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Oct 3, 2010 - production of protease by Bacillus cereus BG1 strain: whole sardinelle powder (WSP), meat sardinelle powder. (MSP) and combined heads ...
Ann Microbiol (2011) 61:273–280 DOI 10.1007/s13213-010-0134-0

ORIGINAL ARTICLE

Enhanced Bacillus cereus BG1 protease production by the use of sardinelle (Sardinella aurita) powder Alya Sellami-Kamoun & Basma Ghorbel-Frikha & Anissa Haddar & Moncef Nasri

Received: 21 February 2010 / Accepted: 16 September 2010 / Published online: 3 October 2010 # Springer-Verlag and the University of Milan 2010

Abstract The present study is concerned with the selection of a new economical medium for growth and production of a calcium-dependent protease by Bacillus cereus BG1 strain.Various fish powders were prepared from sardinelle (Sardinella aurita) and then tested for the growth and the production of protease by Bacillus cereus BG1 strain: whole sardinelle powder (WSP), meat sardinelle powder (MSP) and combined heads and viscera sardinelle powder (CHVSP). Protease synthesis was significantly low when the strain was grown in media containing only fish powders. However, the addition of Ca2+ to the fish media enhanced the production of protease. Maximum activity was obtained on CHVSP followed by WSP. Other metal ions, such as Mg2+, Mn2+ and Ba2+, were also found to enhance protease production. However, in media containing maltose as carbon source and ammonium sulfate as nitrogen source, protease activity was detected only when media were supplemented with CaCl2.These results clearly indicated that the utilization of fish powders, in particular that produced from sardinelle by-products, may result in a cost-effective process suitable for large-scale production of proteases.

Alya Sellami-Kamoun and Basma Ghorbel-Frikha contributed equally to this work. A. Sellami-Kamoun : B. Ghorbel-Frikha : A. Haddar : M. Nasri Laboratoire de Génie Enzymatique et de Microbiologie, Ecole Nationale d’Ingénieurs de Sfax, B.P. “W” 3038, Sfax, Tunisie A. Sellami-Kamoun (*) Faculté des Sciences de Sfax, BP 1171-3000, Sfax, Tunisia e-mail: [email protected]

Keywords Calcium-dependent protease . Bacillus cereus . Sardinella aurita . Fish powder . Heads and viscera

Introduction Each year, large amounts of protein-rich by-products from the seafood industries are discarded. They include bone, skin, fins, internal organs, heads, and some muscles (Nair and Gopakumar 1982). In Tunisia, sardinelle catches are very important and totalled about 13,300 tonnes in 2002 (F. A.O. 2004). During processing, solid wastes including heads and viscera are generated and can amount to 30% of the original material. These wastes, which represent an environmental problem to the fishing industry, constitute an important source of proteins. Traditionally, fish processing by-products have been converted to fish flours for animal feeding (Ström and Eggum 1981), fertilizer or fish silage. However, most of these products possess low economic value. The questions remain on what to do with the large amounts of fish by-products and how to enhance their value? Novel processing methods are needed to convert sardinelle by-products into more profitable and marketable products. One method would be to transform the insoluble fish proteins by-products to a soluble form by proteolytic treatment. The enzymatic breakage of proteins generates peptides and amino acids, which can modify biological and functional characteristics of the proteins, improving their quality (Shahidi 1994; Souissi et al. 2007) and offering interesting opportunities for food applications. Biological activities and functional characteristics of peptides depend on the molecular size, structure and specific amino acids

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(Panyam and Kilara 1996). However, in some cases, extensive enzymatic hydrolysis may on the contrary impair some functional properties of food proteins, or even cause off-flavors in the corresponding hydrolysates. Since they are rich in proteins, sardinelle by-products constitute potential nitrogen and/or carbon sources for microbiological use. Fish protein hydrolysates for maintaining the growth of different microorganisms have been receiving great attention (Clausen et al. 1985; Gildberg et al. 1989; Dufossé et al. 2000; Va’zquez et al. 2008), but only a limited number of reports have been published about the application of such substrates for metabolite production (Coello et al. 2000; Ghorbel et al. 2005). Non-hydrolyzed fish proteins have also been used as carbon and nitrogen sources for growth and metabolite production (Ellouz et al. 2001; Souissi et al. 2008; Manni et al. 2009). The most important industrial enzymes in use today include proteases and carbohydrate-hydrolyzing enzymes. Proteolytic enzymes account for nearly 60% of the industrial enzyme market (Rao et al. 1998). They have diverse applications in a wide variety of industries, such as detergents, food, pharmaceuticals, leather, and silk, and for the recovery of silver from used X-ray films (Kumar and Takagi 1999; Gupta et al. 2002). They are produced by a wide range of microorganisms including bacteria, molds, yeasts and also mammalian tissues. Most of the commercial alkaline proteases were isolated from Bacillus species (Maurer 2004). It is well known that extracellular protease production is greatly influenced by physical factors such as pH, temperature, inoculum density and incubation time, and by the composition of the medium, especially carbon and nitrogen sources (Johnvesly and Naik 2001; Puri et al. 2002). The growth substrates constitute a major cost in the production of bioproducts by fermentation processes. In most instances, the growth medium accounts for approximately 30–40% of the production cost of industrial enzymes (Joo et al. 2004). Considering this fact, the use of cost-effective growth media can significantly reduce the cost of enzyme production (Gessesse and Gashe 1997). Then, processing by-products such as potato peels (Mukherjee et al. 2008) or low cost substrates such as hulled grains of wheat (Haddar et al. 2010) become suitable for metabolite production. In a previous screening for protease-producing strains, B. cereus BG1 strain, producing a calcium-dependent and solvent stable metalloprotease, was isolated from an activated sludge reactor treating fishery wastewaters (Ghorbel et al. 2003). The crude extracellular protease produced by the isolate had optimal activity at 60°C and pH 8.0. The strain exhibited high productivity of the protease in medium containing starch as carbon source (Ghorbel-Frikha et al. 2005).

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The aim of this study was to examine the possible utilization of sardinelle fish powders as bacterial substrate for the production of the Ca-dependent protease by B. cereus BG1.

Materials and methods Materials Sardinelle (Sardinella aurita) were purchased from the fish market at Sfax city, Tunisia. Casein sodium salt from bovine milk was purchased from Sigma (St Louis, MO, USA). Casein peptone and yeast extract were from BioRad (France). Trichloroacetic acid was from Carlo Erba Reactifs. Other chemicals were of analytical grade. Bacterial strain The Bacillus cereus BG1 used in this study was isolated from an activated sludge reactor treating fishing industry wastewaters (Ghorbel et al. 2003). Cultivation and media Inocula were routinely grown in Luria-Bertani (LB) broth medium composed of (g/l): peptone 10; yeast extract 5; NaCl 5 (Miller 1972). The previously optimized medium used for protease production was composed of (g/l): starch 5, CaCl2 2, yeast extract 2, K2HPO4 0.2 and KH2PO4 0.2, pH 8.0 (Ghorbel-Frikha et al. 2005). The initial fish media used for growth and protease production was composed of sardinelle powders buffered at pH 8.0. Media were autoclaved at 120°C for 20 min. Cultivations were performed on a rotatory shaker (200 rpm) for 48 h at 37°C, in 250-ml conical flasks with a working volume of 25 ml. The cultures were centrifuged at 5000g for 5 min, and the supernatants were used for estimation of extracellular proteolytic activity. Various working volumes were tested 20, 25, 40, and 60 ml, and the relative protease activities were determined after 48 h at 37°C. Cell growth determination The growth of the microorganism was estimated by the determination of colony-forming units (CFU/ml). All experiments were carried out in duplicate and repeated at least twice. Preparation of fish substrate To obtain meat fish flour (MSP), heads and viscera were first eliminated. The raw material was then heated until

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boiling. The bones were removed and the cooked meat was pressed to remove water and fat. The resulting pressed product was minced in a meat grinder, and then dried at 80°C during 24–48 h. The dried fish cake was minced again to obtain a fine powder, and then stored in glass bottles at room temperature. In order to obtain whole fish (WSP) or combined head and viscera fish (CHVSP) powders, raw materials were cooked, pressed, minced and then dried (Fig. 1). Chemical composition and mineral content Dry weight of fish powders was determined after heating samples at 105°C to constant weight, and ash content was determined after heating dried samples at 600°C for 2 h. Total nitrogen was determined using the Kjeldahl method and then protein content was estimated. Crude fat was determined gravimetrically after Soxhlet extraction of dried samples with diethyl ether. Assay of proteolytic activity Protease activity was measured by the method of Kembhavi et al. (1993) using casein as a substrate. A 0.5-ml aliquot of the culture supernatant, suitably diluted, was mixed with 0.5 ml 1% (w/v) casein in 100 mM Tris-HCl (pH 8.0) containing 2 mM CaCl2, and incubated for 15 min at 60°C. The reaction was stopped by addition of 0.5 ml trichloroacetic acid (20%, w/v). The mixture was allowed to stand at room temperature for 15 min and then centrifuged at 10,000g for 15 min to remove the precipitate. The acid soluble material was estimated spectrophotometrically at 280 nm in the supernatant. A standard curve was generated using solutions of 0–50 mg/l tyrosine. One unit of protease Sardinelle (S. aurita)

activity was defined as the amount of enzyme required to liberate 1 μg of tyrosine per min under the experimental conditions. Effect of pH on protease activity and stability The pH optimum of the crude preparation was studied over a range pH 6.0–12.0 with casein as substrate. The pH stability of the enzyme was determined by incubating the enzyme in buffers of different pH in the range of 6.0–12.0 for 1 and 3 h, at 50°C. Aliquots were withdrawn and proteolytic activity was determined at pH 8.0 and 60°C. The following buffer systems were used at 100 mM: potassium phosphate buffer for pH 6.0– 8.0; Tris-HCl buffer for pH 7.0–9.0; and glycine-NaOH buffer for pH 9.0–12.0. Hydrolysis specificity The proteins used in this study (casein, bovine serum albumin, gelatin, fibrin, and keratin) were purchased from Sigma-Aldrich except for bovine fibrin (MP Biomedical, St. Louis, MO, USA). Protease activity was assayed by mixing 0.5 ml of the diluted enzyme with 0.5 ml of 100 mM Tris-HCl (pH 8.0) buffer containing substrate at 1% (w/v). The mixture was incubated at 60°C for 15 min, and then the reaction was stopped by adding 0.5 ml of TCA 20% (w/v) and allowed to stand at room temperature for 15 min. The mixture was then centrifuged for 15 min at 10,000g. The absorbance was measured under the corresponding wavelength to the substrate as indicated in Table 2 (below). The relative protease activity toward casein was taken as 100% control.

Results and discussion Whole sardinelle

Heads and viscera

Preparation of sardinelle powder

Meat sardinelle Thermal treatment at boiling temperature, 10-15 min Pressing

Water Lipid Bone

Grinding Drying 24-48 h at 80 ºC Grinding CHVSP

MSP

WSP

Fig. 1 Flow diagram for the preparation of powders from S. aurita. MSP meat sardinelle powder, CHVSP combined heads and viscera sardinelle powder, WSP whole sardinelle powder

Three derived sardinelle powders were prepared and used as growth substrates: WSP, whole sardinelle powder; MSP, meat sardinelle powder and CHVSP, combined heads and viscera sardinelle powder (Fig. 1). The composition of the fish preparations used in this study is given in Table 1. Since sardinelle is a fatty fish, powders prepared as described in “Materials and Methods” have relatively high lipid content. MSP contains higher protein content (76.2%) but lower ash and relatively high lipid content. CHVSP shows more lipids and less protein content than whole and meat sardinelle powders and, due to the included bones, there are more minerals than the other powders.

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Table 1 Chemical composition of sardinelle powders (g/100 g powder) Water Protein Ash Lipids Cl (Nx6.25) WSP 5.6 MSP 1.2 CHVSP 1.3

62.4 76.2 48.6

10.6 19.3 4.5 15.8 20.9 26.9

Na

K

a

60

Ca 50

0.174 0.427 0.362 1.8 0.34 0.51 0.18 0.47 0.19 0.49 0.38 0.79

MSP meat sardinelle powder, CHVSP combined heads and viscera sardinelle powder, WSP whole sardinelle powder

6000

CFU/ml U/ml

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1000

0

Production of protease on sardinelle powders based media In a previous study, Ghorbel-Frikha et al. (2005) reported that the production of neutral protease by BG1 strain is Cadependent. Maximum induction was observed in the presence of 1 g/l CaCl2, while no protease activity was detected in the absence of CaCl2. Fish powders are rich in both organic and inorganic materials. Notably, fish powders contain the essential substances required in microbiological media, such as sources of carbon, nitrogen and minerals. In order to test if powders prepared from sardinelle promote biomass and enzyme synthesis, protease production was assayed in media containing only fish substrates at a concentration of 10 g/l. Growth and protease production on fish media are given in Fig. 2a. All fish substrates tested stimulate the growth of the strain; however, protease synthesis was significantly low. The addition CaCl2 at 1 g/l, to fish media considerably enhanced the enzyme production. The highest level of protease activity was achieved by using CHVSP (5273 U/ml), although this substrate contains less protein (45.5%) than MSP and WSP. However, the level of protease production with MSP remains weak even by the addition of CaCl2. The enhancement of protease synthesis with CHVSP may be explained by the existence in this fish substrate of bioactive substances that induced protease synthesis. Similar to our results, Ellouz et al. (2001) reported highest level of protease production by Bacillus subtilis when the strain was cultivated in medium containing CHVSP. The results obtained showed that sardinelle powders may be used as growth media but need to be enriched by the addition of CaCl2. The fact that the strain was able to grow and produce the enzyme in media containing only fish powders supplemented by CaCl2 indicate that it can obtain its nitrogen and carbon requirements directly from undigested proteins. Optimization of the CHVSP concentration Since CHVSP was the best substrate for protease synthesis by B. cereus BG1 strain, the effect of its concentration on the enzyme production was studied. As shown, the growth

0

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WSP MSP MSP CHVSP CHVSP + CaCl2 + CaCl2 + CaCl2

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0 2

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Fig. 2 Growth and protease production by B. cereus BG1 on sardinelle powders (a) and effect of CHVSP concentration (b). Cultivations were performed for 48 h at 37°C in media consisting of fish powder at 10 g/l, without or with CaCl2 at 1 g/l (a), and in media containing 1 g/l CaCl2 and various concentrations of CHVSP (b ). MSP meat sardinelle powder, CHVSP combined heads and viscera sardinelle powder, WSP whole sardinelle powder. Values are means of three independent experiments. Standard deviations±2.5% (based on three replicates)

and protease production by B. cereus BG1 strain (Fig. 2-b) increased with the increasing of CHVSP concentration. Highest protease production was achieved between 20 and 40 g/l. Effect of metallic ions on protease production Several studies have reported that metal ions can stimulate or inhibit enzyme synthesis. In a previous paper, GhorbelFrikha et al. (2005) reported that protease production by BG1 strain is absolutely Ca-dependent. Other divalent cations were not able to induce protease activity. In this study, the effect of addition of some inorganic salts to CHVSP (20 g/l) was investigated (Fig. 3a). In the absence of divalent cations, protease production was significantly low, about 771 U/ml, although this substrate contained higher ash content (20.9%). Of special interest was the finding that the BG1 protease was strongly

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a

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0 None CaCl2 ZnSO4 MgCl2 CuSO4 MnSO4 BaCl2

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0 Control

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Maltose

Starch

Fig. 3 Effect of salts (a), CaCl2 concentration (b), and carbon sources (c) on biomass and protease production by B. cereus BG1. Cultivations were performed for 48 h at 37°C in media consisting of 20 g/l CHVSP and various salts at 1 g/l (a) or different concentrations of CaCl2 (b) or various carbon sources at 5 g/l were added (+1 g/l CaCl2 ) (c) . Values are means of three independent experiments. Standard deviations±2.5% (based on three replicates)

enhanced by Ca2+, Mg2+, Mn2+ or Ba2+. The highest level of enzyme synthesis was obtained with Ca2+ and was 11fold higher than that without salts. The addition of Zn2+ and Cu2+ considerably reduced the biomass and enzyme production compared to control medium which contained only CHVSP. However, in media containing starch or maltose as carbon source and yeast extract as nitrogen source, protease induction was observed only with Ca2+. No other metal ions were able to induce enzyme production (Ghorbel-Frikha et al. 2005).

The results obtained clearly indicated that CHVSP alone promotes biomass but that protease synthesis needs a supplementation with some metallic ions of which the best one is Ca2+. Optimization of calcium chloride concentration Since the highest protease production level was obtained with calcium ions, it is necessary to study the effect of its concentration on protease production. So, the strain was grown in the CHVSP medium (20 g/l) supplemented with different concentrations of calcium chloride ranging from 0.2 to 2 g/l. As shown in Fig. 3b, the biosynthesis of the enzyme is specifically dependent on the CaCl2 concentration. With 0.2 g/l there was about 400% increase in the production of protease compared to the control medium without cations. Maximum protease production was observed with CaCl21 g/l, about 8,397 U/ml, which was about 11-fold over the medium without CaCl2. Beyond a CaCl2 concentration of 1 g/l, protease synthesis decreased, while B. cereus BG1 growth seems to need very low CaCl2 concentration and did not affected by variation in its concentration (Fig. 3-b). Effect of addition of carbon sources on protease synthesis As it was already shown, the higher level of protease synthesis was obtained with 20 g/l of CHVSP supplemented with 1 g/l CaCl2. Since this fish substrate is a complex source of carbon and nitrogen, we have tested the addition of some carbon sources (5 g/l) on bacterial growth and protease production by B. cereus BG1 strain. As shown in Fig. 3c, although all carbon sources were used efficiently by B. cereus BG1 strain for its growth, protease synthesis decreased by the addition of the tested substrates. Among all carbon sources tested, glucose was found to be the better carbon substrate for BG1 strain growth, but the lower protease synthesis level was obtained with this substrate. We showed in a previous work (Ghorbel-Frikha et al. 2005), that B. cereus BG1 strain was able to grow well and to produce protease at the maximum level of 4,000 U/ml on starch (5 g/l) supplemented with yeast extract at 2 g/l. On glucose, the B. cereus BG1 strain produced protease at a very low level. In this work, we showed that the use of starch simultaneously with CHVSP enhanced the protease production much more, to 6,793 U/ml. Then, CHVSP could be used as a nitrogen supplement for B. cereus BG1 growth and protease production. In this study, we showed that the protease production level was the highest when the strain was cultivated only on CHVSP-based medium supplemented with calcium ions. So, the CHVSP is sufficient for B. cereus BG1 strain to produce high levels of protease.

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Ann Microbiol (2011) 61:273–280 120

a

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0 20

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Fig. 4 Effect of working volume on protease production. Cultivations were performed for 48 h at 37°C in media consisting of 20 g/l CHVSP and 1 g/l CaCl2. The activity of the enzyme obtained with a working volume of 25 ml was taken as 100%

b

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Effect of working volume

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In order to check the effect of working volume on protease production, various working volumes from 20 to 80 ml were assayed. Enzymatic activities were expressed as relative values with reference to 100% activity obtained for a 25-ml working volume. As shown in Fig. 4, a working volume of 20 ml gives a slight increase in protease production compared to that obtained with 25 ml. When the working volume was greater than 25 ml, protease production decrease to reach 68% for an 80-ml working volume. These results indicated that working volume could not be a significant factor for protease production by B. cereus BG1 in the range of tested volumes (20–80 ml).

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Fig. 5 Time courses of protease production and growth of B. cereus BG1 in 100 ml of culture medium consisting of (g/l): CHVSP 20 and CaCl2 1. Protease activity was determined in culture supernatant obtained after removal of cells by centrifugation, as described in “Materials and methods”. Values are means of three independent experiments. Standard deviations were less than 2.5% (based on three replicates)

20 0 6

7

8

9

10

11

12

Fig. 6 Effect of pH on proteolytic activity and stability. a The pH profile was determined in different buffers by varying pH values, at 60°C. The maximum activity obtained at pH 8.0 is considered as 100% activity. b pH stability: the enzyme preparation was pre-incubated for 1 and 3 h at 50°C, in buffers of various pH values and the residual activity was measured at pH 8.0. The activity of the enzyme before incubation was taken as 100%. Buffer solutions used for pH activity and stability are presented in “Materials and methods”

Time course of protease production by B. cereus BG1 Profiles of the cell growth and protease production by BG1 strain in medium containing CHVSP supplemented with CaCl2 are shown in Fig. 5. Results indicate that the B. cereus BG1 grew well in the medium and reached the stationary phase after about 18 h. The biosynthesis of protease by the strain appeared to be growth-related, since the activity was detected from early stages of the growth of the microorganism, and the values increased exponentially at the end of the exponential phase, then continued to increase even during the stationary phase. However, Kembhavi et al. (1993) works reported that protease production ends before the stationary phase.

Table 2 Hydrolysis specificity

Relative activity (%)

Casein

Fibrin

Keratin

BSA

Gelatin

100

5

1.3

20

2

BSA Bovine serum albumin

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Effect of pH on protease activity and stability The relative activity values at various pH are shown in Fig. 6a. The optimum pH for B. cereus BG1 protease activity determined at 60°C was 8.0. Activity declined above pH 8.0 and was 96, 69, and 19.5% of the maximal activity at pH 8.5, 9.0, and 9.5, respectively. The pH stability was checked by incubating the enzyme in buffers of different pH in the range of 6.0–12.0 for 1 and 3 h at 50°C in the presence of 2 mM Ca2+, followed by the estimation of proteolytic activity at pH 8.0. As shown in Fig. 6b, the enzyme was stable between pH 6.0 and 9.0, retaining more than 95% of its initial activity, after incubation at 50°C for 1 and 3 h. The proteolytic activity declined rapidly below pH 9, and was only 19.2 and 11.8% of its initial activity at pH 10.0 after 1 and 3 h, respectively. These results are similar to those found previously (Ghorbel et al. 2003). Hydrolysis specificity Among the tested protein substrates, casein served as the most preferred substrate as shown in Table 2. Bovine serum albumin was weakly hydrolyzed, whereas gelatin, keratin and fibrin were not.

Conclusion This study describes the selection of an economical medium for the growth and protease production by B. cereus BG1 strain. This medium is based on low-cost sardinelle powder. Among all the three sardinelle powders tested, CHVSP supplemented with calcium chloride provided the highest protease production level, followed by WSP. However, the production of protease was significantly low when the strain was cultivated in medium containing MSP. The highest protease production (8,473 U/ml) in medium containing 20 g/l CHVSP and 1 g/l CaCl2 indicates that the strain can obtain its carbon, nitrogen and salts requirements directly from sardinelle heads and viscera substrate. The high level of protease activity obtained in the presence of substrate prepared from whole sardinelle or from heads and viscera, compared to that obtained with meat sardinelle powder, could be due to the presence of bioactive compounds in heads and/or viscera acting as protease inducers. Further works are necessary to characterize bioactive molecules present in heads or viscera sardinelle. The high protease production obtained with cheap product such as fish powders clearly indicated that these substrates could be used in industrial fermentation processes. Furthermore, using medium containing only powder, obtained from fish processing by-products as

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growth substrate, may reduce considerably the cost of protease production. The produced protease by B. cereus BG1 was stable in neutral pH buffers and had a hydrolysis specificity for casein. Acknowledgment This work was funded by “Ministère de l’Enseignement Supérieur, de la Technologie et de la Recherche Scientifique Tunisie”.

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