A Two-step Strategy for High-Value-Added Utilization of Rapeseed Meal by Concurrent Improvement of Phenolic Extraction and Protein Conversion for Microbial Iturin A Production

Rapeseed meal (RSM) is a major by-product of oil extraction from rapeseed, consists mainly of proteins and phenolic compounds. The use of RSM as protein feedstock for microbial fermentation is always hampered by phenolic compounds, which have antioxidant property with health-promoting benefits but inhibit bacterial growth. However, there is still not any good process that simultaneously improve extraction efficiency of phenolic compounds with conversion efficiency of protein residue into microbial production. Here we established a two-step strategy including fungal pretreatment followed by extraction of phenolic compounds. This could not only increase extraction efficiency and antioxidant property of phenolic compounds by about 2-fold, but also improve conversion efficiency of protein residue into iturin A production by Bacillus amyloliquefaciens CX-20 by about 33%. The antioxidant and antibacterial activities of phenolic extracts were influenced by both total phenolic content and profile, while microbial feedstock value of residue was greatly improved because protein content was increased by ∼5% and phenolic content was decreased by ∼60%. Moreover, this two-step process resulted in isolating more proteins from RSM, bringing iturin A production to 1.95 g/L. In conclusion, high-value-added and graded utilization of phenolic extract and protein residue from RSM with zero waste is realized by a two-step strategy, which combines both benefits of fungal pretreatment and phenolic extraction procedures.

Iturin A Induces Resistance and Improves the Quality and Safety of Harvested Cherry Tomato

The soft rot disease caused by Rhizopus stolonifer is an important disease in cherry tomato fruit. In this study, the effect of iturin A on soft rot of cherry tomato and its influence on the storage quality of cherry tomato fruit were investigated. The results showed that 512 μg/mL of iturin A could effectively inhibit the incidence of soft rot of cherry tomato fruit. It was found that iturin A could induce the activity of resistance-related enzymes including phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), peroxidase (POD), glucanase (GLU), and chitinase (CHI), and active oxygen-related enzymes including ascorbate peroxidases (APX), superoxide dismutases (SOD), catalases (CAT), and glutathione reductase (GR) of cherry tomato fruit. In addition, iturin A treatment could slow down the weight loss of cherry tomato and soften the fruit. These results indicated that iturin A could retard the decay and improve the quality of cherry tomato fruit by both the inhibition growth of R. stolonifera and the inducing the resistance.

516 Late-Breaking: Iturin a Protects the Intestinal Mucosal Integrity Against E. coli Containing the Gene Coding for Heat-stable Enterotoxin B

Enterotoxigenic E. coli causes severe infectious diarrhea with high morbidity and mortality in weanling pigs mainly through the production of heat-stable enterotoxins. Iturin A, originally isolated from the insect Hyalophora cecropia, exerts strong antibiotic activity against Gram-positive and Gram-negative bacteria. Thus, our study investigated whether iturin A could protect the intestinal epithelium from the insults of E. coli Rosetta containing the gene coding for heat-stable enterotoxin b (STb-Rosetta). Twenty-eight piglets were divided into four groups and were orally administered PBS (control), 0.16 g/kg BW iturin A, 2 × 109 CFU STb-Rosetta, and 0.16 g/kg BW iturin A + 2 × 109 CFU STb-Rosetta respectively for 8 days. The intestinal epithelial morphology and barrier function were assessed by H&E staining and Ussing chamber, and the growth advantage in enteroids derived from jejunal crypts of piglets in each group was used to evaluate the intestinal stem cell (ISC) activity. The results showed that iturin A increased the average daily gain (ADG) and average daily feed intake (ADFI) of piglets exposed to STb-Rosetta (P < 0.05). Meanwhile, STb-Rosetta-induced decrease in jejunum weight and damage to jejunal morphology and barrier function were significantly improved after iturin A supplementation (P < 0.05). Furthermore, the jejunal crypt cells from piglets in the iturin A + STb-Rosetta group had greater growth advantages of ISCs compared with the STb-Rosetta group, including enteroid forming efficiency, surface area, budding efficiency, and branching coefficient (P < 0.05). Our results indicate that iturin A protects against STb-Rosetta-induced intestinal mucosal injury, which provides an intervention strategy that regulates the function of ISCs in the high turnover-rate crypt-villous axis under ETEC infection. This study was supported by the National Natural Science Foundation of China (31872389, 32072777) and Basic and Applied Basic Research Foundation of Guangdong Province (2019B1515210021).

Activity of Fengycin and Iturin A Isolated From Bacillus subtilis Z-14 on Gaeumannomyces graminis Var. tritici and Soil Microbial Diversity

Bacillus subtilis Z-14 can inhibit phytopathogenic fungi, and is used as a biocontrol agent for wheat take-all disease. The present study used the soil-borne fungus Gaeumannomyces graminis var. tritici (Ggt), which causes wheat take-all disease, and the soil microbial community as indicators, and investigated the antifungal effects of fengycin and iturin A purified from strain Z-14 using high performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, respectively. The results showed that fengycin destroyed the internal structure of Ggt cells by digesting the cytoplasm and organelles, forming vacuoles, and inducing hyphal shrinkage and distortion. Iturin A induced cell wall disappearance, membrane degeneration, intracellular material shrinkage, and hyphal fragmentation. A biocontrol test demonstrated a 100% control effect on wheat take-all when wheat seedlings were treated with fengycin at 100 μg/ml or iturin A at 500 μg/ml. Iturin A and fengycin both reduced the relative abundance of Aspergillus and Gibberella. At the genus level, iturin A reduced the relative abundance of Mortierella and Myrothecium, while fengycin reduced that of Fusarium. Only fengycin treatment for 7 days had a significant effect on soil bacterial diversity.

The influence of cultivation conditions of promising Bacillus subtilis strains on their ability to produce antifungal metabolites

Antifungal compounds, including surfactin and iturin A, are accumulated by B. subtilis BZR336g strain at the cultivation temperature of 20.0-25.0 °C and the nutrient medium acidity pH8.0, for B. subtilis BZR517 strain these parameters are 30.0-35.0 °C and pH8.0-10.0, respectively.

Identification of Protease Inhibition Mechanism by Iturin A against Agriculture Cutworm (Spodoptera litura) by Homology Modeling and Molecular Dynamics

Objective: Spodoptera litura, otherwise known as cutworm, belongs to the Noctuidae tribe, which is a severe scourge for numerous crop systems and is considered one of Asian tropical agriculture's most important insects. The world's leading environmental threats are plant pests, and the already commercialized pesticides are extremely poisonous and non-biodegradable and maybe additional residues harmful to the ecosystem. The increased resistance in pests often demands the need for advanced, active pesticides that are environmentally friendly and biodegradable. Methods: In the current work, the significance of proteases for the Spodoptera litura digestive system has been determined by the use of microbial metabolite protease inhibitor (Iturin A) in silico models. In the present study, we developed a model based on sequence structural alignment of known crystal structure 2D1I protease from Homo sapiens. The model's reliability evaluation was performed using programs such as PROCHECK, WHAT IF, PROSA, Validate 3D, ERRAT, etc. Results: In an attempt to find new inhibitors for Protease docking, the study was carried out with Iturin A. PMDB ID for the produced protease model was submitted to identify new inhibitors for Protease docking, and its accession number is PM0082285. The detailed study of enzyme-inhibitor interactions identified similar core residues; GLU215, LEU216, LYS217, and GLU237 have demonstrated their role in the binding efficacy of ligands. Conclusion: The latest homology modeling and docking experiments on the protease model will provide useful insight knowledge for the logical approach of constructing a wide spectrum of novel insecticide against Spodoptera.