Conversion of Wheat Bran to Xylanases and Dye Adsorbent by Streptomyces thermocarboxydus

Agro-byproducts can be utilized as effective and low-cost nutrient sources for microbial fermentation to produce a variety of usable products. In this study, wheat bran powder (WBP) was found to be the most effective carbon source for xylanase production by Streptomyces thermocarboxydus TKU045. The optimal media for xylanase production was 2% (w/v) WBP, 1.50% (w/v) KNO3, 0.05% (w/v) MgSO4, and 0.10% (w/v) K2HPO4, and the optimal culture conditions were 50 mL (in a 250 mL-volume Erlenmeyer flask), initial pH 9.0, 37 °C, 125 rpm, and 48 h. Accordingly, the highest xylanase activity was 6.393 ± 0.130 U/mL, 6.9-fold higher than that from un-optimized conditions. S. thermocarboxydus TKU045 secreted at least four xylanases with the molecular weights of >180, 36, 29, and 27 kDa when cultured on the WBP-containing medium. The enzyme cocktail produced by S. thermocarboxydus TKU045 was optimally active over a broad range of temperature and pH (40–70 °C and pH 5–8, respectively) and could hydrolyze birchwood xylan to produce xylobiose as the major product. The obtained xylose oligosaccharide (XOS) were investigated for 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and the growth effect of lactic acid bacteria. Finally, the solid waste from the WBP fermentation using S. thermocarboxydus TKU045 revealed the high adsorption of Congo red, Red 7, and Methyl blue. Thus, S. thermocarboxydus TKU045 could be a potential strain to utilize wheat bran to produce xylanases for XOS preparation and dye adsorbent.


Effects of Nitrogen Sources on Xylanase Production
The medium with 2% WBP, 0.05% (w/v) MgSO 4 , and 0.10% (w/v) K 2 HPO 4 was used to optimize the nitrogen source for xylanase production. Different nitrogenous materials such as yeast extract, peptone, beef extract, KNO 3 , and NH 4 NO 3 were supplemented to the medium at the same concentration of 1% (w/v). The culture conditions were 37 • C, and 150 rpm and the xylanase activity of the culture medium was estimated every day. The best nitrogen source was then examined for the optimum concentration for xylanase production. The effect of nitrogen source concentration was investigated in a range of 0-2% (w/v).

Effects of Culture Conditions on Xylanase Production
The WBP-containing medium was used to optimize the culture condition for the xylanase production by S. thermocarboxydus TKU045. The medium included 2% WBP, 1% KNO 3 , 0.05% (w/v) MgSO 4 , and 0.10% (w/v) K 2 HPO 4 . The effect of the initial pH, incubation temperature and shaking speed was examined in the range of pH 7-10, 30-50 • C, and 0 rpm-175 rpm, respectively. The effect of culture medium was investigated in a range of 50-150 mL in a 250 mL flask. After every one day, 1 mL of culture medium was withdrawn to test the xylanase activity. Finally, all the optimum conditions were applied to confirm the efficiency of the optimization process.

Determination of Molecular Weight (MW) of the Xylanases
The MW of xylanases was determined using the sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE) method [53] with some modifications. Briefly, the 10% resolving gel was co-polymerized with 0.05% birchwood xylan (w/v). The culture medium and crude enzyme samples were mixed with the sample buffer and then 5 µL of each sample was loaded on the prepared gel and electrophoresis was conducted at 114 V, and 4 • C. Then, residual SDS was removed by washing the gel in 2% Triton X-100 (prepared in phosphate buffer, 50 mM, pH 7), and then in phosphate buffer alone. The xylanolytic reaction was carried out by incubating the gel in phosphate buffer for 1 h at 37 • C. The staining step was conducted by dipping the gel in 0.01% (w/v) Congo red solution for 30 min, and then the gel was washed by 1 M NaCl solution. The xylanase activity appeared as a clear band against the red background of the gel.

Effects of Temperature and pH
The optimal temperature of the crude enzyme cocktail was explored by immediately incubating it with substrate solution at different temperatures (from 10 to 100 • C) for 30 min. The thermal stability of the crude enzyme cocktail solution was explored by pre-treating at different temperatures (10-100 • C) for 30 min and the residual xylanase activity was measured. The optimal pH of the crude enzyme cocktail was determined by adjusting the pH of the reaction solution. The buffer systems explored were citrate buffer (pH 3-7), phosphate buffer (pH 6-8.5), and carbonate buffer (pH 8.5-11). The pre-treated crude enzyme solution in different pH for 30 min at 4 • C, was then adjusted to pH 7 and its residual xylanase activity was measured to explore the pH stability of the crude enzyme cocktail. SDS, Tween 20, Tween 40, Triton X-100 and SDS were prepared at 5% and 10%. At first, an equivalent volume of the crude enzyme cocktail and each of those chemicals were mixed in a glass tube at 4 • C for 30 min and then the residual xylanase activities of the crude enzyme cocktail were then measured by xylanase assay (as mentioned above).

Growth Effect Assay
The effect of xylose oligosaccharide (XOS) was checked on the growth of L. rhamnosus BCRC 16000, B. bifidum BCRC 14615, L. paracasei subsp. paracasei BCRC 14023, and L. rhamnosus BCRC 10940. XOS was added to MRS medium at different concentrations (0.01%, 0.05%, and 0.10%, w/v). The bacterial strains were grown at 37 • C for 24 h. For B. bifidum BCRC 14615, 0.05% (w/v) cysteine was added to the medium, and the culture was carried out in anaerobic mode. To measure the relative growth cell of the bacteria on MRS culture containing XOS, A 600 nm of the culture MRS medium was used as the control.

Dye Adsorption Assay
The dye adsorption ability of solid waste obtained from the WBP fermentation by S. thermocarboxydus TKU045 was investigated using ten dyes, including Red 6, Yellow 4, Red 40, Red 7, Green 3, Yellow 5, Blue 1, Blue 2, Congo red, and Methyl blue. The mixture of 100 mg of adsorbent and 5 mL of dye solution (0.02%, w/v) was shaken for 60 min, and then the color intensity of the final solution was measured by a spectrophotometer. The adsorption rate (%) was calculated by using the formula: Adsorption rate (%) = (A C − A S )/A C where A C is the absorbance of the control solution (original dye solution), and A S is the absorbance of the experimental group.

The Fourier-Transform Infrared Spectroscopy (FTIR) Analysis
FTIR spectra of unloaded and loaded dye samples were measured in the presence of KBr over the range of 4000-400 cm −1 on Nicolet iS5 FTIR spectrophotometer (Thermo, Waltham, MA, USA) [56].

Statistical Analysis
For the statistical analysis of the results, the data on the experiments were analyzed by Microsoft Excel 2016.

Agro-Byproducts as the Sole Carbon Source for the Xylanase Production
Various carbon sources were investigated for the preparation of an optimum culture medium for the production of xylanase by S. thermocarboxydus TKU045. These included RBP, WBP, SCG, CHP, birchwood xylan, and CMC. As shown in Figure 1a, the highest xylanase activity was observed in the culture supernatant using xylan or WBP as the carbon source (0.952 ± 0.088 U/mL and 0.975 ± 0.085U/mL, respectively) followed by using CHP (0.788 ± 0.047 U/mL) on the 5th day. In contrast, much lower xylanase activity was detected when CMC, RBP, and SCG were used as carbon sources. Xylan is known to be the most effective carbon source for the synthesis of xylanase by Streptomyces [32][33][34][35][36]38,[57][58][59]. However, the high price of xylan may be a deterrent to its use in the production of xylanase. Therefore, the utilization of agro-byproducts can be an effective alternative. Accordingly, various substances have been indicated in the literature as suitable carbon sources for producing xylanase, such as rice bran [37], wheat bran [60][61][62], rice straw [33], and sugar cane bagasse [49]. In this study, WBP exhibited the highest enzyme activity among the tested byproducts and was thus chosen as the most suitable carbon source for xylanase production by S. thermocarboxydus TKU045.

Agro-Byproducts as the Sole Carbon Source for the Xylanase Production
Various carbon sources were investigated for the preparation of an optimum culture medium for the production of xylanase by S. thermocarboxydus TKU045. These included RBP, WBP, SCG, CHP, birchwood xylan, and CMC. As shown in Figure 1a, the highest xylanase activity was observed in the culture supernatant using xylan or WBP as the carbon source (0.952 ± 0.088 U/mL and 0.975 ± 0.085U/mL, respectively) followed by using CHP (0.788 ± 0.047 U/mL) on the 5th day. In contrast, much lower xylanase activity was detected when CMC, RBP, and SCG were used as carbon sources. Xylan is known to be the most effective carbon source for the synthesis of xylanase by Streptomyces [32][33][34][35][36]38,[57][58][59]. However, the high price of xylan may be a deterrent to its use in the production of xylanase. Therefore, the utilization of agro-byproducts can be an effective alternative. Accordingly, various substances have been indicated in the literature as suitable carbon sources for producing xylanase, such as rice bran [37], wheat bran [60][61][62], rice straw [33], and sugar cane bagasse [49]. In this study, WBP exhibited the highest enzyme activity among the tested byproducts and was thus chosen as the most suitable carbon source for xylanase production by S. thermocarboxydus TKU045. To examine the effect of WBP concentration on the production of xylanase, the amount of WBP in the medium was adjusted in a range of 0.25-3% (w/v). As shown in Figure 1b, WBP at 2% was the most effective (2.288 ± 0.198 U/mL) in the production of xylanase, followed by 1.50% and 1% concentration (1.972 ± 0.003 U/mL, and 1.249 ± 0.021 U/mL respectively) on the 5th day. A lower xylanase activity was detected using WBP at other concentrations (0.25%, 0.50%, 2.50%, and 3%). Thus, the optimum carbon source used for xylanase production by S. thermocarboxydus TKU045 was 2% (w/v) WBP.

Effects of Nitrogen Source on Xylanase Production
Different nitrogen sources such as yeast extract, peptone, beef extract, KNO3, and NH4NO3 were supplemented to the WBP-containing medium to assess the suitable nitrogen source for xylanase production by S. thermocarboxydus TKU045. Besides, NB medium supplemented with 2% WBP was also used in this investigation. Interestingly, only KNO3 showed a positive effect on xylanase production, whereas all other nitrogen sources had a negative impact (Figure 2a). The highest xylanase activity was observed on the 4th day using KNO3 as the nitrogen source (4.271 ± 0.131 U/mg). This result was 2.34-folds higher To examine the effect of WBP concentration on the production of xylanase, the amount of WBP in the medium was adjusted in a range of 0.25-3% (w/v). As shown in Figure 1b, WBP at 2% was the most effective (2.288 ± 0.198 U/mL) in the production of xylanase, followed by 1.50% and 1% concentration (1.972 ± 0.003 U/mL, and 1.249 ± 0.021 U/mL respectively) on the 5th day. A lower xylanase activity was detected using WBP at other concentrations (0.25%, 0.50%, 2.50%, and 3%). Thus, the optimum carbon source used for xylanase production by S. thermocarboxydus TKU045 was 2% (w/v) WBP.

Effects of Nitrogen Source on Xylanase Production
Different nitrogen sources such as yeast extract, peptone, beef extract, KNO 3 , and NH 4 NO 3 were supplemented to the WBP-containing medium to assess the suitable nitrogen source for xylanase production by S. thermocarboxydus TKU045. Besides, NB medium supplemented with 2% WBP was also used in this investigation. Interestingly, only KNO 3 showed a positive effect on xylanase production, whereas all other nitrogen sources had a negative impact (Figure 2a). The highest xylanase activity was observed on the 4th day using KNO 3 as the nitrogen source (4.271 ± 0.131 U/mg). This result was 2.34-folds higher than the xylanase activity of liquid supernatant of the control group (1.820 ± 0.211 U/mL) on the 5th day. Also, the cultivation time to achieve the maximum xylanase productivity using KNO 3 was shorter (4 days) than that of other nitrogen sources. Chi et al. (2013) reported that the best xylanase production for S. thermocarboxydus MW8 was observed using soytone as the nitrogen source [32]. However, Sinjaroonsak et al. (2019) found that yeast extract was the most suitable nitrogen source for xylanase production by S. thermocoprophilus TC13W [63]. on the 5th day. Also, the cultivation time to achieve the maximum xylanase productivity using KNO3 was shorter (4 days) than that of other nitrogen sources. Chi et al. (2013) reported that the best xylanase production for S. thermocarboxydus MW8 was observed using soytone as the nitrogen source [32]. However, Sinjaroonsak et al. (2019) found that yeast extract was the most suitable nitrogen source for xylanase production by S. thermocoprophilus TC13W [63]. The effect of KNO3 concentration on the xylanase production was studied by adding 0-2% KNO3 into the WBP-containing medium. As shown in Figure 2b, the maximal xylanase activity of medium with KNO3 at 0%, 0.50%, 1%, 1.50%, and 2% was 1.840 ± 0.013 U/mL (day 5), 4.251 ± 0.206 U/mL (day 4), 4.300 ± 0.357 U/mL (day 4), 4.476 ± 0.393 U/mL (day 4), 5.563 ± 0.137 (day 3), and 5.037 ± 0.045 U/mL (day 4), respectively. This result indicates that KNO3 at 1.50% concentration was the most suitable for xylanase production by S. thermocarboxydus TKU045.

Effects of Culture Conditions on Xylanase Production
The effect of initial pH on xylanase production was studied by adjusting the pH of WBP containing medium to pH 7, 8, 9, and 10. The medium without pH adjustment (at pH 7.4) was also studied. As shown in Figure 3a, the highest xylanase activity was observed on the 4 th day at 4.765 ± 0.185 U/mL (pH 7), 4.884 ± 0.290 U/mL (pH 7.4), 5.190 ± 0.326 U/mL (pH 8), 5.785 ± 0.252 U/mL (pH 9), and 5.710 ± 0.133 U/mL (pH 10). An increase in the initial pH of the medium seemed to promote more xylanase production. At pH 9 and 10, the maximum xylanase activity of medium was significantly higher than that at pH 7 and 7.4. However, the maximum enzyme activity of medium at pH 8 was not significantly different than that at other pH values. This indicated that xylanase is produced media over a wide range of pH (pH 7-10). The optimum pH of the medium for xylanase production was different from that of other Streptomyces strains. Some reports indicated that the optimum pH for the xylanase production was pH 8-9 using strains including Streptomyces sp. RCK-2010 [60], S. cyaneus SN32 [62], and S. griseorubens LH-3 [64]. In contrast, other strains like Streptomyces sp. B6 [35], S. thermocarboxydus MW8 [32], S. olivaceoviridis E-86 [58], and S. rameus L2001 could produce xylanase at optimum levels in the range of pH 6-7.2 [65]. The effect of KNO 3 concentration on the xylanase production was studied by adding 0-2% KNO 3 into the WBP-containing medium. As shown in Figure 2b, the maximal xylanase activity of medium with KNO 3 at 0%, 0.50%, 1%, 1.50%, and 2% was 1.840 ± 0.013 U/mL (day 5), 4.251 ± 0.206 U/mL (day 4), 4.300 ± 0.357 U/mL (day 4), 4.476 ± 0.393 U/mL (day 4), 5.563 ± 0.137 (day 3), and 5.037 ± 0.045 U/mL (day 4), respectively. This result indicates that KNO 3 at 1.50% concentration was the most suitable for xylanase production by S. thermocarboxydus TKU045.

Effects of Culture Conditions on Xylanase Production
The effect of initial pH on xylanase production was studied by adjusting the pH of WBP containing medium to pH 7, 8, 9, and 10. The medium without pH adjustment (at pH 7.4) was also studied. As shown in Figure 3a, the highest xylanase activity was observed on the 4 th day at 4.765 ± 0.185 U/mL (pH 7), 4.884 ± 0.290 U/mL (pH 7.4), 5.190 ± 0.326 U/mL (pH 8), 5.785 ± 0.252 U/mL (pH 9), and 5.710 ± 0.133 U/mL (pH 10). An increase in the initial pH of the medium seemed to promote more xylanase production. At pH 9 and 10, the maximum xylanase activity of medium was significantly higher than that at pH 7 and 7.4. However, the maximum enzyme activity of medium at pH 8 was not significantly different than that at other pH values. This indicated that xylanase is produced media over a wide range of pH (pH 7-10). The optimum pH of the medium for xylanase production was different from that of other Streptomyces strains. Some reports indicated that the optimum pH for the xylanase production was pH 8-9 using strains including Streptomyces sp. RCK-2010 [60], S. cyaneus SN32 [62], and S. griseorubens LH-3 [64]. In contrast, other strains like Streptomyces sp. B6 [35], S. thermocarboxydus MW8 [32], S. olivaceoviridis E-86 [58], and S. rameus L2001 could produce xylanase at optimum levels in the range of pH 6-7.2 [65].  Temperature is one of the key factors that significantly impacts microbial growth and enzyme production. In this study, we investigated a temperature range of 30-50 °C for the optimal temperature for xylanase production by S. thermocarboxydus TKU045. This strain Temperature is one of the key factors that significantly impacts microbial growth and enzyme production. In this study, we investigated a temperature range of 30-50 • C for the optimal temperature for xylanase production by S. thermocarboxydus TKU045. This strain gave the highest xylanase activity of 4.330 ± 0.114 U/mL at 37 • C (Figure 3b). Thus, Polymers 2021, 13, 287 8 of 18 37 • C was selected at the most suitable temperature for the xylanase production by S. thermocarboxydus TKU045. Several studies have shown that an optimum temperature ≥ 40 • C for xylanase production by S. thermocarboxydus MW8 [32], Streptomyces sp. RCK-2010 [60], S. cyaneus SN32 [62], S. thermovulgaris TISTR1948 [66], and S. variabilis MAB3 [67]. However, some Streptomyces strains such as Streptomyces spp. SKK1-8 (30 • C) [57], and Streptomyces sp. P12-137 could also produce xylanase at a lower temperature (28 • C) [68].
The shaking speed of the fermentation flask plays an important role in microbial fermentation as it involves oxygen dissolution and substance transfer in the culture medium. As shown in Figure 3c, S. thermocarboxydus TKU045 exhibited the highest xylanase activity at the shaking speed of 125 rpm (4.803 ± 0.099 U/mL), whereas a lower activity was observed at higher or lower shaking speed. Too high shaking speed may cause higher shear stress, resulting in the decline in enzyme production while too low shaking speed may cause less homogenous mixing and low dissolved oxygen. Several studies have indicated improved production of xylanase by Streptomyces at a higher shaking speed (140 rpm-200 rpm) unlike that of S. thermocarboxydus TKU045 observed in this study [33,34,38,60,[62][63][64][65].
The effect of medium volume on xylanase production was studied by adjusting the volume of WBP containing medium to 50, 75, 100, 125 and 150 mL. As shown in Figure 3d, the highest xylanase activity was observed on the 3rd day (5.934 ± 0.279 U/mL) in 50 mL medium, and less activity was observed in 75 mL, and 100 mL medium on the 4th day (5.189 ± 0.071, and 4.308 ± 0.075 U/mL, respectively), and in 125 mL and 150 mL medium on the 5th day (4.905 ± 0.0195, and 3.447 ± 0.253 U/mL, respectively). Thus, 50 mL was selected as the optimum medium volume for xylanase production by S. thermocarboxydus TKU045.
The optimum components of medium and culture conditions to obtain the highest xylanase productivity by S. thermocarboxydus TKU045 were explored. The medium components were 2% WBP, 1.50% KNO 3 , 0.05% MgSO 4 , and 0.10% K 2 HPO 4 , and the culture conditions were 50/250 mL, pH 9.0, 37 • C, and 125 rpm. To evaluate the efficiency of the optimization process, S. thermocarboxydus TKU045 was cultured at conditions before and after optimization. The conditions before optimization included the medium components (1% xylan, 0.05% MgSO 4 , and 0.10% K 2 HPO 4 ), and culture conditions (100/250 mL, pH 8.0, 37 • C, 150 rpm). The time course of xylanase production by S. thermocarboxydus TKU045 in the conditions before and after optimization is shown in Figure 3e. The maximum xylanase productivity of S. thermocarboxydus TKU045 was 6.393 ± 0.130 U/mL (at 48th h) at the optimized conditions, and 0.932 ± 0.088 U/mL (at 120th h) in the original condition. The results indicated that S. thermocarboxydus TKU045 could increase the xylanase productivity in the optimized conditions by 6.9-fold than that in the original conditions. Moreover, the incubation time for maximum xylanase productivity was shortened from 5 days to about 48 h. To date, an optimal incubation time of 48 h has been reported for only a few Streptomyces strains for xylanase production, (for example Streptomyces sp. RCK-2010 [60], and S. cyaneus SN32 [62]), while many strains required a longer fermentation period such as Streptomyces sp. SWU10 (3 days) [33], S. thermocarboxydus MW8 (4 days) [32], S. matensis DW67 (5 days) [34], S. chartreusis L1105 (7 days) [38], and Streptomyces spp. SKK1-8 (10 days) [57]. The longer fermentation period may increase the production cost, therefore, a shorter fermentation is more advantageous. Besides, wheat bran is a cheap and largely available agro-byproduct, suggesting that it is a suitable carbon source for microbial fermentation processes on a larger scale. Taken together, the conversion of wheat bran by S. thermocarboxydus TKU045 could be considered as a cheap and efficient way in the xylanase production process.

Molecular Weight Determination of the Xylanases
To determine the expression pattern of the xylanases, the secretome of S. thermocarboxydus TKU045 over WBP-containing medium was analyzed by SDS-PAGE containing 0.05% xylan. As shown in Figure 4, at least four different xylanases with >180, 36, 29, and 27 kDa of MW were observed indicating that TKU045 produced multiple xylanases into the culture Polymers 2021, 13, 287 9 of 18 medium to effectively degrade the complex substance such as wheat bran. Interestingly, (NH 4 ) 2 SO 4 , a common chemical for enzyme concentration, could only precipitate the 29 and >180 kDa xylanases. The SDS-PAGE zymography result of the crude enzyme in Figure 4 reveal only >180 and 29 kDa bands of xylanase activity, whereas 36 and 27 kDa bands not present. The production of multiple xylanases has also been observed in several Streptomyces strains for example Streptomyces sp. SWU10, Streptomyces sp. B-12-2, S. actuosus A-151, Streptomyces sp. P12-137, and S. olivaceoviridis E-86 (Table 1). In general, the MW of xylanase from Streptomyces strains is in the range of 15-50 kDa. Thus, the >180 kDa xylanase produced by TKU045 in this study may be an exception. Furthermore, the MW of TKU045 xylanases was remarkably different from that obtained from S. thermocarboxydus HY-5 (43.962 Da) and S. thermocarboxydus MW8 (52 kDa). Therefore, these results might be considered as a novel observation towards xylanase production from Streptomyces.
Polymers 2021, 13, x FOR PEER REVIEW 9 of 18 27 kDa of MW were observed indicating that TKU045 produced multiple xylanases into the culture medium to effectively degrade the complex substance such as wheat bran. Interestingly, (NH4)2SO4, a common chemical for enzyme concentration, could only precipitate the 29 and >180 kDa xylanases. The SDS-PAGE zymography result of the crude enzyme in Figure 4 reveal only >180 and 29 kDa bands of xylanase activity, whereas 36 and 27 kDa bands not present. The production of multiple xylanases has also been observed in several Streptomyces strains for example Streptomyces sp. SWU10, Streptomyces sp. B-12-2, S. actuosus A-151, Streptomyces sp. P12-137, and S. olivaceoviridis E-86 (Table 1). In general, the MW of xylanase from Streptomyces strains is in the range of 15-50 kDa. Thus, the >180 kDa xylanase produced by TKU045 in this study may be an exception. Furthermore, the MW of TKU045 xylanases was remarkably different from that obtained from S. thermocarboxydus HY-5 (43.962 Da) and S. thermocarboxydus MW8 (52 kDa). Therefore, these results might be considered as a novel observation towards xylanase production from Streptomyces. S. thermocarboxydus TKU045 was cultured in the optimized medium at optimal conditions. Then, the culture medium was centrifuged at 13,000 rpm to collect the culture supernatant.  S. thermocarboxydus TKU045 was cultured in the optimized medium at optimal conditions. Then, the culture medium was centrifuged at 13,000 rpm to collect the culture supernatant.

Biochemical Characterization of Crude Enzyme Cocktail
The optimum temperature and pH of S. thermocarboxydus TKU045 s crude enzyme cocktail were determined using birchwood xylan as the substrate. The optimum temperature range of the crude enzyme was determined to be between 40-70 • C (Figure 5a). At temperatures beyond this range, the activity of the crude enzyme was significantly lower than that of optimum temperatures. The crude enzyme was thermally stable up to 60 • C, indicating that S. thermocarboxydus TKU045 s crude enzyme cocktail had high thermo-activity and thermo-stability. Thermo-activity and -stability are the common features of xylanases from Streptomyces. Liu et al. (2020) reported that two xylanases from Streptomyces sp. B6, XynST10 and XynST11, have a high thermo-activity (60 • C, and 60-90 • C, respectively) and thermo-stability (50 and 60 • C, respectively) [35]. The highest xylanase activity of S. thermocarboxydus TKU045 s crude enzyme cocktail was measured at pH 7 (in phosphate buffer). However, more than 80% of the enzyme activity was also observed at pH 5, 6, and 8, indicating the activity of enzyme cocktail over a broad optimum pH from pH 5-pH 8. Likewise, the enzyme cocktail was the most stable in the range of pH 5-pH 8 (Figure 5b). The broad optimal pH and pH stability may provide the enzyme a greater ability to act at various pH conditions, as was observed in the enzyme cocktail from S. thermocarboxydus TKU045. The effect of metal ions on the activity of S. thermocarboxydus TKU045′s crude enzyme cocktail was also studied by adding the respective metal salts in the reaction mixture as described in the method section. As shown in Figure 5c, there was no significant effect of all the tested ion metals (Ba 2+ , Ca 2+ , Cu 2+ , Fe 2+ , Mn 2+ , Mg 2+ , and Zn 2+ ) on the xylanase activity of crude enzyme cocktail at 5 mM and 10 mM concentrations. In contrast, Ca 2+ , Cu 2+ , Fe 2+ , Mn 2+ , Mg 2+ , and Zn 2+ were reported to inhibit S. thermocarboxydus MW8 xylanase [32]. Likewise, Kim et al. (2010) reported that Cu 2+ strongly inhibited S. thermocarboxydus HY-5 xylanase [31]. EDTA showed no apparent effects on the activity of S. thermocarboxydus TKU045′s crude enzyme cocktail, indicating that the xylanases do not need metal ions as the cofactor. Nonionic surfactants (Tween 20, Tween 40, and Triton X-100) did not show any significant effect on the xylanase activity of crude enzyme cocktail at 5% and 10% concentrations. However, SDS, an anionic surfactant strongly inhibited the enzyme activity.
The hydrolysis of xylan by S. thermocarboxydus TKU045 xylanases was analyzed by the TLC method. As shown in Figure 5d, the products of the process could be observed after 30 min of the initiation of hydrolysis, which revealed the highest intensity of the band of xylose dimer, followed by the band of xylose trimer. This indicates that the enzymes rapidly degrade xylan to release xylobiose and xylotriose. The occurrence of xylose The effect of metal ions on the activity of S. thermocarboxydus TKU045 s crude enzyme cocktail was also studied by adding the respective metal salts in the reaction mixture as described in the method section. As shown in Figure 5c, there was no significant effect of all the tested ion metals (Ba 2+ , Ca 2+ , Cu 2+ , Fe 2+ , Mn 2+ , Mg 2+ , and Zn 2+ ) on the xylanase activity of crude enzyme cocktail at 5 mM and 10 mM concentrations. In contrast, Ca 2+ , Cu 2+ , Fe 2+ , Mn 2+ , Mg 2+ , and Zn 2+ were reported to inhibit S. thermocarboxydus MW8 xylanase [32]. Likewise, Kim et al. (2010) reported that Cu 2+ strongly inhibited S. thermocarboxydus HY-5 xylanase [31]. EDTA showed no apparent effects on the activity of S. thermocarboxydus TKU045 s crude enzyme cocktail, indicating that the xylanases do not need metal ions as the cofactor. Nonionic surfactants (Tween 20, Tween 40, and Triton X-100) did not show any significant effect on the xylanase activity of crude enzyme cocktail at 5% and 10% concentrations. However, SDS, an anionic surfactant strongly inhibited the enzyme activity.
The hydrolysis of xylan by S. thermocarboxydus TKU045 xylanases was analyzed by the TLC method. As shown in Figure 5d, the products of the process could be observed after 30 min of the initiation of hydrolysis, which revealed the highest intensity of the band of xylose dimer, followed by the band of xylose trimer. This indicates that the enzymes rapidly degrade xylan to release xylobiose and xylotriose. The occurrence of xylose oligomers with DP ≥ 3 was attributed to endo-type xylanase, whereas the occurrence of xylobiose was attributed to exo-type xylanase. Over time, the intensity of spots of xylose oligomers with DP ≥ 3 decreased and the spot of xylose appeared from 1 h of the initiation of hydrolysis. These results indicate that the cocktail of S. thermocarboxydus TKU045 xylanases has the exo-type activity, which could degrade xylose oligosaccharides with DP ≥ 3 to xylose and xylobiose [5]. Besides, the intensity of xylobiose did not deteriorate during the reaction period, suggesting that the exo-type enzyme(s) of TKU045 xylanases could not act on xylobiose to release xylose monomer. In short, a mixture of xylose oligosaccharides, with DP = 2 as the main component, could be obtained from the xylan hydrolysis catalyzed by the cocktail of S. thermocarboxydus TKU045 xylanases.

Antioxidant Activity and the Growth Effect on Lactic Acid Bacteria of Xylan Hydrolysate
Natural materials have recently been shown to be a very promising source of antioxidants, for example, peptides [53], chitosan-oligosaccharides [70], polyphenols [71], flavonoids [72], and others [71,72]. In the search for a potential antioxidant, XOS was found to possess a good free radical scavenging capacity and thus could be considered as a potential source [73]. In this study, the XOS obtained from the hydrolysis of birchwood xylan with S. thermocarboxydus TKU045's crude enzyme cocktail was studied by the DPPH radical scavenging activity. As shown in Figure 6a, the DPPH radical activity of the XOS was dose-dependent at 1 mg/mL-20 mg/mL of XOS and the maximum scavenging activity was 87-91% at 20 mg/mL-50 mg/mL XOS. Huang et al. (2019) reported that XOS from Moso bamboo prehydrolyzate could achieve 86% DPPH radical scavenging activity [74]. Likewise, the birchwood xylan XOS prepared by Bacillus amyloliquifaciens NRRL B-14393 xylanase exhibited the maximum DPPH radical scavenging activity of 87% [75]. However, the DPPH radical scavenging activity of corncob XOSs prepared by B. aerophilus KGJ2 xylanase could only reach the maximum value of 74.2% [73].
Polymers 2021, 13, x FOR PEER REVIEW 12 of 18 oligomers with DP ≥ 3 was attributed to endo-type xylanase, whereas the occurrence of xylobiose was attributed to exo-type xylanase. Over time, the intensity of spots of xylose oligomers with DP ≥ 3 decreased and the spot of xylose appeared from 1 h of the initiation of hydrolysis. These results indicate that the cocktail of S. thermocarboxydus TKU045 xylanases has the exo-type activity, which could degrade xylose oligosaccharides with DP ≥ 3 to xylose and xylobiose [5]. Besides, the intensity of xylobiose did not deteriorate during the reaction period, suggesting that the exo-type enzyme(s) of TKU045 xylanases could not act on xylobiose to release xylose monomer. In short, a mixture of xylose oligosaccharides, with DP = 2 as the main component, could be obtained from the xylan hydrolysis catalyzed by the cocktail of S. thermocarboxydus TKU045 xylanases.

Antioxidant Activity and the Growth Effect on Lactic Acid Bacteria of Xylan Hydrolysate
Natural materials have recently been shown to be a very promising source of antioxidants, for example, peptides [53], chitosan-oligosaccharides [70], polyphenols [71], flavonoids [72], and others [71,72]. In the search for a potential antioxidant, XOS was found to possess a good free radical scavenging capacity and thus could be considered as a potential source [73]. In this study, the XOS obtained from the hydrolysis of birchwood xylan with S. thermocarboxydus TKU045's crude enzyme cocktail was studied by the DPPH radical scavenging activity. As shown in Figure 6a, the DPPH radical activity of the XOS was dose-dependent at 1 mg/mL-20 mg/mL of XOS and the maximum scavenging activity was 87-91% at 20 mg/mL-50 mg/mL XOS. Huang et al. (2019) reported that XOS from Moso bamboo prehydrolyzate could achieve 86% DPPH radical scavenging activity [74]. Likewise, the birchwood xylan XOS prepared by Bacillus amyloliquifaciens NRRL B-14393 xylanase exhibited the maximum DPPH radical scavenging activity of 87% [75]. However, the DPPH radical scavenging activity of corncob XOSs prepared by B. aerophilus KGJ2 xylanase could only reach the maximum value of 74.2% [73]. XOSs have been described as a potential prebiotic and are gradually being widely recognized. However, in several reports, XOS also show antimicrobial activity. In this study, the growth effect of XOS from the hydrolysis of birchwood xylan with S. thermocarboxydus TKU045's crude enzyme cocktail was examined on four lactic acid bacterial strains, including B. bifidum BCRC 14615, L. rhamnosus BCRC 16000, L. rhamnosus BCRC 10940, and L. paracasei subsp. paracasei BCRC 14023 at the concentration of 0.01-0.10% of XOS. Notably, 0.10% XOS exhibited a growth-inhibiting effect (52%), and 0.01% XOS had XOSs have been described as a potential prebiotic and are gradually being widely recognized. However, in several reports, XOS also show antimicrobial activity. In this study, the growth effect of XOS from the hydrolysis of birchwood xylan with S. thermocarboxydus TKU045's crude enzyme cocktail was examined on four lactic acid bacterial strains, including B. bifidum BCRC 14615, L. rhamnosus BCRC 16000, L. rhamnosus BCRC 10940, and L. paracasei subsp. paracasei BCRC 14023 at the concentration of 0.01-0.10% of XOS. Notably, 0.10% XOS exhibited a growth-inhibiting effect (52%), and 0.01% XOS had a growth-enhancing effect (115%) on B. bifidum BCRC 14615 (Figure 6b), indicating that the growth effect of the XOS may depend on its concentration. In contrast to the prebiotic activity, the antibacterial activity of XOS has been rarely reported. Christakopoulos et al. (2002) reported that acidic XOSs produced from birchwood xylan showed antimicrobial activity against Staphylococcus aureus, B. cereus, Micrococcus flavus, Escherichia coli, and Helicobacter pylori [76]. Likewise, Kallel et al. (2014) reported that XOS from garlic straw-extracted xylan had high antimicrobial activity against Pseudomonas aeruginosa, B. thuringiensis, Enterococcus faecalis, and Klebsiella pneumoniae [77]. In contrast, in the concentration range of 0.01-0.10%, the XOS did not show a significant effect on the growth of L. rhamnosus BCRC 16000, L. rhamnosus BCRC 10940, and L. paracasei subsp. paracasei BCRC 14023 (data not shown). This indicates that the XOS obtained from the hydrolysis of birchwood xylan with S. thermocarboxydus TKU045's crude enzyme cocktail showed the selective growth-enhancing effect on a limited number of microbes such as Bifidobacterium. Similar results of the selectively growth-enhancing effect of XOSs were reported in other studies [78][79][80].

Utilization of Solid Waste from the WBP Fermentation as a Dye Adsorbent
Solid waste (SW) obtained from the WBP fermentation contained residual WBP and biomass of S. thermocarboxydus TKU045. The high dye adsorption efficiency of wheat bran and Streptomyces biomass [81][82][83][84] inspired us to utilize the solid waste from the WBP fermentation as a dye adsorbent. As shown in Figure 7a, SW showed the highest adsorption rate of Congo red (89 ± 4%), Red 7 (88 ± 1%), and Methyl blue (88 ± 1%), whereas < 30% of adsorption rate was observed for Red 6, Red 40, Green 3, Yellow 4, Yellow 5, Blue 1, and Blue 2. Thus, SW holds great potential in removing Congo red, Red 7, and Methyl blue from wastewater. As shown in Figure 7b, the maximum Congo red, Red 7, and Methyl blue adsorption rates of SW were calculated to be 100%, 89%, and 100%, respectively. Further by adding 200 mg of the adsorbent, the Red 7 adsorption rate of SW reached 94% (data not shown). The FTIR spectrum profile reveals several peaks indicating that SW is composed of numerous functional groups that potentially support its binding to the dye (Figure 7c). SW exhibited a broad and strong absorption peak at 3400 cm −1 , corresponding to the associated hydroxyl and phenolic hydroxyl groups in the cellulose. The peaks at 2930 and 2850 cm −1 were attributed to C-H vibrations. The peak at 1740 cm −1 was a carbonyl stretching vibration peak, probably due to the presence of hemicellulose, or lignin. The peak at 1648 cm −1 was attributed to -NH stretching, whereas, the peak at 1550 cm −1 indicated C-N stretching. The peak at 1460 cm −1 indicated the presence of C-H bending, and the peak at 1250 cm −1 was due to the C-O group. The broad peaks at 1160 cm −1 and 1050 cm −1 were assigned to C-O stretching from C-O-H and C-O-C bonds. The FTIR spectrum profiles of dye-loaded SWs showed a similar pattern to that of the original SW except for some slight changes in the intensity, suggesting that the adsorption mechanism of SW belonged to physical adsorption. As such, this finding provides a potential use of the waste from WBP fermentation as a low-cost and efficient sorbent.

Conclusions
In this study, a medium containing WBP as the sole carbon source was optimized for xylanase production by S. thermocarboxydus TKU045. From the WBP-containing medium, four xylanases of MW > 180, 36, 29 and 27 kDa were explored. The enzyme cocktail obtained by growing S. thermocarboxydus TKU045 on the optimal conditions was characterized. The enzyme cocktail generated xylobiose as the primary product. The xylan hydrolysate displayed DPPH radical scavenging activity and affected the growth of B. bifidum BCRC 14615. Finally, the solid waste from the WBP fermentation revealed the dye adsorption ability on Congo red, Red 7, and Methyl blue. Therefore, WBP fermentation by S. thermocarboxydus TKU045 may have potential applications in food, medicinal, and

Conclusions
In this study, a medium containing WBP as the sole carbon source was optimized for xylanase production by S. thermocarboxydus TKU045. From the WBP-containing medium, four xylanases of MW > 180, 36, 29 and 27 kDa were explored. The enzyme cocktail obtained by growing S. thermocarboxydus TKU045 on the optimal conditions was characterized. The enzyme cocktail generated xylobiose as the primary product. The xylan hydrolysate displayed DPPH radical scavenging activity and affected the growth of B. bifidum BCRC 14615. Finally, the solid waste from the WBP fermentation revealed the dye adsorption ability on Congo red, Red 7, and Methyl blue. Therefore, WBP fermentation by S. thermo-