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The present study aims to maximize proteases production by Bacillus mojavensis SA strain and their use to produce bioactive protein hydrolysates from a meat by-product. The production of SA bacteria proteases was maximized using a culture medium based on wheat bran, which offer an advantage in minimizing the production cost and enhancing the enzyme activity by using agro-industrial wastes. The composition of media and cultural conditions for optimal proteases production by B. mojavensis SA strain were investigated. A successful and significant improvement of the alkaline proteases production (four folds) by the SA strain was achieved using the medium composed of (g/l): wheat bran, 50.0; KH PO ,0.5; K HPO , 0.5; CaCl ,2.0; pH 6.0, 2 4 2 4 2 where the growth conditions were monitored at 37 °C with an agitation speed of 200 rpm. Interestingly, the enzyme preparation of B. mojavensis was applied for the preparation of protein hydrolysates from a meat by-product. Hydrolysis was carried out for 180 min at pH 12.0. The resulting hydrolysate displayed an important antioxidant activity as evaluated by the radical scavenging capacity, the reducing power, and the β-carotene bleaching inhibition. The present study showed the high proteases’ producing level by B. mojavensis SA strain in a low-cost fermentation medium (wheat bran) and their potential use in the production of bioactive protein hydrolysate from meat by-products. . . . . . Keywords Low-cost medium Bacillus mojavensis SA Protease Meatby-products Proteinhydrolysates Antioxidant activity Introduction as microbes are easy to manipulate and to produce enzymes with desired characteristics (Ramesh and Lonsane 1990). However, Alkaline proteases produced by microorganisms are of prime the overall production process of extracellular industrial enzymes interest in biotechnological applications, including detergents, is expensive due to the high cost of substrates and media com- foods, pharmaceutical, tannery, and leather industries (Pandey ponents used for bacterial growth (Sharma et al. 2017). This is et al. 2000; Gupta et al. 2002a, b). The use of microorganisms the most critical factor limiting the use of alkaline proteases in for the production of hydrolytic enzymes is in economical bulk, industrial applications. Therefore, developing novel processes for enzymes’ production, with respect to industrial requirements and low production cost, is commercially appreciable. Highlights • Alkaline proteases production using agro-industrial wastes was en- In general, no defined medium has been designed for the com- hanced. mon production of alkaline proteases from different microorgan- • The best medium found to be advantageous for maximum proteases isms. In fact, each strain has its specific conditions for maximum production was based on wheat bran. enzyme production (Gupta et al., 2002a, b). Many researchers • Successful use of Bacillus mojavensis proteases to digest meat by- have been attempted to improve enzyme production using glucose product proteins and produce bioactive protein hydrolysates. or starch, coupled with expensive nitrogen sources such as yeast * Amal Hammami extract, peptone, or casamino acids. Thus, many industrial en- email@example.com deavors have been made to induce enzymes’ production using inexpensive carbon and nitrogen sources (Prakasham et al. 2006; Laboratoire de Génie Enzymatique et de Microbiologie, Ecole Mhamdi et al. 2014). It is, therefore, important to search new low- Nationale d’Ingénieurs de Sfax, Université de Sfax, B.P. 1173-3038, cost substrates that are suitable for proteases activity enhancement. Sfax, Tunisia 474 Ann Microbiol (2018) 68:473–484 The growth and enzyme production of the microorganism Enzyme assay are strongly influenced by medium components, like carbon and nitrogen sources. Besides the nutritional factors, the cul- Proteolytic activity was measured by the method of Kembhavi tural parameters, such as temperature, pH, and incubation et al. (1993). An aliquot of 0.5 ml from the culture superna- time, played a major role in enzymes’ production tant, suitably diluted, was mixed with 0.5 ml of 100 mM KCl- (Mazzucotelli et al. 2014). Therfore, the optimization of media NaOH (pH 12.0) containing 1.0% (w/v) casein. The reaction components and cultural parameters are the primary task in a mixture was then incubated for 15 min at 60 °C and stopped biological process would be crucial. by the addition of 0.5 ml of TCA (20%, w/v). The reaction Besides their role in industrial applications, microbial pro- tubes were allowed to stand at room temperature for 15 min teases have been widely used in the elaboration of bioactive and centrifuged at 12,500 rpm for 15 min to remove the pre- peptide mixtures through fermentation (Jemil et al. 2014)or cipitate. The optical density of the soluble fraction was read at hydrolysis processes (Lassoued et al. 2015; Abdelhedi et al. 280 nm. A standard curve was generated using solutions of 0– 2016). A great deal of interest has been made for antioxidant 50 mg/l of tyrosine. One unit of protease activity against ca- peptides from food proteins, including fish (Abdelhedi et al. sein was defined as the amount of enzyme required to liberate 2016), ham (Mora et al. 2014), and chicken (Centenaro et al. 1 μg of tyrosine per minute under the experimental conditions. 2014) meats. Poultry meat sausage is one of the oldest and Protease activity represented the mean of at least two determi- most consumed meat products in the world, for its sensory nations carried out in duplicate. quality, nutritional value, and economic cost. Due to the im- portant sausages production, the meat processing industries Analysis of different carbon and nitrogen sources generate huge quantities of wastes and by-products. for proteases production Therefore, meat sausage by-products (MSB) represent a po- tential source of proteins that can be used to generate protein Initial medium (M1) consisted of the following composition hydrolysates following proteolysis action. (g/l): hulled grain of wheat, 10; yeast extract (YE), 2.0; CaCl , In the present work, different low-cost and agricultural by- 2.0; K HPO ,0.1;and KH PO , 0.1. The initial pH of the 2 4 2 4 products were screened in view of producing alkaline prote- culture was adjusted at pH = 9.0. The physicochemical com- ases from Bacillus mojavensis SA with the highest yield and position of hulled grain of wheat is illustrated in Table 1. the lowest cost fermentation media. A various number of pa- Different carbon sources with low cost (at 10 g/l), instead rameters affecting the production of alkaline proteases are of hulled grain of wheat, were screened to maximize proteases described. On the other hand, MSB were digested with B. production. By-products from semolina factories such as mojavensis proteases in order to produce protein hydrolysate, hulled grain of wheat, spent grain of corn, soya meal and with antioxidant potential. wheat bran, and others prepared in our laboratory, such as Mirabilis jalaba tuber powder, shrimp wastes powder, feather meal, waste octopus powder, crab flour, corn flour, poultry Materials and methods feathers, and cuttlefish’s waste powder, were tested. After selecting the best carbon source, the effect of increasing con- Chemicals and reagents centration (10–50 g/l) on the protease activity was assessed. On the other hand, different nitrogen sources (used at 2 g/l) Casein, 1,1-diphenyl-2-picrylhydrazyl (DPPH•), butylated such as, casein peptone, soy peptone, ammonium sulfate hydroxyanisole (BHA), β-carotene, and linoleic acid were (NH ) SO , ammonium chloride (NH Cl), sodium nitrate 4 2 4 4 purchased from Sigma Chemical Co. (St. Louis, MO, USA). (NaNO ), potassium nitrate (KNO ), urea (CO (NH ) )and 3 3 2 2 Trichloroacetic acid (TCA), sodium hydroxide, and other soya meal were tested for enzyme production by B. chemicals were of analytical grade. mojavensis SA. All media were autoclaved at 120 °C for 20 min. Microorganism Inocula were routinely grown in Luria-Bertani (LB) broth medium, composed of the following (g/l): peptone, 10.0; yeast Bacillus mojavensis SA, producing stable alkaline proteases, extract, 5.0 and NaCl, 5.0 (Miller 1972). Then, the culture was isolated from slaughterhouse waste water. It was identi- medium was inoculated with a level of 0.1% and cultivations fied on the basis of 16S rRNA gene sequencing and assigned were conducted in 250-ml Erlenmeyer flasks, with a working by the accession number MF407277.1. The strain was con- volume of 25 ml, and incubated at 37 °C on a rotatory shaker served in 30% (v/v) glycerol/LB broth at − 80 °C and replicat- at 200 rpm for 24 h. Thereafter, cultures were centrifuged, and ed in Luria-Bertani (LB) medium for routine using. The opti- the cell-free supernatants were used for the proteolytic activity mal proteases activity conditions have been previously studied estimation. All experiments were carried out in duplicate and in a previous work (Hammami et al. 2017). repeated at least twice. Ann Microbiol (2018) 68:473–484 475 Table 1 Chemical composition Hulled grain of wheat Wheat bran Soya meal Meat sausage of hulled grain of wheat, wheat by-product bran, soya meal, and meat by- product Proteins (%) 18.17 ± 0.1 21.13 ± 0.62 56.52 ± 0.06 41.69 ± 0.52 Fat (%) 4.52 ± 0.17 5.45 ± 0.13 4.39 ± 0.1 32.71 ± 1.50 Ash (%) 2.41 ± 0.07 11.66 ± 0.06 8.62 ± 0.021 15.57 ± 0.10 Carbohydrates (%) 72.63 ± 5.18 60.48 ± 2.71 32.65 ± 1.74 6.51 ± 0.08 Analytical results are reported, on dry matter basis Effect of salts’ concentrations viable count (spread plate method) determination. In parallel, the proteases production at different time intervals was deter- The effect of various salts (K HPO ,KH PO ,and CaCl )on mined after removal of cells by centrifugation. 2 4 2 4 2 enzyme activity and the biomass concentration (CFU) was studied by varying their concentrations. After 24 h of bacteria Determination of chemical composition cell growth, the supernatant of the fermented substrate extract was used for the enzyme activity assay, as previously The moisture and ash content were determined according to described. the A.O.A.C. standard methods numbers 930.15 and 942.05 (A.O.A.C. 2000), respectively. The protein content was deter- Effect of additives on enzymes’ production mined by estimating its total nitrogen content by the Kjeldahl method according to the A.O.A.C. method number 984.13 To determine the effect of metal ions (MnSO ,MgSO , (A.O.A.C. 2000). A factor of 6.25 was used to convert the 4 4 CuSO ,NaCl,BaCl and ZnCl ) on the enzyme activity pro- nitrogen value to protein. Fat was determined gravimetrically 4 2 2 duction, different ions were individually added at a concentra- after Soxhlet extraction of dried samples with hexane. tion of 5 mM to the media cell culture. Each culture was Carbohydrates content was estimated as described by incubated at 37 °C for 24 h at 200 rpm and the supernatant Dubois et al. (1956). All measurements were performed in was analyzed for protease activity. triplicate. Effect of experimental conditions on enzymes Preparation of protein hydrolysate from meat production sausage by-product using SA proteases by the in vitro enzymatic hydrolysis technique The effect of pH, temperature, and agitation rate on the en- zymes production was assessed. Initially, the medium pH was The meat sausage by-product (MSB), which corresponds to adjusted at different values of 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 and the product generated during the manufacture of poultry sau- 12.0, and then incubated at 37 °C for 24 h. Similarly, the sages with technological defects, was collected from a local ability of B. mojavensis SA to grow and produce enzymes, processing industry (Chahia, Sfax, Tunisia) and minced into at different temperatures (30, 37 and 40 °C) was investigated. small pieces. The chemical composition of MSB (g/100 g The optimal temperature for enzyme production was deter- MSB) is presented in Table 1. mined. On the other hand, to study the agitation rate effect, First, the raw material (50 %, w/v) was cooked in distilled bacterial cultivation was carried out at different agitation rates water at 95 °C for 15 min and then the mixture was homoge- (150 and 200 rpm) at 37 °C for 24 h. For each experiment, nized in a Moulinex® blender for 5 min. Hydrolysis was after 24 h of culture time, the supernatant was analyzed for carried out under optimal conditions of alkaline proteases ac- protease activity. Three independent experiments were per- tivity (pH 12.0; 50 °C) (Hammami et al. 2017). Crude en- formed for each parameter. zymes were added with an enzyme/substrate ratio of 3 units of enzyme per milligram of protein. During hydrolysis, the pH Time course of enzymes production by B. mojavensis was maintained constant at the desired value by the addition of SA 4 N NaOH solution. After the achievement of the digestion process, the reaction was stopped by heating the solution at To study the relation between enzymes production and the 95 °C for 20 min, for enzyme inactivation. The obtained prod- growth profile of the bacterium, a volume of 100 ml of the uct was centrifuged at 8000 rpm for 20 min and the superna- final production media was inoculated and the culture was tant was freeze-dried (Bioblock Scientific Christ ALPHA 1-2, monitored under the optimized conditions, using 1-l flasks. IllKrich-Cedex, France). The resulting hydrolysate from meat The growth was measured at a regular interval of time by total by-product (MBH) was stored at − 20 °C for further use. The 476 Ann Microbiol (2018) 68:473–484 undigested meat by-product (UMB) was treated under the 700 nm after 10 min. Values presented are the mean of tripli- same conditions without enzyme addition and serves as cate analyses. control. The hydrolysis degree (HD), defined as the percent ratio of Antioxidant assay using β-carotene bleaching method the number of peptide bonds cleaved to the total number of peptide bonds in the substrate per mass unit, was calculated The ability of MBH and UMB to prevent β-carotene from from the amount of NaOH solution added to keep the pH bleaching was assessed as described by Koleva et al. (2002). constant during the hydrolysis (Adler-Nissan 1986). First, the emulsion of β-carotene/linoleic acid was freshly The protein fraction contained in the hydrolysate was pre- prepared by dissolving 0.5 mg of β-carotene, 25 μlof linoleic cipitated using ammonium sulfate (saturation up to 80%). acid and 200 μl of Tween 40 in 1 ml of chloroform. The Then, the precipitate was dissolved in distilled water and dia- chloroform was then completely evaporated under vacuum lyzed for 48 h against water. Dialyzed proteins were freeze- in a rotatory evaporator at 40 °C. Then, 100 ml of distilled dried to be further analyzed. water were added and the resulting mixture was vigorously stirred. Thereafter, 2.5 ml of the β-carotene/linoleic acid emul- sion were transferred to test tubes containing 0.5 ml of each Determination of antioxidant activities sample (from 1 to 5 mg/ml). Control tube was prepared in the same conditions by adding 0.5 ml of H O to the emulsion DPPH� free radical scavenging activity instead of sample. The absorbance of each test tube was mea- sured at 470 nm before and after incubation for 1 h at 50 °C. The DPPH• (2,2-diphenyl-1-picrylhydrazyl) free radical scav- BHA was used as a positive standard. The antioxidant activity enging activity of the different samples was determined as was evaluated using the following formula: described by Bersuder et al. (1998). A volume of 500 μlof hi MBH and UMB at different concentrations (1 to 5 mg/ml) was 0 0 Antioxidant activityðÞ % ¼ 1−ðÞ A −A = A −A 100 0 t 0 t addedto375 μl of absolute ethanol and 125 μlof0.2‰ DPPH• solution. The mixtures were then kept for 60 min in where A and A represent the absorbances of the test sample 0 t dark at room temperature, and the reduction of DPPH• radical measured before and after incubation, respectively, and A ′ was measured at 517 nm using a UV-visible spectrophotome- and A ′ represent the absorbances of the control measured ter (T70, UV/VIS spectrometer, PG Instruments Ltd., China). before and after incubation, respectively. The test was carried Lower absorbance of the reaction mixture indicated higher out in triplicate. DPPH• radical scavenging activity. The control was conduct- ed in the same manner, except that distilled water was used Statistical analysis instead of sample. BHA was used as positive control. The DPPH• radical scavenging activity was calculated as follows: All experiments were carried out at least in triplicate, and Scavenging activityðÞ % ¼½ ðÞ A −A þ A 100 =A average values with standard deviation errors were reported. C S B C Significance between values was analyzed using the SPSS where A , A ,and A represent the absorbances of the control, C S B software package (SPSS, ver. 17.0 for windows professional the sample reaction, and the blank tubes, respectively. The test edition). A one-way analysis of variance (ANOVA) was then was carried out in triplicate. performed and followed by Duncan’s test to estimate the sig- nificance at the 5% probability level. Reducing power assay 3+ 2+ The capacity to convert Fe into Fe was evaluated accord- Results and discussion ing to the method of Yildirim et al. (2001). A sample solution (0.5 ml) of each hydrolysate at different concentrations (from Effect of different carbon sources on the proteases 1 to 5 mg/ml) was mixed with 1.25 ml of 0.2 M phosphate activity buffer (pH 6.6) and 1.25 ml of 1% (w/v) potassium ferricya- nide solution. The reaction mixtures were incubated at 50 °C Because carbon is considered the primary nutrient for bacteria for 30 min. After incubation, 0.5 ml of 10% TCA was added growth, different low-cost complex carbon sources, added at and the reaction mixtures were then centrifuged at 3000 rpm 10 g/l in M1 medium, were analyzed. Data reported in Fig. 1 for 10 min. Finally, 1.25 ml of the supernatant solution, from showed that the maximum proteases production (~ 400 U/ml) each sample mixture, was mixed with 1.25 ml of phosphate was obtained using wheat bran and soya meal, followed by buffer and then 0.25 ml of 0.1% ferric chloride was added. corn flour (254.54 ± 0.01 U/ml). However, poultry feathers The absorbance of the resulting solutions was measured at were ineffective for proteases production. Ann Microbiol (2018) 68:473–484 477 Fig. 1 Effect of different carbon sources on the biomass and the a a production of proteases by B. mojavensis SA. Cultivations were performed for 24 h at 37 °C in b bc basal medium consisting of (g/l): carbon source 10, yeast extract 2; KH PO 0.1; K HPO 0.1, and 2 4 2 4 CaCl (pH 9.0). Data are expressed as mean ± SD. Different letters with different carbon sources indicate significant differences in the protease activity at P <0.05 Wheat bran and soya meal, being the best carbon sources, increase of wheat bran concentration (Fig. 2a).However,the were examined at different concentrations (10–50 g/l), as soya meal concentration correlated negatively with the proteo- shown in Fig. 2. It was found that proteolytic activity was lytic activity, which did not exceed 330 U/ml at 10 mg/ml (P highly improved as the concentration of wheat bran increased < 0.05). Hence, wheat bran served as the better low-cost car- and reached 745 and 883 U/ml at 40 and 50 g/l, respectively. It bon source for the proteases’ production. Previous study re- was further noted that bacterial growth increased with the ported the use of wheat bran as a carbon source for enzyme Fig. 2 a, b Effect of increasing Proteolytic activity 1000 35 concentration of carbon sources 900 Biomass (wheat bran or soya meal) on proteases production by B. mojavensis SA. Different letters 25 with different concentrations of carbon sources indicate significant differences at P <0.05 0 0 10 20 30 40 50 Concentration of wheat bran (g/l) (a) 400 450 Proteolytic activity Biomass 0 0 10 20 30 40 50 Concentration of soya meal (g/l) (b) Protease activity (U/ml) Protease activity (U/ml) Biomass (10 CFU/ml) Biomass (10 CFU/ml) 478 Ann Microbiol (2018) 68:473–484 production (Uyar 2004; Meena et al. 2013). Proteases’ pro- showed that wheat bran could play the role of, both, carbon duction from Bacillus species using various agricultural resi- and nitrogen source. dues was widely described in literature, including the hulled The chemical composition of wheat bran is given in Table grain of wheat (Kumar et al. 2014), soybean meal (Joo et al. 1. Data showed that wheat bran contained high level of protein 2002), wheat flower (Joo and Chang 2005) and rice bran (21.13%) and sugar (60.48%). High ash (11.66%) and low (Naidu and Devi 2005). Such products constitute better sub- lipid content (5.45%) were further noted. Thus, wheat bran strates for enzyme production than simple sugars like glucose, could be considered as a complete medium containing the maltose, and sucrose, which can induce catabolic repression essential substances required for a microbial growth, and mechanism and greatly decrease proteases’ production thereby it could play the role of carbon and nitrogen source. (Mehta et al. 2006; Sinsuwan et al. 2015). In fact, in the ab- Hence, wheat bran may be considered as a cost-effective sub- sence of glucose, the protease synthesis could be repressed strate for the production of proteases. when the energy status of the cells would be high (Sharma In the same context, Chu (2007), Tari et al. (2006) and et al. 2017). In this context, proteases production by Jaswal et al. (2008) have reported, respectively, that Bacillus Pseudomonas aeruginosa (MCM B-327) in soya bean and sp. APP1, Bacillus sp. L21 and Bacillus circulans can tryptone-based media was suppressed by 95 and 60%, follow- considerably grow and produce proteases, when using soya ing its supplementation with glucose and fructose, respective- bean meal as an organic nitrogen source. Zambare et al. (2011) ly (Zambare et al. 2011). similarly proved that maximum proteases production by P. aeruginosa MCM B-327 was obtained with a combination of soybean meal and tryptone. In another study, protein hy- Effect of nitrogen sources drolysates from different fish species such as tuna, cod, salm- on, and unspecified fish have been used as nitrogenous Besides the carbon source, nitrogen serves as an important sources for microbial growth (Dufossé et al. 2001). nutrient for the proteases production. The requirement of a specific nitrogen supplement differs from a microorganism to another. To this end, casein peptone, soya peptone, ammo- Effect of oligo-elements addition nium sulfate, ammonium chloride, sodium nitrate, potassium nitrate, urea and soya meal were investigated in M1 medium The influence of K HPO and KH PO concentration on the 2 4 2 4 containing wheat bran at 50 g/l, as a carbon source (Fig. 3). cell growth and alkaline protease production by SA strain was Data show that all the tested nitrogen sources allowed an im- investigated (Fig. 4). After 24 h of incubation, the maximum portant proteases production, but they still lower than the yeast alkaline protease production (1259 U/ml) was observed in the extract. On the contrary, ammonium chloride and ammonium medium containing 0.5 g/l of both K HPO and KH PO . 2 4 2 4 sulfate have been reported to suppress alkaline proteases pro- Increasing K HPO and KH PO concentrations to 2 g/l led 2 4 2 4 duction by Bacillus sp. 2–5 (Darani et al. 2008). More inter- to a drastic reduction in the proteases production, while the estingly, proteases production in the absence of yeast extract bacterial growth increased. Similarly, Shirato and Nagatsu in the culture medium, based on the wheat bran alone, gave (1965) found that 0.8 g/l of KH PO have the greatest effect 2 4 the best proteases activity level with an activity close to on the production of alkaline proteases by Streptomyces 1000 U/ml and a biomass of 320.10 CFU/ml. These findings griseus. Fig. 3 Effect of different nitrogen sources on the proteases production by B. mojavensis SA. b Cultures were conducted at 37 °C in the basal medium containing cd cd wheat burn at 50 g/l and supplemented or not with one of nitrogen sources (2 g/l). Different letters with different nitrogen sources indicate significant differences at P <0.05 Ann Microbiol (2018) 68:473–484 479 Fig. 4 Proteases production by B. a b d mojavensis SA on medium containing wheat bran at 50 g/l supplemented with various concentrations of K HPO and 2 4 KH PO . Different letters with 2 4 different concentrations of K HPO and KH PO indicate c 2 4 2 4 correlated negatively differences at P <0.05 Moreover, the effect of CaCl concentration was investi- and Mg are known by their positive effect on the alkaline gated (Table 2). An addition of 2 g/l of CaCl was found to protease activity from B. circulans (Rao et al. 2009)and give the greatest effect for the production of alkaline prote- Bacillus licheniformis MP1 (Jellouli et al. 2011). Basu et al. ases, whereas, the activity was gradually reduced when the (2008) reported the activation of protease from Aspergillus +2 +2 +2 salt concentration increased above 2.5 g/l. A slight activity niger AB by metal ions, such as Ca ,Fe ,Zn , and reduction occurred in the absence of CaCl . It has been report- Mg . ed that an increased salt concentration creates changes in the lipid composition of cell membranes, leading to the growth Factors affecting the production of proteases by SA rate decrease along with the enzyme production (Sandhya et al. 2005). Temperature, agitation rate, and initial pH value of culture medium were investigated as the most important physical fac- Metal ion requirements for proteases production tors affecting the bacterial growth and enzyme production (Srividya and Mala 2011;Pathak and Deshmukh 2012)and The results illustrated in Table 3 show the effect of different results are summarized in Table 4. ions, supplemented at 0.5 g/l to the culture media, on the Proteases production by B. mojavensis SA was studied at proteases production and cell growth. The activity depended three different temperatures 30, 37, and 40 °C. Culture per- on the nature of the element used, and the maximum protease formed at 37 °C showed the maximum production level. yield, of 1356 U/ml, was obtained using the medium culture However, at 40 °C, the activity lost more than 83% after devoid of ions, as compared to all the other mixtures. 24 h of incubation, compared to the maximum level. Hence, 2+ 2+ However, the presence of Zn and Cu totally inhibited bac- B. mojavensis SA strain could be classified as a mesophilic terial growth and, thus, the proteases production. Ibrahim et al. bacterium. Similarly, the optimum temperature for protease (2015) reported that the supplementation of the culture medi- production by a number of Bacillus species such as Bacillus 2+ 2+ 2+ 2+ um with Zn ,Cu ,Fe ,andCo caused severe inhibition subtilis strain (Abusham et al. 2009), Bacillus aquimaris of proteases production by Bacillus sp. NPST-AK15, particu- larly at high cation concentrations. Various metal ions have Table 3 Effect of metal ions, added at 0.5 g/l, on the proteases activity and biomass been reported to affect the activity of proteases. In fact, Ca Metal ion Activity (U/ml) Biomass (CFU/ml × 10)pH Table 2 Effect of CaCl level on the proteases activity and biomass Control 1356.70 ± 44.90 412 8.32 2+ 9 [Ca ] (g/l) Activity (U/ml) Biomass (CFU/ml × 10)pH MgSO 1175.66 ± 28.40 496 9.10 c d NaCl 953.87 ± 0.02 479 9.13 0.0 1028.00 ± 0.51 342 8.70 b e 1.0 1129.45 ± 22.62 404 8.51 MnSO 649.39 ± 3.81 338 9.09 a b 2.0 1259.71 ± 12.85 412 8.32 BaCl 1269.03 ± 11.60 367 9.01 d f 2.5 1191.09 ± 0.00 387 8.76 ZnCl 0.00 130 7.96 3.0 728.25 ± 25.71 733 8.61 CuSO 0.00 40 7.60 The initial pH of the culture was adjusted at pH = 9.0. pHf is the final pH The initial pH of the culture was adjusted at pH = 9.0. pH is the final pH value of the medium measured at the end of the reaction. Data are value of the medium measured at the end of the reaction. Data are expressed as mean ± SD. Different letters in the same column indicate expressed as mean ± SD. Different letters in the same column indicate significant differences at P <0.05 significant differences at P <0.05 480 Ann Microbiol (2018) 68:473–484 Table 4 Effect of temperature, Parameters rpm/ Activity (U/ml) Biomass (CFU/ml × 10)pH agitation rate and pH on the f °C proteases activity and biomass after 24 h of incubation time Temperature (°C) 30 1198.18 ± 12.85 550 7.37 37 1467.27 ± 95.13a 420 8.32 40 240.00 ± 10.28c 80 8.97 Agitation rate (rpm) 150 989.27 ± 15.42 460 8.71 200 1388.38 ± 23.14 420 8.32 pH 6834.54±2.57 220 8.11 7 850.90 ± 15.42 250 8.43 8 1067.27 ± 23.14 390 8.6 9 1463.63 ± 17.99 420 8.32 10 1432.72 ± 10.28 370 8.62 11 836.36 ± 5.14 200 8.77 12 705.45 ± 0.10 190 8.9 For the effect of temperature and the agitation rate, the initial pH of the culture was adjusted at pH = 9.0. pHi and pH represent the initial and the final pH values of the medium, respectively. Data are expressed as mean ± SD. Different letters in the same column indicate significant differences at P <0.05 VITP4(ShivanandandJayaraman 2009), Bacillus by the studied isolate. This finding was in agreement with that proteolyticus CFR3001 (Bhaskar et al. 2007) and Bacillus reported by Ibrahim et al. (2015) and Joshi et al. (2008)for amovivorus (Sharmin et al. 2005) was found to be at 37 °C. Bacillus sp. NPST-AK15 and halophilic bacterium These findings may be explained by the effect of temperature MBIC3303, respectively. On the contrary, Nadeem et al. on monitoring enzyme synthesis at mRNA transcription (2008) found that maximum alkaline protease production by (Votruba et al. 1991) and, thus, regulating the production of B. licheniformis was obtained with agitation culture speed of intracellular and extracellular enzymes. For extracellular en- 140 rpm. zymes, temperature influences their secretion, possibly by The pH of culture is furthermore an important parameter to changing the physical properties of the cell membrane. be optimized. In fact, pH affects all enzymatic processes and Similarly, temperature strongly affects the secretion of prote- transportation of various components across the cell mem- ases, influencing the rates of biochemical reactions. brane (Sharma et al. 2017). As proton motive force in Moreover, the effect of the agitation rate on the proteases chemiosmosis is affected by the medium pH value, it is pos- production of B. mojavensis SA was studied (Table 4). sible that under optimum pH range, the relative metabolic Maximum protease production (around 1400 U/ml) was ob- efficiency will be high (Singh et al. 2010). With respect to tained after SA culture incubation at 37 °C and 200 rpm dur- pH, B. mojavensis SA could grow and produce alkaline pro- ing 24 h. This activity was, however, markedly decreased at teases over a wide pH range from 6.0 to 12.0, with optimum 150 rpm of agitation speed. These results indicated the impor- production obtained at pH 9.0, confirming the alkali nature of tance of high aeration level for alkaline proteases’ production the SA proteases (Table 4). Under both acidic and alkaline pH Fig. 5 Time course of proteases 450 1800 Growth production and growth of B. mojavensis SA in the final 400 1600 Protease activity medium. Shaking cultivation was 350 1400 carried at 37 °C. Proteolytic activity was determined in culture 300 1200 filtrate obtained after removal of 250 1000 cells by centrifugation 200 800 150 600 100 400 50 200 0 0 0 1020304050 Time (h) CFU (10 ) Activity (U/ml) Ann Microbiol (2018) 68:473–484 481 Fig. 6 Kinetic hydrolysis curve of MBH elaborated by the action of SA proteases 0 30 60 90 120 150 180 Time (min) conditions, the bacterial growth and proteases production Preparation of protein hydrolysate from MSB were significantly reduced, compared to those obtained at pH values, ranged between 8.0 and 10.0 (P < 0.05). Similar Hydrolysate (MBH) from MSB was prepared using the crude results have been reported by Chu (2007), Thumar and Singh enzymes of B. mojavensis SA under optimal conditions of (2007), and Bhaskar et al. (2007), who found that the alkaline protease activity (pH 12.0, 50 °C). The kinetic curve of hy- proteases from Bacillus sp. strain APP1, Streptomyces drolysis is presented in Fig. 6. Hydrolysis profile was charac- clavuligerus strain Mit-1, and B. proteolyticus CFR3001, re- terized by a high initial rate during the first minutes, which spectively, were produced at pH 9.0. Higher initial pH values, was subsequently decreased with reaction time and then the about 10.0 for B. licheniformis TISTR 1010 (Vaithanomsat et enzymatic reaction reached a steady-state phase after 120 min. al. 2008), 10.5 for B. circulans (Jaswal et al. 2008), and 10.7 The shape of hydrolysis curve could be explained by the ac- for Bacillus sp. 2–5 (Darani et al. 2008), have been reported tion of inhibitory peptides, which were continuously solubi- for maximum proteases production. lized during the hydrolysis and they may inhibit the enzyme action. The obtained HD value of 12% after 180 min of hy- drolysis time reflected the presence of short peptides in MBH, Time course of proteases production by B. mojavensis which could serve as natural bioactive compounds (antioxi- SA and cell growth dant, antibacterial, etc.). Microbial proteases have been widely used to obtain protein hydrolysates from many sources (Nasri Generally, the time required for the optimum proteases pro- et al. 2013;Lassoued et al. 2015). duction by bacteria is comprised between 48 h to 9 days (Sharma et al. 2017). The effect of incubation time on prote- Antioxidant potential of MBH ases production and cell growth of B. mojavensis SA under the optimal conditions (pH 9.0, 37 °C and 200 rpm) is displayed Peptides contained in the MBH were concentrated by the ad- in Fig. 5. As it can be seen, the proteolytic activity started to dition of ammonium sulfate (80%) followed by a dialyze step appear after 10 h of incubation, then, it was gradually in- for 48 h to remove salts. The same treatment was applied for creased during the exponential phase of bacterial growth and the undigested meat by-product (UMB). reached its maximum after 24 h (1456 U/ml). After 32 h of culture period, the activity gradually decreased to 653 U/ml at 44 h, during the stationary phase. In fact, the proteases pro- DPPH� radical scavenging assay duction can be both growth and non-growth dependent. First, the protease production profile was found to be associated The results presented in Fig. 7a indicated that MBH exhibited with growth, then the enzyme production peak occurred at a significant radical scavenging activity that increased with the mid exponential phase (9 h), and thereafter the enzyme increasing the concentration to reach 96% at 5 mg/ml, while denaturation occurred, while the biomass reached its maxi- the UMB did not show any antioxidant effect (P <0.05). mum level (Soares et al. 2005). Accordingly, Thumar and These results suggest that MBH contained some peptides that Singh (2007)and Lazimetal. (2009) showed that the maximal were hydrogen donors, which could react with free radicals to protease production started in early stationary phase of convert them to more stable products and terminate the radical bacterial growth and then decreased with increasing chain reaction (Khantaphant and Benjakul 2008). Previous incubation time. studies have reported that DPPH• radical scavenging activity Hydrolysis degree (%) 482 Ann Microbiol (2018) 68:473–484 MBH UMB BHA indicate that MBH, with an HD of 12%, contained biopeptides that could serve as electron donors. It has been reported by Abdelhedi et al. (2016) that the highest reducing power was observed for the hydrolysate having a medium HD of 13.7%. Antioxidant activity measured by the β-carotene bleaching method In an oil-water emulsion-based system, linoleic acid acts as a Concentration (mg/ml) (a) free radical producer that generates peroxyl radicals under thermally induced oxidation. The preservation of the β- 3.5 MBH UMB BHA carotene color can be hindered by the presence of a stronger free radical scavenger. The antioxidant activity of MBH and 2.5 UMB measured by β-carotene bleaching inhibition were re- ported in Fig. 7c. Except UMB, the MBH and BHA inhibited the oxidation of β-carotene in a dose-dependent way (47 and 1.5 99% at 5 mg/ml, respectively). These results demonstrated that MBH may contain peptides with hydrophobic character 0.5 that can donate hydrogen atoms to peroxyl radicals of linoleic acid in an emulsified mixture. Concentration (mg/ml) (b) The overall results proved that MBH contains peptides that exhibited interesting antioxidant activities, in terms of its rad- 120 ical scavenging activity, reducing power and β-carotene MBH UMB BHA bleaching protection. Conclusion 20 Due to the increasing economic relevance of enzymes, a part of this study was conducted in an attempt to screen a variety of fermentation parameters, including medium composition and Concentration (mg/ml) (c) culture conditions in order to maximize alkaline proteases pro- Fig. 7 DPPH& free radical scavenging effect (a), reducing power (b)and duction from B. mojavensis SA. In addition, the use of agro- antioxidant activity using the β-carotene bleaching method (c)of MBH industrial wastes (wheat bran and soya meal) as components in and UMB used at different concentrations. All values are given in mean ± the bacterial growth medium offers an advantage in minimiz- standard deviation (SD) of three determinations. MBH and UMB ing the production cost and maximizing the unities of prote- represent meat sausage by-product hydrolysate and undigested meat by-product, respectively. BHA (butylated hydroxyanisole) was used as ases. The highest production level was obtained after 24 h positive control under mesophilic conditions. This result would facilitate the economic design of large-scale fermentation operation system increased with the smallest peptides content present in the in bioreactor in batch/fed batch process. The second part of protein hydrolysate (Nasri et al. 2014). this study revealed the successful use of SA strain to produce bioactive protein hydrolysate from a meat sausage by-product. The resulting hydrolysate exhibited strong antioxidant activi- Reducing power activity ties. Therefore, this study provides a possible biotechnological application of B. mojavensis in food processing technologies. The reducing power assay is often used to evaluate the ability 3+ of antioxidant to donate an electron Fe ion (Khantaphant Funding This work was funded by the Ministry of Higher Education and and Benjakul 2008). The reducing power of MBH and Scientific Research, Tunisia. UMB as well as BHA, at different concentrations is shown in Fig. 7b. As expected, the reducing power values of MBH Compliance with ethical standards increased with increasing its concentration. 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Annals of Microbiology – Springer Journals
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