INTRODUCTION:

In Algeria; the lack of sanitary controls, the poor packaging of food and the absence of strict regulations as well as the harvesting and storage conditions increase the rate of contamination by molds and by its geographical location and its characteristics of regional diversity, Algeria offers a wide variety of medicinal plants. In this context we tried to use the exuded gum Acacia tortilis (Forssk) for the storage of grains of common wheat¹.

The different parts of Acacia: leaves, pods, gum exudate and bark are very useful for medical, pastoral and commercial purposes.

The tannin from the bark is used as a color, the pods and gum as food, and the leaves to fertilize the soil. In medicine, Acacia tortilis is useful for the treatment of skin allergies, diabetes, hypertension².

MATERIALS AND METHODS:

Preparation and sampling the gum arabic:

Because it is characterized by abundance; We have chosen to take samples in the Tindouf region; The gum arabic "exudate of solidified sap", produced naturally or following an incision on branch and stem of the tree, was harvested at coordinates (27° 43'33.31 "N 8° 4'15.54"O).

The gum arabic was taken to the laboratory, cleaned with distilled water and wiped with a clean cotton cloth, then dried.

The dried gum arabic was crushed successively in a porcelain mortar. The powders obtained were sieved using a sieve of 160 μm mesh diameter and then chemically characterized.

Screening phytochemical:

Phytochemical screening by color reactions^{3, 4, 5} (table1).

Extracts reparation:

The gum powder is dissolved in sterile distilled water using a vortex shaker to obtain the following concentrations (1g / ml).

Soft wheat sampling:

Sampling is carried out according to ISO 13690. 22 samples of common wheat imported from France were taken during the 2014 period, at the port of Mostaganem, Algeria; and then transported to the laboratory. Each sample was designed in 3 sub-samples.

Storage tests for common wheat by coating with aqueous extract of the gum arabic studied:

The test is carried out as follows; each sample is moistened with 1 kg of soft wheat per one liter of this concentration (1g/ml) of the aqueous extract of the gum used to coat the wheat grains with the gum. In flacons; the sample is then dried and stored for a period of 5 years (from March 2014 until March 2019). The effectiveness of these methods is assessed by mycological analyzes of all samples before and after coating (figure 1).

Table 1 Different reagents and reactions for phytochemical screening of gum.

Components		Reagents and reactions		Results	References
Alkaloids		The reagent of Dragendorff	H₂SO₄	Orange precipitate	[6] [7]
Reducing compounds		Fehling liquor	Heater	Brick red precipitate	[8]
Cardiac glycosides		Chloroform	H₂SO₄	Reddish-brown	[9]
Tannins	Catechic tannins	Solution of Fecl₃ diluted to 1%		Dark green coloring	[8]
	Gallic tannins			Blue-green coloring
Flavonoids		The reaction to cyanidin	Isoamyl alcohol	Orange or purplish pink	[6]
Saponins		Solution to analyze prepare by decoction in aqueous, hydroalcoholic medium		Foam	[8]
Sterols and tri terpenes		The reaction of Liebermann		Purple or purple ring turning blue then green	[9]
Coumarins		NH₄OH	Ammonia	Fluorescence is observed under UV at 366 nm	[9]
Free quinones		Petroleum	NaOH 1/10	Turns yellow, red or purple	[10]
Cyanogenic compounds		CHCl₃	Filter paper strip impregnated with picrate sodium.	Turning red on the strip indicates

Fig. 1 Comparison between (A) Wheat grains coated with gum and (B) Wheat grain none coated.

Mycological analysis:

Isolation and count:

Fungi isolation, from soft wheat samples (with and without the gum), was carried out according to the method proposed by Mills and al. (1978)¹¹. 100 randomly selected wheat grains from each sample were placed in 10 sterile Petri dishes containing sterile filter paper, soaked with 5 ml of sterile 7.5% aqueous sodium chloride solution.

Strains’ identification and purification:

After the purification of the isolated strains on an acidified PDA and Sabouraud¹², the identification was based on the technology of Pitt and Hocking 2009.

RESULTS AND DISCUSSIONS:

Phytochemical screening of gum:

From the results obtained in the table 2, we noted that gum arabic, is very rich in reducing compounds, saponins, polyphenols and As less we find coumarins, flavonoids; similarly we recorded an absence gallic tannins, sterols and triterpenes, free quinones and cyanogenic compounds.

Our phytochemical study carried out on acacia gum Arabic showed results which are confirmed with other works; Saini and al. (2008)¹³ have found that both species of Acacia gave positive result for alkaloid.

Table 2 Results of phytochemical screening of gum.

Components		Results
Alkaloids		+
Reducing compounds		++
Cardiac glycosides		++
Tannins	Catechic tannins	+
	Gallic tannins	-
Flavonoids		+
Saponins		+++
Coumarins		+
Free quinones		-
Cyanogenic compounds		-
- : absent, + : present

The mathanolic extracts of Acacia gum gave positive result for saponin and flavonoid. Similarly, the study conducted by Sharma and al. (2017)¹⁴ revealed the presence of alkaloids, flavonoids and glucosides in the gum and two authors have shown^{15, 16} presence of tannins, steroids, cardiac glycosides, flavonoids, saponins, anthraquinone, alkaloids and volatile oil.

Estimation:

The samples are found to be contaminated by 7 different genera of molds (figure 2), of which

Fig 2. Some grains contaminated with mold.

Aspergillus (represented by 9 species figure 3) is the most dominant with a frequency of appearance more than 49% and for the genus Penicillium (represented by 4 species figure 4) contaminates 33, 65% in PDA.

The genera of the mucorals, Monilia, Eurotium, Alternaria and Fusarium contaminate samples with proportions respectively as follows: 5.53%, 1.64%, 6.38%, 2.57% and 0.78%. The figure 5 shows the frequency of contamination of Molds in the soft wheat samples.

According to Wicklow (1998)¹⁷, shows that sporulation and dissemination of spores at high speed is the great common characteristic of fungal species in poorly hydrated foodstuffs. These results are in agreement with several studies which have found similar results in corn silage and sorghum^{18, 19}.

The results of the mycological analysis showed a clear dominance of the genus Aspergillus. This dominance seems to be favored by high level of humidity grain, slight acidity, a well-defined broken grain ratio.

The dominance of the genus Aspergillus in the contaminated flora of cereals has been reported in several works^{20, 21, 22}.

Thus, the species of the genus Aspergillus are considered as storage molds²³, Aspergillus flavus is the most frequent species followed by A. parasiticus and A. niger.

The rate of contamination by the genus Penicillium was found to be inversely proportional to the high humidity and the duration of storage. However, as reported by Pitt and Miscamble (1995)²¹, the other genera of mucus and Monilia sp isolated are naturally present on crops at field level and in the soil²³.

Cahagnier (1996)²⁴ reports that the presence of mucorals in cereal stocks is a sign of poor storage conditions.

Fig. 3 Frequency of appearance of Aspergillus.

Fig. 4 Frequency of appearance of Penicillium.

Fig. 5 Frequency of contamination of Molds in the soft wheat samples.

Mycological analysis:

High levels of contamination were noted in the control compare with other coated samples, the coated seeds gave overall a lower contamination rate than that obtained for the control (figure 5). Indeed, the extract of gum arabic exerted a pronounced antifungal activity on the fungal strains tested with a more or less significant rate of inhibition which resulted either in the absence or in slowing down of mycelial growth on the grains containing these different plant extracts. The pronounced antifungal activity of this gum is thought to be due to the chemical composition²⁵.

Fig. 5 The evolution of the frequency of appearance of molds of the grains coated with the extract of the gum Arabic powder and the control.

This confirms that the bioactive substances in plants are considered to be potentially effective against molds. This antifungal activity is probably due to the richness of Acacia gum in active compounds revealed by phytochemical screening, which confirms the presence of flavonoids, saponosides, sterols, steroids and tannins, these compounds have antifungal actions according to several authors^26,27,28.

Thanks to the composition which is much diversified of the gum arabic perhaps to act on several levels on the sensitive mold. The mechanism of action of extracts on the fungal flora is not clear, but the majority of reports indicate that their activity is done on the one hand by:

-Morphological modifications of the hyphae²⁹

-Direct disturbance of the fungal cell membrane³⁰

And on the other hand; the nature of the active ingredient; flavonoids, known for their multiple biological properties such as antifungal activity^{31, 32, 33, 34, 35, 36, 37, 38} have described the antifungal effects of flavonoids against grain mold.

James Bound et al. (2016)³⁹ have shown the antifungal activity of four triterpenoid saponins and the lack of genotoxic effects and have shown the antifungal activities of the new phenol and alcohol terpenes against Aspergillus flavus, Aspergillus ochraceus, Fusarium oxysporum, Saccharomyces cerevisiae and Candida albicans.

The majority of polyphenols have very powerful antifungal activity. Orturno (2005)⁴⁰ demonstrated the activity of the flavanones glycosides and polymethoxyflavones of Citrus parasidi, and of Citrus sinensis on Penicillium digitatum. Also, the flavonoids of Conyza aegyptica L. have a fungicidal and fungistatic action on different fungal agents: Microsporum canis, M. gypseum, Trichophyton mentagrophytes, Candida zeylanoïds⁴¹.

According to Moghtet S and al (2018)⁴², the phenolic compounds caused swelling of the hyphae and inhibition of plasma permeability around the hyphae. This impermeability destroys the cell walls. Gum arabic also contains several types of enzymes such as oxidases, peroxidases and pectinases, some of which have antimicrobial properties^3,43,44.

CONCLUSION:

The current study showed that gum of Acacia tortilis is rich in phytochemicals.

This plant showed potent antifungal activity. This would be useful for using gum arabic in wheat grain storage silos against mold growth.

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Received on 04.03.2020 Modified on 18.04.2020

Research J. Pharm. and Tech. 2020; 13(12):5668-5672.

DOI: 10.5958/0974-360X.2020.00987.7