Bacillus velezensis strain NA16 shows high poultry feather-degrading efficiency, protease and amino acid production.

Isolated Bacillus velezensis strain NA16, which produces proteases, amino acids and the transcription levels of different keratinolytic enzymes and disulfide reductase genes in whole gene sequencing, was evaluated during feather degradation. The result shows under optimum fermentation conditions, chicken feather fermentation showed total amino acid concentration of 7599 mg/L, degradation efficiency of 99.3% at 72 h, and protease activity of 1058 U/mL and keratinase activity of 288 U/mL at 48 h. Goose feather fermentation showed total amino acid concentration of 4918 mg/L (96 h), and degradation efficiency was 98.9% at 120 h. Chicken feather fermentation broth at 72 h showed high levels of 17 amino acids, particularly phenylalanine (1050 ± 1.90 mg/L), valine (960 ± 1.04 mg/L), and glutamic (950 ± 3.00 mg/L). Scanning electron microscopy and Fourier transform infrared analysis revealed the essential role of peptide bond cleavage in structural changes and degradation of feathers. Protein purification and zymographic analyses revealed a key role in feather degradation of the 39-kDa protein encoded by gene1031, identified as an S8 family serine peptidase. Whole genome sequencing of NA16 revealed 26 metalloproteinase genes and 22 serine protease genes. Among the proteins, S8 family serine peptidase (gene1031, gene1428) and S9 family peptidase (gene3132) were shown by transcription analysis to play major roles in chicken feather degradation. These findings revealed the transcription levels of different families of keratinolytic enzymes in the degradation of feather keratin by microorganisms, and suggested potential applications of NA16 in feather waste management and amino acid production.

Complete genome sequencing and investigation on the fiber-degrading potential of Bacillus amyloliquefaciens strain TL106 from the tibetan pig.

BACKGROUND: Cellulolytic microorganisms are considered a key player in the degradation of feed fiber. These microorganisms can be isolated from various resources, such as animal gut, plant surfaces, soil and oceans. A new strain of Bacillus amyloliquefaciens, TL106, was isolated from faeces of a healthy Tibetan pigs. This strain can produce cellulase and shows strong antimicrobial activity in mice. Thus, in this study, to better understand the strain of B. amyloliquefaciens TL106 on degradation of cellulose, the genome of the strain TL106 was completely sequenced and analyzed. In addition, we also explored the cellulose degradation ability of strain TL106 in vitro. RESULTS: TL106 was completely sequenced with the third generation high-throughput DNA sequencing. In vitro analysis with enzymatic hydrolysis identified the activity of cellulose degradation. TL106 consisted of one circular chromosome with 3,980,960 bp and one plasmid with 16,916 bp, the genome total length was 3.99 Mb and total of 4,130 genes were predicted. Several genes of cellulases and hemicellulase were blasted in Genbank, including ß-glucosidase, endoglucanase, ß-glucanase and xylanase genes. Additionally, the activities of amylase (20.25 U/mL), cellulase (20.86 U/mL), xylanase (39.71 U/mL) and ß-glucanase (36.13 U/mL) in the fermentation supernatant of strain TL106 were higher. In the study of degradation characteristics, we found that strain TL106 had a better degradation effect on crude fiber, neutral detergent fiber, acid detergent fiber, starch, arabinoxylan and ß-glucan of wheat and highland barley . CONCLUSIONS: The genome of B. amyloliquefaciens TL106 contained several genes of cellulases and hemicellulases, can produce carbohydrate-active enzymes, amylase, cellulase, xylanase and ß-glucanase. The supernatant of fermented had activities of strain TL106. It could degrade the fiber fraction and non-starch polysaccharides (arabinoxylans and ß-glucan) of wheat and highland barley. The present study demonstrated that the degradation activity of TL106 to crude fiber which can potentially be applied as a feed additive to potentiate the digestion of plant feed by monogastric animals.

Secretory expression of ß-mannanase in Saccharomyces cerevisiae and its high efficiency for hydrolysis of mannans to mannooligosaccharides.

Degradation of mannans is a key process in the production of foods and prebiotics. ß-Mannanase is the key enzyme that hydrolyzes 1,4-ß-D-mannosidic linkages in mannans. Heterogeneous expression of ß-mannanase in Pichia pastoris systems is widely used; however, Saccharomyces cerevisiae expression systems are more reliable and safer. We optimized ß-mannanase gene from Aspergillus sulphureus and expressed it in five S. cerevisiae strains. Haploid and diploid strains, and strains with constitutive promoter TEF1 or inducible promoter GAL1, were tested for enzyme expression in synthetic auxotrophic or complex medium. Highest efficiency expression was observed for haploid strain BY4741 integrated with ß-mannanase gene under constitutive promoter TEF1, cultured in complex medium. In fed-batch culture in a fermentor, enzyme activity reached ~ 24 U/mL after 36 h, and production efficiency reached 16 U/mL/day. Optimal enzyme pH was 2.0-7.0, and optimal temperature was 60 °C. In studies of ß-mannanase kinetic parameters for two substrates, locust bean gum galactomannan (LBG) gave Km = 24.13 mg/mL and Vmax = 715 U/mg, while konjac glucomannan (KGM) gave Km = 33 mg/mL and Vmax = 625 U/mg. One-hour hydrolysis efficiency values were 57% for 1% LBG, 74% for 1% KGM, 39% for 10% LBG, and 53% for 10% KGM. HPLC analysis revealed that the major hydrolysis products were the oligosaccharides mannose, mannobiose, mannotriose, mannotetraose, mannopentaose, and mannohexaose. Our findings show that this ß-mannanase has high efficiency for hydrolysis of mannans to mannooligosaccharides, a type of prebiotic, suggesting strong potential application in food industries.

Effects of xylanase supplementation to wheat-based diets on growth performance, nutrient digestibility and gut microbes in weanling pigs.

OBJECTIVE: This study was designed to investigate the effects of an Aspergillus sulphureus xylanase expressed in Pichia pastoris on the growth performance, nutrient digestibility and gut microbes in weanling pigs. METHODS: A total of 180 weanling pigs (initial body weights were 8.47±1.40 kg) were assigned randomly to 5 dietary treatments. Each treatment had 6 replicates with 6 pigs per replicate. The experimental diets were wheat based with supplementation of 0, 500, 1,000, 2,000, and 4,000 U xylanase/kg. The experiment lasted 28 days (early phase, d 0 to 14; late phase, d 15 to 28). RESULTS: In the early phase, compared to the control, average daily gain (ADG) was higher for pigs fed diets supplemented with xylanase and there was a quadratic response in ADG (p<0.05). In the entire phase, ADG was higher for the pigs fed 1,000 or 2,000 U/kg xylanase compared to the control (p<0.05). The gain to feed ratio was higher for pigs fed diets supplemented with 1,000 or 2,000 U/kg xylanase compared to the control (p<0.05). Increasing the amount of xylanase improved the apparent total tract digestibility of dry matter, crude protein, neutral detergent fiber, calcium, and phosphorus during both periods (p<0.05). Xylanase supplementation (2,000 U/kg) decreased the proportion of Lachnospiraceae (by 50%) in Firmicutes, but increased Prevotellaceae (by 175%) in Bacteroidetes and almost diminished Enterobacteriaceae (Escherichia-Shigella) in Proteobacteria. CONCLUSION: Xylanase supplementation increased growth performance and nutrient digestibility up to 2,000 U/kg. Supplementation of xylanase (2,000 U/kg) decreased the richness of gut bacteria but diminished the growth of harmful pathogenic bacteria, such as Escherichia-Shigella, in the colon.

miR-24 suppression of POZ/BTB and AT-hook-containing zinc finger protein 1 (PATZ1) protects endothelial cell from diabetic damage.

The regulatory transcriptional factor PATZ1 is abnormally up-regulated in diabetic endothelial cells (ECs) where it acts as an anti-angiogenic factor via modulation of fatty acid-binding protein 4 (FABP4) signaling. The aim of the present work was to elucidate the upstream molecular events regulating PATZ1 expression in diabetic angiogenesis. The bioinformatics search for microRNAs (miRNAs) able to potentially target PATZ1 led to the identification of several miRNAs. Among them we focused on the miR-24 since the multiple targets of miR-24, which have so far been identified in beta cells, cardiomyocytes and macrophages, are all involved in diabetic complications. miR-24 expression was significantly impaired in the ECs isolated from diabetic hearts. Functionally, endothelial migration was profoundly inhibited by miR-24 suppression in Ctrl ECs, whereas miR-24 overexpression by mimics treatment effectively restored the migration rate in diabetic ECs. Mechanistically, miR-24 directly targeted the 3'untranslated region (3'UTR) of PATZ1, and miR-24 accumulation potentiated endothelial migration by reducing the mRNA stability of PATZ1. Together, these results suggest a novel mechanism regulating endothelial PATZ1 expression based on the down-regulation of miR-24 expression caused by hyperglycemia. Interfering with PATZ1 expression via miRNAs or miRNA mimics could potentially represent a new way to target endothelial PATZ1-dependent signaling of vascular dysfunction in diabetes.

Directed evolution of a ß-1,3-1,4-glucanase from Bacillus subtilis MA139 for improving thermal stability and other characteristics.

In order to improve some characteristics of a ß-1,3-1,4-glucanase from Bacillus subtilis MA139, directed evolution was conducted in this study. After error-prone PCR, the ß-1,3-1,4-glucanase gene, glu-opt, was cloned into the vector pBGP1 and transformed into Pichia pastoris X-33 to construct a mutant library. Three variants named as 7-32, 7-87, and 7-115 were screened from 8000 colonies. Amino-acid sequence analysis showed that these mutants had one or two amino-acid substitutions (7-32: T113S, 7-87: M44V/N53H, and 7-115: N157D). The variants were over-expressed in P. pastoris by methanol induction. After purification of the enzyme proteins, the characteristics of the variants were analyzed in detail. It indicated that these mutant enzymes had broader ranges of pH value and better pH stability than the wild-type enzyme. The mutant enzyme 7-87 had the best ability to tolerate an acid environment (pH 2.0), while the wild-type enzyme had no activity under this condition. Moreover, all these mutants demonstrated improved thermal stability. In particular, the mutant enzyme 7-32 had residual enzymatic activity of 60% and 40% after being incubated at 80 °C and 90 °C for 10 min. While, the wild-type enzyme had no residual enzymatic activity after being incubated at 80 °C for 4 min. In addition, the mutant enzymes had better tolerance to some chemicals than the wild-type enzyme. The improved stability could enhance the prospects for this enzyme to have use in the feed industry to reduce the effects of the anti-nutritional factor ß-glucan.

Cloning and expression of a xylanase xynB from Aspergillus niger IA-001 in Pichia pastoris.

The high-level expression of the xylanase GH11 gene from Aspergillus niger IA-001 called xynB was successfully completed in Pichia pastoris. The xynB gene encoding a mature xylanase of 225 amino acid was subcloned into the pPICZαA vector and was transformed into P. pastoris X-33 under the control of the alcohol oxidase I (AOX1) promoter. The xynB gene was ligated with a sequence encoding modified α-factor signal peptide (pPICZαmA) and the recombinant xylanase activity, which was measured 1280 U ml(-1), was 1.5-fold higher than when it was inserted into pPICZαA and was 19.39-fold greater than the native xylanase in the original strain. In a 10 L fermenter, the recombinant xylanase activity measured 10,035 U ml(-1) after 114 h. The SDS-PAGE analysis revealed that the purified xynB protein migrated as a single band with an apparent molecular weight of 24 kDa. The specific activity, using beechwood xylan as a substrate, was 1916 U mg(-1). The xylanase activity was optimal at pH 5.0 and at 50 °C. In addition, the xynB was active over a pH range of 2.2 to 10.0. The apparent Km and Vmax values were 4.429 mg ml(-1) and 1429 U mg(-1), respectively.

Multi-copy expression of a protease-resistant xylanase with high xylan degradation ability and its application in broilers fed wheat-based diets.

The acidic thermostable xylanase (AT-xynA) has great potential in the feed industry, but its low activity is not conductive to large-scale production, and its application in poultry diets still needs to be further evaluated. In Experiment1, AT-xynA activity increased 3.10 times by constructing multi-copy strains, and the highest activity reached 10,018.29 ± 91.18 U/mL. AT-xynA showed protease resistance, high specificity for xylan substrates, xylobiose and xylotriose were the main hydrolysates. In Experiment2, 192 broilers were assigned into 3 treatments including a wheat-based diet, and the diets supplemented with AT-xynA during the entire period (XY-42) or exclusively during the early stage (XY-21). AT-xynA improved growth performance, while the performance of XY-21 and XY-42 was identical. To further clarify the mechanism underlying the particular effectiveness of AT-xynA during the early stage, 128 broilers were allotted into 2 treatments including a wheat-based diet and the diet supplemented with AT-xynA for 42 d in Experiment3. AT-xynA improved intestinal digestive function and microbiota composition, the benefits were stronger in younger broilers than older ones. Overall, the activity of AT-xynA exhibiting protease resistance and high xylan degradation ability increased by constructing multi-copy strains, and AT-xynA was particularly effective in improving broiler performance during the early stage.

Effects of crude protease produced by Bacillus subtilis (MAFIC Y7) on growth performance, immune indices, and anti-inflammatory responses of broilers fed soybean meal- or cottonseed meal-based diets.

A strain of Bacillus subtilis (MAFIC Y7) was isolated from the intestine of Tibetan pigs and was able to express high protease activity. The aim of this study was to characterize the proteases produced by MAFIC Y7, and to investigate the effects of protease addition on growth performance, ileal amino acid digestibility, and serum immunoglobulin and immune factors of broilers fed SBM-based diets, or on growth performance, carcass characteristics, and intestinal morphology of broilers fed CSM-based diets. B. subtilis (MAFIC Y7) expressed protease showed its optimal enzyme activity at 50 °C and pH 7.0. The coated crude enzyme (CCE) showed greater stability at pH 3.0 than its uncoated counterpart. Experiment 1 was conducted with six diets based on three levels of crude protein (CP)-CPlow, CPmedium, and CPhigh-with or without CCE. In CPlow, CCE increased gain:feed (G:F) (days 1 to 21, days 1 to 42) by 8%, 3%, respectively, and enhanced apparent ileal digestibility (AID) of crude protein and lysine (on day 42) by 8.8%, 4.6%, respectively, compared with diets containing no CCE (P < 0.05). CCE increased G:F from days 1 to 21 from 0.63 to 0.68, improved G:F and average daily gain (ADG) during days 1 to 42, and enhanced AID of crude protein, lysine, cysteine, and isoleucine on day 42 compared with the unsupplemented treatments (in CPmedium, P < 0.05). CCE increased serum IgA (on day 21), serum IgA and IgG and increased serum IL-10 (on day 42), but decreased serum tumor necrosis factor-α (TNF-α; on day 21), and serum IL-8 and TNF-α (on day 42) compared with unsupplemented treatments. At CPhigh, CCE decreased serum levels of IL-6 and TNF-α (on day 21), and IL-8 and TNF-α (on day 42) compared with unsupplemented treatments (in CPhigh, P < 0.05). In experiment 2, CSM-based diets with two lysine-to-protein ratios (5.2% or 5.5%) with or without CCE. In the high Lys diet (5.5% Lys:protein), CCE increased ADG and G:F, increased carcass, but decreased abdominal fat compared with the unsupplemented treatment (P < 0.05). In the 5.2% Lys:protein dietary treatment, CCE improved duodenal villus height compared with the unsupplemented treatment (P < 0.05). Supplementation of protease produced by MAFIC Y7 was associated with lower inflammatory responses in SBM diets (CPmedium or CPhigh) and improved ADG in broilers fed CPmedium or CPhigh. The proteases improved ADG and the efficiency of CSM use when the ratio of Lys to protein was 5.5%.

The aim of this study was to investigate the effects of Bacillus subtilis (MAFIC Y7)-expressed protease on reducing inflammatory responses of soybean meal (SBM) diets and improving the efficiency of cottonseed meal (CSM) in broilers. Experiment 1 was conducted with six dietary treatments based on three levels of crude protein (CP)­CPlow, CPmedium, and CPhigh­without or with proteases (0 or 4,000 U/kg). Supplementation of proteases significantly improved growth performance, gain:feed (G:F), and apparent ileal digestibility of crude protein and amino acids (cysteine, isoleucine, and histidine) in broilers fed CPmedium treatment (P < 0.05). Proteases inhibited inflammatory responses in SBM-based diets by decreasing serum tumor necrosis factor-α (TNF-α) (in CPmedium and CPhigh), and interleukin (IL)-6 (in CPhigh); and IL-8 and TNF-α (in CPmedium and CPhigh) on day 21. In experiment 2, broilers were fed with CSM-based diets with two ratios of lysine-to-protein (5.2% or 5.5%) with or without proteases. Proteases in the diet of 5.5% Lys to protein ratio increased growth performance and G:F compared to diets without proteases (P < 0.05). Proteases produced by MAFIC Y7 improved growth performance and G:F in CPmedium. Supplementation of protease was associated with lower inflammatory responses of broilers fed SBM-based diets (CPmedium or CPhigh) and improved the efficiency of CSM use when the ratio of lysine-to-protein was 5.5%.

Effect of dietary supplementation with recombinant human lysozyme on growth performance, antioxidative characteristics, and intestinal health in broiler chickens.

Lysozyme is often used as a feed additive to act as an antibacterial protein that boosts the immune system of livestock and poultry while protecting against pathogens. To investigate the effects of recombinant human lysozyme (rhLYZ) from Pichia pastoris and chlortetracycline on broiler chicken's production performance, antioxidant characteristics, and intestinal microbiota, a total of 200, 1-d-old male Arbor Acres broiler chickens (46.53 ±â€…0.42 g) were selected for a 42-d experiment. Dietary treatments included a basal diet of corn-soybean meal supplemented with either 0 mg/kg (CON), 50 mg/kg aureomycin (ANT), 20 mg/kg rhLYZ (LOW), 60 mg/kg rhLYZ (MEDIUM), or 180 mg/kg rhLYZ (HIGH). Compared with CON, MEDIUM diet increased (P < 0.05) average daily gain (67.40 g) of broilers from day 22 to 42. In the early (1.29) and overall phases (1.69), MEDIUM led to a reduction (P < 0.05) in the feed conversion ratio of broiler chickens. Furthermore, in comparison to the CON and ANT, MEDIUM exhibited reduced (P < 0.05) levels of INF-γ and tumor necrosis factor-α in the serum. In the cecum, the abundance of Monoglobus and Family_XIII_AD3011_group was lower (P < 0.05) in the MEDIUM treatment compared to CON. Overall, supplementation of 60 mg/kg of rhLYZ improved growth performance, nutrient utilization efficiency, and serum immune function, while also influencing the composition of intestinal microbiota. This suggests lysozyme's potential to replace antibiotic additives in feed.

The aim of this study was to explore the effects of recombinant human lysozyme (rhLYZ) produced from Pichia pastoris and chlortetracycline on broiler chicken performance, antioxidant properties, and gut microbiota. A 42-d experiment was conducted, involving 200 1-d-old male Arbor Acres broiler chickens. We provided different diets: a standard diet (CON), a diet with 50 mg/kg aureomycin (ANT), a diet with 20 mg/kg rhLYZ (LOW), a diet with 60 mg/kg rhLYZ (MEDIUM), or a diet with 180 mg/kg rhLYZ (HIGH). The results showed that, compared to the control group, the MEDIUM group significantly increased the average daily gain of broilers to 67.40 g from day 22 to 42. Additionally, the MEDIUM group exhibited a reduced feed conversion ratio during both the early and overall growth stages of the chickens. Furthermore, serum levels of INF-γ and tumor necrosis factor-α were lower in the MEDIUM group compared to both the CON and ANT groups. In the cecum, the abundance of Monoglobus and Family_XIII_AD3011_group was also lower in the MEDIUM treatment compared to the CON group. Overall, supplementation with 60 mg/kg of rhLYZ improved growth performance, nutrient utilization efficiency, and serum immune function in broiler chickens while also influencing the composition of their intestinal microbiota. This suggests the potential of lysozyme as a replacement for antibiotic additives in feed.

Expression and Characterization of Laccase Lac1 from Coriolopsis trogii Strain Mafic-2001 in Pichia pastoris and Its Degradation of Lignin.

The laccase gene (Lac1) was cloned from Coriolopsis trogii strain Mafic-2001. Full-length sequence of Lac1 containing 11 exons and 10 introns is composed of 2140 nucleotides (nts). mRNA of Lac1 encoded for a protein of 517 aa. Nucleotide sequence of the laccase was optimized and expressed in Pichia pastoris X-33. SDS-PAGE analysis showed that the molecular weight of the purified recombinant laccase rLac1 was about 70 kDa. The optimum temperature and pH of rLac1 were 40 ℃ and 3.0, respectively. rLac1 showed high residual activity (90%) in the solutions after 1 h incubation at the pH ranging from 2.5 to 8.0. rLac1 maintained over 60% of laccase activity at the temperatures ranging from 20 to 60 °C, and kept higher than 50% of its activity at 40 °C for 2 h. The activity of rLac1 was promoted by Cu2+ and inhibited by Fe2+. Under optimal conditions, lignin degradation rates of rLac1 on the substrates of rice straw, corn stover, and palm kernel cake were 50.24%, 55.49%, and 24.43% (the lignin contents of substrates untreated with rLac1 were 100%), respectively. Treated with rLac1, the structures of agricultural residues (rice straw, corn stover, and palm kernel cake) were obviously loosened which was reflected by the analysis of scanning electron microscopy and Fourier transform infrared spectroscopy. Based on the specific activity of rLac1 on the degradation of lignin, rLac1 from Coriolopsis trogii strain Mafic-2001 has the potential for in-depth utilization of agricultural residues.

Genomic insights into antimicrobial potential and optimization of fermentation conditions of pig-derived Bacillus subtilis BS21.

Bacillus spp. have been widely used as probiotic supplements in animal feed as alternatives to antibiotics. In the present study, we screened a Bacillus subtilis strain named BS21 from pig feces. Antimicrobial activities, whole genome mining and UHPLC-MS/MS analysis were used to explore its antimicrobial mechanism. Strain BS21 showed Significant growth inhibition against a variety of animal pathogens, including Escherichia coli, Salmonella enterica Pullorum, Salmonella enterica Typhimurium, Citrobacter rodentium, Shigella flexneri and Staphylococcus aureus. Seven gene clusters involved in antimicrobial biosynthesis of secondary metabolites were encoded by strain BS21 genome, including four non-ribosomal peptides (bacillibactin, fengycin, surfactin and zwittermicin A), one ribosomal peptide (subtilosin A), one dipeptide (bacilysin) and one polyketide (bacillaene). Among them, production of surfactin, fengycin, bacillibactin, bacilysin and bacillaene was detected in the supernatant of B. subtilis strain BS21. To develop the potential application of BS21 in animal production, medium components and fermentation parameters optimization was carried out using response surface methodology (RSM). Production of antimicrobial secondary metabolites of strain BS21 was increased by 43.4%, and the best medium formula after optimization was corn flour 2%, soybean meal 1.7% and NaCl 0.5% with optimum culture parameters of initial pH 7.0, temperature 30°C, rotating speed at 220 rpm for 26 h. Our results suggested that strain BS21 has the potential for large-scale production and application as a potential source of probiotics and alternative to antibiotics for animal production.

Expression and characterization of α-L-arabinofuranosidase derived from Aspergillus awamori and its enzymatic degradation of corn byproducts with xylanase.

In this study, α-L-arabinofuranosidase (AF) from Aspergillus awamori was heterologously expressed in Pichia pastoris X33, with a 1-fold increase in AF activity after codon and vector optimization. AF remained stable at 60-65 °C and displayed a broad pH stability range of 2.5-8.0. It also demonstrated considerable resistance to pepsin and trypsin. Furthermore, compared with xylanase alone, AF with xylanase exhibited a marked synergistic effect in the degradation of expanded corn bran, corn bran, and corn distillers' dried grains with solubles, reducing sugars by 3.6-fold, 1.4-fold, and 6.5-fold, respectively, with the degree of synergy increasing to 4.61, 2.44, and 5.4, respectively, while in vitro dry matter digestibility values were 17.6%, 5.2%, and 8.8%, respectively. After enzymatic saccharification, corn byproducts were converted to prebiotic xylo-oligosaccharides and arabinoses, thereby demonstrating the favorable properties of AF in the degradation of corn biomass and its byproducts.

The Effects of Dietary Bacillus amyloliquefaciens TL106 Supplementation, as an Alternative to Antibiotics, on Growth Performance, Intestinal Immunity, Epithelial Barrier Integrity, and Intestinal Microbiota in Broilers.

A total of 240 1-day-old Arbor Acres male broilers were randomly divided into five dietary treatments (control feed (CON), supplemented with 75 mg/kg aureomycin (ANT), supplemented with 7.5 × 108 CFU/kg (Ba1) and 2.5 × 109 CFU/kg (Ba1), and 7.5 × 109 CFU/kg (Ba3) Bacillus amyloliquefaciens TL106, respectively) to investigate the probiotic effect of TL106 instead of antibiotics in broilers. On days 1−21, the average daily gain of broilers in the Ba groups was increased compared with the CON group (p < 0.05). In addition, the feed/gain ratio of broilers in the Ba groups was lower than that of broilers in the CON and ANT groups on days 22−42 and days 1−42 (p < 0.05). Compared with the CON group, dietary TL106 increased the digestibility of crude fiber and crude protein (p < 0.05), and the effect was similar to that of the ANT group. The levels of IL-1ß, IFN-γ, and IL-6 in serum, jejunum, and ileum of broilers fed TL106 were decreased compared with the control group (p < 0.05). The mRNA expression of tight junction proteins in broilers of ANT and Ba groups was higher than the control group (p < 0.05). After 21 days, villus height and the ratio of villus height to crypt depth of duodenum and jejunum of broilers fed TL106 were higher than the control group (p < 0.05). The concentrations of short-chain fatty acids such as lactate, acetate, propionate, and butyrate in cecal digesta of broilers dietary TL106 were higher than the control group (p < 0.05). The supplementation with TL106 altered the compositions and diversity of the cecal microbiota of broilers. Moreover, supplementation with TL106 improved the ratio of Firmicutes to Bacteroidetes and decreased the relative abundance of Proteobacteria on days 21 and 28, while the abundance of Peptostreptococcaceae, Ruminococcaceae and Lactobacillaceae was increased. On days 35 and 42, broilers fed TL106 had an increased total abundance of Firmicutes and Bacteroidetes and decreased abundances of Lactobacillaceae, while the abundance of Barnesiellaceae was increased. In conclusion, dietary supplementation with TL106 improved the broiler's growth performance, immune response capacity, gut health, modulated development, and composition of the gut microbiota in broilers. It is suggested that Bacillus amyloliquefaciens TL106 may be a suitable alternative to in-feed antibiotics to improve broiler health and performance.

Characterization of a novel GH10 xylanase with a carbohydrate binding module from Aspergillus sulphureus and its synergistic hydrolysis activity with cellulase.

A study was carried out to investigate the characterization of a novel Aspergillus sulphureus JCM01963 xylanase (AS-xyn10A) with a carbohydrate binding module (CBM) and its application in degrading alkali pretreated corncob, rapeseed meal and corn stover alone and in combination with a commercial cellulase. In this study, the 3D structure of AS-xyn10A, which contained a CBM at C-terminal. AS-xyn10A and its CBM-truncated variant (AS-xyn10A-dC) was codon-optimized and over-expressed in Komagaella phaffii X-33 (syn. Pichia pastoris) and characterized with optimal condition at 70 °C and pH 5.0, respectively. AS-xyn10A displayed high activity to xylan extracted from corn stover, corncob, and rapeseed meal. The concentration of hydrolyzed xylo-oligosaccharides (XOSs) reached 1592.26 µg/mL, 1149.92 µg/mL, and 621.86 µg/mL, respectively. Xylobiose was the main product (~70%) in the hydrolysis mixture. AS-xyn10A significantly synergized with cellulase to improve the hydrolysis efficiency of corn stover, corncob, and rapeseed meal to glucose. The degree of synergy (DS) was 1.32, 1.31, and 1.30, respectively. Simultaneously, XOSs hydrolyzed with AS-xyn10A and cellulase was improved by 46.48%, 66.13% and 141.45%, respectively. In addition, CBM variant decreased the yields of xylo-oligosaccharide and glucose in rapeseed meal degradation. This study provided a novel GH10 endo-xylanase, which has potential applications in hydrolysis of biomass.

High efficient degradation of glucan/glucomannan to cello-/mannan-oligosaccharide by endoglucanase via tetrasaccharide as intermediate.

Here, we report an efficient endoglucanase from Aureobasidium pullulans (termed ApCel5A) was expressed in Pichia pastoris. ApCel5A shows two different enzyme activities of endoglucanase (1270 U/mg) and mannanase (31.2 U/mg). Through engineering the signal peptide and fed-batch fermentation, the enzyme activity of endoglucanase was improved to 6.63-folds, totally. Its efficient synergism with Celluclast 1.5 L, excellent tolerance to low pH (2.5), cholate and protease suggests potential application in bioresources, food and feed industries. Site-directed mutagenesis experiments present that ApCel5A residues Glu245 and Glu358 are key catalytic sites, while Asp118, Asp122, Asp198 and Asp314 play an auxiliary role. More importantly, ApCel5A display high degradation efficiency of glucan and glucomannan substrates by using tetrasaccharide contained reducing end of glucose residue as an intermediate. This study elucidated the effective methods to improve an endoglucanase expression and detailed catalytic mechanism for degradation of various substrates, which provides a new insight for endoglucanase application.

Effects of Xylanase in Corn- or Wheat-Based Diets on Cecal Microbiota of Broilers.

Xylanase has been demonstrated to improve growth performance of broilers fed wheat- or corn-based diets due to its ability to degrade arabinoxylans (AX). However, content and structure of AX in corn and wheat are different, comparing effects of xylanase on cecal microbiota of broilers fed corn- or wheat-based diets could further elaborate the mechanism of the specificity of xylanase for different cereal grains. Thus, a total of 192 one-day-old broilers were randomly allotted into four dietary treatments, including wheat-soybean basal diet, wheat-soybean basal diet with 4,000U/kg xylanase, corn-soybean basal diet, and corn-soybean basal diet with 4,000U/kg xylanase to evaluate interactive effects of xylanase in corn- or wheat-based diets on broilers cecal microbiota during a 6-week production period. The results indicated that bacterial community clustering was mainly due to cereal grains rather than xylanase supplementation. Compared with broilers fed wheat-based diets, corn-based diets increased alpha-diversity and separated from wheat-based diets (p<0.05). Xylanase modulated the abundance of specific bacteria without changing overall microbial structure. In broilers fed wheat-based diets, xylanase increased the abundance of Lactobacillus, Bifidobacterium, and some butyrate-producing bacteria, and decreased the abundance of non-starch polysaccharides-degrading (NSP) bacteria, such as Ruminococcaceae and Bacteroidetes (p<0.05). In broilers fed corn-based diets, xylanase decreased the abundance of harmful bacteria (such as genus Faecalitalea and Escherichia-Shigella) and promoted the abundance of beneficial bacteria (such as Anaerofustis and Lachnospiraceae_UCG_010) in the cecum (p<0.05). Overall, xylanase supplementation to wheat- or corn-based diets improved broilers performance and cecal microbiota composition. Xylanase supplementation to wheat-based diets increased the abundance of butyrate-producing bacteria and decreased the abundance of NSP-degrading bacteria. Moreover, positive effects of xylanase on cecal microbiota of broilers fed corn-based diets were mostly related to the inhibition of potentially pathogenic bacteria, and xylanase supplementation to corn-based diets slightly affected the abundance of butyrate-producing bacteria and NSP-degrading bacterium, the difference might be related to lower content of AX in corn compared to wheat.

The enzymatic characters of heterologous expressed novel ß-1, 4-glucosidase originated from Aspergillus fresenii.

ß-1, 4-glucosidases generate glucose from cellobiose and oligosaccharides, enhancing the productivity in biorefinery and the bioconversion process as well as the nutritional value in food and feed. With the high-throughput sequencing technique, a novel ß-1, 4-glucosidase, named bgl T2, containing 861 amino acid residues, was found from Aspergillus fresenii. bgl T2 belongs to the glycosyl hydrolase (GH) family 3. The bgl T2 that expressed by Komagataella phaffii X33 presented the highest activity at 55 °C and pH 5.5. The half-lives of bgl T2 under 50 °C, 55 °C, 60 °C, and 65 °C were 9 min 36 s, 4 min 22 s, 117 s, and 68 s, respectively. The bgl T2 was stable between pH 3.0 to pH 8.0. The Michaelis constant (K m) and the theoretical maximum rate (V max) of bgl T2 were 0.0007 mol/L and 9 × 10-8 mol/L/s, respectively. In a 5 L fermentation vessel, the recombinant K.phaffii X33 could yield a ß-1, 4-glucosidase activity of 4.45 U/mL after 96 h methanol inducement. As an important member of cellulases, the novel bgl T2 might contribute to bioenergy, food processing, feed enrichment, and nutritional study, etc. This study also developed a path to obtain new enzymes depending on high-throughput sequencing technique.

Administration of Saccharomyces boulardii mafic-1701 improves feed conversion ratio, promotes antioxidant capacity, alleviates intestinal inflammation and modulates gut microbiota in weaned piglets.

BACKGROUND: Probiotics are used as a means to improve animal health and intestinal development. Saccharomyces boulardii is a well-known probiotic; however, few studies have examined the effects of S. boulardii on weaned piglet performance. Therefore, this 28-day study compared the effects of S. boulardii mafic-1701 and aureomycin in diets for weaned piglets on growth performance, antioxidant parameters, inflammation and intestinal microbiota. One hundred and eight piglets, weaned at 28 d of age (8.5 ± 1.1 kg), were randomly divided into the three dietary treatment groups with six pens and six piglets per pen (half male and half female). The dietary treatment groups were as follows: 1) basal diet (CON); 2) basal diet supplemented with 75 mg/kg aureomycin (ANT); 3) basal diet supplemented with 1 × 108 CFU/kg S. boulardii mafic-1701 (SB). RESULTS: Compared to CON group, SB group had higher feed efficiency (P < 0.05) in the last 14 d and lower diarrhea rate (P <  0.05) over the entire 28 d. Total superoxide dismutase in serum was markedly increased in SB group (P < 0.05). Moreover, compared with CON group, SB group decreased the levels of pro-inflammatory cytokines interleukin-6 (P <  0.01) and Tumor necrosis factor-α (P < 0.05) in jejunum. Supplementation of S. boulardii mafic-1701 increased the abundance of Ruminococcaceae_UCG_009 and Turicibacter (P < 0.05), whereas the abundance of unclassified_Clostridiaceae_4 was decreased (P < 0.05). Furthermore, S. boulardii mafic-1701 administration increased cecal concentration of microbial metabolites, isobutyrate and valerate (P < 0.05). CONCLUSIONS: The improvement in feed conversion ratio, reduction in diarrhea rate in weaned piglets provided diets supplemented with S. boulardii mafic-1701 may be associated with enhanced antioxidant activity, anti-inflammatory responses and improved intestinal microbial ecology.

High-level expression of an acidic thermostable xylanase in Pichia pastoris and its application in weaned piglets.

An acidic thermostable xylanase (AT-xynA) which was stable at low pH and high temperature was considered to have great potential in animal feed. For large-scale production, AT-xynA activity was enhanced about 1-fold in Pichia pastoris by constructing a double-copy expression strain in this study. Furthermore, impacts of different AT-xynA levels on growth performance, nutrient digestibility, short-chain fatty acids, and bacterial community in weaned piglets were determined. Compared with the control group, ADFI and ADG were higher for the pigs fed 4,000 or 6,000 U/kg AT-xynA (P < 0.05). AT-xynA supplementation also significantly increased the digestibility of OM, GE, and DM (P < 0.05). AT-xynA supplementation increased the concentrations of acetate in ileal (P < 0.01) and cecal digesta (P < 0.05). Isobutyrate (P < 0.05) and valerate (P < 0.05) concentrations in colonic digesta also significantly increased compared with the control group. AT-xynA supplementation increased the abundance of Lactobacillus in the ileal, cecal, and colonic digesta of weaned piglets (P < 0.05). AT-xynA alleviated anti-nutritional effects of nonstarch polysaccharides (NSP) by preventing the growth of Pateurella and Leptotrichia in the ileum (P < 0.05). AT-xynA increased the abundance of NSP-degrading bacteria, such as Ruminococcaceae, Prevotella in the cecum and colon (P < 0.05). In summary, AT-xynA addition could improve the growth performance of weaned piglets by altering gut microbiota.