Removal of ochratoxin A by a carboxypeptidase and peptides present in liquid cultures of Bacillus subtilis CW14

2018 ◽  
Vol 11 (4) ◽  
pp. 559-570 ◽  
Author(s):  
H.N. Hu ◽  
X. Jia ◽  
Y.P. Wang ◽  
Z.H. Liang
Keyword(s):  
Bacillus Subtilis ◽  
Ochratoxin A ◽  
Culture Supernatant ◽  
Physical Adsorption ◽  
Proteinase K ◽  
Small Peptides ◽  
Liquid Cultures ◽  
Ochratoxin Α ◽  
Peptide Complexes

Ochratoxin A (OTA) is an important mycotoxin that contaminates a variety of agricultural products. The cell-free supernatant of Bacillus subtilis CW14 liquid cultures were reported previously to be capable of removing OTA efficiently. In this work, we examined several substances that are probably involved in this removal of OTA using in vitro experiments. The strain CW14 culture supernatant that was separated by ultrafiltration showed that the fractions collected at >10 kDa and <3 kDa had a significant ability to reduce OTA (84.9 and 74.8%, respectively) when incubated with 6 μg/ml OTA at 37 °C for 24 h. A putative metalloenzyme was responsible for the activity of the >10-kDa fraction, which was confirmed by the detrimental effects of heat treatments or addition of SDS, proteinase K, or EDTA. Subsequently, a carboxypeptidase (CP) gene that was likely related to the enzymatic conversion of OTA by the >10-kDa fraction was cloned from the B. subtilis CW14 genome, and over-expressed in Escherichia coli. The recombinant CP degraded 71.3% of OTA at 37 °C for 24 h, and ochratoxin α (OTα) was confirmed as a degradation product. From the <3-kDa fraction, some small peptides (1.7 kDa >Mw >0.7 kDa) were purified and decreased OTA by 45.0% under the same conditions, but no product was detected. These peptides were presumed to be capable of binding OTA due to their affinity with the OTA molecule, and the OTA-peptide complexes escaped from the extraction procedures for OTA quantification. These results indicated there was a probable synergistic effect that was involved in removal of OTA by the strain CW14 culture supernatant, which included enzymatic degradation by a CP and physical adsorption by some small peptides.

Degradation of zearalenone in swine feed and feed ingredients by Bacillus subtilis ANSB01G

2014 ◽  
Vol 7 (2) ◽  
pp. 143-151 ◽  
Author(s):  
Y.P. Lei ◽  
L.H. Zhao ◽  
Q.G. Ma ◽  
J.Y. Zhang ◽  
T. Zhou ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Culture Supernatant ◽  
Antimicrobial Activities ◽  
Proteinase K ◽  
Rrna Gene ◽  
Distillers Grains With Solubles ◽  
Dried Distillers Grains ◽  
Cell Extracts ◽  
Gene Sequence Analysis ◽  
And Staphylococcus Aureus

Zearalenone (ZEA) and its derivatives are mycotoxins that can cause oestrogenic effects and impair the reproductive physiology of animals, especially in female swine. Strategies to reduce or eliminate ZEA contamination in foods and feeds are very much needed. Among 36 bacterial isolates obtained from a variety of animal intestinal chyme, mouldy foods and feeds, soils, etc., five isolates demonstrated the ability to reduce more than 50% of ZEA in a liquid medium; ANSB01G isolate taken from normal broiler intestinal chyme reduced ZEA the most, by 88.65%. Using physiological, biochemical, and 16S rRNA gene sequence analysis methods, the ANSB01G isolate was identified as Bacillus subtilis. Under simulated intestinal tract conditions, the ANSB01G B. subtilis isolate degraded 84.58, 66.34 and 83.04% of ZEA in naturally contaminated maize, dried distillers’ grains with solubles, and swine complete feed, respectively. The highest degradation of ZEA occurred when the mycotoxin was co-incubated with the whole bacterial culture, resulting in a reduction of 88.65%, followed by 75.60% using culture supernatant, 26.11% using cell extracts, and 15.06% using viable cells. Treatments consisting of both heating and addition of proteinase K significantly reduced the rate of ZEA degradation in the culture supernatant, indicating that the ZEA degradation might be enzymatic. B. subtilis ANSB01G displayed resistance to simulated gastrointestinal tract environments and antimicrobial activities against several common bacterial pathogens, including Escherichia coli, Salmonella typhimurium and Staphylococcus aureus. These properties of B. subtilis ANSB01G suggest the possibility of its potential to effectively degrade ZEA in feed and to develop functional feed products for livestock industries.

scholarly journals Heterologous Expression and Characterization of A Novel Ochratoxin A Degrading Enzyme, N-acyl-L-amino Acid Amidohydrolase, from Alcaligenes faecalis

Toxins ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 518
Author(s):  
Honghai Zhang ◽  
Yunpeng Zhang ◽  
Tie Yin ◽  
Jing Wang ◽  
Xiaolin Zhang
Keyword(s):  
Amino Acid ◽  
Ochratoxin A ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Alcaligenes Faecalis ◽  
Degrading Enzyme ◽  
Sds Page ◽  
Ochratoxin Α ◽  
Acid Amidohydrolase

Ochratoxin A (OTA) is a well-known, natural contaminant in foods and feeds because of its toxic effects, such as nephrotoxicity in various animals. Recent studies have revealed that Alcaligenes faecalis could generate enzymes to efficiently degrade OTA to ochratoxin α (OTα) in vitro. In an effort to obtain the OTA degrading mechanism, we purified and identified a novel degrading enzyme, N-acyl-L-amino acid amidohydrolase (AfOTase), from A. faecalis DSM 16503 via mass spectrometry. The same gene of the enzyme was also encountered in other A. faecalis strains. AfOTase belongs to peptidase family M20 and contains metal ions at the active site. In this study, recombination AfOTase was expressed and characterized in Escherichia coli. The molecular mass of recombinant rAfOTase was approximately 47.0 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited a wide temperature range (30–70 °C) and pH adaptation (4.5–9.0) and the optimal temperature and pH were 50 °C and 6.5, respectively.

scholarly journals In-Vitro Application of a Qatari Burkholderia cepacia strain (QBC03) in the Biocontrol of Mycotoxigenic Fungi and in the Reduction of Ochratoxin A biosynthesis by Aspergillus carbonarius

Toxins ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 700 ◽  
Author(s):  
Randa Zeidan ◽  
Zahoor Ul-Hassan ◽  
Roda Al-Thani ◽  
Quirico Migheli ◽  
Samir Jaoua
Keyword(s):  
Antifungal Activity ◽  
Ochratoxin A ◽  
Burkholderia Cepacia ◽  
Culture Supernatant ◽  
Animal Health ◽  
Aspergillus Carbonarius ◽  
Penicillium Verrucosum ◽  
Wide Range ◽  
Mycotoxigenic Fungi

Mycotoxins are secondary metabolites produced by certain filamentous fungi, causing human and animal health issues upon the ingestion of contaminated food and feed. Among the safest approaches to the control of mycotoxigenic fungi and mycotoxin detoxification is the application of microbial biocontrol agents. Burkholderia cepacia is known for producing metabolites active against a broad number of pathogenic fungi. In this study, the antifungal potential of a Qatari strain of Burkholderia cepacia (QBC03) was explored. QBC03 exhibited antifungal activity against a wide range of mycotoxigenic, as well as phytopathogenic, fungal genera and species. The QBC03 culture supernatant significantly inhibited the growth of Aspergillus carbonarius, Fusarium culmorum and Penicillium verrucosum in PDA medium, as well as A. carbonarius and P. verrucosum biomass in PDB medium. The QBC03 culture supernatant was found to dramatically reduce the synthesis of ochratoxin A (OTA) by A. carbonarius, in addition to inducing mycelia malformation. The antifungal activity of QBC03’s culture extract was retained following thermal treatment at 100 °C for 30 min. The findings of the present study advocate that QBC03 is a suitable biocontrol agent against toxigenic fungi, due to the inhibitory activity of its thermostable metabolites.

Degradation of ochratoxin A by supernatant and ochratoxinase of Aspergillus niger W-35 isolated from cereals

2020 ◽  
Vol 13 (2) ◽  
pp. 287-298
Author(s):  
M. Zhao ◽  
X.Y. Wang ◽  
S.H. Xu ◽  
G.Q. Yuan ◽  
X.J. Shi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Food Safety ◽  
Aspergillus Niger ◽  
Ochratoxin A ◽  
Promising Strategy ◽  
Ochratoxin Α ◽  
Food And Feed ◽  
Commercial Feeds ◽  
Penicillium Spp

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus spp. and Penicillium spp. and poses a threat to food safety. Biodegradation may be a promising strategy for reducing the OTA contamination in the future. In this study, Aspergillus niger strain W-35 was isolated from cereals and studied for its ability to degrade OTA. Results showed that the supernatant of W-35 could degrade OTA both in vitro and in commercial feeds after incubation at 37 °C for 12 h by 78.0 and 37.0%, respectively. Ochratoxin α (OTα) was assayed as a degradation product by HPLC-FLD. Furthermore, an enzyme specific for OTA degradation (ochratoxinase, OTase) obtained from W-35 was successfully expressed in Escherichia coli BL21, and degraded OTA at a rate of 85.1% for 12 h. These results indicated that this OTA degradation is enzymatic and that the responsible enzyme is extracellular OTase. Reliable degradation of OTA has the potential for wide-ranging applications in the food and feed industries.

scholarly journals Bacillus subtilis RZ001 improves intestinal integrity and alleviates colitis by inhibiting the Notch signalling pathway and activating ATOH-1

2020 ◽  
Vol 78 (2) ◽  
Author(s):  
Yanru Li ◽  
Tengxun Zhang ◽  
Congcong Guo ◽  
Meng Geng ◽  
Sailun Gai ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Culture Supernatant ◽  
Inflammatory Diseases ◽  
Notch Signalling ◽  
The Body ◽  
Signalling Pathway ◽  
Subtilis Strain ◽  
Notch Signalling Pathway ◽  
Adult Mice

ABSTRACT Intestinal mucosal barriers help the body resist many intestinal inflammatory diseases, such as inflammatory bowel disease (IBD). In this study, we identified a novel bacterium promoting the repair of intestinal mucosa and investigated the potential mechanisms underlying its activity. Culture supernatant of Bacillus subtilis RZ001 upregulated the expression of mucin 2 (MUC2) and tight junction (TJ) proteins in HT-29 cells in vitro. Oral administration of B. subtilis RZ001 may have significantly reduced symptoms such as the dextran sulfate sodium (DSS)-induced decrease in body weight, shortening of colon length and overproduction of proinflammatory factors. The number of goblet cells and levels of MUC2 and TJ proteins were significantly increased in adult mice fed with B. subtilis RZ001. B. subtilis RZ001 cells upregulated the levels of MUC2 in the intestinal organoids. Furthermore, culture supernatant of B. subtilis RZ001 could suppress the Notch signalling pathway and activate the expression of atonal homolog 1 (Atoh1). The transcription factor Atoh1 is required for intestinal secretory cell differentiation and activates transcription of MUC2 via binding to E-boxes on the MUC2 promoter. Taken together, B. subtilis strain RZ001 has the potential for treating IBD. The present study is helpful to elucidate the mechanisms of B. subtilis action.

scholarly journals Strains of Aureobasidium pullulans Can Lower Ochratoxin A Contamination in Wine Grapes

2008 ◽  
Vol 98 (12) ◽  
pp. 1261-1270 ◽  
Author(s):  
D. V. de Felice ◽  
M. Solfrizzo ◽  
F. De Curtis ◽  
G. Lima ◽  
A. Visconti ◽  
...  
Keyword(s):  
Ochratoxin A ◽  
Biocontrol Agent ◽  
Degradation Products ◽  
Positive Influence ◽  
Biocontrol Agents ◽  
Amide Bond ◽  
Wine Grapes ◽  
Crop Species ◽  
Ochratoxin Α

Wine contamination with ochratoxin A (OTA) is due to the attack of wine grapes by ochratoxigenic Aspergillus carbonarius and Aspergillus spp. section Nigri. Four A. pullulans strains, AU14-3-1, AU18-3B, AU34-2, and LS30, are resistant to and actively degrade ochratoxin A in vitro. The less toxic ochratoxin α and the aminoacid L-β-phenylalanine were the major degradation products, deriving from the cleavage of the amide bond linking these two moieties of OTA. The same strains were studied further as biocontrol agents of A. carbonarius on wine grapes in laboratory experiments. Three of the four strains significantly prevented infections by A. carbonarius. Berries pretreated with the biocontrol agents and infected with A. carbonarius contained lower amounts of OTA as compared to the untreated infected control berries. Two of these strains were shown to degrade OTA to ochratoxin α in fresh grape must, but the mechanisms of the decrease of OTA accumulation in infected berries pretreated with the biocontrol agents remain to be elucidated. Assessment of one strain carried out in the vineyard during the growing season of 2006 showed that the tested strain was an effective biocontrol agent, reducing both severity of Aspergillus rots and OTA accumulation in wine grapes. To our knowledge this is the first report describing the positive influence of biocontrol agents on OTA accumulation in this crop species.

scholarly journals Metabolites of bovine-associated non-aureus staphylococci influence expression of Staphylococcus aureus agr-related genes in vitro

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Bruno Toledo-Silva ◽  
Fernando Nogueira de Souza ◽  
Sofie Piepers ◽  
Kristien Mertens ◽  
Freddy Haesebrouck ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Virulence Genes ◽  
Culture Supernatant ◽  
Bovine Milk ◽  
Microbial Interactions ◽  
Proteinase K ◽  
In Vitro Growth ◽  
Isolation And Identification ◽  
And Control

AbstractCommunications via quorum sensing (QS) between non-aureus staphylococci (NAS) and Staphylococcus (S.) aureus in the bovine mammary gland remains largely unexplored. We determined whether 34 S. chromogenes, 11 S. epidermidis, and 14 S. simulans isolates originating from bovine milk samples and teat apices were able to regulate the QS of S. aureus, and if so, how in vitro growth inhibition of S. aureus by NAS, or NAS metabolites, or NAS cells themselves play a role in this process. In co-culture with S. aureus we observed that these 3 NAS species in general downregulated the expression of rnaIII, the effector molecule of the QS system, but this effect was more pronounced in S. chromogenes and S. simulans isolates than in S. epidermidis isolates. In vitro growth inhibition of S. aureus by NAS resulted in a small underestimation of the downregulating effect of NAS on rnaIII expression of S. aureus. Additionally, the culture supernatant of these NAS isolates and supernatant treated with proteinase K expressed greater regulatory activity over S. aureus virulence genes rnaIII, hla, and spa than washed NAS cells suspended in sterile water. These microbial interactions may influence S. aureus virulence and pathogenesis within the host. Isolation and identification of NAS metabolites affecting the QS system of S. aureus might help to develop alternative strategies for treatment and control of S. aureus mastitis.

Structure elucidation and in vitro cytotoxicity of ochratoxin α amide, a new degradation product of ochratoxin A

2015 ◽  
Vol 31 (2) ◽  
pp. 83-90 ◽  
Author(s):  
Andrea Bittner ◽  
Benedikt Cramer ◽  
Henning Harrer ◽  
Hans-Ulrich Humpf
Keyword(s):  
Ochratoxin A ◽  
Degradation Product ◽  
Structure Elucidation ◽  
In Vitro Cytotoxicity ◽  
Ochratoxin Α
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