Determination of Iron and Zinc Absorption by Local Isolates of Saccharomyces cerevisiae to Produce Iron and Zinc in Organic Form

Iron (Fe) and Zinc (Zn) play important role in health both of live stock and human. Fe and Zn in organic form were claimed increasing their viabilities. They bind to certain amino acid formed as a product of microbial metabolism. The Amount Fe and Zn absorbed may indicated the Fe and Zn organic produced. The aim of the study is to determine the absorption of microelement of Fe and Zn by local isolates of Saccharomyces cerevisiae to produce Fe and Zn in organic forms. S. cerevisae BCC F0205, BCC F0206, and BCC F0214 were treated with Fe or Zn 10 ppm to obtain S. cerevisae which has the highest of total concentration of Fe and Zn. Selected isolate was then treated with Fe or Zn respectively 2.5, 5, 10 ppm and their combination. Fe and Zn absorbed by isolates were measured by Atomic Absorption Spectrophotometer (AAS). The results show that S. cerevisae BCC F0205, BCC F0206, and BCC F0214 treated with 10 ppm Fe or Zn contained total concentration of Fe respectively 1.57, 3.07, 2.24 ppm and total concentration of Zn respectively 2.34, 3.20, 3.13 ppm. Then, S. cerevisae BCC F0206 treated with 2.5, 5, 10 ppm Fe or Zn, absorbed Fe 1.45, 0.50, 0.94 ppm and Zn 0.73, 0.38, 0.53 ppm respectively. Furthermore, combination of Fe and Zn (2.5:2.5, 5:5, 10:10 ppm) produce absorption of Fe 3.10, 2.13, 3.67 ppm and Zn 0.11, 0.10, 0.28 ppm per gram S. cerevisae BCC F0206. Percentages of absorption by S. cerevisae are up to 100% for Fe and up to 47.20% for Zn. In conclusion, this study indicated that S. cerevisae BCC F0206 absorb Fe and Zn higher than BCC F0205 and BCC F0214 and there are antagonistic interactions found between Fe and Zn in this research.


Introduction
Iron (Fe) is a micronutrient that has been used critically in blood cells and enzymes formations.In a body, around 55% of Fe form complex compounds with haemoglobin that play in metabolism process (King, 2006).In addition, Fe also form ferritin, transferrin and haemosiderin as nutrient reservation placed in hepatocytes in liver tissue (Nadadur et al. 2008).Other f u n c t i o n s o f F e a r e t o a c t i v a t e reticuloendothelial system that will prevent liver dysfunction (Knutson & Wessling-Resnick 2003, Maliken et al. 2013) and play in nervous and immune systems (Arredondo & Nunez 2005).If deficiency of Fe is occurred, it will cause anaemia and trigger other diseases due to there are interactions with heavy metals, lead (Pb) and cadmium (Cd), which will shift the binding of Fe with a number of proteins (WHO et al. 2001).
Not only Fe, but Zinc (Zn) is also a micronutrient that plays an important role in enzyme formations and metabolic process.More than 300 enzymes containing Zn are used in carbohydrate metabolism as source of energy and in catalytic system, especially on synthesis of DNA, RNA and protein (Sillerova et al. 2012).Then, Zn is also important in cell signalling, apoptosis, lipid metabolism, immune and brain system, as well as development and growth (Ackland & Michalczyk. 2006).If concentration of Zn in the body is less, it will cause problems in reproductive and immune systems (Widhyari, 2012;King, 2000, Kumar et al. 2011).Moreover, Zn deficiency will affect to decrease absorption of water and electrolytes substances by intestine and also to increase secretion that will cause dehydration (Roy et al. 2006).
To overcome deficiency of minerals has been done which is provision of salt mixed with feed.In other studies, administrations of mineral blocks are able to increase body weight of cow up to 370g/day compared to cows without mineral blocks (Addai, 2014).Currently, the application of Saccharomyces cereisiae is used widely for feed supplement.Perone et al. (2013), revealed that S cerevisiae could trigger bone development by measuring closure of distal radius growth plate and other parameters such as alkaline phosphatase, calcium and magnesium for some horses at 24 months of age.In other cases, provision of S cerevisiae in pigs (pregnancy lactation) could reduce weaning to estrus internal (WEI) and increase IgG in colostrum (Jang et al. 2013) while provision of S cerevisiae to laying hens improved egg weight and increased efficiency of feeding.S. cerevisiae could be applied as a medium for preparation of organic minerals which are more effective to absorb by livestock (Hegoczki, 1994).Therefore, the aim of study is to determine the absorption of microelement Fe and Zn by local isolates of Saccharomyces cerevisiae to produce Fe and Zn in organic forms.

Preparation of Saccharomyces cerevisae
All isolates of S. cerevisae were obtained from BBlitvet Culture Collection (BCC), namely BCC F0205, BCC F0206 and BCC F0214.Isolates were grown in Sabouraud dextrose agar (SDA) medium

Screening of S. cerevisiae isolates containing the highest concentration of Fe and Zn
Each S cerevisae was treated with 10 ppm of Fe or Zn and incubated at 37°C for 48 h.Then, the total of Fe or Zn per gram S. cerevisae was measured by AAS.Each treatment was made three replications.The isolate that had the highest concentration of Fe or Zn was selected for further experiment.

Optimization Fe or Zn absorption
Suspension of S. cerevisiae with 7 concentration 10 CFU /ml in 200 ml of PDB medium were treated as shown in Table 1, and incubated at 37°C for 48 h with shaking 150 rpm ,1 h for 4 times.The ability of selected isolate to absorb Fe and Zn was measured by AAS (Munawar, 2011).The absorbed concentration of Fe or Zn by selected isolate can be calculated as followed: 76 Biosfera 32 (2) Mei 2015

Determination of Fe and Zn concentration
S. cerevisae was collected by centrifugation at 3000 rpm for 15 minutes.These pellets obtained were weighed and soaked with 10ml concentrate nitric acid (HNO ) for 24 hours.The solutions were 3 heated approximately 2 -4 hours to obtain colorless solutions and 2 ml remaining volume.Furthermore, the solutions were filtered by Whatman filter paper No. 42, poured in a flask 10 ml, and added HNO 10% 3 until volume reached 10 ml.Finally, the solutions were transferred into a test tube and measured concentration of Zn and Fe by AAS (Munawar, 2011).

Screening of S. cerevisiae isolates containing the highest concentration of Fe and Zn
S. cerevisae was reported able to decrease aflatoxin B1 residue in chicken feed (Kusumaningtyas et al., 2006a;Kusumaningtyas et al., 2006b).The study was conducted to explore other potentials of S. cerevisae to change Fe and Zn in inorganic form to organic form by passing in S. cerevisae metabolism.Organic minerals from S. cerevisae are relatively more secure t h a n i n o r g a n i c m i n e r a l s a s f e e d supplements.Minerals in inorganic form are difficult to be absorbed by intestine because they can form complex with other substance and quickly secreted along with faeces.In other hands, organic minerals, called also bioplex, are more stable and will not interfere with absorption process of amino acids (Blazejak & 2004;Korniewicz et Reinhard al. 2007).
Naturally, S. cerevisae use Zn and Fe for cell growth and development.A single cell 7 needs 1.5 x 10 zinc atoms for growing optimally so that if the amount is less, the cell growth will be inhibited (Simm et al. 2007).S. cerevisae also releases Fe compounds proven by presence of additional Fe in various food products contaminated by S. cerevisae (Aguilar et al. 2009).Natural concentrations of Fe and Zn were also found in all isolates (Figure 1).The graphs show that each isolate has different natural content of Fe and Zn.Concentrations of Fe and Zn in all isolates are respectively around 0.72 to 2.66 ppm and 0.49 to 1.80 ppm.These differences of natural mineral content may cause by genetic and physiology of isolates.Unfortunately, the experiments conducted previously have not given satisfactory explanation of the differences of natural mineral content.Figure 1 show that S. cerevisae BCC F0206 content of highest of natural level of Fe and Zn with concentration Fe is 2.66 ppm and Zn is 1.80 ppm.After treatment, Fe and Zn in all isolates increased significantly at around 1.57 to 3.01 ppm for Fe and 2.34 to 3.20 ppm for Zn.This indicated that all isolates have ability to absorb Fe and Zn differently from media.S. cerevisae BCC F0205 is isolate which has the highest ability to absorb Fe and Zn (Figure 1).Enhancement of absorption is almost 2 times for Fe (0.72 ppm to 1.57 ppm) and almost 5 times for Zn, (0.47 ppm to 2.34 ppm).However, factually, total concentration of Fe and Zn in BCC F0205 is still lower than other two isolates.For instance, BCC F0206 contained total concentration 3.07 ppm for Fe and 3.20 ppm for Zn, and BCC F0214 contained total concentration 2.24 ppm for Fe and 3.13 ppm for Zn .Thus, BCC F0206 is the most potent to generate organic form of Fe and Zn because this isolate has natural content and absorption ability was higher than the others.S. cerevisae BCC F0206 was used in the next treatment to find out its maximum absorption by addition various concentration of Fe and Zn to medium.

Absorption ability of S. cerevisae with various concentrations of Fe and Zn in medium
In figure 2, the graph show that SFe1 absorbed Fe around 0.64 to 2.65 ppm (mean 1.45 ppm ).These ranges of concentrations are higher than the other treatments, SFe2 (0.03 to 1.11 ppm, mean 0.50 ppm) and SFe3 (0.02 to 1.69 ppm, mean 0.94 ppm), although the existing concentration of Fe in media is lower than both SFe2 and SFe3.Therefore, S. cerevisae BCC F0206 absorbed Fe optimally in media containing Fe 2.5 ppm.Then, in figure 2, S. cerevisae BCC F0206 is able to absorb Zn from media.SZn1 absorb Zn higher than SZn2 and SZn3.SZn1 absorbed Zn from 0.38 to 1.18 ppm (mean 0.70 ppm ), but for SZn2 and SZn3 respectively capable to absorb from 0.25 to 0.50 ppm (mean 0.38 ppm) and from 0.39 to 0.79 ppm (mean 0.53 ppm).Overall, BCC F0206 absorbed Zn optimally in media containing Zn 2.5 ppm.
Figure 3 Mean of Zn concentration absorbed by S. cerevisae BCC F0206 from media PDB containing Zn : 2.5, 5, 10 ppm According to the results, the availability concentration of Fe or Zn in media can affect the absorption of Fe and Zn by S. cerevisae BCC F0206.Varieties of these data are strongly influenced not only by genetic and physiology of isolate but also by environment conditions such as mineral content in media.

Effect of interaction between Fe and Zn to absorption
In this part, the experiment did further to determine whether interaction is synergistic or antagonistic.Figure 4 show that the more concentrations of Fe and Zn were added in media, the more concentration was absorbed by S. cerevisae BCC F0206.The ranges of absorbed concentrations are from 3.10 to 3.67 ppm for Fe and from 0.11 to 0.28 ppm for Zn.However, Fe or Zn was absorbed differently when they were added together into media compared with they were added separately.The highest absorption of Fe in single treatment is 1.67 ppm while the highest absorption of Fe in combination treatment is 3.67 ppm obviously there is enhancement more than 2 times.In other hand, in Zn cases, the highest absorption of Zn in single treatment is 0.79 ppm while the highest absorption of Fe in combination treatment is 0.28 ppm so that there is decline more than 3 times.These results show that there are antagonistic interactions between Fe and Zn during absorption.In addition, the same results were also reported by Evans & & Johnson (1980); Gordon (1983) that supremacy of Fe can inhibit Zn but others reported that there is no interaction between Fe and Zn (Momcilovic at al. 1977;Gruden & Momcovic 1979;Pallauf & Kirchgessner 1974;Fairweather-Tait et al. 1984) o and incubated at 25 C for 48 h.Furthermore, each isolate was inoculated into 200 ml of Potato dextrose broth (PDB) medium and o incubated at 25 C for 48 h.Concentration of Fe and Zn per gram S cerevisae naturally was measured by AAS.Then, concentration 7 of S. cerevisae was adjusted to 10 CFU/ml in 200 ml of PDB medium for next treatments.

77Figure 1
Figure 1 Total concentrations of Fe (a) and Zn (b) from S cerevisae naturally and after treatment with 10 ppm of Fe or Zn

Table 1
Composition of treatment on selected isolates ConclusionSaccharomyses. cerevisae BCC F0206 absorb Fe and Zn higher than BCC F0205 and BCC F0214.The percentages of Fe absorbed are up to 100% and Zn absorbed is up to 47.20% by S. cerevisae BCC F0206.There are antagonistic interactions between Fe and Zn in this study so to obtain absorption of Zn optimally, it should be better added separately. Figure 4. Mean of Fe and Zn concentration absorbed by S. cerevisae BCC F0206 from media PDB containing combination Fe and Zn: 2.5: 2.5, 5:5, 10:10 ppmTable 2 percentages of absorption of Fe and Zn by S. cerevisae BCC F0206