In previous shake-flasks experiments with A. niger 426 at 28 °C and pH 4.5, which were conducted on 10 g of orange or grapefruit rind added to a nutrient solution but without any addition of inducer, naringinase production was observed on both substrates. Maximal enzyme production was observed on grapefruit rind (1.92 ± 0.23 U/mL) against 1.05 ± 0.26 U/mL of activity when orange rind was tested. These results were supported by Mendoza-Cal et al. [21] who also yielded higher amounts of naringinase using grapefruit rind instead of orange rind as substrate. Puri et al. [2] reported that citrus peel powder contains high proportions of polyphenols, which act as inducers on narigninase production. The increased production of naringinase with A. niger on grapefruit rind is probably due to its very high naringin content and very low content of naringinase inhibitor compounds, such as monosaccharides when compared with orange rind [6, 22]. When the levels of monosaccharides decrease, naringinase synthesis is induced by naringin, leading to an increased naringinase production. This induction mechanism may help A. niger to degrade naringin to access further other nutrient supplies in the media, especially carbon [23]. Because of its high level of flavonoids, grapefruit rind was selected as one of the substrates for more in-depth studies of naringinase production. This is advantageous from an industrial point of view because naringinase can be produced cheaply by using agricultural waste.
3.1 Effect of substrate on naringinase production
The effect of the inclusion of naringin (10 g / L) was investigated. Since the variation in pH and temperature of the initial medium can have significant effects on the growth and production of naringinase by species of different fungi, including Aspergillus, we chose the acid pH 4.5, below or higher than this pH range, which can result in a drastic reduction in naringinase activity. Since the molecular charges and consequently the molecular interactions and functions are directly related to the pH of the medium; so any change in average pH affects many biological functions. Therefore, at 28 ° C and pH 4.5, the time of 24 and 120 hours of SSF by A. niger showed greater production of naringinase, 22.4 ± 0.73 U / mL, respectively (Fig. 1). The central point was composed of a ternary mixture of 33.33% of each support substrate (grapefruit peel, rice bran, and wheat bran). After 120 h, 97% of the carbon source coinsured in 5 mL of fermentation extract had been consumed and the enzyme formation was reduced. In this way, all of the following crops were carried out for only 5 days.
Table 1 summarizes the different combinations of grapefruit peel, rice bran, and wheat bran used for A. niger crops, and the maximum activities, in terms of naringinase for EMD 1. The maximum values of naringinase obtained in the experiments varied from 1.6 (run 1) to 22.6 U / mL (run 7), (Table 1).
The special cubic model was determined to estimate naringinase titers in terms of the concentrations of the components grapefruit rind (x1), rice bran (x2), and wheat bran (x3) in the cultivation medium, as described by Eq. (2), in which terms with an asterisk are significant (p < 0.05). See equation 2 in the supplementary files.
A preliminary analysis of this data revealed the significance of the agro-industrial residues used as substrates for naringinase production. All linear blendings showed a statistically significant positive influence on naringinase activity titers since p-values were smaller than 0.05 for each of the components. As linear blending, wheat bran was significant in the highest level; which means that an increase of this substrate in the culture medium could improve the results. However, the quadratic term x1x2 (grapefruit rind and rice bran) was not significant, with p > 0.05. The most significant effect on naringinase activity was composed of a ternary mixture of each supporting substrate (x1x2x3), followed by the quadratic term (x1x3). The determination coefficient for naringinase production was 99.96% for EMD 1. According to Silva et al. [24] values of R2 > 90% are very good in the experimental design of bioprocesses.
Since the least favorable condition was grapefruit rind as a sole substrate (1.6 U/mL, Table 1) and due to the presence of citric acid in grapefruit juice, it was decided to study the effect of citric acid and sugars on naringinase activity. Norouzian et al. [25] reported that citric acid at 0.02 M non-competitively inhibited naringinase activity. Repression of naringinase activity by glucose and sucrose was also reported by Puri et al. [6], although these carbon sources supported excellent growth.
A quantitative HPLC analysis of substrates sugars found that grapefruit rind presented high amounts of glucose and sucrose (625.299 and 4.273 mg/g of dry substrate, respectively), (Table 2). Unlike wheat bran that presented the lowest amount of glucose (12.2 mg/g of the dry substrate). The number of sugars present in grapefruit rind might suggest a change in the metabolism of A. niger and a subsequent decrease in naringinase activity, this might also be due to citric acid amount 305.55 ± 0.172 mg/g of dry substrate, which represents 1.59 M present in 10 g of grapefruit rind, and could cause inhibition of naringinase biosynthesis. Although, grapefruit rind has a high content of naringin and rutin, 8.884 ± 0.464 and 5.503 ± 0.65 mg/g of dry substrate, respectively, which act as flavonoids inducers for naringinase production, so it can’t be discarded as a substrate in the mixture.
The models used to describe the effects of each component on naringinase production (Eq. (2)) were used to generate the contour plots shown in Fig. 2a. These results revealed a broad region of elevated naringinase production. Using our data, a predictive analysis estimated the maximum naringinase activity to be 23.94 U/mL in cultures that contained 2.3 g of grapefruit rind, 2.5 g of rice bran, and 5.2 g of wheat bran. Additional cultivation was performed to validate the proposed model and yielded a mean naringinase activity of 23.2 ± 0.02 U/mL. This result corresponded to 97% of the expected value, validating the effectiveness of the predictive model and confirming the substrate's proportions.
3.2 Comparison of different inducers on naringinase production
For a variety of applications, to increase the production of naringinase, an inducible enzyme, optimize the culture conditions, especially by performing the continuous or gradual addition of a type of inducer [26]. For the production of naringinase, the inducers reported are generally natural substrates or substrate analogs for the enzyme, such as naringin [6, 27], rutin [28] and hesperidin [29].
To evaluate the effect of flavonoid concentration on the production of naringinase by A. niger, the addition of naringin, rutin, and hesperidin in various concentrations was performed based on the best-selected condition of the EMD 1 culture. Naringinase activity was observed in all media (Fig. 3), maximum naringinase was produced with rutin (27.48 ± 1.23 U / mL) followed by naringin (23.45 ± 0.96 U / mL) and hesperidin (23.22 ± 1.12 U / mL) at a concentration of 10 g / L. The increase in the concentration of the inducers had no significant effect on the production of the enzyme. Custodio et al. [30] also reported that rutin (0.5%, w / v) was considered the most effective inducer of α-L-ramnosidase production among quercitrin, naringin, naringenin, hesperetin, and hesperidin. On the other hand, Kumar et al. [28] reported a naringinase activity in different five media, that the enzyme naringinase had maximum production in naringin (7.48 IU / ml) while rutin (3.71 IU / ml). It is also worth mentioning that our products obtained in this work were much higher than the findings by Kumar et al. [28]. These same authors observed that with rutin and naringenin, naringinase was produced on the 8th day, but the enzyme activities were not distinguished on the 11th day.
To improve naringinase yield using citrus residues, Borkar et al. [15] studying the fermentative production of naringinase from A. niger van Tieghem MTCC 2425 with an inducer concentration of 14.9 g L -1 verified an ideal naringinase activity of 545.2 IU g -1 and concluded that the pH and the medium temperature act synergistically for the production of the enzyme, while the antagonistic behavior of the temperature and the concentration of the inducer at higher levels leads to a decrease in enzyme activity [15]. On the other hand, they obtained the activity of the enzyme can be completely reserved for up to 5 months at 4 ° C, same in the literature, naringinase from this species favored 45 °C for naringinase activity [3]. However, researchers noted that scaling parameters vary significantly about microbial types, such as bacteria or fungi. About the fungus Aspergillus niger, studies that identify the most influential factors among the different media and process parameters during fermentation to produce naringinase are still scarce.
3.3 Effect of a mixture of inducers
To select the best mixture of inducers, fermentation experiments were performed based on the best condition selected from EMD 1 cultivation using 10 g/L of inducers concentration (Table 1, EMD 2). Maximum naringinase activities can be found in the central points that are composed of a ternary mixture of each inducer. According to the analysis of variance (ANOVA), the regression was statistically significant (p < 0.05). The high value of the coefficient of determination (R2 = 0.994) indicates that 99.4% of the variability of the responses can be explained by the model. The value of the adjusted determination coefficient (adjusted R2 = 0.977) is also high, showing a high significance of the model.
In this way, the mathematical model representing naringinase production by adding inducers naringin (x1), rutin (x2) and hesperidin (x3) in the experimental region considered here can be expressed as: see equation 3 in the supplementary files.
Similar to Eq. (2), Eq. (3) indicated that the most significant effect on naringinase activity was composed of a ternary mixture of each inducer, resulting in a naringinase activity of 28.16 U/mL (Table 1). Because x2 > x3 > x1 we would conclude that rutin (x2) increases naringinase production. Although all the linear terms, as well as the cubic term, had significant effects on the maximum attainable value of naringinase production, it can be seen that the effect of the quadratic term was not significant (p > 0.05). Furthermore, because x2x3 is negative, blending rutin and hesperidin would have an antagonistic effect, which means that the proportion of rutin in the blending should be greater than hesperidin. These results can provide a reference for inducer selection for similar studies in the future. To our knowledge, this is the first time that a mixture of inducers combined with the analysis of responses by Response Surface Methodology and optimization through a desirability function is used for producing naringinase.
According to Bokkenheurser et al. [31], rutin can be hydrolyzed to the monosaccharide 3-glucosylquercetin, which could be further hydrolyzed to the aglycone, quercetin by the β-D-glucosidase portion of naringinase.
A quantitative HPLC analysis of the fermentation broth revealed the presence of the inducer rutin in Controls 4-7, which represents cultivations without inoculation, and its aglycone quercetin in Runs 4-7 of the EMD 2 (Table 3). This indicates that A. niger successfully metabolized rutin. Additionally, rutin was detected at a concentration of 2.435 ± 0.422 mg/mL in Control 5 of EMD 2 even though it was not added. A possible explanation could be that rutin is coming from grapefruit rind, which has a rutin concentration of 5.503 ± 0.65 mg/g of the dry substrate (Table 2). This highlights the importance of agroindustrial residues application since this reduces the costs of production by the addition of flavonoids. This is advantageous on two levels, first because grapefruit rind is an inexpensive carbon source compared to other carbon sources and second its application on naringinase production could solve environmental problems resulting from grapefruit waste.
For better industry usage of this waste, it could be done a pre-treatment for monosaccharides inhibitor removal, but costs have to be taken into account so the process would remain financially advantageous.
The graph of the response surface (Fig. 2b) showed that maximum naringinase production could be achieved with a mixture of naringin, rutin, and hesperidin at a concentration of 2.5, 4.5, 3.0 g/L, respectively; furthermore, the inducer rutin would increase enzyme activity. The maximum predicted value was 27.86 U/mL. The mean value of the experimental validation of the optimized condition (28.10 ± 0.45 U/mL) was in excellent correlation with the predicted value, confirming the validity of the model. This activity is comparatively higher than those obtained by Petri et al. [32]. After medium optimization, these authors found a final α-L-rhamnosidase activity of 3.02 U/mL contained in 5 mL of enzymatic extract from SSF by A. niger 426.