Substrate Report for: Fumonisin-B1

Fumonisins (FBs) are toxic mycotoxins that commonly exist in food and feed. FBs can induce many aspects of toxicity, leading to adverse effects on human and animal health; therefore, investigating methods to reduce fumonisin contamination is necessary. In our study, we generated a recombinant fusion enzyme called FUMDI by linking the carboxylesterase gene (fumD) and the aminotransferase gene (fumI) by overlapping polymerase chain reaction (PCR). The fusion enzyme FUMDI was successfully, secretively expressed in the host Pichia pastoris (P. pastoris) GS115, and its expression was optimized. Our results demonstrated that the fusion enzyme FUMDI had high biodegradation activity of fumonisin B1 (FB1) and other common FBs, such as fumonisin B2 (FB2) and fumonisin B3 (FB3), and almost completely degraded 5 microg/mL of each toxin within 24 h. We also found that FUMDI enzyme and its reaction products had no negative effect on cell viability and did not induce cell apoptosis, oxidative stress, or endoplasmic reticulum (ER) stress in a human gastric epithelial cell line (GES-1). The results indicated that these FBs degradation products cannot have adverse effects in a cell model. In conclusion, a safe and efficient fumonisin-degrading enzyme was discovered, which could be a new a technical method for hazard control of FBs in the future.

Mycotoxin intoxication is in general an acknowledged and tackled issue in animals. However, in several parts of the world, mycotoxicoses in humans still remain a relevant issue. The efficacy of two mycotoxin detoxifying animal feed additives, an aflatoxin bentonite clay binder and a fumonisin esterase, was investigated in a human child gut model, i.e. the in vitro Simulator of the Human Intestinal Microbial Ecosystem (SHIME(a)). Additionally, the effect of the detoxifiers on gut microbiota was examined in the SHIME. After an initial two weeks of system stabilisation, aflatoxin B1 (AFB1) and fumonisin B1 (FB1) were added to the SHIME diet during one week. Next, the two detoxifiers and mycotoxins were added to the system for an additional week. The AFB1, FB1, hydrolysed FB1 (HFB1), partially hydrolysed FB1a and FB1b concentrations were determined in SHIME samples using a validated ultra-performance liquid chromatography-tandem mass spectrometry method. The short-chain fatty acid (SCFA) concentrations were determined by a validated gas chromatography-mass spectrometry method. Colonic bacterial communities were analysed using metabarcoding, targeting the hypervariable V1-V3 regions of the 16S rRNA genes. The AFB1 and FB1 concentrations significantly decreased after the addition of the detoxifiers. Likewise, the concentration of HFB1 significantly increased. Concentrations of SCFAs remained generally stable throughout the experiment. No major changes in bacterial composition occurred during the experiment. The results demonstrate the promising effect of these detoxifiers in reducing AFB1 and FB1 concentrations in the human intestinal environment, without compromising the gastrointestinal microbiota.

Enzymatic detoxification has become a promising approach for control of mycotoxins postharvest in grains through modification of chemical structures determining their toxicity. In the present study fumonisin esterase FumD (EC 3.1.1.87) (FUMzyme((a)); BIOMIN, Tulln, Austria), hydrolysing fumonisin (FB) mycotoxins by de-esterification, was utilised to develop an enzymatic reduction method in a maize kernel enzyme incubation mixture. Efficacy of the FumD FB reduction method in "low" and "high" FB contaminated home-grown maize was compared by monitoring FB(1) hydrolysis to the hydrolysed FB(1) (HFB(1)) product utilising a validated LC-MS/MS analytical method. The method was further evaluated in terms of enzyme activity and treatment duration by assessing enzyme kinetic parameters and the relative distribution of HFB(1) between maize kernels and the residual aqueous environment. FumD treatments resulted in significant reduction (<=80%) in "low" (<=1000 U/L, p < 0.05) and "high" (100 U/L, p < 0.05; <=1000 U/L, p < 0.0001) FB contaminated maize after 1 h respectively, with an approximate 1:1 micromol conversion ratio of FB(1) into the formation of HFB(1). Enzyme kinetic parameters indicated that, depending on the activity of FumD utilised, a significantly (p < 0.05) higher FB(1) conversion rate was noticed in "high" FB contaminated maize. The FumD FB reduction method in maize could find application in commercial maize-based practices as well as in communities utilising home-grown maize as a main dietary staple and known to be exposed above the tolerable daily intake levels.

Fumonisins (FBs) are toxic mycotoxins that commonly exist in food and feed. FBs can induce many aspects of toxicity, leading to adverse effects on human and animal health; therefore, investigating methods to reduce fumonisin contamination is necessary. In our study, we generated a recombinant fusion enzyme called FUMDI by linking the carboxylesterase gene (fumD) and the aminotransferase gene (fumI) by overlapping polymerase chain reaction (PCR). The fusion enzyme FUMDI was successfully, secretively expressed in the host Pichia pastoris (P. pastoris) GS115, and its expression was optimized. Our results demonstrated that the fusion enzyme FUMDI had high biodegradation activity of fumonisin B1 (FB1) and other common FBs, such as fumonisin B2 (FB2) and fumonisin B3 (FB3), and almost completely degraded 5 microg/mL of each toxin within 24 h. We also found that FUMDI enzyme and its reaction products had no negative effect on cell viability and did not induce cell apoptosis, oxidative stress, or endoplasmic reticulum (ER) stress in a human gastric epithelial cell line (GES-1). The results indicated that these FBs degradation products cannot have adverse effects in a cell model. In conclusion, a safe and efficient fumonisin-degrading enzyme was discovered, which could be a new a technical method for hazard control of FBs in the future.

Fumonisin B(1) (FB(1)) has the highest natural contamination rate among all fumonisin analogs and can inhibit food intake and weight gain of pigs. Under laboratory conditions, carboxylesterase FumDSB has a high FB(1) degradation rate and excellent pH and thermal stability. The present study sought to estimate the effects of FumDSB on growing pigs from the perspective of a brain-intestinal axis. Twenty-four growing pigs of similar weight were divided into Control, FB(1) (5mg FB(1)/kg feed), and FumDSB (5mg FB(1)/kg and 0.1% FumDSB in the feed) groups. After 42 days of feeding, hypothalamus and jejunum samples were collected for quantitative real-time fluorescence, western blotting, and immunohistochemistry. The results showed that FB(1) consumption can destruct the tissue structure of hypothalamus and jejunum, affect the expression and distribution of several appetite-related neuropeptides and inflammatory cytokines, thereby inducing neuroinflammatory responses and affecting food intake and weight gain. However, these anorexia effects and inflammatory responses are alleviated when FumDSB is added to the feed. In short, FumDSB can alleviate the inflammatory response induced by FB(1) in growing pigs.

Fumonisins have posed hazardous threat to human and animal health worldwide. Enzymatic degradation is a desirable detoxification approach but is severely hindered by serious shortage of detoxification enzymes. After mining enzymes by bioinformatics analysis, a novel carboxylesterase FumDSB from Sphingomonadales bacterium was expressed in Escherichia coli, and confirmed to catalyze fumonisin B1 to produce hydrolyzed fumonisin B1 by liquid chromatography mass spectrometry for the first time. FumDSB showed high sequence novelty, sharing only ~34% sequence identity with three reported fumonisin detoxification carboxylesterases. Besides, FumDSB displayed its high degrading activity at 30-40 degreesC within a broad pH range from 6.0 to 9.0, which is perfectly suitable to be used in animal physiological condition. It also exhibited excellent pH stability and moderate thermostability. This study provides a FB1 detoxification carboxylesterase which could be further used as a potential food and feed additive.

Fumonisin B(1) (FB(1)) is the most common food-borne mycotoxin produced by the Fusarium species, posing a potential threat to human and animal health. Pigs are more sensitive to FB(1) ingested from feed compared to other farmed livestock. Enzymatic degradation is an ideal detoxification method that has attracted much attention. This study aimed to explore the functional characteristics of the carboxylesterase FumDSB in growing pigs from the perspective of brain-gut regulation. A total of 24 growing pigs were divided into three groups. The control group was fed a basal diet, the FB(1) group was supplemented with FB(1) at 5 mg/kg feed, and the FumDSB group received added FumDSB based on the diet of the FB(1) group. After 35 days of animal trials, samples from the hypothalamus and jejunum were analyzed through HE staining, qRT-PCR and immunohistochemistry. The results demonstrated that the ingestion of FB(1) can reduce the feed intake and weight gain of growing pigs, indicating that several appetite-related brain-gut peptides (including NPY, PYY, ghrelin and obestatin, etc.) play important roles in the anorexia response induced by FB(1). After adding FumDSB as detoxifying enzymes, however, the anorexia effects of FB(1) were alleviated, and the expression and distribution of the corresponding brain-gut peptides exhibited a certain degree of regulation. In conclusion, the addition of FumDSB can reduce the anorexia effects of FB(1) by regulating several brain-gut peptides in both the hypothalamus and the jejunum of growing pigs.

Mycotoxin intoxication is in general an acknowledged and tackled issue in animals. However, in several parts of the world, mycotoxicoses in humans still remain a relevant issue. The efficacy of two mycotoxin detoxifying animal feed additives, an aflatoxin bentonite clay binder and a fumonisin esterase, was investigated in a human child gut model, i.e. the in vitro Simulator of the Human Intestinal Microbial Ecosystem (SHIME(a)). Additionally, the effect of the detoxifiers on gut microbiota was examined in the SHIME. After an initial two weeks of system stabilisation, aflatoxin B1 (AFB1) and fumonisin B1 (FB1) were added to the SHIME diet during one week. Next, the two detoxifiers and mycotoxins were added to the system for an additional week. The AFB1, FB1, hydrolysed FB1 (HFB1), partially hydrolysed FB1a and FB1b concentrations were determined in SHIME samples using a validated ultra-performance liquid chromatography-tandem mass spectrometry method. The short-chain fatty acid (SCFA) concentrations were determined by a validated gas chromatography-mass spectrometry method. Colonic bacterial communities were analysed using metabarcoding, targeting the hypervariable V1-V3 regions of the 16S rRNA genes. The AFB1 and FB1 concentrations significantly decreased after the addition of the detoxifiers. Likewise, the concentration of HFB1 significantly increased. Concentrations of SCFAs remained generally stable throughout the experiment. No major changes in bacterial composition occurred during the experiment. The results demonstrate the promising effect of these detoxifiers in reducing AFB1 and FB1 concentrations in the human intestinal environment, without compromising the gastrointestinal microbiota.

Enzymatic detoxification has become a promising approach for control of mycotoxins postharvest in grains through modification of chemical structures determining their toxicity. In the present study fumonisin esterase FumD (EC 3.1.1.87) (FUMzyme((a)); BIOMIN, Tulln, Austria), hydrolysing fumonisin (FB) mycotoxins by de-esterification, was utilised to develop an enzymatic reduction method in a maize kernel enzyme incubation mixture. Efficacy of the FumD FB reduction method in "low" and "high" FB contaminated home-grown maize was compared by monitoring FB(1) hydrolysis to the hydrolysed FB(1) (HFB(1)) product utilising a validated LC-MS/MS analytical method. The method was further evaluated in terms of enzyme activity and treatment duration by assessing enzyme kinetic parameters and the relative distribution of HFB(1) between maize kernels and the residual aqueous environment. FumD treatments resulted in significant reduction (<=80%) in "low" (<=1000 U/L, p < 0.05) and "high" (100 U/L, p < 0.05; <=1000 U/L, p < 0.0001) FB contaminated maize after 1 h respectively, with an approximate 1:1 micromol conversion ratio of FB(1) into the formation of HFB(1). Enzyme kinetic parameters indicated that, depending on the activity of FumD utilised, a significantly (p < 0.05) higher FB(1) conversion rate was noticed in "high" FB contaminated maize. The FumD FB reduction method in maize could find application in commercial maize-based practices as well as in communities utilising home-grown maize as a main dietary staple and known to be exposed above the tolerable daily intake levels.

The mycotoxin fumonisin B(1) (FB(1)) is a frequent contaminant of feed and causes various adverse health effects in domestic animals. Hence, effective strategies are needed to prevent the impact of fumonisins on livestock productivity. Here we evaluated the capability of the fumonisin carboxylesterase FumD to degrade FB(1) to its less toxic metabolite hydrolyzed FB(1) (HFB(1)) in the gastrointestinal tract of turkeys and pigs. First, an ex vivo pig model was used to examine the activity of FumD under digestive conditions. Within 2 h of incubation with FumD, FB(1) was completely degraded to HFB(1) in the duodenum and jejunum, respectively. To test the efficacy of the commercial application of FumD (FUMzyme) in vivo, female turkeys (n = 5) received either basal feed (CON), fumonisin-contaminated feed (15 mg/kg FB(1)+FB(2); FB) or fumonisin-contaminated feed supplemented with FUMzyme (15 U/kg; FB+FUMzyme) for 14 days ad libitum. Addition of FUMzyme resulted in significantly decreased levels of FB(1) in excreta, whereas HFB(1) concentrations were significantly increased. Compared to the FB group (0.24 +/- 0.02), the mean serum sphinganine-to-sphingosine (Sa/So) ratio was significantly reduced in the FB+FUMzyme group (0.19 +/- 0.02), thus resembling values of the CON group (0.16 +/- 0.02). Similarly, exposure of piglets (n = 10) to 2 mg/kg FB(1)+FB(2) for 42 days caused significantly elevated serum Sa/So ratios (0.39 +/- 0.15) compared to the CON group (0.14 +/- 0.01). Supplementation with FUMzyme (60 U/kg) resulted in gastrointestinal degradation of FB(1) and unaffected Sa/So ratios (0.16 +/- 0.02). Thus, the carboxylesterase FumD represents an effective strategy to detoxify FB(1) in the digestive tract of turkeys and pigs.

Detoxification of the mycotoxin fumonisin B(1) comprises at least two enzymatic steps, an initial deesterification reaction, followed by deamination of the resulting hydrolyzed fumonisin B(1). In this study, two genes that are responsible for degradation of fumonisin B(1) by the bacterium Sphingopyxis sp. MTA144 were identified within a gene cluster, assumed to be associated with fumonisin degradation. The first gene encodes a protein which shows similarity to carboxylesterases, type B. The second gene encodes a polypeptide homologous to aminotransferases, class III. The two genes were isolated and expressed heterologously. The effect of the recombinant enzymes on fumonisin B(1) and hydrolyzed fumonisin B(1) was determined. The recombinant carboxylesterase was shown to catalyze the deesterification of fumonisin B(1) to hydrolyzed fumonisin B(1). The heterologously expressed aminotransferase was shown to deaminate hydrolyzed fumonisin B(1) in the presence of pyruvate and pyridoxal phosphate. We propose that the consecutive action of these two enzymes is sufficient for fumonisin B(1) detoxification. The results of this work provide a basis for the development of an enzymatic detoxification process for fumonisin B(1) in food and animal feed, especially under oxygen limited conditions, as they are found, e.g. in ensilaged forage or in the intestinal tract of animals.