Applications of Some Natural Active Extracts for Cereal Storage Improvement
Abdelazim SAA1, Ahmed NB2 and Mohamed GS3
1 Department of Field Crop Technology Research, Agriculture Research Centre, Egypt.
2Department of Food Toxicology and Contaminants, National Research Centre, Egypt
3Arid Lands Cultivation Research Institute, City of Scientific Research and Technological Application, Egypt
Submission: July 02, 2018; Published: November 16, 2018
*Corresponding author: Abdelazim Sayed Abdelazim, Department of Field Crop Technology Research, Food Technology Research Institute, Agri culture Research Centre, Giza, Egypt.
How to cite this article: Abdelazim SAA, Ahmed NB, Mohamed GS. Applications of Some Natural Active Extracts for Cereal Storage Improvement. Nutri Food Sci Int J. 2018; 7(5): 555723. DOI: 10.19080/NFSIJ.2018.07.555723.
Abstract
The impact of some plant extract as natural antioxidant antimicrobial agents and their toxic (Arabic gum, pomegranate peels and Propolis) were evaluated. Phenolic contentwas228.2±17mg GAE/g in Pomegranate peels extract and 87.1±2.3, 9.7±0.1mg GAE/g for Propolis and Arabic gum, respectively. Total flavonoid ranged from 24.41±1.7 to 42.15±2.83mg Catechol/g in Propolis and pomegranate peels respectively, while it was not present in the Arabic gum. Our results show the presence of a strong association between the scavenging activity against the stable ABTS, DPPH, FRAP radical and total phenol for extracts. Propolis was the most effective extract in either minimal inhibition concentration (MIC) or minimal bacterial concentrations (MBC) values of Propolis were ranged (1.2-1.5mg/ml between 1.5-1.9mg/ml respectively) which considered closed to the standard antibiotic values. Data represented in the order of the antifungal activities for use substrates as Propolis >pomegranate peels > Arabic gum. The high antibacterial impact of Propolis was recorded on the bacterial strain of staphylococcus aureus. The pomegranate peel extract had the higher reducing effect on Aflatoxin B1 than Propolis extract and Arabic gum extract (36.39±3.84, 14.93±2.31, 5.70±4.37(ng/g) respectively, however, for AFG1 the pomegranate peels recorded higher reducing ratio more than the two other extracts. The coating rice with pomegranate peels extract had the highest resistance toward the fungal growth during storage than Propolis and Arabic gum extract, respectively. The amount evaluated of aflatoxins on the rice grains recorded a variation in the extract's ability to limit the aflatoxins presence on grains. Were ordering as pomegranate peels extract > Propolis extract > Arabic gum extract. We can confirm the possibility of using these substances in functional foods.
Keywords: Arabic gum; Pomegranate peels; Propolis; Natural antioxidant; Antifungal agents; Aflatoxin B1and Aflatoxin G1
Introduction
Mycotoxins, which are fungal secondary metabolites produced during toxigenic fungi metabolism process, are very harmful compound could be found in the feed and food products [1]. Mycotoxin may also the main causes of various types of a health hazard for humans and animals. It consists of a large group of compounds, recently it has reached about four hundred types of toxic materials. Each species of fungi had the ability to excrete one or more of toxic substances, in the same times, one toxin may produce by more than one fungi. It may contaminate food and feed chains which deem the main reason of toxic impacts in the higher creatures when consumed impure commodities [2]. However, like all over the world, Mycotoxins are a public health worry for Egyptian populations. Cereals, crops, Agricultural products are the major food products suffering from mycotoxin contamination.
Toxigenic fungi are at most out righted by Aspergillus, Penicillium, Alternaria, and Fusarium, however, Aspergillus among the others is very significant fungi, it takes into consideration as a contaminant in either food/feed materials, and it is a plant pathogen. Aspergillus and Penicillium fungi are mycotoxins producer. Otherwise, toxigenic fungi which recorded to be high grown on cereals are Alternaria, Penicillium, Fusarium, Cladosporium, Stachybotrys, Trichoderma, Acremonium and Paecilomyces [3,4]. Ultimately, there are four groups of aflatoxins AFB1, AFB2, AFG1, and AFG2 and its two metabol ism derivatives AFM1 and AFM2; from which AFB1 is the extreme potency aflatoxins that incentive the health problems to human and animal. Aflatoxins are a toxic compound to human and animal and the main reason for different [5].
Aflatoxins are particularly a problem in hot and dry cultivated areas, aflatoxins expansion is exacerbated due to the factors affecting of its existence which involves the drought, pests’ infection, and the delayed harvest. As one more point, insufficient drying and poor post-harvest handling are also included as a factors increase the aflatoxin infection [1]. Furthermore, Mycotoxin production by fungi could happen in the storage area causing spoilage in cereals, subsequently actually creating a health problem when it consumed by animals or human. Another hazard factor is the cropping system where previous crop and soil plowing could effect on the resultant crop safety [6].
Aflatoxin B1 has classified as group A carcinogens, according to IARC classification report for carcinogenic substances. The modern suggested techniques for aflatoxins reduction are the exclusion of aflatoxin-producing fungi by non-toxigenic strains either simultaneously or with an interval time difference. Different techniques for aflatoxin reducing has applied in the feed using aflatoxin’s binders and additives in animal feed. Also, chemical treatment of contaminated crops is a wonderful method for mycotoxin decreases like the ozone fumigation and treatment with ammonia. Recently, biocontrol of toxigenic fungi to suppress its growth was utilized to reduce the mycotoxin excretion amounts. Moreover, application of probiotic bacteria to reduce aflatoxin uptake record a positive result in many cases. Use of the superabsorbent polymers and the essential oils in mycotoxin binding reported as a successful application, avoiding mycotoxin contamination-hazard in animal feed [7].
Antioxidants are components which prohibit the oxidation process either in food substances or body fluids. The free radical activity has been involved in cancer, tissue damages, aging, and destruction of DNA. Recently, there has been an increase to examine more substances that can defend against these reactive materials species and play a significant role in disease inhibition [8]. Antioxidants play an important role in decreasing mycotoxins impacts in food and feed products [5,9]. Many substances had antioxidation effects such as phenolic components in plant materials, also, flavonoids, plant extracts, and food residual sources. The plant extracts considered a rich material in bioactive substances, it could use to include in the bio-films which applied to reduce mycotoxin contamination that happens in cereal and crops [10].
The pomegranate (Punicagranatum L.) is a shrub plant mainly grew in the hot and moderate weather. It is widely spread in North Africa, Mediterranean area, India, and Southern East area of Asia. Pomegranate fruits are rich in bioactive substances including the phytochemicals like tannins, anthocyanins, and ellagic acid, and other phenolics. It has been utilized widely in folk medicine in many countries all over the world. Anthocyanin in pomegranate has also is a natural dye which preferred in some types of food products. Moreover, the Propolis is a natural sticky component possessed by the bees from different plants and flowers roots. Chemically the propolis takes notice as a better source of natural antioxidants and antibacterial [11]. Propolis includes an assortment of chemical components like coumarins, steroids, polyphenols, sesquiterpene, quinines, and inorganic materials. Phenolic compounds are the most significant compounds found in propolis,in particular flavonoids and phenolic acids [12]. Propolis has described having various biological effectiveness, including the antibacterial, antifungal, anticancer, antiviral and antitumor characteristics. Consequently, propolis utilized in pharmaceuticals and nutraceuticals to promote health and prevent diseases like pre-cancer, inflammation, hemorrhagic, and chronic diseases [13]. Arabic Gum is an edible, dried, the sticky exudate substance from the specific types of tree stems and branches. Oral intake of Arabic gum has reported to prolonging various health benefits, it may serve as an antioxidant, anticancer, and antitumor compound [14]. Gum Arabic deems an essentially effective due to the powerful antioxidant characteristics.
The aim of the present study was to evaluate the antimicrobial, antifungal, antioxidant, and ant mycotoxigenic potency of Pomegranate peels, Propolis, and Arabic gum against some types of food harmful. These hazards included the toxigenic fungi, pathogenic bacteria, and mycotoxins. The result of this study will assist to increase the shelf-life of food and feed products encompass the cereals and its based products. This study is the utilized of food grade extracts with lower cost to enhance the main food products safety and quality. The extraction applied in this study not only safe, cheap, and easy applied; it will also increase the nutritional values of the final products.
Materials and Methods
Collection of plant materials and sources
Arabic gum (AG)obtained from the Alsamandal for import and export, Batch No.2120, Manufacturing date 10/10/2016, Expired 30/9/2020, From Sudan. Pomegranate peels (PGP) isolated from the Manfaloty Pomegranate fruits purchased from the local market, after collecting it washed, dried, ground and stored in the freezing conditions until applied. Propolis (PP) purchased from the Agricultural Research Center, Beni-Suef Governorate. The bacterial strains (Bacillus cereus ATCC 11778, Salmonella typhi ATCC 14028, E. coli ATCC11229 ,Pseudomonase aerugnosa ATCC 9027,Enterococcus faecalies ATCC 47077, Staphylococcus aureus NCTC 10788) and The fungl strains (Aspergillus paracitcus ATCC 15517, Aspergillus niger ITEM 3856, Aspergillus parasiticus ITEM 11, Aspergillus carbonarius ITEM5010, Aspergillus flavus ITEM 698, Aspergillus oryzae ITEMB5,Penicillium citrinum NRRL 1841T,Alternaria alternata ATTC 96151 , Fusarium oxysporum ITEM 12591,Fusarium graminearum ITEM 124, Fusarium solani ITEM 250 and Penicillium chrysogenum ATCC 10106)
Methods
Preparation of extracts: Extracts of pomegranate peels (PGE), propolis (PPE), and Arabic gum (AGE)prepared using isopropyl: water (1:1 v/v) as eco-friendly solvents. Waterisopropyl (v/v) was added to the finely ground powders at a ratio of 1:10 (w/v). The mixture stirred using an overhead stirrer for 50 min at 45 °C. The extracts, then separated from the residue by filtration through Whatman No.4 filter paper. The filtrate concentrated under vacuum at 45 °C using a rotary evaporator. The extra solvent was removed by freeze-drying and the dried powder stored at -20 °C until further analyses
Total phenolic content: The Folin-Ciocalteu reagent assay was used to determine the total phenolic content of extracts as described by Ozsoy et al., [15]. The total phenolic content expressed in mg Gallic acid equivalents (GAE)/g of sample (mg/g). All determinations performed in triplicate.
Total flavonoid content: The total flavonoid contents of the pomegranate peels (PGE), propolais (PPE), and Arabic gum (AGE) extracts determined by a colorimetric method described by Sakanaka et al. [16], the absorbance measured at 510nm. The results were expressed as mg cat./g of sample. All determinations were performed in triplicate.
DPPH radical scavenging activity: The free radical scavenging activity of pomegranate peels (PGE), propolais (PPE), and Arabic gum (AGE) extracts measured by the DPPH method as proposed by Turkmen et al. [17]. The measured conditions recorded at 517nm with a spectrophotometer.
ABTS cation decolorization assay: ABTS radical assay used to evaluate the ability to scavenge free ABTS radicals, based on the protocol of Re et al. [18]. Absorbance readings measured using a spectrophotometer at 734nm. Results were expressed as μ mol of trolox equivalents (TE) per g sample from a standard curve developed with Trolox. All determinations performed in triplicate.
Ferric reducing ability (FRAP)assay: The FRAP assay was done according to according to Hwang & Do-Thi [19]. Readings of the colored product, absorbent measured at 593nm. The standard curve was prepared using Trolox and the results were expressed as mMTorlox equivalent (TE/g sample).
Determination of minimal inhibition concentration of extracts: The minimal inhibitory concentration (MIC) value is determined as the lowest concentration of extract that didn’t give any visible bacterial growth. Each assay carried out in triplicate. The MIC test quantified the antimicrobial activity of the extract, it was done as the method described by Sakanaka et al., [16].
Determination of minimal fungicidal concentration of extracts: The hypha growth inhibition test can be used to determine the antifungal activity of the extracts against fungal strains as previously described by [20]. The concentration required to give 50% inhibition of hyphen growth IC50will be calculated from the regression equation. Nystatin can be used as a positive control.
Determination of several types of extraction on fungal growth: Yeast extract sucrose (YES) used to examine the effect of each extraction method which may include several components of the fungal growth of Aspergillus parasiticus ATTC 15517, also the powder of the three materials examined by using 0.5g of each as additional material in liquid media. The liquid media of YES consists of 15g yeast extract, 150g sugar, and completely to one literusing de-ionize double distilled water. The inhibition effect represented as the loss of the fungal growth in the presence of extracts against the control growth.
Preparation of standards for aflatoxin: The standard of Aflatoxins received as dry films or crystals to container of dry aflatoxins B, G, add a volume of the following solvents: methanol: acetonitrile (9:1) calculated to give a concentration as ng/ml.
HPLC Chromatography: One hundred microliters of the samples were injected into the HPLC column. The mobile phase was an acetonitrile: water: acetic acid solution (51:47:2, v/v). The flow rate was 1ml/min. High performance liquid chromatography (HPLC) system consisted of Waters Binary Pump Model1525, Model Waters 1500 Rheodyne Manual Injector, Waters 2475 Multi-Wavelength Fluorescence Detector, and a data workstation with software Breeze. A Phenomenex Column C18, dimensions: 250× 4.6mm, particle size: 5μm, from Waters Corporation (USA) as well as Microfiber Filters, 11cm, product ID: 31955, VICAM Company (Sweden), were used.
Detoxification methods: The extracts prepared by using aqueous isopropyl, the extracted materials concentrated to near dried, then freeze-dried used to convert it as powder materials, 5mg of each extract concentrate was dissolved in 0.5mL in Dimethyl sulphoxide (DMSO), this value used to inject in the plate wells or in the plate disks as a detoxification application against either pathogenic bacteria or toxigenic fungi [21,22].
Total fungal count of toxigenic fungi inoculated to rice with several treatments before and after 60 days of storage: To apply the results of the study, the antifungal ability of each extract of PGE, PPE, and AGE were applied on rice grain samples. A rice samples equal to 150g were brushed with a water solution of each extract for 3 min. After that, the samples were dried in hot air oven at 45 °C for 5-6 h to reach 11% moisture content. The samples of the three extracts treatments, side of the control without treatment were packed in a sterilized polyethylene bag. Each type of extract treatment was inoculated by nine strains of mycotoxigenic fungi (one strain/one bag/one treatment). The initial count of each inoculated strain at 25 °C was recorded as before treatment and at the end of the storage period (60 days) the final count of each strain recorded as after treatment value.
Application of some natural active extracts on rice grains as cereal food preservatives: To apply the results of the study, the antifungal ability of each extract of PGE, PPE, and AGE were applied on rice grain samples. A rice samples equal to 150g were brushed with a water solution of each extract for 3 min. After that, the samples were dried in hot air oven at 45 °C for 5-6 h to reach 11% moisture content. The samples of the three extracts treatments, side of the control without treatment were packed in a sterilized polyethylene bag. The bags of the three treatments and the control were inoculated with a high producing toxic strain of Aspergillus parasiticus ITEM 11 and incubated at 25 °C for 15 days. The result of aflatoxins amount that excreted on the rice seeds were evaluated using HPLC techniques.
Statistical evaluation
The obtained results were evaluated statistically using analysis of variance (One-way ANOVA analysis) using SPSS 16.0 as reported by McClave & Benson [23].
Results and Discussion
Total phenolic and flavonoid content
The total phenolic content (TPC) and total flavonoid contents (TFC) of the three extracts comprised PGE, PPE, and AGE have evaluated. The results were recorded as shown in Figure 1. The TPH was 228.2±17, 87.1±2.3, 9.7± 0.1mg GAE/g for PGE, PPE, and AGE, respectively. However, The TFC recorded at values of 24.41±1.7 and 42.15±2.83mg Catechol/g for Propolis and pomegranate peels, respectively; otherwise, it was not detected in the Arabic gum. Health benefits of the bioactive molecules included the flavonoids was exhibited and its potent antioxidant impact had proved [24]. The TPH and the TF were significantly different among different extracts as reported in Figure 1. Our results are compatible with several recent studies [24-26], these studies reported the highest values of TPH in pomegranate peels and Propolis. There are various reports of TPH and TFC in pomegranate peels with a wide difference in the published results is remarked. This could be the result of geographical, cultivar [27- 29]. Variations in the results might be also due to the different extraction procedure used in the experiments, particularly due to the type and the concentration of the starting extraction solvent. Fawole et al., [28] noted that the aqueous ethanol was effective in the TPH extraction of PGP with values ranged from 180 to 290mg GAE/g. The TPH in PP ranged from 79.8 to 156.3μg/g, the average being 119.5μg/g. As a result of this discussion, aqueous solvent extraction technique could be a superior system for the effective bioactive compound extraction from PGP (Figure 1).
The antioxidant activity of the PGE, PPE, and AGE extracts using DPPH, ABTS, and FRAP assays
Antioxidant activity of three extracts of PGE, PPE, and AGE measured using three parallel test assays, the results are presented in Table 1. The values indicate that; the IC50 of extracts is ordering descending at P=0.05 from PGE to AGE. The antioxidant potency of PGE, PPE, and AGE evaluated using three different assays, these were DPPH, ABTS, and FRAP reducing ability. Among the extracts analyzed, PGE recorded the greatest antioxidant activity by the three methods. Low IC50 corresponds to a strong inhibitory capacity of DPPH, ABTS, FRAP radical, the highest IC50 is recorded for the AG.
IC50: Concentration of sample required for 50% inhibition. Results are expressed as mean of three experiments ± SD.
In a previous study; the PGE had the highest antioxidant activity among the peels, pulp and seed fractions of 28 kinds of fruits commonly consumed in China, as determined by FRAP assay that described in the study of Guo et al., [30]. While Pagliarulo, et al. [31] demonstrated that the methanol extract of pomegranate peels had the highest antioxidant activity among ethyl acetate, methanol, and water extracts. Moreover, Li et al. [32] reported the aqueous methanol to be more effective than each solvent alone. The antioxidant activity potential of Propolis may be due to the presence of diverse phytochemical compounds, mainly flavonoids and phenolic acids [25]. The decrease of DPPH radical by the different plant extracts has been attributed by many authors, at the presence of phenolic compounds which yield simply to reduce protons [32]. Our results recorded the presence of a strong association between the scavenging activities against the stable ABTS, DPPH, FRAP radical and total phenol for extracts. This could the main reason in the antimicrobial, antifungal and antimycotoxigenic impact of these types of extracts. These results are in agreement with results of Abdelazim [33] who found that Silymarin can be used as natural antioxidants and antimicrobial activity in the food industry (Table 1).
Minimal inhibitory and minimal bacterial concentration
The MIC values of the three extracts for the raw materials of PGPE, PPE, and AGE evaluated to explain the antimicrobial impact. Moreover, minimal bacterial concentration (MBC) were determined to explore the ability of these extracts to apply as antibacterial components in food productions. Four pathogenesis bacterial strains were applied to examine the inhibition of its growth in the presence of these GRAS extracts, Amoxicillin antibiotic was utilized as a standard reference to compare the antibacterial effects. Represented as the data in Table 2, Propolis was the most effective extract in either minimal inhibition concentration. MIC and MBC values of propolis were ranged between (1.2-1.5mg/ml and1.5-1.9mg/ml respectively) which considered closed to the standard antibiotic values. Among the three extracts, Arabic gum extraction showed the lowest values either for MIC or the MBC on the pathogenic bacterial strains in this experiment. The results of MIC and MBC values of the three types of the extracts were applied against three types of pathogenic strains, these strains were Pseudomonase aerugnosa ATCC 9027, Enterococcus faecalies ATCC47077, and Staphylococcus aureus NCTC 10788. The strain of Enterococcus faecalies ATCC47077 recorded a high resistance value either in MIC or in MBC for the three types of extracts.
On the other hands, some of the previous studies reported an antibacterial activity of ethanolic extracts of the Propolis, the results manifested a high antibacterial action against grampositive bacteria with a MIC ranging from 0.2 to 0.78 mg/mL, also it was reported an elevated antibacterial activity against gramnegative bacteria Salmonellae Terica [25,26] (Table 2).
*MIC minimal inhibitory concentration
**MBCminimal bacterial concentration
Antifungal effect of different extraction types on Aspergillus parasiticus
Antifungal effect of several types of the extraction methods of Arabic gum, Propolis, and pomegranate peels were applied on YES media against the fungal growth of Aspergillus parasiticus ATTC 15517 showed in Table 3).The extracts were ass to the YES liquid media with a concentration of 10mg/ml for each material, also the powder of the three materials was examined by using 0.5g of each as additional material in liquid media. The results represented a good inhibition impact of the pomegranate peels on the fungal growth for the aqueous extraction methods as well as for the powder addition in the media (Table 3).
*Data were represented as mean of values ±SD.The weight of fungal growth on liquid media is recorded in grams
Antifungal activity of pomegranate peels, propolis extracts and Arabic gum against tested fungus strains (mm) showed in Table 4. In order to the need to ensure the results, the antifungal of these plant materials, extracts were tested by other antifungal method. Plate inhibition zone assay of the extracts against four toxigenic fungi strains were applied to explore the variations recorded between the several materials in the antifungal effect, this will be useful to determine the suitable amounts which aimed to be applied as food additive to enhance the good qualities of food against the fungal contaminations. The data represented in the order of the antifungal activities for used substrates as Propolis > Pomegranate peels>Arabic gum. The extract of Propolis showed a closed result to the standard antifungal compound (Nystatine). The high antifungal impact of Propolis was recorded on the Fusarium fungi (Table 4).
*Data were represented as mean of values ±SD. Each value represents the mean of three replicates.
Antibacterial activity of the extracts of pomegranate peels, propolis and arabic gum extracts
Antibacterial characteristics of the three materials, extracts were measured as an inhibition zone on them against the bacterial growth of four pathogenic strains on potato dextrose agar supplemented by Nystatine as antifungal antibiotic to suppress the fungi and the results showed in Table 5. Plate inhibition zone assay of the extracts against four bacterial strains were applied to explore the variations recorded between the several materials in the antibacterial impact, this will help to evaluate the application of these extracts during the food manufacturing to support the good qualities of food products as well as its expected application in food commodities coating against the bacterial spoilage. The data represented in the order of antibacterial activities for use substrates as Propolis >pomegranate peels>Arabic gum. The extract of Propolis showed a closed result to the standard antifungal compound (Amoxicillin). The high antibacterial impact of Propolis was recorded on the bacterial strain of Streptococcus aureus (Table 5).
Data were represented as mean of values ±SD.Each value represents the mean of three replicates.
In a previous study, The PP reported to had an antibacterial activity against some strains of testing pathogens such as; Salmonella enteritidis ATCC 13076, Staphylococcus aureus ATCC 13565 Escherichia coli ATTC 25923, Listeria monocytogenes and Bacillus subtilis NCTC 8236 [34]. Antimicrobial activity of PP also reported by using several types of solvents (Ethanol, Acetone, Toluene). Otherwise, the change in the pH values of the resultant extract at three different pH values (6,7,8) showed a widely range of PP antibacterial activity [35]. This result agrees with Chacko and Estherlydia & Labyad et al., [36,37], they described exhibited antibacterial activity of PGP against various Gram-positive and Gram-negative bacteria of pomegranate peels was characterized by the highest antibacterial activity against of Shigellastrain of pathogenic bacterial [38].
Notwithstanding; Rather & Rajagopal [39] extracted phenolics from dried powdered PP with various solvents (methanol, ethyl acetate, chloroform, and water) and revealed the antimicrobial activity of methanolic extract against Escherichia coli, Pseudomonas sp., Klebsiella sp., vancomycin and methicillin resistant Staphylococcus aureus.
Anti-aflatoxigenic effect of Pomegranate peels, propolis and arabic gum extracts against aflatoxins in liquid media
Reducing the impact of iso-propanol-water extracts of Pomegranate peels, Propolis and Arabic gum on aflatoxin B1 and aflatoxin G1 were exanimated in the YES fungal growth of liquid media, the amount of AFB1 and AFG1 were determined in the control flask of Aspergillus paraciticus strain ATCC 11517 and the results illustrated in Figure 2. The toxin amount was indicated the control aflatoxins amount of AFB1 and AFG1. While, other flasks were supported by the extracted materials at a concentration of 10mg/ml in the growth media. Using the apparatus of HPLC, the reducing amount of aflatoxins was recorded against the amount that excreted in the control flask. The reducing amount here was reflected either the inhibition effect of the extracted material on the fungal excretion metabolites or it could be referred to the chemical reaction happen during the extraction present in growth media. The PGE had the higher reducing effect on Aflatoxin B1 and G1 more than PPE and AGE (36.39 ±3.84, 14.93±2.31, 5.70± 4.37(ng/g)) and (34.37±0.6 than 21.04±0.18 and 17.34±0.88 (ng/g)) respectively, however, for AFG1 the recorded the higher reducing ratio for each of the extracts compared to control. The AGE recorded as the lower effect than PGEand PPE) 52.9±0.88than 42.56±0.6 and 50.53±0.18 (ng/g( respectively on both Aflatoxin B1and Aflatoxin G1. Notwithstanding, the recovery for the aflatoxins from AG powder precipitated in the YES media after the experimental time was not applied to explain if the toxin could recover from the powder or not. Maybe the effect of the pomegranate peel extract is higher than the other extracts to cause the high content of polyphenolic compound (Figure 2).
Effect of natural extracts on the growth of fungi during storage period of rice
To gain the benefits of using these extracts as antimicrobial and antifungal components, it has applied as a brushing coat on the rice grains then the grains dried before packed in polyethylene bags. These bags stored for 60 days before the fungal count calculated. At the initial time; the grains inoculated by one strain of fungi for each bag and the log CFU/g of rice recorded as (before value), while at the end of the storing period the log CFU/g of rice evaluated as (after value).The reducing values of the log CFU/g indicated the power of the extract type as anti-mycotic material which help to enhancement the grain safety during the storage period. The results in Table 6 & Figure 3 showed a high powerful of each extract to reduce the log CFU/g of fungi during the storage period. The PGE coating material appeared as an excellent coated material against the nine strains of toxigenic fungi. while AGE exhibited the lowest type of coated material to resist the fungal growth on the stored rice. These results support the application of PGE as a cheap source of bioactive substances to reduce the fungal infection in the stored grain when it is applying as coating film. The results gained from this experiment was in agreement with [10] (Table 6).
Value represented at the mean value of five replecates. The LSD= 2.189
Effect of natural extracts on the reduce Aflatoxins during storage period of rice
As an application to convert the results of this experiment to increase the agriculture, sustainable production and increase the food safety, the extracts of PGE, PPE, and AGE had applied to coat the rice grains and inoculated by A. parasiticus ITEM 11 strain which recorded as a high producing strain of the four types of aflatoxins. According to the recorded results; PGE was the effective extract that able to reduce the toxin excretion on the stored rice grain during the optimum time of aflatoxin excretion by the fungi (about 15 days). The amount evaluated of aflatoxins on the rice grains recorded a variation in the extracts ability to limit the aflatoxins presence on grains. The reducing ratio were ordering as PGE, PPE, AGE, respectively (Figure 4).
Conclusion
Pomegranate peels, Propolis and Arabic gum had considerable levels of polyphenolic compounds. Their chemical characteristics induce in general a strong bactericidal action against Pseudomonase aerugnosa ATCC 9027 and Staphylococcus aureus NCTC 10788, Propolis, Pomegranate peels and Arabic gum had a reducing effect on Aflatoxin B1 and Aflatoxin G1 (ng/g). Presented results of this study demonstrated high biological potential of these extracts. Statistical analysis showed high correlation among the contents of investigating classes of compounds and biological activity of prepared extracts. All in all, these results confirmed that extract of Pomegranate peels, Propolis and Arabic gum might indeed be a potential source of biological compounds. We can could be reported can use the Propolis, Pomegranate peels and Arabic gum as plant natural antioxidant, antimicrobial agents and their toxics and as functional foods.
References
- Demissie N (2018) A Review of Aflatoxin: Occurrence, Prevention, and Gaps in Both Food and Feed Safety. Nov Tech Nutri Food Sci 8(2): 190- 197.
- Waliyar F, Siambi M, Denloye, S. and Jones R. (2008) Institutionalizing Mycotoxin Testing in Africa. In: Leslie (Ed.), pp. 367-375
- Nooh A, Amra H, Youssef MM, El-Banna AA (2014) Mycotoxin and toxigenic fungi occurrence in Egyptian maize. Int J Adv Res 2: 521-532.
- Badr AN, FL Antonio AA Hassan, Taha H (2017) Ochratoxin a occurrence on Egyptian wheat during seasons (2009-2014). Asian J Sci Res 10(3): 178-185.
- Badr AN, Fatma N, Shehata MGH.A. Amra, (2017) Anti-mycotic and anti-mycotoxigenic properties of Egyptian dill. J Applied Sci 17(4): 184-195.
- Groth CG, Bäckman L, Morales JM, Calne R, Kreis H, eta l. (1999) Sirolimus (rapamycin)-based Therapy In Human Renal Transplantation: Similar Efficacy and Different Toxicity Compared with Cyclosporine1, 2. Transplantation 67(7): 1036-1042.
- Zahra P, Hedayat H (2017) Application of Modern Agricultural Practices to Reduce Aflatoxins Exposure in Developing Countries. Nutrition and Food Toxicology 2(3): 346-347.
- Kapadiya DB, Dabhi BK and Kishor kumar DA (2016) Spices and Herbs as a Source of Natural Antioxidants for Food. Int J Curr Microbiol App Sci 5(7): 280-288.
- Abdel-Razek AG, Noah Badr A, Shehata MG (2017) Characterization of Olive Oil By- products: Antioxidant Activity, Its Ability to Reduce Aflatoxigenic Fungi Hazard and Its Aflatoxins. ARRB 14(5): 1-14.
- Shehata MG, Badr AN, Abdel-Razek AG, Hassanein MM, Hassan AA (2017) Oil- bioactive Films as an Antifungal Application to Save Postharvest Food Crops. ARRB 16(4): 1-16.
- Narbona Em, Garcı´a-Garcı´a E, Va´zquez-Arau´jo L, Carbonell Barrachina AA (2010) Volatile composition of functional ‘a la Piedra’turro´n with propolis. International Journal of Food Science and Technology 45(3): 569-577.
- Falcao SI, Vilas-Boas M, Estevinho LM, Barros C, Domingues MRM, et al. (2010) Phenolic characterization of Northeast Portuguese propolis: usual and unusual compounds. Anal Bioanal Chem 396(2): 887-897.
- Sforcin JM, Bankova V (2011) Propolis: Is there a potential for the development of new drugs? J Ethnopharmacol 133: 253-260.
- Ali A, Cheong CK, Zahid N (2014) Composite effect of Propolis and gum arabic to control postharvest anthracnose and maintain quality of papaya during storage. Int J Agric Biol 16: 1117-1122.
- Ozsoy N, Can A, Yanardag R, Akev N (2008) Antioxidant activity of Smilax excelsa L. leaf extracts.Food Chemistry 110: 571-583.
- Sakanaka S, Tachibana Y, Okada Y (2005) Preparation and antioxidant properties of extracts of Japanese persimmon leaf tea (kakinoha-cha). Food Chemistry 89(4): 569-575.
- Turkmen N, Sari F, Velioglu YS (2005) The effect of cooking methods on total phenolics and antioxidant activity of selected green vegetables. Food Chemistry 93(4): 713-718.
- Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, et al. (1999) Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med 26(9-10): 1231-1237.
- Hwang ES, Do-Thi N (2014) Effects of extraction and processing methods on antioxidant compound contents and radical scavenging activities of laver (Porphyra tenera). Prev Nutr Food Sci 19(1): 40-48.
- Picman AK, Schneider EF, Gershenzon J (1990) Antifungal activities of sunflower terpenoids. Biochemical Systematics and Ecology, 18(5): 325-328.
- Esper RH, Gonçalez E, Marques MO, Felicio RC, Felicio JD (2014) Potential of essential oils for protection of grains contaminated by aflatoxin produced by Aspergillus flavus. Front Microbiol 5: 269.
- Balouiri M, Sadiki M, Ibnsouda SK (2016) Methods for in vitro evaluating antimicrobial activity: A review. J Pharma Anal 6(2): 71-79.
- McClave JT, Benson PG (1991) Statistical for Business and Economics. Max Well Macmillan International (edn.), Dellen Publishing Co., USA., pp: 272-295.
- Orak HH, Aktas T, Yagar H, Isbilir SS, Ekinci N, et al. (2012) Effects of hot air and freeze drying methods on antioxidant activity, colour and some nutritional characteristics of strawberry tree (Arbutus unedo L) fruit. Food Sci Technol Int 18: 391-402.
- Mohdaly Adel AA, Mahmoud AA, Mohamed HHR, Iryna Smetanska, Mohamed FR (2015) Phenolic Extract from Propolis and Bee Pollen: Composition, Antioxidant and Antibacterial Activities. Journal of Food Biochemistry 39(5): 538-547.
- Ramadan MF, Mohdaly AA, Assiri AM, Tadros M, Niemeyer B (2016) Functional characteristics, nutritional value and industrial applications of Madhuca longifolia seeds: an overview. J Food Sci Technol 53(5): 2149-2157.
- Lu Y, Ma W, Hu R, Dai X, Pan Y (2008) Ionic liquid-based microwaveassisted extraction of phenolic alkaloids from the medicinal plant Nelumbo nucifera Gaertn. J Chromatogr A1208(1-2): 42-46.
- Fawole OA, Makunga NP, Opara UL (2012) Antibacterial, antioxidant and tyrosinase- inhibition activities of pomegranate fruit peel methanolic extract. BMC Complementary and Alternative Medicine 12: 200.
- Hasnaoui N, Wathelet B, Jimenez-Araujo (2014) Valorization of pomegranate peel from 12 cultivars: dietary fibre composition, antioxidant capacity and functional properties. Food Chemistry 160: 196-203.
- Guo C, Yang J, Wei J, Li Y, Xu J, et al. (2003) The antioxidant activities of peels,pulp and seed fractions of common fruit as determined by FRAP assay. Nutrition Research 23(12): 1719-1726.
- Pagliarulo C, De Vito V, Picariello G, Colicchio R, Pastore G, et al. (2016) Inhibitory effect of pomegranate (Punica granatum L.) polyphenol extracts on the bacterial growth and survival of clinical isolates of pathogenic Staphylococcus aureus and Escherichia coli. Food Chem 190: 824-831.
- Li Y, Guo C, Yang J, Wei J, Xu J, et al. (2006) Evaluation of antioxidant properties of pomegranate peel extract in comparison with pomegranate pulp extract. Food Chemistry 96(2): 254-260.
- Abdelazim SAA (2017) Effect of Silymarin as Natural Antioxidants and Antimicrobial Activity. Egypt J Agric Res 95(2): 725-737.
- Attalla KM, Ayman AO, Karem MM (2007) Antibacterial activities of bee venom, propolis, and royal jelly produced by three honey bee, Apis mellifera L., hybrids reared in the same environmental conditions. Annals of Agric Sci Moshtohor 45(2): 895-902.
- Salman HD (2016) Antibacterial Activity of Propolis Extracted in Three Different Solvents and in Three Different pH values on some Pathogenic Bacteria. International Journal of Pharm Tech Research 9(10): 258-266.
- Chacko CM, Estherlydia D (2014) Antimicrobial evaluation of jams made from indigenous fruit peels. International Journal of Advanced Research 2(1): 202-207.
- Labyad N, Basma D, Nouha SE, Marwa MA, Masoud K (2016) Phytochemical antioxidant and antimicrobial study of Libyan propolis ethanolic extract. International Journal of Herbal Medicine, 4(5): 01- 04.
- Vodnar DC, Călinoiu LF, Dulf, FV, ŞTefănescu BE, CriŞan D (2017) Identification of the bioactive compounds and antioxidant, antimutagenic and antimicrobial activities of thermally processed agro-industrial waste. Food Chemistry 231: 131-140.
- Rather RA, Swetha C, Rajagopal K (2010) Screening of peel extracts as antioxidants, anticancer agents and antimicrobials. Advances in Bioresearch 1(1): 29-33.
- Abuarra A, Hashim R, Bauk S, Kandaiya S, Tousi ET (2014) Fabrication and characterization of gum Arabic bonded Rhizophora spp. particleboards. Materials and Design 60: 108-115.
- Anu Aravind A, Asha K, Ramesh kumar K (2016) Phytochemical analysis and antioxidant potential of the leaves of Garcinia travancorica Bedd. Nat Prod Res 30(2): 232-236.
- Fumonisin B (2002) Some traditional herbal medicines, some mycotoxins, naphthalene and styrene. IARC monograph sonthee valuation of carcinogenic risks to humans.
- IARC (2012) Monographs on the evaluation of carcinogenic risks to humans: chemical agents and related occupations. A review of human carcinogens. Lyon, France: International Agency for Research on Cancer 100F: 224-248.
- Ismaiel AA, Papenbrock J (2015) Mycotoxins: Producing Fungi and Mechanisms of Phytotoxicity. Agriculture 5(3): 492-537.
- Keno T, Azmach G, Wegary D, Worku M, Tadesse B, et al. (2018) Major Biotic Maize Production Stresses in Ethiopia and their Management through Host Resistance. African Journal of Agricultural Research 13(21) : 1042-1052.
- Noah BA, Nada F, Shehata MG, Amra HA (2017) Anti-mycotic and Anti-mycotoxigenic properties of Egyptian dill. Journal of Applied Sciences17: 184-195.
- Waliyar F, Kumar PL, Traoré A, Ntare BR, Diarra B, et al. (2008) Preand post- harvest management of aflatoxin contamination in peanuts. Mycotoxins: detection methods, management, public health and agricultural trade. CABI, Wallingford, UK, pp. 209-218.