Major phytochemical components of the AECP:
Data presented in (Table 1) revealed that the AECP has the high content from TPC (209±3.21), TFC (16.4±1.18), TAC (13.27±1.16), and tannins (5.71±1.31), which includes many bioactive compounds, the presence of these compounds makes carob (pods) of therapeutic value, as well as, this results are in good agreement with a previous (Nawel 2017; Ayache et al. 2020). Content of TPC, TFC, and TAC is affected by genetic, geographic factors, storage settings, soil and methods of analysis (El Turhan 2004; Bouzdoudi et al. 2016). On the other hand, DPPH free radical scavenging activity was 57.3 %. Phenolic compounds are known to have antioxidant activity and it is likely that the activity of these extracts is due to these compounds. These results confirmed by FTIR analysis (Fig. 1) showed many functional groups such presented lowest Transmittance (T) at 3430 cm-1, 1630.5 cm-1 and 1389 cm-1 (OH stretching, N-H and –C=C-, and-C-H) for alcohols, phenols, alkanes and alkenes (Mellado-Mojica et al. 2016; Christou et al. 2018).
Evaluation of AECP on growth toxigenic Aspergillus fungi:
Data presented in (Fig. 2) show the inhibition zone (mm) of fungal growth after treatment with AECP at the concentration1.5, 2.5 and 5 mg/ml. The obtained data indicated that the highest level of inhibition was detected at the concentration 5mg/ml of AECP, also, the inhibition increased with increasing the used concentration. In addition the strain of A.ochraceus is more affected by AECP than A. aflatoxiformans. The highest level of inhibition at 5 mg/ml was recorded with A. ochraceus (15.7±0.4) then A. aflatoxiformans (13.4±0.5). Moreover, the lowest inhibition was shown with A. flavus at three treatments. The inhibitory effect of EACP against mycelial growth of Aspergillus ssp. was analyzed statistically as shown in (Table 2). The ANOVA analysis show significantly both of the concentration EACP and types of fungi. In general, active compounds in AECP have several mechanisms against fungal, through inhibition of chitin and β-glucan synthesis. In addition, lipid membrane dissolution, and RNA and DNA synthesis alteration. Several previous studies reported that CP contains many compounds phenolics that have antifungal activity such as protocatechuic acid, gallic acid, cinnamic acid and rutin. Lakkab et al, (2019) reported that the GA was the major phenolic acid present in CP. Moreover, Goulas and Georgiou found that the main components of water CP extract are myricetin 94.5%, gallic acid 80%, epicatechin 77.3%, and rutin 67% (Lakkab et al. 2019; Darwish et al. 2021), which effect on fungal cells through division and synthesis of RNA leading to inhibition of growth.
Inhibitor effect of AECP on the biosynthesis of AFs and OTA.
Data presented in (Fig 3) shown the inhibition percentages of AFs and OTA produced by A. parasiticus and A. ochraceus, respectively in YES media treated by AECP. Data reflected that the inhibition percentages increased directly with increasing concentration added from AECP. OTA recorded the highest inhibition levels of 18.6, 40.7, 77.8% at 1.5, 2.5, and 5 mg/ml AECP, at the same concentrations the lowest percentages were 12.8, 22.6 and 61.6% with AFB1. In case YES was treated by 5mg/ml from AECP the AFG1, AFG2, and AFB2 inhibition were 72.5, 67.3, and 70.05%, respectively. AECP reduced of mycelium weight to 63.9% and 54.8% for A. ochraceous and A. parasiticus, respectively at the concentration 2.5mg/ml (Fig. 4). The phenolic compounds' hydroxyl groups may create hydrogen bonds with some active enzymes, resulting in deactivation and suppression of fungal biomass and the formation of mycotoxins. In addition, AECP causes cytotoxicity in fungi by affecting cell membrane permeability and functions, so that AECP can inactivate biotransformation pathways needed for synthesis AFs and OTA from A. parasiticus and A. ochraceus, respectively (Loi et al. 2020). Previous studies used plant extracts to prevent or control the production of AFs; Karapynar (1989) used the crude extracts from mint, sage, bay, anise, and ground red pepper to control the growth of A. parasiticus and its AFs production in vitr. Another study by Satish et al. (2007) tested aqueous extracts of fifty-two plants from different families for their antifungal, they found that the potential against eight of Aspergillus spp. Similarly, Pundir and Jain (2010) studied the efficacy of 22 plant extracts against food-associated fungi and found that clove and ginger are more effective than other plant extracts]. The alkaloids such as piperine and piperlongumine have an effect on the biosynthesis of OTA -producing from Aspergilli: A. auricomus, A. sclerotiorum, and two isolates of A. alliaceous. The antitoxigenic potential of the spices was tested against the OTA-producing strain of A. ochraceus Wilhelm. Clove completely inhibited the mycelial growth of the fungi A. ochraceus, as well as, garlic and laurel completely inhibited the OTA production. Cinnamon and anis inhibited the synthesis of OTA when used at concentration 3% and mint starting from 4% (Pereira et al. 2006). Finally, AFs production could be disrupted if any step in the aflatoxin biosynthetic pathway is completely blocked by a specific inhibitor. aflD (nor-1) gene expression that represents the early enzymatic steps in the aflatoxin biosynthetic pathway could be an appropriate target for inhibiting aflatoxin biosynthesis. Disruption or deletion of the aflD (nor-1) gene leads to the accumulation of norsolorinic acid and blocks the synthesis of all aflatoxins and their intermediates beyond norsolorinic acid. Ren et al. (2020) and Buitimea-Cantua et al. (2020) suggest inhibits AFs biosynthesis by A. flavus via enhancing fungal oxidative stress response. Additionally, inactivating some enzymes in biosynthesis of AFs, in addition to confusion in the expression of aflD, aflM, aflR, and aflS genes of AFs biosynthetic pathway.
Influence of AECP on accumulation of AFs and OTA during storage of RPKs:
This study was carried out to investigate the effect of AECP at 1.5, 2.5, and 5 mg/ml on the accumulation of total AFs and OTA produced by A. parasiticus and A. ochraceus during storage of RPKs for 90 days. Concerning AFs, HPLC analysis did not detect any amount of AFs in infected RPKs by A. parasiticus samples were stored for 15 days after being treated at 1.5, 2.5, and 5mg/ml for AECP, storage period extended to 45 days with treatment at 5mg/ml without any amount from AFs (Fig. 5). Whereas extension of the storage causes resulted in an intense accumulation of total AFs in the treated sample and positive control sample both, with differences in concentrations, this is probably due to the increase in the number of spores of A. parasiticus capable of producing toxin. Also, some spores can recover their activity with increasing storage period, and another possibility on the probably for consumption the content of the active compounds in the AECP (Mahmoud 1999; Passone et al. 2008. The amount of OTA in infected RPKs by A. ochraceus during storage 90 days showed in (Fig. 6). The results indicated that OTA formed after 15, 30, and 45 days from storage after treated RPKs by AECP at 1.5, 2.5, and 5 mg/ml, respectively. On the other hand, the concentration of OTA in the positive control sample was (26.5µg/kg), while RPKs with 1.5, 2.5, and 5 mg/ml were 21.35, 7.5, and 3.31µg/kg at the end of the storage period after 90 days.
Finally, the percentages of inhibition AFB1, AFG1, AFB2, AFG2, and OTA for each type separately in RPKs treated by AECP after storage for 90 days compared with positive control samples calculated as (Fig. 8). The percentages of inhibition AFB1 in RPKs were lowest percentages with all treatment were 12.3, 50.5, and 76.5% with adding 1.5, 2.5, and 5mg/ml AECP. On the other hand, OTA had the highest inhibition percentages, which reached 19.3, 71.6, and 87.5% with the same treatments, while AFG1, AFG2 and AFB2 inhibited to 85.1 5, 86.5. and 84.2% with applied 5mg/ml from AECP.
AFB1 occur more frequently as contaminants, and are also believed to be more potent, than G aflatoxins. From a genomic perspective, the inability to produce G aflatoxins is reported to result from a deletion between the norB (aflF) and cypA (aflU) genes, upstream in the AFs biosynthesis pathway (Passone et al. 2013). Previous studies have reported that some phenolic antioxidants, such as gallic, 4-hydroxybenzoic, and chlorogenic acid tended to inhibit OTA production, for example, Romero et al. (2010) agreement with (Palumbo et al. 2007; Bisogno et al. 2007) notice a significant reduction in growth rate and OTA production with 250 mg/L of caffeic acid, rutin, and quercetin