*3.1. Solid State Fermentation (SSF) and Crude β-Galactosidase Production*

The leading target of this study was to evaluate the hydrolytic activity of *A. awamori* on CW to obtain a nutrient-rich supplement deriving from lactose hydrolysis, that will substitute synthetic media in a following bioconversion process. Therefore, SSF optimization to enhance β-galactosidase production using WB as a single substrate was initially undertaken, based also on previous studies that have outlined that WB reinforced β-galactosidase production [36]. This has been attributed to the appropriate ratio of hemicellulose to sugars, that is defined as a stimulus factor for galactosidase production [37]. Figures 1 and 2 demonstrate the effect of initial moisture content, ranging from 60 to 75%, along with incubation time (1–5 days). Maximum production of β-galactosidase reached 148 U/g (db) at 70% of initial moisture after 70 h of fermentation. Earlier reports highlighted that increased moisture levels enhanced β-galactosidase yield in *A. tubingensis* [29]. The latter usually associates with the fact that moisture crucially affects nutrient solubility within the substrate [38]. As it can be easily observed, the production rate exhibits an increasing trend (Figure 1), during the first three days of fermentation followed by a decrease after approximately 70 h (three days) of incubation. Similar results were also obtained in studies using *A. tubigensis* and *A. awamori*, respectively [14,29], whereby prolonged fermentation times entailed higher β-galactosidase activities. For instance, Nizamuddin et al. [30] demonstrated optimum β-galactosidase production by *A. oryzae* after seven days of incubation, Raol et al. [29] found maximum enzyme activity by *A. tubingensis* at seven

days, whereas Cardoso et al. [13] performed SSF for six days to produce β-galactosidase production by *A. lacticoffeatus*. produce β-galactosidase production by *A. lacticoffeatus*. 160 60%

rate exhibits an increasing trend (Figure 1), during the first three days of fermentation followed by a decrease after approximately 70 h (three days) of incubation. Similar results were also obtained in studies using *A. tubigensis* and *A. awamori*, respectively [14,29], whereby prolonged fermentation times entailed higher β-galactosidase activities. For instance, Nizamuddin et al. [30] demonstrated optimum β-galactosidase production by *A. oryzae* after seven days of incubation, Raol et al. [29] found maximum enzyme activity by *A. tubingensis* at seven days, whereas Cardoso et al. [13] performed SSF for six days to

rate exhibits an increasing trend (Figure 1), during the first three days of fermentation followed by a decrease after approximately 70 h (three days) of incubation. Similar results were also obtained in studies using *A. tubigensis* and *A. awamori*, respectively [14,29], whereby prolonged fermentation times entailed higher β-galactosidase activities. For instance, Nizamuddin et al. [30] demonstrated optimum β-galactosidase production by *A. oryzae* after seven days of incubation, Raol et al. [29] found maximum enzyme activity by *A. tubingensis* at seven days, whereas Cardoso et al. [13] performed SSF for six days to

*Fermentation* **2021**, *7*, x FOR PEER REVIEW 5 of 13

produce β-galactosidase production by *A. lacticoffeatus*.

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140

**Figure 1.** Effect of solid state fermentation (SSF) time in crude β-galactosidase production by *A. awamori*, at different initial moisture contents. **Figure 1.** Effect of solid state fermentation (SSF) time in crude β-galactosidase production by *A. awamori*, at different initial moisture contents. *awamori*, at different initial moisture contents.

0 20 40 60 80 100 120 Fermentation time (h) **Figure 2.** Effect of solid state fermentation (SSF) time in crude protease production by *A. awamori*, at different initial moisture contents. **Figure 2.** Effect of solid state fermentation (SSF) time in crude protease production by *A. awamori*, at different initial moisture contents.

**Figure 2.** Effect of solid state fermentation (SSF) time in crude protease production by *A. awamori*, at different initial moisture contents. Recently, Vidya et al. [14] studied α- and β-galactosidase production from *A. awamori* (MTCC 548), whereby the purified enzyme exhibited 25.5–176.5 U/mg of activity, respectively. On top of that, the authors reported β-xylosidase and β-glucosidase activities, suggesting the ample substrate specificity. Several preceding studies had also suggested multi-enzyme production by *A. awamori* including glucoamylase and protease [16,39]. Therefore, proteolytic activity was also undertaken (Figure 2), reaching the highest value after 70 h of fermentation (30.9 U/g). Similarly, Wang et al. [40] reported protease activities up to 40 U/g, (db) after 120 h employing similar SSF conditions. Evidently, it could be speculated that the addition of CW in SSF cultures, induced the secretion of β-galactosidases considering that fungal strains tend to adapt in the environmental niches and develop mechanisms for the production of specific enzymes. Moreo-Recently, Vidya et al. [14] studied α- and β-galactosidase production from *A. awamori* (MTCC 548), whereby the purified enzyme exhibited 25.5–176.5 U/mg of activity, respectively. On top of that, the authors reported β-xylosidase and β-glucosidase activities, suggesting the ample substrate specificity. Several preceding studies had also suggested multi-enzyme production by *A. awamori* including glucoamylase and protease [16,39]. Therefore, proteolytic activity was also undertaken (Figure 2), reaching the highest value after 70 h of fermentation (30.9 U/g). Similarly, Wang et al. [40] reported protease activities up to 40 U/g, (db) after 120 h employing similar SSF conditions. Evidently, it could be speculated that the addition of CW in SSF cultures, induced the secretion of β-galactosidases considering that fungal strains tend to adapt in the environmental niches and develop mechanisms for the production of specific enzymes. Moreover, this could be attributed to the low pH during fermentation, that could potentially Recently, Vidya et al. [14] studied α- and β-galactosidase production from *A. awamori* (MTCC 548), whereby the purified enzyme exhibited 25.5–176.5 U/mg of activity, respectively. On top of that, the authors reported β-xylosidase and β-glucosidase activities, suggesting the ample substrate specificity. Several preceding studies had also suggested multi-enzyme production by *A. awamori* including glucoamylase and protease [16,39]. Therefore, proteolytic activity was also undertaken (Figure 2), reaching the highest value after 70 h of fermentation (30.9 U/g). Similarly, Wang et al. [40] reported protease activities up to 40 U/g, (db) after 120 h employing similar SSF conditions. Evidently, it could be speculated that the addition of CW in SSF cultures, induced the secretion of β-galactosidases considering that fungal strains tend to adapt in the environmental niches and develop mechanisms for the production of specific enzymes. Moreover, this could be attributed to the low pH during fermentation, that could potentially enhance *Aspergillus* β-galactosidase production [11,13,41] Ultimately, SSF time for crude β-galactosidase and protease was standardized at 70 h to obtain maximal activities, that would be implemented in subsequent hydrolytic reactions of CW.

## ver, this could be attributed to the low pH during fermentation, that could potentially *3.2. Cheese Whey Hydrolysis Study*

CW hydrolysis was performed using the crude enzymatic extracts obtained from SSF cultures. Figure 3a illustrates the results obtained from different hydrolysis temperatures, whereby it can be observed that 60–65 ◦C was the optimum hydrolysis temperature of *A. awamori*. Ultimately, at the end of the bioprocess, crude enzymes hydrolyzed >90% of the initial whey lactose (Figure 3a). On the other hand, the optimum proteolytic activity was observed at 55 ◦C, as it has been earlier indicated by Tsakona et al. [16]. More particularly, as displayed in Figure 3b, FAN production increased along with the increase in temperature up to 55 ◦C, followed by a gradual reduction with further temperature increments (Figure 3b). Based on our results, significant differences (*p* < 0.05) were observed on the performed hydrolyses, at almost all evaluated temperatures. Likewise, no significant differences (*p* > 0.05) were observed on hydrolysis experiments carried out at 60 and 65 ◦C. Previous studies have also demonstrated processing of cheese whey via the implementation of microbial β-galactosidase to generate value-added products [42,43]. Generally, temperatures ranging between 50 and 60 ◦C and acidic pH values (3.5–4.5) have been reported as the optimal conditions for fungal β-galactosidase activity [13]. Additionally, Silvério et al. [44] recently studied β-galactosidase production in several *Aspergillus* species, aiming to synthesize potential prebiotics, whereby an increased enzyme activity in the range of 50–60 ◦C was noted. The current observation highlights the significant potential of the enzymes, since thermal stability is of imperative practical use for diverse bioprocesses, preventing various contaminations [45,46]. Furthermore, the results obtained postulate that the enzyme is more accessible during the first hours of hydrolysis. More specifically, a higher hydrolysis rate during the first 12 h entailed 30–50% of lactose hydrolysis, followed by a decreased rate at prolonged incubation time. Several studies also coincide with such findings where hydrolysis products decreased or even restricted lactose hydrolysis reaction [41,47,48]. Indeed, it has been previously established that at high galactose concentrations, β-galactosidase activity is impaired since the conformational modification of the enzyme's active site reduces the affinity for its substrate [49,50]. Moreover, galactose could also act as a competitive inhibitor of β-galactosidase via the formation of galactosyl–enzyme intermediate products [51]. >90% of the initial whey lactose (Figure 3a). On the other hand, the optimum proteolytic activity was observed at 55 °C, as it has been earlier indicated by Tsakona et al. [16]. More particularly, as displayed in Figure 3b, FAN production increased along with the increase in temperature up to 55 °C, followed by a gradual reduction with further temperature increments (Figure 3b). Based on our results, significant differences (*p* < 0.05) were observed on the performed hydrolyses, at almost all evaluated temperatures. Likewise, no significant differences (*p* > 0.05) were observed on hydrolysis experiments carried out at 60 and 65 °C. Previous studies have also demonstrated processing of cheese whey via the implementation of microbial β-galactosidase to generate value-added products [42,43]. Generally, temperatures ranging between 50 and 60 °C and acidic pH values (3.5–4.5) have been reported as the optimal conditions for fungal β-galactosidase activity [13]. Additionally, Silvério et al. [44] recently studied β-galactosidase production in several *Aspergillus* species, aiming to synthesize potential prebiotics, whereby an increased enzyme activity in the range of 50–60 °C was noted. The current observation highlights the significant potential of the enzymes, since thermal stability is of imperative practical use for diverse bioprocesses, preventing various contaminations [45,46]. Furthermore, the results obtained postulate that the enzyme is more accessible during the first hours of hydrolysis. More specifically, a higher hydrolysis rate during the first 12 h entailed 30–50% of lactose hydrolysis, followed by a decreased rate at prolonged incubation time. Several studies also coincide with such findings where hydrolysis products decreased or even restricted lactose hydrolysis reaction [41,47,48]. Indeed, it has been previously established that at high galactose concentrations, β-galactosidase activity is impaired since the conformational modification of the enzyme's active site reduces the affinity for its substrate [49,50]. Moreover, galactose could also act as a competitive inhibitor of β-galactosidase via the formation of galactosyl–enzyme intermediate products [51].

enhance *Aspergillus* β-galactosidase production [11,13,41] Ultimately, SSF time for crude β-galactosidase and protease was standardized at 70 h to obtain maximal activities, that

CW hydrolysis was performed using the crude enzymatic extracts obtained from SSF cultures. Figure 3a illustrates the results obtained from different hydrolysis temperatures, whereby it can be observed that 60–65 °C was the optimum hydrolysis temperature of *A. awamori*. Ultimately, at the end of the bioprocess, crude enzymes hydrolyzed

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*3.2. Cheese Whey Hydrolysis Study* 

would be implemented in subsequent hydrolytic reactions of CW.

**Figure 3.** Effect of temperature on cheese whey (CW) hydrolysis using crude β-galactosidase and proteases. Kinetics of (**a**) lactose hydrolysis and (**b**) free amino nitrogen (FAN) produc-**Figure 3.** Effect of temperature on cheese whey (CW) hydrolysis using crude β-galactosidase and proteases. Kinetics of (**a**) lactose hydrolysis and (**b**) free amino nitrogen (FAN) production.

In an effort to further optimize whey hydrolysis, trials were also performed to evaluate the effect of different initial enzymatic activities on lactose breakdown and FAN production. Initial enzymatic activities of 7.5, 11, 15 U/mL were employed, and the results are illustrated in Figure 4. Figure 4a presents the kinetic profile of lactose hydrolysis, whereas Figure 4b presents FAN production in specific timepoints. Evidently, the use of 15 U/mL resulted in accelerated rates and complete lactose hydrolysis at 36 h and the production of 583.13 mg/L FAN. On the other hand, initial enzymatic activities of 7.5 and 11 U/mL yielded 87 and 93% of hydrolysis, respectively, at the same time point, providing lower productivities. Even though the degree of hydrolysis seems to follow a dose-dependent trend, apparently much higher concentrations do not significantly alter the hydrolysis efficiency, although

tion.

complete hydrolysis is performed significantly earlier at higher initial enzymatic activities. Worth noting, FAN production increased almost two-fold at higher initial enzymatic activities. Rosolen et al. [52] also presented similar efficiency levels on whey lactose hydrolysis by *A. oryzae*, regardless of the enzyme concentrations used (3, 6 and 9 U/mL). This observation probably also indicates the saturation of lactose at high β-galactosidase concentrations [53]. Thus, as in previous studies, our results imply that CW lactose hydrolysis is not strictly proportional with enzyme concentration [7,54]. However, complete hydrolysis was performed in almost half the time, using 15 U/mL, compared with the case of 7.5 U/mL. Nonetheless, in the event that scale up should be considered, lactose hydrolysis efficiency and FAN production should coincide with the feasibility of the process to highlight the most favorable operating conditions, which will be designated by the end target products. at higher initial enzymatic activities. Worth noting, FAN production increased almost two-fold at higher initial enzymatic activities. Rosolen et al. [52] also presented similar efficiency levels on whey lactose hydrolysis by *A. oryzae*, regardless of the enzyme concentrations used (3, 6 and 9 U/mL). This observation probably also indicates the saturation of lactose at high β-galactosidase concentrations [53]. Thus, as in previous studies, our results imply that CW lactose hydrolysis is not strictly proportional with enzyme concentration [7,54]. However, complete hydrolysis was performed in almost half the time, using 15 U/mL, compared with the case of 7.5 U/mL. Nonetheless, in the event that scale up should be considered, lactose hydrolysis efficiency and FAN production should coincide with the feasibility of the process to highlight the most favorable operating conditions, which will be designated by the end target products.

In an effort to further optimize whey hydrolysis, trials were also performed to evaluate the effect of different initial enzymatic activities on lactose breakdown and FAN production. Initial enzymatic activities of 7.5, 11, 15 U/mL were employed, and the results are illustrated in Figure 4. Figure 4a presents the kinetic profile of lactose hydrolysis, whereas Figure 4b presents FAN production in specific timepoints. Evidently, the use of 15 U/mL resulted in accelerated rates and complete lactose hydrolysis at 36 h and the production of 583.13 mg/L FAN. On the other hand, initial enzymatic activities of 7.5 and 11 U/mL yielded 87 and 93% of hydrolysis, respectively, at the same time point, providing lower productivities. Even though the degree of hydrolysis seems to follow a dose-dependent trend, apparently much higher concentrations do not significantly alter the hydrolysis efficiency, although complete hydrolysis is performed significantly earlier

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**Figure 4.** Effect of different initial enzymatic activity of crude β-galactosidase on cheese whey (CW) hydrolysis. Kinetics of (**a**) lactose hydrolysis and (**b**) free amino nitrogen (FAN) production. **Figure 4.** Effect of different initial enzymatic activity of crude β-galactosidase on cheese whey (CW) hydrolysis. Kinetics of (**a**) lactose hydrolysis and (**b**) free amino nitrogen (FAN) production.

#### *3.3. Bacterial Cellulose Production 3.3. Bacterial Cellulose Production*

CW constitutes a renewable, zero-cost substrate suitable for microbial fermentation, mostly requiring minimal pretreatment. However, often lactose does not undergo fermentation by several microorganisms including acetic acid bacteria. Previous reports demonstrated low BC-production from unhydrolyzed CW, thus hindering further implementation. Thus, pretreatment is often essential to overcome such limitations. Besides this, only limited studies have evaluated CW for BC production [55,56]. Based on similar literature reports, BC production is species and strain dependent. Evidently, the results of the current work confirmed the ability of *A. xylinum* to use CW hydrolysate under three different fermentation schemes. Hydrolysates derived from 11.25 (Hydrolysate A) and 7.5 U/mL (Hydrolysate B) crude β-galactosidase, respectively, were used to evaluate BC production, and the results are presented in Table 1. Different nitrogen concentrations were used, based on previous observations where elevated levels of nitrogen content CW constitutes a renewable, zero-cost substrate suitable for microbial fermentation, mostly requiring minimal pretreatment. However, often lactose does not undergo fermentation by several microorganisms including acetic acid bacteria. Previous reports demonstrated low BC-production from unhydrolyzed CW, thus hindering further implementation. Thus, pretreatment is often essential to overcome such limitations. Besides this, only limited studies have evaluated CW for BC production [55,56]. Based on similar literature reports, BC production is species and strain dependent. Evidently, the results of the current work confirmed the ability of *A. xylinum* to use CW hydrolysate under three different fermentation schemes. Hydrolysates derived from 11.25 (Hydrolysate A) and 7.5 U/mL (Hydrolysate B) crude β-galactosidase, respectively, were used to evaluate BC production, and the results are presented in Table 1. Different nitrogen concentrations were used, based on previous observations where elevated levels of nitrogen content induced cell proliferation at the expense of BC production [32]. As it can be seen in Figure 5a, the consumption of 13.91 g/L of glucose and 224.79 mg/L of FAN resulted in the production of 7.05 g/L BC (Hydrolysate A). Hydrolysate B followed a similar trend with respect to glucose consumption rate. The consumption of 13.41 g/L of glucose and 132.64 mg/L FAN, resulted in 5.78 g/L of BC production (Figure 5b). However, it is worth noting that in both experiments a considerable amount of sugars remained unfermented by *A. xylinum*. Therefore, a third treatment was deployed using diluted hydrolysate (hydrolysate C) (1:1 CW:H2O) in order to evaluate the BC production yield on approximately 25 g/L total sugar content and 260 mg/L FAN concentration (Figure 5c). In fact, in these experimental conditions, almost complete glucose consumption was attained along with the consumption of 114.94 mg/L FAN, achieving a final BC concentration of 5.59 g/L.

**Figure 5.** Bacterial cellulose (BC) production and kinetics of sugars and free amino nitrogen (FAN) consumption, using different cheese whey (CW) hydrolysates. (**a**) Hydrolysate A; (**b**) Hydrolysate B; (**c**) Hydrolysate C. **Figure 5.** Bacterial cellulose (BC) production and kinetics of sugars and free amino nitrogen (FAN) consumption, using different cheese whey (CW) hydrolysates. (**a**) Hydrolysate A; (**b**) Hydrolysate B; (**c**) Hydrolysate C.

The above results are in accordance with other studies describing the utilization of

ysate (20 g/L reducing sugars) as the sole carbon source using the strain *Komagataeibacter rhaeticus* P 1463. Additionally, Salari et al. [57] recently referred to a BC production of 3.5 g/L within 14 days of fermentation by *Gluconacetobacter xylinum* PTCC 1734 in static cultures, using an equimolar glucose/galactose mixture from hydrolyzed CW. In all the conducted experiments, a considerable increase in BC production was observed when compared with the results obtained by media with lower amount of FAN concentration. On the other hand, BC production levels by unhydrolyzed whey were quite close to those previously reported [58]. More specifically, as it is presented in Table 1, *A. xylinum* consumed 19.49 mg/L of FAN, producing 0.58 g/L of BC in unhydrolyzed CW (cheese whey A) (Table 1). Likewise, significant differences (*p* < 0.05) on BC production were observed, when different fermentation media were applied, whereas significantly higher concentrations were produced using all types of CW hydrolysates, compared to sole CW (Table

induced cell proliferation at the expense of BC production [32]. As it can be seen in Figure 5a, the consumption of 13.91 g/L of glucose and 224.79 mg/L of FAN resulted in the production of 7.05 g/L BC (Hydrolysate A). Hydrolysate B followed a similar trend with respect to glucose consumption rate. The consumption of 13.41 g/L of glucose and 132.64 mg/L FAN, resulted in 5.78 g/L of BC production (Figure 5b). However, it is worth noting that in both experiments a considerable amount of sugars remained unfermented by *A. xylinum*. Therefore, a third treatment was deployed using diluted hydrolysate (hydrolysate C) (1:1 CW:H2O) in order to evaluate the BC production yield on approximately 25 g/L total sugar content and 260 mg/L FAN concentration (Figure 5c). In fact, in these experimental conditions, almost complete glucose consumption was attained along with the consumption of 114.94 mg/L FAN, achieving a final BC concentration of 5.59 g/L.

**Table 1.** Experimental schemes of cheese whey and cheese whey hydrolysates fermentation by *A. xylinum.*

Hydrolysate A 45.04 ± 2.60 23.31 ± 0.77 9.40 ± 0.24 520.05 ± 0.34 224.79 ± 8.39 7.05 ± 0.14 A 0.71 Hydrolysate B 45.80 ± 0.77 22.35 ± 0.46 8.93 ± 0.19 331.36 ± 12.96 132.64 ± 5.21 5.78 ± 0.35 A,B 0.58 Hydrolysate C 24.68 ± 0.69 11.48 ± 0.48 1.00 + 0.08 259.25 ± 5.70 114.94 ± 4.73 5.59 ± 0.22 B 0.56 Cheese whey A 50.00 ± 1.22 2.51 ± 0.23 - 56.00 ± 3.35 19.49 ± 2.78 0.58 ± 0.01 a 0.06 Cheese whey B 24.45 ± 1.03 1.28 ± 0.10 - 22.98 ± 5.70 22.98 ± 0.00 0.71 ± 0.05 a,b 0.07 Cheese whey C 24.29 ± 1.18 1.39 ± 0.14 - 250.00 ± 10.06 81.33 ± 4.65 1.07 ± 0.09 b 0.11 \* Different letters (A,B, a, b) within each group (hydrolysates and cheese whey) indicate significant differences (*p* < 0.05).

**Initial FAN (mg/L)** 

**FAN Consumption (mg/L)** 

Recently, Kumar et al. [59] demonstrated the production of 1.4 g/L of BC under static culture conditions in whey medium by *Acetobacter pasteurianus*. The formation of BC in these cases is mostly due to the presence of several other compounds such as the residual carbon present in the initial inocula. In addition to this, higher BC production was observed using diluted CW, which is consistent which similar studies [60]. In specific, *A.* 

**BC Production \* (g/L)** 

**BC Productivity (g/L/d)** 

**Residual Glucose (g/L)** 

1).

**Initial Glucose (g/L)** 

**Initial Total Sugars (g/L)** 

FAN: free amino nitrogen; BC: bacterial cellulose.

**Fermentation Media** 


**Table 1.** Experimental schemes of cheese whey and cheese whey hydrolysates fermentation by *A. xylinum*.

\* Different letters (A, B, a, b) within each group (hydrolysates and cheese whey) indicate significant differences (*p* < 0.05). FAN: free amino nitrogen; BC: bacterial cellulose.

> The above results are in accordance with other studies describing the utilization of several monosaccharides and disaccharides as carbon sources to generate BC by various *Acetobacter* spp. strains. Semjonovs et al. [55] reported a high BC yield with CW hydrolysate (20 g/L reducing sugars) as the sole carbon source using the strain *Komagataeibacter rhaeticus* P 1463. Additionally, Salari et al. [57] recently referred to a BC production of 3.5 g/L within 14 days of fermentation by *Gluconacetobacter xylinum* PTCC 1734 in static cultures, using an equimolar glucose/galactose mixture from hydrolyzed CW. In all the conducted experiments, a considerable increase in BC production was observed when compared with the results obtained by media with lower amount of FAN concentration. On the other hand, BC production levels by unhydrolyzed whey were quite close to those previously reported [58]. More specifically, as it is presented in Table 1, *A. xylinum* consumed 19.49 mg/L of FAN, producing 0.58 g/L of BC in unhydrolyzed CW (cheese whey A) (Table 1). Likewise, significant differences (*p* < 0.05) on BC production were observed, when different fermentation media were applied, whereas significantly higher concentrations were produced using all types of CW hydrolysates, compared to sole CW (Table 1).

> Recently, Kumar et al. [59] demonstrated the production of 1.4 g/L of BC under static culture conditions in whey medium by *Acetobacter pasteurianus*. The formation of BC in these cases is mostly due to the presence of several other compounds such as the residual carbon present in the initial inocula. In addition to this, higher BC production was observed using diluted CW, which is consistent which similar studies [60]. In specific, *A. xylinum* produced 0.71 g/L and 1.07 g/L BC, when diluted CW (cheese whey B) and diluted CW supplemented with yeast medium (cheese whey C) were, respectively, applied (Table 1). In general, lactose as a sole carbon source is reported as a weak substrate for BC production leading to 0.04–0.07 g/L [39,61], while BC production by unhydrolyzed CW is recorded slightly higher ranging from 0.15 to 0.78 g/L [58,60]. Our results (using unhydrolyzed CW) are in agreement with those previously reported, whereas BC production was significant higher using CW hydrolysates. Overall, in this study, high production of BC was achieved using CW hydrolysates compared even to BC production using conventional synthetic HS medium. These findings are exceptionally promising pointing out potential for a cost-effective bioprocess.
