A novel ß-fructofuranosidase produced by Penicillium citreonigrum URM 4459: purification and biochemical features.

Fructooligosaccharides (FOS) are prebiotics of interest to the food industry. These compounds can be produced through the transfructosylation reaction by the enzyme fructofuranosidase. This enzyme is widely produced by fungi in a medium rich in sugar. Therefore, in this work, the main objectives were production, purification, biochemical characterization of a novel fructofuranosidase enzyme by Penicillium citreonigrum URM 4459 and synthesize and evaluate the antibacterial potential of fructooligosaccharides. With respect to sucrose hydrolysis, the optimal pH was 5.5, the apparent Km for purified FFase was 3.8 mM, the molecular mass was 43.0 kDa, estimated by gel filtration on Superdex increase G75 controlled by AKTA Avant 25 and confirmed by 10% SDS-PAGE under denaturing condition. Also, the isoelectric point was 4.9. The fractions obtained with enzymatic activities, both stable at acidic pH and high temperatures, as well as being able to produce FOS. Regarding antibacterial activity, the FOS produced in this study showed better results than commercial FOS and other carbon sources. Thus, this work presents relevant data for the use of P. citreonigum to produce fructofuranosidase and consequently FOS and can be used in the food and pharmaceutical industry.

Whole Genome Analysis Revealed the Genes Responsible for Citreoviridin Biosynthesis in Penicillium citreonigrum.

Citreoviridin (CTV) is a mycotoxin that is produced by Aspergillus terreus, Eupenicillium ochrosalmoneum and Penicillium citreonigrum, and CTV has been detected in a wide range of cereal grains throughout the world. Furthermore, it is especially a serious problem in regions where rice is consumed as a staple food. Moreover, CTV is a well-known yellow rice toxin, and outbreaks of beriberi have occurred due to consumption of rice that is contaminated by CTV even in the recent years. Although CTV biosynthetic genes of A. terreus have been described, those of P. citreonigrum remain unclear, which is concerning since P. citreonigrum is the main cause of CTV contamination in rice. In the present study, we determined the draft genome of the P. citreonigrum strain IMI92228 and revealed the presence of all four genes that form a gene cluster and that are homologous to the CTV biosynthesis genes of A. terreus. The expression of these four homologous genes were highly correlated with CTV production, suggesting that they may play an important role in CTV biosynthesis in P. citreonigrum. We concluded that the gene cluster is a CTV biosynthesis cluster of P. citreonigrum. The findings will contribute to the understanding of the biosynthetic pathway of CTV and will ultimately lead to improvements in the CTV management of agricultural products.

Process development for the production of prebiotic fructo-oligosaccharides by penicillium citreonigrum.

A new isolated P. citreonigrum URM 4459 was selected to produce fructooligosaccharides (FOS) in an efficient, economical and fast one-step fermentation. Optimal culture conditions were stablished by experimental design. Experiments run in bioreactor resulted in a high yield, content, productivity and purity of FOS (0.65 ±â€¯0.06 gFOS/ginitial Sucrose, 126.3 ±â€¯0.1 g/L, 2.28 ±â€¯0.08 g/L.h and 61 ±â€¯0%). The FOS mixture was purified up to 92% (w/w) with an activated charcoal column. FOS produced were able to promote lactobacilli and bifidobacteria growth. Higher bacteria cell density was obtained for microbial-FOS mixtures than commercial Raftilose® P95. Some strains grew even faster in the FOS mixture produced than in all other carbon sources. FOS were resistant to the simulated gastrointestinal conditions. A high amount of a reducing trisaccharide was identified in the FOS produced mixture, possibly neokestose, which may explain the great prebiotic potential of the FOS.

Selective cytotoxic eremophilane-type sesquiterpenes from Penicillium citreonigrum.

One new eremophilane-type sesquiterpene (1, citreopenin) was isolated from Penicillium citreonigrum (HQ738282), and the structure was elucidated by a combination of spectroscopic data interpretation and single-crystal X-ray diffraction analysis using Cu Kα radiation (CCDC 1030588). Compound 1 showed weak activity against KB-VIN (IC50 = 11.0 ± 0.156 µM), while the known compound 3 exhibited selective cytotoxicity against MDA-MB-231 triple-negative breast cancer (TNBC) (IC50 = 5.42 ± 0.167 µM).

Purification and full characterisation of citreoviridin produced by Penicillium citreonigrum in yeast extract sucrose (YES) medium.

The mycotoxin citreoviridin has been associated with the 'yellow rice' disease, which caused cardiac beriberi in Japan. In Brazil, the consumption of contaminated rice was suspected to be involved in a recent beriberi outbreak. In this work, citreoviridin was produced by Penicillium citreonigrum, cultivated in 500 ml yeast extract sucrose (YES) liquid medium for 8 days at 25ºC, and the toxin extracted with chloroform from the liquid medium and the mycelium. A total of 15.3 g of crude extract was obtained from 48 culture flasks, with an estimated citreoviridin contend of 5.54 g, 74.3% being present in the mycelia. Semi-preparative HPLC of the crude extract yielded 27.1% citreoviridin. The HPLC-purified citreoviridin fraction was fully characterised by UV/VIS, FT-IR, (1)H- and (13)C-NMR, LC-MS/MS and LC-MSD TOF, and purity confirmed by gravimetric analysis. Isocitreoviridin was also produced by P. citreonigrum, accounting for about 10% of the citreoviridin present in the crude extract, most transformed into citreoviridin after 10 months under freezing conditions protected from light. Citreoviridin was shown to be stable under the same conditions, although it can suffer isomerisation after a longer storage period. Isomerisation is a potential source of variability in toxicological studies and purity of the material should be checked before study initiation.

Halogenated metabolites isolated from Penicillium citreonigrum.

Three chromone analogs, 1-3, a chlorinated alkaloid sclerotioramine (4), together with two 11-noreremophilane-type sesquiterpenes with a conjugated enolic OH group and a brominated one, 5 and 6, respectively, were isolated from Penicillium citreonigrum (HQ738282). Compounds 1, 5, and 6 were new. Biological tests revealed that 4 exhibited a significant activity (IC50 7.32 µg/ml), and 6 showed a moderate activity (IC50 16.31 µg/ml) in vitro against HepG2 cell line, and 4 also displayed an activity comparable to that of acarbose against α-glucosidase.