Lactase production by Saccharomyces fragilis IZ 275 using different carbon sources

This study sought to create a better fermentation medium to maximize lactase production by Saccharomyces fragilis IZ 275 using different carbon sources, including reconstituted powdered cheese whey. A factorial design 2 4 was applied to evaluate the significant effects of variables which compose the fermentation medium. Then, a steepest descent-ascent design was applied to obtain the maximum activity. A Rotational Central Composite Design (RCCD) 2 4 was made to optimize the fermentation medium. We verified that the cheese whey, a by-product of the dairy industry, can be employed as an excellent fermentation medium by yeast, within the bioeconomy concept and used by the dairy industry as product with additional value. The employed methodology is an efficient tool in the optimization process for β-galactosidase production. In the optimized fermentation medium, the maximum production of β-galactosidase (54.68 U/mL) by S. fragilis IZ 275 is obtained with 14 g/L sucrose, 17.7 g/L reconstituted powdered cheese whey, 5.14 g/L yeast extract and 8.85 g/L peptone. Rotacional (DCCR) lacticínios, como excelente meio de fermentação por leveduras, dentro do conceito de bioeconomia com valor agregado. O emprego da metodologia é uma ferramenta eficiente para otimizar o processo de produção da β-galactosidase. No meio de fermentação otimizado, a máxima produção de β-galactosidase (54,68 U/mL) por S. fragilis IZ 275 é obtida com 14 g/L sacarose, 17,7 g/L soro de queijo em pó reconstituído, 5,14 g/L extrato de levedura e 8,85 g/L peptona.


INTRODUCTION
β-D-galactosidase (EC 3.2.1.23; β-D-galactoside galactohydrolase) or lacatase has several applications in the food industry (Jones et al., 2017). It is an intracellular enzyme that hydrolyzes lactose, a disaccharide present in milk and dairy products, into its two monosaccharides units, galactose and glucose, which are easily absorbed in most organisms, including humans (Anisha, 2017;Panesar et al., 2006). Some people are lactose intolerant and cannot digest lactose properly due to an inactive intestinal lactase enzyme. Common symptoms of lactose intolerance are intestinal-dysfunction gas, abdominal pain, and diarrhea. The sugar is found in mammalian milk at a concentration of 3-8% (w/v), and has low solubility and sweetness. (Perini et al., 2013;Cardoso et al., 2017).
One of the main sources for the production of lactase are yeasts (Mlichová and Rosenberg, 2006) by the submerged fermentation process (Anisha, 2017). Several factors influence the production of lactase, such as temperature, pH, incubation time and fermentation medium. The optimization of the fermentation medium is critical for β-galactosidase production (Jones et al., 2017). and some works have described these factors that influence the β-galactosidase production (Karlapudi et al., 2018;Venkateswarulu et al., 2017). However, the best composition of fermentation medium for maximizing the lactase production by yeast has not been yet developed.
Cheese whey produced during cheese-making or during the coagulation of milk casein process presents as principal components, lactose (70-72% of the total solids), whey proteins (8%-10%) and minerals (12-15%) (Yadav et al., 2015). Over the last few decades, the dairy industry has explored different alternatives to exploit the valuable components of cheese whey. The world production of whey (El-Tanboly et al., 2017) is estimated to be around 180 to 190 x 10 6 ton / year, and causes serious socio-economic and environmental problems, since much of this amount is discarded in the environment. Furthermore, Lopes et al. (2018) found that, in comparison to domestic sewage, cheese whey can be 100 times more polluting. One alternative to help remediate these problems would be the application of cheese whey as the fermentation medium for lactase production by microorganisms within the concept of circular economy or bioeconomy (Ranta et al., 2018;López-Gómez et al., 2019). This will promote the integration of economic activities and environmental wellbeing in a sustainable way.
Considering these aspects, we studied the creation of a better composition of fermentation medium to maximize lactase production by Saccharomyces fragilis IZ 275 using different carbon sources, including reconstituted powdered cheese whey.

Microorganism and inoculum
Saccharomyces fragilis IZ 275 yeast was used for the study and collected in the Collection of Tropical Cultures (WDCM 69 885 number). The yeast was maintained in tubes containing Where A was the absorbance at 420 nm, dilution factor was the fold dilution of the enzyme solution, enzyme solution was the amount of enzyme (mL) undergoing the reaction, ɛ was the extinction coefficient (determined from the o-nitrophenol standard curve) and time was the incubation time (15 min).

Experiment 1: screening experiments to investigate the composition of medium fermentation
First, initial experiments were performed to evaluate the significant effects of variables which compose the fermentation medium used for β-galactosidase production by Saccharomyces fragilis IZ 275 yeast. A factorial design 2 4 was applied with eight variables and three replicates at the central point totaling 19 assays. The coded independent variables (x1, x2, x3, x4, x5, x6, x7 and x8) and uncoded variables (X1 = g/L lactose, X2 = g/L sucrose, X3 = g/L glucose, X4 = g/L cheese whey, X5 = g/L yeast extract, X6 = g/L peptone, X7 = g/L MgSO4 and X8 = g/L K2HPO4) are shown in Table 1 with their variation levels.

Experiment 2: Method of steepest ascent-descent design to investigate the maximum increase of β-galactosidase activity
From the results of factorial design and to obtain the maximum increase of β-galactosidase activity, a steepest descent-ascent design was applied (Montgomery, 2011). The independent variables were stabilized as follows: sucrose and cheese whey ranged from 6 to 16 g/L; yeast extract, peptone and MgSO4 ranged from 2 to 8 g/L; the variables lactose and glucose ranged from null to 10 g/L and K2PHO4 ranged from null to 5 g/L (Table 2).

Experiment 3: Rotational Central Composite Design (RCCD) 2 4 to optimize the medium fermentation to β-galactosidase production and model validation
From the results of steepest ascent-descent design and to optimize the fermentation medium for β-galactosidase production, a third experiment was performed. In this third step, a Rotational Central Composite Design (RCCD) 2 4 was applied with two central points and eight axial points, for orthogonal, totaling 26 assays. The coded independent variables (x1, x2, x3, x4) and uncoded variables (X1 = sucrose g/L, X2 = cheese whey g/L, X3 = yeast extract g/L and X4 = peptone g/L) are shown in Table 3 with their variation levels. The coded independent variables (x5, x6, x7, x8) and uncoded variables (X5 = lactose g/L, X6 = glucose g/L, X7 = MgSO4 g/L and X8 = K2HPO4 = g/L) that did not show significance in the factorial design were stabilized according to the steepest ascent-descent design (Table 2).
Where Y2 (response function), x1, x2, x3 and x4 (coded variables), β (estimated coefficients for each term of the response surface model) and e = pure error. The response functions (Y2) were used to perform regression analyses and analysis of variance (ANOVA) for the regression. The equation model was fitted to experimental data to yield the proposed model. Response surface graphs were generated. All executed analysis and response surfaces were performed with STATISTICA 7.0 software (StatSoft Inc., 2007). After response surface analysis for maximum β-galactosidase activity, the proposed model was validated by performing new assays in triplicate. The results (Yexp.) were compared with the estimated response (y^1) by Student's t-test (p < 0.05).

Factorial design 2 4 to investigate the composition of fermentation medium
The first experiment was performed using a factorial design of 2 4 for evaluating the effects of significant variables which compose the fermentation medium of Saccharomyces fragillis IZ 275 during β-galactosidase production. According to ANOVA and regression analysis, only the independent variables X4 (cheese whey) and X5 (yeast extract) were shown to have a significant effect on the response function Y1 (β-galactosidase activity, U/mL). None of the other independent variables had a significant effect and the coefficient of determination (R 2 ) was 0.75. The proposed model could be described as follows: Y1 = 6.23 + 5.58 x4 + 8.72x5. Thus, the β-galactosidase activity was 20.53 U/mL. The highest β-galactosidase activity (Y1) was obtained in the assay 16 (Y1 = 28.42 U/mL) (Table 4) when the independent variables were shown in the maximum levels. This correspond to using X1 (lactose); X2 (sucrose); X3 (glucose) and X4 (cheese whey) at a concentration of 10 g/L and while using X5 (yeast extract), X6 (peptone), X7 (MgSO4) and X8 (K2HPO4) at 5 g/L concentration. This observation suggested that, although not significant to the model, the investigated variables influenced the β-galactosidase activity. It was decided to make a new regression analysis and ANOVA including only the significant independent variables. Our results confirmed that the response function Y1 decreased (data not shown), indicating that the other independent variables (x1, x2, x3, x6, x7 and x8) were not in their optimal regions earlier and they were important to explain the model. The complete model can now be described as follows: Y1 = 6.23 + 0.21x1 + 3.23x2 + 0.94x3 + 5.58x4 + 8.72x5 + 3.81x6 + 4.70x7 -0.55x8.

Method of steepest ascent-descent design to investigate the maximum increase in the β-galactosidase production
According to the results of the steepest ascent-descent design (Table 2), it was observed that assay 4 yielded maximum value for β-galactosidase activity (39.81 U/mL). This corresponds when, the concentration of independent variables sucrose (X2) and cheese whey (X4) were 14 g/L, yeast extract (X5), peptone (X6) and MgSO4 (X7) were 7 g/L and lactose (X1), glucose (X3) and K2HPO4 (X8) were at a concentration of 10 g/L, 10 g/L and 5 g/L, respectively i.e. when present at their maximum levels. Also, the values of β-galactosidase activity were lower (1.57 U/mL and 2.46 U/mL, respectively) when the levels of independent variables decreased, as shown in assays 6 and 16. The results from the steepest ascent-descent design and from factorial design 2 4 suggest that the non-significant independent variables (lactose, sucrose, glucose, peptone, MgSO4 and K2HPO4) contribute to the increase in β-galactosidase production. Therefore, to optimize the composition of fermentation medium for the βgalactosidase production and the model validation, all variables were considered.
After analyzing the mathematical model, response function Y2 and response surface (Figure 1a), it was observed that there was a region with maximum β-galactosidase activity when x1(X1) was -1.48 (10.29 g/L) and x2 is between -1 and +1 or X2 was between 11.5 and 16.5 g/L, suggesting that the cheese whey, rich in lactose, promotes the production of enzyme by Saccharomyces fragilis IZ 275. In Figure 1b, there is a region in which the β-galactosidase activity is greater when x1 and x3 is -1.48 or X1 is 10.29 g/L and x3 is 5.14 g/L. Figure 1c indicates two regions with maximum β-galactosidase activity when x1 is +1.48 or 17.70 g/L and x4 is -1.48 or 5.14 g/L and the other regions in which x1 is -1.48 (10.29 g/L) and x4 +1.48 (8.85 g/L). In Figure 1d, there is a region which the maximum β-galactosidase activity is observed when cheese whey, x2, ranged from 0 to 1.48, regardless of yeast extract concentration; In Figure 1e, the maximum β-galactosidase activity is observed when the x2 ranged from 0 (center point) to + 1.48 and in the Figure 1f when peptone, x4, is 1.48 or X4 is 8.85 g/L.
The data obtained from the present study draws attention towards two points: 1) the variables studied are critical for lactase production by Saccharomyces fragilis IZ 275; and, 2) cheese whey, a by-product of the milk and dairy industry, is an important medium for the Lactase production by Saccharomyces fragilis IZ 275 … Rev. Ambient. Água vol. 15 n. 3, e2474 -Taubaté 2020 growth of yeast. Thus, cheese whey could be re-used in the fermentation industry instead of being discarded as a pollutant as per the concept of the circular economy or bioeconomy (Ranta et al., 2018;López-Gómez et al., 2019). The maximum β-galactosidase activity (28.42 U/mL) obtained in factorial design was observed with 10 g/L of lactose, sucrose, glucose and cheese whey and of variables yeast extract, peptone, MgSO4 and K2HPO4 5 g/L. In the steepest ascentdescent design, the maximum β-galactosidase activity obtained (39.81 U/mL) for sucrose and cheese whey were 14 g/L, yeast extract, peptone and MgSO4 were 7 g/L, lactose and glucose were 10 g/L, and for K2HPO4 was 5 g/L . In the optimized conditions, the maximum βgalactosidase activity was 52.84 U/mL and obtained with 17.7 cheese whey, 14 g/L of sucrose, 5.14 g/L of yeast extract, 7 g/L peptone and MgSO4, 10 g/L lactose and glucose and 5 g/L K2HPO4. The study clearly showed that the Central Rotational Composite Design (CRCC) and Response Surface (RSM) are efficient tools to optimize the composition of fermentation medium and lactase production increased 53.78% after optimization. (1) 16.5 (-1) 11.5 (-1) 5.75 (-1) 5.75 10 10 7 5 51.09 10 (1) 16.5 (-1) 11.5 (-1) 5.75 (1) 8.25 10 10 7 5 39.92 11 (1) 16.5 (-1) 11.5 (1) 8.25 (-1) 5.75 10 10 7 5 41.23 12 (1) 16.5 (-1) 11.5 (1) 8.25 (1)  X1 (sucrose, g/L); X2 (cheese whey, g/L), X3 (yeast extract, g/L), X4 (peptone, g/L). It is very important to determine the composition of fermentation medium for maximum lactase production. There are few studies described in the literature that show the βgalactosidase production using different fermentations. Bosso et al. (2019) worked with microfiltrated cheese whey permeate as substrate for Saccharomyces fragilis IZ 275 yeast for the production of beta-galactosidase. Kumari et al. (2019) concluded that the use of cheese whey for β-galactosidase production improves the economics of the process, and the problems associated with its disposal. Manera et al. (2008) obtained by CRCC the maximum β-