ube 579 Orginal scientific paper BIOTECHNOLOGICAL PRODUCTION OF ORGANICALLY BONDED E INTIAL OLIGO AND TRACE ELEMENTS (EOTEs): FROM CONVENTIONAL TOWARD MODERN BIOTECHNOLOGY? Miroslav M. VRVIC'* and Valerija F. MATIC? 'Paculty of Chemistry, University of Belgrade. Belgrade *Department of Chemistry, Institute of Chemistry, Technology and Metallurgy, University of Belgrade, Belgrade, Serbia Natiram si sequémur ducem nunquain aberébimus! [M-T Cicero (106-43B.C.),De off. 128, 100} (We'll never go astray if nature is our guide?”) Vrvié M. Miroslav and Valerija F. Matié (2006): Biotechnological production of organically bonded essential oligo and wace elements (EOTES): From conventional toward modern hiotechnology? ~ Mikrobiologija, Vol. 43, No.1. 1-14, Beograd. Bioavailability of EOTEs is the key for their absorption in the digestive tract, transport and inclusion in the metabolism resp. their activity and it directly depends on the nature of the compound in which they are contained. This fact is the base for development of our biotechnological procedures for obtaining organically bonded EOTEs among which are: selenium, chromium(III), _ calcium, magnesium, calcium+magnesium, iron(II), zine, potassium and silicon. Our strategy relates to the conventional biotechnologies for obtaining biomass (complex bioligands-“bilogical agents”) enriched by organically bonded EOTEs or application of biotechnological products * Corresponding author: Faculty of Chemistry, 11001 Belgrade, Studentski tre 16. P.O.Box 158, Serbia, Phone: +381-11+ 637-273, Fax: +381-11-636-061. E-mail: mmvchem‘disezampro.yuMIKROBIOLOGUIA, Vol. 43, No. 1, 1-14. 2006. as ligands and more rarely secretion of compounds containing them which is shown in this paper, as a review of results of our about twenty years long work in this field Based on natural organic complex ligands (baker's and brewing yeast and bacteria) several products (selenium, chromium and calcium) of these our own biotechnologies are marketed as food supplements and non-prescription drugs only for oral application at present. Modern biotechnology which understands application of genetically engineered microorganisms (GEM) is not our trend for the future in this domain. . Key words: bioavailability, organically bonded EOTEs [Se. C(I, Ca, Mg, CaiMg, Fe(II), Zn, K and Si], bioligands, complex bioligands-“biological agents” (baker's, brewing yeast and bacterial macrocapsules), conventional biotechnological processes and products, “traps” of modern biotechnology INTRODUCTION EOTEs status is not clearly defined: from micronutritients and food supplements vie OTC (over-the-counter i.c. non-prescription drugs) to drugs. The difference relates first of all to doses and is defined by regulations in individual countries (SCHRUVER ef al., 1993). There is also non-coordination about oligo and trace (micro and ultramicro) determinants (Rousstiet, 1991). Therefore. under oligo-elements are understood here those which RDA (Recommended Dietary Allowance) is exceeding 100 mg, while for trace elements this value is less than 100 mg. In any case all EOTEs are nutraceuticals from the category of minerals, either ingredients or formulated produets-preparations are concerned, and their production is a new industrial sector positioned between the food industry and pharmaceutical products (RoBEREROID, 2000). Research and development in this domain have besides the fundamental and applicable importance also. great commercial effects. So, for example, the market annual growth rate of minerals in the USA for the period 1994-1998 was 8.6% and until 2003 it is estimated to be 4.0% (Jarvis, 2000), PATH TO BIOTECHNOLOGICAL OBTAINING OF ORGANICALLY BONDED EOTEs Bioavailability and selection of ligands It is known that EOTEs bioavailability is considerably increased if they are organically bonded to natural ligands, in relation to inorganic and organicMM, VRVIC, ef uf: ORGANICALLY BONDED LOTEs forms with synthetic ligands, which results in higher efficiency, a smaller dose and better tolerance of the preparation (ARNAUD ef al., 1995; BURK ef al., 1985), This refers both to humans and to animals. On selection of the natural organic molecule-ligand to which element it will be bonded, the HSAB (hard and soft acids and bases) (PEARSON. 1968) conception is applied giving the useful information on “candidates” for ligands. As potential ligands in our studies are considered: baker's and brewing yeast biomass (Saccharomyces serevisiae steains as “biological agents”). baker's yeast autolysate and small molecules-biotechnological products (organi¢ acids such as acetic and lactic). Macrocapsules of unpathogenie bacterial strains (gents Bacillus) are potential new ligands we work with EOTEs and interactions with bioligands Biomass is a complex bioligand with which EOTEs interact during the process of growing and multiplication resp. in which they are incorporated from the medium into which were added in the form of inorganic compounds. The main places and the methods of their interactions with yeast microorganisms cells are as follows (HUGHES, ef al., 1989): 1. Capsules and cell wall-adsorption, complexing and covalent bonding 2. Nuclei acids-complexing, and 3. Cytosol-complexing and covalent bonding, Interactions of some EOTEs [selenium and chromium(II1)] with baker's yeast biomass are known as well as the conventional biotechnological procedures for obtaining the biomass enriched with these elements (VRviC, ef al., 1988). Incorporation of EOTEs in the brewing yeast biomass as a new complex bioligand should have been developed by analogy with known procedures assuming that the products shall have higher bioavailability (Vevic, e¢ al., 1995) When working with biomass as complex bioligands. HSAB approach gives only rough estimates due to which a greater number of experiments are required in the stage of optimization of the medium. Baker’s yeast autolysate which chemical composition can be simply defined with free amino-acid ligands dominating (minimum bidentate) and small peptides and organic acids, particularly monocarboxylic and hydroxymonocarboxylic as bioligands are much more simple for initial considerations and the application of HSAB criteria gives still better results, To have the thorough knowledge of chemical and biochemical properties [especially chemical speciation in human body, including computer simulation (HrRcBERG, 1988)] and physiological-biochemical role of EOTEs which should be obtained in the organically bonded form with some of the available ligands is the essential for development of the procedure. The situation4 MIKROBIOLOGUA, Vol. 43, No.1, L-14, 2006, becomes additionally complicated when EOTEs exist in a range of oxidation states at which bioavailability depends on valence and for which the typical example is iron(II) and iron (II), when iron(II) is’also expressly subjected to hydrolysis (HeRCBERG, 1988). Covalent bonding of EOTEs (non-metals) into organic biomass molecules by assimilation and metabolization of the elements from an inorganic form found in the medium is chemically and biochemically as well as biotechnologically complex case which is still more multiplexed if the element has redox properties and if changes the oxidation state in the process of biosynthesis. It is the case with selenium. As selenium source in the medium there is sodium selenite (Se*") which is included by reductional assimilation in the serine biochemical pathway of selenoamino acids biosynthesis (predominantly selenocysteine and selenomethionine containing formal oxidation state Se”) resp. proteins. On the other hand, sodium selenite is the oxidant due to which a smaller portion is reduced in the presence of the organic substance into elementary selenium (Se®) depositing on the surface of the yeast cells (NAZARENKO, ef al, 1971; Ziveovic, er al., 1991; Vucrnic, ef ul., 1991). All the above stated indicates that the most complete answers in the initial stages of the process development resp. defining of aims and scopes can be given by bioinorganie chemistry (KaIM, et al.. 1994; Witsams, ef al.. 1996). Our rules: bioavailability, ligands and EOTEs At selection of bioligands i.e. chemical form of organically bonded desired EOTEs (metals first of all), except the known bioavailability, the principles we follow are: |. To use ligands present at the place of absorption-digestive tract; and 2. The most advantageous are ligands and chemical forms found in victuals for it is known that EOTEs from them have high bioavailability Respecting the aforegoing principles—our rules (“in place-forms”) . we are striving toward realization of the natural biocompatibility. However, guiding by the rule No, 2 is sometimes doubted due to non-precise data as it was the case with long years of mistaken notion that spinach is a good source of iron (MILLER, 1996). In such cases bioavailability should be checked. For some EOTEs there are standard methods on animals. Such is the case with iron (DeuTsctt, 1996) which was used for iron(II) malic chelate formulated in OTC while as ligand we are using the natural malic acid (approx. 90%) obtained from fruit (VRvic et al., 2000), to confirm assumed bioavailability (Mien er al., 2001). Digestibility of the obtained organically-bonded EOTEs and their formulations determined in vitro (pepsin digestion) is the datum used as a good one for estimation of bioavailability (PorkoNsAK ef al., 1995).M.M. VRVIC. ef al: ORGANICALLY BONDED EOTES sl Conventional biotechnological processes and organically bonded EOTEs The basic assumption for development of the procedure in which biomass is a complex bioligand is the conventional biotechnology for obtaining dry inactive baker's yeast biomass with utilization of the industrial baker's and brewing yeast strains in the batch and fed-batch processes. In case of obtaining nutraceutical beverages based on the fermented malt extract with organically bonded selenium where the aerobically obtained biomass of the brewing (beer) yeast enriched with organically bonded selenium is used in the malt extract fermentation process during which the yeast extracellulary excretes selenoamino acids and proteins containing them, a complete bio/technological line is used for beer production including the propagation station (Vucenc ef al, 1993). Thus, application of our strategy for biotechnological obtaining of organically bonded EOTEs understands the conventional biotechnology and processing equipment ee eg Development of the biotechnological process itself has a usual course: shake Mask testing technique (choice of the microorganism strain, optimization of the medium composition and establishing of the basic biochemical engineering conditions~ temperature, inoculum, aeration and duration of the process), laboratory fermentor with mechanical stirrer (defining of biochemical engineering. parameters—aeration, temperature regime, pH and foam control, as well as the basic “down-stream” data) and scale-up on pilot fermentor of the overall volume 150 L (optimization of the whole process including the spray-drying as finalization for obtaining a dry inactive biomass with organically bonded EOTEs). After this, transition is made to the industrial fermentation in bioreactors of the overall volume 10 (only batch process), 50 and 150 m' (BLANCH ef al,, 1997) Considering obtaining of organically bonded EOTEs with bioligands from baker's yeast autolysate and organic acids which are biotechnological products, after classical laboratory research-development experiments, the procedures are transferred onto the pilot batch plant (100 ky product per batch) and realized on the standard industrial equipment for the batch obtaining of salts of organic acids (about 500 ke. per batch) Problem of analytical procedures A special problem which should be solved on the development path of biotechnological processes are analytical procedures. First, for monitoring incorporation of EOTEs and then, what is much more important and complex, determination of the content of organically bonded element and its speciation and distribution in the biomass cells which mostly is not sufficiently and completely described (MckENzIE ef al., 1988: CHAPPUIS, 1995). In that respect, selenium is6 MIKROBIOLOGIA, Vol. 43, No. 1. 1-14, 2006, characteristical in the dry inactive yeast biomass (ERMAxov ef al, 1974; Vakimo et al., 1983; OuvanG ef al., 1988) for which we developed the stated analytical procedures (Vucenic ef al.. 1991; Vewic ef af, 1989). The essential selenium speciation is represented by three forms: absorbed as SeOy””, deposited elementary and organically bonded. The sum of the first two is the total inorganic selenium and its concentration is maximum 10%, usually it is less than 5% of the total amount of selenium with a most identical shares. Organically bonded selenium (minimum 90% and the most frequently over 95%) is found, as stated above, in selenoamino acids, which are free (selenomethionine+selenocysteine about 4% of organic selenium in the ratio approx. 2:1) and included in proteins (approx. 60%) and other organic selenium compounds (approx. 35%). For defining the distribution of organic selenium in the yeast cell fractions. we developed a special procedure, by means of which, after the mechanical disintegration and enzyme hydrolysis (RN-ase and DN-ase). the following fractions are obtained: I-cell wall (37.2-38.0% of the total organic selenium), H-nuclei¢ acids (0.1-0.3%), I+ cytosol (3.2-4.0%) and [V-proteins (57.7- 58.3%). All data about the distribution of the organically bonded selenium refer to a dry inactive biomass of the baker's yeast enriched by the organically bonded selenium. Last stage: formulation and application The last stage on the development path of organically bonded ETOEs. prior to their utilization by consumers-release to the market, is their formulation in preparations (tablets, pills, dragees, capsules, instant products, syrups) which contain only minerals, their compounds or combinations with other nutraceuticals ingredients which are in our products mostly vitamins (A, C, E, folic acid), which is the routine job of the pharmaceutical technology (Vevic. 2001). Some of our organically bonded EOTEs are applied also as raw materials, most often in the food fortification industry-obtaining of functional food (for example enriching of children’s biscuits with organically bonded calcium) (VRwic ef al.. 2001). (These products rank. according to the Yugoslav regulations, into the category of (natural) dietary products being equivalent to food supplements and (natural) OTC, while iron subject to the individual dose can also be a drug] Some of our EOTEs nutraceuticals are the raw materials for preparation of the fodder premix (Vavic ef al, 2001) “Stage after” “Stage after"-checking the known and discovering new action effects resp. expansion of the indication area of the obtained organically bonded EOTE by conducting tests on animals and target groups of healthy volunteers, and by theMM. VRVIC. ef ali: ORGANICALLY BONDED EOTEs — 7 clinical tests. is the greatest challenge for researchers and _ practitioners biochemists, nutritionists, physiologists. pharmacologists and physicians of various specialties both in the human and in the animal domain (our selected results MILE ef al. 2001, Diune ef al, 1991; Dsune et al., 1992; Vunin ef al., 2000; Diorpsevic et al., 1995; Vavic er al., 1999), CONVENTIONAL BIOTECHNOLOGY AND OBTAINING OF ORGANICALLY BONDED EOTEs The summarized results of our work on biotechnological obtaining of organically bonded EOTEs are shown in Table 1. Ligands (in table denoted as microbial) in case of magnesium, magnesium in the composition of calcium+magnesium, iron(I, zine and potassium are the autolysate of the baker's yeast, for calcium biotechnologically obtained acetic and factic acids, and for silicon [in the last years actual essential trace element aimed to safeguard bone integrity and cognitive tissues (BENDER ef al., 1997)] the macrocapsules of non-pathogenic strains Bacillus sp. isolated from the soil and oil shale (Vevic ef al., 2001; Dractrinovic ef al, 2000). As the source of EOTEs in all cases are used the synthetically obtained inorganic compounds except in case of calcium and silicon where the source of this elements is the natural material-calcite of high purity resp. fine previously chemically prepared river's sand, Lab. 1. ~ Ow Own Bio Technologies for Production of Natural Organically Bonded EOTEs for Application in Human and Animal Nuvvition and Medicine No Fore Cina pplication es “aa ofdevcopment Sete Bikers yea Tora supplement Gastar 2 Selenium Then (beer yea” Food supplements intake + Sleniam eer yest Naaceuiel ben Ines Market 4 Cvomuntt) Beer yeast al suplercas Indus Make 5 Chromiunutth) Bakers yeast Fenn supplements tnhsiaMarkt Feud G Microbial additive/supplements: Ureluettial ati 7 Magnesiun crbi Fava suppemensOTC Pit Coleman — Microbial Fowl supplemenisOTC Pat 9 tenth Mierobal Feed va 10 fae _ tewalnupplemensit'1 Lah 11 Potasiom Meobial Fowl supreme Lab 12 Silicon Food supplementsMIKROBIOLOC Bioavailability of selenium from the dry inactive biomass of the baker's yeast enriched by organically bonded selenium (“selenium baker's yeast") is known (Stmonorr ef al., (1991). however organically bonded selenium in the beer yeast [“selenium beer yeast"-2 mg of organically bonded Se/g of dry mass(DM)] is for the first time obtained and tested in our laboratories (VRANILS ef al., 1996); it was proved that in relation to “selenium baker's yeast” (2 my of organically bonded Se/g DM, production only in batch process) it has 16 times more free amino acids and thereby also selenoamino acids, for which is known to become directly absorbed from the digestive tract, the higher is digestibility too and this bioavailability (PorkoNAK ef a/., 1995), so that the situation is analogous with “selenium beer” (VUCETIC ef al., 993). The situation is just the same with bioavailability of organically bonded chromium(III) in a dry inactive beer yeast biomass (“chromium beer yeast"-2 mg of organically bonded Cr/g DM, production only in batch prosess). We proved that in it more than 95% chrome (VRvi e¢ al., 1995) is contained in the complex soluble coordination compound with organic ligands (nicotinic acid-vitamin Bs and tripeptid glutation)~GTF (glucose tolerance factor), which has a role like insulin, more precisely, it is the mediator between insulin and insulin cell receptors (KATZ e/ al., 1994; KAMEN, 1990). which we also proved (DiorDuevié et al., 1995). Better properties of the beer yeast as the “biological agent” justify the higher price (as a consequence of less yields in the production) of nutraceuticals on the basis of this complex bioligands. Application of the baker's yeast autolysate (the content of free amino acids in autolysate with which we work is about 230 g/kg DM) as bioligand for the stated elements comes out of the greater affinity of the numbered metals for N-electron donors (HSAB principle) (PEARSON, 1968) and application of our rules: 1. In the digestive tract where from adsorption of EOTES is carried out are present the amino acids which are N-donors (Guyton ef al, 1999) and 2. Victuals of animal origin in-which proteins dominate (also N-donors from peptide bond, amino and guanidine groups and pyrrolidine and indole rings of aminoacids) are rich with these elements which out of these chemical forms have a high bioavailability (Suretz et al., 1994), We are developing an oral iron(II) preparation with these ligands because of its great economic significance for survival of piglets in the first weeks of age, by prevention of sideropenic anaemia (Menon at ef al.. 1997), How useful HSAB approach is when operating with chemically defined bioligands shows the example of calcium which in contrast to magnesium has greater affinity to Q-electron donors, which means to carbonic and hydroxycarbonic acids (Vrvic er al., 1999). On the other hand bifidogenic bacteria in intestine and colon, especially with babies, exogencously produceMM. VRVIC, ef al: ORGANICALLY BONDED EOTEs 9 acetic and lactic acids in the mole ratio 3:2. which enable the best adsorption of calcium and vitamin D (GourNtR-Chattau ef al., 1994; De Vers ef al, 1968; Vavic et al, 2000). Adsorption of calcium is better also from lactic-acidie beverages (GoURNIER-CHATEAU ef al., 1994). So, if calcium is in the form of calcium acetate lactate (hydrate) at which the mole ratio acetate:lactate=3:2 then it shall have high bioavailability and biocompatibility shall be achieved. These are the reasons why we developed, by explicit utilization of the theoretical principles and consistently respecting our rules. the procedure for the production of organically bonded calcium (Orgacaleium®) in this form (calcium content about 20 %) (Vavic ef al., 2001: Vevic ef al., 1999) The procedure for the production of nutraceutical calcium+magnesium which is under development now and which contains Orgacalcium® and organically bonded magnesium using the autolysate as bioligands, at which the mass ratio Ca:Mg=2:1, has practical reasons. It was shown that this ratio is the most efficient in the prevention of osteoporosis as world-wide problem in women in the postfertile period (Vevic ef ul., 1999; PrussMan ef al., 1998: Liver. 1991). Finally, organically bonded silicon to the bacterial macrocapsules (also biological agents") which’ is of polysaccharide nature (DRacutinovic ef al, 2000; Isobe ef al., 1997) promises a possibility of development of nutraceuticals which will ensure passing of the element in soluble form through the barrier of low pH into the stomach and to be further adsorbed with satisfactory bioavailability because the path of all inorganic silicon compounds due to its chemical nature end is just in the stomach, by conversion into biologically inert insoluble hydrated silicon dioxide (VoRONKoV ef al., 1978). This would be the first natural preparation of the organically bonded silicon TOWARD MODERN BIOTECHNOLOGY ? Contribution of a modern technology in this field could be expected in the direction of the increase of the microorganisms biomass capacity, yeast first of all. for organically bonded of some EOTEs [for example chromium(III), zine or copper] and obtaining of extracellular active EOTEs compounds which understands GEM (Hucues et al., 1989; Misart: ef af, 2001; Watsu er al., 1994; Bakr et al., 1989) application. Our future operations are not directed toward state-of-the-art biotechnology (except classic genetic methods: selection, adaptation. adaptive selection and physical and chemical mutagenesis) _ since we are aware of all “traps” borne by application of genetically modified microorganisms. Also, nutraceutical products obtained by means of or with GEM participation due to regulations, particularly in Europe, which are becoming more strict in the apparent labeling of these products, would cause problems on the market because_ MIKROBIOLOGUA, Vol 43, No.1, 1-14, 2006, public opinion is becoming less friendly toward the genetic engineering, first of all from ecological and ethics reasons. This is particularly accentuated when victuals are concerned from the understandable fear for health resulting in an almost “pandemic” orientation to “healthy foods” as a life style (Sprinciiam, 1999; HenizepereR, 1998) which is also our motto in research, development and production stated at the beginning of the paper, CONCLUSIONS, Conventional biotechnology offers wide possibilities for obtaining of physiologically-biochemically active organically bonded EOTEs with high bioavailability and biocompatibility using natural ligands-yeast biomass and bacterial macrocapsules (complex ligands-“biological agents”), baker's yeast autolysate and small molecules (organic acids) obtained biotechnologically. The procedures for the industrial obtaining of these nutraceuticals (already developed or in some of the developing stages) as ingredients or products, humane or animal application, do not require any additional equipment except the one for the production of dry inactive baker's yeast, beer, and salts of organic acids and for formulation of preparations. the regular equipment of the pharmaceutical technology. On the industrial level we possess biotechnologies which are in exploitation resp. the products are marketed for obtaining organically bonded: selenium (three technologies, four groups of products), chromium(III) (two technologies, two groups of products) and calcium (one technology. two groups of products). On the pilot level are organically bonded: magnesium, calcium+magnesium and iron(II). Organically bonded: zine, potassium and silicon are in the laboratory stage of development. The modern technology which understands utilization of GEM is not our orientation in the future (except an application of methods of the classic genetics) first of all due to less inclination of the public opinion toward genetic manipulations, particularly toward food which bears the label of genetically modified victuals Acknowledgements.-We wish to express our appreciation to the Ministry of Science and Environmental Protection of the Republic of Serbia for their support of part of this work (Projects No. 142018B and TR 6845B) The most sincere gratitude does to the company NRK ENGINEERING”, Ltd. from Belgrade for their continuing, particularly financial, support to our research and development endeavorings in the domain of biotechnology and application of organically bonded elements of life.MM. VRVIC, ef al: ORGANICALLY BONDED EOTES u REFERENCES ARNAUD. J. annd BELLEVILLE-NABET, F, (1998), in Oligo Elements in Nutrition and in Therapeutics, Chappuis, P. and Fayier. A.. eds. 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Vin LAZIC, M. L. and Vavic, M. M. (991). in .thsiracts of the International Symposium on Selenium. Maksimovig, Z. J. ed.. Serbian Academy of Sciences and Arts (SASA), Belgrade, Yugoslavia, pp Meeting i M. J Received May 4, 2001 Accepted December 1. 2006u 2006. BIOTEHNOLOSKA PROIZVODNJA ORGANSKI VEZANIH ESENCIJALNIH OLIGO I MIKROELEMENATA (EOM. 2D KONVENCIONALNE KA MODERNOJ BIOTEHNOLOGIJI ? Miroslav M. VRVIC'? i Valerija F. MATIC? ‘ Hemijski fakultet, Univerzitet u Beogradu. Beograd ? Centar za hemiju, Institut za hemiju, tehnologiju i metalurgiju, Univerzitet u Beogradu, Beograd, Srbija lzvod Biloska dostupnost EOME je kljué njihove apsorpcije u digestivnom traktu, transporta i ukljucivanja u metabolizam, odnosno njihove aktivnosti i direktno zavisi od prirode jedinjenja koje ih sadrzi. Ova Cinjenica je osnova za razvoj nasih biotehnoloskih postupaka za dobijanje organski vezanih EOME medju kojima su: selen, hrom(II1), kalcijum, magnezijum, kaleijum+magnezijum, gvordje({1), cink i silicijum Naga strategija odnosi se na konvencionalne biotehnologije za dobijanje biomase (kompleksni bioligandi-“bioloski agensi”) obogaéene organski vezanim EOME ili na primenu biotehnoloskih proizvoda kao liganada i, retko, izludivanje jedinjenja koja ih sadrze, sto je prikazano u ovom radu, kao pregled rezultata nagih radova na ovom polju, koji traju oko dvadeset godina. Na bazi prirodnih organskih kompleksnih liganada (pekarski i pivski kvasac i bakterije) i_nekih EOME (selen, hrom i kalcijum), primenom sopstvenih biotehnologija na t2istu se nalazi nekoliko proizvoda iz Kiase dijetetskih proizvoda (food supplements) i tekova bez recepta (OTC). za sada, samo za oralnu primenu Moderna biotehnologija, koja podrazumeva primenu —_genetski modifikovanih mikroorganizama (GMM), nije na’ trend za buduénost uw ovoj oblasti. Primljeno 4. maja 2001 Odobreno I. decembra 2006,