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%.

Sorption of phenols and flavonoids on activated charcoal improves protein metabolism, antioxidant status, immunity, and intestinal morphology in broilers.

Previous studies have revealed that activated charcoal sorption of Chinese herbal extracts is more effective than activated charcoal. The present study was designed to investigate whether phenols and flavonoids have an effect on nutrient metabolism, antioxidant activity, immunity, and intestinal morphology in broilers. Seven diets [basal diet (CON); CON supplemented with 450 mg/kg of activated charcoal (AC); CON supplemented with 250, 500, 750, 1,000, or 7,500 mg/kg of phenolic acids and flavonoids (PF) to AC (PFAC)]. PFAC was the complex of AC sorption of PF in the ratio of 9:1. These dietary treatments for broilers lasted for 42 days. Results showed that at d 21, all doses of PFAC altered serum levels of total protein, albumin, and creatinine compared to AC (p < 0.05). Both PFAC and AC altered HDL-, LDL-, and VLDL-cholesterol levels compared to CON (p < 0.05). PFAC at 500 mg/kg (450 mg/kg AC+ 50 mg/kg phenolic acids and flavonoids) increased serum IgA and IgM (p < 0.05), but AC at 450 mg/kg did not, compared to CON. At d 42, breast and thigh muscles of PFAC-treated broilers had higher free radical scavenging activities compared to CON (p < 0.05), but AC had no such effect. PFAC at 500 mg/kg increased villus height in the duodenum, jejunum, and ileum compared to CON (p < 0.05), but AC had no such response. PFAC at 500 mg/kg effectively improved protein and lipid metabolism, antioxidant status, and intestinal morphology, but AC had no such effect at a similar dose. Excessive PFAC (7,500 mg/kg) showed no significant side effects on broiler growth, liver damage, or hematology. These results suggest that phenols and flavonoids, in cooperation with activated charcoal, provide the majority of the functions of the herbal extract from multiple Chinese medicinal herbs.

Cooperative Interaction of Phenolic Acids and Flavonoids Contained in Activated Charcoal with Herb Extracts, Involving Cholesterol, Bile Acid, and FXR/PXR Activation in Broilers Fed with Mycotoxin-Containing Diets.

The charcoal-herb extract complex (CHC) is a product of activated charcoal sorption of herb extracts that contain phenolic acids and flavonoids. The effective dose of CHC to promote animal growth is about one tenth of effective dosage of activated charcoal. The purpose of this study was to evaluate potential cooperative interactions between activated charcoal and herb extracts. Two feeding experiments were conducted. In Experiment 1, a responsive dose of CHC to broiler growth was determined to be 250 mg/kg of the diet. In Experiment 2, CHC increased growth performance and improved meat quality, but decreased indices of oxidative stress and inflammation as compared with similar doses of activated charcoal or herb extracts. CHC also increased concentrations of serum cholesterol, bile acid in the gallbladder, and bile acid in feces. The herb extracts present in CHC were largely represented by phenolic acids (PAs, caffeic acid, and vanillin) and flavonoids (FVs, daidzein, and quercetin-D-glucoside) in the detoxification activity of CHC in a mouse rescue test when the mice were gavaged with T-2 mycotoxin. PAs and FVs significantly increased the expression of CYP7A1, PXR, CYP3A37, Slco1B3, and Bsep in chicken primary hepatocytes. In conclusion, CHC integrated the cooperative interactions of activated charcoal and herb extracts via the FXR/RXR-PXR pathway to detoxify mycotoxins.

Beneficial Alteration in Growth Performance, Immune Status, and Intestinal Microbiota by Supplementation of Activated Charcoal-Herb Extractum Complex in Broilers.

This study aimed to examine the effects of activated charcoal-herb extractum complex (CHC) on the growth performance of broilers, inflammatory status, microbiota, and their relationships. A total of 864 1-day-old Arbor Acres male broilers (41.83 ± 0.64 g) were distributed to eight dietary treatments with six replicates (18 birds per replicate), which were a corn-soybean meal-based diet (NCON); basal diets supplemented with 250, 500, 750, or 1,000 mg/kg CHC, and three positive controls; basal diets supplemented with 200 mg/kg antibacterial peptide (AMP), 200 mg/kg calsporin (Probio) or 500 mg/kg montmorillonite. The study period was 42 days including the starter (day 0-21) and grower (day 22-42) phases. Compared with the NCON group, CHC supplementation (optimal dose of 500 mg/kg) increased (p < 0.05) growth performance and tended to increase feed conversion rate in broilers. CHC (optimal dose of 500 mg/kg) decreased the level of the interleukin-1ß (IL-1ß) and interferon-γ (IFN-γ) in serum and improved the levels of immunoglobulins A (IgA) and immunoglobulins A (IgM) in serum, and secretory immunoglobulin A (SIgA) in the mucosa of duodenum and jejunum (p < 0.05). In the ileum, CHC supplementation decreased community abundance represented by lower Sobs, Chao 1, Ace, and Shannon compared with NCON (p < 0.05). At the phylum level, CHC supplementation increased the abundance of Firmicutes, while decreasing the abundance of Bacteroidetes in ileum and cecum (p < 0.05). At the genus level, compared with the NCON group, CHC markedly reduced (p < 0.05) the abundances of pathogenic bacteria Alistipes in the ileum, which were negatively associated with the levels of SIgA and IL-1ß in ileum mucosa. In conclusion, CHC had beneficial effects on growth performance, immune status, and intestinal microbiota composition. CHC had dual functions of absorption like clays and antibacterial like antibacterial peptides.