Improving of hydrolases biosythesis by solid-state fermentation of Penicillium camemberti on rapeseed cake

The study show usefulness of rapeseed cake, rich in fats and proteins byproduct generated after oil production, which may be used as a microbial medium for lipase and protease biosynthesis. Of 26 different filamentous fungi screened by solid-state fermentation, Penicillium camemberti AM83 was found to abundantly produce lipase and protease. Various process parameters were then optimized to maximize lipase and protease secretion, including carbon and nitrogen source, C/N ratio, metal ions, temperature, moisture content, initial pH, and inoculum size. Lipase production increased approximately 11.2-fold in solid-state cultures on rapeseed cake supplemented with lactose and calcium chloride, alkalinized to pH 8, hydrated to 80%, and inoculated with 1.2 × 106 spores/mL. Similarly, protease production increased approximately 8.4-fold in optimized cultures inoculated with 3.2 × 108 spores/mL, and grown on rapeseed cake with lactose and ammonium sulfate at pH 9 and moisture content 60%. The results highlight the potential economic value of solid-state fermentation on rapeseed cake to produce industrial hydrolases.

Process optimization. The effect of carbon and nitrogen sources was assessed by supplementing solid-state cultures with 2% (w/w) simple or complex carbon (arabinose, mannose, ribose, soluble starch, lactose, fructose, saccharose, maltose, glucose, xylose, glycogen, olive oil) and organic and inorganic nitrogen (peptone, yeast extract, soy flour, ammonium nitrate, ammonium sulfate, sodium nitrate, ammonium tartrate, and diammonium citrate). The impact of the C/N ratio (C: lactose for lipases and proteases; N: peptone for lipases and ammonium sulfate for proteases) was also evaluated, along with that of 0.2% w/w metal ions (CaCl 2 , NaCl, sodium citrate, MgSO 4 , MnSO 4 , and ZnSO 4 ) and surfactant (Triton X-100). Incubation temperature was tested from 20 to 40 °C, while initial pH was assessed at pH 3-5 in 0.1 M citrate buffer, pH 6-8 in phosphate buffer, and pH 9-11 in Tris base buffer. Moisture content was tested at 40%, 50%, 60%, 70%, 80%, and 90%, while inoculum size was examined using inocula with corresponding suspension of spores: 1.2 × 10 6 , 2.3 × 10 7 and 3.2 × 10 8 spores/mL. Enzyme extraction. Samples of solid-state media (5 g) were vortexed for 5 min at 3,500 rpm in phosphate buffer pH 7.2, centrifuged at 6,700 × g for 10 min at room temperature, and assayed for lipase and protease activity. The total protein content in crude extract was estimated by the method of Bradford, using a bovine serum albumine as a standard. In control experiments, which constituted oilseed cakes without microorganism, analogous procedure was conducted.
Lipase activity assay. Lipase activity was determined in a spectrophotometric assay with p-nitrophenyl palmitate (pNPP, Sigma) as a substrate according to the Mahdi et al. 18 with some modifications. The enzyme reaction mixture contained 75 μL of substrate (1 mM) dissolved in isopropanol, 50 μL of crude enzyme filled to 3 mL by 50 mM Tris-HCl buffer (pH 8) and incubated at 37 °C for 10 min. The reaction was interrupted by addition of 1 mL cooled ethanol. The activity was measured at 410 nm. One enzyme unit (U) was defined as an amount of enzyme that released 1 μM p-nitrophenol per minute. Lipase activity was calculated using p-nitrophenol standard curve. Lipase activity was expressed in units/gram of rapessed cake/mg of protein contained in crude extract.
Protease activity assay. Protease activity was determined according to the Afifi et al. 19 with some modifications. Protease activity in crude extract was measured in a mixture containing 1 mL of casein solution (0.6% in 0.1 M Tris-HCl buffer, pH 8), 1 mL of phosphate buffer, 0.5 mL of crude enzyme. The reaction was performed for 30 min at 37 °C, and then 5 mL of 0.11 M TCA was added to the mixture to terminate the reaction. Subsequently, mixture was filtered and the optical density was measured at 275 nm. One unit of enzyme was defined as an amount of the enzyme required to liberate 1 μg of tyrosine per minute. Protease activity was expressed in units/ gram of rapessed cake/mg of protein contained in crude extract. Statistical analysis. Statistical analyses (standard deviation and t-test for unpaired samples) were performed by Statistica (StatSoftPoland, ver. 13.1). The tests were two-tailed, alpha level was equal 0.05. Error bars are the mean ± standard deviation from three independent experiments.

Results and Discussion
Strain selection. In initial experiments, twenty six filamentous fungi, mainly Aspergillus, Fusarium, Penicillium as well as Botrytis, Mucor, Papulari, Poria, Sclerophama, Spicoria, and Syncephalastrum were tested in solid-state fermentation on rapeseed cake to identify the most prolific producer of lipase and protease. As can be seen in Fig. 1, lipase secretion was highest in P. camemberti AM83, B. cirenea AM235, F. oxysporum AM21, and P. thomi AM91, while protease production was highest in P. camemberti AM83, F. semitectum AM20, and A. ochraceus AM456. Notably, efficient producers of lipase were weak producers of protease and vice versa, with the exception of P. camemberti AM83, which abundantly produced both enzymes. Hence, P. camemberti AM83 was selected for further optimization since it abundantly produces both lipase and protease. In order to underline the distinctive biosynthesis of the desired hydrolases by P. camemberti AM83, the hydrolytic activity of all screened fungi in relation to this strain has been represented ( Fig. 1).
Various physical and chemical factors are well-known to affect enzyme production from microbial cultures. Accordingly, we found that lipase production increased from 0 to 48 h of culture, but decreased thereafter. In contrast, protease production increased gradually and peaked at 120 h (data not shown). This pattern is probably due to the abundance of lipids in rapeseed cake, which are thus metabolized first (2 nd day), while proteins are metabolized later (5 th day), presumably after lipids have been depleted.
While solid-state fermentation is widely used to produce enzymes by fungi, we are aware of only one report to date describing lipase production in solid-state (wheat bran) cultures of P. camemberti 20 . In addition, protease production in this species has not been investigated, although lipase and protease production by solid-state fermentation on various substrates has been reported for other Penicillium species. For example, P. simplicissimum 21,22 , P. verrucosum 23 , and Penicillium sp. [24][25][26] have been grown on soybean meal, while P. simplicissimum 27 , P. brevicompactum 28 , and P. restrictum [29][30][31] have been cultivated on babassu cake.
We found that lactose was the optimal carbon source to produce lipase from P. camemberti, as was noted by Tan et al. 32 . Lactose was also tested in other Penicillium species by Rehman 33 and Bancerz et al. 34 , although only Amin and Bhatti 35 found that it increased lipase production. In other cases, olive oil 20,29-31,33,35 and soluble starch 29,31 were found to stimulate lipase production in solid-state cultures. We also noted that lactose increased protease production, although protease biosynthesis increased in Bacillus cereus 36 and Aspergillus versicolor CJS-98 37 after supplementation with maltose. Nitrogen source. Both organic and inorganic nitrogen relieve catabolite repression and induce hydrolase synthesis. Inorganic nitrogen is rapidly consumed by microorganisms, while organic sources provide amino acids and growth factors. Most of the nitrogen sources tested were found to induce lipase production (Fig. 3), while ammonium ions (NH 4 + ) stimulated protease production. In particular, ammonium sulfate increased protease activity to 2130 U/mg (5.6-fold).
Similarly, various nitrogen sources have been shown to affect lipase production, although effects vary among strains. In P. camemberti, we found peptone to be most suitable, in line with published data 29,30 . Of note, Rehman 33 reported that peptone reduced lipase production in P. notatum. On the other hand, ammonium sulfate induced lipase production in other Penicillium strains, as reported by Amin and Bhatti 35 , Malilas et al. 20 , and Tan et al. 32 . In any case, we found that ammonium sulfate maximized protease production, in contrast to previous studies indicating that peptone or yeast extract was more advantageous 19,30,37,38 .   Fig. 4, the optimal C/N ratio was 3:1 for both lipase and protease production. We note, however, that while lactose was optimal as carbon source for producing both enzymes, peptone was the most suitable nitrogen source for producing lipase, while ammonium sulfate was optimal for producing protease. The value of C/N ratio had a significant impact on lipase and protease activity as was reported by Rigo et al. 24 , Dai and Xia 39 , Palma et al. 30 , and Lima 40 .
Metal ions and surfactant. Lipases are usually used in processes that require detergents, although such detergents may actually inhibit lipase activity. Indeed, we found that supplementation with 0.2% Triton X-100, a non-ionic surfactant widely used to extract membrane components, inhibited P. camemberti AM83 lipase and protease activity by 0.9 and 0.5-fold (from 22 to 3 U/mg and from 382 to 195 U/mg). Similarly, metal ions may directly or indirectly impact enzymatic activities. We found that while NaCl did not affect lipase and protease activity, calcium ions stimulated lipase activity approximately 4.0-fold (to 98 U/mg) while magnesium ions increased protease biosynthesis 5.4-fold (to 2059 U/mg). Zinc ions was found to inhibit protease activity, but stimulate lipase activity, while manganese ions stimulated both lipase and protease biosynthesis (Fig. 5).
The observed stimulatory effect of calcium ions on P. camemberti AM83 lipase was reported for other Penicillium species, including P. chrysogenum 9′ 34 , P. candidum 41 , and P. expansum 42 . However, Ca 2+ also inhibited lipase activity in P. citrinum 43 and P. camemberti Thom PG-3 32 . Consistent with Lima et al. 44 , we also noted that Zn 2+ boosted lipase activity, although it inhibited P. chrysogenum 9′ lipase 34 . However, zinc ions completely inhibited protease activity, while magnesium ions increased it. These results correlate with those of Abidi et al. 45 , Afifi et al. 19 , Papagianni and Sergelidis 46 , and Zhu et al. 47 , who reported similar effects in solid-state cultures of P. italicum and in submerged cultures of P. chrysogenum, P. nalgiovense, and P. chrysogenum FS010.
Incubation temperature. The optimum temperature for lipase and protease biosynthesis was 30 °C, based on solid-state fermentation tested in the temperature range between 20 °C and 40 °C (Fig. 6).
The optimal incubation temperature for lipase production in P. camemberti AM83 is similar to that of P. fellutanum growing on canola cake 35 , P. camemberti KCCM 11268 growing on wheat bran 20 , and P. simplicissimum growing on babassu cake 27 . For protease production, the same optimal temperature was observed for P. verrucosum cultivated on babassu and canola cake 48 as well as for P. chrysogenum and P. chrysogenum IHH 5 49 in submerged cultures.
Moisture content. On one hand, excessively low moisture content would decrease the solubility of solid media, diminish swelling, and generate higher water tension. On the other hand, excessively high moisture content would reduce porosity, limit oxygen transfer, and increase the risk of contamination. Hence, we investigated lipase and protease production in P. camemberti AM83 cultured on rapeseed cake with 40 to 90% moisture content   (Fig. 7). Lipase and protease activities were highest in media with 80% and 60% water, respectively. Lipase activity was lowest in media with 40-60% moisture, while protease activity sharply decreased in media with 70% moisture and was lowest in media hydrated to 90%.
A level comparable to revealed moisture content was reported by Rigo et al. 24 . Similarly, the optimal moisture content for lipase synthesis was estimated by Gutarra 27 and Silva et al. 28 to be 70% for P. simplicissimum and P. brevicompactum in solid-state cultures on babassu cake and castor meal. For protease production, we found that 60%, a level similar to moisture was optimal (55%) reported by Germano et al. 26 for Penicillium sp. growing on soybean cake and that (50%) reported by Chutmanop et al. 50 for Aspergillus oryzae growing on rice bran.
pH. Initial pH is a critical factor in any fermentation process. Accordingly, we found that lipase activity was lowest at pH 3-5 (Fig. 8), but gradually increased with pH, peaking at pH 8 before sharply decreasing at pH 9-11. Similarly, protease production was negligible at pH 3-5, but increased at pH 6-8 before peaking at pH 9. Taken together, the results indicate that lipase and protease were more efficiently produced by P. camemberti AM83 in alkaline pH.   Most Penicillium lipases appear to have suitable pH value between 4 and 7 24,33,35,[51][52][53] . Rarely, the optimum pH is 9, as was observed for lipases from P. simplicissimum and P. candidum 41,51 . For P. camemberti AM83, we found that the optimal initial pH was 8, as was also observed for P. aurantiogriseum 44 . For protease production, the same optimal initial pH was reported for P. chrysogenum FS010 47 . We note that for a majority of Penicillium proteases, the optimal pH is 8 in submerged cultures 19,[54][55][56] , but is currently unknown for solid-state cultures.
Inoculum size. In solid-state fermentation, a suitable inoculum size is required to optimize growth. Too small inocula result in inefficient cell proliferation and production of active metabolites, while too large inocula would rapidly deplete nutrients and prevent sustained growth. As can be seen in Fig. 9, large inocula generally inhibited lipase production, but stimulated protease secretion.
Optimized culture conditions. Optimal culture parameters were combined in final fermentation runs.
To maximize lipase production, media were hydrated to 80%, alkalinized to initial pH 8, supplemented with 2% w/w lactose and calcium chloride, and inoculated with a suspension of spores 1.2 × 10 6 spores/mL. An 11.2-fold increase in lipase production was observed as a result (Table 1). Similarly, protease production increased 8.4-fold when rapeseed cakes were supplemented 2% w/w lactose and ammonium sulfate, alkalinized to initial pH 9, hydrated to 60%, and inoculated with a suspension of spores 3.2 × 10 8 spores/mL. Chemical composition of solid media is an essential factor for the production of enzymes. In rapeseed cake high content of carbohydrates and proteins was determined (~30% and ~40% respectively), which makes it attractive medium for microorganism growth. Besides, protein-rich byproduct is an inducer of proteases biosynthesis. The amount of the residual oil is also significant (~15%) 9,59 . The main factor, which can affect on lipases biosynthesis meaningfully is lipids content and fatty acid composition. In rapeseed cake significant majority are unsaturated fatty acids (90-94%). In this agroindustrial side stream quantity of oleic acid prevails (~60%) and it provides ~20% of linoleic acid 59 .

Conclusion
The scientific significance of the research concerns a number of different aspects. First, production of industrially important enzymes through solid-state fermentation is economically justifiable technique. Second, sustainable development of rapeseed cake, one of the major byproducts of the oleochemical industry in Europe, is undoubtedly important issue from the biorefinery point of view. Third, an initial screening of 26 fungal strains identified Penicillium camemberti with GRAS status (Generally Recognised As Safe) used in cheese production as an effective producer of lipase and protease from solid-state rapeseed cake. During the studies, influence of culture conditions on hydrolases biosynthesis was determined. Carbon source and pH proved to be a key factor of lipase production by P. camemberti. Significant influence of nitrogen and metal ion sources on protease activity was established. However, strong impact revealed also conditions such as temperature of incubation and moisture content. After optimization of culture conditions, lipase activity increased from 22 U/mg to 247 U/mg (11.2-fold), while protease production increased from 382 U/mg to 3556 U/mg (8.4-fold). The results suggest that agroindustrial byproducts such as rapeseed cake has potential as an alternative cost-effective substrate for producing industrial enzymes.