Effectiveness of neutral electrolyzed water and copper oxychloride on fungi spores isolated from tropical fruits.

The aim of this study was to assess the antifungal effectiveness of neutral electrolyzed water (NEW) to inhibit the spore germination of post-harvest fungi common in fruits, determine the required available chlorine concentration (ACC) of NEW and to compare it with copper oxychloride (CO) and sterile distilled water (SDW) in vitro. This study evaluated the biological effectiveness of NEW to inactivate pure cultures of 11 different fungi obtained from post-harvest tropical fruits with anthracnose, rottenness or necrosis symptoms. A conidial solution of 1 × 104 spores/mL per culture was prepared and treated with a low, medium and high ACC of NEW (pH 7.0 ± 0.05, 12, 33 and 53 mg/L of ACC and ORP of 850 mV), CO at 0.3 g/L, or sterile distilled water as a control, for 3-, 5- and 10-min contact time. Spore germination of Alternaria alternata, Botrytis cinerea, Cladosporium australiense, Colletotrichum gloeosporioides and C. siamense, Fusarium solani and F. oxysporum, and Lasiodiplodia theobromae was inhibited in 100% by NEW at 12, 33 and 53 ppm ACC; 3,5 and 10 min contact time. Aspergillus niger and A. tamarii required 53 mg/L ACC to inhibit 100% of spore germination. NEW at 33 and 12 mg/L inhibited around 50% and <25% of A. niger spore germination, respectively. NEW at 53 mg/L ACC was the most efficient treatment against Rhizopus stolonifer but only inhibited spore germination in ∼25%. CO inhibited spore germination by 100% of A. alternata, B. cinerea, C. australiense, C. gloeosporioides, C. siamense and L. theobromae. However, CO inhibited <25% of spore germination of F. solani, F. oxysporum, A. niger, A. tamarii and R. stolonifer. NEW and CO had a significant effect on every fungus compared to a SDW treatment. SDW was the least effective treatment, followed by CO. NEW at 12 mg/L and 33 mg/L ACC were equally effective in eliminating the fungi, and more effective than CO. NEW at a concentration of 53 mg/L ACC was the most effective treatment. Results obtained in this study show that NEW has effectively inhibited spore germination of these species, and this treatment could be used as a substitute an ecological novel alternative to CO to avoid spore growth in the above-mentioned fruits.

Ameliorative Effects of Neutral Electrolyzed Water on Growth Performance, Biochemical Constituents, and Histopathological Changes in Turkey Poults during Aflatoxicosis.

Different in vitro and in silico approaches from our research group have demonstrated that neutral electrolyzed water (NEW) can be used to detoxify aflatoxins. The objective of this investigation was to evaluate the ability of NEW to detoxify B-aflatoxins (AFB1 and AFB2) in contaminated maize and to confirm detoxification in an in vivo experimental model. Batches of aflatoxin-contaminated maize were detoxified with NEW and mixed in commercial feed. A total of 240 6-day-old female large white Nicholas-700 turkey poults were randomly divided into four treatments of six replicates each (10 turkeys per replicate), which were fed ad libitum for two weeks with the following dietary treatments: (1) control feed containing aflatoxin-free maize (CONTROL); (2) feed containing the aflatoxin-contaminated maize (AF); (3) feed containing the aflatoxin-contaminated maize detoxified with NEW (AF + NEW); and (4) control feed containing aflatoxin-free maize treated with NEW (NEW). Compared to the control groups, turkey poults of the AF group significantly reduced body weight gain and increased feed conversion ratio and mortality rate; whereas turkey poults of the AF + NEW group did not present significant differences on productive parameters. In addition, alterations in serum biochemical constituents, enzyme activities, relative organ weight, gross morphological changes and histopathological studies were significantly mitigated by the aflatoxin-detoxification procedure. From these results, it is concluded that the treatment of aflatoxin-contaminated maize with NEW provided reasonable protection against the effects caused by aflatoxins in young turkey poults.

A Theoretical Study of 8-Chloro-9-Hydroxy-Aflatoxin B1, the Conversion Product of Aflatoxin B1 by Neutral Electrolyzed Water.

Theoretical studies of 8-chloro-9-hydroxy-aflatoxin B1 (2) were carried out by Density Functional Theory (DFT). This molecule is the reaction product of the treatment of aflatoxin B1 (1) with hypochlorous acid, from neutral electrolyzed water. Determination of the structural, electronic and spectroscopic properties of the reaction product allowed its theoretical characterization. In order to elucidate the formation process of 2, two reaction pathways were evaluated-the first one considering only ionic species (Cl⁺ and OH(-)) and the second one taking into account the entire hypochlorous acid molecule (HOCl). Both pathways were studied theoretically in gas and solution phases. In the first suggested pathway, the reaction involves the addition of chlorenium ion to 1 forming a non-classic carbocation assisted by anchimeric effect of the nearest aromatic system, and then a nucleophilic attack to the intermediate by the hydroxide ion. In the second studied pathway, as a first step, the attack of the double bond from the furanic moiety of 1 to the hypochlorous acid is considered, accomplishing the same non-classical carbocation, and again in the second step, a nucleophilic attack by the hydroxide ion. In order to validate both reaction pathways, the atomic charges, the highest occupied molecular orbital and the lowest unoccupied molecular orbital were obtained for both substrate and product. The corresponding data imply that the C9 atom is the more suitable site of the substrate to interact with the hydroxide ion. It was demonstrated by theoretical calculations that a vicinal and anti chlorohydrin is produced in the terminal furan ring. Data of the studied compound indicate an important reduction in the cytotoxic and genotoxic potential of the target molecule, as demonstrated previously by our research group using different in vitro assays.

Effectiveness of Neutral Electrolyzed Water on Incidence of Fungal Rot on Tomato Fruits ( Solanum lycopersicum L.).

We assessed the effect of neutral electrolyzed water (NEW) on the incidence of rot on tomato ( Solanum lycopersicum L.) fruits inoculated with Fusarium oxysporum , Galactomyces geotrichum , and Alternaria sp. at sites with lesions. The inoculated fruits were treated with NEW at 10, 30, and 60 mg liter-1 active chlorine, with copper oxychloride fungicide, and with sterile distilled water (control) for 3, 5, and 10 min. In the experiment with F. oxysporum , 50 to 80% of the control fruits and 50 to 60% of the fruits treated with the fungicide exhibited symptoms of rot at the inoculated sites. The lowest incidence recorded was 30% for fruits treated with NEW at 60 mg liter-1 active chlorine with an immersion time of 5 min. In the experiment with G. geotrichum , incidence of rot on control fruits was 70 to 90%, and for treatment with fungicide rot incidence was 50 to 90%. NEW at 60 mg liter-1 active chlorine significantly reduced incidence of symptomatic fruit: only 30% of the inoculated fruits washed for 5 min had damage from rot. In the experiment with Alternaria sp., 60 to 90% of the fruits in the control group and 60 to 70% of the fruits in the fungicide group were symptomatic. The lowest incidence was recorded for the treatment in which the fruits were submerged in NEW with 60 mg liter-1 active chlorine for 3 min. In this group, 40 to 50% of the fruits exhibited symptoms of rot. These results were obtained 8 days after inoculation. NEW, with 60 mg liter-1 active chlorine, significantly reduced incidence of rot symptoms on fruits inoculated with one of the experimental fungi relative to the control (P ≤ 0.05). NEW at 60 mg liter-1 is effective in the control of fungal rot in tomatoes.

Detoxification of Aflatoxin-Contaminated Maize by Neutral Electrolyzed Oxidizing Water.

Aflatoxins, a group of extremely toxic mycotoxins produced by Aspergillus flavus, A. parasiticus and A. nomius, can occur as natural contaminants of certain agricultural commodities, particularly maize. These toxins have been shown to be hepatotoxic, carcinogenic, mutagenic and cause severe human and animal diseases. The effectiveness of neutral electrolyzed oxidizing water (NEW) on aflatoxin detoxification was investigated in HepG2 cells using several validation methodologies such as the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide assay, the induction of lipid peroxidation, the oxidative damage by means of glutathione modulation, the Ames test and the alkaline Comet assay. Our results showed that, after the aflatoxin-contaminated maize containing 360 ng/g was soaked in NEW (60 mg/L available chlorine, pH 7.01) during 15 min at room temperature, the aflatoxin content did not decrease as confirmed by the immunoaffinity column and ultra performance liquid chromatography methods. Aflatoxin fluorescence strength of detoxified samples was similar to untreated samples. However, aflatoxin-associated cytotoxicity and OPEN ACCESS Toxins 2015, 7 4295 genotoxicity effects were markedly reduced upon treatment. According to these results, NEW can be effectively used to detoxify aflatoxin-contaminated maize.