PRODUCTION OF Lactomicrosel® AND nanosize (100-500 NM) selenium SPHERES by PROBIOTIC lactic ACID bacteria

Péter Eszenyi1, Attila Sztrik1, Beáta Babka1, József Prokisch1 1Institute of Bio and Environmental Energetics University of Debrecen, Centre for Agricultural Sciences and

Engineering H-4032 Debrecen, Böszörményi út 138. Hungary

Abstract. Selenium is well known as an essential trace element since the 20th century, but it can be overdosed easily because of its toxicity. According to the present regulations only the potentially most harmful, inorganic selenium salts can be added to any comestibles. Elemental selenium is considered as the least toxical selenium of all forms and in the same time supplementation with its nano-size particles has the same or better bioavailability compared to salts. In our experiments we managed to produce nano-size (100-500nm) elemental selenium by useing probiotic yogurt bacteria in a fermentation procedure. We tested the effects of nanoselenium both on plants and animals and we compared the characteristics of conventionally used and nanoselenium in biological systems. In addition we developed the laboratory technology of product purification and extraction from bacteria.

Keywords: selenium, nanosphere, antioxidant, food supplement, lactic acid bacteria

1. Introduction One of the major projects, regarding to dietary supplementation, in our lab focuses on the deeper

recognition of an essential trace element, the selenium, which was first well known about being toxical. Choosing this element has the good reason, because selenium is in the centre of attention of many researches, and selenium has a very iportant direct and indirect role in many selenium deficiency diseases. For example the different kind of cardiovascular [1] and tumoral diseases, Keshan, Kashin-Beck and scientists found correlation between selenium deficiency and Down and Arzheimer diseases.

Our fundamental purpose was to produce a non-toxical selenium form by the means of granted microbes, which are already in use and allowed to use in food industry to put out yogurts. Our product gained from lactic acid bacteria can be batched in higher concentrations this way, without any non-desired side effects, enhancing resistivity toward illnesses, increasing blood antioxidant level and boosting the quality and quantity of agricultural products as a fertilizer or forage supplement. Our experiments were based on the patent of Dr. Jozsef Prokisch in 2008, which relating to elemental selenium nanospheres and their production [2].

2. Elemental selenium nanospheres Researchers just started to recognise in the recent years the importance of the ability of certain

microorganisms to produce nano.sized metabolic products in the course of their metabolism. For example those thermophyl and psychrotolerant metal-reducer bacteria that produce magnetite crystals [3].Foremost [4] reported about the production of elemental selenium nanospheres, that our laboratory also focuses on, in case of specific bacteria. In 2004 scientists described that a selenium reducer anaerob bacteria, that inspires toxical selenium oxianions is able to produce and accumulate extracellular elemental selenium [Se(0)]. In their experiments they later used three, phisiologically and phylogenetically diverse bacterium species in order to look into the phenomenon. Sulfurospirillum barnesii, Bacillus selenitireducens and Selenihalanaerobacter shriftii. During the cell growth they were using selenium oxianions as electron

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2011 International Conference on Food Engineering and Biotechnology IPCBEE vol.9 (2011) © (2011)IACSIT Press, Singapoore

acceptors, following this each microorganisms produced extracellular, uniform sized and shaped approx. 300nm large selenium spheres with monoclinic crystal structure. Not only extracellular but also intracellular elemental selenium production was observed. Optical properties of elemental selenium produced this way were very distinct compared to the chemically synthesized selenium, we can conclude that elemental selenium production in microbial way results more unique, standard structured nanospheres. Positive effects of chemically synthesized selenium nanospheres were examined in several experiments succesfully. Wang et al. [5] were led to the conclusion in 2007 based on animal tests, nanoselenium (Nano-Se) is a very effective antioxidant, without high toxicity properties which is typical for other selenium forms. Nanoselenium has at least the same effect on activating glutathione peroxidase and thioredoxin reductase enzymes as selenoproteines has, but according to the LD50 this form is less toxical, it doesn’t trigger acute liver injury and short term toxications. Furthermore nanoselenium accumulates less in the treated mice and activates glutathione-S-transferase more effectively than selenoproteins do, independently from the level of toxication.

Studiing behavior of selenium during yoghurt producing process led researchers in our laboratory to the discovery that certain bacteria species are able to transform the selenite in toxical concentration into nano sized, elemental selenium spheres via a hardly known biochemical pathway. This is the first technology in which lactic acid and other probiotic bacteria produce elemental selenium in laboratory environment [2]. In our tests we mainly used Lactobacillus sp., Bifidobacter sp., Streptococcus thermofilus and different kind of mixtures from various strains. During the electronmicroscopic analysis (1. picture) it became clear that each bacteria strain produce selenium spheres in different size ranges as follows: Lactobacillus sp. :100-200nm, Bifidobacter sp. 400-500nm, Streptococcus thermofilus: 50-100nm. By the selection of bacteria used for production of high purity elemental red and grey selenium it was a very important factor to choose bacteria being already used in the food industry because our new product in this manner meets the strict quality requirements regarding for food supplements, additives. This technology seems to be more effective than the chemical synthesis, because it results relatively regular and uniform sized, high purity selenium spheres (100-500 nm, bacterium depending), production process is cheaper and faster or rather parameters can be controlled better. One of our future plans is to share this novel nanoselenium as food supplements, forage additives or plant nutritions.

3. Materials and methods We used flame emission atomic absorption spectrometer (Thermo ICE 3000) and atomic fluorescence

spectrometer (PSA Thermo, Excalibur) to determine the final selenium concentration of our ready nanoselenium samples. In sample preparation process in order to digest the samples we added 3ml cc. HNO3 and 5ml 30% H2O2 to 1ml sample and heated at 120oC for 60 minutes. We filtered the digested samples and adjusted up to 50ml with purified water. In the purified selenium samples we measured 200-500 mg/L selenium concentration, while in the liofilised LactoMicroSel samples we measured 1000-3000 mg/Kg concentrations. For detailed examination of our samples we used scanning electron microscope. Dr. Daróczi Lajos at the Department of Solid State Physics, University of Debrecen, made possible our experiments. Size of the nanoselenium was determined by our Particle Size Analyzer (Malvern, Mastersizer 2000).

4. Results

1. Picture

Electron microscopic picture of 250 nm sized selenium nanospheres

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4.1. Production of elemental (red) selenium As the results of our fermentation process we have two products, first is the purified elemental

nanoselenium, that is stored in purified water in the end, the second is the lacto selenium (LactoMicroSel) which is produced by lactic acid bacteria in the yoghurt making process. The basic principles of these processes are almost the same, with the only difference that the Lactomicrosel is not purified from the medium (milk) and the selenium spheres are not released from the bacteria cells, we utilize this product as yoghurt or yoghurt powder. Both methods are focused on red selenium production, but we are able to produce gray selenium with the help of Bifidobacterium bifidum.

4.2. Production of Lactomicrosel In the production of Lactomicrosel the applied medium was fresh milk. Before the innoculation the milk

was skimmed with an industrial milk centrifuge, the residual fat content of milk was less than 0.3 %. The selenium concentration of medium was set to 200 mg/l with NaHSeO3. The media was inoculated with a mixture of Lactobacillus acidophillus, Streptococcus thermophilus and Lactobacillus casei. The mixture was fermented on 37-38 oC for 24 hours. Till the end of the fermentation the color of suspension became red. The yogurt was centrifuged, and the solid phase was dried on 50 oC (2/A. Picture):. The dried red material was grinded and packed (2/B. Picture):. The selenium concentration of the obtained Lactomicrosel was measured by atomic absorption spectrometry that is usually 1200-3300 mg/kg.

4.3. Production of purified nanoselenium suspension (3/A. Picture): Culture medium used for culturing microorganism may contain carbon source, nitrogen source, inorganic

ions and, if necessary, other organic materials. The applied organic micronutrient source advantageously contains sufficient quantity of desired materials, such as vitamin B1 or L-isoleucine, or, alternatively, yeast extract max be used. In addition, potassium phosphate, magnesium sulphate, ferrous ions and manganous ions can be added. The medium may also contain buffer and antifoaming agent. Culturing is carried out under aerobic conditions for 4-24 hours, advantageously for 4-8 hours. Fermentation may be carried out in any known manner, for example by surface or submerged culturing. Incubation temperature may be between about 35 °C and 45 °C, depending on the employed strain. pH is adjusted to between about 5 and 8 for the culture duration. For adjusting the pH, inorganic or organic acidic or basic materials as well as ammonia gas may be used. According to an advantageous embodiment, a sterile solution of sodium selenite is added to the sterilized solution of agar-free culture broth for providing the sufficient selenium concentration. The commercially available media the MRS or M17 are suitable for the Lactic acid bacteria or the S. thermophilus respectively.

4.4. Fermentation Culture broth is incubated for 4-24 hours at the optimum temperature of the used bacteria, which is

advantageously 37 °C for lactic acid bacteria, and in the case of S. thermophilus, at 42 °C, 20 respectively.

4.5. Recovery and purification of selenium for the nanosize selenium suspension

2/A. Dried selenium rich yoghurt, 2/B. Grinded selenium rich yoghurt powder

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For the recovery of selenium from the medium and/or producing bacteria, bacteria are advantageously lysed enzymatically or killed by chemical (hydrochloric acid or sodium hydroxide) or physical means (freezing, drying or osmosis) to make the cellular content available. The most effective method was the hidrolysis with concentrated hydrochloric acid for 7 days on room temperature. The elemental selenium nanospheres can be recovered in pure form (3/B. Picture) by repeated aqueous washing, sedimentation or centrifugation.

5. Discussion Simplicity and rapidity of the technology and safety of the employed strains makes significant progress

over the art in producing selenium nanospheres. Homogeneity and quality of the shape and size of elemental selenium nanoparticles produced chemically by other technology used in the art does not come close to the characteristics of the material prepared by the fermentation technology of the technology using e.g. lactic acid bacteria. The process of the technology is suitable for producing elemental selenium nanospheres sized 100- 500 nm, wherein the size distribution of nanospheres is generally characterized by a percentage deviation from the mean size of 5-20%. Advantageously, the microorganism used in the process of the technology may be selected from the group consisting of the following species: Lactobacillus bulgaricus, Lactobacillus acidophillus, Bifidobacterium bifidum, Streptococcus thermophilus, Lactobacillus casei, Lactobacillus rhamnosus and Bifidobacterium longum.

Advantageously, when used as a food additive, the nanospheres produced according to the processes of the technology need not be fully purified or purified at all because the medium and the bacteria applied in the production process may be fully suitable for human or animal consumption. The technology further concerns isolated grey elemental selenium nanospheres directly obtainable by a process according to the technology. The technology also relates to the use of elemental selenium nanospheres obtainable by a process according to the technology in the food, microelectronic or optical industry.

6. Conclusions A novel technology for producing selenium nanospheres in homogeneous in form and size within a short

period of time (4-24 hours) has been developed. Elements with metallic properties have not been previously produced by fermentation using non-toxic aerobic bacteria, advantageously using bacteria permitted for use in the food industry. Selenium produced this way is exceptionally good raw material for forming nanosurfaces because of its homogeneous particle size distribution and regular, spherical shape. The technology developed is a manufacturing process which enables forming of a suspension as well as a powder containing valuable selenium spheres having unique characteristics. Material prepared in such a way can be used in the food industry as food or feed additive, in microelectronics as special semiconductor as well as in optical applications. The relative simplicity of the technology developed allows for significant decline in prices which can further broaden the range of useful high quality raw materials available.

7. Acknowledgements The authors wish to thank. Dr. Lajos Daróczi at the Department of Solid State Physics, University of

Debrecen for the scanning electron microscopic pictures and Kinga Nagy for the help in the atomic absorption spectrometer measurements, Nanofood Lab., University of Debrecen.

3/A Selenium spheres releasing from bacteria;

3/B Purified selenium suspension

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8. References [1] Am J Clin Nutr, Selenium and coronary heart disease: a meta-analysis. American Society for Nutrition

2006;84:762–73.

[2] Prokisch J., Zommara M. Vörös és szürke elemi szelén nanogömbök és technológia előállításukra (szabadalom), Magyar Szabadalmi Hivatal. 2008, NSZO: C12P-003/00

[3] Moon J. W., Roh Y., Lauf R. J., Vali H., Yeary L. W., Phelps T. J. Microbial preparation of metal-substituted magnetite nanoparticles. Journal of microbiological methods. 2007, 70(1), 150-58.

[4] Oremland R. S., Herbel M. J., Blum J. S., Langley S., Beveridge T. J., Ajayan P. M., Sutto T., Ellis A. V., Curran S. Structural and spectral features of selenium nanospheres produced by Se-respiring bacteria. Applied and environmental microbiology. 2004, 70(1), 52-60

[5] Wang H., Zhang J., Yu H. Elemental selenium at nano size possesses lower toxicity without compromising the fundamental effect on selenoenzymes: Comparison with selenomethionine in mice. Free Radical Biology & Medicine. 2007, 42, 1524–33.

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