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1 Available online freely at Bioscience Research Print ISSN: Online ISSN: Journal by Innovative Scientific Information & Services Network RESEARCH ARTICLE BIOSCIENCE RESEARCH, (3): OPEN ACCESS The effect of mung bean (Phaseolus radiatus L.) sprout on lovastatin and red pigments production of red mold rice Elok Zubaidah, Lestari Puji Astuti and Teti Estiasih * Department of Food Science and Technology, Faculty of Agricultural Technology, Brawijaya University, Jl. Veteran, Malang, Indonesia. *Correspondence: teties@yahoo.co.id; teties@ub.ac.id Accepted: 18 May 2018 Published online: 13 Aug Red mold rice, known as angkak, is a natural pigment product of Monascus purpureus fermented rice. Red mold rice produces lovastatin, a health beneficial compound, and yellow, orange, and red pigments. Mungbean sprout is supposed to enhance lovastatin and red pigments production during red mold rice fermentation. This study aimed to determine the effect of adding mung bean sprout at different concentration level (0, 2.5, 5, 7.5, 10, 12.5 and 15% w/w) on red pigments and lovastatin production of red mold rice. Water solubility at different temperature (25, 60, 80, 100 C) and stability of red pigments toward temperature and ph were also evaluated. The result showed that red pigments and lovastatin production increased to mung bean sprout concentration 5% (1.92 AU/g), but higher concentration did not show further increase. In high mung bean sprout concentration, the high availability of protein possibly stimulated the growing cell and reduced the production of pigments. Similarly, lovastatin production increased to concentration of mung bean sprout 5% (23.46 mg/100 g) and then decreased in higher concentration. Lovastatin production was supposed to relate to micronutrient contents and amino acids in mung bean sprout. Increasing temperature enhanced the water solubility of red pigments. Red pigments were unstable at high temperature and more stable at ph 7 than ph 3. Keywords: lovastatin, mung bean sprout, red mold rice, red pigments INTRODUCTION Red mold rice (angkak) is a product of rice fermentation using Monascus sp especially M. purpureus. Angkak is originating from China and has been widely used as a natural food coloring for fish, china cheese, red wine, and sausage. Beside red pigment, fermentation of rice by Monascus purpureus also produces many secondary metabolites such as pigments, lovastatin, mevinolin, citrinin, and vitamins (Panda et al. 2010; Feng et al. 2012). Angkak pigment also has antioxidant activity with its activity depends on red intensity of the pigments (Chairote et al. 2009). Monacolin K (lovastatin) from Monascus purpureus competitively inhibits a rate limiting enzyme, HMG-CoA reductase, which catalyzes the reduction of HMG-CoA to mevalonate during cholesterol biosynthesis (Seema, 2016). Lovastatin also exhibits the ability to inhibit mammary carcinoma cells (Herman et al. 2002), potential treatment of autoimmune (Bartosikpsujek et al. 2010)), protective effect on endothelial cells (Chu et al. 2016) by oxidized LDL (Ma et al. 2009), heals osteoporosis (Ibrahim et al., 2014), and prevents inflammatory response (Chauhan et al. 2004). Beside lovastatin, Monascus purpureus also produces some pigments including two yellow pigments, monascin and ankaflavin; two orange

2 pigments, rubropunctatin and monascorubrin); and two red pigments, rubropunctamine and monascorubramine were identified, which are well-known as Monascus pigments fundamental compound types. Solid-state fermentation and liquid-state fermentation are two major processes for Monascus pigments production. The advantage of solid state fermentation by using rice is it can be directly used as food colorants. Some factors affecting the growth of Monascus purpureus on rice and pigment production such as carbon source, nitrogen source, ph, temperature, minerals, oxygen partial pressure, other microorganisms, etc (Feng et al., 2012). These pigments exhibit antimicrobial activity (Vendruscolo et al. 2016). Fermenting medium is a critical factor of Monascus purpureus growth that also affects the production of pigment. The metal ions Zn 2+ and Mg 2+, and certain amino acids (glycine, leucine, tryptophan) might improve transferring coefficient of carbon sources. Zn ion also probably act as a co-factor for enzyme involved in the carbohydrate and nitrogen metabolism of Monascus spp (Feng et al., 2012). Therefore, enrichment of rice as fermenting media is important to stimulate the production of Monascus pigment and other secondary metabolites. Combination of rice with other substrate to enrich amino acid and mineral might increase the production of red pigments and lovastatin. One source of amino acids is mung bean sprout. Mung bean is the sources of carbohydrate, protein, Ca, P, and niacin (Goyal et al., 2014). Germination of mung bean increases the level of amino acids due to proteolytic cleavage of proteins (Tang et al., 2014). Dahiya et al., (2014) showed that mung bean-based products had significant amount of Zn. The aim of this study is to evaluate the effects of different concentration of mung bean sprout on red pigments and lovastatin production and, also evaluate red pigments solubility and stability at different temperature and ph. MATERIALS AND METHODS M. purpureus culture preparation Culture of M. purpureus was obtained from Laboratory of Food Microbiology, Faculty of Agricultural Technology, Brawijaya University (East Java, Indonesia). M. purpureus culture was maintained on Potato Dextrose Agar slant and sub-cultured monthly. M. purpureus starter was prepared by inoculating M. purpureus culture stock on Potato Dextrose Agar (PDA) slant and incubated for 7 days at 30 ºC. Mung bean germination Mung bean was soaked in water (1:1) for 12 hours. After draining, the beans were germinated in a container covered by wet dark cloth. The germination was conducted for 36 hours with watering for every 4 hours. Mung bean sprout is crushed with a mortar and mixed with rice as main fermenting medium. Fermenting medium preparation Rice (IR36 variety) was soaked into distilled water (1:1) for 8 hours and after draining, it was put into jars with weight of 40 g for each jar. Mung bean sprout was added into each jar as much as 0; 2.5; 5; 7.5; 10; and 12.5 % (w/w) and thoroughly mixed. The jars were sterilized at 121 ºC for 15 minutes. After cooling, the medium was inoculated by 8 ml of starter culture containing 4x10 7 spores/ml and then incubated at 30 ºC for 14 days. At the end of fermentation, the red mold rice (angkak) was dried in an oven for 12 hours at 60 ºC. Analysis of red pigment intensity This analysis was referred to method of Dikshit and Tallagaprada (2016). Red mold rice powder (0.05 g) was added to 10 ml 96% methanol in a glass tube to extract the pigments. Extraction was carried out for 24 hours in a shaker, and afterward the mixture was vortexed and filtered by filter paper. The absorbance of the filtrate was measured using UV-vis spectrophotometer (Shimadzu) at 500 nm as the maximum wavelength for red pigment. Absorbance of methanol was also measured as a blank. Analysis of Lovastatin Analysis of lovastatin by spectrophotometric method was according to Danuri (2008). The standard was dissolve in water at concentration of 120, 200, 500, and 1000 ppm. The absorbance of lovastatin solution was measured at 237 nm using UV-vis spectrophotometer. Red mold rice 1 mg was mixed with 9 ml of ethanol 75% and then vortexed for a few minutes. The mixture was centrifuged at 9,520 rpm for 15 minutes. The supernatant was removed and retained, while the pellet was extracted with another 9 ml ethanol 75%. Each supernatant was then mixed together, and the absorbance was measured at 237 nm. Lovastatin content was expressed as mg lovastatin/100 g. Bioscience Research, 2018 volume 15(3):

3 Solubility of red pigments Water solubility of red Monascus pigments at different temperature (25ºC, 60ºC, 80ºC and 100ºC) was evaluated. Test tube was filled with 10 ml water at different temperature, and 50 mg of red mold rice was added and mixed for 30 second. The solution was filtered by filter paper and the absorbance of the filtrate was measured at 500 nm. Stability of red pigments at various temperature Red mold rice powder 600 mg was dissolved in 100 ml water and then filtered. 10 ml of angkak filtered solution was poured into each test tube. Each tube was heated at temperature of 25, 70, 121, and 180ºC for 1 hour. After heating, its absorbance was measured at 500 nm. Stability of red pigments at various ph Red mold rice powder 600 mg was dissolved in 100 ml water and then filtered. A series of test tubes was poured by 1 ml of filtrate. The ph was adjusted into ph 3 and 7 by adding 9 ml of Nacitrate buffer ph 3 and other test tube was added by 9 ml of Na-phosphate buffer ph 7. The solution was left for 12 hours, and the absorbance was measured at 500 nm every 4 hour. Statistical Analysis The data was analyzed using analysis of variance (ANOVA) with 95% of confidence interval and a standard error of using SPSS software. RESULTS Red pigments intensity M. purpureus pigments are a mixture of red, orange and yellow compunds, produce at least six major related pigments which can be categorized into 3 groups based on color. The red pigments named rubropunctamine and monascorubramine, are most abundant, the orange pigments are rubropunctatin and monascorubrin, and yellow pigments are monascin and ankaflavin (Arunachalam and Narmadhapriya, 2011). The composition of the pigments produced by Monascus sp. was influenced by strain and types of media used. Mung bean sprout, which was mixed with rice as main substrate, increased the intensity of red pigments (Fig. 1). Mung bean sprout are rich in vitamin B1 (Goyal et al., 2014), zinc (Dahiya et al., 2014), and amino acids (Tang et al., 2014). According to Yanli et al. (2012), Monascus pigment biosynthesis is considered generally follow polyketide pathway, and only orange pigments (rubropuntatin and monascorubrin) are biosynthesized. Other pigments are chemically transformed from them. Zn 2+ and combination of amino acids could improve carbon source transfer during Monascus spp. fermentation (Yanli et al. 2012). Red pigments (monascorubramine and rubropuntamine) are produced under nearly neutral ph and high concentration of nitrogen sources (Wang et al. 2016). Mung bean sprout was contributed to the availability of nitrogen from free amino acid and protein remaining. According to Yanli et al. (2012), organic nitrogen sources such as urea (CO(NH2)2), peptone, monosodium glutamate (MSG), and amino acids, are good nitrogen sources for both growth and pigment production of Monascus spp. Mung bean sprout up to concentration of 5% increased the production of red pigments (1.92 AU/g) (Fig. 1). Further concentration did not reveal an increase in red pigments intensity, even the intensity was lowered. According to Wang et al. (2016), production of Monascus pigments and cell growth had been separated. Orange pigments are produced intracellular, and accumulation of intracellular orange pigments during cell growth is strongly associated to cell growth. Protein inhibitor was required to inhibit the growth of cell thus stimulate the production of extracellular pigments and presumably the bioconversion of orange into red pigments. Therefore, in high mung bean sprout concentration, the high availability of protein possibly stimulated the growing cell and reduced the production of pigments. During mung bean sprouting, carbohydrate and fat content decreased, but the protein contents increased (Syed et al., 2011). The apparent increase was observed in ash, meanwhile the nutritional value of mung bean such as polyunsaturated fatty acids, amino acids, vitamin (B1, B2, B6, C, E) has been improved (Harmuth et al.1987). A decrease in red pigments intensity occurs in mung bean sprout concentration more than 5%. It presumably related to higher concentration of minerals from mung bean sprout such as calcium, iron and phosphorus. Germination increased the availability of minerals such as iron and zinc (Luo and Xie, 2014). Bioscience Research, 2018 volume 15(3):

4 Figure. 1. Effect of mung bean sprout on red pigment intensity Such minerals are required for Monascus purpureus growth, but high concentration might hinder cell growth itself (Feng et al.,2012). Yanli et al., (2012) indicated that ion Zn 2+ inhibited and promoted the growth and pigmentation of Monascus spp. High Zn 2+ concentration tended to inhibit the growth and Monascus pigment production. Carbon sources have different and complex effects on both Monascus growth and its pigment production. Compared with glucose, maltose was a fitter material for monascorubramine (red pigments) production by M. purpureus (Yanli et al., 2012). Degradation of starch during mung bean sprouting was gradually including starch hydrolysis into maltose. According to Grewal and Jood (2009), germination mobilized and hydrolyzed seed polysaccharides by amylolytic enzymes, leading to more available sugars. Mung bean sprout contributed to the availability of more simple carbon sources of Monascus fermentation. Starch content of fermenting medium decreased by increasing mung bean sprout because lower starch level of mung bean sprout than rice. Starch is a carbon source for cell growth. According to Timotius (2005), tapioca starch, which is a cheap source of carbon, also enhanced biomass and pigment production in a manner similar with glucose. Lovastatin content Both Monascus pigments and monacolin K (lovastatin or mevinolin) are polyketides (Su et al., 2003). Monacolin K was formed in the middle and end phase of fermentation because it was a secondary metabolite (Lee et al. 2006). Monacolin L and J are intermediates in lovastatin biosynthetic pathway. Firstly, monacolin L is synthetized from nine molecules of acetate and then converted to monacolin J by hydroxylation. Monacolin J is further transformed to lovastatin (Barrios-Gonzalez and Miranda. 2010). Monacolin production by Monascus spp is strongly influenced by environmental factors, particularly nitrogen sources (Zhang et al., 2017). Lovastatin content of red mold rice increased to 5% mung bean sprout concentration and then decreased in the higher concentration (Fig. 2). This is presumably related to micronutrient contents and amino acids in mung bean sprout. Adding mung bean sprout increased nitrogen of Monascus fermenting substrates. Nitrogen sources affect the production of lovastatin. Su et al., (2003) showed that adding MSG reduced the yield of monacolin K to zero, whereas adding ammonium sulfate had no effect on monacolin K yield. Chairote et al., (2008) showed that monacolin K could be obtained without adding soybean milk into Thai glutinous rice as main substrate. The addition of soybean milk darkened the color rather than increased monacolin K. However, the effects of amino acids on the secondary metabolites of Monascus are multifaceted, the impact mechanisms are still unclear (Zhang et al., 2017). Zhang et al., (2017) reported that glutamic acid stimulated monacolin K production via the upregulation of the expression levels of eight genes. Bioscience Research, 2018 volume 15(3):

5 Figure. 2. The effect of mung bean sprout on lovastatin content Glutamic acid may not only provide energy for growth and metabolism, but also acetyl coenzyme A as a substrate for monacolin K synthesis. According to Mubarak (2005), germinated mung bean had glutamic acid 21.5 g/16 g N. Mung bean sprout might act as a source of glutamic acid for monacolin K synthesis. Mung bean sprout contributed to available minerals for lovastatin synthesis. Germination increased the availability of zinc (Luo and Xie, 2014). According to Jia et al., (2009), Fe 2+, Ca 2+, Zn 2+, Mg 2+, and Mn 2+ promoted the cell growth and lovastatin biosynthesis of Aspergillus terreus in different extents. Zn 2+ exhibited the highest production of biomass and lovastatin, but excessive Zn 2+ inhibited the cell growth. Their results revealed that the divalent metal ions Zn 2+ or Fe 2+ influence the production by regulating the action of key enzymes in lovastatin biosynthesis. It seemed that high mung bean sprouts inhibited production of lovastatin, presumably related to high Zn 2+ concentration in the substrates. Substrate with too much or too less moisture content is unsuitable for the growth of Monascus (Lee et al., 2006; Lee and Pan, 2012). Adding mung bean sprout into rice would increase moisture content of the substrates. Lee et al., (2006) reported that adding dioscorea into rice increased moisture content and, also increased the production of monacolin K, therefore extending growth and monacolin K production time of Monascus with dioscorea. Perhaps, excessive moisture also decreased the production of monacolin K. In high mung bean sprout, the production of lovastatin also decreased (Fig. 2) that might also relate to high moisture content of the substrates. Feng et al., (2015) showed a correlation between glycerol concentration as a carbon source, and type of monacolin produced. In addition to glycerol, the main source of carbon in Monascus fermentation is starch from rice or other materials. The composition of starch or the type of carbon source would directly affect the growth of Monascus species (Lee and Pan, 2012). Mung bean sprout has less starch than rice, therefore in the high concentration of mung bean sprout would reduce the starch and decrease the cell growth. Therefore, higher mung bean sprout than 5% also decreased lovastatin production. Monascus produces three kinds of described polyketides: citrinin, red pigments, and monacolin K (Vendruscolo et al., 2016). Level of lovastatin have a positive correlation with the intensity of the red pigments. Lovastatin and pigments have the same precursor, tetraketide (one molecule of acetyl CoA and three malonyl CoA molecules), which can be synthesized into lovastatin and pigments (Kraboun et al., 2013). This study showed that adding mung bean sprout 5% is the best for red pigments and lovastatin production. Red mold rice mixed with 5% mung bean sprout were further evaluated for the solubility and stability to ph and temperature. Water solubility of red pigments at various temperature Long et al., (2018) revealed Monascus Bioscience Research, 2018 volume 15(3):

6 pigments based on solubility is divided into water soluble pigments and ethanol-soluble pigments. Water is widely used in foods therefore water solubility of red pigments is very important to evaluate. Increasing temperature enhanced the water solubility of red pigments (Fig. 3). Fig. 3 shows that red pigments are more soluble in water at high temperatures. Based on solubility, Monascus pigments are grouped into water-soluble and water-insoluble pigments, most of the pigments are soluble in ethanol (Babitha et al., 2006; Feng et al., 2012). Stability of red pigments at various temperature and ph Some pigments of Monascus spp. are unstable at high temperatures, light, presence of oxygen, metal ions, and ph changes. Color degradation is common for natural pigments and is therefore a major concern in coloring foods (Vendruscolo et al., 2016). In this study, to evaluate the stability of red pigments in some applications, Monascus pigments were incubated at different ph and temperatures. Stability was measured spectrophotometrically at 500 nm. Monascus pigments show lower absorbance after heating (Fig. 4), that means red pigments were unstable at high temperature. This result was in accordance to that reported by Vendruscolo et al., (2016). Instability of pigments at high temperature might be due to degradation of chromophore group containing double bonds. Torres et al., (2016) revealed that chromophore is responsible to the characteristics of color. Stability of Monascus pigments is greatly increased by adding amino acids (Buhler et al., 2015). In this study, the stability of red pigments was evaluated at ph 3 and ph 7 that represented acidic and neutral ph which usually applied in food processing. Red pigments were more stable at ph 7 than ph 3 (Fig. 5). Monascus pigments are more sensitive to acidic ph and more stable in alkaline or neutral ph. At low ph, the reduction in the pigment color is related to abundant H + ions, that leads to chromophore groups degradation thus decreasing the color of pigments (Fabre, 1993). According to Julio et al., (2005) red pigments of Monascus spp. decreased more rapidly in low ph. Acid accelerates interaction between water with pigments that breaks ester linkage in red pigments, rubropunctamine or monascorubramine. This effect is not found when the pigments are dissolved in ethanol. The stability toward ph and temperature of Monascus pigments were further evaluated. It was found that these pigments were unstable at low ph and high temperatures. Therefore, they are suitable for low temperature processing below 60 C and near neutral ph, such as refrigerated foods. Figure. 3. Red pigment water solubility at various temperature Bioscience Research, 2018 volume 15(3):

7 Figure. 4. Red pigment stability at various temperature Figure. 5. Red pigment stability at ph 3 and 7 CONCLUSION Mung bean sprout increased production of red pigments and lovastatin in concentration dependent manner. Concentration of mung bean sprout 5% stimulated the highest production of red pigments and lovastatin. Mung bean sprout was a source of protein for production of red pigments and mineral especially Zn and amino acid for production of lovastatin. Higher level of mung bean sprout did not reveal further increase in red pigments and lovastatin due to inhibitory effect of excessive protein for production of red pigments and moisture content for production of lovastatin. Increasing temperature enhanced the water solubility of red pigments. Red pigments were unstable at high temperature and more stable at ph 7 than ph 3. Instability of pigments at high temperature might be due to degradation of chromophore group containing double bonds. CONFLICT OF INTEREST The authors declared that present study was performed in absence of any conflict of interest. ACKNOWLEGEMENT The authors would like to thank to all laboratory technicians who assisted this experiment. AUTHOR CONTRIBUTIONS EZB designed the experiment, control the progress of experiment, and problem solving related to experiment. LPA performed the experiment and data analysis. TES wrote the manuscript, data analysis, figures preparation, and was responsible for submission, corrections, and correspondence. All authors read and approved the final version. Copyrights: author (s). This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author(s) and source are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or Bioscience Research, 2018 volume 15(3):

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