Protect peanut kernels from Aspergillus spp and their mycotoxins during storage by aqueous extract of carob pulp

Recently, the trend of many studies to replace synthetic antifungal commonly used during storage with natural antifungal; Plant extracts has been one of the most promising strategies for this purpose. Therefore, this study aimed to evaluate the aqueous extract of the carob pulp (AECP) at 1.5, 2.5, and 5 mg/ml on the growth of some toxigenic Aspergillus species, and its effectiveness as an anti-mycotoxigenic (aflatoxins (AFs) and ochratoxin A (OTA) during storage red peanut kernels (RPKs) for 90 days. The results indicate that the increased concentration of AECP, the area of inhibition zone increased with all treatments. As well, the strain of Aspergillus ochraceus was more affected by AECP than Aspergillus aflatoxiformans followed by Aspergillus parasiticus which recorded (15.7 ± 0.4 mm), (13.4 ± 0.5 mm), and (11.9 ± 0.4 mm) at 5 mg/ml. Respectively. While, the lowest inhibition was shown with Aspergillus flavus at three concentrations of AECP. The impact of AECP on the accumulation of total AFs and OTA during storage of RPKs infected by A. parasiticus and A. ochraceus, respectively, for 90 days. The results showed that the active compounds in the AECP at 5 mg/ml can protect RPKs for 45 days without detecting any amounts of AFs and OTA. After 90 days, the inhibition percentages for the four types of aflatoxins (AFB1, AFG1, AFB2, AFG2) and OTA compared with positive control samples were 85.1, 76.5, 86.5, 84.2, and 87.5%, respectively. Finally, AFB1 recorded the lowest inhibition percentages of all treatments; on the contrary, it was OTA.


Determination of total phenolic content (TPC)
The total phenolic content in aqueous extract for carob pulp (CP) was determined using the Folin-Ciocalteau method as follows: Briefly, 1 ml of extract in a volumetric flask was diluted with distilled water to 46 ml. 1 ml of Folin-Ciocalteu reagent was added and the contents of the flask were blended completely. After 3 min, 3 ml of Na 2 CO 3 (2%) was added, then mixture left for 2 h. The absorbance of the mixture was measured at 760 nm. TPC was calibrated as mg Gallic acid equivalents (GAE) 100 g −1 carob pod by the calibration curve of a Gallic acid equivalent (GAE) standard [18].

Determination of total flavonoid content (TFC)
The TFC was measured according to the method Yan-Hwa et al. (2000) as follows: 1 ml of extract was diluted with 4.3 ml of 80% aqueous ethanol containing 0.1 ml of 10% Al (NO 3 ) 3 and 0.1 ml of 1 M aqueous CH 3 COOK. After 40 min at room temperature, the absorbance was determined spectrophotometrically at 415 nm. The total flavonoid content was measured by us using a calibration curve of a quercetin equivalent (QE) standard [19].

Quantification of the total alkaloid content (TAC)
Total alkaloid content was determined by using the method developed by [20] which is based on the reaction between alkaloid with bromocresol green (BCG), resulting in a yellow-colored product. This method has the benefits of sensitivity and stability. In brief, 0.1 ml of AECP was mixed with 2 N HCl, and one milliliter of this solution was transferred to a separatory funnel and washed (three times) using 10 ml chloroform. The pH of this solution was adjusted to neutral with 0.1 N NaOH. Then 5 ml of BCG solution and 5 ml of phosphate buffer were added to this solution. After that, the mixture was shaken and the complex formed was extracted with 1, 2, 3, and 4 ml chloroform using vigorous shaking. The extracts were collected in a 10 ml volumetric flask and diluted to volume with chloroform. The absorbance of the complex in chloroform was measured at 470 nm. Alkaloids (mg/ml) = (Abs-0.048)/0.021 [20].

Determination of antioxidant activities
Determination of DPPH radical scavenging activity: The antioxidant activity of the extracts, based on the scavenging activity of the stable DPPH free radical was determined by the method described by Lee et al. [21].

Determination total content of tannins
The condensed tannins were estimated according to Julkunen-Titto. (1985) and expressed as g of catechin equivalents per 100 g of dry weight [22].

Assay of antifungal activity
The antifungal activity was tested on the three strains of fungi using agar well diffusion technique. The strains were cultivated on PDA slants at 28 °C for 7 days. Spores were harvested by adding 10 ml of sterile distilled water containing 0.05% Tween 20 and scraping the surface of the culture to free the spores. One ml of spore suspension was inoculated into each plate. Wells of 5 mm diameter was made on the PDA surface and filled with the three graduals with three concentrations of (1.5, 2.5 and 5 mg/ml), obtained from diluting the stock solutions were used. Wells containing with (100 µl pure solvent (water) were used as a negative control, while wells containing with Nystatin (1000 Unit/ ml were considered as positive control. The inoculated plates were incubated at 27 °C for 72 h, and then the antifungal activity was assessed by measuring the zone of inhibition (mm). The results average was calculated from at least three replicates for each treatment [23].

Control of producing AFs and OTA by AECP in YES medium
The yeast extract sucrose (YES) culture medium (2% yeast extract and 15% sucrose/liter distilled water) was used in this experiment, 100 ml of YES was poured into 250 ml Erlenmeyer flask and autoclaved at 121 °C for 15 min, then cooled and inoculated with 1 ml of spore suspension containing 10 6 spores/ml of A. parasiticus (ATCC 26692) and A. ochraceus both separately, then treated by gradual with concentrations (1.5, 2.5 and 5 mg/ml) from AECP as treatment samples, while Three replications from YES treated by spores of A. parasiticus (ATCC 26692) and A. ochraceus only as positive samples, and flasks were incubated at 28 °C for 7 days. After the end of the incubation period, the AFs were extracted then determined using HPLC as follows: AFs were extracted from the YES medium by chloroform (20 ml twice with 10 ml YES media) then homogenization for 3 min in a separation funnel. Finally, filtration of the chloroform phase, then evaporated to dryness to use with HPLC. The percentage of inhibition was calculated as the following equation.
where: C is mean concentrations of toxins (AFB 1 , AFB 2 , AFG 1 and AFG 2 ) in the positive samples that inoculated by spores of A. parasiticus only. T is concentrations of the toxin in the sample containing spores of fungus and AECP (treatment sample) [24].

Extraction and determination of OTA
Ten milliliters of YES medium were filtered, and then extracted with 20 ml of chloroform in separation funnel. The chloroform phase was filtered and concentrated then dryness under nitrogen to dry film; it was dissolved in 1 ml water/acetonitrile (3: 1 v/v) and mixed well by vortex for 30 s. HPLC analysis according to Ammar and El-Desouky. (2016). On the other hand, the percentage of inhibition OTA was calculated according to the above equation [25].

Statistical analysis
General Linear Model procedure of the SPSS ver. 18 (IBM Corp, NY) was used to statistically analyse. The significance of the differences among treatment groups was determined by Waller-Duncan k-ratio. All statements of significance were depended on the probability of P ≤ 0.05 was considered to be statistically significant. Values represent averages ± standard deviations for triplicate experiments.

Major phytochemical components of the AECP
Data presented in (Table 1) revealed that the AECP has the high content from TPC (209 ± 3.21 mg/100 g), TFC (16.4 ± 1.18 mg/100 g), TAC (13.27 ± 1.16 mg/100 g), and tannins (5.71 ± 1.31 mg/100 g), 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 [28,29]. Content of TPC, TFC, and TAC is affected by genetic, geographic factors, storage settings, soil and methods of analysis [30,31]. On the other hand, DPPH free radical scavenging activity was 57.3%. 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 [32,33]. Phenolic compounds in the CP include primarily hydroxybenzoic acids, flavonols, flavan-3-ols and gallotannins. In addition, Gallic acid and its derivatives such as methyl gallate comprise the  [17,34,35]. These compounds have bioactive effects and health benefits in various therapeutic areas, as well as the prevention of certain diseases such as cancer, diabetes and diarrhea. Additionally, carob has antioxidant activities are of particular interest in human cells, so these natural antioxidants can replace the synthetic antioxidants used in foods, which can be harmful components due to their toxicity and carcinogenic effects [36,37].

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 concentration 1.5, 2.5 and 5 mg/ml. The obtained data indicated that the highest level of inhibition was detected at the concentration 5 mg/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 mm) then A. aflatoxiformans (13.4 ± 0.5 mm). 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 shows significantly both of the concentration EACP and types of fungi. In general, the 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. [38] Reported that the GA was the major phenolic acid present in CP. Moreover, Goulas and Georgiou found that the main components of the water CP extract are myricetin 94.5%, gallic acid 80%, epicatechin 77.3%, and rutin 67% [17,38]. Which effect on fungal cells through the division and synthesis of RNA leading to inhibition of growth, as kaempferol has been inhibits the expression of protein. Flavones have antifungal activity against pathogenic fungi, may due to Reactive oxygen species (ROS) accumulation is considered one of the primary biochemical causes of apoptosis. Unsuitable regulation of ROS levels can damage, fungal cells, caused to untypical fungal growth and consequential apoptotic-like cell death. As well as, changes in the Mitochondrial membrane like opening of transition pores in the membrane and release of apoptogenic factors into the cytosol, are considered another characteristic of apoptosis [39,40].

Inhibitory effect of AECP on the biosynthesis of AFs and OTA
The data presented in (Fig. 3) show the inhibition percentages of AF and OTA produced by A. parasiticus and A. ochraceus, respectively in YES media treated by AECP. The data reflected that the inhibition percentages increased directly with increasing concentration added from AECP. The highest inhibition was recorded with OTA was 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 AFB 1. In case YES was treated with 5 mg/ml from AECP the AFG 1 , AFG 2 , and AFB 2 inhibition were 72.5, 67.3, and 70.05%, respectively. AECP decreased mycelial weight to 63.9% and 54.8% for A. ochraceous and A. parasiticus, respectively at the concentration 2.5 mg/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 [41]. Previous studies used plant extracts to prevent or control the production of AFs; [42] 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 vitro. Another study by [43] tested aqueous extracts of fifty-two plants from different families for their antifungal, they found that the potential against eight of Aspergillus spp [42,43]. 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 [44]. 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 antioxygenic 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% [45]. 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

A. parasiƟcus A . ochraceus
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. In this regard Ren et al. [46], and Buitimea-Cantua et al. [47] reported that the biosynthesis of AFs was inhibited by repression of AFs structural genes which including (aflK and aflLa) may be joint with the down-regulation of the transcription regulator genes (aflR and aflS) in the cluster. Additionally, the suppression of many key AFs genes including aflR, aflD, aflM, and aflP may also be associated with the decreased expression of the global regulator gene veA [46,47].

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 5 mg/ml for AECP, storage period extended to 45 days with treatment at 5 mg/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 [48,49]. 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 (26.5 µg/kg), while RPKs with 1.5, 2.5, and 5 mg/ml were 21.35, 7.5, and 3.31 µg/kg at the end of the storage period after 90 days. The RPKs treated by AECP at 5 mg/ml and infected by A. ochraceus shown as (Fig. 7). Finally, the percentages of inhibition AFB 1 , AFG 1 , AFB 2 , AFG 2 , 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 AFB 1 in RPKs were lowest percentages with all treatment were 12.3, 50.5, and 76.5% with adding 1.5, 2.5, and 5 mg/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 AFG 1 , AFG 2 and AFB 2 inhibited to 85.1 5, 86.5 and 84.2% with applied 5 mg/ml from AECP. AFB 1 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 [50]. Previous studies have reported that some phenolic antioxidants, such as gallic, 4-hydroxybenzoic, and chlorogenic acid tended to inhibit OTA production, Total AFs (µg/kg)

Conclusion
In conclusion, this study demonstrated that AECP has the many bioactive compounds (TFC, TPC and TAC), which able to protect of RPKs from toxigenic Aspergillus spp strains. By inhibiting the growth of these strains and forming AFs and OTA during storage RPKs at ambient temperature. The results of the study showed that the AECP at 5 mg/ml is effective and better their antifungal activity against all tested mycotoxigenic fungi, as well against accumulation of AFs and OTA for 45 days of storage of infected RPKs by mycotoxigenic fungi. Whereas RPKs are stored for 90 days, the inhibition percentages of the four types of aflatoxins ranged from 76.5 to 86.5%. However, the percent inhibition of AFB 1 was lowest for all treatments. On the other hand, the inhibitory effect increased to 87.5% for OTA. Finally, this is the first study to use AECP in monitoring AFs and OTA during peanut storage. Still necessary are further studies to assess the sensory evaluation, and impact of roasting process after treatment by AECP. a commercial scale.