Thermal stability of natural pigments produced by Monascus purpureus in submerged fermentation

Abstract The major aim of the current study was to assess thermal stability of red pigments produced by Monascus purpureus ATCC 16362/PTCC 5303 in submerged fermentation. Natural pigments were produced by Monascus purpureus using stirred tank bioreactor. Stability of Monascus purpureus pigments was assessed under various temperature (50.2–97.8°C), salt (0%–2.5%), and pH (4.3–7.7) values. Thermal degradation constant and half‐life value of the red Monascus purpureus pigments were analyzed using response surface methodology followed by a first‐order kinetic reaction. Results of this study showed that pH, temperature, and salt content could affect red color stability of Monascus purpureus. The pigment showed various stabilities in various thermal conditions (temperature, salt, and pH). At high temperatures, degradation constant of the red pigments increased with decreasing pH, revealing that the Monascus red pigment was destroyed at lower pH values and salt could affect stability of the red pigments at lower temperatures.

Most strains belong to three species of M. pilosus, M. purpureus, and M. ruber (Pan and Hsu, 2014). For centuries, fermented rice products have been used in Asia and Indonesia as dietary staples and food (Domínguez-Espinosa and Webb, 2003). Monascus pigments (Mps) are used for controlling blood cholesterol, diabetes, and obesity and preventing cancers (Hong et al., 2008;Lee et al., 2011;Lee & Pan., 2012;Shi & Pan, 2011). These pigments also include antioxidant, antimicrobial, and antifungal characteristics. Furthermore, Mps are used as nitrite or nitrate replacements of meats (Estiasih et al., 2020;Feng et al., 2012;Nateghi et al., 2020;Seong et al., 2017). The most important pigment produced by Monascus sp. is red. This pigment contains monacolin K, which includes several characteristics in red koji (Lee et al., 2001).
Naturally, Mps are dissolved in water and fat (Carvalho et al., 2005), stable against pH 2-10 and high heats and usually affected by the production processes (Tseng et al., 2000). Monascus pigments produced by Monascus ruber include moderate stability when exposed at low pH or high temperatures (Silveira et al., 2013).
Thermal processing is one of the most important methods of food preservation. Extreme thermal processes such as pasteurization, sterilization, and baking can affect stability of Mps (Velmurugan et al., 2011). Monascus pigments might tolerate pasteurization conditions. However, further studies are necessary to verify the interaction of Mps with food components on their stability (Silveira et al., 2013).
Degradation constant (D c ) is an important factor to show instability of pigments during processing (Fernández-López et al., 2013;Silveira et al., 2013;Vendruscolo et al., 2013). Design of kinetic models for Dc of Mp under processing conditions is necessary to prepare food products. However, availability of information on reaction kinetics is quite limited and modeling of the degradation kinetics for Mps in temperature ranges of heat processing is necessary. Based on the above-mentioned facts, the aim of the current study was to investigate effects of salt content, temperature, and pH on the stability of Mps produced by Monascus purpureus ATCC 16362 in submerged fermentation using response surface methodology (RSM).

| Preparation of fungal strains
Monascus purpureus ATCC 16362/PTCC 5303 was provided by the

Microbial Collection Center of Iran Scientific and Industrial Research
Organization, Tehran, Iran. Mycelia were cultured on yeast powdersoluble starch (YpSS) and resuspended every 30 days in fresh culture media and incubated at 30°C for 7 days. These were stored at 4°C until use (Keivani et al., 2020).

| Preparation of spore suspensions
To prepare spore suspensions, the fungal strain was cultured on YpSS agar media and incubated at 30°C for 7-10 days and then rinsed with 5 ml of sterile distilled water (DW). Fungal cells in suspensions were counted using light microscope and cell-counter slides (Keivani et al., 2020).

| Preparation and inoculation of seed media
Seed culture media were prepared with specific compounds at pH 6 and sterilized using autoclave at 121°C for 15 min. Spore suspensions were used for the inoculation of seed media and incubated (Raiman Zist Fanavar, Iran) at 30°C for 30 hr at 120 rpm (Keivani et al., 2020).

| Preparation and inoculation of the major culture media
Using seed culture media as the final media of 10 5 spores, inoculation was carried out in the major media optimized at 30°C for 21 days at 40 rpm (pH 4) using stirred tank bioreactor (MiniX, Yekta Tech, Iran) (Keivani et al., 2020).

| Extraction of Monascus red pigments
Briefly, Mps were separated from the biomass using Whatman No.
1 filter papers. The mixing ratio of filtered materials to ethanol was 10:100. This was ultrasounded for 30 min using ultrasound device and transferred to shaking incubator for 1 hr at 180 rpm to extract red pigments (Aruldass et al., 2018).

| Kinetic calculations
The first-order kinetic model could calculate heat Dcs. This parameter was expressed using Equation (1), and the regression lines were achieved by plotting logarithms of the remaining pigments. (1) where A was the absorbance (UA 500nm ), t was the time (h), and D C was the Dc (h −1 ). If boundary conditions in this formula linearized to A = A 0 at t = 0 and A = A when t = t, results are shown in Equation (2).
where A 0 was the initial absorbance (UA 500 nm ). Half-life value (t 1/2 ) was calculated using various values of D C as follows: where A/A 0 was 2.
Effects of the significant independent variables were assessed in terms of D C (Y 1 ) and t 1/2 (Y 2 ) of the Mps using RSM at a temperature range of 50.2-97.8°C, pH range of 4.3-7.7, and salt content of 0%-2.5%.
The CCD with 20 experiments (14 axial points and six replicates at the center point, α = 1.7) was used (Table 2). Response model was expressed using coded variables (Vendruscolo et al., 2013). The Mp solutions were heated for 2 hr using water bath (Table 2), and data were collected within 15-min time intervals for the calculation of Dc and t 1/2 .
Results presented in terms of A/A 0 showed changes in Mp absorption. Since the graph was logarithmic, ln (A/A 0 ) was used to linearize the graph. Based on Table 2, the highest Dc was linked to treatment 10 (97.8°C, pH 6, 1.27% salt) and the lowest Dc was associated with treatment 3 (60°C, pH 7, 0.55% salt). Regression analysis was used first for each isothermal experiment.  Table 2. Clearly, Mp degradation increased with increasing temperatures. Furthermore, relationships revealed that degradation of the pigments was followed by a kinetic model with a good regres-  Figure 2). This heat-induced pigment degradation has been reported in red and orange pigments extracted from M. ruber (Vendruscolo et al., 2013).
Stability of the red pigments of Monascus added to rice has studied and achieved similar results in this model. Decreased color stability due to high temperatures is expected in most natural colors, but changes in pH can include various effects on natural colors (Priatni, 2015). The 3-D diagram showed that at 88°C, the Dc of aqueous of Mps was more stable at 0.55 compared to 2% salt, although, at 60°C, the Dc of aqueous of Mps was more stable at 2 compared to 0.55% salt (Figure 3). Furthermore, red pigments of salt-containing solutions of red pigments of Monascus sp. could affect the pigment stability. Red pigments of Monascus sp. were more sensitive to pH, compared to that yellow and orange pigments were. Greater stability of the yellow pigments could occur due to two reasons. The first reason was that the yellow pigments were less susceptible to red pigments and its true stability and the second reason was that degradation of yellow pigments and production of yellow compounds because of the color degradation caused further persistence of the yellow pigments (Francis, 1987).
Half-life value (t 1/2) assessments showed stability of the Monascus red pigments under process conditions. The t 1/2 of Mps was inversely linked to Dc. Half-life value of Mps was another factor to show stability of Monascus red pigments. Values of t 1/2 were calculated as follows: where t 1/2 was the half-life value, A was the variable temperature, B was the variable pH, and C was the salt content. As  shown in Equation 5, temperature and salt content were linearly linked to negative coefficient of decrease, pH was linearly linked to positive coefficient of additive effects, and temperature interacted with salt content of additive effects. Moreover, square of the three factors was individually linked to negative coefficients, including decreasing effects on t 1/2 of the Mps.
The variance analysis showed significant effects of temperature, pH, and salt content on the t 1/2 of Mps (p < .01), insignificance of lack of fit (p = .9325) and R 2 of .8949 (Table 3). The most effective independent variable on t 1/2 was temperature.
Effects of temperature linearly and square of temperature, salt, and pH on t 1/2 of Mps were significant; adjusted R 2 was .8204, and predicted R 2 was .7289. The coefficient of variation in this model was 11.06%.

ACK N OWLED G M ENTS
The authors would like to thank Plant Improvement and Seed Production Research Center, Isfahan (Khorasgan) Branch, Isfahan, Islamic Azad University, Isfahan, Iran.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

I N FO R M E D CO N S E NT
Written informed consent was obtained from all study participants.

TA B L E 3
Effects of temperature (A), pH (B), and salt (C) and their interactions on Dc and t 1/2 of the red pigments produced by Monascus purpureus using RSM

DATA AVA I L A B I L I T Y S TAT E M E N T
The raw/processed data required to reproduce these findings cannot be shared at this time as the data also form part of an ongoing study.