Microbial enzymes are being increasingly used as therapeutics that play an important role in alleviating the burden of several digestive and malabsorption disorders. Use of microbial enzymes to substitute pancreatic enzymes has been considered safe with very less side effects and also proven economic [1]. Further, microbial derived digestive enzymes were proven to exert efficient digestive property at very low doses and also can possess a broader pH range of activity than animal and plant based counterparts [2]. Furthermore, GRAS (generally regarded as safe) strains of bacteria and probiotics with extracellular digestive enzyme activity were also extensively studied for their oral administration in order to improve digestion in the host [3].

Several studies showed that lipase producing probiotics can be used to reduce cholesterol levels, to overcome malabsorption, aid in the proper digestion of fats in the diet in addition to adjunct therapy in the management and prevention of mental depression [4-9]. Further, probiotics have proven to be useful in the management of colorectal cancer as evaluated in mice system and also possess anticancer properties [10-12]. Interestingly, the treatment of steatorrhea by lipase supplementation therapy has become more successful in the last decade [13]. To consider, in an in vivo study, acid resistant lipase from fungi was used to treat steatorrhea in dogs. In the study 4800 U of fungal lipase was used and a significant reduction in stool bulking and fat excretion was observed [14]. Further, microbial proteases are known for efficient proteolytic activity and are extensively used in food processing for gluten intolerance, cow’s milk allergy, soy allergy and also in other dietary protein intolerances [15,16]. Addition of microbial beta-galactosidases directly to milk represents a potential enzyme replacement therapy for primary lactase deficiency [17]. In addition, the use of probiotic fermented milk products also found beneficial in improving lactose digestion [18]. Further, biotechnological application of phytase in food is gaining importance, since phytate in the diet act as an anti-nutrient [19]. The addition of phytase or fermenting foods with phytase producing strains of GRAS and probiotics can improve nutritional value of plant based foods by improving protein digestion and also mineral bioavailability through phytate hydrolysis [20].

Compared to enzyme preparations, the use of whole microbial cells that secret extracellular digestive enzyme was found beneficial. Because, enzyme preparations must be supplemented at each meal time to ensure the digestion of ingested diet. In addition, there are several problems associated with the use of enzyme preparations that mainly includes in vivo efficacy, efficient delivery against gastric condition in the stomach, formulation and dosage [2,21,22].

Most of the commercially available digestive enzymes are from Aspergillus and Bacillus spp. Bacillus spp. continue to be dominant bacterial workhorses in microbial fermentations due to their stability in wide range of pH and temperature [23]. The GRAS strains of Bacillus mainly include Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, Bacillus pumilus, Bacillus clausii and Bacillus coagulans [24]. Given this, in the present study, the four isolates namely: Bacillus subtilis GS 1, B. cereus GS 3, B. cereus GS 199 and B. subtilis GS 547 isolated from curd samples were evaluated and optimized for their extracellular digestive enzyme activity.

Inoculum preparation

In the present study, the four isolates namely Bacillus subtilis GS 1, Bacillus cereus GS 3, Bacillus cereus GS 199, and Bacillus subtilis GS 547 isolated from curd samples were tested for their extracellular hydrolytic enzyme activity. The isolates were grown overnight in nutrient broth at 37°C. After incubation, the cells were harvested by spinning at 7000 rpm/10 min, washed thrice and resuspended in sterile distilled water to obtain OD 1.2 at A₆₀₀ and used as inoculum.

Quantitative assay for amylase, protease, lipase and phytase from Bacillus spp.

Spectrophotometric assay of Amylase by DNS method: The amylase was produced by submerged fermentation in the medium containing lactose 40 gl^-1, yeast extract 20 gl^-1, KH2PO4 0.05 gl^-1, MnCl2 .4H2O 0.015 gl^-1, MgSO4.7H2O 0.25 gl^-1, CaCl2.2H2O 0.05 gl^-1, FeSO4.7H2O 0.01 gl-l [25]. 100 mL of autoclaved production medium was inoculated with 2% inoculum and incubated at 37°C/24 h with continuous shaking (200 rpm). After incubation, the broth was centrifuged at 7000 rpm/15 min in a cooling centrifuge. Then the supernatant obtained was used as source for estimation of amylase activity by DNS (3, 5-dinitro salicylic acid) method by monitoring the amount of reducing sugar liberated from starch. To the one milliliter of crude enzyme, 0.5 mL of 1% soluble starch (prepared in 0.1 M phosphate buffer of pH 6) was added and incubated at 37°C for 20 min. Further, 1 mL of DNS reagent was added and boiled for 10 min to stop the reaction. Then, the final volume was made up to 5 mL by adding distilled water and absorbance was measured at 540 nm. One unit of amylase activity was defined as the amount of enzyme per mL of culture supernatant that released 1 μg of maltose per minute. In the assay, heat killed culture supernatant was served as control. The experiment was repeated in triplicates and results were expressed as mean ± standard deviation.

Estimation of Bacillus spp. lipase activity using copper soap method: The lipase enzyme was produced by submerged fermentation and quantified using olive oil as a substrate according to Veerapagu. In brief, after 72 h of submerged fermentation, the broth was centrifuged at 1000 rpm/20 min/4°C and supernatant was used as source of enzymes. To the 1 mL of culture supernatant, 2.5 mL of olive oil was added and incubated for 5 min at 37°C. Enzyme activity was arrested by adding 1 mL of 6N HCl and 5 mL benzene. Further, 4 mL of upper layer was carefully collected and to which 1 mL of cupric acetate pyridine was added. In the assay, reaction mixture with heat inactivated supernatant served as control. The lipase activity was determined by measuring the absorbance of free fatty acids dissolved in benzene at 715 nm and compared with oleic acid standard curve. One unit of lipase activity is defined as the amount of enzyme that liberated 1 μmol FFA in 1 min at 37°C.

Spectrophotometric assay of extracellular protease activity of Bacillus spp: The submerged fermentation for protease production was carried out according to Ikram-Ul-Haq and Mukthar (2006) and protease activity was assayed by Lowry’s method. In brief, after 24 h of incubation at 37°C with continuous shaking at 200 rpm, the fermentation broth was centrifuged at 5000 rpm for 10 min and supernatant was assayed for protease activity. The reaction mixture containing 1 mL of casein (1% solution in 0.1 M phosphate buffer of pH 6) and 1 mL of culture supernatant was incubated at 37°C/30 min. Control tubes contained supernatants boiled for 10 min. The enzyme activity was arrested by adding 5 mL of 5% trichloro acetic acid and further incubated at 37°C/10 min. Then the reaction mixture was centrifuged at 8000 rpm/10 min to remove insoluble particles and to which 1 mL of 1:1 Folin and Ciocalteau reagent and water was added. After 30 min of incubation, absorbance was measured at 700 nm. The amount of amino acids released was compared with tyrosine standard curve for the determination of protease activity.

Spectrophotometric assay of extracellular phytase activity of Bacillus spp: The extra cellular phytase was produced as described by Sreeramulu et al., with slight modification [26]. After fermentation for 72 h, the broth was centrifuged at 6000 rpm/30 min/4°C and supernatant was used for phytase assay. The assay mixture consisted of 1 mL of acetate buffer (pH 5.5) containing 6.82 mM sodium phytate, 0.2 mL of culture supernatant and 0.2 mL 100 mM MgSO₄ and incubated at 37°C for 30 min. Reaction mixture containing heat inactivated culture supernatant served as control. Immediately after stopping the reaction by adding 1 mL of 10% trichloroacetic acid, 1 mL of Taussky shorr color reagent solution prepared as described by Tungala was added and absorbance was measured at 660 nm. Then the absorption values were compared with the standard graph of potassium dihydrogen phosphate (0.1 to 0.5 mg/mL) to determine phytase activity. One unit of phytase activity was defined as the amount of enzyme required to liberate 1 mole of phosphate per min under assay conditions.

The total protein content was estimated by Lowry’s method.

Optimisation of pH, temperature and substrate concentration for amylase, lipase, protease and phytase from Bacillus spp.

The optimum pH for all the four enzyme assay was determined by incubating the enzyme–substrate at various pH from 3 to 10 in the following buffers: 50 mM sodium acetate buffer (pH 3–5), 50 mM potassium phosphate buffer (pH 5–7), 50 mM Tris–HCl buffer (pH 7–9), and 50 mM glycine–NaOH buffer (pH 9–10). Enzyme activity in each buffer was measured using the standard assays as described previously. Further, to determine the optimum temperature for the amylase, lipase, protease and phytase, the reaction mixtures were incubated at various temperature ranged from 10 to 60°C and then absorbance were measured according to the standard assays. Furthermore, to determine the effect of substrate concentration on enzyme activities, culture supernatants were incubated with various volumes of specific substrate solutions viz., starch, olive oil, casein and sodium phytate for amylase, lipase, protease and phytase respectively.

All the experiments were conducted in triplicates and results were expressed as mean ± standard deviation. The data were analyzed using two-tailed paired t-test and for all tests, the level of significance was set at p<0.05 (GraphPad Prism version 5.02 for Windows, GraphPad Software, San Diego, California, USA).

In the present study the extracellular amylase, lipase, protease and phytase activity of four Bacillus spp. were evaluated and optimized. The total protein content and specific activity of all the four extracellular digestive enzymes evaluated (Table 1).

Table 1: Total protein and specific activity.

Amylases break down α-linked sugar units in the ingested diet to glucose, maltose, and maltotriose which further undergo fermentation to produce various short chain fatty acids. In case of pancreatic insufficiency, microbial amylases are of best choice in improving the carbohydrate digestion. In the present study, the extracellular amylase activity of the four Bacillus spp. were studied and optimized for various physical parameters. As shown in the Figure 1, the amylolytic activity of the tested isolates were ranged between 87 and 128 U. Amongst, Bacillus subtilis GS 1 showed highest activity (127.66 ± 8.02U, p<0.05). Further, in optimization studies, the four Bacillus spp. tested showed optimum activity at 40°C ranging between 87 and 130 U/mL and subsequently decreased at 50°C with activity ranging from 50 to 73 U/mL. Whereas in another study, Bacillus amylase activity as 34 U/mL at 37°C/pH 7 [25], however, Abd-Elhalem et al. reported that the amylase activity of B. amyloliquefaciens was 72.5 U/mL with an increased activity at 50°C [27]. During optimization for pH, B. subtilis GS 1 and B. cereus GS 3 showed maximum activity at pH 6 while B. subtilis GS 547 and B. cereus GS 199 showed highest activity at pH 7. Further, the V_max value for B. subtilis GS 1 was 149 ± 4.05 U/mL at a substrate concentration 2 mg/mL/1 mL enzyme solution while for B. cereus GS 3 the V_max was 122 ± 3.87 U/mL at 1 mg/mL/ 1 mL enzyme solution. However, for B. subtilis GS 547 and B. cereus GS 199 V_max values were 108 ± 5.32 and 95 ± 3.69 U/mL, respectively at a substrate concentration 1.5 mg/mL/ 1 mL enzyme solution. In another study, V_max for B. subtilis was 100 U/ mL which was in agreement with the present study [28] (Figures 1-4).

Figure 1: Extracellular digestive enzyme activity of B. subtilis GS 1, B. cereus GS 3, B. cereus GS 199 and B. subtilis GS 547.

Figure 2: Temperature optimization for amylase activity.

Figure 3: pH optimization for amylase activity by Bacillus spp.

Figure 4: Effect of substrate concentration on amylase activity.

Lipase hydrolyzes fats into fatty acids and glycerols. In the present study, the crude lipase activity of the four tested Bacillus spp. ranged between 27 and 40 U/mL using olive oil as substrate. Amongst, Bacillus subtilis GS 547 showed highest activity 40 ± 2.13 U/ml (p<0.05). However, Bacillus subtilis showed 6.921 U/mL in soyabean oil, while Bacillus amyloliquefaciens exhibited 6.506 U/mL using ground nut oil as substrate at optimum pH 7.5 and temperature 45°C [29]. On storage at temperature above 40°C, pancreatic lipases lose their activity, however, microbial lipases are more resistant to heat inactivation and therefore, thermal stability is one of their desirable characteristics and which was corroborated in the present study [30]. The maximum lipolytic activity was found to be 40°C for all the four isolates tested and indicating the thermal stability at a temperature range between 20 to 50°C and decreased beyond 60°C. Further, optimum pH was 6 for all the isolates except B. subtilis GS 2 (pH 7), however, lipase activity was stable at a pH range between 5 and 8 for all the four isolates tested. Further, the maximum rate of reaction V_max for B. cereus 3 and B. subtilis 547 were found to be 31 ± 1.42 and 41 ± 2.36 U/mL respectively. Whereas for B. subtilis GS 1 and B. cereus GS 199 V_max values were 32 ±3.4 and 34 ± 2.1 U/mL respectively at a substrate concentration 0.5 mL/mL (v/v)/1 mL enzyme solution. However, Hasan et al. (2007) reported the V_max value of B. subtilis as 0.416 U/mL/min which was quite lower the maximum rate of reaction of the four tested Bacillus spp. (Figures 5-7).

Figure 5: Temperature optimization for lipase activity.

Figure 6: pH optimization for lipase activity.

Figure 7: Effect of substrate concentration on lipase activity.

The enzyme protease breakdown protein into smaller fragments as peptides and further to amino acids. In the present study, the protease activity of four tested Bacillus spp. were ranged between 120 and 137 U/mL. Amongst, B. subtilis GS 1 showed the highest enzyme activity 157 ± 3.60 U/ml (p<0.05) at 37°C. Further, the optimum temperature was found to be 40°C and moreover, enzyme activity was stable in the temperature range 20 to 50°C for all the isolates and was in agreement with the report of Kotlar et al. [31]. Further, pH 8 was optimum and the activity was stabilized between pH 6 and 9 for all the tested isolates. Furthermore, the maximum rate of reaction V_max were 160 ± 2.34, 145 ± 1.45 and 130 ± 3.72 U/mL for B. subtilis GS 1, B. cereus GS 3 and GS 9 respectively at a substrate concentration 2 mg/mL/1 mL enzyme solution. However, Nadeem et al. reported V_max value of 61.58 U/ml for B. licheniformis [32]. The stability at wide range of temperature and pH suggested that the protease from tested Bacillus spp. may function at different pH conditions in vivo and also can withstand varying temperature during processing (Figures 8-10).

Figure 8: Temperature optimization for protease activity.

Figure 9: pH optimization for protease activity.

Figure 10: Effect of substrate concentration on protease activity.

Phytase catalyzes the hydrolysis of phytic acid into myo-inositol and inorganic phosphates. In the present study, all the four tested Bacillus spp. showed phytase activity using sodium phytate as substrate. As shown in the Figure 1, the phytase activity of the four isolates ranged between 54 to 81 U/mL. Out of four isolates evaluated, B. cereus GS 3 showed highest activity (81.07 ± 2.47 U/mL, p<0.05). In addition, optimum temperature was 40°C, although activity remained fairly stable over temperature range of 30 to 50°C. Interestingly, the activity was found to stabilize at different pH range from 4 to 7 with an optimum pH 5 except for B. subtilis GS 547 (pH 6). Further, V_max values for B. subtilis GS 1 and B. cereus GS 3 were 67.64 and 85.87 U/ mL for 4.8 mg/mL/1 mL enzyme solution. Whereas for B. cereus GS 199 and B. subtilis GS 547, the V_max values were 60.75 and 93.47 U/ mL for 6 mg/mL/1 mL enzyme solution. However, in an another study, the optimum pH and temperature for phytase from B. subtilis were 7 and 60°C respectively and K_m and V_max values for sodium phytate were 0.42 mM and 4.35 μmol/min, respectively. The activity of phytase at optimum pH 4 indicated that the enzyme from the four tested isolates could be used in vivo to improve the bioavailability of phosphate from ingested food (Figures 11-13).

Figure 11: Temperature optimization for phytase activity.

Figure 12: pH optimization for phytase activity.

Figure 13: Effect of substrate concentration on phytase activity.

In the present study, B. subtilis GS 1, B. cereus GS 3, B. cereus GS 199 and B. subtilis GS 547 were evaluated and optimized for their extracellular digestive enzymes viz., amylase, lipase, protease and phytase in vitro. The digestive enzymes of all the four isolates were active at wide temperature and pH range. Further, purification and characterization of these digestive enzymes would pave for the development of therapeutic enzyme formulations to alleviate the burden of pancreatic insufficiency and other digestive disorders. In addition, further in vitro and in vivo evaluation of the safety of tested Bacillus spp. and their validation as GRAS (generally regarded as safe) would also helpful in oral administration of these isolates as source of digestive enzymes in improving digestion.

The authors sincerely acknowledge the funding support by Department of Biotechnology, Government of India, India, R&D project No.BT/PR7089/ FNS/20/734/2013.