Maurice Tibiru Apaliya

The Possible Mechanisms Involved in Degradation of Patulin by Pichia caribbica

In this work, we examined the mechanisms involved in the degradation of patulin by Pichia caribbica. Our results indicate that cell-free filtrate of P. caribbica reduced patutlin content. The heat-killed cells could not degrade patulin. However, the live cells significantly reduced the concentration of the patulin. In furtherance to this, it was observed that patulin was not detected in the broken yeast cells and cell wall. The addition of cycloheximide to the P. caribbica cells decreased the capacity of degradation of patulin. Proteomics analyses revealed that patulin treatment resulted in an upregulated protein which was involved in metabolism and stress response processes. Our results suggested that the mechanism of degradation of patulin by P. caribbica was not absorption; the presence of patulin can induce P. caribbica to produce associated intracellular and extracellular enzymes, both of which have the ability to degrade patulin. The result provides a new possible method that used the enzymes produced by yeast to detoxify patulin in food and feed.

Biodegradation of zearalenone by Saccharomyces cerevisiae: Possible involvement of ZEN responsive proteins of the yeast.

UNLABELLED The mycotoxin zearalenone, also known as F-2 mycotoxin or RAL is a potent estrogenic metabolite produced by some Gibberella and Fusarium species. It is a common contaminant of cereal crops, livestock and poultry products. However, detoxification of zearalenone (ZEN) remains a challenge. Recently, biological approach for ZEN detoxification is being explored. In this study, we investigated the biodegradation of ZEN by using Saccharomyces cerevisiae and the possible mechanisms involved. The findings revealed that, after 48h of incubation of S. cerevisiae in combination with ZEN, the ZEN was completely degraded by S. cerevisiae. On the contrary, heat-killed cells and cell-free culture filtrates of S. cerevisiae could not degrade ZEN. Furthermore, addition of cycloheximide to S. cerevisiae combined with ZEN at time 0h prevented ZEN degradation, while addition of cycloheximide at 12h significantly slowed down degradation. The results also indicated cellular proteomics of S. cerevisiae. Several differential proteins were identified, most of which were related to basic metabolism. BIOLOGICAL SIGNIFICANCE The findings revealed that, after 48h of incubating ZEN together with S. cerevisiae, ZEN was completely degraded by S. cerevisiae. The mechanisms involved in the degradation of ZEN by S. cerevisiae may be the production of associated intracellular and extracellular enzymes, which have the ability to degrade ZEN. In addition, there were some functional proteins produced by S. cerevisiae, indicating that the basic metabolism of S. cerevisiae was improved when ZEN was added. This novel discovery by the authors, will greatly contribute to the field of biodegradation of mycotoxin by antagonists. The authors also believed this innovation will open the grounds for further research and improvement of S. cerevisiae in the field of biodegradation.

Biocontrol agents increase the specific rate of patulin production by Penicillium expansum but decrease the disease and total patulin contamination of apples

Synthetic fungicides are commonly employed for the control of postharvest diseases of fruits. However, due to health concerns about the use of these chemicals, alternative control methods including biocontrol based on antagonistic yeasts are gaining in popularity. In this study, we investigated the effects of two biocontrol yeasts, Rhodotorula mucilaginosa strain 3617 and Rhodotorula kratochvilovae strain LS11, on blue mold and patulin (PAT) contamination caused by Penicillium expansum strains PY and FS7 in artificially inoculated Fuji apples stored at 20°C for 9 days. To correlate the development of the P. expansum strains in yeast-treated and untreated apples with PAT production, we quantified their biomass in the infected fruits using a recently published quantitative real-time polymerase chain reaction method based on specific primers for patF, a gene from P. expansum that is involved in PAT biosynthesis. Both yeasts significantly reduced the disease incidence caused by the two strains of P. expansum up to 5–7 days of incubation, and lowered their biomass and the progression of symptoms up to 9 days. Interestingly, both yeasts strains increased the rate of PAT production (expressed as ng patulin/μg fungal DNA) by the two pathogenic strains. Nevertheless, both biocontrol agents reduced the total PAT contamination, especially in the case of P. expansum strain FS7, the higher PAT producer of the two tested P. expansum strains. Comparing between the yeast strains, R. kratochvilovae LS11 was more effective than R. mucilaginosa 3617 for the control of P. expansum.

Impact of extraction parameters and their optimization on the nutraceuticals and antioxidant properties of aqueous extract mulberry leaf

ABSTRACT An investigation into the efficient use of water as a solvent and the influence of extraction temperature, extraction time, water to leaf powder ratio, particle size, and extraction cycle on the nutraceutical and antioxidant profile of aqueous mulberry leaf extract were conducted using a single-factor experiment approach. All the assessed extracting parameters showed a significant effect on the nutraceutical compounds and antioxidant properties. The optimum extraction conditions were as follows: extraction temperature of 70°C, extraction time of 40 min, water to leaf powder ratio of 40:1 ml/g, particle size of 25 µm, and two extraction cycles. Based on these optimal conditions, chlorogenic acid (62.10 mg/g), caffeic acid (32.21 mg/g), kaempferol-7-O-glucoside (19.30 mg/g), quercetin-3-rutinose (15.69 mg/g), quercetin-3-O-glucoside (32.38 mg/g), kaempferol-3-(6-rhamnosylglucoside) (42.52 mg/g), quercetin-3-(6-malonylglucoside) (65.19 mg/g), kaempferol-3-glucoside (66.27 mg/g), kaempferol-3-(6-malonylglucoside) (50.18 mg/g), 1-deoxynojirimycin (15.58 mg/g), and gamma-aminobutyric acid (5.05 mg/g) were obtained. The optimal aqueous extract had high antioxidant properties of 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (39.98 mM/g), cupric ion reducing capacity (58.93 mM/g), 1,1-diphenyl-2-picrylhydrazyl (101.33 mM/g), and ferric reducing antioxidant power capacity (233.77 mM/g) of dried mulberry leaf extract.

The mechanisms involved in ochratoxin A elimination by Yarrowia lipolytica Y-2: The mechanisms involved in ochratoxin A elimination

Biological control of mycotoxin in cereals, fruits and vegetables have emerged as a promising method. In a previous study, Yarrowia lipolytica Y‐2 isolated by our research team showed biocontrol effect on the post‐harvest decay of grapes and ochratoxin A (OTA) elimination in polytoma medium. The aim of this study was to elucidate the possible mechanisms of OTA elimination by Y. lipolytica Y‐2. The results indicated that OTA elimination by Y. lipolytica Y‐2 was attributed to the degradation action of intracellular enzymes but not extracellular enzymes. A degradation product was identified as ochratoxin alpha (OTα) by liquid chromatography‐tandem mass spectrometry. The intracellular enzymes precipitated with 65% saturation of ammonium sulphate degrade OTA the most quickly and 97.2% OTA was degraded within 4 h. Analysis of this fraction showed that two proteins of carboxypeptidase were expressed in Y. lipolytica Y‐2 but not in Y. lipolytica Polh without the ability to degrade OTA. The results of the protein identification combined with product identification indicated that OTA was degraded to OTα by Y. lipolytica Y‐2 through the hydrolysis activity of carboxypeptidases. Additionally, many proteins of Y. lipolytica Y‐2 involved in stress response and reactive O₂ species elimination also played essential role in OTA degradation.

Screening of Deoxynivalenol Producing Strains and Elucidation of Possible Toxigenic Molecular Mechanism

In this study, seven strains of Fusarium graminearum were isolated from wheat, of which six were identified to produce deoxynivalenol and the production of deoxynivalenol was assessed. F. graminearum strain Fg1 was noted to produce 1.0 μg/g deoxynivalenol during the incubation period in the Czapek yeast broth, while none was detected in F. graminearum strain Fg2. Hence, the differences in proteomes and transcriptomes of Fg1 and Fg2 were compared to analyze the mechanism underlying deoxynivalenol production. Among the 66 significantly differentially expressed proteins in Fg1, 39 and 27 were more or less abundant expressed. Functional analysis suggested that the enzymes involved in the methylerythritol 4-phosphate and mevalonate pathways, which provide a substrate for biosynthesis of farnesyl pyrophosphate, a precursor of DON, were activated in Fg1. The transcriptomics data demonstrated that the expression level of a majority of genes, including trichothecene biosynthetic genes, protein kinases, and transcription factors, involved in trichothecene biosynthesis was higher in Fg1 than in Fg2. The results also revealed differential expression profiles of deoxynivalenol biosynthesis genes in strains Fg1 and Fg2, which emphasized their deoxynivalenol producing ability and the underlying mechanism.

Proteomics profile of Hanseniaspora uvarum enhanced with trehalose involved in the biocontrol efficacy of grape berry

This present study tested the extent to which 2% w/v trehalose enhanced the proteins expression profile of Hanseniaspora uvarum Y3. Furthermore, it explored the relative gene expression of stilbene synthase (StSy), one of the vital defense-related genes found in the skin of grapes. The proteomics profile revealed that 29 proteins were differentially expressed out of which 26 were significantly up-regulated and 3 were download-regulated. The pathogenesis related (PR) and other protein spots were visible at 97.4 kDa and 14.4 kDa. Peroxiredoxin TSA1 and superoxide dismutase were the main proteins involved in defense response and both proteins were significantly up-regulated. The carbohydrate and energy metabolism proteins were also significantly up-regulated. The results revealed that the treatments were associated with substantial increase in peroxidase activity compared to the control. StSy relative gene expression level was observed to increase by 2.5-fold in grapes treated with the pre-enhanced H. uvarum compared to the control.

Effect of pulsed light treatment on the phytochemical, volatile, and sensorial attributes of lactic-acid-fermented mulberry juice

ABSTRACT Lactic-acid-fermented mulberry juice (LFMJ) was subjected to pulsed light (PL) treatment at exposure time of 2, 4, and 8 s at high insensitive pulses of 14.0 J/cm2. The effect of PL treatment on the microbial inactivation, physicochemical, phytochemical, volatile, and sensory characteristics of LFMJ was evaluated. It was found that the PL was able to reduce the microbial load to acceptable levels (1.02 ± 0.04 log10 cfu/mL) with no significant impact on the physicochemical properties of LFMJ. It was also observed that the PL treatment caused a slight decrease in anthocyanin concentration at 8 s exposure time. The color difference (∆E) of the juice treated for 2 and 4 s fell below the slightly noticeable range 0.5<ΔE<1.5 while ∆E values for the 8 s (0.55 ± 0.02) and the thermal (0.50 ± 0.02) treated samples were slightly noticeable. The volatile profile and odor activity values were positively affected by increasing the exposure time. The results depict that, under the present experimental conditions, the application of the PL resulted in a fermented juice with superior quality attributes as compared to the thermal treated juice.

The Response of Rhodotorula mucilaginosa to Patulin Based on Lysine Crotonylation

Patulin (PAT) is a mycotoxin produced by some Penicillium, Aspergillus, and Byssochlamys species. Rhodotorula mucilaginosa is able to degrade PAT in vivo as well as in vitro, up till date, the process and molecular mechanism(s) involved patulin degradation still remains unknown. Protein lysine crotonylation (Kcr) plays an important role in regulating chromatin dynamics, gene expression, and metabolic pathways in mammals and eukaryotes. Investigation of the Kcr changes accompanying degradation of patulin in R. mucilaginosa were observed to investigate the mechanisms of patulin inhibition. Tandem mass tag (TMT) labeling and Kcro affinity enrichment, followed by high-resolution LC-MS/MS analysis, were used to perform quantitative lysine crotonylome analysis on R. mucilaginosa. Consequently, 1691 lysine crotonylation sites in 629 protein groups were identified, among which we quantified 1457 sites in 562 proteins. Among the quantified proteins, 79 and 46 crotonylated proteins were up-regulated and down-regulated, respectively. The differentially up expressed modified proteins were mainly involved in tricarboxylic acid cycle and gluconeogenic pathway. The differentially down expressed Kcr proteins were mainly classified to ribosome and carbohydrate transport and metabolism. Bioinformatic analyses were performed to annotate the quantifiable lysine crotonylated targets. Moreover, interaction networks and high confidence domain architectures of crotonylated proteins were investigated with the aid of bioinformatic tools, and these results showed that there was an increase in the number of yeasts with crotonylated proteins. The results also provided information on the various roles of crotonylation, which are involved in PAT degradation.

Integration of transcriptome and proteome data reveals ochratoxin A biosynthesis regulated by pH in Penicillium citrinum

Ochratoxin A (OTA) has been found in a wide range of commodities and is highly toxic to both humans and animals. Therefore, a good understanding of the mechanisms of OTA production by fungi will contribute to the development of eco-friendly methods to mitigate this toxin. In this study, the results showed that Penicillium citrinum X9-4, which was isolated from infected grapes in our laboratory, produced the highest amount of OTA at pH 5 in culture media, and toxin production was restrained under an acidic environment (pH 3). Then, differentially expressed proteins of P. citrinum X9-4 cultured under these two conditions were analyzed by proteomic technology. Additionally, through the analysis of the transcriptome data of P. citrinum cultured at pH 3 and 5, the differentially expressed genes have been found to be involved in many metabolic pathways including amino acid transport and metabolism, transport and metabolism of carbohydrates, inorganic ion transport and metabolism, biosynthesis of secondary metabolites, and energy and supply metabolism, which are likely to be involved in the regulation of OTA biosynthesis. It was also revealed that the expression levels of some OTA synthesis-related enzymes, such as acetyltransferase, acyl coenzyme A oxidase, alcohol oxidase, cytochrome P450, acetyl xylan esterase, and turn ketol enzyme, and genes for the regulation of toxin synthesis pathways were reduced under acidic culture conditions.