Qiugang Ma

Aflatoxin B1 Degradation by Stenotrophomonas Maltophilia and Other Microbes Selected Using Coumarin Medium

Aflatoxin B1 (AFB1) is one of the most harmful mycotoxins in animal production and food industry. A safe, effective and environmentally sound detoxification method is needed for controlling this toxin. In this study, 65 samples were screened from various sources with vast microbial populations using a newly developed medium containing coumarin as the sole carbon source. Twenty five single-colony bacterial isolates showing AFB1 reduction activity in a liquid culture medium were selected from the screen. Isolate 35-3, obtained from tapir feces and identified to be Stenotrophomonas maltophilia, reduced AFB1 by 82.5% after incubation in the liquid medium at 37 °C for 72 h. The culture supernatant of isolate 35-3 was able to degrade AFB1 effectively, whereas the viable cells and cell extracts were far less effective. Factors influencing AFB1 degradation by the culture supernatant were investigated. Activity was reduced to 60.8% and 63.5% at 20 °C and 30 °C, respectively, from 78.7% at 37 °C. The highest degradation rate was 84.8% at pH 8 and the lowest was only 14.3% at pH 4.0. Ions Mg2+ and Cu2+ were activators for AFB1 degradation, however ion Zn2+ was a strong inhibitor. Treatments with proteinase K, proteinase K plus SDS and heating significantly reduced or eradicated the degradation activity of the culture supernatant. The results indicated that the degradation of AFB1 by S. maltophilia 35-3 was enzymatic and could have a great potential in industrial applications.

Preparation, purification and characteristics of an aflatoxin degradation enzyme from Myxococcus fulvus ANSM068.

Aims:  To prepare, purify and characterize an extracellular enzyme from Myxococcus fulvus ANSM068, designated as myxobacteria aflatoxin degradation enzyme (MADE), which possesses degradation activity against aflatoxin B1 (AFB1), G1 (AFG1) and M1 (AFM1) in solution.

Development of breast muscle and meat quality in Arbor Acres broilers, Jingxing 100 crossbred chickens and Beijing fatty chickens

The objective of this experiment was to examine development of breast muscle and myofiber of M. pectoralis superficialis in three chicken breeds. Commercial broiler chickens (Arbor Acres broilers, AA), crossbred chickens (Jingxing 100 crossbred chickens, JXC) and Chinese native chickens (Beijing fatty chickens, BJF) were grown up to 98d to estimate myofiber density, and size (area, and diameter of myofibers) in P. superficialis. At 42, 56, 70, 84, and 98d of age, Pectoralis muscle was used to evaluate breast muscle weight, breast yield, and tenderness (shear force value). Results indicate that commercial broilers have higher breast weight, and higher shear force value than crossbred chickens and Chinese native chickens, that may be due to an increased myofiber diameter and area in Pectoralis muscle. It is suggested that histological properties of myofibers play an important role in increasing the shear force value of meat.

In Vitro Efficacy of Myxococcus fulvus ANSM068 to Biotransform Aflatoxin B1

Aflatoxin B1 (AFB1) is commonly found in cereals and animal feeds and causes a significant threat to the food industry and animal production. Several microbial isolates with high AFB1 transformation ability have been identified in our previous studies. The aim of this research was to characterize one of those isolates, Myxococcus fulvus ANSM068, and to explore its biotransformation mechanism. The bacterial isolate of M. fulvus ANSM068, isolated from deer feces, was able to transform AFB1 by 80.7% in liquid VY/2 medium after incubation at 30 °C for 72 h. The supernatant of the bacterial culture was more effective in transforming AFB1 as compared to the cells alone and the cell extract. The transformation activity was significantly reduced and eradicated after the culture supernatant was treated with proteinase K, proteinase K plus SDS and heating. Culture conditions, including nitrogen source, initial pH and incubation temperature were evaluated for an optimal AFB1 transformation. Liquid chromatography mass spectrometry (LCMS) analyses showed that AFB1 was transformed to a structurally different compound. Infrared analysis (IR) indicated that the lactone ring on the AFB1 molecule was modified by the culture supernatant. Chromatographies on DEAE-Ion exchange and Sephadex-Molecular sieve and SDS-PAGE electrophoresis were used to determine active components from the culture supernatant, indicating that enzyme(s) were responsible for the AFB1 biotransformation. This is the first report on AFB1 transformation by a strain of myxobacteria through enzymatic reaction(s).

Effects of Lipoic Acid on Immune Function, the Antioxidant Defense System, and Inflammation-Related Genes Expression of Broiler Chickens Fed Aflatoxin Contaminated Diets

This study was designed to evaluate the effect of low level of Aflatoxin B1 (AFB1) on oxidative stress, immune reaction and inflammation response and the possible ameliorating effects of dietary alpha-lipoic acid (α-LA) in broilers. Birds were randomly allocated into three groups and assigned to receive different diets: basal diet, diet containing 74 μg/kg AFB1, and 300 mg/kg α-LA supplementation in diet containing 74 μg/kg AFB1 for three weeks. The results showed that the serum levels of malondialdehyde, tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ) in the AFB1-treated group were significantly increased than the control group. In addition, the increased expressions of interleukin 6 (IL6), TNFα and IFNγ were observed in birds exposed to the AFB1-contaminated diet. These degenerative changes were inhibited by α-LA-supplement. The activities of total superoxide dismutase and glutathione peroxidase, the levels of humoral immunity, and the expressions of nuclear factor-κB p65 and heme oxygenase-1, however, were not affected by AFB1. The results suggest that α-LA alleviates AFB1 induced oxidative stress and immune changes and modulates the inflammatory response at least partly through changes in the expression of proinflammatory cytokines of spleen such as IL6 and TNFα in broiler chickens.

Dietary Lipoic Acid Influences Antioxidant Capability and Oxidative Status of Broilers

The effects of lipoic acid (LA) on the antioxidant status of broilers were investigated. Birds (1 day old) were randomly assigned to four groups and fed corn-soybean diets supplemented with 0, 100, 200, 300 mg/kg LA, respectively. The feeding program included a starter diet from 1 to 21 days of age and a grower diet from 22 to 42 days of age. Serum, liver and muscle samples were collected at 42 days of age. For antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity in serum, liver and breast muscle significantly increased in chickens fed with LA. The concentration of malondiadehyde (MDA), an indicator of lipid peroxidation, was significantly lower in serum, liver and leg muscle in birds that received LA than in the control group. Treatments with LA significantly increased glutathione (GSH) content in liver and increased α-tocopherol content in leg muscle as compared to the control. These results indicate that dietary supplementation with 300 mg/kg LA may enhance antioxidant capability and depress oxidative stress in broilers.

Protective effect of Bacillus subtilis ANSB060 on egg quality, biochemical and histopathological changes in layers exposed to aflatoxin B1

Bacillus subtilis ANSB060 from the fish gut had strong ability to detoxify aflatoxins. The aim of this research was to investigate the protective effect of B. subtilis ANSB060 (ANSB060) on egg quality and biochemical and histopathological changes of liver and kidney in laying hens when exposed to aflatoxin B(1). Treatments (C20, C40, and C60) were prepared by substituting corn contaminated by aflatoxin B(1) (AFB1) at different proportions (20, 40, and 60%) for normal corn in basic diets. The aflatoxin degradation enzyme (E) treatments (E20, E40, and E60) were mixed with the fermentation liquor of ANSB060 with C20, C40, and C60, respectively. The results showed that ANSB060 can improve the eggshell strength in E60 compared with C60 (P ≤ 0.05), and toxin reduced the content of total protein (in groups C20, C40, and C60) and albumin (in C20 and C40; P < 0.05) and heightened the activities of GPT (in C60) and GOT (in C40 and C60) in serum (P < 0.05). In the liver, AFB1 inhibited the activity of superoxide dismutase and glutathione peroxidase (C40 and C60; P < 0.05) and increased the content of malonaldehyde (in C40 and C60), which induced the damage in the liver and kidney as shown in the photomicrographs of hematoxylin and eosin-stained sections. The addition of ANSB060 can enhance the activity of antioxidant enzymes, and it recovered the protein synthesis in liver. Moreover, ANSB060 also ameliorated the damage of liver and kidney tissue and restored them to normal. Hence, ANSB060 had the ability to inhibit the damage induced by AFB1; it will have a great potential in industrial applications.

Molecular Mechanisms of Lipoic Acid Protection against Aflatoxin B1-Induced Liver Oxidative Damage and Inflammatory Responses in Broilers

Alpha-lipoic acid (α-LA) was evaluated in this study for its molecular mechanisms against liver oxidative damage and inflammatory responses induced by aflatoxin B1 (AFB1). Birds were randomly allocated into four groups with different diets for three weeks: a basal diet, a 300 mg/kg α-LA supplementation in a basal diet, a diet containing 74 μg/kg AFB1, and 300 mg/kg α-LA supplementation in a diet containing 74 μg/kg AFB1. In the AFB1 group, the expression of GSH-PX mRNA was down-regulated (p < 0.05), and the levels of lipid peroxide and nitric oxide were increased (p < 0.05) in the chicken livers compared to those of the control group. Additionally, the mRNA level of the pro-inflammatory factor interleukin-6 was up-regulated significantly (p < 0.05), the protein expressions of both the nuclear factor kappa B (NF-κB) p65 and the inducible nitric oxide synthase were enhanced significantly (p < 0.05) in the AFB1 group. All of these negative effects were inhibited by α-LA. These results indicate that α-LA may be effective in preventing hepatic oxidative stress, down-regulating the expression of hepatic pro-inflammatory cytokines, as well as inhibiting NF-κB expression.

Evaluation of the compositional and nutritional values of phytase transgenic corn to conventional corn in roosters

Three experiments were conducted to evaluate the compositional and nutritional values of corn grains [phytase transgenic corn (PTC) and isogenic conventional corn (CC)] and compare the efficacy of corn-based phytase and extraneous microbial phytase for enhancing the utilization of phytate phosphorus (P) in single corn or corn-soybean mixed meals (corn:soybean = 2.5:1, wt:wt) fed to roosters. Following a 48-h fasting period, 16 roosters were given 50 g of each sample via crop intubation and excreta were collected for 48 h. Nitrogen-free and phosphorus-free diets were used to evaluate endogenous amino acid and endogenous P losses, respectively. Chemical composition was not different between PTC and CC, whereas the phytase content for PTC was greater than CC (8,047 vs. 37 FTU/kg of corn, DM basis; P < 0.001). No difference was observed in the TME and true amino acid availability values between the PTC and CC in roosters. The true P utilization for PTC was greater than CC (37.92 vs. 55.85%; P < 0.001), and CC and PTC contained 0.13 and 0.19% available P (AP, DM basis; P < 0.001), respectively. There was no difference in P utilization (72.76 vs. 70.23%; P > 0.05) between roosters fed PTC and extraneous microbial phytase in equivalent FTU/kg of diets. The results of this study indicated that the chemical composition, TME, and true amino acid availability in PTC are essentially equivalent to that in CC, and the true P utilization for roosters is higher in PTC than in CC. Corn expressing phytase is as efficacious as equivalent microbial phytase when supplemented in corn-soybean diets for chickens.

Ameliorating Effects of Bacillus subtilis ANSB060 on Growth Performance, Antioxidant Functions, and Aflatoxin Residues in Ducks Fed Diets Contaminated with Aflatoxins

Bacillus subtilis ANSB060 isolated from fish gut is very effective in detoxifying aflatoxins in feed and feed ingredients. The purpose of this research was to investigate the effects of B. subtilis ANSB060 on growth performance, body antioxidant functions, and aflatoxin residues in ducks fed moldy maize naturally contaminated with aflatoxins. A total of 1500 18-d-old male Cherry Valley ducks with similar body weight were randomly assigned to five treatments with six replicates of 50 ducks per repeat. The experiment design consisted of five dietary treatments labeled as C0 (basal diet containing 60% normal maize), M0 (basal diet containing 60% moldy maize contaminated with aflatoxins substituted for normal maize), M500, M1000, and M2000 (M0 +500, 1000 or 2000 g/t aflatoxin biodegradation preparation mainly consisted of B. subtilis ANSB060). The results showed that ducks fed 22.44 ± 2.46 μg/kg of AFB1 (M0) exhibited a decreasing tendency in average daily gain (ADG) and total superoxide dismutase (T-SOD) activity in serum, and T-SOD and glutathione peroxidase (GSH-Px) activities in the liver significantly decreased along with the appearance of AFB1 and AFM1 compared with those in Group C0. The supplementation of B. subtilis ANSB060 into aflatoxin-contaminated diets increased the ADG of ducks (p > 0.05), significantly improved antioxidant enzyme activities, and reduced aflatoxin accumulation in duck liver. In conclusion, Bacillus subtilis ANSB060 in diets showed an ameliorating effect to duck aflatoxicosis and may be a promising feed additive.