Wheat Bran Hydrolysate Culture Medium Design for Talaromyces purpureogenus CFRM02 Pigment Production and Colour Characteristics

Wheat bran hydrolysate (WBH) in combination with carbon and nitrogen was utilized as substrate for pigment production by Talaromyces purpureogenus CFRM02. Pigment yield was significantly increased (≈threefold: OD units and ≈twofold: redness [a* value]) by xylose supplementation with WBH compared to other carbon sources, whereas 1% xylose supplementation increased pigment production (1.57 ± 0.05 OD units and 49 ± 1.62 a* value). Pigment yield was low when WBH supplemented with 0.3% nitrogen sources. However, significant increase (≈2–2.5 fold, OD units and a* value) was observed, when yeast extract (1.2%), nitrate of sodium (1.2%) and potassium (1.6%) were supplemented. Accordingly, 16 WBH media were designed by supplementing carbon and nitrogen. Interestingly, the pigment production was significantly increased (1.59 OD units and 32 a* value) in the medium supplemented with 4% carbon and 0.9–1.2% nitrogen. T. purpureogenus CFRM02 was able to co-utilize xylose, fructose and glucose in WBH medium. The CIE Lab values indicated that pigment characteristics differed significantly among the media. Apparently, T. purpureogenus CFRM02 possesses alternative gene(s) or pathway(s) for xylose metabolism and channelled towards pigment biosynthesis. Comparative results revealed that 1% xylose supplementation to WBH makes the fermentation process economically competitive for pigment production.


Introduction
Pigments have a broad range of application in food industries, that includes their utilization as additives, bioactives and colour intensifiers [1][2][3]. Undesirable health effects upon excessive consumption of synthetic pigments have encouraged research on pigments from natural sources [4]. Pigment-producing microorganisms have gained significant attention due to the existing genetic diversity [5] and various industrial applications. The production of natural pigments from microorganisms is highly desirable due to their controlled condition of growth, yield and health benefits [6].
The fungi, yeasts and bacteria are known to produce pigment molecules like carotenoids, polyketides, azophilones, melanins, quinones, flavins, prodigiosins, monascins, violacein or indigo [7,8]. Among microorganisms, the fungi like Monascus, Paecilomyces and Penicillium/Talaromyces are highly researched because of high yield of pigments [9,10]. The water solubility characteristics [11,12] of these pigments increased their demand to produce in industrial scale. Because the water solubility conditions of pigment is being crucial for application in food industries, while the presence of mycotoxins has restricted from commercialization and utilization as food colourant [12][13][14].
However, studies have identified Monascus-like red pigments produced by some species of Penicillium or Talaromyces [15]. Species of Talaromyces like T. purpurogenus, T. purpureogenus, T. marneffei, T. albobiverticillius and T. minioluteus secrete large amounts of red pigment [14,16]. The genus Talaromyces/Penicillium has gained more attention at industrial scale, as a cell factory for the process of pigment production [10,17,18]. The pigments produced by Talaromyces can also be used in textile industries for colouring the cloths like wool and silk [19]. Like Monascus pigments, Talaromyces pigments possess a wide range of biological activities like antioxidant, antibacterial. The safety efficacy was proven and considered industrially important in the food industry owing to lack of mycotoxin production [1][2][3]. The first commercial food colourant product, Arpink Red TM, produced by P. oxalicum was marketed by Ascolor Biotech (Czech Republic) [10,20]. Now, the researches have focused their attention on industrial-scale production of Talaromyces pigments using agricultural waste due to their potency to produce wide range of pigments [10,12,18]. To create the sustainable industry-scale fermentation process, identification of low-cost substrate is of particular interest to maximize pigment yields [21][22][23]. There are few reports on the production of Talaromyces pigment utilizing various agro substrates like corncob, broken wheat, orange peels, brown rice and cassava waste and sesame cake [10,18,22,24,25]. Hydrolysates derived from these lignocellulosic materials generally contain a mixture of carbohydrates pentoses and hexoses [26]. Hence, it is necessary to understand the capability of microorganism to co-utilize pentoses and hexoses in order to maximize process on the produced pigmented molecules. It is also that growth and pigment production in Talaromyces are profoundly affected by media components like carbon and nitrogen sources [10,24].
Wheat bran (WB) is the main by-product of wheat processing industry to produce flour. Annually, 150 million tons of WB is produced and only a small part is utilized [27]. Generally, WB comprises about 56.0% carbohydrate 13.2-18.4% protein. The carbohydrate in WB comprises starch, hemicellulose and cellulose [26,28], whereas there is no report on the utilization of wheat bran hydrolysate (WBH) for pigment production and deals primarily with the colour characteristics of derivatives. Hence, developing a new bioprocess requires several steps that includes designing and feasible process conditions to achieve high yields, using low-cost fermentative substrate like wheat bran and its hydrolysate. The present study aimed to utilize WBH as a substrate, supplemented with carbon and nitrogen for pigment production by T. purpureogenus CFRM02 and analysis of colour characteristics.

Material
The culture media potato dextrose agar (PDA), potato dextrose broth (PDB), peptone, yeast extract and sugars were obtained from Hi-Media Laboratories (Mumbai, India). Sodium nitrate and potassium nitrate were obtained from SRL Chemicals (Mumbai, India). The WB was obtained from the Flour Milling Backing and Confectionary Technology Department, CFTRI (Mysore, India). The particle size of wheat bran was approximately 0.5 to 0.7 mm. Previously isolated T. purpureogenus CFRM02 culture [29] was maintained on PDA at 4 °C.

Solid-State Fermentation
The solid-state fermentation media was prepared by adding distilled water to the Erlenmeyer flasks containing 10 g of WB. The moisture condition was adjusted by varying the volume of distilled water 10 ml (1:1), 20 ml (1:2) and 30 ml (1:3 w/v). These flasks were autoclaved at 121 °C for 15 min. After cooling, inoculated with 1 ml of spore suspension (~ 1.93 × 10 6 cfu) and kept for incubation (Adolf Khuner Therm-Lab, Switzerland) at 30 °C for 10 days.

Preparation of WBH
The WBH was prepared by acid hydrolysis. The WB (100 g) was added to 1 l distilled water containing 0.25% H 2 SO 4 [30]. This was autoclaved at 121 °C for 15 min. After cooling, filtered through muslin cloth and stored at − 20 °C in dark for further use. The pH of filtrate was adjusted prior to the autoclave by adding 0.1 N NaOH or HCl.

Submerged Fermentation
The 250-ml Erlenmeyer flasks containing 100 ml of culture medium were autoclaved at 121 °C for 15 min and inoculated with 1 ml of spore suspension. The initial pH of the medium was adjusted to 5.5 by adding 0.1 N NaOH or 0.1 N HCl prior to autoclave. The culture flasks were illuminated 12-h day and night condition at 30 °C for 10 days at 110 rpm on rotary shaker (Adolf Khuner Therm-Lab, Switzerland). To determine the dry biomass, whole flask cultures were poured through pre-dried (100 °C) reweighed Whatman No.1 filter paper. Retained mycelia material was washed with distilled water then ethanol until colourless and dried at 100 °C to constant weight (48 h).

Culture Media Formulation
The WBH obtained by acid hydrolysis was used for fermentation process as (i) WBH without nutrient supplementation, (ii) WBH supplemented with carbon sources, (iii) WBH supplementation adjusted to a xylose concentration, (iv) WBH supplemented with nitrogen sources, (v) WBH adjusted to nitrogen concentrations, (vi) WBH supplemented with adjusted carbon and nitrogen sources [24]. A synthetic PDB was used as a control.

Carbon Source Supplementation
Six different carbon sources (glucose, fructose, sucrose, lactose, maltose and xylose) were supplemented to the WBH. One gram of each carbon source was added to the flasks containing WBH [24]. The flask containing only WBH without carbon sources was prepared for comparison as control. The medium was autoclaved and inoculated with 1 ml spore suspension to each flask. These flasks were kept in rotary shaker incubator at 30 °C, 120 rpm for 10 days (Adolf Khuner Therm-Lab, Switzerland). Furthermore, the optimum concentration of xylose was evaluated for the pigment production. Accordingly, 1, 2, 3 and 4 g of xylose was added to the 100 ml of WBH separately. After autoclave, the medium was inoculated with 1 ml spore suspension and incubated at 30 °C, 120 rpm for 10 days (Adolf Khuner Therm-Lab, Switzerland).

Nitrogen Source Supplementation
Five different nitrogen sources such as inorganic (KNO 3, NH 4 NO 3 and NaNO 3 ) and organic (peptone and yeast extract) were used for the media formulation [24]. To each Erlenmeyer flask containing 100 ml of WBH medium, 0.3 gm of nitrogen sources was added individually. After autoclave 1 ml spore suspension was added to each flask. These flasks were kept in rotary shaker incubator at 30 °C, 120 rpm for 10 days (Adolf Khuner Therm-Lab, Switzerland). Mixture of nitrogen source consisting of yeast extract (0.3%), NH 4 NO 3 (0.4%) and NaNO 3 (0.3%) added to the WBH was recorded as N1. Further incremental addition (multiple of 2, 3 and 4) of nitrogen mixture to the WBH was recorded as N2, N3 and N4 ( Table 2). The culture medium was autoclaved and inoculated with 1 ml spore suspension.

Combined Effect of Carbon and Nitrogen
The combination of carbon and nitrogen sources is presented in Table 3 for the optimization of pigment yield [24]. The above described submerged fermentation culture conditions were followed.

Pigment Quantification and Colour Analysis
Pigment production was quantified after culture broth was filtered through Whatman No. 1 filter paper. The culture filtrates were directly used for quantification of pigment after appropriate dilution. The absorbance was recorded (UV-Visible 2450, Shimadzu Spectrophotometer, Japan) at 494 nm to quantify the pigments as absorption units (OD/ AU ml −1 ) [9]. The colour values were measured in accordance with CIE L * , a * , b * colour measuring system. The lightness (L * ), redness (+ ve a * ) and yellowness (+ ve b * ) values of culture filtrate were estimated using colour measuring system. The samples were analyzed by placing in the port of 1-inch diameter (Lab Scan XE Hunter Lab Instruments, VA, USA). Appropriate control and blank were maintained for quantification and colour analysis [1]. The chroma (C * ) and hue angle (h * ) were calculated by the following equations.

Statistical Analysis
Data were expressed as the mean ± standard deviation of triplicate measurements. Results were processed by oneway analysis of variance (ANOVA) using the SPSS statistical package (version 16.0) software. Statistical differences between means were determined by analysis of variance and Duncan's test. Differences at p < 0.05 were considered significant.

Solid-State Fermentation
It was observed after 10 days of solid-state fermentation on WB at 30 °C, T. purpureogenus CFRM02 culture with deep green spores. The production of red colour pigment was negligible under various concentrations (w/v) of moisture condition (Fig S1). Similarly, the pigment production by Penicillium sp. was less in solid-state fermentation compared to submerged fermentation [9]. Kantifedaki et al. [17] have also reported lower level of pigment production by P. purpurogenum CBS 113139 during solid-state fermentation using orange peel waste. Moreover, the medium used for fermentation process significantly affects the product yield in terms of biomass and pigment. It was confirmed that solid-state fermentation is not appropriate to produce T. purpureogenus CFRM02 pigment by utilizing WB.

Submerged Fermentation
Consequently, submerged fermentation conditions were adopted to produce T. purpureogenus CFRM02 pigment in higher quantities. It was observed that the pH plays an essential role for the production of secondary metabolites and biomass yield [31]. The fungus grown in the PDB medium at different pH condition (Fig S2) has produced more red pigment (0.362 ± 0.00 OD units/ml) at pH 6 and 8 (0.346 ± 0.03 OD units/ml). Even though the OD value (0.552 ± 0.05 OD unit/ml) of the culture broth was higher at pH 10, the pigment was not produced (Fig S2). The higher OD values of culture broth of pH 10 was due to the interference of the other metabolites produced during the growth. This was evidenced by the analysis of T. purpureogenus CFRM02 culture broth containing pigment through UV-visible spectra and CIE Lab colour values. The prominent peak at 494 nm (λ max ) was observed in the spectrum of the culture broth of pH 6 and 8 ( Fig S2). Furthermore, the CIE Lab colour values for pH 6 and 8 in terms of redness (a * value) were 16.32 ± 1.40 and 11.65 ± 1.82, respectively. The biomass production was ranging between 2.63 ± 0.2 and 3.77 ± 0.6 g/l. Even though the higher biomass yield was observed at pH 10, the pigment production was not increased significantly. Similarly, the temperature also affects the production of secondary metabolites. The higher pigment production was observed in the culture medium grown at 30 °C (Fig S3). Similar studies with Monascus spp., Penicillium spp., Talaromyces spp. the optimal pH was found to be 5.5-6.5 and optimal temperature was 24-30 °C for pigment production. The pH affected the growth and pigment production of the fungus. This may be due to the stress condition induced by the changes in the pH. Similarly due to the changes in pH level and alteration of osmolarity, the growth and pigment production in T. albobiverticillius 30548 was affected [31]. Accordingly, for further cultivation of fungus on WBH medium and other nutrient supplementation, the pH and temperature were adjusted to 6 and 30 °C, respectively.

Cost-effective Medium
The main carbohydrate in WB comprises starch, hemicellulose and cellulose. The main monosaccharides L-arabinose, D-xylose and D-glucose are released by the hydrolyzation process [26]. The hydrolysate of WB can be used for the production of T. purpureogenus CFRM02 pigment as waste raw material to develop the cost-effective bioprocess. It is also that the pigment production in Penicillium sp. was influenced by the presence of nitrogen in the medium [10]. Accordingly, the study was designed in four tasks. Initially, the WBH was evaluated with addition of individual carbon and nitrogen source after acid hydrolysis (Tables 1 and 2). The conditions of xylose levels and combination of C/N were evaluated to obtain a liquor to be used as a fermentation medium.

Supplementation of Carbon Source to WBH
Carbon source type had a significant effect on pH, biomass and pigment yield as shown in Fig. 1. The pH of the fermented broth was in the range of 4.5 to 6.5. The higher biomass observed in the fructose (15.5 ± 1.3 g/l) containing media compared to other sugars, whereas the significant increase in the pigment (0.945 ± 0.14 OD units/ml) production was observed in the xylose medium (Fig. 1A), while there was no significant difference in the pigment production in other carbon-containing medium (Fig. 1A). Similarly, the highest redness a‫٭‬ value was found in basal medium supplemented with 1% xylose (49.55 ± 1.62), whereas the least was in sucrose (5.30 ± 1.13). The production of pigments by T. amestolkiae DPUA 1275 was explained utilizing glucose as carbon source and careful selection of nitrogen source [32]. Carbon source addition (glucose, fructose, sucrose, xylose, maltose and lactose) to the WBH basal medium significantly affected the biomass yield (Fig. 1). The biomass yield in the basal medium containing the carbon sources, except xylose (10.90 ± 1.4 g/l), was significantly higher than the basal medium (10.44 ± 0.5 g/l) alone, but with higher pigment yields. It is due to the fact that simple sugars have induced more of the growth and inhibited the secondary metabolite production [10]. In all the fermented broth containing different carbon source, the L*, a* and b* values were positive, indicating yellowness and redness hue (Table 1 and Fig S4). The lightness values were in the range of 57-88. The hue angle of the pigment produced was in the range of 66-78 indicating the minor shade changes in the red colour characteristics of the pigment (Table 1). But the significant difference was observed in the chroma value of the pigment produced in the medium containing xylose (72.61 ± 1.81). This revealed the saturation intensity of the pigment produced in the 1% xylose medium (Table 1) and correlated with the UV-visible spectrum colour appearance in the culture flask (Fig S4).

Supplementation of Xylose to WBH
Since 1% xylose has significantly induced the pigment production, further to observe the effect of higher concentration of xylose, the concentration was gradually increased to 4%. The results indicated that the increased concentration of xylose was inversely proportional to the pigment production (Fig. 1B). One gram of xylose has shown significant increase in red pigment production in the medium (49.55 ± 2.29) and least in 4 g xylose composition (3.83 ± 0.78). Similar results were reported that higher concentration of xylose did not induce the pigment production in T. atroroseus GH2 utilizing the corn cob hydrolysate [30]. However, the higher concentration of xylose has positively affected the growth of fungus (Fig. 1B). The biomass yield was highest in 2 g (14.9 ± 0.7 g/l) and 4 g xylose composition (14.5 ± 1.6 g/l), whereas the least biomass yield was in 1 g xylose composition (9.15 ± 0.3 g/l). The medium containing 1 g of xylose has shown maximum absorbance (1.565 ± 0.07) at 494 nm indicated the higher pigment yield while less pigment yield was observed in the medium containing 4 g xylose (Fig S5). It was also observed that the colour characteristics of the pigments were significantly affected by varying xylose (1-4%) concentration, even though the L*, a* and b* values were positive, indicating yellowness and redness ( Table 1). The a* value (redness), 50 to 4 and chroma intensity, 73 to 32 were significantly decreased and inversely proportional to the xylose concentration ( Table 1). The increase in b* value (yellowness), 31 to 53 and hue angle 47° to 83° towards 90° indicted that higher concentration of xylose induced yellow pigment production.
The influence of xylose over the glucose on pigment production was observed by transferring the T. atroroseus GH2 to a medium with xylose as carbon source than glucose [30]. The production of pigment was reported negligible or low in P. purpurogenum, when cultured using orange peel waste. The orange peel medium mainly contained glucose, fructose and sucrose. Thus, carbon composition could be the reason for the low production of pigment [17]. Addition of xylose to waste stream cellulose medium induced higher production by P. resticulosum [24]. The higher pigment production was obtained when it was grown in xylose rather than in favourable carbon sources. Apparently, T. purpureogenus CFRM02 contain alternative gene(s)/pathway(s) for metabolizing xylose. Therefore, most of the assimilated xylose might be channelled towards the biosynthesis of pigment rather than towards central carbon metabolism.

Supplementation of Nitrogen to WBH
It was reported that the red pigment produced by Talaromyces is similar to Monascus purpureus pigments. The yield and characteristics of the pigments were affected by the nitrogen source in the growth medium [33]. Accordingly, the effect of nitrogen source was evaluated. The pH of the fermented medium was in the range of 5-7. The individual nitrogen source added to the basal medium significantly affected the yield of biomass (Fig. 1C). The significant increase in the biomass was observed in the medium containing NaNO 3 (25.91 ± 5.9 g/l) compared to other nitrogen sources. The biomass yield was less in the medium containing peptone and NH 4 NO 3 , while the yeast extract positively influenced the biomass yield (20.78 ± 2.0 g/l). Even though the biomass yield was significantly affected by type of nitrogen sources, the pigment production in all the nitrogen-containing medium (inorganic or organic) remained almost the same (Fig. 1C). Similar type of behaviour was observed by Penicillium sp. for pigment production utilizing waste stream cellulose as culture medium [24]. The nitrogen sources added individually to the medium did not affect the colour characteristics (Table 2). In all the fermented broth containing different nitrogen source, the L*, a* and b* values were positive, indicating yellowness and redness ( Table 2). The lightness values were 85 to 88. The hue angle of the pigment produced in the medium was in the range of 74 to 84° indicating the minor shade changes in the red colour characteristics of the pigment (Table 2), whereas the chroma value was ranging from 28 to 36.

Supplementation NaNO 3 and Yeast Extract to WBH
Since NaNO 3 and yeast extract have positively affected the growth, these two nitrogen sources were selected for further process of pigment production along with KNO 3 for potassium ion. Interestingly, the pigment production was increased, when these nitrogen sources were added to the medium (Fig. 1D). Furthermore, significant increase in biomass and pigment yield was also observed by increasing the concentration of these nitrogen (0.3 to 0.12%) sources (Fig. 1D). The nitrogen sources added to the medium influenced colour characteristics of the pigment. In all the fermented broth containing different nitrogen source, the L*, a* and b* values were positive, indicating yellowness and redness (Table 2), whereas the b* values (71 to 83) were higher compared to a* values (13 to 27). These results indicted the nitrogen sources influence more yellow pigment production compared carbon sources ( Table 2). There were significant changes in the colour characteristics of the pigment as the nitrogen concentration increased from 0.3 to 1.2%. The lightness values ranged from 59 to 68. The hue angle was ranging from 72 to 80° indicating production of orangish red colour characteristic (Table 2).

Combined Effect of Carbon and Nitrogen
It was reported earlier that the type of pigment or hue produced by ascomycetous filamentous fungi might vary within the same species by growing in different media [34]. Herein also, the culture medium influenced the pigment yield and characteristics of T. purpureogenus CFRM02. Hence, the sixteen different WBH mediums were prepared for the cultivation of T. purpureogenus CFRM02 by addition of carbon and nitrogen (Fig S6). Fermentation broths were used for pigment estimation and analysis of colour characteristics. Interestingly, it was observed that the significant increase in the higher pigment production in the medium containing high xylose concentration (Fig. 2), whereas the production of pigment was higher in the WBH medium containing 1% xylose only ( Fig. 1A and B). Similarly, the biomass yield was more when high concentration of carbon and nitrogen was added to the medium (Fig. 2). The values of L*, a* and b* were all positive, indicating yellowness and redness ( Table 3). The significant difference was observed in the L*, a* and b* values. The lightness values were ranging from 52 to 80. The a* values were ranging from 7 to 33 indicating the light red to middle red intensity. The b* values were ranging from 41 to 75 indicating middle reddish-orange to deep reddish-orange pigment. The a* value was higher (33 ± 3) in the medium containing 1-2% carbon and nitrogen sources as presented in Table 3, while the hue angle was ranging from 65 to 82° corresponding to Fig. 2 Effect of the concentration of xylose (1-4%) and combination of nitrogen sources on the pH, pigment and biomass production. The N and C correspond to the nitrogen and carbon at different concentrations in the WBH medium. The N nitrogen sources, N1 = 0.3%YE, 0.4% KNO 3 , 0.3% NaNO 3 , N2 = N1 X 2, N3 = N1 X 3 and N4 = N1 X 4. The C1-C4 correspond to 1-4% of xylose concentration. The mean values within the bar and lines with different superscript were significantly (p < 0.05) different light red and middle orange. This indicated changes in the hue of the pigment produced from red to orange to yellow in the culture medium of the T. purpureogenus CFRM02. The chroma values varied in a range from 42 (grey) to 78 (bright) according to the medium used for cultivation. On the basis of the hue angle and chroma values, the pigments derived with carbon and nitrogen source had various shades of red. The higher chroma values were observed in the higher concentration of the xylose that suggested the brightest colour hue. These results suggested that higher chroma values can be observed by increasing the concentration of the xylose ( Table 3). The colours varied according to the concentration of the carbon and nitrogen source used in the fermentation medium.
Studies have reported that organic nitrogen sources are better than inorganic ones to promote pigment production by Talaromyces/Penicillium [32]. However, in this study, significant changes were not observed in the pigment production and colour characteristics when supplemented with organic and inorganic nitrogen ( Table 2). There are no more data on the CIE Lab colour characteristics of T. purpureogenus CFRM02 especially from the prospective of food use. Moreover, our previous studies on the toxicity evaluation of the T. purpureogenus CFRM02 pigment confirmed its nontoxic effect [1,3]. The colour hues of T. purpureogenus CFRM02 pigment was similar to colour hues of the red and orange Monascus pigments [33]. This signifies the contribution of the present findings and the reddish-orange hues of T. purpureogenus CFRM02 pigments can be worth to consider in food use.

Conclusions
This study showed the promising use of WBH for the production of pigments by T. purpureogenus CFRM02. The WBH supplemented with xylose (1%) was the best medium for pigment production, comparable to the obtained with a synthetic medium, whereas the colour characteristics with the WBH medium differed from the medium used as a control. In general, the evidence from this study validates that carbon and nitrogen composition significantly affects the microorganism growth and the pigment production by T. purpureogenus CFRM02. The carbon and nitrogen sources have influenced the L*, a*, b*, chroma and hue angle values. Characterization of agro-industrial wastes is crucial in order to induce the microorganism's secondary metabolism and to control the pigmented molecules production. The ability of T. purpureogenus CFRM02 to grow and produce pigments using WBH medium makes it a promising and economically competitive for large-scale fermentation process.

Supplementary Information
The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s12155-021-10368-z. Table 3 The combined effect of carbon and nitrogen concentration supplementation to WBH medium on T. purpureogenus CFRM02 pigment characteristics and the CIELAB value The N and C correspond to the nitrogen and carbon at different concentrations in the WBH medium The N nitrogen sources, N1 = 0.3%YE, 0.4% KNO 3 , 0.3% NaNO 3 , N2 = N1 X 2, N3 = N1 X 3 and N4 = N1 X 4 The C1-C4 correspond to 1-4% of xylose concentration The mean values within the column with different superscript were significantly (p < 0.05) different L lightness a value b value h hue angle c chroma