Biochemical Composition, Antimicrobial and Antifungal Activities Assessment of the Fermented Medicinal Plants Extract Using Lactic Acid Bacteria

doi:10.21203/rs.3.rs-1143205/v1

To prevent foodborne diseases and extend shelf-life, antimicrobial agents may be used in food to inhibit the growth of undesired microorganisms. The present study was aimed to determine the antimicrobial and antifungal activities of the fermented medicinal plants extract using Lactobacillus acidophilus ATCC 4356. The fermentation kinetic parameters, biochemical composition and the volatile compounds of the fermented plant extract was assessed. The results showed that, the fermented plants beverage exhibited high content in polyphenols, flavonoids, and tannins (152.7 mg AGE/ L; 93.6 mg RE/ L; and 62.1mg CE/ L, respectively) to compare with the extract without fermentation. The GC-MS headspace analyses showed the presence of twenty-four interesting volatile compounds. The richness of the fermented plants extracts of polyphenols content and the bioactive compound such as Eucalyptol, Camphene, α-Phellandrene, α-Terpinene improve their biological activity. In addition, the fermented plants extract exhibited a high antimicrobial potential against resistance pathogenic bacteria and fungi determined by different methods. The maximum inhibition showed in the fermented plants beverage against Escherichia coli 25922/3, Pseudomonas aeruginosa 27853 ATCC, Staphylococcus aureus 29213 ATCC, Enterococcus aerogenes 13048 ATCC, Phytophthora infestans P3 4/91 R+, Phytophthora infestans P4 20/01 R, Phytophthora infestans (GL-1). The obtained results support the hypothesis of using whey as a functional ingredient to improve food preservation. The bioprocesses of fermentation technology enhance antimicrobial and antifungal activities which could be used in different industrial applications.

General Microbiology

Fermented Plants extract

Lactobacillus acidophilus

Biochemical composition

antimicrobial activity

antifungal activity

Aromatic and medicinal plants are particularly valuable for selective bioprocesses because they already contain many bioactive compounds, including phenolic compounds, carotenoids, anthocyanins and tocopherols (Naczk and Shahidi, 2006). In addition, the content of these compounds can be increased by the metabolic activity of the microorganisms involved in the fermentation process. In particular, plants can be used as an ideal substrate for the growth of LAB (Yoon et al. 2006; Andersen et al., 2012; Filho et al., 2017). More specifically, the fermentation technology breaks down or converts the substrates such as the medicinal plants into compatible components under the action of microbial enzymes, thereby improving the substrate properties via the production and enhancing the extraction of bioactive compounds (Parvez, Malik, Ah Kang and Kim, 2006). Many of the changes occur during fermentation, leading to a modified the properties of products such as bioactivity, digestibility and therefore the aromatic compounds changed, new compounds were generated from the fermentation (Zhang et al. 2012). Lactobacillus and other lactic acid bacteria (LAB) are commonly used in dairy and also nondairy fermentations because of their ability to metabolize different substrates (Freire et al. 2017; Luana et al. 2014). LAB produces metabolites, such as volatile and nonvolatile compounds, during the fermentation process, which contribute to their use in the food industry. Fermentation-mediated bio-activation of the medicinal herbs results in improved therapeutic potencies and efficacies and decreased toxicities where the microbial population plays a pivotal role (Lin Wang Lee and Su, 2008; Miyake et al. 2005; Nakano et al. 2006; Wu et al. 2013). Fermentation improves the pharmacological properties of herbal medicines mainly through the modification of naturally occurring molecules such as isoflavones, saponins, phytosterols, and phenols that exert beneficial health-promoting and disease-preventing effects, in keeping with the ‘theory of the oriental medicine’ (Choi and Kang, 2003). For the past few years, with the rapid progress in microbial fermentation technologies and in-depth research on the modernization of herbal medicines, the microbial fermentation and transformation of herbal drugs have gained considerable interest and appeared as new approaches to produce novel active compounds with potent medicinal values (Wu et al. 2013). The fermentation of medicinal herbs, a decomposition process, is performed by using microorganisms such as bacteria and fungi. Fermentation is considered to be one of the most useful techniques of biocatalytical process which is a feasible method for the production of new, active, and less toxic bioactive products that would be otherwise troublesome to generate from either biological systems or chemical synthesis (Rasor et al. 2001).

The rapid acidification of the food matrix occurs through conversion of fermentable carbohydrates, mainly into lactic acid. Reduced pH and the production of antimicrobial agents (such as bacteriocins) decrease the growth and development of pathogenic microorganisms and food spoilers, extending the shelf life and ensuring food safety of the final product (Smid et al. 2014). On another level as important as health, biological control is also a very promising field of action where fermented extracts will play a crucial role.

The fungi are the most destructive among all phytopathogens which can cause about 65% loss of plant host species (Fisher et al. 2012). According to FAOSTAT1, fungi affected most of the economically important crops globally (www. fao.org). Under the present climate change scenario, incidences of several fungal diseases have escalated (Garcia-Solache et al. 2010). Therefore, management of these fungal diseases is very crucial to safeguard a constant supply of food to the increasing world population.

Therefore, the management of these fungal and microbial diseases are very crucial to ensure a constant supply of food to the growing world population. For example, Phytophtora infestans causes serious losses of potato crops worldwide and is probably the most important pathogen of potato and tomato today. To control the fungal diseases, a number of agrochemicals including synthetic fungicides are applied widely and repeatedly (Scarpino et al. 2015). Numerous studies have suggested the role of organic acid such as lactic acid and acetic acid in the antibacterial and antifungal activity of fermented beverages (Greenwalt et al. 1998; Blanc 1996; Velicanski et al. 2014; Steinkraus et al. 1996; Cetojevic Simin et al. 2008, Sreeramulu et al. 2001). Bacteriocins, enzymes, and other protein, volatile compounds, and compounds produced by bacteria have a role in the antimicrobial activity of this beverage. Bacteriocins are proteinaceous compounds that exhibit bactericidal activity against species closely related to the producer strain. Bacteriocins alter the membrane potential by corrupting the potassium ion and ATP and cause cell failure to balance intracellular pH (Sezer and Guven 2013). For the reasons, we are developing a new strategy using medicinal plants fermented by LAB as an alternative to the chemical agent against pathogenic microorganisms.

The objective of the study was to investigate the antimicrobial and antifungal activities of the fermented medicinal plants extract using Lactobacillus acidophilus ATCC 4356. The fermentation kinetic and Biochemical composition and identification of the volatile compounds before and after fermentation was explored.

Plant’s materiel

The plant materials used in this study; ficus indica, Linum usitatissimum, Lavandula multifida, Periploca laevigata, Foeniculum Vulgare and Thymus algeriensis were collected from Orbata National Park Mountain (Gafsa, Tunisia) with coordinates: N 34° 22′49.8′′and E 9° 3′23.4′′, Nigella sativa, Linum usitatissimum, Zingiber officinalis, and Vitis vinifera were purchased from commercial central market of Tunisia (Gadhoumi et al. 2021). A voucher specimen was deposited in the Laboratory of Extremophile plants, Center of Biotechnology at the Ecopark of Borj-cédria, and identified by prof. Abderrazak Smaoui. The selected plant materials were harvested and mixed with sugarcane molasses in sterile distillated water (table 1).

Microorganisms

The fungi Phytophthora infestanes (GL-1: 01114 mold type von Großlüsecotz, German; P3: R+ producent, Meppen, German; P4:20101 R- wild type (unknown) obtained from Julius Kühn institute in Braunschweih, German), Colletotrichum higginsianum (originally isolated in Japan, via the Department of Biology, Friedrich-Alexander-Universität (Erlangen, Germany)); Fusarium oxysporum 39/1201 (St.9336 from the Technische Universität Berlin in Germany); Aspergilus niger DSM 246 from DSMZ in Braunschweig, Germany.

The bacteria strains; Escherichia coli 25922 ATCC, Escherichia coli K12, Escherichia coli 8739 ATCC, Escherichia coli 25922/3, Pseudomonas aeruginosa DSM1128, Pseudomonas aeruginosa 27853 ATCC, Staphylococcus aureus 25923 ATCC, Staphylococcus aureus 29213 ATCC, Serratia marcescens (Enterobacteriaceae) 13880 ATCC, Enterococcus aerogenes 13048 ATCC, Enterococcus faecalis 29212 ATCC were obtained from the microbiology Laboratory of the Department of Agriculture and Food Sciences, University of Neubrandenburg, Germany.

The culture of Lactobacillus acidophilus ATCC 4356 obtained from the microbiology Laboratory of the Department of Agriculture and Food Sciences, University of Neubrandenburg, Germany.

Fermentation condition

Two equivalent batches were prepared using plants row materiel. The selected plant materials were crushed and mixed with sugarcane molasses in sterile distillated water were sterilized by pasteurization (80°C in 15 min) (table 1). After pasteurization, the first Batches were filtered through 0.2-μm filters and concentrated using freeze drying and preserved at + 4°C until analysis. The second Batches were fermented using lactic acid bacteria (LAB) cultures: Lactobacillus acidophilus ATCC 4356. Before fermentation, For the purpose of fermentation a ready to use (RTU), the LAB cultures were activated to the stationary phase by cultivation at 37°C for 24 h and used for the inoculation with Log 6 - 8 CFU/ml at pH = 3.5 were added to each batch at the rate of 10 ml/l. After two weeks of fermentation, the beverages were sterilized by filtration through 0.2-μm filters and concentrated using freeze drying and preserved at + 4°C until analysis (Gadhoumi et al. 2021).

Fermentative parameters

Acidity was measured by titration using 0.1 M NaOH solution and phenolphthalein as indicator. The pH was measured using a calibrated Hanna pH meter. Total sugars were assessed by an ATC refractometer (Brix 0-32%). The determination of reducing sugars was assesses using the DNS method described by Song et al. (2016). The total proteins were determined according to the Bradford method (1976). The results were expressed as the means of three replicates.

Total Phenolic content

The polyphenols content was determinate using the Folin–Ciocalteu reagent, using the method described by Velioglu et al. (1998). 0.1 ml of extract was added to 1 ml of distilled water and 0.2 ml of the Folin–Ciocalteu reagent (diluted 1/10). The mixture solution was shaken and allowed to stand for 5 min. After that, 1 ml of Na2CO3 (6%) was added to the mixture. After 30 min of incubation in dark, the absorbance was read at 760 nm. Total polyphenol content was expressed in µg gallic acid equivalents per g dry residue (mg GAE/ L).

Flavonoid content

The flavonoids content was determinate using the method described by Tlili et al. (20013), using the AlCl₃ reagent. A volume of 1.5 ml of diluted extract was mixed with 1.5 ml of AlCl3 solution (10% in methanol). After incubation for 30 min at room temperature, the absorbance was read at 415 nm. The results were expressed in µg rutin equivalents per g dry residue (mg RE/L).

Condensed tannins contents

The determination of the condensed tannins content in the different extracts according to the method described by Tlili et al. (2013). A volume of 400 μl of diluted extract or standard, 3 ml of vanillin (4%) dissolved in methanol and 1.5 ml of concentrated sulfuric acid. After 15 min of incubation at ambient temperature, the absorbance is measured at 500 nm. The concentration of condensed tannins was expressed in µg catechin equivalent per gram dry residue (mg CE / L).

Headspace solid-phase microextraction of volatile compounds

The volatile compounds analyzed by Headspace Solid-Phase Microextraction (HS-SPME) coupled with Gas Chromatography-Mass Spectrometry (GC-MS) as previously described Navarini et al. (1999). The GC-MS headspace analysis was performed with an iron sources temperature of 240°C and an ionization voltage of 70eV. The mass spectrometer was operated in scan mode from m/z 50 to 350. Peak areas were determined for each compound by integrating a selected ion unique to that compound. The volatile compounds were identified by matching their mass spectra with those in the NIST1.l Library of MS spectra. The Kovats retention index (RI) was calculated with a homologous series of n-alkanes (C6-C28) under the same conditions applied for the sample analyses. The volatile compounds (OAV > 1) are considered to contribute to the aroma of fermented beverages (You et al. 2013; Tian et al. 2017). The different fermented beverages were analyzed in triplicate.

Antimicrobial activity and antifungal activity determinate by Disc diffusion methods:

The antimicrobial and antifungal activity of the different extracts was determined by the diffusion method in agar medium cited by Celiktas et al. (2007). with a slight modification, this method was employed to determine inhibition diameter of fermented extract against pathogen bacteria strains. In the Square Petri Dish surface, mueller hinton agar MHA solid plate was swabbed with 100 µL of the Culture bacterial suspension or spores 10^5-7 CFU/mL with each tested microorganism and kept 1h in 4 C° for bacterial fixation. Sterile discs of extracts at the rate of 20 µl per disc of concentration were deposited sterile on the agar surface. The dishes were incubated for 24 hours at 37 ° C for the antimicrobial activity. In a normal atmosphere for not demanding bacteria, and in an atmosphere containing 5% CO2 for bacteria demanding. The inhibition diameter was measured (mm) three times in triplicate. 50µg/disk of Streptomycin (sigma chemical) was used as positive control.

Antimicrobial and Antifungal susceptibility testing

Antifungal and antibacterial susceptibility testing was performed by a broth microdilution method in accordance with the National Committee for Clinical Laboratory Standards (NCCLS, 1997). The final concentrations of the antifungal and antimicrobial agents were 0.1 to 20 μg/ml. Test strains were suspended in NB to give a final density (bacteria or spores) of Log 6-7 CFU/ml and these were confirmed by viable counts. Plates were incubated at 35°C for 48 h for the bacteria and 3 to 6 days for the fungal. MICs of all the samples were defined as the lowest concentrations resulting in 80% inhibition of growth compared to that of the growth control. The wells were then examined for evidence of growth and MICs values were determined as the lowest extracts concentration that inhibited visible growth of the tested microorganism which was indicated by the presence of a white “pellet” on the well bottom. MIC ranges were obtained for each species-extracts combination tested. MICs for 50 and 90% of the isolates of each species tested (MIC₅₀ and MIC₉₀, respectively)

Antifungal activity: Inhibitory Testing Using Growth Assays

This method described by (Thanusu et al. 2010), can be used to generally test growth inhibitory effects of fermented beverages towards fungi. To study growth inhibition, the optical density of the cultures is followed and visualized by microscopic means. Depicts growth inhibition of strain of gungy in incubated with 20 µg of the fermented extract. Prepare spore solution of different fungal strain as follows: 1) Streak spores from a single colony on a CM agar plate and incubate until the plate is abundantly covered with sporulated mycelium (3–6 days, 25–37°); 2) harvested spores from CM agar plate add 10 mL of saline solution to the plate and carefully release spores by scraping over the surface plate with a sterile cotton stick; 3) Pipette spore solution from the plate into a sterile 15-mL tube; 4) If required, remove mycelial debris (vegetative mycelium, conidiophores) by filtration through a sterile myracloth filter; 5) Count spores using a microscope counting chamber; 6) Prepare different concentration of a spore solution; 7) Start the growth inhibition assays with letting the spores germinate first: Fill each well of a flat-bottom 96 well plate with 300 µL sterile medium, 100 µL 2× MM and 50 µL spore solution; 8) Prepare a fermented beverages stock plate for efficient and fast addition of the fermented beverages by using a V-bottom 96 well plate; 9) Prepare serial dilutions of the fermented beverages and add each concentration (20µg per well; 10) Every beverage should be tested in triplicate; 11) Pipette also 50 µL of water or any other solvent used as negative control to at least three wells; 12) Incubate the growth inhibition plate at 30 °C; 13) Visualize growth of fungi in the 96 well plate of each condition via microscopy.

Statistical analyses:

The data were expressed as mean values and the standard deviations (SD) were calculated. The comparison between the average variables was performed by Duncan's multiple-range tests and the differences considered significant when p <0.05. The Statistica version 7.0 software was used for data processing.

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).

Volatile compounds

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.

During the fermentation process, the soluble components of protein, free amino acid, carbohydrate, free sugars were readily available, and the consumption of these nutrients by microorganisms led pH levels to drop (Di Cagno et al. 2017). As shown in Fig. 2. Lactic acid fermentation is a metabolic process carried out by lactic acid bacteria to convert carbohydrates from plants into lactic acid or a mixture of lactic acid, acetic acid, ethanol and CO₂. During the fermentation process the decrease of sugar due to their biotransformation inorganic acid. The production of these compounds can extend the shelf life by limiting the growth of contaminating and pathogenic microorganisms. In this sense, the growth and subsequent disappearance of some bacteria is common due to intra and interspecific competition, as well as competition regarding the substrate, thence involving production of organic acids (Stoyanova et al. 2012). In this way, the fluctuation of pH values is a recurrent finding by several authors (Di Cagno et al. 2011; Begunova et al. 2020). Numerous studies have suggested the role of organic acid such as lactic acid and acetic acid in the antibacterial and antifungal activity of fermented beverages (Greenwalt et al. 1998; Sreeramulu et al. 2001).

In addition, Polyphenols constitute one of the most preponderate groups of secondary metabolites in plants, including a wide variety of bioactive molecules that contain at least one aromatic ring with one or more hydroxyl groups in addition to other substitutes with large spectra of biological activities (Tlili et al. 2013). They are divided on different groups such as; flavonoids, tannins and phenolic acids (Tlili et al. 2013). After fermentation, the results indicated in the Fig. 2. showed that the content of total polyphenols, flavonoids and tannins increased to compared with the plants extract without fermentation which confirms that lactic fermentation increases the extraction of secondary metabolites such as polyphenols compounds. During fermentation many changes of composition occur, leading to a modified ratio of nutrients and anti-nutrients and therefore the properties of the product, such as bioactivity and digestibility are modified (Barba et al. 2015). Several authors indicated that the lactic fermentation of plants has been shown to increase the concentration of several phenolic compounds (Teplova et al. 2018; Lukianov et al. 2018; Gadhoumi et al. 2021). We reported that the richness of our fermented medicinal plants extracts with phenolic compound could be an important starting point to explore their biological activities such as antioxidant activity (Gadhoumi et al. 2021).

Our results report that the microbial activity during the fermentation processes the aromatic compound profiles of fermented plants extract changed to compare with plants extract without fermentation. Several authors suggested that, among the aromatic compounds, organic acid is regarded as the predominant compounds in the aromatic profile of fermented plants extract and are a common terminal end product in the catabolism of the sugar and the free amino acids (Gadhoumi et al. 2021; Di Cagno et al. 2017). Also, the aromatic compound characterization of each extract is a result of the diverse routes used by microorganism to metabolize the substrate releasing volatile compounds. Esters are considered to be important volatile components in fermented plants extract. The ester compounds are generated by the esterification of free acids with alcohol (Lukianov et al. 2018), in our results, in the fermented plants extract we find the Propanoic acid propyl ester produces by the esterification reaction. The major compound detected by GC-MS from the plant’s materiel such as α -PINENE, Camphene, α-Phellandrene, α-Terpinene, and Eucalyptol has a high antimicrobial and antifungal activity (Lukianov et al. 2018). The formation of medicinal plants enhances the extraction of bioactive compounds such as the volatile compounds produced by the plants (Gadhoumi et al. 2021).

The richness of the fermented plants, beverages in polyphenols content and the bioactive compound such as Eucalyptol, Camphene, α-Phellandrene, α-Terpinene improve their biological activity. Bacteriocins, enzymes, and other protein compounds produced by bacteria and plants have a role in the antimicrobial activity of this fermented plants extract. Several proteinaceous compounds exhibit bactericidal activity against pathogenic bacteria species closely related to the producer strain such as Bacteriocins. These compounds alter the membrane potential and cause cell failure to balance intracellular pH (Sezer et al. 2013).

Moreover, the three-test used during this study; Disc diffusion methods, susceptibility testing, and Inhibitory Testing Using Growth Assays confirmed the high antimicrobial and fungal activity of the fermented plants extract against pathogenic bacteria and fungi. Several authors mention that different bacteria and fungi process high resistance to antibiotic and fungicide such as Phytophthora infestans exhibited high resistance to the fungicide has harmful effects on plants such as tomatoes and potatoes (Fry et al. 2008). In our study, the fermented plants extract exhibited a high antimicrobial and antifungal activity against resistant bacteria and fungi who is in accordance with the results reported by Jiang et al. (2021). The fermentation technology based in lactic fermentation and medicinal plants can be used as a solution for the pathogenic bacteria and fungi. It has been proposed that the antimicrobial effects involve the inhibition of different mechanism cellular, followed by an increase in plasma membrane permeability and finally ion leakage from the cells which causes pH imbalance (Kang et al. 2006). The antimicrobial and antifungal potency of the fermented plants extract is believed to be due to the bioactive compounds such as tannins, saponins, phenolic compounds and flavonoids (Omafuvbe et al. 2009). It is interesting to note that even crude extracts of the medicinal plants improved by the fermentation technology showed good activity against resistant strains where modern antibiotic therapy has failed. As per our results suggesting that this fermented extract inhibited growth of the test microorganisms at lower concentrations. The fermentation would be an opportunity for improving antimicrobial and antifungal activity of medicinal plants, which would open more applications. However, further studies are required to elucidate the biochemical composition and the mechanism of action.

To conclude, this study has demonstrated for the first time the effects of fermented plants extract using lactic acid bacteria against resistant pathogenic bacteria and fungal determined by Inhibitory Testing Using Growth Assays, disc diffusion methods and Susceptibility testing (MICs) according to National Committee for Clinical Laboratory Standards. In our study, the fermented plants extract revealed an enhanced level of polyphenols, flavonoids and tannins. In addition, the detection of aromatic compounds in the fermented plants extract showed interesting, volatile compounds, which could give pleasant-positive aroma attributes to the flavor of the beverages and high antimicrobial activity. It is recognized that medicinal plants, as well as lactic acid bacteria, can be sources of antimicrobial compounds which can be applied for food preservation. The results showed that, the lactic fermentation enhances the extraction of polyphenols content, aromatic compound and increased the antimicrobial and antifungal activity potential against resistance pathogenic microorganism such as Phytophthora infestans (GL-1). However, further studies are required to elucidate the biochemical composition and the mechanism of action.

Acknowledgements

We are grateful and provide sincere thanks to honorable Vice Chancellor University of Applied Sciences. Neubrandenburg, Germany for the infrastructure and facilities. This study supported by a grant from the Ministry of Tunisia, Laboratory of Aromatic and Medicinal Plants, Center of Biotechnology (LR15CBBC06) at the Ecopark of Borj-cédria. BP-901, 2050 Hammam-Lif. Tunisia.

Funding

This study supported by University of Tunis EL Manar, University of Applied Sciences, Neubrandenburg, Germany, for the infrastructure and facilities, and Laboratory of Aromatic and Medicinal Plants, Center of Biotechnology (LR15CBBC06) at the Ecopark of Borj-cédria. BP-901, 2050 Hammam-Lif. Tunisia.

Conflict of Interest

The authors of this manuscript certify that they have NO affiliation with or involvement in any organization or entity with any financial interest. I certify that I am submitting the manuscript on behalf of all the authors.

Author contribution

Hamza Gadhoumi and Akrem el hayouni, Martinez-Rojas Enriqueta, and Moufida saidani Tounsi designed the study and performed the experiments. Hamza Gadhoumi and Martinez-Rojas Enriqueta performed the experiment and carried out data analyses. Hamza Gadhoumi and Akrem El Hayouni contributed to drafting the manuscript. All authors have read and approved the final version of submitted manuscript.

Authors' information

Affiliations

Faculty of Sciences of Tunis, University of Tunis El Manar, Tunis, Tunisia.

Hamza Gadhoumi

Laboratory of Aromatic and Medicinal Plants, Center of Biotechnology at the Ecopark of Borj-cédria. BP-901, 2050 Hammam-Lif, Tunisia.

Hamza Gadhoumi, Moufida Saidani Tounsi, and EL Akrem Hayouni

Department of Agriculture and Food Sciences, Neubrandenburg University of Applied Sciences. Neubrandenburg, Germany.

Martinez-Rojas Enriqueta

Corresponding author

Correspondence to Hamza Gadhoumi

Ethics approval This article does not contain any studies with human participants performed by any of the authors.

Consent for publication All authors have seen the manuscript and approved to submit Archives of Microbiology.

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Table.1. Plant’s material used for the fermentation process

Plants	Botanical family	Weight (g per l)	Used organ
*Nigella sativa*	Ranunculaceae	5 g	seeds
*Foeniculum Vulgare*	Apiaceae	28 g	seeds
*ficus indica*	Cactaceae	90 g	Fruits
*Linum usitatissimum*	Linaceae	10 g	seeds
*Vitis* *vinifera*	Vitaceae	5 g	seeds
*Lavandula multifida*	Lamiaceae	18 g	leaves
*Periploca laevigata*	Apocynaceae	10 g	root
*Thymus algeriensis*	Lamiaceae	33 g	leaves
*Zingiber* *officinalis*	Zingiberaceae	20 g	root
30 g organic sugar cane molasses solubilized in 1 L distilled water in each bottle.

Table 2: Aromatic compounds identified from fermented beverages extracts of by GC-MS head space.

		After fermentation			Before fermentation
Peak #	compounds	Ret Time	Area	% Area	Area	% Area
1	2-methyl-Butyraldehyde	3.631	3166922	2.37	NF	NF
2	n-Propyl acetate	4.282	2752332	2.06	NF	NF
3	1-Butanol. 3-methyl-. formate	4.676	2205104	1.65	NF	NF
4	Propanoic acid	4.975	1216092	0.91	NF	NF
5	Propanoic acid. propyl ester	5.857	724850	0.54	NF	NF
6	2-Oxopentanedioic acid	5.996	1244074	0.93	NF	NF
7	Lactic acid	7.841	566468	0.42	NF	NF
8	α -PINENE	8.138	480350	0.36	6944145	6.14
9	Camphene	8.414	449670	0.34	5106354	4.51
10	α-Phellandrene	9.404	NF	NF	640494	0.57
11	α-Terpinene	9.633	841209	0.63	1637160	1.45
12	p-Cimene	9.790	2889327	2.16	1222297	1.08
13	D-Limonene	9.879	NF	NF	1266200	1.12
14	Eucalyptol	9.950	82174283	61.57	71285193	63.01
15	gamma. -Terpinene	10.522	887853	0.67	1849510	1.63
16	Terpinolene	11.298	2302629	1.73	481400	0.43
17	Fenchone	11.300	NF	NF	1254048	1.11
18	Linalol	11.554	604976	0.45	NF	NF
19	Camphor	12.844	12301015	9.22	17312969	15.30
20	endo-Borneol	13.341	3391528	2.54	1011447	0.89
21	Terpinen-4-ol	13.556	15049231	11.28	1439575	1.27
22	α-Terpineol	13.885	218307	0.16	NF	NF
23	Bornyl acetate	15.400	NF	NF	1257857	1.11
24	Caryophyllene	17.123	NF	NF	415800	0.37

Table 3: Antimicrobial activity of fermented beverages (mm), Method: Disc diffusion assay:

Bacteria	Inhibition zone (mm)
Bacteria	Before fermentation	After fermentation	Streptomycin
*Escherichia coli 25922 ATCC*	6 ± 2*	13.4 ± 2**	28.2 ± 6
*Escherichia coli K12*	6 ± 3*	10 ± 2**	13 ± 2
*Escherichia coli 8739 ATCC*	6.5± 4*	14.4 ± 3**	30.2 ± 4
*Escherichia coli 25922/3*	6.2± 3*	16.3 ± 3**	32.4 ± 4
*Pseudomonas aeruginosa DSM1128*	---*	10 ± 2**	12 ± 3
*Pseudomonas aeruginosa 27853 ATCC*	7.5 ± 2*	11.5 ± 2**	18.1 ± 3
*Staphylococcus aureus 25923 ATCC*	6.2 ± 3*	9.1 ± 2**	25.4 ± 4
*Staphylococcus aureus 29213 ATCC*	6.5 ± 1*	14.3 ± 1**	23.8 ± 5
*Serratia marcescens 13880 ATCC*	6.2 ± 3*	13.6 ± 4**	24.1 ± 4
*Enterococcus aerogenes 13048 ATCC*	6.6 ± 2*	14.5 ± 5**	26.6 ± 3
*Enterococcus faecalis 29212 ATCC*	7.6 ± 4*	12 ± 4**	23.5 ± 2
*Champignons*
*Fusarium oxysporum 39/1201*	7 ± 1*	8 ± 2**	14 ± 2
*Aspergillus niger DSM 246*	6 ± 0.5*	9 ± 3**	16 ± 1.5
*Phytophthora infestans P3 4/91 R+*	---*	14 ± 1**	13 ± 2
*Phytophthora infestans P4 20/01 R*	6 ± 1*	10 ± 2**	10 ± 1
*Phytophthora infestans (GL-1)*	---*	8 ± 2.5**	11.5 ± 0.5

Results are expressed as mean of 3 experiments of three beverages. Data bearing different lowercase letters indicate a significant difference between the beverages (p < 0.05).

Table 4: Antifungal and antibacterial susceptibility testing

Strain	Before fermentation		After fermentation		Control
	MIC₅₀	MIC₉₀	MIC₅₀	MIC₉₀	MIC₅₀	MIC₉₀
Bacterial strains	8**	18.5**	2.6*	9*	2	6
Escherichia coli 25922 ATCC	8**	16**	1.8*	7.5*	1.6	6
Escherichia coli K12	10**	18**	3*	6*	1.6	8
Escherichia coli 8739 ATCC	12**	>20**	4*	8*	1.6	6.5
Escherichia coli 25922/3	10**	18**	2.6*	8.5*	2	4
Pseudomonas aeruginosa DSM1128	9**	16**	6*	9*	1.8	4
Pseudomonas aeruginosa 27853 ATCC	4**	14.5**	4.5*	9*	1.6	6
Staphylococcus aureus 25923 ATCC	6**	14**	8*	6*	1.4	8
Staphylococcus aureus 29213 ATCC	8**	18**	4*	9.5*	1.2	8
Serratia marcescens 13880 ATCC	10**	>20**	4.5*	9*	1.2	6.5
Enterococcus aerogenes 13048 ATCC	9**	19**	2.8*	8.5*	1.6	6
Enterococcus faecalis 29212 ATCC	6**	14**	4*	10*	1.6	8
Fungal strains
Fusarium oxysporum 39/120	14**	18**	9*	16.5*	4	11
Aspergillus niger DSM 246	16**	19.6**	8*	16*	6	11
Phytophthora infestans P3 4/91 R+	16**	>20**	10*	18*	6.5	13
Phytophthora infestans P4 20/01 R	16**	>20**	13*	17*	8	13
Phytophthora infestans (GL-1)	18**	>20**	13*	17.6*	8	13

All tests were performed in duplicate and repeated twice. Statistically, Data bearing different lowercase letters indicate a significant difference between the beverages (p < 0.05). MIC: minimal inhibitory concentration; values given as µg/ml for the fermented plants extract, plants extract without fermentation and antibiotics.

Table .5. Antifungal activity: Inhibitory Testing Using Growth Assays.

		Inhibitory Testing Using Growth Assays
Spores number CFU/ml		10¹⁰	10⁹	10⁸	10⁷	10⁶	10⁵	10⁴
*Fusarium oxysporum 39/1201*	FP-ext	+++	+--	---	---	---	---	---
	P-ext	+++	+++	+++	+++	+++	++-	---
	Fungicide	+--	---	---	---	---	---	---
*Aspergillus niger DSM 246*	FP-ext	---	---	---	---	---	---	---
	P-ext	+++	+++	+++	+++	+++	++-	---
	Fungicide	+--	---	---	---	---	---	---
*Phytophthora infestans P3 4/91 R+*	FP-ext	---	---	---	---	---	---	---
	P-ext	+++	+++	+++	+++	+++	++-	---
	Fungicide	+--	---	---	---	---	---	---
*Phytophthora infestans P4 20/01 R*	FP-ext	---	---	---	---	---	---	---
	P-ext	+++	+++	+++	+++	+++	++-	---
	Fungicide	++-	---	---	---	---	---	---
*Phytophthora infestans (GL-1)*	FP-ext	+++	++-	+--	---	---	---	---
	P-ext	+++	+++	+++	+++	+++	++-	+--
	Fungicide	++-	+--	---	---	---	---	---

Results are expressed as mean of 3 experiments of three beverages. FP-ext; fermented plants extract, P-ext; plants extract without fermentation, Fungicide; trailed by fungicide

+; Growth

-; Inhibition

Biochemical Composition, Antimicrobial and Antifungal Activities Assessment of the Fermented Medicinal Plants Extract Using Lactic Acid Bacteria

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Abstract

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Materials And Methods

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