RESUMO
The enzymatic biodegradation of mycotoxins in food and feed has attracted the most interest in recent years. In this paper, the laccase gene from Bacillus swezeyi was cloned and expressed in Escherichia coli BL 21(D3). The sequence analysis indicated that the gene consisted of 1533 bp. The purified B. swezeyi laccase was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis -12% with an estimated molecular weight of 56.7 kDa. The enzyme is thermo-alkali-tolerant, displaying the optimal degradation of zearalenone (ZEN) and aflatoxin B1 (AFB1) at pH 8 and 9, with incubation temperatures of 55 and 50 °C, respectively, within 24 h. The degradation potentials of the 50 µg of the enzyme against ZEN (5.0 µg/mL) and AFB1 (2.5 µg/mL) were 99.60 and 96.73%, respectively, within 24 h. To the best of our knowledge, this is the first study revealing the recombinant production of laccase from B. swezeyi, its biochemical properties, and potential use in ZEN and AFB1 degradation in vitro and in vivo.
Assuntos
Aflatoxina B1 , Bacillus , Proteínas de Bactérias , Estabilidade Enzimática , Lacase , Proteínas Recombinantes , Zearalenona , Lacase/genética , Lacase/metabolismo , Lacase/química , Aflatoxina B1/metabolismo , Aflatoxina B1/química , Zearalenona/metabolismo , Zearalenona/química , Bacillus/enzimologia , Bacillus/genética , Bacillus/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Concentração de Íons de Hidrogênio , Temperatura , Peso Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Clonagem Molecular , Álcalis/metabolismo , Álcalis/químicaRESUMO
Zearalenone (ZEN) is a widespread mycotoxin that causes serious damage to animal husbandry and poses a threat to human health. A screen of ZEN-degrading soil bacteria yielded Bacillus subtilis YT-4, which yielded 80% ZEN degradation after 6 h and 95% after 36 h. The gene sequence encoding the degradative enzyme ZENY was mined from the genome of YT-4 and expressed in yeast. ZENY is an α/ß-hydrolase with an optimal enzyme activity at 37 °C and pH 8. By breaking the lactone ring of ZEN, it produces ZENY-C18H24O5 with a molecular weight of 320.16 g/mol. Sequence comparison and molecular docking analyses identified the catalytic ZENY triad 99S-245H-123E and the primary ZEN-binding mode within the hydrophobic pocket of the enzyme. To improve the thermal stability of the enzyme for industrial applications, we introduced a mutation at the N-terminus, specifically replacing the fifth residue N with V, and achieved a 25% improvement in stability at 45 °C. These findings aim to achieve ZEN biodegradation and provide insight into the structure and function of ZEN hydrolases.
Assuntos
Zearalenona , Animais , Humanos , Zearalenona/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Simulação de Acoplamento Molecular , Hidrolases/genética , MutaçãoRESUMO
Protein oxidation caused by food processing is harmful to human health. A large number of studies have focused on the effects of hot processing on protein oxidation of meat products. As an important protein source for human beings, the effects of hot processing on protein oxidation in flour products are also worthy of further study. This study investigated the influences on the protein oxidation of wheat dough under baking (0-30 min, 200 °C or 20 min, 80-230 °C) and frying (0-18 min, 180 °C or 10 min, 140-200 °C). With the increase in baking and frying time and temperature, we found that the color of the dough deepened, the secondary structure of the protein changed from α-helix to ß-sheet and ß-turn, the content of carbonyl and advanced glycation end products (AGEs) increased, and the content of free sulfhydryl (SH) and free amino groups decreased. Furthermore, baking and frying resulted in a decrease in some special amino acid components in the dough, and an increase in the content of amino acid oxidation products, dityrosine, kynurenine, and N'-formylkynurenine. Moreover, the nutritional value evaluation results showed that excessive baking and frying reduced the free radical scavenging rate and digestibility of the dough. These results suggest that frying and baking can cause protein oxidation in the dough, resulting in the accumulation of protein oxidation products and decreased nutritional value. Therefore, it is necessary to reduce excessive processing or take reasonable intervention measures to reduce the effects of thermal processing on protein oxidation of flour products.
RESUMO
Mycotoxins are toxic secondary metabolites mainly produced by filamentous fungal species that commonly contaminate food and feed. Aflatoxin B1 (AFB1) is extremely toxic and seriously threatens the health of humans and animals. In this work, the Bacillus megaterium HNGD-A6 was obtained and showed a 94.66% removal ability of AFB1 by employing extracellular enzymes as the degrading active substance. The degradation products were P1 (AFD1, C16H14O5) and P2 (C14H16N2O2), and their toxicity was greatly reduced compared to that of AFB1. The AttM gene was mined by BlastP comparison and successfully expressed in Escherichia coli BL21. AttM could degrade 86.78% of AFB1 at pH 8.5 and 80 °C, as well as 81.32% of ochratoxin A and 67.82% of zearalenone. The ability of AttM to degrade a wide range of toxins and its resistance to high temperatures offer the possibility of its use in food or feed applications.
Assuntos
Bacillus megaterium , Micotoxinas , Zearalenona , Animais , Humanos , Aflatoxina B1/toxicidade , Bacillus megaterium/genética , Zearalenona/metabolismoRESUMO
Removal of aflatoxin is an urgent issue in agricultural products. A porous graphitic carbon nitride/graphene oxide hydrogel microsphere (CN/GO/SA) was synthesized and used to degrade AFB1 in peanut oil. CN/GO/SA was characterized by scanning electron micrograph (SEM), X-ray diffraction (XRD) and FT-IR. The introduction of GO significantly improved the adsorption capacity and visible light activity of photocatalysts. About 98.4% AFB1 in peanut oil was removed by 20% CN/GO/SA under visible light for 120 min. â§O2- and h+ were the main active species during photoreaction, and five degradation products were identified by UPLC-Q-Orbitrap MS analysis. At the same time, the quality of treated peanut oil was still acceptable. More importantly, CN/GO/SA showed excellent cycle stability, and the degradation rate of AFB1 in peanut oil remained above 95% after five-time recycling. This work provides a practical way for developing efficient and sustainable photocatalysts to degrade mycotoxins in edible oil.
Assuntos
Aflatoxina B1 , Hidrogéis , Óleo de Amendoim , Aflatoxina B1/análise , Espectroscopia de Infravermelho com Transformada de Fourier , Porosidade , MicroesferasRESUMO
Ochratoxins (OTs) is an extremely toxic mycotoxin in which Ochratoxin A (OTA) is the most toxic and prevalent in the ochratoxin family. OTA is among the five most critical mycotoxins that are subject to legal regulations. Animals and humans may be exposed to OTA through dietary intake, inhalation, and dermal contact. OTA is considered nephrotoxic, genotoxic, cytotoxic, teratogenic, carcinogenic, mutagenic, immunotoxic, and myelotoxic. So, intake of OTA contaminated foods and feeds can impact the productivity of animals and health of people. According to this review, several studies have reported on the approaches that have been established for OTA removal. This review focused on the control approaches to mitigate OTA contamination, OTA bio-detoxification materials and their applicable techniques, recombinant strains for OTA bio-detoxification, and their detoxification effects, recombinant OTA-degrading enzymes and their sources, recombinant fusion enzymes for OTA, ZEN and AFB1 mycotoxins detoxification, as well as the current application and commercialized OTA bio-detoxification products. However, there is no single technique that has been approved to detoxify OTA by 100% to date. Some preferred current strategies for OTA bio-detoxification have been recombinant degrading enzymes and genetic engineering technology due to their efficiency and safety. Therefore, prospective studies should focus on standardizing pure enzymes from genetically engineered microbial strains that have great potential for OTA detoxification.