Characterization of Bacillus cereus isolated from fermented cabbage and conventional optimization of extracellular protease production
K C.E., P A.G, S R., R S.I.
bacillus cereus, characterization, fermentation, growth phases, protease, proteolytic activity
K C.E., P A.G, S R., R S.I.. Characterization of Bacillus cereus isolated from fermented cabbage and conventional optimization of extracellular protease production. The Internet Journal of Microbiology. 2009 Volume 8 Number 1.
A strain of
The biosynthesis of proteolytic enzymes by microorganisms is not only of scientific but also of great practical importance. Bacteria, moulds and yeast are some of the microorganisms that are able to produce proteases. Proteases are the most important kind of enzymes from an industrial point of view; they execute a large variety of functions and have important biotechnological applications. They could be useful in leather processing, laundry detergents, producing of protein hydrolysates and food processing (Salem S.R. et al., 2009). Proteases are commonly classified according to their optimum pH: acidic protease, neutral protease and alkaline protease. There have been extensive researches on the properties and functionalities of acidic or alkaline proteases (Dunn-Coleman N. et al.).
Among bacteria, Bacillus strains are the most important producers of commercial proteases (Ghorbel-Frikha B. et al., 2005), being specific producers of extracellular protease. Strains of
Taken into account the potential uses of the highly demanded Bacillus proteases, there is a need for the research of new strains of bacteria that produce enzymes with novel properties and the development of low cost industrial medium formulations. In commercial practice, the optimization of medium composition is done to maintain a balance between the various medium components, thus minimizing the amount of unutilized components at the end of fermentation. Recently, in a previous work (Pérez Borla et al., 2009) through the utilization of a simple and reliable method, three proteolytic bacteria were isolated from fermented cabbage:
Materials and Methods
Strain and culture conditions
Effect of physicochemical conditions on microorganism growth
Temperature effect on microorganism growth
To investigate growth at different temperatures,
pH effect on microorganism growth
Incubation media modifiers effect on microorganism growth
The effect of different modifiers on
Extracellular protease activity: qualitative and quantitative assay
For enzymatic extract preparation,
The protease activity was assayed through quantitative and qualitative enzymatic assays at regular intervals of 0, 1, 2, 5, 6, 7, 8 and 24 hours. Protease activity was measured using azocasein as the substrate. The assay mixture contained 100 µl of protease source and 500 µl of 2% (w/v) azocasein (Sigma-A-2765 Lot.043K7021) in 10 mM Tris-HCl, pH 7.5. After incubation in a thermal bath (Lauda E-300) at 37ºC for 90 min, the reaction was stopped by adding 600 µl of 20% (w/v) trichloroacetic acid (TCA) and keeping the mixture at room temperature. A vortex mixture was used to insure complete mixing at various stages of these assay procedures. The mixture was centrifuged at 10.000 rpm for 5 min. 1 ml aliquot of the cell free supernatant was taken. The absorbance of the supernatant was determined at 335 nm by using of precision quartz cells (Hellma- Type Nº 10-QS-light path 10 mm). Reaction blank was prepared by adding the TCA to the substrate solution immediately before the enzyme preparation was added. Proteolytic activity was defined as the difference between the sample and blank absorbance at 335 nm. One unit of absolute proteolytic activity (UA) was the amount of enzyme that caused a change of absorbance of 0.01 at 335 nm in 90 min at 37ºC. One unit of relative proteolytic activity unit (U) was defined as an increase of one absorbance unit at 335 nm in 90 min at 37ºC per growth (absorbance of culture measured at 600 nm).
Qualitative proteolytic activity assay was evaluated in Modiefied Basal Medium (MM) supplemented with 6.2 g/l protein of skim milk plates (5 g/l of casein) (Pérez Borla et al., 2009). Wells 5 mm in diameter were cut under sterile conditions into these agar plates. A 20 µl aliquot of the growth culture was placed into the well. These plates were then incubated aerobically at 32ºC during 48 hours and bacterial proteolytic activity was observed daily. Proteolytic activity of
The protease substrate specificity of
Morphological and physiological characteristics of the culture:
To analyze motility,
The growth and the proteolytic activity of
Effect of physicochemical conditions on microorganism growth:
Temperature effect on microorganism growth:
Figure 1 shows the time course of patterns of cell growth for
The maximum optical density (0.9) was observed at 32ºC so this temperature was considered optimal for the cell growth.
pH effect on microorganism growth
To establish the optimum pH growth,
At the latency phase similar cell growth profiles were obtained for pH 7.0 and 5.7.
Incubation media modifiers effect on microorganism growth
Figure 3 shows the effect of different modifiers in MB at optimal conditions of temperature and pH; 32°C and 7.0 respectively. The use of glucose as modifier showed an increase in the optical cells density, reaching the maximum in the seventh hour of growth. This result indicated that the exponential phase was prolonged compared to control sample (only MB). The other modifiers (Ca2+, K+ and Peptone) presented an inhibitor effect in the cell growth compared to control sample.
Extracellular protease activity: qualitative and quantitative assay
Extracellular proteolytic activity of the
Maximum relative activity of
Figure 5B shows the absolute proteolytic activity during Bacillus growth. The protease activity began at the lag phase extending its protease action during the exponential phase. The highest absolute proteolytic activity was found at the exponential phase. At it was expected, non absolute proteolytic activity was observed during the stationary phase. The maximum cell concentration was at 6 h of incubation (Figure 1) and the highest enzyme activity was at 5 h with values of absorbance (600 nm) of 0.879 and 11.84 UA, respectively.
Parallel to these measures, we determined the proteolytic activity halos in MM plates supplemented with milk (Figure 5C). Aliquots of cell culture incubated in MB were placed in MM plates wells. Under these conditions, the maximum proteolytic halos observed took place during the lag and exponential phases. Notorious small halos were detected in the stationary phase, inducing on that a lower protease activity was present at this phase, indicating a poor protease activity at this phase.
When spores were inoculated again in MM agar plates supplemented with milk, they found adequate conditions to begin their vegetative growth producing proteolytic enzymes, breaking proteins and obtaining energy. To support this hypothesis, the halo diameter square was quantified but using only cell free culture supernatants (protease source) obtained at the same Bacillus growth times as Figure 5C. The values in terms of the diameter square of the clear zone surrounding the protease source were lower that those which appeared by inoculation of the active culture (Figure 5D). As a constant, the highest activity of protease was found, from the lag phase to the end of the exponential phase. In the stationary growth period a minimal halo was detected and it was smaller compared to the halo found when culture was inoculated. The halo which appeared in the stationary phase could be present by the enzymatic hydrolysis of the nitrogen source present in the MB (yeast extract).
The results on the pH effect on protease activity were shown in Figure 6 A and B. As was mentioned, 7.0 was the optimum pH for
The effect of growth temperature on specific extracellular proteolytic activity is shown in Figure 7 A and B. Different information was obtained if the activity was expressed in a relative or absolute way. At 10ºC Bacillus grew significantly slower showing the maximum relative extracellular proteolytic activity compared with Bacillus growing at 32°C, (Figure 7 A). Once again, at 10ºC the microorganisms could deplete the substrate nutrients gradually showing a remaining proteolytic activity during the stationary phase.
If absolute activity was recorder, the maximum extracellular proteolytic activity was observed at 32 and 40ºC, while at 32ºC the activity was expressed early in the lag phase, at 40ºC the maximum activity expression was during the exponential phase.
The effect of different modifiers on the extracellular protease production from
At 20ºC, and similar to the modifier effects observed at 10ºC, a positive effect in the protease expression was observed. The activity increase ranged between seven and ten times compared with the control sample (MB). All modifiers expressed the protease activity during the lag and exponential phase. With glucose addition the maximum activity was present in the late exponential phase, maintaining a high protease activity (14 -15 UA) during the stationary phase. It was probably that microorganisms use the glucose as fast carbon source of MB. When an important biomass was reaches, the microorganisms use its enzyme battery to hydrolyzed the protein substrate of MB toward obtains energy.
At 32ºC only the MB enrichment with peptone appears to cause an accelerated protease expression reaching at 2 h its maximum activity (19 UA), after that the activity gradually decrease during time. K+ improved also the proteolyic expression compared with MB reaching the highest value in the late exponential phase. For all modifiers added a significant protease activity was observed during the stationary phase.
At 40ºC any of the modifiers improved the proteolytic expression. For the control culture (MB) and with modifiers, the activity expression occurred during the late exponential phase.
In general, Ca2+ ions did not exert an enzyme stabilization role without effects in the protease activity at any of the assayed temperatures.
The hydrolysis of tested protein substrates by
a The presence of clear halos in the culture medium indicated the presence of hydrolysis. The values shown are means of three independent determinations.
Morphological and physiological characteristics of the culture
The fermentation process developed in MB at 20, 32 and 40ºC (pH 7.0) could be divided in four distinct phases, each of them with well-defined particularities: Phase I, vegetative growth; Phase II, transition to sporulation; Phase III, sporulation; Phase IV, spore maturation with cell lysis. Phase I corresponds to the beginning of the fermentation process and was characterized by a rapid period of adaptation of the cells to the culture medium, resulting in a short lag phase, due to the use of active inoculums, with reactivated vegetative cells only (Table 2, Picture 1). Phase II occurred between the 5 and the 8 hours of process and showed an abrupt morphology change forced by the beginning of the bacterium sporulation. Cell size presented a marked reduction without arrangements in pairs and chains. After that, the cells started to group and form small flacks (Picture 2). Phase III presented an intense sporulation of the culture. Although most of the cells were within large clumps (Picture 3). Phase IV was the last phase of the cellular cycle and was defined as the period of complete maturation of spores and partial lysis of sporulated cells population. The clumps size diminished while cell lysis increased continuously (Picture 4). The main characteristics of each phase are summarized in Table 2.
The pH behavior at each phase during
Swim motility of
The strain of
The optimal pH for the microorganism growth 7, while the optimal pH for the enzyme production was found at pH 8. This result is accordance with several earlier reports showing pH optima for
Despite of the strain was isolated from fermented cabbage, which juice had an acid pH between 3 and 4 (Pérez Borla et al., 2009), these pH ranges were not the optimal for
Generally food preservation processes exposes bacteria to both lethal and sub-lethal stresses. Bacteria may have different mechanisms for surviving these extreme environmental stresses (Davidson P.M. et al., 2002).
In this context, the formation of endospores in response to acid stress during cabbage fermentation is a survival strategy for
Our experimental results showed that the maximum amount of enzyme was produced by the bacterium in its exponential growth phase and a reduction in enzyme expression was noted beyond this period, precisely in the stationary phase. Similar results reported previously other authors (Gopal Joshu et al., 2007). Although the rapid proteolytic activity expression constituted a technologic advantage, in Fig. 5A when proteolytic activity was expressed relative to the bacterial growth, the reduced number of microorganisms could be the responsible for the enhanced proteolytic activity as this was not a benefit. For a technological point of view, an elevated enzyme concentration was desirable. The enzyme presents a significant absolute extracellular proteolytic activity at the beginning of the fermentation during the lag and exponential phases; following occurs a diminution of the absolute extracellular protease activity. Two independent facts could explain this behavior: the extracellular protease production is controlled by the level of a repressor which is directly or indirectly an intermediate in many pathways related to a feedback inhibition; or the low level of nutrients in the medium provoke a depletion in the enzyme activity, due to the morphological changes from vegetative to sporulated cell forms that not produce extracellular proteases. It must be emphasize that only vegetative cells are capable to liberate protease into the medium. The proteolytic activity of culture assayed in MM enrichment with milk indicates that the enzyme presents activity in the stationary phase inclusively; indicating that if the MM is complement with related substrate, then there is no drain of protease production during the stationary phase. This fact could indicate that limited substrate conditions observed during the stationary phase (Figure 5A and B) could be responsible for the absence of proteolytic activity in this period.
Also the influence of pH on
Based on the obtained results, the effect of the MB supplementation with different modifiers on the protease activity shows a variation with the incubation temperature. The results indicate that the production of protease required the presence of the assayed modifiers as a function of the temperature. At 10 and 20ºC all modifiers positively affected protease expression, but at 32 and 40ºC, the microorganism appeared not to require any modifiers to express the proteolytic activity. In contrast, other microorganisms show only very low level of specific-activity in no-supplemented synthetic medium (Nicodéme M. et al., 2005). In a previous work, Ghorbel- Frinkha
Growth temperature controlled the production of the protease by
In future works we will emphasize the search of microorganisms that resist several technological stress condition during the different process and could produce desirable metabolic products. Proteases produced by
Many researchers are interested in the synthesis of peptides or aminoacids using proteases, extracted from diverse sources. Microorganisms are a potentially important source of enzyme production. The relationship between the kinetic growth of the microorganisms and the culture conditions can not be extended proportionally to the proteolytic activity. Therefore, optimization of the protease production implicates the well knowledge of the physical and chemical factors that influence the growing of the microorganisms and the studied of the factor that affect directly or indirectly the protease activity.
In spite of the fact that several studies refer to
This work was partially supported by grants from CONICET, UNMdP and Secyt, Agencia. Buenos Aires, Argentina.