Determination of Proteolytic Activities of Bacillus species Isolated From Traditional Fermented Oil Bean Seed (UGBA: Pentaclethra macrophylla, Benth)

Eighty samples of traditional fermented “ugba” ( Pentaclethra macrophylla , Benth) wraps collected from eight markets in Umuahia were analyzed for proteolytic activities (by zone of inhibition in mm) of Bacillus species using two protein-based substrates: Skim Milk Agar and Nutrient Agar supplemented with 1% gelatin. 51 isolates were identified as B. subtilis , 18 as B. licheniformis and 11 as B. pumilus with Ariam Market having the highest mean count (mm) of 1.93±0.16. B. subtilis had the highest proteolytic activity (28.00±0.55) on Skim Milk Agar (P=.05) but together with B. licheniformis had the same level of proteolytic activity (26.00±0.00 and 26.00±0.55 respectively at P=.05) which is higher than B. pumilis (13.00±0.55) on Nutrient agar supplemented with 1% gelatin. Original However, B. licheniformis and B. pumulis had better proteolytic activities (26.00±0.55 and 13.00±0.55: P=.05) respectively on Nutrient agar supplemented with 1% gelatin than on Skim Milk Agar. The two potential protease producers that showed higher proteolytic activity were incubated at high temperature and result showed that B. subtilis proteolytic activity was optimum at (29.00±0.71) at 50°C while that of B. licheniformis was optimum (28.00±0.00) at 40°C (P=.05). Antimicrobial susceptibility of the Bacillus species showed that the three Bacillus species were most sensitive to Chloramphenicol (21.00±0.51) without any statistical difference followed by Gentamycin (20.00±0.31) but completely resistant to Rifampicin (0.00±0.0; P=.05). B. subtilis showed better proteolytic activity at a higher temperature and will be better suited for production processes in industries where thermophilic temperatures are needed.


INTRODUCTION
"Ugba", a fermented product of African oil bean seed (Pentaclethra macrophylla, Benth) is one of the common, fermented legumes, predominantly consumed by the Ibos and other smaller ethnic groups of the South Eastern Nigeria [1]. Oil bean seed belongs to the leguminous family mimosa cease found mostly in tropical Africa, it is cultivated in forest areas, with about eight flat glossy brown edible seeds per pod. The pods explode at maturity and disperse the seeds [2].
Fermentation detoxifies the African oil bean seed with subsequent increase in nutrient availability and digestibility [3]. Ugba is widely consumed as a snack, side dish or food condiment [4]. Ugba production is locally done through a mixed wild bacteria fermentation of the sliced, boiled and soaked African oil bean seeds are bitter and possess anti-nutritional factors amongst which pancine, cyanide, oxalates, saponin, phytic acid, phytate and tannins [5,6]. Microbial population of Ugba is introduced through the air, water, utensil, banana leaves or the handler; no starter culture is used for the traditional method [3]. Microorganisms excrete a wide variety of proteolytic enzymes, which are also found in systems. They are molecules of small size, compact, spherical structures that catalyzes the peptide bond cleavage in protein [7]. Commercially they are very important and isolated from various living sources such as plants, animals, bacteria and fungi [8].
Proteases from microbial sources are preferred over the enzymes from plant or animal sources since they possess all most, all the characteristics desired for their biotechnological application [9]. Among bacteria, Bacillus species are specific producers of extra-cellular proteases. These proteases have wide applications in pharmaceutical, leather, laundry, food and waste processing industries [10].
The main fermenting microorganisms in the fermentation of African oil bean seeds have been identified to be proteolytic Bacillus species, which include B. subtilis (most predominantly), B. licheniformis, B. megaterium, B. macerans, B. circulans and B. pumilus [11,2]. During fermentation, these microorganisms use the nutritional components of the seeds, converting them into products that contribute to the chemical composition and taste of the condiment/ugba [12]. Although, yeasts and other bacteria are also seen, only part of them can be considered to play a substantial role in fermentation processes. For instance, non-fermenting species may just be ubiquitous contaminants, although they may affect the flavour of the final product when occurring in high numbers. The Bacillus species is of particular interest due to its prevalence in these fermented foods [13]. In addition, use of B. subtilis in the food industry is recommended by the US FDA as one of the GRAS (Generally Recognized as Safe) organisms [14]. This research work initiated in 2014 was aimed at: i. Isolation and identification of proteolytic Bacillus species from traditional fermented oil bean seed (ugba); ii. Comparing the proteolytic activities of Bacillus species on two different media and iii. Determining the effect of temperature on the proteolytic activities of the isolates and their antibiotic sensitivity profile.

Sample Preparation and Inoculation
The samples were aseptically transferred to a sterile porcelain mortar, mashed to paste and 1g was serially diluted using sterile peptone water. 1ml aliquot of appropriate dilution was then inoculated on nutrient agar in triplicates using pour plate method. The plates were incubated at 30°C for 24 hours [1]. Thereafter, the mean colony forming unit per gram (cfu/g) was calculated.
Representative colonies were isolated, purified by repeated streaking. The cultures were identified using biochemical methods according to Bergey's Manual of Determinative Bacteriology. Bacterial colonies were isolated and characterized by their morphological and physiological properties [15].

Growth in 5% and 7% NaCl
Five percent NaCl was prepared by adding 2.8 g of nutrient agar and additional 5g of NaCl to 100 ml of distilled water and sterilized by autoclaving at 121°C for 15 minutes. 7% NaCl was also prepared by adding additional 7 g of NaCl and 2.8 g of nutrient agar to 100 ml of distilled water, sterilized by autoclaving at 121°C for 15 minutes, and allowed to cool. The two media were poured on different Petri dish and labeled. The bacterial isolates were spread onto surface of the two different media by repeated streaking and incubated at 37° for 24 hours [16,17]. Growth on the plates was determined by the presence of colonies on the plates.

Growth at 50°C
The bacterial isolates were sub-cultured onto Nutrient agar and incubated at 50°C for 24 hours [1].

Growth on Nutrient Agar Supplemented with 1% Gelatin
Gelatin was used to investigate the presence of proteolytic microorganisms as evidenced by the liquefaction of gelatin. The nutrient agar was supplemented with 1% (1 g) gelatin and sterilized in autoclave at 121°C for 15 minutes. The Nutrient gelatin composition include: 12% gelatin, 0.3% beef extract and 0.5% gelatin peptone [17]. A loopful of the culture growth was placed on the centre of Nutrient agar supplemented with 1% gelatin (Na+G). The plates were then incubated at 37°C for 24 hours. The bacterial growth and enzymatic activity were monitored on the media (agar plates) from the first 12, 16 and 24 hours of incubation. Proteolytic activities of the bacterial isolates were detected by observing the presence of a clear zone around the growing colony [13] in replicates of five: R 1 , R 2 , R 3 , R 4 , R 5 , on each medium.

Effect of Temperature on Proteolytic Activity
This was determined by growing the isolates that showed the highest enzyme activity at different temperatures (20,30,40,50 and 60°C) at a constant pH 8.0 in skim milk agar. The protease activity was recorded according to the diameter of the clear zone on the media (agar plates) after overnight incubation [18].

Preparation of Turbidity Standard Equivalent to McFarland 0.5
One percent (1%) v/v solution of sulphuric acid was prepared by adding 1ml of concentrated sulphuric acid to 99ml of water. 1% of barium chloride was also prepared by dissolving 0.5 g of dehydrated Barium chloride in 50ml of distilled water. 0.6ml of Barium chloride solution was added to 99.4 ml of the sulphuric acid solution and mixed properly. The solution was preserved in the fridge.

Determination of Antimicrobial Activity
Antimicrobial activity was carried out on the isolates on Mueller Hinton Agar. The overnight bacterial cultures were spread onto surfaces of the set Petri dishes using sterile swab sticks. Then, antimicrobial impregnated discs were placed on the surface of the agar using a sterile forcep and incubated at 37°C for 24 hours [19]. The presence of zones of inhibition of growth were observed and recorded by measuring the diameter (mm) and the susceptibility was interpreted using Interpretative Range Table.

RESULTS AND DISCUSSION
In this study, eighty samples of traditional fermented oil bean seed (ugba: Pentaclethra macrophylla) collected from eight different market places in Abia State were analysed for the presence of Bacillus species with proteolytic activities on Skim Milk Agar (SMA) and Nutrient Agar supplemented with 1% gelatin. Eighty Bacillus species were isolated from the samples out of which 51(63.8%) were identified as B. subtilis, 11(13.8%) were identified as B. pumilus while 18(22.5%) isolates were identified as B. licheniformis ( Table 1). The three B. species were isolated from the 80 samples analysed and they represent a group of phenotypically related species known as the B. subtilis spectrum. When subjected to a battery of tests, strains representing these species share many common properties and show relatively few characteristics by which they can be separated [20]. Based on cell morphology, all the eighty isolates were rods and endospore formers. [12] reported that the endospore forming bacteria were present throughout the 72 hours of fermentation period of "ugba", whereas the non-spore forming bacilli were found only at the beginning of the fermentation.
The colony forming units per gram result showed that the highest values was obtained from the Ariam Market (coded "E"; 1.93x10 5 ) while the least value (1.70x10 5 ) was from Ahiaeke Market (coded "A"; Table 2). Variation in the mean count values recorded against the eight markets could be due to differences in the varieties of the "ugba" seeds used in the fermentation. In earlier works, [21] found that other bacteria progressively decreased in total count and the disappearance of these bacteria could be due to the production of the antibiotic (Bacitracin) by B. subtilis, which may have inhibited the growth of these bacteria. It was noted that the disappearance of Micrococcus species especially at 96 hours of fermentation may be due to the presence of this antibiotic. [21] noted that although, there were other bacteria identified in the fermenting slices of "ugba" such as coagulase negative Staphylococcus species, Micrococcus species, Leuconostoc mesenteriodes, Lactobacillus plantarum, Streptococcus lactis, Proteus species, Enterobacter species and E. coli, their counts decreased after 72 hours of fermentation while the main fermenting microorganisms have been identified to be proteolytic Bacillus species. [22] demonstrated that proteolytic microorganisms have been detected in appreciable numbers from 10 4 to 10 6 cfu/g and the highest mean count result from this work fell within this range of values. However, previous reports suggested that the predominance of the Bacillus species may be due to the fact that the other bacteria grow on the little carbohydrate present in the seeds, majority of which have been lost due to leaching during preparatory stages. Additionally, pre-fermentation treatment (boiling) must have assisted in initiating the hydrolysis of the complex proteins found in the seeds.
Two different protein-based media were used to evaluate the proteolytic activity (PA) of the three species and B. subtilis had the best PA on SMA (P=.05) among the three. Although B. subtilis and B. licheniformis had the same level of PA (P=.05) on Nutrient agar supplemented with 1% gelatin (NA+G), this value is higher (P=.05) than that of B. pumilis (12mm) on NA+G. The proteinase enzyme is considered the most important enzyme in ugba fermentation. This result suggested that the ability of the three species of Bacillus to produce enzyme (protease) at varying ranges (according to the zone of clearance on the media) depends on the genetic constituents of each species. By this result, B. subtilis has better proteases that could hydrolyse the complex proteins found in the seeds of oil bean plant. Serine and metallo-proteases are the principal classes of proteases found in several species of Bacillus. We noticed (Fig. 1) that SMA had the same level of effect on the proteolytic activities of B. subtilis (P=.05), but has less effect on the proteolytic activities of B. licheniformis and B. pumilis (P=.05). It is possible that gelatin, as a high molecular weight protein, induces an increase in the protease production to degrade the substrate to an available form for the microorganism. [23] noted that the use of gelatin in the culture media provided a qualitative assay, simple, inexpensive, straight forward method to assess the presence of proteolytic activity of a given colony. [17] evaluated the proteolytic activities of Bacillus using five different proteinbased media to evaluate whether this might affect the proteolytic activity of Bacillus species. These media include: skim milk agar; skim milk agar supplemented with 0.02% sodium azide; soya protein agar; nutrient agar supplemented with soya protein; nutrient agar supplemented with 1% gelatin. They suggested that skim milk agar and nutrient agar supplemented with soya protein or gelatin were good for the detection of protease activity, but also suggested that sodium azide can prevent microbial growth, thus reducing protease production.
During the Bacillus growth, cell morphology was examined; all bacterial cells appeared in vegetative forms during the first 12 hours, but spore formation was observed between 16 and 24 hours of the incubation period. According to [13] this suggested that protease production reached the highest level during the exponential phase and continued constant level when spores were formed (stationary phase).
Evaluation of the effect of temperature on the proteolytic activities of the isolates was carried out on the two isolates that performed best ab initio on SMA and NA + G respectively (B. subtilis and B. licheniformis) and result showed that the proteolytic activity of B. subtilis peaked at 50°C (28 mm) while that of B. licheniformis was maximum (28.00 mm) at 40°C (Fig. 2). Both species had the same proteolytic activities (P=.05) at the two different optimum temperatures. This result implies that B. subtilis will perform better in industries that operate at temperatures above 40°C. Similar result was obtained by [18] with the maximum activity at 40°C for B. licheniformis and 50°C for B. subtilis with a reduction in activity for both species at 60°C, respectively. [18] commented that temperature is one of the critical parameter which has to be controlled and maintained in an optimum condition for maximal enzyme production. [24,25] showed that 50°C and 35+2°C was optimum for B. subtilis and B. licheniformis respectively. It was clear that the Bacillus species, usually being mesophilic can survive high temperatures by forming spores.
This result suggested that one feature of an extreme survival strategy employed by these species is the formation of endospores. This allowed the bacterium to produce a dormant and highly resistant cell to preserve the cell's genetic material in times of extreme stress. Therefore, alkaliphilic Bacillus species have important industrial application due to their ability to produce alkaline enzymes such as protease. These species produce extracellular enzymes that are resistant to high temperature conditions. Many obligately or facultatively alkaliphilic Bacillus strains have been isolated for use in industries and biotechnology.
The antibiotic susceptibility test ( P=.05) but was completely resistant to Rifampicin. B. subtilis was also completely resistant to Rifampicin but most sensitive to Chloramphenicol (P=.05). It's noteworthy that the three Bacillus species were completely resistant to Rifampicin but most sensitive to Chloramphenicol followed by Gentamycin (P=.05). The high level of resistance exhibited to Rifampicin by the three species of Bacillus could be genetically mediated as there was no inhibition from the antibiotic-an indication that the three species are closely related in genetic make-up. The high performance of Chloramphenicol and Gentamycin to the three isolates is highly appreciated especially at a time like this when many bacteria are developing resistance to many antibiotics. Although the three Bacillus species are "GRAS", (generally recognized as safe) in food industry and agriculture; they can cause disease in the debilitated or immuno-compromised individuals. Some recent studies revealed that these species of Bacillus spp can cause infectious disease to man and animals ranging from skin infection to life threatening bacteremia in the immunocompromised individual [26].

CONCLUSION
The present study has shown that Bacillus species are the predominant organisms in the traditional fermented ugba as they persisted throughout the fermentation stages. The isolates performed well in protease production while the two media tested showed good protease producing activity and B. licheniformis having best activity at 50°C. B. licheniformis and B. subtilis exhibited good abilities to survive high thermal temperatures and this has made them useful in the industries where thermophilic condition is needed especially as these isolates are sourced locally from traditional fermented foods which are readily available. The isolates may be useful in the industries such as foods, medicals, pharmaceutical and cosmetics.