX showed higher body weights and lower levels of DAI and spleen index. Tissue damages induced by DSS were also alleviated by OX treatment. The colitis mice with OX treatment exhibited lower levels of AST, ALT, BUN, CR, MDA and a down-regulated expression of IL-6 and IL-1ß, while the activity of SOD was up-regulated. Furthermore, OX improved the relative abundance of gut microbiota and restored the proportions of Bacteroidetes and Muribaculaceae. We found that oligosaccharides from XSJ alleviated the symptoms of colitis mice through its inhibitory effects on inflammation and oxidative stress, and also regulated the composition of intestinal flora, which indicates a beneficial role for patients with colitis.

Effects of Cinnamon Powder on Glucose Metabolism in Diabetic Mice and the Molecular Mechanisms.

The liver is the primary organ regulating glucose metabolism. In our recent study, cinnamon improved liver function in diabetic mice. However, it is not clear whether cinnamon can reduce the glycemia of diabetic animals by regulating liver glucose metabolism. The purpose of this study was to investigate the hypoglycemic mechanism of cinnamon powder (CP) from the perspective of regulating liver glucose metabolism. To achieve this, different doses of CP (200, 400, or 800 mg/kg body weight) were given to diabetic mice by gavage once per day for 8 weeks. These mice were compared with healthy controls, untreated diabetic mice, and diabetic mice treated with metformin (the main first-line drug for type 2 diabetes). CP treatment effectively reduced fasting blood glucose levels and food intake, improved glucose tolerance and fasting serum insulin levels, and decreased glycated serum protein levels in diabetic mice. Furthermore, treatment with CP increased liver glycogen content and reduced the level of the gluconeogenesis precursor pyruvate in the liver. Data obtained by qPCR and western blotting suggested that CP improved glucose metabolism disorders by regulating AMPKα/PGC1α-mediated hepatic gluconeogenesis and PI3K/AKT-mediated hepatic glycogen synthesis. CP exhibits good hypoglycemic effects by improving hepatic glycogen synthesis and controlling hepatic gluconeogenesis. Therefore, CP may be applied as a functional food to decrease blood glucose.

Antibacterial Effects of Ramulus mori Oligosaccharides against Streptococcus mutans.

Ramulus mori has been widely used in traditional Chinese medicine because of its physiological activities, including antibacterial, anti-inflammatory, and antioxidant activities. Antimicrobial properties of Ramulus mori extract have been well described. However, no information is available regarding on Ramulus mori oligosaccharides (RMOS). The aim of this study was to investigate the effects of RMOS on the growth and virulence properties of the cariogenic bacterium Streptococcus mutans. The effects of RMOS on the biofilm structure and virulence gene expression of S. mutans were also evaluated, and the results were compared with the effects of commercial prebiotic galactooligosaccharides. RMOS were found to have an antibacterial effect against S. mutans, resulting in significant reductions in acid production, lactate dehydrogenase activity, adhesion, insoluble extracellular polysaccharide production, glucosyltransferase activity, and biofilm formation in a dose-dependent manner. Moreover, the biofilm structure was visibly damaged. A quantitative real-time PCR assay revealed downregulation of virulence gene-regulated acid production, polysaccharide production, adhesion, biofilm formation, and quorum sensing. These findings suggest that RMOS may be a promising natural product for the prevention of dental caries.

Analysis of Akkermansia muciniphila in Mulberry Galacto-Oligosaccharide Medium via Comparative Transcriptomics.

Akkermansia muciniphila is a common member of the human gut microbiota and belongs to the phylum Verrucomicrobia. Decreased levels of A. muciniphila are associated with many diseases, so it is thought to be a beneficial resident of the intestinal mucosal layer. In this study, we found that different prebiotics promoted the proliferation of A. muciniphila, and mulberry galacto-oligosaccharide (MGO) had the greatest effect. We cultured A. muciniphila in a brian heart infusion (BHI) medium containing 5% galactooligosaccharides (GOS), mulberry polysaccharide solution (MPS), and MGO, and transcriptomic analyses were performed. The results revealed that, after 6 days of cultivation, the numbers of upregulated functional genes (based on Gene Ontology) were approximately 0.7 and 19% higher with MPS and MGO, respectively, than with GOS. Analysis using the Kyoto Encyclopedia of Genes and Genomes showed that, when A. muciniphila was cultured with MGO, genes that were upregulated were enriched in the carbohydrate metabolism, the metabolism of cofactors and vitamins, the energy metabolism, the amino acid metabolism, and the lipid metabolism. Upregulated genes included galM and pfkA in the galactose metabolism, and pgi, pfk, fbaA, tpiA, gapA, pgk, gpml, eno, pyk, and lpd in the glycolysis/gluconeogenesis pathway. Real-time quantitative PCR results were consistent with the RNA-Seq data. This work provides valuable knowledge which can be available for the functional application of A. muciniphila and MGO.

Diverse Galactooligosaccharides Differentially Reduce LPS-Induced Inflammation in Macrophages.

The effects of natural and synthetic galactooligosaccharides (GOS) on inflammation were explored by investigating the structure-activity relationship between the degree of GOS polymerization and in vitro anti-inflammatory activity, together with the potential underlying mechanism of their anti-inflammatory effects. The results demonstrated that GOS had strong anti-inflammatory effects in lipopolysaccharide (LPS)-induced RAW264.7 macrophages, including the inhibition of nitric oxide production and the reduced expression of pro-inflammatory mediators (interleukin-1ß, interleukin-6, and tumor necrosis factor α), induced nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), and proteins related to the Toll-like receptor 4 (TLR4)/nuclear factor (NF)-κB signaling pathway. GOS4, which has the highest degree of polymerization, exerted the strongest anti-inflammatory activity among the GOS examined. More importantly, our findings confirmed the anti-inflammatory effects of GOS on RAW264.7 macrophages via the TLR4/NF-κB pathway. Our experimental results could provide further support for the exploration of GOS in human nutrition and health.

The transported active mulberry leaf phenolics inhibited adipogenesis through PPAR-γ and Leptin signaling pathway.

The effective components of mulberry leaf polyphenols (MLPs) should be absorbed and transported by the intestinal cells before regulating lipid metabolism. The Caco-2 intestinal epithelial cell and 3 T3-L1 adipocytes were coupled to screen the effective components of MLPs that are being absorbed and transported by intestinal cells. The regulation and molecular mechanism by which the effective components affect adipogenesis were analyzed in this study. Among the 12 main components identified, five main compounds were well absorbed with Papp in the order of benzoic acid > chlorogenic acid > astragaloside > hyperoside > rutin. Chlorogenic acid and benzoic acid were mainly absorbed through passive diffusion, while rutin, astragaloside, and hyperoside were mainly by active transport, of which chlorogenic and rutin absorption were mediated by the efflux protein, P-glycoprotein (P-pg). Based on the transport volume of 2 mg/ml MLPs within 2 h, 25% of the maximum transported MLPs (TMLPs) was a safe concentration for 3 T3-L1 preadipocytes. Except for astragaloside, the other four components showed a significant inhibitory effect on lipid droplets, TG and TC, and chlorogenic acid and benzoic acid had the strongest effect. Additionally, we observed a synergistic effect as TMLPs were the most effective. We hypothesized that TMLPs, chlorogenic acid and benzoic acid suppressed adipogenesis and regulated lipid metabolism by inhibiting PPAR-γ, C/EBP-α, and FAS mRNA while promoting ADIPO and Leptin mRNA expression. PRACTICAL APPLICATIONS: The absorption and adipogenesis inhibition effect of mulberry leaf phenolics were evaluated in this study. The results provided guideline for the development of functional foods in regulating lipid metabolism.

Analysis of Lactobacillus rhamnosus GG in Mulberry Galacto-Oligosaccharide Medium by Comparative Transcriptomics and Metabolomics.

Lactobacillus rhamnosus GG (LGG) has strong acid resistance and can survive passing through the stomach to colonize the intestines, where it promotes the growth of beneficial bacteria. Prebiotics such as mulberry galacto-oligosaccharide (MGO), mulberry polysaccharide solution (MPS), and galactooligosaccharides (GOS) promote LGG proliferation, and MGO has the greatest effect. After culturing LGG with prebiotics, changes in gene expression were studied at the transcriptomic and metabolomic levels. The results showed that, in the stable 24-h growth period of cultivation, ~63 and 132% more differential genes were found after MPS and MGO were added to the MRS medium, respectively, than after GOS was added, and the numbers of up-regulated genes were about 18 and 66% higher with MPS and MGO, respectively, than GOS. Analysis using the KEGG database revealed that, when LGG was cultured with MGO, 120 genes that were up-regulated as the growth rate increased were mainly enriched in pathways such as membrane transport, amino acid metabolism, and carbohydrate metabolism. The genes gatB and gatC were up-regulated for galactose metabolism, and bglA was up-regulated in the glycolysis/gluconeogenesis pathway. The qRT-RCR results, which were in agreement with the RNA-seq, indicated the genes involved in the proliferation effect of LGG were up-regulated. UDP-glucose may be a key metabolite for MGO to promote LGG proliferation.

Preparation, structural characterization and prebiotic potential of mulberry leaf oligosaccharides.

The present study shows the purification of a main oligosaccharide fraction (MLO 1-2) from the enzymatic hydrolysate of mulberry leaf polysaccharides by DEAE-52 cellulose and gel column chromatography. The physicochemical properties of MLO 1-2 were characterized. The structure of MLO 1-2 was obtained as follows: α-(2-OAc)-Manp-1 → 2-ß-Glcp-1 → 4-ß-Glcp-1 → 4-α-Glcp-1 → 2-α-Glcp-1 → 2-α-Galp-1 → 2-ß-Galp-1 → 2-ß-Galp-1, which was elucidated by methylation and NMR analysis. The molecular weight of MLO 1-2 showed no significant change after simulated saliva, gastric and intestinal digestion. This indicated that MLO 1-2 could pass through the digestive system without being degraded to safely reach the colon to regulate the gut microbiota. Additionally, MLO 1-2, more than glucose or galactooligosaccharides, promoted the proliferation of Bifidobacterium bifidum, B. adolescentis, Lacticaseibacillus rhamnosus and Lactobacillus acidophilus. Furthermore, the acetic and lactic acid concentrations of bacterial cultures inoculated with MLO 1-2 were higher than those inoculated with glucose and galactooligosaccharide (GOS). These results suggest that MLO 1-2 could be an excellent prebiotic for intestinal flora regulation and the promotion of gut health.

Antibacterial Mechanism of Cinnamaldehyde: Modulation of Biosynthesis of Phosphatidylethanolamine and Phosphatidylglycerol in Staphylococcus aureus and Escherichia coli.

Cinnamaldehyde is a natural antimicrobial food preservative. Previous studies have suggested that cinnamaldehyde interacts with the cell membrane, but the molecular targets of cinnamaldehyde action on foodborne pathogens are still unclear. In this study, the structural changes of Staphylococcus aureus and Escherichia coli cells were observed after cinnamaldehyde treatment. Then, quantitative real-time polymerase chain reaction (PCR) and parallel reaction monitoring were used for determining the effects of cinnamaldehyde treatment of these bacteria on the expression of genes and proteins associated with glycerophospholipid biosynthesis. Changes in fatty acids (raw materials for the biosynthesis of glycerophospholipids) and glycerophospholipids in S. aureus and E. coli after cinnamaldehyde treatment were analyzed to confirm the results of gene and protein expression experiments. Cinnamaldehyde regulated the glycerophospholipid biosynthesis pathways of these foodborne pathogens, mainly targeting phosphatidylglycerol and phosphatidylethanolamine, which resulted in the disruption of cell membrane integrity.

The hypoglycemic effect of freeze-dried fermented mulberry mixed with soybean on type 2 diabetes mellitus.

Mulberry has significant hypoglycemic effect and can be used as an auxiliary food for people with type 2 diabetes. However, it is rich in carbohydrate and cannot be consumed directly by diabetic patients. In the study, we fermented the mulberry to reduce the content of glucose and fructose, and added the soybean to reduce the loss of probiotics during fermentation and then determined its hypoglycemic effect. We induced type 2 diabetes mellitus (T2DM) mice by streptozotocin and measured its blood glucose, serum biochemistry, hepatic and pancreatic histopathology, and the diversity of the gut microbiota. After 5 weeks of oral DFMS administration, the glucose tolerance was improved significantly in T2DM mice. Furthermore, there were also significant increases in superoxide dismutase activity and glutathione concentration, and marked reductions in the concentrations of malondialdehyde and free fatty acids. Moreover, DFMS also prevented histopathological changes and the increases in the activities of alanine transaminase and aspartate transaminase. DFMS treatment also markedly increased the richness of the gut microbial community. The abundance of Bacteroidetes was increased, and those of Proteobacteria, Escherichia-Shigella, and Lactobacillus were reduced. In summary, DFMS has a clear hypoglycemic effect in mice with T2DM.

Effects of enzymatic hydrolysis on the structural, rheological, and functional properties of mulberry leaf polysaccharide.

Effects of enzymatic hydrolysis on the structural, rheological, and functional properties of mulberry leaf polysaccharide (MLP) were characterized in this study. The enzymatic hydrolysis of MLP raised the carbonyl, carboxyl, and hydroxyl groups from 7.21 ± 0.86 to 10.08 ± 0.28 CO/100 Glu, 9.40 ± 0.13 to 17.55 ± 0.34 COOH/100 Glu, and 5.71 ± 0.33 to 8.14 ± 0.24 OH/100 Glu, respectively. Meanwhile, an increase in thixotropic performance and structure-recovery capacities were observed in hydrolyzed MLP, while the molecular weight, surface tension, apparent viscosity, and thermal stability were decreased. An improved antioxidant activity of MLP was also achieved after the enzymatic degradation. Moreover, the hydrolyzed MLP showed greater ability to promote the growths of Bifidobacterium bifidum, Bifidobacterium adolescentis, Lactobacillus rhamnosus, and Lactobacillus acidophilus and the production of acetic acid, butyric acid, and lactic acid. The results demonstrate that enzymatic modification is a useful approach for polysaccharide processing.

Mulberry leaf polyphenols attenuated postprandial glucose absorption via inhibition of disaccharidases activity and glucose transport in Caco-2 cells.

The present study attempted to evaluate the mechanism of action and bioactivity of mulberry leaf polyphenols (MLPs) in type-2 diabetes prevention via inhibition of disaccharidase and glucose transport. MLPs were purified with D101 resin and the main composition was determined as chlorogenic acid, rutin, benzoic acid and hyperoside. MLPs demonstrated a strong inhibitory effect on disaccharidases derived from both mouse and Caco-2 cells, and the order of IC50 value was: murine sucrase (7.065 mg mL-1) > murine maltase (4.037 mg mL-1) > Caco-2 cell maltase (0.732 mg mL-1) > Caco-2 cell sucrase (0.146 mg mL-1). MLPs showed the strongest inhibitory effect on sucrase derived from Caco-2 cells and played a role in lowering postprandial glucose mainly by inhibiting sucrase activity. The Caco-2 monolayer cell model was established to simulate the glucose transport process in the human small intestine. We found that within the concentration range of 0.5-2 mg mL-1, MLPs significantly inhibited glucose transport, and the inhibition rate increased with time and dose. The effect of phlorizin (SGLT1 inhibitor) in the control group showed a similar effect on glucose transport, revealing that MLPs may inhibit glucose transport mainly by inhibiting the SGLT1 transporter. RT-qPCR analysis confirmed that MLPs inhibited glucose absorption by suppressing the SGLT1-GLUT2 pathway via downregulation of the mRNA expression of phospholipase, protein kinase A and protein kinase C.

Destruction of the cell membrane and inhibition of cell phosphatidic acid biosynthesis in Staphylococcus aureus: an explanation for the antibacterial mechanism of morusin.

Morusin is a prenylated flavonoid found in mulberry that shows antimicrobial activity against foodborne pathogens. The MIC values of morusin toward S. aureus ATCC 6538 and S. aureus ATCC 25923 were both 14.9 µmol L-1. This study further investigated the antimicrobial mechanism of morusin in inhibiting the growth of Staphylococcus aureus ATCC 6538. Scanning electron microscopy and transmission electron microscopy revealed that morusin disrupted the integrity of the bacterial cell membrane. Morusin may also affect the phospholipid-repair system of bacteria, which repairs membrane structures. To test this hypothesis, quantitative real-time PCR was used to examine the effect of morusin treatment of S. aureus on the regulation of genes associated with the cell phosphatidic acid biosynthesis pathway. Gas chromatography-mass spectrometry was used to investigate the fatty acid components, which are used to synthesize bacterial phosphatidic acids. In summary, the results revealed that morusin showed a potent antibacterial effect by disrupting the cell membrane architecture and inhibiting the phosphatidic acid biosynthesis pathway of S. aureus.

Effect of 1-Deoxynojirimycin Isolated from Mulberry Leaves on Glucose Metabolism and Gut Microbiota in a Streptozotocin-Induced Diabetic Mouse Model.

1-Deoxynojirimycin (DNJ) exerts hypoglycemic effects. However, the traditional method for DNJ extraction is inefficient, and the hypoglycemic mechanism of DNJ remains unclear. In this study, the mixed fermentation by Lactobacillus fermentum and Saccharomyces cerevisiae was used to enhance DNJ extraction efficiency. It was found that this strategy was more efficient than the traditional method as the yield improved from the original 3.24 mg/g to 5.97 mg/g. The purified DNJ significantly decreased serum glucose (P < 0.01) and insulin levels (P < 0.05), improved serum lipid levels (P < 0.05), and reversed insulin resistance (P < 0.05) in diabetic mice. These changes were caused by up-regulating the protein expression of insulin receptor and glycolysis enzymes (GK, PK, and PFK) (P < 0.05) and down-regulating the protein expression of insulin receptor substrate-1 and gluconeogenesis enzymes (PCB, PEPCK, FBPase, and G-6-Pase) (P < 0.05), thus alleviating glucose tolerance. Additionally, DNJ treatment relieved gut dysbiosis in diabetic mice by promoting the growth of Lactobacillus, Lachnospiraceae NK4A136 group, Oscillibacter, norank Lachnospiraceae, Alistipes, and Bifidobacterium (P < 0.05) and suppressing the growth of Ruminococcaceae UCG-014, Weissella, Ruminococcus, Prevotellaceae Ga6A1 group, Anaerostipes, Klebsiella, Prevotellaceae UCG-001, and Bacteroidales S24-7 group (P < 0.05).

Purification, Characterization, Prebiotic Preparations and Antioxidant Activity of Oligosaccharides from Mulberries.

A water-soluble oligosaccharide termed EMOS-1a was prepared by enzymatic hydrolysis of polysaccharides purified from mulberries by column chromatography. The chemical structure of the purified fraction was investigated by ultraviolet spectroscopy, Fourier-transform infrared spectroscopy, and gas chromatography-mass spectrometry, which indicated that galactose was the main constituent of EMOS-1a. Chemical analyses showed that the uronic acid and sulfate content of EMOS-1a were 5.6% and 8.35%, respectively, while gel permeation chromatography showed that EMOS-1a had an average molecular weight of 987 Da. The antioxidant activities of EMOS-1a were next investigated, and EMOS-1a exhibited concentration-dependent 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, Trolox equivalent antioxidant capacity, and ferric reducing antioxidant power. The level of proliferation of Lactobacillus rhamnosus reached 1420 ± 16% when 4% (w/v) EMOS-1a was added, where the number of colonies in MRS (de Man, Rogosa, and Sharpe) medium with no added oligosaccharide was defined as 100% proliferation. These results indicate that the oligosaccharide EMOS-1a could be used as a natural antioxidant in prebiotic preparations.

Identification and molecular characterization of bacteriophage phiAxp-2 of Achromobacter xylosoxidans.

A novel Achromobacter xylosoxidans bacteriophage, phiAxp-2, was isolated from hospital sewage in China. The phage was morphologically and microbiologically characterized, and its one-step growth curve, host range, genomic sequence, and receptor were determined. Its morphology showed that phiAxp-2 belongs to the family Siphoviridae. Microbiological characterization demonstrated that pH 7 is most suitable for phage phiAxp-2; its titer decreased when the temperature exceeded 50 °C; phiAxp-2 is sensitive to ethanol and isopropanol; and the presence of calcium and magnesium ions is necessary to accelerate cell lysis and improve the formation of phiAxp-2 plaques. Genomic sequencing and a bioinformatic analysis showed that phage phiAxp-2 is a novel bacteriophage, consisting of a circular, double-stranded 62,220-bp DNA molecule with a GC content of 60.11% that encodes 86 putative open reading frames (ORFs). The lipopolysaccharide of A. xylosoxidans is involved in the adsorption of phiAxp-2.

Corrigendum: Survey and Visual Detection of Zaire Ebolavirus in Clinical Samples Targeting the Nucleoprotein Gene in Sierra Leone.

[This corrects the article on p. 1332 in vol. 6, PMID: 26648918.].

Isolation and characterization of a bacteriophage phiEap-2 infecting multidrug resistant Enterobacter aerogenes.

Enterobacter aerogenes (Enterobacteriaceae) is an important opportunistic pathogen that causes hospital-acquired pneumonia, bacteremia, and urinary tract infections. Recently, multidrug-resistant E. aerogenes have been a public health problem. To develop an effective antimicrobial agent, bacteriophage phiEap-2 was isolated from sewage and its genome was sequenced because of its ability to lyse the multidrug-resistant clinical E. aerogenes strain 3-SP. Morphological observations suggested that the phage belongs to the Siphoviridae family. Comparative genome analysis revealed that phage phiEap-2 is related to the Salmonella phage FSL SP-031 (KC139518). All of the structural gene products (except capsid protein) encoded by phiEap-2 had orthologous gene products in FSL SP-031 and Serratia phage Eta (KC460990). Here, we report the complete genome sequence of phiEap-2 and major findings from the genomic analysis. Knowledge of this phage might be helpful for developing therapeutic strategies against E. aerogenes.