Production of red yeast biomass with accumulated metals

Heavy metals are natural components of the Earth’s crust. As trace elements, some heavy metals (e. g., copper, selenium, zinc) are essential to maintain the metabolism of the human body. However, at higher concentrations they can lead to poisoning. A special case of antioxidant/prooxidant behavior of carotenoids emerge in the presence of metals (e. g. metal-induced lipid peroxidation). In this case metal ions (Fe2+ or Cu2+) react with hydroperoxides, via a Fenton-type reaction, to initiate free radical chain processes. There are several studies which indicate that P-carotene offers protection against metal-induced lipid oxidation. Presence of carotenoid in the reaction system not only decreases the free radical concentration, but also the reduction of Fe3+ to Fe2+ by carotenoids may occur. Recently free radical scavenging and antioxidant activities of metabolites produced by carotenogenic yeasts of Rhodotorula sp. and Sporobolomyces sp. grown under heavy metal presence were studied using various EPR experiments (Rapta et al., 2005). Since carotenogenic yeast differ each to other in resistance against the heavy metals due to their individual protective system, quenching properties and antioxidant activities of carotenoids yeasts were modulated by metal ions variously. Thus, activated biosynthesis of carotenoides by yeasts exposed to heavy metal presence could be in part explained by their scavenger characters (Rapta et al., 2005) as a protection against the harmful effect of the environment.

Several divalent cations (Ba, Fe, Mg, Ca, Zn and Co) have been demonstrated to act as stimulants for growth of R. glutinis. Trace elements have been shown to exert a selective influence on the carotenoid profile in R. graminis — Al3+ and Zn2+ had a stimulatory effect on beta-carotene synthesis, while Zn2+ and Mn2+ had a inhibitory effect on torulene and torularhodin synthesis (Buzzini et al, 2005). The observed effect of trace elements on the biosynthesis of specific carotenoids in red yeasts may be explained by hypothesizing a possible activation or inhibition mechanism by selected metal ions on specific carotenogenic enzymes, in particular, on specific desaturases involved in carotenoid biosynthesis. In a recent study, calcium, zink and ferrous salts were shown to have a stimulatory effect on volumetric production as well as cellular accumulation of carotenoids from the yeast R. glutinis (Bhosale & Gadre, 2001). Divalent cation salts increased the total carotenoid content (mg/L) about two times. It can be assumed that this positive response was due to a stimulatory effect of cations on carotenoid-synthesizing enzymes, or to the generation of active oxygen radicalcals in the culture broth. In contrast, the addition of manganese salt in the presence of generators of oxygen radicals had an inhibitory effect on carotenoid formation in X. dendrorhous since manganese acts as a scavenger; however, this effect could be concentration dependent as manganese is also known to act as a cofactor for enzymes involved in carotenoid biosynthesis and thus enhances carotenoid accumulation at certain concentrations (Frengova & Beshkova, 2009).

Astaxanthin content was decreased significantly at >1 mg/L FeCl3 and growth of P. rhodozyma was poor at an FeCl3 concentration of <0.1-1.0 mg/L (An et al., 2001). Carotenoid production decreased in yeast with increasing Mn2+ concentration (0-10 mg/l) when succinate was used as the sole C source, but not when growth took place in the presence of glucose. The week oxygen radical scavengers Zn2+ and Cu2+ had no effect on carotenoid production by P. rhodozyma, whereas Cu2+ below 3.2 |iM increased the astaxanthin content of cells P. rhodozyma but at the expense of a slightly decreased growth. In yeast, there are at least two intracellular enzyme systems requiring copper: cytochrome-c — oxidase and superoxide dismutase. These enzymes are probably related to the increased astaxanthin production seen in concentrations of Cu2+ below 3.2 |iM. Copper deficit decreases the activity of antioxidant enzyme Cu, Zn-superoxide dismutase, as reported previously and may induce oxidative stress and astaxanthin synthesis because of diminished antioxidant defences. In contrast, iron below 1 |iM decreased both the growth and astaxanthin content of cells P. rhodozyma (Flores-Cotera & Sanchez, 2001).

Selenium (Se) is a key trace element required in small amounts in humans and animals for the function of a number of Se-dependent enzymes; however, this element can also be toxic in larger doses. Se is incorporated into proteins to provide selenoproteins, which are important antioxidant enzymes; other selenoproteins participate in the regulation of thyroid function and play a role in the immune system (Wang & Xu, 2008). Organically bound Se is considered as more bioavailable and suitable for dietary application than sodium selenite or podium selenate, the two inorganic forms of Se commonly used in the feed industry. Yeasts

naturally incorporate Se into the biomass where it is stored as selenomethionine. The organic form of Se produced in yeasts is of the similar type as that obtained from food. Recently preparation of antioxidant formula based on carotenoid forming yeasts Rhodotorula glutinis and Sporobolomyces roseus that also efficiently accumulated selenium from the growth medium was reported (Breierova et al., 2008).

In the presence of Se, carotenogenic yeast strains produced less carotene pigments. The results obtained indicate that the most dramatic change was observed in the significantly lowered levels of P-carotene, while torularhodin and torulene contents decreased to a lesser extent (Breierova et al., 2008). Previously, it has been shown that Cd, Ni, and Zn induce the opposite effect and stimulate production of P-carotene. It was found that direct incorporation of Se into yeast cells during cultivation in Se-rich medium can not be used for preparation Se-enriched yeast biomass. Instead, cultivation of the yeasts and a subsequent treatment with sodium selenite during 24h should be applied. A non-lethal and simultaneously maximum tolerated concentration of Se was determined based on the growth curves of the individual strains. A 60-ppm concentration was used with all strains, and the distribution of Se in the cells, on the surface of cells, and in the exopolymers was analyzed. The maximum Se sorption was observed with the cells of species Rhodotorula glutinis CCY 20-2-26 (17 mg/ g dry weight), while its exopolymers accumulated only 7% of the total adsorbed Se. The remaining Se was sorbed onto the fibrillar part of the cell wall and into the cells. Similarly, two other studied strains, CCY 19-6-4 and CCY 20-2-33, sorbed Se primarily into cells (63-74%) and the fibrillar part of cell wall (2-22%), whereas exopolymers bound only 12-32% of the total sorbed amount. The yeasts with high content of the carotenoid pigments and selenium may be used for the preparation of a new type of antioxidant formula that could be directly applied for various human and animal diets. Such a formula can only be produced by separate processes of the cultivation of red yeasts and a subsequent sorption of selenium into the cells (Breierova et al., 2008).

In general, there have been several reports on the enhancement of volumetric production (mg/l) as well as cellular accumulation (mg/ g) of microbial carotenoid upon supplementation of metal ions (copper, zinc, ferrous, calcium, cobalt, alluminium) in yeasts and molds (Bhosale, 2004; Buzzini et al., 2005). Trace elements have been shown to exert a selective influence on the carotenoid profile in red yeasts. It may be explained by hypothesizing a possible activation or inhibition mechanism by selected metal ions on specific carotenogenic enzymes, in particular, on specific desaturases involved in carotenoid biosynthesis, in agreement with previous studies reporting activation or inhibition by metal ions in microbial desaturases (Buzzini et al., 2005). The other explanation is based on observations that presence of heavy metals results in formation of various active oxygen radicals what, in a turn, induces generation of protective carotenoid metabolites that reduce negative behaviour of free radicals. Such strategy has been applied in several pigment­forming microorganisms to increase the yield of microbial pigments (Rapta et al., 2005; Breierova et al., 2008).

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