Monitoring of biochemical and fermentation parameters
As reported in fig 1 and fig 2, all samples obtained from sugarcane molasses and plants raw material, fermented by the lactic acid bacteria, showed a substantial decrease of the pH and a subsequent increase of the titratable acidity due to the liberation of organic acids such as lactic, acetic, formic, malic acids…. However, there was an increase in pH values up to the end of fermentation (pH = 3). In addition, the results illustrated in the fig.2 showed that the level of the total sugar and reducing sugar, total proteins and free amino acid decreased (38 mg/l, 18 mg/l, 20 mg/l, and 8 mg/l, respectively) after fermentation in the fermented plants extract to compared with the plant extract without fermentation. In this study, we used the lactic fermentation technology applied for fermented the medicinal plants in order to enhance the extract of bioactive metabolite has a high antimicrobial and antifungal activity. The lactic fermentation process influence on physicol-chemical parameters the pH decreased and acidity increased of the obtained fermented plants extract. The biochemical analyses flavonoids, tannins and total polyphenols content of the obtained fermented plants extract Fig.2 showed that, the fermented beverages using LAB after two weeks of fermentation increase the levels of total phenolics, flavonoids contents and tannin content (152.7; 93.6; and 62.1, respectively) to compare the levels of total phenolics, flavonoids contents and tannin content in the extract before fermentation (93.2; 38.4; and 28.11, respectively).
During the fermentation process the decrease of carbohydrate, total protein and free amino acid due to their biotransformation inorganic acid. These metabolisms and biotransformation in the beverages can be proving the presence of new volatile compounds. The results indicated in the table 2, shown twenty-four volatile compounds were identified with remarkable differences between the fermented plants extract and the plant extract without fermentation (Table.2). The GC-MS profiling showed that, some compounds were detected only in fermented plants extract such as 2-methyl-Butyraldehyde, n-Propyl acetate, 1-Butanol. 3-methyl-. Formate, Propanoic acid, Propanoic acid. propyl ester, 2-Oxopentanedioic acid, and Lactic acid, other compounds were detected only in plants extract such as α-Phellandrene, D-Limonene, Fenchone, Bornyl acetate, and Caryophyllene. Among the volatile compounds detected some are typically produced by plants and other compounds are generated by the LAB metabolism. There were great variations in the composition of aroma components. The major compounds detected were (Eucalyptol (61 %), Terpinen-4-ol, endo-Borneol, and p-Cimene were found in the two extracts. The results showed that the fermentation technology enhance the secondary metabolite extraction which give new aromatic compounds to compare at the results before fermentation. In addetion, our results suggest that the volatile compounds produced after fermentation by LAB could give positive flavor attributes to the flavor of the fermented plants extract. On the other hand, the primary metabolic actions of the used selected strain in fermented plants extract include their ability to predominantly ferment carbohydrates and, to a lesser degree, degrade proteins and fats in the substrate. This leads to the production of a broad range of bioactive metabolites, mainly organic acids (for example, lactic, acetic, malic, formic, propionic), peptides (bacteriocin), free amino acids, along with many volatile and non-volatile low-molecular-mass compounds, such as ketones and esters. During this study the aromatic compounds changed after fermentation new volatile compound generated by the lactic acid bacteria such as 2-methyl-Butyraldehyde, n-Propyl acetate, 1-Butanol. 3-methyl-. Formate, Propanoic acid, Propanoic acid. propyl ester, 2-Oxopentanedioic acid, and Lactic acid, other from plant materials (α -PINENE, Camphene, α-Phellandrene, α-Terpinene, p-Cimene, D-Limonene, Eucalyptol, gamma. -Terpinene, Terpinolene, Fenchone, Linalol, Camphor…etc) table.2.
Antimicrobial activity and antifungal activity determinate by Disc diffusion methods
In this study, we have tested the fermented plants extract for their antimicrobial activity against resistant strains. The antimicrobial activity tested by disc diffusion method of obtaining beverages against resistance strains, presented in the table 3. The result showed that the high antimicrobial activity with a maximum zone of inhibition against Escherichia coli 25922 ATCC, Escherichia coli 8739 ATCC, Escherichia coli 25922/3, Pseudomonas aeruginosa DSM1128, Staphylococcus aureus 29213 ATCC, Enterococcus aerogenes 13048 ATCC, Enterococcus faecalis 29212 ATCC (13.4 ± 2, 14.4 ± 3, 16 ± 3, 10 ± 2, 14.3 ± 1, 14.5 ± 5, and 12 ± 4 mm, respectively). Results indicate that, the fermented beverages exhibited a great potential of antibacterial activity against all tested strains to compare with the extract without fermentation. In addition, the antifungal activity tested by disc diffusion method of obtaining beverages against resistance fungi strains, presented in the table 2. The result showed that the fermented plants extract exhibited high antifungal activity with a maximum zone of inhibition against Fusarium oxysporum, Aspergillus niger, Phytophthora infestans (GL-1), Phytophthora infestans P3 4/91 R+, and Phytophthora infestans P4 20/01 R (8 ± 2, 9 ± 3, 14 ± 1, 10 ± 2, and 8 ± 2.5, respectively) to compared with the plants extract without fermentation. However, the fermentation showed a significant (p≤0.05) increase in antifungal activity of plants. This high antimicrobial activity due to the effect of lactic acid Bacteria increased and enhance the extraction of bioactive metabolites from the medicinal plants have a high antimicrobial activity.
Antimicrobial and Antifungal susceptibility testing
Susceptibility testing was done by broth microdilution in accordance with the National Committee for Clinical Laboratory Standards (CLSI). Table 4 summarizes the MIC50 and MIC90 values against the resistance bacterial and fungal. The results showed that the fermented plants extract exhibited highest antimicrobial and antifungal activity more than the plants extract without fermentation. The antimicrobial test showed that the MIC50 and MIC90 values against resistance bacteria (1.8 µg to 10 µg, respectively, from the fermented plants extract and 4 µg to >20µg, respectively, for the plants extract without fermentation) were lower or equivalent to the corresponding values of the control antibiotics.
The antifungal activity tested against fungi showed that significantly different between the fermented plant extract and the plants extract without fermentation (p < 0.05) to compare with the control antibiotics. In addition, similar results for the susceptibility antifungal activity testing were also observed in the table 4. The MIC50 and MIC90 values of the fermented plants extract exhibited high activity against Fusarium oxysporum, Aspergillus niger, Phytophthora infestans (GL-1), Phytophthora infestans P3 4/91 R+, and Phytophthora infestans P4 20/01 R (9 to 16.5, 8 to 16, 10 to 18, 13 to 17 and 13 to 17.6 µg, respectively) to compared with the plants extract without fermentation (9 µg to 18 µg, respectively, for the fermented plants extract) to compared with the plants extract without fermentation (14 µg to >20µg, respectively).
Antifungal activity: Inhibitory Testing Using Growth Assays
In this study, the Antifungal activity of the fermented plant extract was tested against five stains have a high antifungal resistance: Fusarium oxysporum, Aspergillus niger, Phytophthora infestans (GL-1), Phytophthora infestans P3 4/91 R+, and Phytophthora infestans P4 20/01 R. The screening of the antifungal activity evaluated Using Growth Assays illustrated in the table 5 showed that, the fermented plants extract exhibited high inhibition of fungal at the different concentration of spores to compare with plant extract without fermentation table 2. In addition, the fermented plants extract exhibited total inhibition of the different fungi at the concentration of spore’s log (8) CFU/ml, to compare with the plants extract the fungal inhibition showed at log (4) CFU/ml. This data was confirmed by the morphological study illustrated in the fig. 3. That's the microscopic observation showed that the fermented plants extract total inhibition of fungi growth Fig.3 to compared with the plants extract without fermentation. This study confirmed the other data obtained by disc diffusion methods and the Susceptibility test.