**3. Groups of microorganisms demonstrating greatest potential for the production of health-promoting properties in SSF**

#### **3.1** *Bacillus* **genus**

It was demonstrated that the representatives of the *Bacillus* genus were able to produce fibrinolytic enzymes by SSF [52–58, 60]. That ability is mostly assigned to the expression of *fibE* gene which encodes enzyme called subtilisin [88] which solubilises blood clots. Gene expression was not investigated in cited papers so the ability to produce those enzymes by tested strains could be the result of other genes expression.

*Bacillus* genus is successfully used in solid-state fermentation to improve antimicrobial activity of fermented food. Rochín-Medina et al. [19] who tried to determine optimal bioprocessing conditions for SSF of spent coffee grounds by *Bacillus clausii* achieved increase of flavonoid and total phenolic contents by 13 and 36%,

respectively. SSF also enhanced antibacterial activity of obtained extracts. That phenomenon could be explained by the fact that *Bacillus sp*. strains could metabolise fibre which releases phenolic compounds as a result of lignocellulolytic activity, and demonstrate strong correlation between the increase of phenolic compounds and the synthesis of cellulases and pectinases [19]. Those enzymes can break down plant cell wall components which leads to the hydrolysis of ester bonds that bind phenolic compounds [19].

The representatives of the *Bacillus* genus are able to produce surfactants as well (**Table 1**). There are various enzymes involved in the production of surfactin which form multienzyme peptide synthetase complex. One of those proteins is Srf (which consists of three units A, B and C). There are also two other genes involved in that process – *sfp* and *com*X. However, the role and interactions of protein and genes was described in more details in other review papers [89], therefore, we did not discuss that phenomenon in details.

#### **3.2 Other bacteria**

There are also other bacteria that were tested against the production of substances holding the potential for application in healthcare. For instance, there are several species that are able to produce fibrinolytic enzymes which could serve as potential anticoagulants: *Paenibacillus* sp. IND8 [61], *Pseudoalteromonas* sp. IND11 [62], *Xanthomonas oryzae* IND3 [63] or *Citrobacter freundii nov. haritD11* [50, 51]. In the case of *Paenibacillus* its ability for producing such enzymes could be assigned to the expression of *PPFE-I* gene [90]. It seems that the synthesis of protein encoded by that gene could be stimulated by Zn2+, Mg2+ and Fe+ so the concentration of these ions could be considered in future studies focusing on optimisation of enzyme production. In the case of *Citrobacter freundii*, molecular mechanism seems much simpler because so far, only *chiX* gene was assigned to its ability for chitinase production [91]. It is still unclear which genes are involved in the production of anticoagulant agent by *Pseudoalteromonas* or *Xanthomonas oryzae* so it is an aspect that could be investigated in the future, since the quantity of the enzyme was very prominent – up to 1,388 U/ml.

It was also demonstrated that *Streptococcus hygroscopicus* is able to produce immunosuppressant in SSF based on agricultural waste with added supplements [49] and it seems that a*roA*, *fkbN,* and *luxR* genes are mostly responsible for that phenomenon [92]. On the other hand, molecular mechanisms standing behind the ability of *Yarrowia lipolytica* W29 (ATCC 20460) to produce γ-decalactone [11] which analogues demonstrate antiviral and antifungal properties [93] is simpler because it involves only *POX2* overexpression [94]. Similarly, in the case of *Lactobacillus plantarum* CECT 748 [95] which increased the concentration of particular phenolic compounds, probably only *est\_1092* gene was involved – it encodes phenolic esterase [96].

None of the cited paper evaluated molecular mechanisms involved in processes carried out by tested strains.

#### **3.3 Fungi**

The majority of research involving SSF is carried out with various fungi (**Table 1**). Many of analysed examples focused on the increase of phenolic compounds. One of the genera which were involved in that process was *Trichoderma* [13, 16, 17]. Those fungi are known to produce cellulolytic, ligninolytic and xylanolytic enzymes [97] and it has been demonstrated in other studies that cellulase could significantly increase concentrations of various polyphenols e.g. caffeic acid, vanillin, p-coumaric acid, and ferulic acid [98]. In fact, similar strategies

**339**

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

*Pleurotus sapidus* – ligninolytic enzymes [12], *Trametes versicolor*

case of *I. obliqus*; wheat bran in the case of *X. nigripes*.

during terpene transformation nor determined gene expression.

that encodes for cyclosporin synthetase (*simA* gene) [107].

production in moulds is still unknown.

Based on cited papers it might be stated that *Penicillium brevicopactum* is most common in the research regarding production of mycophenolic acid in SSF [38–43]. Those fungi produce polyketide synthase encoded by *mpaC* gene along with other enzymes such us: protein transacylase, β- ketoacylsynthase, acyltransferase, acyl carrier protein, and methyltransferase (MT) domains [106]. There is another taxon which is able to produce immunosuppressants, namely cyclosporin A, and it is *Tolypocladium inflatum* [44–48] which has got nonribosomal peptide synthetase

*Mucor subtilissimus* UCP 1262 [65] and *Fusarium oxysporum* [66, 67] were shown to produce fibrinolytic enzymes. It has been already demonstrated that *FP* gene is responsible for encoding fibrinolytic protease in *Fusarium* sp. [108] but in the case of *Mucor* sp. it remains unknown. Molecular background of *A. kawachii* KCCM 32819 production of short chain fatty acids could be the same as for *A. nidulans* and other filamentous ascomycetes – *farB* gene is mostly responsible for that ability, however, *farA* participates as well [109]. Genetic background of biosurfactant

might apply to *Aspergillus* spp. [22, 37, 99] because representatives of that genus could produce enzymes demonstrating such activities [98] as well. *Rhizopus oryzae* [36, 100] synthesises cellulase, xylanase and pectinase [101]. Other fungi applied in other cited studies could produce the following enzymes that participate in polyphenol increase: *Lentinus edodes* [15] – xylanase and cellulase [102],

Genetic background of the synthesis of abovementioned enzymes by *Trichoderma* spp. and *Aspergillus* spp. was revised by Amore et al. [103] while in the case of *R. oryzae* and *P. chrysopsorium* it was described by Battaglia et al. [104] so we did not provide details of those processes, especially that many

There are also some fungi which could be natural sources of polyphenols: *Taiwanofungus camphoratus* [21], *Inonotus obliquus* [23], and *Xylaria nigripes* [24]. In all cases SSF increased their anti-inflammatory properties by increasing concentration of particular phenolic compounds which could also originate from substrates that were used for their cultivation and those were: spent substrate of *Pleurotus eryngii*, sunflower seed hulls, corn and rice grain, white birch and mulberry in the

Another significant group of bioactive compounds which concentration was increased by SSF was terpenes (**Table 1**) and those processes were carried out by various fungi: *Fusarium sambucinum* B10 [10]*, Penicillium expansum* KACC 40815 [70], *Diaporthe* sp. KY113119 [25], *Antrodia camphorata* [30], *Saccharomyces cerevisiae* AXAZ-1 and *Kluveromyces marxianus* IMB3 [27], *Trichoderma viride* EMCC-107 [28], and *Aspergillus niger* van Tieghem [29]. *A. camphorate* is the natural source of terpenes and their concentration was increased by selecting millet as the main substrate for SSF. *K. marxianus* and *S. cerevisiae* could probably increase the concentration of tested compounds by releasing terpenes from their glycosidic forms by β-glucosidases. In the case of *Fusarium* spp., *Aspergillus* spp. and *Penicillium* spp. it was demonstrated that those genera could produce several sesquiterpene synthases [105]. As in the case of polyphenols, molecular background of terpene transformations are very complex so we decided not to describe it in the current chapter, but refer to the review of Quin et al. [105] instead. It must be highlighted that except for the study regarding *P. expansum* KACC 40815 demonstrating that terpenoid cyclase was mostly involved in described processes [70], none of the authors investigated enzymatic activity

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

TV-6 – β-glucosidase [20].

genes are involved.

#### *The Application of Solid State Fermentation for Obtaining Substances Useful in Healthcare DOI: http://dx.doi.org/10.5772/intechopen.94296*

might apply to *Aspergillus* spp. [22, 37, 99] because representatives of that genus could produce enzymes demonstrating such activities [98] as well. *Rhizopus oryzae* [36, 100] synthesises cellulase, xylanase and pectinase [101]. Other fungi applied in other cited studies could produce the following enzymes that participate in polyphenol increase: *Lentinus edodes* [15] – xylanase and cellulase [102], *Pleurotus sapidus* – ligninolytic enzymes [12], *Trametes versicolor* TV-6 – β-glucosidase [20].

Genetic background of the synthesis of abovementioned enzymes by *Trichoderma* spp. and *Aspergillus* spp. was revised by Amore et al. [103] while in the case of *R. oryzae* and *P. chrysopsorium* it was described by Battaglia et al. [104] so we did not provide details of those processes, especially that many genes are involved.

There are also some fungi which could be natural sources of polyphenols: *Taiwanofungus camphoratus* [21], *Inonotus obliquus* [23], and *Xylaria nigripes* [24]. In all cases SSF increased their anti-inflammatory properties by increasing concentration of particular phenolic compounds which could also originate from substrates that were used for their cultivation and those were: spent substrate of *Pleurotus eryngii*, sunflower seed hulls, corn and rice grain, white birch and mulberry in the case of *I. obliqus*; wheat bran in the case of *X. nigripes*.

Another significant group of bioactive compounds which concentration was increased by SSF was terpenes (**Table 1**) and those processes were carried out by various fungi: *Fusarium sambucinum* B10 [10]*, Penicillium expansum* KACC 40815 [70], *Diaporthe* sp. KY113119 [25], *Antrodia camphorata* [30], *Saccharomyces cerevisiae* AXAZ-1 and *Kluveromyces marxianus* IMB3 [27], *Trichoderma viride* EMCC-107 [28], and *Aspergillus niger* van Tieghem [29]. *A. camphorate* is the natural source of terpenes and their concentration was increased by selecting millet as the main substrate for SSF. *K. marxianus* and *S. cerevisiae* could probably increase the concentration of tested compounds by releasing terpenes from their glycosidic forms by β-glucosidases. In the case of *Fusarium* spp., *Aspergillus* spp. and *Penicillium* spp. it was demonstrated that those genera could produce several sesquiterpene synthases [105]. As in the case of polyphenols, molecular background of terpene transformations are very complex so we decided not to describe it in the current chapter, but refer to the review of Quin et al. [105] instead. It must be highlighted that except for the study regarding *P. expansum* KACC 40815 demonstrating that terpenoid cyclase was mostly involved in described processes [70], none of the authors investigated enzymatic activity during terpene transformation nor determined gene expression.

Based on cited papers it might be stated that *Penicillium brevicopactum* is most common in the research regarding production of mycophenolic acid in SSF [38–43]. Those fungi produce polyketide synthase encoded by *mpaC* gene along with other enzymes such us: protein transacylase, β- ketoacylsynthase, acyltransferase, acyl carrier protein, and methyltransferase (MT) domains [106]. There is another taxon which is able to produce immunosuppressants, namely cyclosporin A, and it is *Tolypocladium inflatum* [44–48] which has got nonribosomal peptide synthetase that encodes for cyclosporin synthetase (*simA* gene) [107].

*Mucor subtilissimus* UCP 1262 [65] and *Fusarium oxysporum* [66, 67] were shown to produce fibrinolytic enzymes. It has been already demonstrated that *FP* gene is responsible for encoding fibrinolytic protease in *Fusarium* sp. [108] but in the case of *Mucor* sp. it remains unknown. Molecular background of *A. kawachii* KCCM 32819 production of short chain fatty acids could be the same as for *A. nidulans* and other filamentous ascomycetes – *farB* gene is mostly responsible for that ability, however, *farA* participates as well [109]. Genetic background of biosurfactant production in moulds is still unknown.

*Biotechnological Applications of Biomass*

phenolic compounds [19].

that phenomenon in details.

carried out by tested strains.

**3.2 Other bacteria**

respectively. SSF also enhanced antibacterial activity of obtained extracts. That phenomenon could be explained by the fact that *Bacillus sp*. strains could metabolise fibre which releases phenolic compounds as a result of lignocellulolytic activity, and demonstrate strong correlation between the increase of phenolic compounds and the synthesis of cellulases and pectinases [19]. Those enzymes can break down plant cell wall components which leads to the hydrolysis of ester bonds that bind

The representatives of the *Bacillus* genus are able to produce surfactants as well (**Table 1**). There are various enzymes involved in the production of surfactin which form multienzyme peptide synthetase complex. One of those proteins is Srf (which consists of three units A, B and C). There are also two other genes involved in that process – *sfp* and *com*X. However, the role and interactions of protein and genes was described in more details in other review papers [89], therefore, we did not discuss

There are also other bacteria that were tested against the production of substances holding the potential for application in healthcare. For instance, there are several species that are able to produce fibrinolytic enzymes which could serve as potential anticoagulants: *Paenibacillus* sp. IND8 [61], *Pseudoalteromonas* sp. IND11 [62], *Xanthomonas oryzae* IND3 [63] or *Citrobacter freundii nov. haritD11* [50, 51]. In the case of *Paenibacillus* its ability for producing such enzymes could be assigned to the expression of *PPFE-I* gene [90]. It seems that the synthesis of

so the

protein encoded by that gene could be stimulated by Zn2+, Mg2+ and Fe+

of the enzyme was very prominent – up to 1,388 U/ml.

concentration of these ions could be considered in future studies focusing on optimisation of enzyme production. In the case of *Citrobacter freundii*, molecular mechanism seems much simpler because so far, only *chiX* gene was assigned to its ability for chitinase production [91]. It is still unclear which genes are involved in the production of anticoagulant agent by *Pseudoalteromonas* or *Xanthomonas oryzae* so it is an aspect that could be investigated in the future, since the quantity

It was also demonstrated that *Streptococcus hygroscopicus* is able to produce immunosuppressant in SSF based on agricultural waste with added supplements [49] and it seems that a*roA*, *fkbN,* and *luxR* genes are mostly responsible for that phenomenon [92]. On the other hand, molecular mechanisms standing behind the ability of *Yarrowia lipolytica* W29 (ATCC 20460) to produce γ-decalactone [11] which analogues demonstrate antiviral and antifungal properties [93] is simpler because it involves only *POX2* overexpression [94]. Similarly, in the case of *Lactobacillus plantarum* CECT 748 [95] which increased the concentration of particular phenolic compounds, probably only *est\_1092* gene was involved – it encodes phenolic esterase [96]. None of the cited paper evaluated molecular mechanisms involved in processes

The majority of research involving SSF is carried out with various fungi (**Table 1**). Many of analysed examples focused on the increase of phenolic compounds. One of the genera which were involved in that process was *Trichoderma* [13, 16, 17]. Those fungi are known to produce cellulolytic, ligninolytic and xylanolytic enzymes [97] and it has been demonstrated in other studies that cellulase could significantly increase concentrations of various polyphenols e.g. caffeic acid, vanillin, p-coumaric acid, and ferulic acid [98]. In fact, similar strategies

**338**

**3.3 Fungi**

#### **3.4 Modified strains**

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