**4. Optimisation of SSF conditions**

One of the approaches that were applied for the optimisation of the concentration of bioactive substances by SSF is to provide pre-treatment to the main substrate. Heat-treatment was one of the main methods e.g. autoclaving, moistening in boiling water, cooking with deionised water, drying, freezing, freeze-drying, vacuum-drying and roasting. It inactivates native microorganisms and enzymes.

Once the main substrate is prepared, there are other experimental conditions that need to be optimised. In cited papers the major factor contributing to obtained results was moisture which was at least 50% [69, 110]. The next crucial factor was medium composition. In few cases authors used solid media commonly used for the cultivation of microorganisms [2, 70]. However, in the majority of cases authors added some nutrients to agricultural waste to provide better results of releasing bioactive compounds. In the case of mycophenolic acid produced *P. brevicompactum* MTCC 8010 from rice bran those supplements were: peptone, KH2PO4, glycine and methionine [41]. On the other hand, in the case of *P. brevicompactum* ATCC 16024, the addition of mannitol or (NH4)2HPO4 to pearl barley did not enhance the MA synthesis [39]. Surprisingly, the quantity of MA was higher in the latter case – 5.47 g/kg of the substrate in comparison to 4.5 g/kg under optimised conditions. It seems that pearl barley has got chemical composition which is more preferable for obtaining MA.

On the other hand, Plackett-Burman design was applied for the optimisation of cyclosporin A production by *Tolypocladium inflatum* MTCC 557 which resulted in 8,166 mg/kg [45] which is 1.26-fold higher than in studies carried out by Survase et al. [44, 45] who applied the same fungal strain or even 45.62-fold higher than reported by Nisha and Ramasamy [46] who used *T. inflatum* ATTC 34921 strain. In the last study authors firstly used Plackett-Burman design for the selection of nutrients and later on, they used half-factorial central composite rotatable design (CCRD) of response surface methodology (RSM) to select optimum concentrations of those substances which resulted in more than 10-fold increase of tested compound [47].

When the production of fibrinolytic enzyme was optimised with RSM, its activity increased 3 times in the case of *Pseudoalteromonas* sp. IND11 [62]; 4 times in the case of *Xanthomonas oryzae* sp. IND3 [63]; while when central composite design (CCD) was used, 4.5-fold increase was noted when *Paenibacillus* sp. IND8 was used [61]. In the case of *Xanthomonas oryzae* sp. IND3, CCD was additionally used for estimating optimal values of the following variables: sucrose, yeast extract, and pH of the medium [63]. Among all mentioned microorganisms *Paenibacillus* sp. IND8 produced greatest quantities of the enzyme – 4,418 U/ml.

**341**

*The Application of Solid State Fermentation for Obtaining Substances Useful in Healthcare*

that abovementioned bacteria. When authors used two-level factorial design in the case of *Bacillus* sp. IND12 examining the impact of moisture, sucrose, and MgSO4 levels added to the cow dung, maximum enzyme activity reached 4,143 U/g [54]. Further, orthogonal design (corn steep powder, sucrose and MgSO4 · 7H2O) provided even greater enzyme activity (5,865 U/ml) in 100 l fermenter when applying *Bacillus subtilis* WR350 [57]. However, the greatest activity was achieved when *Bacillus halodurans* IND18 was used. It produced 6,851 U/g when two-level full factorial design was applied and the optimum conditions were as follows: 1% peptone, 80% moisture and pH 8.32, using wheat

Ghribi et al. [4] took a different approach for the optimisation of surfactin production – firstly, authors applied Plackett-Burman design to assess which of the five variables were the most important and then they optimised the process with CCD involving three selected variables. They found parameters (temperature – 37°C, inoculum age – 14 h, and moisture – 88%) that were the most favourable for the production of surfactin and increased its yield by 2-fold (up to 2 g/l). Sun et al. [3, 111] carried out step-by-step optimisation and found out that the addition of attapulgite by 1.96-fold (4.3782 g/kg). This would suggest that *B. natto* NT-6 was the

Optimization of halotolerant chitinase was carried out using RSM –Box Behnken

method which involved *Citrobacter freundii* and that process slightly improved enzyme production in comparison to initial optimisation experiments from 112.43 U/g dry substance to 124.73 U/g dry substance [51]. That optimisation was mainly focused on the ratio of main substrates (wheat bran and shrimp shellfish), temperature and moisture content. Similarly, minor changes were observed when wheat bran and powdered fish scales were used for the statistical optimisation of

Solid-state fermentation could provide various substances useful in healthcare: antimicrobials, immunosuppressants, anticoagulants, substances holding anti-inflammatory properties and anticancer agents. It seems that polyphenols and terpenes are especially versatile in their applications. The majority of studies involved various fungi mainly due to their enzymatic activity which supports the release of bioactive compounds from agricultural waste. Molecular mechanisms of those processes are usually very complex; however, they remain unknown for some fungi. Further studies are necessary to assess which genes could be expressed during those processes because those could be used for modification of microorganisms to increase their yield. It is also important to bear in mind that SSF requires the presence of various supplements and fermentation could be optimised by statistical tools, especially Response Surface Methodology and Central Composite Design. In

most suitable for that application among all tested strains.

some cases step-by-step optimisation could be sufficient.

The authors declare no conflict of interest.

*DOI: http://dx.doi.org/10.5772/intechopen.94296*

bran as the main substrate [58].

chitinase production [50].

**5. Conclusions**

**Conflict of interest**

Various *Bacillus* strains were used for the production of fibrinolytic enzyme (**Table 1**) and those proved to be more efficient enzyme producer *The Application of Solid State Fermentation for Obtaining Substances Useful in Healthcare DOI: http://dx.doi.org/10.5772/intechopen.94296*

that abovementioned bacteria. When authors used two-level factorial design in the case of *Bacillus* sp. IND12 examining the impact of moisture, sucrose, and MgSO4 levels added to the cow dung, maximum enzyme activity reached 4,143 U/g [54]. Further, orthogonal design (corn steep powder, sucrose and MgSO4 · 7H2O) provided even greater enzyme activity (5,865 U/ml) in 100 l fermenter when applying *Bacillus subtilis* WR350 [57]. However, the greatest activity was achieved when *Bacillus halodurans* IND18 was used. It produced 6,851 U/g when two-level full factorial design was applied and the optimum conditions were as follows: 1% peptone, 80% moisture and pH 8.32, using wheat bran as the main substrate [58].

Ghribi et al. [4] took a different approach for the optimisation of surfactin production – firstly, authors applied Plackett-Burman design to assess which of the five variables were the most important and then they optimised the process with CCD involving three selected variables. They found parameters (temperature – 37°C, inoculum age – 14 h, and moisture – 88%) that were the most favourable for the production of surfactin and increased its yield by 2-fold (up to 2 g/l). Sun et al. [3, 111] carried out step-by-step optimisation and found out that the addition of attapulgite by 1.96-fold (4.3782 g/kg). This would suggest that *B. natto* NT-6 was the most suitable for that application among all tested strains.

Optimization of halotolerant chitinase was carried out using RSM –Box Behnken method which involved *Citrobacter freundii* and that process slightly improved enzyme production in comparison to initial optimisation experiments from 112.43 U/g dry substance to 124.73 U/g dry substance [51]. That optimisation was mainly focused on the ratio of main substrates (wheat bran and shrimp shellfish), temperature and moisture content. Similarly, minor changes were observed when wheat bran and powdered fish scales were used for the statistical optimisation of chitinase production [50].

### **5. Conclusions**

*Biotechnological Applications of Biomass*

which resulted in much higher yields [44, 45].

**4. Optimisation of SSF conditions**

preferable for obtaining MA.

compound [47].

It has been demonstrated that in some cases the yield of microbial metabolites significantly increased when the strain was subjected to gamma ray – it increased the quantity of obtained mycophenolic acid produced by two strains of *Penicillium requeforti* [43] in comparison of other cited studies which involved unmodified *Penicillium brevicompactum* [39, 41]. On the other hand, UV radiation was used for the modification of *Tolypocladium inflatum* which was used for the production of Cyclosporin A [47], however, in the case of that substance there were other studies

One of the approaches that were applied for the optimisation of the concentration of bioactive substances by SSF is to provide pre-treatment to the main substrate. Heat-treatment was one of the main methods e.g. autoclaving, moistening in boiling water, cooking with deionised water, drying, freezing, freeze-drying, vacuum-drying and roasting. It inactivates native microorganisms and enzymes. Once the main substrate is prepared, there are other experimental conditions that need to be optimised. In cited papers the major factor contributing to obtained results was moisture which was at least 50% [69, 110]. The next crucial factor was medium composition. In few cases authors used solid media commonly used for the cultivation of microorganisms [2, 70]. However, in the majority of cases authors added some nutrients to agricultural waste to provide better results of releasing bioactive compounds. In the case of mycophenolic acid produced *P. brevicompactum* MTCC 8010 from rice bran those supplements were: peptone, KH2PO4, glycine and methionine [41]. On the other hand, in the case of *P. brevicompactum* ATCC 16024, the addition of mannitol or (NH4)2HPO4 to pearl barley did not enhance the MA synthesis [39]. Surprisingly, the quantity of MA was higher in the latter case – 5.47 g/kg of the substrate in comparison to 4.5 g/kg under optimised conditions. It seems that pearl barley has got chemical composition which is more

On the other hand, Plackett-Burman design was applied for the optimisation of cyclosporin A production by *Tolypocladium inflatum* MTCC 557 which resulted in 8,166 mg/kg [45] which is 1.26-fold higher than in studies carried out by Survase et al. [44, 45] who applied the same fungal strain or even 45.62-fold higher than reported by Nisha and Ramasamy [46] who used *T. inflatum* ATTC 34921 strain. In the last study authors firstly used Plackett-Burman design for the selection of nutrients and later on, they used half-factorial central composite rotatable design (CCRD) of response surface methodology (RSM) to select optimum concentrations of those substances which resulted in more than 10-fold increase of tested

When the production of fibrinolytic enzyme was optimised with RSM, its activity increased 3 times in the case of *Pseudoalteromonas* sp. IND11 [62]; 4 times in the case of *Xanthomonas oryzae* sp. IND3 [63]; while when central composite design (CCD) was used, 4.5-fold increase was noted when *Paenibacillus* sp. IND8 was used [61]. In the case of *Xanthomonas oryzae* sp. IND3, CCD was additionally used for estimating optimal values of the following variables: sucrose, yeast extract, and pH of the medium [63]. Among all mentioned microorganisms *Paenibacillus* sp. IND8

Various *Bacillus* strains were used for the production of fibrinolytic enzyme (**Table 1**) and those proved to be more efficient enzyme producer

produced greatest quantities of the enzyme – 4,418 U/ml.

**3.4 Modified strains**

**340**

Solid-state fermentation could provide various substances useful in healthcare: antimicrobials, immunosuppressants, anticoagulants, substances holding anti-inflammatory properties and anticancer agents. It seems that polyphenols and terpenes are especially versatile in their applications. The majority of studies involved various fungi mainly due to their enzymatic activity which supports the release of bioactive compounds from agricultural waste. Molecular mechanisms of those processes are usually very complex; however, they remain unknown for some fungi. Further studies are necessary to assess which genes could be expressed during those processes because those could be used for modification of microorganisms to increase their yield. It is also important to bear in mind that SSF requires the presence of various supplements and fermentation could be optimised by statistical tools, especially Response Surface Methodology and Central Composite Design. In some cases step-by-step optimisation could be sufficient.
