Zinc caproate: Ecofriendly synthesis, structural characterization, and antibacterial action.

Disease-causing microorganisms such as Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) are among the primary contributors to morbidity and mortality of diarrhea in humans. Considering the challenges associated with antibiotic use, including antimicrobial resistance, this study aimed to develop a novel zinc-based agent for bacterial inactivation. To this end, zinc caproate (ZnCA) was synthesized using caproic acid (CA) and zinc oxide (ZnO) in anhydrous ethanol via the solvothermal method. Structural characterization techniques, including Fourier-transform infrared spectroscopy, single crystal X-ray diffraction analysis, and nuclear magnetic resonance spectroscopy, revealed the bidentate bridging coordination of zinc atoms with CA. The resulting two-dimensional ZnCA network was found to be composed of a distinct lamellar pattern, without any evident inter-layer interactions. Powder X-ray diffraction analysis, elemental analysis, and melting point analysis confirmed that ZnCA had an average particle size of 1.320 µm, a melting point of 147.2 °C, and a purity exceeding 98 %. Remarkably, ZnCA demonstrated potent antibacterial activity against E. coli and S. aureus, which exceeded the antibacterial efficacy of ZnO. ZnCA exerted its antibacterial effects by inhibiting biofilm formation, disrupting cell membrane integrity, increasing cell membrane permeability, and altering intracellular Ca2+-Mg2+-ATPase activity. These findings highlight the potential of ZnCA as a promising antibiotic substitute for the treatment of diarrhea in humans.

Investigations of Ascorbic Acid Synthesis and Distribution in Broiler Tissues at Different Post-Hatch Days.

Ascorbic acid (AA) is an indispensable nutrient required to sustain optimal poultry health and performance, which is commonly excluded from the diet of broilers. To investigate the synthesis and distribution of AA during broiler growth and clarify its possible turnover, 144 1 d old healthy Arbor Acres broilers with a body weight of approximately 41 g were randomly assigned to eight groups of 18 broilers each. The kidney, liver, ileum, and spleen of one bird from each group were collected every week until 42 d to detect the synthesis capacity, tissue distribution, and transporter gene expression of AA. The results showed that kidney L-gulonolactone oxidase (GLO) activity responded quadratically (p < 0.001), with maximum activity observed at 7 to 21 d old. Hepatic total AA and dehydroascrobate (DHA) concentration increased linearly (p < 0.001) with age, as did splenic total AA (p < 0.001). In the ileum, mRNA expression of sodium vitamin C transporter 1/2 (SVCT1/2) decreased with the growing age of the broilers (p < 0.05). The expression of SVCT1 in the kidney was not influenced by the growing age of the broilers. The progressive buildup of AA in the liver and spleen of broilers as they age implies an amplified demand for this nutrient. The waning synthesis capacity over time, however, raises concerns regarding the possible inadequacy of AA in the latter growth phase of broilers. The addition of AA to the broilers' diet might have the potential to optimize their performance. However, the effectiveness of such dietary supplementation requires further investigation.

Rehmannia glutinosa Polysaccharides: Optimization of the Decolorization Process and Antioxidant and Anti-Inflammatory Effects in LPS-Stimulated Porcine Intestinal Epithelial Cells.

Polysaccharide decolorization has a major effect on polysaccharide function. In the present study, the decolorization of Rehmannia glutinosa polysaccharides (RGP) is optimized using two methods-the AB-8 macroporous resin (RGP-1) method and the H2O2 (RGP-2) method. The optimal decolorization parameters for the AB-8 macroporous resin method were as follows: temperature, 50 °C; macroporous resin addition, 8.4%; decolorization duration, 64 min; and pH, 5. Under these conditions, the overall score was 65.29 ± 3.4%. The optimal decolorization conditions for the H2O2 method were as follows: temperature, 51 °C; H2O2 addition, 9.5%; decolorization duration, 2 h; and pH, 8.6. Under these conditions, the overall score was 79.29 ± 4.8%. Two pure polysaccharides (RGP-1-A and RGP-2-A) were isolated from RGP-1 and RGP-2. Subsequently, their antioxidant and anti-inflammatory effects and mechanisms were evaluated. RGP treatment activated the Nrf2/Keap1 pathway and significantly increased the activity of antioxidant enzymes (p < 0.05). It also inhibited the expression of pro-inflammatory factors and suppressed the TLR4/NF-κB pathway (p < 0.05). RGP-1-A had a significantly better protective effect than RGP-2-A, likely owing to the sulfate and uronic groups it contains. Together, the findings indicate that RGP can act as a natural agent for the prevention of oxidation and inflammation-related diseases.

Structural Characteristics of Rehmannia glutinosa Polysaccharides Treated Using Different Decolorization Processes and Their Antioxidant Effects in Intestinal Epithelial Cells.

Polysaccharide decolorization is a key determinant of polysaccharide structure. In this study, two purified Rehmannia glutinosa polysaccharides, RGP-1-A and RGP-2-A, were obtained after decolorization using the AB-8 macroporous resin and H2O2, respectively. RGP-1-A (molecular weight (Mw) = 18,964 Da) and RGP-2-A (Mw = 3305 Da) were acidic and neutral heteropolysaccharides, respectively, and were both polycrystalline in structure. FTIR analysis revealed that RGP-1-A was a sulfate polysaccharide, while RGP-2-A had no sulfate group. Experiments on IPEC-1 cells showed that RGPs alleviated oxidative stress by regulating the Nrf2/Keap1 pathway. These findings were confirmed by the upregulation of Nrf2, NQO1, and HO-1; the subsequent increase in the levels of antioxidant indicators (SOD, LDH, CAT, and MDA); and the restoration of mitochondrial membrane potential. Overall, the antioxidant capacity of RGP-1-A was significantly higher than that of RGP-2-A. These results suggest that RGPs may be a potential natural antioxidant and could be developed into functional foods.

Marginal Zinc Deficiency Aggravated Intestinal Barrier Dysfunction and Inflammation through ETEC Virulence Factors in a Mouse Model of Diarrhea.

Zinc is both essential and inhibitory for the pathogenesis of enterotoxigenic Escherichia coli (ETEC). However, the accurate effects and underlying mechanism of marginal zinc deficiency on ETEC infection are not fully understood. Here, a marginal zinc-deficient mouse model was established by feeding mice with a marginal zinc-deficient diet, and ETEC k88 was further administrated to mice after antibiotic disruption of the normal microbiota. Marginal zinc deficiency aggravated growth impairment, diarrhea, intestinal morphology, intestinal permeability, and inflammation induced by ETEC k88 infection. In line with the above observations, marginal zinc deficiency also increased the intestinal ETEC shedding, though the concentration of ETEC in the intestinal content was not different or even decreased in the stool. Moreover, marginal zinc deficiency failed to change the host's zinc levels, as evidenced by the fact that the serum zinc levels and zinc-receptor GPR39 expression in jejunum were not significantly different in mice with ETEC challenge. Finally, marginal zinc deficiency upregulated the relative expression of virulence genes involved in heat-labile and heat-stable enterotoxins, motility, cellular adhesion, and biofilm formation in the cecum content of mice with ETEC infection. These findings demonstrated that marginal zinc deficiency likely regulates ETEC infection through the virulence factors, whereas it is not correlated with host zinc levels.

Effect of Changes in Photoperiods on Melatonin Expression and Gut Health Parameters in Laying Ducks.

We investigated the effect of photoperiod on ileal morphology, barrier function, short-chain fatty acid (SCFA) contents, microbial flora, melatonin expression, and synthesis in laying ducks. After adaption, a total of 180 Jinding laying ducks (252 days old) were randomly divided into three treatments, receiving 12L (hours of light):12D (hours of darkness), 16L:8D, or 20L:4D. Each treatment had six replicates with 10 birds each. The formal experiment lasted 58 days. Compared with 12L:12D, the significantly higher values of villus height and goblet cell percentage (GCP) were observed in 16L:8D treatment, accompanied with the higher mRNA relative expression of zonula occludens-1, zonula occludens-2, zonula occludens-3, claudin-1, occludin, and mucin 2 (P < 0.05). Besides, significantly higher values of acetate and propionate, butyrate and total SCFA concentrations were simultaneously observed in ileal chyme of 16L:8D treatment (P < 0.05). For the ileal microbial community, the results of principal coordinate analysis (PCoA) visually presented that three photoperiod groups were mainly scattered into three clusters, indicating that the microbiota composition in different photoperiod treatments were quite dissimilar. Lower values of Shannon indicators were observed in the 20L:4D treatment (P < 0.05), meaning that the microbiota α-diversity decreased in the 20-h photoperiod. The relative abundance of Actinobacteria, Fusobacteria, and Proteobacteria at phylum level and Fusobacterium, Clostridium_sensu_stricto_1, and Pectobacterium at genus level kept an appropriate balance in the 16L:8D photoperiod. Melatonin level in serum decreased with the increasing photoperiods at 6:00 and 12:00, which was consistent with melatonin receptor expressions in the hypothalamus and ileal tissue. Meanwhile, the adenosine 3',5'-cyclic phosphate (cAMP) contents were significantly downregulated in the pineal gland (P < 0.05), in response to the increase in photoperiod. In conclusion, an appropriate photoperiod could improve ileal morphology, barrier function, SCFA profile, and microbial flora, which may be attributed to the appropriate regulation of the circadian rhythm through melatonin as well as its receptor expression, and 16 h could be an adequate photoperiod for laying ducks.

Structural Characteristics of Inulin and Microcrystalline Cellulose and Their Effect on Ameliorating Colitis and Altering Colonic Microbiota in Dextran Sodium Sulfate-Induced Colitic Mice.

Several studies have reported that dietary fibers (DFs) from plants may exert beneficial effects on inflammatory bowel disease. In the present study, we investigated the structural differences of soluble DF (inulin) and insoluble DF (microcrystalline cellulose, MCC) and their effects on the intestinal barrier integrity, gut microbiota community, and inflammation response in mice with dextran sodium sulfate (DSS)-induced colitis. Mice were fed for 21 days with diets containing inulin or MCC (2.5 g/kg body weight), and colitis was induced by administration of DSS (4% w/v) in drinking water during the last 8 days of experimentation. The results showed that inulin and MCC differ in morphology and structure. MCC exhibited a smaller particle size, a larger specific surface area, and higher thermal stability than inulin. In addition, both inulin and MCC restored various physical signs (body weight, colon weight and length, disease activity index score, and infiltration of inflammatory cells), gut barrier function (as evidenced by the increased expression of claudin-3, claudin-7, ZO-2, occludin, JAM-2, and MUC-3 and the decreased activity of myeloperoxidase activity), downregulation of mRNA expression of proinflammatory cytokines (caspase-1, NLPR3, TLR4, TNF-α, and IL-1ß), and modulation of colon microbiota community. Taken together, the present study demonstrates that DFs differ in morphology and structure and ameliorate DSS-induced colitis in mice by blocking proinflammatory cytokines, reinforcing gut barrier integrity, and modulating gut microbiota. Therefore, DFs, especially inulin, are promising dietary supplements to alleviate intestinal inflammation.

Zinc Laurate Protects against Intestinal Barrier Dysfunction and Inflammation Induced by ETEC in a Mice Model.

Enterotoxigenic Escherichia coli (ETEC) infection is one of the most common bacterial causes of diarrhea in children and young farm animals. Medium-chain fatty acids (MCFAs) have been widely used for their antibacterial and immune functions. However, there is limited information regarding the role of MCFAs chelated with Zn in diarrhea induced by ETEC infection. Here, zinc laurate (ZnLa) was used to evaluate its protective effect in a mice diarrhea model induced by ETEC. A total of 45 ICR-weaned female mice were randomly assigned to marginal zinc deficiency (dZn), dZn, and ETEC infection groups (dZn+ETEC); ETEC infection was co-treated with a low, middle, or high dose of ZnLa (ZnLa LOW+ETEC, ZnLa MID+ETEC, and ZnLa HIGH+ETEC), respectively, to explore the effect and its mechanism of ZnLa on diarrhea and intestinal health of mice challenged with ETEC. To further compare the antibacterial efficiency of ZnLa and ZnSO4 in mice with ETEC infection, a total of 36 ICR-weaned female mice were randomly divided into ZnLa, ZnLa+ETEC, ZnSO4, and ZnSO4 and ETEC infection groups (ZnSO4+ETEC); moreover, the growth curve of ETEC also compared ZnLa and ZnSO4 in vitro. Mice pretreated with ZnLa were effectively guarded against body weight losses and increases in diarrhea scores induced by ETEC. ZnLa pretreatment also prevented intestinal barrier damage and ion transport in mice challenged with ETEC, as evidenced by the fact that the intestinal villus height and the ratio of villus height and crypt depth, tight junction protein, and Na+ absorption were higher, whereas intestinal permeability and anion secretion were lower in mice pretreated with ZnLa. In addition, ZnLa conferred effective protection against ETEC-induced intestinal inflammatory responses, as the increases in protein and mRNAs of proinflammatory cytokines were prevented in serum and jejunum, which was likely associated with the TLR4/MYD88/NF-κB signaling pathway. The increase in ETEC shedding and virulence-related gene expression was prevented in mice with ZnLa pretreatment. Finally, the growth of ETEC and virulence-related gene expression were lower in the ZnLa group than in ZnSO4 with an equal concentration of zinc. These findings suggest that ZnLa is a promising prevention strategy to remedy ETEC infection.

Preparation of ZIF@ADH/NAD-MSN/LDH Core Shell Nanocomposites for the Enhancement of Coenzyme Catalyzed Double Enzyme Cascade.

The field of enzyme cascades in limited microscale or nanoscale environments has undergone a quick growth and attracted increasing interests in the field of rapid development of systems chemistry. In this study, alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH), and mesoporous silica nanoparticles (MSN) immobilized nicotinamide adenine dinucleotide (NAD+) were successfully immobilized on the zeolitic imidazolate frameworks (ZIFs). This immobilized product was named ZIF@ADH/NAD-MSN/LDH, and the effect of the multi-enzyme cascade was studied by measuring the catalytic synthesis of lactic acid. The loading efficiency of the enzyme in the in-situ co-immobilization method reached 92.65%. The synthesis rate of lactic acid was increased to 70.10%, which was about 2.82 times that of the free enzyme under the optimal conditions (40 °C, pH = 8). Additionally, ZIF@ADH/NAD-MSN/LDH had experimental stability (71.67% relative activity after four experiments) and storage stability (93.45% relative activity after three weeks of storage at 4 °C; 76.89% relative activity after incubation in acetonitrile-aqueous solution for 1 h; 27.42% relative activity after incubation in 15% N, N-Dimethylformamide (DMF) solution for 1 h). In summary, in this paper, the cyclic regeneration of coenzymes was achieved, and the reaction efficiency of the multi-enzyme biocatalytic cascade was improved due to the reduction of substrate diffusion.

Effect of molecular weight on the antibacterial activity of polysaccharides produced by Chaetomium globosum CGMCC 6882.

This study investigated the effect of molecular weight on antibacterial activity of polysaccharides. Results showed that low molecular weight (3.105 × 104 Da) polysaccharide (GCP-2) had higher inhibitory effects against Escherichia coli and Staphylococcus aureus than high molecular weight (5.340 × 104 Da) polysaccharide (GCP-1). Meanwhile, antibacterial activities of GCP-2 and GCP-1 against S. aureus were higher than those of E. coli. Minimum inhibitory concentrations (MICs) of GCP-1 against E. coli and S. aureus were 2.0 mg/mL and 1.2 mg/mL, and MICs of GCP-2 against E. coli and S. aureus were 1.75 mg/mL and 0.85 mg/mL, respectively. Antibacterial mechanisms investigation revealed that GCP-2 and GCP-1 influenced cell membrane integrity, Ca2+-Mg2+-ATPase activity on cell membrane and calcium ions in cytoplasm of E. coli and S. aureus, but not cell wall. Present work provided important implications for future studies on development of antibacterial polysaccharides based on molecular weight feature.

Anethole Attenuates Enterotoxigenic Escherichia coli-Induced Intestinal Barrier Disruption and Intestinal Inflammation via Modification of TLR Signaling and Intestinal Microbiota.

This study aimed to investigate the effects of dietary anethole supplementation on the growth performance, intestinal barrier function, inflammatory response, and intestinal microbiota of piglets challenged with enterotoxigenic Escherichia coli K88. Thirty-six weaned piglets (24 ± 1 days old) were randomly allocated into four treatment groups: (1) sham challenge (CON); (2) Escherichia coli K88 challenge (ETEC); (3) Escherichia coli K88 challenge + antibiotics (ATB); and (4) Escherichia coli K88 challenge + anethole (AN). On day 12, the piglets in the ETEC, ATB, and AN group were challenged with 10 mL E. coli K88 (5 × 109 CFU/mL), whereas the piglets in the CON group were orally injected with 10 mL nutrient broth. On day 19, all the piglets were euthanized for sample collection. The results showed that the feed conversion ratio (FCR) was increased in the Escherichia coli K88-challenged piglets, which was reversed by the administration of antibiotics or anethole (P < 0.05). The duodenum and jejunum of the piglets in ETEC group exhibited greater villous atrophy and intestinal morphology disruption than those of the piglets in CON, ATB, and AN groups (P < 0.05). Administration of anethole protected intestinal barrier function and upregulated mucosal layer (mRNA expression of mucin-1 in the jejunum) and tight junction proteins (protein abundance of ZO-1 and Claudin-1 in the ileum) of the piglets challenged with Escherichia coli K88 (P < 0.05). In addition, administration of antibiotics or anethole numerically reduced the plasma concentrations of IL-1ß and TNF-α (P < 0.1) and decreased the mRNA expression of TLR5, TLR9, MyD88, IL-1ß, TNF-α, IL-6, and IL-10 in the jejunum of the piglets after challenge with Escherichia coli K88 (P < 0.05). Dietary anethole supplementation enriched the abundance of beneficial flora in the intestines of the piglets. In summary, anethole can improve the growth performance of weaned piglets infected by ETEC through attenuating intestinal barrier disruption and intestinal inflammation.

Effects of maternal supplementation with fully oxidised ß-carotene on the reproductive performance and immune response of sows, as well as the growth performance of nursing piglets.

The present study was conducted to evaluate the impact of dietary fully oxidised ß-carotene (OxBC, C40H60O15) supplementation during the perinatal period on immune status and productivity in a sow model. At day 85 of pregnancy, 150 sows were allocated to one of three dietary treatments with fifty sows per treatment. The three experimental diets were supplemented with 0, 4 or 8 mg/kg OxBC in the basal diet. The feeding trial was conducted from gestation day 85 until day 21 of lactation. Dietary OxBC supplementation greatly enhanced colostrum IgM, IgA and IgG levels, and the IgM and IgG content of 14-d milk. Dietary OxBC supplementation decreased the TNF-α and IL-8 levels in colostrum, as well as the TNF-α and IL-18 levels in 14-d milk. There was also a tendency towards an increase in the soluble CD14 level in 14-d milk. Although dietary treatments did not affect average daily feed intake nor backfat thickness loss during lactation, dietary OxBC supplementation tended to enhance litter weight and individual piglet weight at weaning. There was a trend towards increased lactose concentration in 14-d milk with increasing dietary OxBC. It is concluded that dietary supplementation with OxBC during the perinatal period enhances the lactose concentration of sow milk and the immune status of sows, which is reflected by improved cytokine status and immunoglobulin concentrations in colostrum and milk, and thus tending to increase litter weight and individual piglet weight at weaning. The results also provide a scientific nutritional reference for perinatal mothers due to the biological similarity between pigs and humans.

Modification of sweet potato (Ipomoea batatas Lam.) residues soluble dietary fiber following twin-screw extrusion.

Effect of twin-screw extrusion on soluble dietary fiber (SDF) from sweet potato residues (SPRs) were investigated using optimized conditions, at screw speed of 180 rpm, feed rate at 17 Hz, feed moisture at 40% and extrusion temperature at 150 °C. Extruded SDF, showed higher SDF levels (9.63%-29.25%), cholesterol and sodiumcholate adsorption capacity, radical scavenging capacity, and inhibition of digestive enzymes. Moreover, extrusion effectively reduced particle size and molecularweight of SDF, modulated monosaccharide ratios, and increased water retention capacity (WRC), oil retention capacity (ORC), swelling capacity (SC) and glucose absorption capacity (GAC). Additionally, scanning electron microscopy (SEM) demonstrated decomposition of macromolecules of SDF to smaller fractions and formation of a porous morphology following extrusion. Furthermore, the extruded SDF increased thermal stability as determined by differential scanning calorimetry (DSC). Overall, the SDF from SPRs with improved functional and physiochemical properties could be used as a functional additive in diverse food products.

Antibacterial mechanism of the polysaccharide produced by Chaetomium globosum CGMCC 6882 against Staphylococcus aureus.

GCP, a polysaccharide produced by endophytic fungus Chaetomium globosum CGMCC 6882, was found to be antibacterial against Staphylococcus aureus via disrupting cell permeability. However, the antibacterial mechanism of GCP has not been studied before. In present work, results showed that GCP could retard the growth of S. aureus by inducing the depolarization of cell membrane, decreasing the activity of Ca2+-Mg2+-ATPase on cell membrane, and increasing the accumulation of calcium ions in cytoplasm. Moreover, we found that GCP could also inhibit the synthesis of whole cell proteins of S. aureus. Overall, this study proved that the antibacterial mechanism of GCP could be diversified and more studies are needed in the investigation of the antibacterial mechanisms of various polysaccharides.

The Effects of Dietary Supplementation of Saccharomyces cerevisiae Fermentation Product During Late Pregnancy and Lactation on Sow Productivity, Colostrum and Milk Composition, and Antioxidant Status of Sows in a Subtropical Climate.

This study aimed to evaluate the effects of dietary supplementation of Saccharomyces cerevisiae fermentation product (SCFP) during late pregnancy and lactation on sow productivity, colostrum and milk composition, and antioxidant status of sows in a subtropical climate. The study was a 2 × 2 factorial treatment design where the first factor was environmental THI level [Low THI (08:00-19:00: 70.76 ± 0.45, 19:00-08:00: 67.91 ± 0.18, L-THI) or High THI (08:00-19:00: 75.14 ± 0.98, 19:00-08:00: 68.35 ± 0.18, H-THI], and the second factor was dietary treatment (supplemented with or without 3 kg/t SCFP). A total of 120 sows were randomly allotted to the four treatments (n = 30). The feeding trial was conducted from 85-days post-breeding until 21-days post-partum. Compared with L-THI group, sows from H-THI group had lesser individual piglet birth weight, individual piglet weight at weaning, preweaning average daily gain of piglets, average daily feed intake of sows during lactation, and protein percentage in 14-days milk. Additionally, sows from H-THI group had lesser antioxidant status, indicated by lesser serum total antioxidant capacity (T-AOC), and superoxide dismutase (SOD) activity at parturition; lesser serum T-AOC and glutathione peroxidase (GSH-Px) activity at 14-days post-partum, as well as lesser SOD activity in colostrum. Compared with sows fed the control diet, sows fed the SCFP diet had greater number of piglets weaned, litter weight at weaning, and preweaning average daily gain of piglets. Moreover, sows fed the SCFP diet had improved antioxidant status as indicated by higher serum T-AOC at parturition, and lesser malondialdehyde (MDA) content in colostrum and 21-days milk. In conclusion, H-THI negatively affected the productivity, milk composition, antioxidant status, and lactation feed intake of sows. Dietary supplementation of SCFP partially alleviated the adverse effects of H-THI, by improving lactation performance and antioxidant status of sows without influencing reproductive performance and colostrum and milk composition in a subtropical climate.

Antibacterial activity of xanthan-oligosaccharide against Staphylococcus aureus via targeting biofilm and cell membrane.

Previously, an antioxidant xanthan-oligosaccharide (LW-XG) was successfully produced via bio-degradation of commercial xanthan. In present work, the antibacterial activity and mechanism action of LW-XG against Staphylococcus aureus were studied. Inhibition zone of LW-XG in agar diffusion test was evident and its minimal inhibitory concentration (MIC) against S. aureus was 0.63 mg/mL. Inhibitory mechanism investigation showed that LW-XG increased the cell membrane permeability of S. aureus. Meanwhile, we found that LW-XG could retard the formation of S. aureus biofilm and lower the transcriptional levels of genes (fnbA, fnbB and clfB) related to biofilm formation. Furthermore, LW-XG decreased the Ca2+-Mg2+-ATPase activity on S. aureus cell membrane and promoted the accumulation of calcium ions in cytoplasm. Overall, LW-XG could inhibit the growth of S. aureus and be regarded as a promising antibacterial substitute in food and pharmaceutical industries.

Characterization and Antioxidant Activity of a Low-Molecular-Weight Xanthan Gum.

In the present work, a low-molecular-weight xanthan gum (LW-XG) was successfully obtained via biodegradation of commercial xanthan by the endophytic fungus Chaetomiumglobosum CGMCC 6882. The monosaccharide composition of LW-XG was glucose, mannose, and glucuronic acid in a molar ratio of 1.63:1.5:1.0. The molecular weight of LW-XG was 4.07 × 104 Da and much smaller than that of commercial xanthan (2.95 × 106 Da). Antioxidant assays showed that LW-XG had a good scavenging ability on DPPH radicals, superoxide anions, and hydroxyl radicals and good ferric reducing power. Moreover, LW-XG exhibited excellent protective effect on H2O2-injured Caco-2 cells. Results of this work suggested that LW-XG could be used in foods or pharmaceuticals to alleviate and resist the oxidative damage induced by the overproduction of reactive oxygen species.

Enhancing thermostability of Yarrowia lipolytica lipase 2 through engineering multiple disulfide bonds and mitigating reduced lipase production associated with disulfide bonds.

The limited thermostability of Yarrowia lipolytica lipase 2 (Lip2) hampers its industrial application. To improve its thermostability, we combined single disulfide bonds which our group identified previously. In this study, combining different regional disulfide bonds had greater effect than combining same regional disulfide bonds. Furthermore, mutants with 4, 5, and 6 disulfide bonds exhibited dramatically enhanced thermostability. Compared with the wild-type, sextuple mutant 6s displayed a 22.53 and 31.23 ℃ increase in the melting temperature (Tm) and the half loss temperature at 15 min (T15 50), respectively, with greater pH stability and a wider reaction pH range. Molecular dynamics simulation revealed that multiple disulfide bonds resulted in more rigid structures of mutants 4s, 5s and 6s, and prolonged enzyme unfolding times. Moreover, secretions of mutants 5s and 6s were significantly increased by 60% and 80% by co-expressing with the chaperone protein disulfide isomerase (PDI), which mitigated the reduced production issue caused by multiple disulfide bonds. Results of this study indicated that enhanced heat endurance giving more potential for industrial application.

Enhanced expression of lipase I from Galactomyces geotrichum by codon optimisation in Pichia pastoris.

Relatively poor heterologous protein yields have limited the commerical applications of Galactomyces geotrichum lipase I (GGl I) efficacy trials. To address this, we have redesigned the GGl I gene to preferentially match codon frequencies of Pichia pastoris (P. pastoris) while retaining the same amino acid sequence. The wild type and codon optimised GGl I (GGl I-wt and GGl I-op) were synthesised and cloned into pPICZαA with an N-terminal 6 × His tag sequence and expressed in P. pastoris X 33. The hydrolytic activity of GGl I-op was 150 U/mL, whereas the activity of the GGl I-wt could not be detected. GGl I-op recombinant proteins were purified by Ni-affinity chromatography and then characterised. The identity and purity of GGl I were confirmed by SDS-PAGE, MALDI-TOF mass spectrometry and Western blot analysis. Enzymatic deglycosylation was used to show that the lipase is a glycosylated protein, containing ∼10% sugar. The molecular weight (MW) of the GGl I secreted by recombinant P. pastoris was approximated at 63 kDa. The optimum pH and temperature of the recombinant lipase were 8.0 and 35 °C, respectively. The enzyme was active over a broad pH range (7.0-9.0) and temperature range (20 °C-45 °C). The lipase showed high activity toward medium- and long-chain fatty acid methyl esters (C8-C16) and retained much of its activity in the presence of Tween-80 and Trition X-100. Lipase activity was stimulated by Mg2+, Ca2+, Mn2+ and Cu2+ and inhibited by Fe2+, Fe3+, Zn2+ and Co2+. This lipase may prove useful to the detergent industry and in organic synthesis reactions.

Optimisation of combi-lipases from Aspergillus niger for the synergistic and efficient hydrolysis of soybean oil.

The enzymatic properties of four lipases (A, B, C and D) from different strains of Aspergillus niger, were investigated, and a 3-factor mixture design and triangular surface analysis were performed to screen the optimal combi-lipase by observing synergistic effects. Lipases B and D differed in optimal pH, temperature and substrate specificity. A combi-lipase with 31.2% lipase B and 68.8% lipase D (w/w, equal to units of 30.36% and 69.64%) exhibited optimal hydrolytic activity on soybean oil, which exceeded the sum of the combined activities of individual lipases (P < 0.05). Free fatty acid from the hydrolyzed soybean oil indicated that the synergistic effect of the combi-lipase resulted from the different fatty acid specificities of the two lipases. Overall, combi-lipase afforded an effective route for the application of lipase enzymes to animal feeds.