Cost-effective production of stable prodigiosin by Serratia marcescens UCP 1549 and application in soap coloring

Prodigiosin is a microbial red pigment, mainly produced by the bacterium Serratia marcescens, considered as a promising antimicrobial, immunosuppressive and antiproliferative compound. However, its industrial commercialization is still limited because of the high cost of production mainly due to the use of expensive substrates. Hence, this work aimed to the sustainable production of prodigiosin by S. marcescens UCP 1549, using corn bran as alternative and low-cost substrate, and its application in soap coloring. According to the results, both bacterial growth and red pigment production occurred, achieving 7.24 g/L and 1.68 g/L of biomass and pigment yield, respectively. Positive result in the presumptive test indicated red pigment as prodigiosin, which was confirmed by UV-Visible spectrophotometry (maximum absorbance peak at 535 nm), TLC (Rf 0.9) and the functional groups identified by FTIR spectroscopy. Prodigiosin showed color stability at different pH values and NaCl concentration. The application of pigment in soap coloring was effective, suggesting its promising potential in the cosmetics industry as natural colorant. The results showed the biotechnological potential of S. marcescens UCP 1549 in the biotransformation of corn bran into prodigiosin, allowing a more economically and competitively industrial bioprocess.


Introduction
Worldwide demand for microbial pigments has increased in recent years due to their better biodegradability and compatibility with the environment, as well as less toxicity and allergic reaction, when compared to those of synthetic origin (Venil, et al., 2020;Nawaz, et al., 2021;Jeong, et al., 2022). Therefore, their biotechnological production has risen exponentially, aiming at the enormous potential for application in diverse areas, such as the food, pharmaceutical, cosmetic and textile industries (Sánchez-Muñoz, et al., 2020).
In this context, prodigiosin is a natural red pigment, that can be produced by various bacterial species, standing out Serratia marcescens as the main microorganism producing (dos Santos, et al., 2021;Sebastian, et al., 2022). It is a secondary metabolite with tripyrrole structure and chemical formula C20H25N30, that exhibits a wide spectrum of biological properties (Araujo, Fukushima, & Campos-Takaki, 2010;Liu, et al., 2021). Prodigiosin is well-known as a promising therapeutic agent due to its antimicrobial, antimalarial, immunosuppressive and antitumor activities (Lapenda, et al., 2015;Yip, et al., 2019).
Despite of its promising applications, the large-scale production of prodigiosin remains limited due to the high cost of production, mainly caused by the use of expensive conventional substrates. Hence, several researches have been addressed to obtain prodigiosin using industrial by-products and waste, as alternative sources of carbon and nitrogen, being a promising and cost-effective strategy (Elkenawy, et al., 2017;Arivizhivendhan, et al., 2018;Nguyen, et al., 2020;Nguyen, et al., 2022).

Microorganisms and preparation of inoculum
The bacterium S. marcescens UCP 1549, isolated from semi-arid soil and identified by Araujo, Fukushima, & Campos-Takaki (2010), was kindly provided by the Culture Collection UCP -Catholic University of Pernambuco (Recife-PE, Brazil), registered to the World Federation for Culture Collections (WFCC) under number 927. This strain was maintained in the solid Luria Bertani (LB) medium (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L and agar 15 g/L) at 5 °C.
Stored culture of S. marcescens was transferred first to LB medium and incubated for 18 h at 28ºC. Then, two colonies were transferred to 50 ml of LB broth and incubated at 28ºC and 150 rpm in an orbital shaker. Once the optical density at 600 nm reached 0.8-1.0, this culture was used as inoculum (Montero- Rodríguez, et al., 2018).

Agro-industrial substrate
In this study, corn bran was used as low-cost substrate, previously obtained in a local market in city of Recife (Pernambuco, Brazil). It was sieved using Tyler series sieves -mesh 16 and 32-to obtain a fine and homogeneous powder, in order to facilitate dissolution in the culture medium.

Prodigiosin production
Fermentation was carried out in 250 ml Erlenmeyer flasks containing 100 ml of saline solution (KH2PO4 3 g/L, K2HPO4 7 g/L, MgSO4 .7H2O 0.2 g/L, (NH4)2SO4 1 g/L) and 1 % corn bran, with pH adjusted to 7. The flaks were sterilized in an autoclave at 121°C for 15 min, inoculated with 5 % of the pre-inoculum of S. marcescens grown in medium LB and then incubated at 31ºC and 100 rpm in an orbital shaker for 72 h.

Determination of biomass yield
After 72 h of cultivation, the fermentation was centrifuged at 10000 g for 15 min. The supernatant was discarded and the pelletized cells were washed twice with distilled water by centrifugation at 10000 g for 20 min. Then the biomass was frozen, lyophilized and quantified as g/L.

Purification and identification of red pigment
Preliminary identification of the crude red pigment was performed after its solubilization in 95% ethanol. The colored solution was divided into two portions: one portion was acidified with a drop of concentrated HCl and the other was alkalized Research, Society and Development, v. 11, n. 4, e9711427078, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org /10.33448/rsd-v11i4.27078 4 with a drop of concentrated NaOH solution (Gerber & Lechevalier, 1976). The red or pink color in acidic condition and the yellow or brown color in alkaline condition confirmed a positive presumptive test for prodigiosin (Sathishkumar & Aparana, 2014;Mansi & Shah, 2015).
Then, the red pigment was dissolved in 3 ml of methanol and subjected to purification by column exclusion chromatography (column 22 x 1 cm) filled with Sephadex LH-20 activated at 800 °C for 1 h, as an absorbent. The elution was carried out by the solvent system chloroform: methanol (1:1, v/v) and then modified to chloroform: methanol: acetone (4:2:3, v/v), aiming maximum removal of impurities (Lins, et al., 2014). The red fraction was collected and analyzed by UV-Vis spectrophotometry, and the absorbance range in the range 400-700 nm was determined. Prodigiosin production was confirmed by the presence of a maximum absorbance peak at 535 nm (Araujo, Fukushima, & Campos-Takaki, 2010).
In addition, the red fraction was subjected to thin layer chromatography (TLC), using aluminum plates coated with silica gel, which after application of the sample was placed in a glass vat containing the chloroform: methanol solvent system: (Araujo, Fukushima, & Campos-Takaki, 2010;Priya, et al., 2013). The retention factor (Rf) was calculated according to the formula Rf: distance travelled by the compound/ distance travelled by the solvent front and then, it was compared to the standard prodigiosin Rf referred in the literature (Lins, et al., 2014).
The purified red pigment was submitted to Fourier transform infrared (FT-IR) spectroscopic analysis on the Shimadzu equipment, IR-TRACER 100, using an attenuated total reflection (ATR) accessory consisting of a mixed "diamond/ZnSe" crystal. The peaks obtained were compared with the literature to confirm the presence of prodigiosin

Pigment stability
Color stability of the prodigiosin produced by S. marcescens was investigated following the methodology proposed by Perumal, et al. (2009) and Velmurugan, et al. (2011), with modifications. Briefly, glass test tubes containing 10 ml of the ethanol extract of prodigiosin were adjusted to pH 2, 4, 6, 8, 10, 12 and 14, homogenized for 10 min and the absorbance measured using a UV-visible spectrophotometer. Another set of tubes containing 10 ml of extract were amended with 0.1, 0.2, 0.5, 1 and 5% (v/v) salt solution (NaCl) and kept at rest for 1 h to determine absorbance. Relative color stability (%) was calculated according to Equation 1.

%S = (A1 x 100)/ A0
( 1) where A0 is pigment absorbance before treatment and A1 is absorbance after treatment. Absorbance of the pigment was measured by spectrophotometry at 535 nm.

Application of prodigiosin in soap coloring
The potential application in soap coloring of the pigment produced by S. marcescens was investigated. For this, 40 g of white soap were fractionated and melted over a low fire with 50 ml of distilled water in a container. When fully melted, 0.3 g of the pigment diluted in water was added for 3 min. Then, the mixture was placed in a mold and allowed to cool to room temperature until it solidified (Meenakshi et al., 2018). The test was done in triplicate.

Production of biomass and pigment
Several synthetic media have been widely used for the growth of prodigiosin-producing microorganisms (Bhagwat & Padalia, 2018;Yip, et al., 2019). However, the high cost of conventional substrates justifies the need for the formulation of cost-effective production medium that allows the bacterial growth and the biosynthesis of this useful pigment. In this sense, low-cost substrates such as brown sugar, cassava wastewater and ram horn peptone have been previously used (Araujo, Fukushima, & Campos-Takaki, 2010;Aruldass, et al., 2014;Kurbanoglu, et al., 2015).
In this study, the use of corn bran reached a biomass yield of 7.24 g/L, which was higher than that obtained by the same strain in medium containing corn steep liquor (3.47 g/L) and cassava wastewater (3.65 g/L) (Araujo, Fukushima, & Campos-Takaki, 2010). In addition, the pigment yield was 1.68 g/L, a result similar or superior to that previously obtained by S. marcescens strains in different agro-industrial based medium (Table 1).

Relative stability of prodigiosin
The potential application of natural pigments in various industrial fields depends on the stability against variables or extreme conditions. In this sense, prodigiosin produced by S. marcescens UCP 1549 was subjected to several physicalchemical treatments and the results are shown in Figure 4. Source: Authors.
As evidenced, prodigiosin showed excellent color stability when subjected to different values of salinity ( Figure 4B).
However, in the case of pH, it showed less stability in alkaline conditions (pH 8-12), where there was a color change from pink to yellow, and consequently, a decrease in absorbance at 535 nm ( Figure 4A). These results corroborated that obtained previously in the presumptive test, where in basic pH there was also a color change of the ethanol extract of the red pigment ( Figure 1), confirming the presence of prodigiosin. Yuan, et al. (2005) obtained similar results when they verified the stability of prodigiosin produced by Pseudomonas sp. after pH 2 and 5, while the pigment showed instability in alkaline conditions. (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org /10.33448/rsd-v11i4.27078 8 The stability of pigments of natural origin such as the prodigiosin produced by S. marcescens UCP 1549, confirm its potential for application as an alternative to synthetic dyes, in several industrial processes, where they are generally subjected to adverse conditions, such as high temperature and acidic pH.

Application of prodigiosin in soap coloring
There is a growing interest in the use of natural dyes in various industrial areas due to the prohibition on the use of some synthetic dyes with demonstrated carcinogenicity of the precursor or product, as well as the toxic effect of their industrial residues on ecosystems. The lower toxicity and allergic reactions of natural pigments make them more compatible with human use, due to the reduction of exposure to harmful chemicals (Venil, Dufossé & Devi, 2020;Nawaz, et al., 2021).
In this study, prodigiosin produced by S. marcescens UCP 1549 in low-cost medium containing corn bran was applied in soap coloring, as shown in Figure 5. The effectiveness of the coloring demonstrates the potential application of this pigment as a natural dye in the cosmetic industry. Previously, Rodríguez (2017) demonstrated the effectiveness of prodigiosin produced by this strain, in textile dyeing and candle coloring.

Conclusion
In this study, Serratia marcescens UCP 1549 demonstrated its ability to use corn bran as the sole carbon source for the cost-effective production of prodigiosin. The pigment demonstrated color stability at different values of pH and NaCl concentrations, as well as effectiveness in soap coloring, confirming its potential application in the cosmetic industry.
However, some analyzes will soon be carried out in order to confirm the antimicrobial properties as well as the low toxicity of soaps colored with prodigiosin, adding value to the cosmetic and personal hygiene products industry.