Abstract

Kawal is a condiment produced by the traditional fermentation of Sicklepod (Senna obtusifolia) leaves. Many people from the different part of Chad use it for flavoring as well as a substitute of meat. The fermentation techniques used in kawal production are often applied on a small scale and still mainly at the household level, characterized by the use of simple and rudiment equipment. This study was conducted firstly to investigate the Kawal traditional methods production. Secondly, to evaluate probiotic potential of Bacillus species isolated in order to develop starter cultures for kawal production. Protease, amylase and phytase activities of Bacillus strains were screened. The candidate were screened in vitro for antimicrobial activity against, Micococcus luteus LMG3293, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 8739 and Salmonella spp. Biofilm synthesis, Antibiotics sensitivity, tolerance to simulated gastric and intestinal juice were also evaluated. The majority of selected strains formed bioﬁlms, produced amylase and protease, some of the strain produced phytase by forming clearing zones around the growth of the colonies. For biochemical conditions for simulated gastric and intestinal juice, among the selected strains tested, some had the highest survival in simulated gastric juice and better viability in simulated intestinal juice. The selected strains showed sensitive to a panel of antibiotics and they presented resistance to penicillin G, oxacycline and bacitracin. Some strains showed an inhibitory effect on all of the indicator bacteria tested. Overall, some of these selected strains could be used as potential microbial starters for the controlled fermentation of kawal.

Keywords: Kawal, Bacillus, Senna obtusifolia, Fermented food, Traditional, Probiotic.

Received: 2 August 2019 / Revised: 4 September 2019 / Accepted: 10 October 2019/ Published: 21 November 2019

Contribution/ Originality

This study is one of the very few studies, which have investigated the traditionnel technologies, and probiotic properties of Bacillus strains isolated from kawal, towards use as a starter culture for fermenting Sicklepod (Senna obtusifolia) leaves.

1. INTRODUCTION

Food quality is generally influenced by the processing techniques used. It was found to be largely influenced by a variety of factors and micro-organisms (Mohamadou et al., 2008). Since old time, various methods have been used to process and to preserve foods. Fermentation is a process that has been used by humans for thousands of years, with major roles in food preservation. This process is generally carried out to bring diversity into the kinds of foods and beverages available; make otherwise inedible foods edible; flavor dishes; enhance the nutritional value; decrease toxicity; preserve food; and decrease cooking times and energy requirements (Steinkraus, 2018). Many different products around the world are a result of fermentation, either occurring naturally or through addition of a starter culture (Marshall and Mejia, 2011). The African continent has a wide variety of traditional fermented foods ; especially those based on plant substrate materials. These usually foods have a large impact on the nutritional health and social economic status of Africa’ people (Franz et al., 2014). Fermentation of African foods is usually performed using minimal technology often on a small scale and household basis, characterized by the use of simple, non-sterile equipment, chance or natural inoculas, unregulated conditions, sensory fluctuations and poor durability (Oguntoyinbo et al., 2016; Wafula et al., 2016). Bacillus spp. are commonly involved in African vegetable fermentation via alkaline hydrolysis of the proteins to amino acids and ammonia. Bacillus species are one of the most investigated bacteria for Africa vegetable alkaline fermented food and their utlisation as stater culture (Parkouda et al., 2010).

In Chad and other African countries, fermented fresh leaves of Sicklepod( Senna obtusifolia ) name kawal, is used in relatively large quantities in the preparation of sauces as a meat substitute or as a spice by some people. Kawal is known to be rich in proteins and contains many nutrients such as essential amino acids, minerals and various organic acids (Mbaiguinam et al., 2005; Nuha et al., 2010). The women rural are often responsible for kawal processing. This production constitutes an importante economical source for the producers. The traditional methods used in the production varies amount producers and often still, at the household level under uncontrolled fermentation, and used rudiment equipment with poor hygenic conditions.

In many developed countries, traditional methods are now replaced with specific technologies for production of fermented foods, and an emerging industrial practice allows for higher quality standardization of food products in the market place (Borresen et al., 2012).

In order to enhance the fermentation process, the kawal production need to make better, like introducing starters culture of microorganisms with a potential technologies and probiotics proprieties. Bacillus species principally B. subtilis group are one of the most investigated bacteria for alkaline food fermentaton. This group, characterized by their ability to produce a high level of enzymes, antimicrobial compounds and others potential Probiotic (Savadogo et al., 2011; Olmos and Paniagua-Michel, 2014; Taalé et al., 2015).

In this sense, B. subtilis group isolated is a potential candidate to be used as starter cultures for production of kawal. This will guarantee the end- product safety and improve keeping the quality. Thus, the objective of this work was to study the technology of kawal in different places of Chad, and then to establish a general flow diagram of production as well as to screen probiotic properties of Bacillus strains, predominant microflora isolated from Kawal to be used as starter cultures.

2. MATERIALS AND METHODS

2.1. Investigation on Kawal Technologies

The survey was conducted to understand the kawal production technologies. It took place in the form of interview and observation during process of production. In total 186 interviewed person, among them 62 producers in plain activity, 54 who practiced technology in the past and 70 kawal producers and sellers were identified by the the surveys. Different productions sites in different places of Chad were identified. For that, a survey was conducted in the Abeché, East regions and Central region including Mongo, and Chari Baguirmi localities.The study concerned the areas of high production and consumption of kawal in Chad. Others sites were divided by considering the large production of kawal in Chad involved by this survey included Sarh and Mayo-kebi area Figure 1. A flow diagram of each site was established, and then a general diagram for all production sites was proposed.

Figure-1. Study area.

2.2. Microbiological Analysis

2.2.1. Sampling and Bacillus sp.Characterization

Dry fermented kawal samples were collected from different kawal site productions and markets places at East, Central and South of Chad. A total 30 kawal samples (30 to 100 g) were collected in a sterilize plastic sampling bag and kept in coolers with ice and transported within 24 h to the laboratory for microbialogical analysis. Bacillus species were isolated over Plate Count Agar (PCA) (Biokar, France) following heat treatment of the homogenate (80 °C for 10 min) and incubation at 30 °C for 24 h. Then the isolated strains were sub-cultured onto tryptone Soy Agar (TSA) by the streaking method and incubated at 30°C. The strains were identiﬁed and selected according to their phenotypic characters : colony and cellular morphology, ability to grow under aerobic conditions, Gram and catalase tests.

2.2.2. Evaluation of Potential Probiotics of Bacillus Strains

In vitro, experiments were used to investigate the selected Bacillus strains for their probiotic properties Simulated gastric and intestinal juice, Antimicrobial activity, Resistance to antibiotics, Biofilm synthesis and Enzyme production : amylase, protease and phytase (Fakhry et al., 2008; Merghni et al., 2014; Prieto et al., 2014; AlGburi et al., 2016; Latorre et al., 2016).

2.2.2.1. Antimicrobial Activity Assay

Antimicrobial activity of isolates was determined by the agar well diffusion assay (AWDA) in triplicate, as previously reported by Mishra and Prasad (2005) against indicator bacteria. These indicator strains included two Gram-positive and two Gram-negative pathogens (Micococcus luteus LMG3293, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 87 39 and Salmonella spp. (isolated from local food)).The indicator bacteria were grown for 24 h in 10 mL of nutrient broth and then spread on nutrient agar plates and incubated at 30°C for 24 h. The colonies from pure culture were suspended and diluted with 0.85 % sterilized saline water. 1 mL of broth indicator strains was spread over Muller Hinton Agar (MHA) plates. Then, four wells with a diameter of 6 mm were made in each MHA plates, and100 μL of antimicrobial filtrate containing the test compound from potential probiotic strains were introduced into the wells. For negative controls, well with DMSO was used as descriped by Nitiema et al. (2012). Plates were incubated at 37 °C for 24 h. After incubation, the plates were observed for growth inhibition zones formed around the well.

2.2.2.2. Antibiotic Susceptibility Assay

The antibiotic susceptibility test of studied Bacillus was carried out as described by Weber et al. (1988) using agar spread diffusion method. Overnight cultures of the each collected bacterial strains incubated at 37°C in TSB were diluted and adjusted to approximately 106 CFU/ mL and spread on Mueller-Hinton agar plates. Then serial antibiotics : ampicillin 10 µg, tetracycline 30 µg, erythromycin 15 µg, chloramphenicol 30 µg, kanamycin 30 µg, streptomycin 10 µg, oxacycline 30 µg, gentamycine 10 µg, amoxiciline 10 µg, imipenem 10 µg, Bacitracine10 µg and penicillin 10 IU, were dispensed onto the plates and incubated for 24 h at 37˚C as suggested by the National Committee for Clinical Laboratory Standards (NCCLS). The results were determined according to the inhibition zone diameter and expressed as susceptible, ≥ 16-mm diameter ; intermediate, 13- to 15-mm diameter ; resistant ≤ 12-mm that suggested by Weber et al. (1988) for Bacillus spp.

2.2.2.3. Determination of Biofilm Formation

The determination of biofilm synthesis was performed using a crystal violet staining method as described by Latorre et al. (2016) with slight modifications to observe bioﬁlm formation and adherence to a surface. Indeed, 10 μL of overnight cultures of the Bacillus isolates growth in nutrient broth were inoculated into 0.5 mL Casein-Mannitol broth in 1.5 mL polypropylene tubes and incubated at 37°C for 12 h without shaking. After incubation the liquid was removed and the tubes were rinsed with distilled water. Then, 1 mL of crystal violet solution (1 % w/v) was added to the polypropylene tubes to fix the cells adhered to form a purple pellicule on the airsurface of the polypropylene tube. After 30 min, the tubes were washed with distilled water, after removing crystal violet. The measurement of biofilm synthesis was based on color intensity and size of the adherent crystal violet ring with a score ranging from negative (−) to strong (+++) biofilm formation. All the strains were tested in triplicate.

2.2.2.4. Tolerance to Simulated Gastric and Intestinal Juices

Resistance to artiﬁcial gastric juice of the Bacillus sp. was studied as previously reported by Charteris et al. (1998) the survival of Bacillus strains in simulated gastric juices was prepared by dissolving pepsin (0.3 %, w/v) in PBS buffer solution and adjusting the pH to 2.0 with hydrochloric acid and sterilization by ﬁltration through a 0.22 μm pore ﬁlter. Briefly, each bacterial suspension (appropriate dilutions) was added to simulated gastric (pH 2.0) and to control (pH 7.0, without gastric juices). Viable count was done at 0.5, 1, 1.5, 2 and 3 h of incubation in the juice and spread onto nutrient agar plate and incubated at 30°C for 24 h.

Simulated small intestinal juice was prepared, by suspending pancreatin and bile salts in PB to ﬁnal concentrations of 1 g /L and 4.5 g /L, respectively as previously reported by Huang and Adams (2004) and adding bile salts (4.5 % w v-1). The pH was adjusted to 7.0 with 1 N sodium hydroxide (NaOH). The mixture was sterilized by ﬁltering through 0.45 µm ﬁlter. The bacteria suspension was inoculated in this juice and incubated for 1 h, 2 h and 3 h. The survivability of of Bacillus species were determined by spread-plate technique after serial dilution.

2.2.2.5.Phytate Degrading Test

Phytase screening was carried out on agar plates, using phytate specific medium (Ehon et al., 2015) containing [Phytase screening was carried out on agar plates, using phytate specipic medium [18] containing [glucose 15g, (NH 4 )2 SO4 5, KCl 0.5 g, MgSO4 0.1 G .7H2O, NaCl 0.1g, 0.01 % CaCl 20.1g. 2H2O, FeSO 4 0.01 g, MnSO 4, 0.01 g, pH 6.5 with sodium phytate 5g and 20 g of noble agar] per 1000 mL of distilled water. The reaction mixture was autoclaved at 121°C for 15 min and poured into petri dishes. Then, 7 µL of bacterial suspension were spotted onto phytate agar medium. The Petri dishes were incubated at 37° C for 1- 3 days to observe the clear zones of hydrolysis around the colonies.

2.2.2.6. Determination of Amylase Activity

Amyliotic activity of the isolates was determined as described by Ibrahim et al. (2012) and Latorre et al. (2016) using basal medium with following composition: 10 g of tryptone, 5 g of KH2PO4 , 10 g of yeast extract, 15 g of noble agar, supplemented with 3 g of soluble starch and 1000 mL of distilled water. The pH of the reaction mixture was adjusted to pH 7 then the components of the starch agar medium were autoclaved at 121°C for 15 min and poured in Petri dishes. Each tested Bacillus strain was inoculated and incubated at 37°C for 24-48 h. The Petri dishes were flooded with Gram’s iodine solution. The results were reported amylase positive, if the clear zone was appeared around inoculated colonies and amylase negative if the clear zone did not appear.

2.2.2.7. Determination of Protease Activity

Detection of the proteolytic activity of Bacillus strains, was performed as described by Latorre et al. (2016) with minor modifications. Protease production was screened on skim milk agar media. The skim milk agar media containing 25 g of skim milk, 25 g of noble agar and 1000 mL of distilled water was prepared. The mixture was autoclaved at 121°C for 15 min and poured into plates. Each selected Bacillus strain was inoculated on Petri dishes and incubated at 37°C for 24 h. Proteolysis activity was characterized by the formation of clear zone around the colonies.

2.3. Statistical Analysis

All data obtained from technologies questionnaire and resultats of microbiological analysis in laboratory were analysed using Excel (Microsoft office 2013) and SPSS for Windows, version 17.0. The results were expressed as mean ± standard deviation (SD) and significant difference was accepted at 𝑃 <0.05 using analysis of variance (ANOVA).

3. RESULTS AND DISCUSSION

3.1. Kawal Production Technology

The production of kawal is a traditional process involving different unit operations with the most important being fermentations and drying after harvesting of the leaves. The technologies used in kawal production are often applied on a small scale and still mainly at the household level, characterized by the use of simple and rudimentary equipments under poor hygenic conditions. These technologies remain traditional with the empirical knowledge and the know-how is transmitted from mother to daughter and from generation to generation. Different varieties of technologies processes in kawal production depending on the sites and producers Figure 3 was observed in this study and the essentiel operation are the fermentation and drying. The flow general chart of the processing line of Senna obtusifolia fresh leaves to Kawal food condiment is presented in Figure 4.

Figure-2. A ; B ; and C : Different varieties of technologies processes in kawal production from different sites.

Figure-3. Flow general chart for Kawal production in Chad.

3.1.1. Preparation of Raw Material

The raw material (Senna obtusifolia leaves) was reaped in the rainy season when the plant is fully grown and pre-processed before the real production step. The pre-processing step consists of a selection by manually sorting. The green leaves are cleaned and the impurities, such as flower petals and leaves of other plant, worm, insects or leaves damaged by insects were remove.

3.1.2. Pounding

The unwashed, healthy green leaves are beaten manually in a wooden pestle and mortar to give a green paste. The leaves are usually pounded to destroy the filament, reducing and harmonizing until they formed a paste, without loss of juice. This hydration creates favorable conditions for the metabolic and microbial enzymes activities during the fermentation.

3.1.3. Fermentation

After a complete pounding, the paste is placed immediately in a plastic bag or earthenware. Meanwhile, a pit is dug in the ground in a shaded cool place, then the whole containers is fitted into the pit, leaving only the neck of the container above ground level. The containers is packed with cover to ferment at room temperature. Processing techniques used in kawal fermentation and procedures differ with localities, environmental factors and the types of processing equipment and technologies available. The duration of fermentation depends on the local conditions, the producer and the organoleptic quality of final product desired. However, for most producers, the fermentation was done at one step for 3 - 4 weeks according to the intensity of the desired ammonia-like flavour displayed by the fermented. This fermentation method is very popular in many parts of Chad. For others, the fermentation takes about 2 weeks or more and every 3-4 days the jar is opened and the contents are mixed by hand and repacked. Sometimes, after the first pounding of the process, the paste is not completly pounding, in this case at the end of the fermentation the pastes were removed and pounded again. At the end of the fermentation the product acquired a very pronounced and stronger ammonia-like odour.

3.1.4. Drying

At the end of the fermentation, the fermented paste is removed, moulded into small balls and sun-dried for 3 - 5 days according to the intensity of sunshine.

The process used in the production are often applied on a small scale by rural woman, characterized by the use of simple and rudimentary equipments, uncontrol fermentation with the empirical knowledge and the know-how is transmitted from mother to daughter and from generation to generation. The technology of kawal like most of the Africa fermented food remains traditional. In many developed countries, different traditional methods are now replaced with specific technologies for the production of fermented foods, and an emerging industrial practice allows for higher quality standardization of food products in the market place (Borresen et al., 2012). In recent times in Africa, a number of research works focused on developing starter cultures to upgrade the processing of traditionally fermented foods like dawadawa (Amoa-Awua et al., 2006). In the same line, this study aimed to the standardizing of the production of kawal, a traditional condiment produced by spontaneous fermentation of Senna obbtusifolia leaves. This study was to isolate and investigate Bacillus strains from kawal for potential starter culture selection.

3.2. Bacterial Isolation and Characterization

Ninety-seven (97) bacteria were isolated from the kawal. According the morphological and biochemical characters obtained, all isolates were belong to Bacillus spp. These strains were isolated by heat treatment of kawal samples. They were unicellular rod-shaped bacteria, rough, opaque colonies, Gram positive, catalase positive and negative for others, and able to grow under aerobic conditions. They were endospore forming, and motile. According the phenotypic characters of the strains, we retained only 17 strains for probiotic properties evaluation.

3.3. Antimicrobial Activity

The selected Bacillus strains were screened for their antibiotic producing and inhibitory effect against Gram-negatives and Gram-positive bacterias. Majority of potential probiotic strains tested (58.82%) had activity against at minmum on one of the indicator bacteria : Micococcus luteus LMG3293, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 8739and Salmonella spp. (isolated from local food). However, this selected strains exhibited antagonistic activity, but varied in their degree of inhibition and spectrum of activity. In particular, the strains SS6, SA9 and SG2 showed high inhibitory activity against the indicator bacteria, Micococcus luteus LMG3293 and Staphylococcus aureus ATCC 25923 respectively with inhibition zone more than ranged 3 mm. Some of the Bacillus strains showed moderate inhibition (2.1 to 3 mm) to the indicator bacteria. The others strains were least effective against the indicator bacteria. However, the weak inhibitions were observed with others against the indicators bacteria, as indicated in the Table 1.

Table-1. Antimicrobial activity of Bacillus Isolates using the agar-solid diffusion method.

Strain	Indicators bacteria
Strain	E. coli ATCC 87 39	Salmonella sp.	S. aureus ATCC 25923	M. luteus LMG3293
SM2	+	+	++	+
SM1	-	+	+	+
SG6	++	++	+	++
SL2	-	-	-	-
SS6	+	++	++	+++
SM6	-	+	-	-
SS2	-	-	-	+
SG2	+	+	+++	+
SG5	-	-	-	-
SG3	++	+	++	+
SN1	-	-	-	-
SL5	-	-	-	-
SK1	+	+	++	++
SK2	-	-	-	+
SA4	-	-	+	-
SA9	+	+	++	+++
SB12	-	-	-	-

Mean of inhibition zone : +,0.1 to 2 mm ; ++, 2.1 to 3 mm ; +++ >3 mm ; - no antimicrobial activity.

This research was also conducted to evaluate the antibacterial activity of the Bacillus strains isolated from kawal. The results of tests show good antibacterial activity of the tested strains against the indicator bacteria (Micococcus luteus LMG3293, Staphylococcus aureus ATCC 25923, Salmonella sp. and Escherichia coli ATCC 87 39) which resulted in inhibition zones Table 2. However, the strains SM2, SG6 and SK2 respectively showed high activity against the M. luteus LMG3293 and S. aureus ATCC 25923 with large inhibition zones more than 3 mm. The majority of the strains iolated were most effective against M. luteus LMG3293 and S. aureus ATCC 25923. This inhibition may be due to the production of one or more antibacterial substances or active compounds. Several studies have reported about the genus Bacillus showing antagonism against Gram-negatives and Gram-positive bacterias (Korzybski et al., 1978; Zweerink and Edison, 1987; Savadogo et al., 2011; Taalé et al., 2015). have reported isolation, characterization and pathways of antibacterial substances produce by Bacillus strains isolated from fermented food. Currently, the scientific works have been consecrated to the exploitation of antimicrobial metabolites of certain bacteria to improve the sanitary quality of foods (Desmazeaud and Cogan, 1996). Bioprotection of food is based on the use of beneficial microorganisms that can control the presence of pathogenic bacteria by the production of specific metabolites.

3.4. Antibiotic Sensitivity of Bacillus Strains

The sensitivity of the Bacillus strains to a panel of antibiotics was determined using the agar disc-diffusion assay as recommended by the Use of the National Committee for Clinical Laboratory Standards guidelines for disk diffusion susceptibility testing in New York state laboratories. Ampicillin 10 µg, tetracycline 30 µg, erythromycin 15 µg, chloramphenicol 30 µg, kanamycin 30 µg, streptomycin 10 µg, oxacycline 30 µg, gentamycine 10 µg, amoxicilin 10 µg, imipenum 10 µg, Bacitracine10 µg and penicillin 10 IU were the antibiotics used in this study. The test results Table 2 revealed that the majority of the strains evaluated for the antibiotic susceptibility test had the same level of highest sensitivity to amoxicillin, kanamycin, chloramphenicol, streptomycin, and tetracyclin. All the Bacillus sp. tested were more susceptible to imipenum, erythromycin and gentamycin. Many isolates were resistant to penicillin G, oxacycline and bacitracin compared to their sensitivity proﬁles to other tested antibiotics.

Table-2. Susceptibility test of Bacillus sp. to antibiotics with diameter of inhibition (mm).

Antibiotic discs	Dose (µg)	Inhibition zone diameters by strains
		BS1	SM1	SG1	SG2	SKB
Ampicillin	10	20 ± 0.4	2 1 ± 0.4	20.4 ± 0.4	18 ± 1.2	21 ± 0.2
Chloramphenicol	30	29.5 ± 0.1	26 ± 0.3	24 ± 0.9	29 ± 2.1	25. ± 4.1
Tetracyclin	30	19 ± 1.4	25 ± 1.2	12.3 ± 0.3	30± 0.9	19.3 ± 06.
Erythromycin	15	34 ± 0.0	36 ± 0.5	33.5 ± 0.5	32 ± 0.6	30 ± 9.0
Kanamycin	30	25 ± 0.6	26 ± 1.1	22 ± 1.5	26.6 ± 0.4	24 ± 3.0
Penicillin	10	08 ± 1.3	06 ± 0.0	06 ± 0.0	08 ± 0.1	06 ± 0.0
Streptomycin	10	16 ± 0.7	15 ± 1.6	17 ± 1.7	16 ± 0.4	15 ± 0.5
Oxacycline	30	07 ± 0.2	06 ± 0.1	07 ± 0.2	06 ± 0.0	08± 0.9
Gentamycin	10	32 ± 0.4	35 ± 0.1	30 ± 0.4	35.2 ± 0.4	31 ± 0.8
Amoxicilin	30	22 ± 0.6	18± 2.4	15.6 ± 0.9	20 ± 1.3	24 ± 1.1
Imipenem	10	51 ± 2.1	53 ± 0.2	50.8 ± 0.1	51.6 ± 0.1	52 ± 3.2
Bacitracine	10	06 ± 0.2	06 ± 0.0	12 ± 0.4	06 ± 0.0	06 ± 0.0

Diameter of inhibition zone : susceptible, ≥ 16-mm diameter ; intermediate, 13- to 15-mm diameter ; resistant ≤ 12-mm.

Resistance profile of isolated, the present study showed that all the Bacillus isolates were highly susceptible to imipenum, erythromycin and gentamycin, and the results show that many of these Bacillus sp. tested were resistant to β-lactam antibiotics (penicillin G, oxacycline and bacitracin). The hight resistance of Bacillus strains to penicillin G, oxacycline and bacitracin probably in part by means of β-lactamase synthesis, on the other hand by biofilm production, that protects the bacteria (Philippon et al., 1998; Chen et al., 2004). The isolates strains were categorized as intermediately susceptible to amoxicillin, kanamycin, chloramphenicol, streptomycin, and tetracyclin. This observation is similar with that reported by Coonrod et al. (1971) to provide detailed antibiotic profiles of large number of well-characterized Bacillus spp. Coonrod et al., reported that tetracycline, chloramphenicol, kanamycin, and gentamicin inhibited almost all Bacillus strains regardless of species.

3.5. Screening for Protease and Amylase Activities

The Bacillus strains were screened for protease and amylase respectively on skim milk agar media and on basal medium supplemented with soluble starch in order to detect their potential enzyme activity. Results showed that all selected strains are able to assimilate the protein and starch except two investigated becteria that were amylase negative. However The selected strains BL1 and BS23 showed the highest amylase and protease activities. These Bacillus strains tested gave different production levels of the enzymes studied, indicated by the presence of halos around the colonies Table 3.

3.6. Phytate Degrading Bacteria

All isolates were tested for their ability to phytase degradation, using sodium phytate media. 65.76 % of the strains produced zone of clearing on sodium phytate agar medium. Among them the strains BS23, SK23 and SB8 showed good phytase activities and could produce medium clear halo zone on phytase specific agar medium around colonies. The iolate BL1 was found to be the best strains and could produce large clear halo zone around the colony. Enzyme activities of Bacillus sp. selected as well as size of zone of clearance are shown in Table 3.

Table-3. Screening of different enzymes and biofilm formation of Bacillus sp. Isolates.

Enzymes and biofilm formation
Strains	Protease	amylase	Phytase	Biofilm
BS23	+++	+++	++	+++
BS42	++	++	+	+
BS12	++	+	+	++
BS5	++	++	+	+
SM2	+	+	+	+
BL1	+++	+++	+++	++
SM3	+	-	-	+
SS7	+	+	+	+
BA21	++	++	-	++
AM9	+	+	-	+
SK11	+++	+	-	+
SK23	++	+++	++	+++
SB8	+	+++	++	+++
SL6	++	-	-	-
GS4	+	+	+	++
SL9	+	+	-	-
SA11	+	+	-	+

Highly (+++), medium (++), small (+) and (-) unable to synthese.

The production of enzymes, protease, amylase and phytase by Bacillus spp. showed different production levels of the studied enzymes indicated by the presence of halos diameters. The ability of Bacillus spp. to produce different hydrolytic enzymes is an interesting healthy and technological property. Phytic acid is an antinutritional factor that forms a complexe with proteins and also chelate metals making them unavailable to the organism (Quax, 1997; Nakamura et al., 2000). The presence of phytate in Senna obtusifolia leaves will be harmful for human health. Phytic acid is known as a food inhibitor which chelates micronutrient and prevents it to be bioavailabe for monogastric animals, including humans, because they lack enzyme phytase in their digestive tract (Gupta et al., 2015). Fermentation of Senna obtusifolia leaves to produce kawal was found to cause highly significant decrease in phytic acid content as reported by Algadi and Yousif (2015). The involvement of Bacillus genus with phytase activities in kawal fermentation could play a major role on phytate degradation. As concern amylase, this enzyme hydrolyzes the starch to produce sugar used by microorganisms involving in fermentation as energy source (Ketiku and Oyenuga, 1972) and improves digestibility of fermented food. In the alkaline or vegetable fermented food, the Bacillus genus hydrolyzes proteins to peptides and free amino acids and they also have a meat-like flavour, which contributes to food enjoyment (Sarkar et al., 2002; Ouoba et al., 2003).

3.7. Biofilm Synthesis

The ability of the tested strains to synthese biofilm was determined in liquid medium. All of Bacillus spp. strains tested were able to adhere to the polypropylene tubes giving a purple pellicule on the airsurface of the polypropylene tube, except to strains. However, the color intensity and size of the adherent crystal violet differ from one to another strain. In fact, BS23, BL1, BA21 and GS4 strains were identified as highly adherent, giving a large purple (+++). BS12, SK23, and BS5 strains were identified as faintly adherent showing a medium purple pellicule (++). Others strains were identified with a very small purple pellicule (+). Whereas the strains SL6 and SL9 were unable to synthese biofilms in liquid Table 3.

The biofilm production ability of the Bacillus strains isolates was further assessed by generation of an adherent crystal violet-stained ring in polypropylene tubes. Majority of the screened Bacillus spp. strains produced biofilms. We found in this study a difference in the adherent crystal violet-stained ring in polypropylene tubes between the colonies. However among the isolates formed biofilm, some of the strains were identified as highly, medium or small adherent, others were unable to synthese biofilms. Biofilm synthesis, facilites Bacillus spp. adherence to intestinal epithelial cells and served as a mechanism of survival for some Bacillus to the harsh environmental conditions of the gastrointestinal tract (Barbosa et al., 2005). These properties could facilite intestinal colonisation and also prevent enteropathogen (Barbosa et al., 2005; Fakhry et al., 2008; Latorre et al., 2016).

3.8. Simulated Gastric and Intestinal Juices

One of the required criteria of probiotics microorganisms is their ability to survive in harsh environment of the gastrointestinal juice. Therefore, the survival ability of Bacillus strains was researched under simulated gastric juice at pH 2.0 and simulated intestinal juice (pH 7.0) conditions. The survival rate was compared with bacterial suspension (concentration between 1.0 x 108 and 1.0 x 109 CFU) before treatment and the viable counts of bacteria after incubation in gastric fluid and intestinal fluid for 3 h. Seventeen Bacillus strains were examined for gastrointestinal juice in this research. The results obtained after growing the strains in the harsh condition medium are shown in Figures 4 and 5. The viable counts of these 17 strains tested decreased at pH 2.0 of simulated gastric juice after 3 h compared with the control (S8). The strain S7 was the most acid tolerant of simulated gastric juice at 3h of incubation, with a survival rate of 7.05 log (CFU mL-1juice) among the seven strains (S1, S2, S3, S4, S5, S6 and S7) showed in Figure 4. The viabilities of the certains strains after incubation in intestinal juice at pH 7.0 for 3 h showed survival rates of 7.21 ± 0.11, 6.9 ± 0.13, 6.70 ± 0.1, 6.60 ± 0.1, 6.54 ± 0.1, 6.13 ± 0.1, and 5.80 ± 0.13 log (CFU mL-1juice), respectively for the strains SM3, SA1, LK3, KA, AKL, KBK, and KGA and considered intrinsically tolerant to gastrointestinal transit. All the strain tested showed decreasing numbers, except the control strain LK6 and S8 showed very good survival at 3 h of incubation in simple medium without simulated gastrointestinal juice Figures 4 and 5.

Figure-4. Tolerance of Bacillus strains to simulated gastric juice.

Figure-5. Tolerance of Bacillus strains to simulated intestinal juice.

The isolates of Bacillus sp. were examined under harsh environment of gastrointestinal conditions. Hence, evaluation of the survivability of the strains was carried out with regard to the cell counts in suspension obtained after the dilution as initial cells before the gastrointestinal treatment. The results obtained for simulated gastric and simulated intestinal conditions, indicates that all the cell densities of the strains tested reduced after treatment during the first 180 min. In that case the selected strains showed their capability to survive in simulated gastrointestinal conditions and showed differentes variations in the surviviabilities throught the treatment period. Among the strains tested, most of them gave the highest surival rate more than 7.0 log (CFU mL-1) in gastric juice after 120 min Figure 4. At the same time the strains selected had better viability in simulated intestinal juice for 3 hours with survival rate 7,21 log (CFU mL-1 juice) Figure 5. The excellent resistance to stimulated gastric juice and stimulated intestinal juice of the Bacillus strains selected are similar with previous results obtained by Wang et al. (2010) and Hong et al. (2005). This ﬁnding suggests that these isolates have the potential to survive in the human gastrointestinal tract and can likely survive the passage through the stomach and small and large intestine (Jena et al., 2013).

4. CONCLUSION

Different varieties of technologies processes in kawal production were identified in this study. The kawal production is still traditional with rudimentary equipment, natural fermentation, no standards, conducing to the variability of the nutritional and hygienic qualities as well as the stability of the final product. This alkaline fermentation of Senna obtusifolia leaves is a proteolotic enzyme process, and most important metabolism throughout the fermentation. The Bacillus spp. Screened showed high production levels of the studied enzymes. These selected Bacillus strains were very good proteolytic, phytasic and amylolytic properties as well as important antimicrobial activities against most common food spoilage and foodborne pathogens bacteria. The isolates susceptible to antibiotic tested, among them the isolates were susceptible to all types of antibiotic tested, biofilm synthesis and survive in harsh conditions. These isolates constitute then suitable candidates for the selection of starter cultures for controlled production of kawal.

Funding: This study received financial support from our Laboratory.

Competing Interests: The authors declare that they have no competing interests.

Acknowledgement: All authors contributed equally to the conception and design of the study.

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