Development of a zinc-enriched yeast strain for nutritional applications using low-cost ingredients

Human health is increasingly threatened by diseases caused by deficiencies in trace elements, essential for maintaining health. The World Health Organization (WHO) classifies trace elements into three categories: essential (e.g., iodine, iron, copper), important but less critical (e.g., manganese, silicon), and rare but potentially toxic (e.g., fluorine, lead). These elements are vital in gene regulation, nucleic acid metabolism, and other physiological functions¹. Food alone cannot meet these nutritional needs, necessitating supplements to maintain a balanced intake of trace elements, especially for children who require more micronutrients².

Currently, over two billion people suffer from micronutrient deficiencies. Zinc is a vital micronutrient in protein, lipid, nucleic acid metabolism, gene transcription, reproduction, immune function, and wound healing. It affects macrophages, neutrophils, natural killer cells, and complement activity at the cellular level. Zinc is a cofactor for many enzymes involved in various biochemical reactions. It plays a crucial role in supporting enzyme activity and catalyzing chemical reactions in foods such as meat, fish, legumes, and nuts; its absorption varies by carrier³. Zinc deficiency is a widespread health issue, particularly in developing countries. Currently, two main supplements are available on the market: inorganic and organic. Trace elements in inorganic forms, such as zinc sulfate, zinc oxide, and zinc chloride, are often mineral salts⁴. It is challenging to produce them on a large scale, and they are unstable with low zinc digestibility. Microorganism-derived organic zinc supplements usually refer to supplements that contain zinc obtained from organic sources such as yeast, plants, or other microorganisms. These supplements are often marketed as natural or herbal alternatives to mineral zinc supplements. In general, compared to mineral zinc supplements, they have higher bioavailability⁵. Additionally, organic zinc supplements are generally easier to digest and are less likely to cause digestive issues. In recent years, microorganisms, particularly yeasts, have been utilized to produce organic trace element supplements that are more digestible and absorbable. These supplements are also cost-effective to produce and can efficiently replace other inorganic supplements. Yeasts can absorb metals into their cell structure through absorption and bioaccumulation⁶. Yeast cells possess specific transporters and mechanisms that enable them to actively absorb and accumulate essential trace elements, thereby maintaining their intracellular concentration within an optimal range⁷. For yeasts to utilize this mechanism, they must possess the capability to grow and survive in environments with high concentrations of zinc metal. A low-cost culture medium with a readily available carbon and nitrogen source is also crucial for yeast growth⁸. In a study conducted by Maaria, zinc-enriched yeast was produced as a food supplement by Saccharomyces cerevisiae. They compared the in vitro model of zinc digestion released from yeast with intestinal zinc digestion. They concluded that the bioavailability of organic zinc is significantly higher than that of inorganic zinc, and zinc released from yeast was available for biological processes within enterocytes, leading to the regulation of metallothionein mRNA. Various factors affect the production of zinc-rich yeast⁹. Esmaeili and Davoodi studied the output of selenium-enriched yeast using the Plackett–Burman design method. They investigated the effects of different culture conditions, including temperature, stirring speed, fermentation time, inoculum rate, selenium source concentration, and the timing of selenium addition. The findings indicated that temperature, stirring speed, fermentation duration, inoculation rate, and selenium source concentration all play a role in the efficiency of selenium biotransformation and the formation of organic selenium¹⁰. Some research has demonstrated that the zinc found in zinc-enriched yeast has greater bioavailability than other supplements, such as zinc sulfate and zinc gluconate. In a recent study involving two groups of mice, one group was randomly treated with zinc-enriched Candida tropicalis yeast, while the other received zinc gluconate. Blood tests and their analysis revealed that zinc was present in C. tropicalis sp. T-A exhibited better bioavailability than zinc gluconate in rats¹¹.

This study aimed to identify a zinc-tolerant yeast strain capable of high intracellular zinc accumulation and optimize its biomass production using low-cost carbon and nitrogen sources. Our findings highlight the potential of a yeast-based approach to serve as a sustainable and cost-effective source of organic, fermented zinc supplements, which may offer superior digestibility and absorption compared to conventional mineral fortification methods.

Culture conditions and bioaccumulation analysis

50 yeast strains from the microbial collection of Environmental Biotechnology Laboratory, University of Tehran, Iran were obtained. These strains were previously isolated from food and environmental sources. In this study, the yeasts were screened for growth ability and resistance in culture media containing different concentrations of zinc metal. Yeast strains were cultured in Yeast extract Peptone Dextrose (YPD) agar medium containing glucose 20 g/L, peptone 20 g/L, yeast extract 10 g/L, agar 20 g/L, along with zinc metal salts (Merck, Germany) in concentrations of 2000, 3000, 4000, and 50 g/L¹². Note: At this stage, extremely high zinc concentrations (50 g/L) were intended for preliminary screening to identify isolates with exceptional tolerance. In subsequent bioaccumulation and optimization experiments, more biologically relevant concentrations (1 to 2 g/L) were applied. The yeast strains were incubated for two days at 32 °C. Those yeasts that grew in different concentrations of zinc metal were selected and then cultivated in YPD broth medium for 48 h at 32 °C and centrifuged at 4000 rpm for 15 min. Their dry weight was measured, and the yeast with the highest biomass dry weight was selected for further analysis¹³.

The bioaccumulation of zinc metal in yeasts was measured using an Inductively Coupled Plasma Mass Spectrometry (ICP-MS) assay. This method involved culturing the yeast strains in a YPD medium containing 2 g/L of zinc salt. The cultures were incubated in a shaker incubator at 30 °C and 150 rpm for 48 h. After incubation, the cell biomass was harvested by centrifugation. The supernatant was collected, and the remaining biomass was washed twice with deionized water.

The washed biomass was treated with 5 mL of 1 M hydrochloric acid (HCl) to measure zinc metal accumulated in yeast. This mixture was then heated in a water bath (bain-marie) at 40 °C and 60–70 °C, each for one hour. The concentration of the extracted zinc metal was then measured using an ICP-MS device¹⁴. Transmission electron microscopy (TEM) was used to observe zinc accumulation in yeast cells. To prepare yeast cells, strains were grown in a YPD medium containing 2 g/L of zinc chloride and incubated at 32 °C for 2 days. The biomass was then collected by centrifugation at 8000 g for 15 min and washed twice with phosphate buffer. The biomass was fixed by adding 2% (v/v) glutaraldehyde in a 1.5 mM phosphate buffer and incubated for 24 h. Thin sections were prepared by embedding the samples in LR White resin. The samples were then analyzed using a transmission electron microscope (Philips EM208S, Netherlands) operating at 100 kV¹⁵.

Molecular identification of selected strain

For DNA extraction, the selected yeast was cultured in YPD broth for 24 h. The microbial biomass was collected and washed twice with distilled water. The DNA was extracted using a CinnaGen extraction kit (Iran). Polymerase Chain Reaction (PCR) was used to amplify the ITS1-4 gene using the ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) primers¹⁶. The samples were then sent to Microsynth (Switzerland) for Sanger sequencing. The DNA sequences were compared to the Gene Bank database using the BLAST search tool to identify the yeast strain¹⁷.

Selecting the appropriate sources of carbon, nitrogen, and salts

The effects of different carbon sources (molasses, sucrose, dextrin, mannose) and nitrogen sources (corn steep liquor, yeast extract, peptone, ammonium nitrate), as well as mineral salts (MgSO₄, K₂HPO₄), were assessed on the production of cell biomass for the selected yeast strain¹⁸. The experiment was conducted in 50 mL Erlenmeyer flasks, using 20 mL of various prepared culture medium combinations. The experiment was conducted in two replicates, following the described methodology.

The goal was to determine which combinations of carbon sources, nitrogen sources, and mineral salts had the most tremendous impact on the growth and biomass production of the yeast strain under investigation. The optimal carbon and nitrogen sources were selected based on the biomass produced, measured by dry weight, and compared to the YPD culture medium. Additionally, a comparison was made between the growth of the selected yeast strain and that of Saccharomyces cerevisiae S288C, which served as a control in both YPD medium and the medium with selective carbon and nitrogen sources.

Saccharomyces cerevisiae was chosen as a scientifically relevant reference because it is the industry standard for producing enriched yeast supplements. It has GRAS (Generally Recognized as Safe) status, and its mechanisms of zinc homeostasis are well-documented in the scientific literature. This comparison provides a benchmark to evaluate the practical potential of the newly isolated strains¹⁴.

Culture medium optimization using BBD type’s response surface method (RSM)

To investigate the independent variables that influence culture media, including carbon source, nitrogen source, and mineral salts, a Response Surface Methodology (RSM) approach was employed using a Box-Behnken Design (BBD). This analysis was conducted using Design Expert software (version 12). All experimental runs were prepared with a volume of 20 mL in 50 mL flasks, following the values determined by the software. The cultures were incubated at 32 °C with a shaking speed of 150 rpm for 48 h, after which the dry weight of the biomass was measured. All results were recorded in the software for subsequent analysis¹⁹.

The biomass dry weight obtained from each experimental run was entered into the Design Expert software to determine each variable’s optimal point and perform further analysis. The software generated equations for each response variable, regression coefficients, standard deviation, accuracy, p-values, three-dimensional plots, and contour charts. These were examined to compare the effect of each variable and the interactions between the variables²⁰.

Investigating the bioavailability of zinc metal by gastric and intestinal simulation

A simulation was conducted to investigate the bioavailability of zinc metal in the stomach and intestinal environment. A 20 mL solution containing 0.5 g/L of pepsin was prepared in a 0.5% w/v saline mixture for the stomach-simulating solution. The pH of this solution was then adjusted to 2 using 1 M HCl²¹. A separate solution was prepared to simulate the intestinal conditions, containing 0.1% pancreatin and 0.8% bile salt in a 0.5% w/v mixture. The pH of this intestinal simulation solution was adjusted to 7.5. These two solutions, mimicking the stomach and intestinal environments, were used to assess the bioavailability of zinc metal under these differing physiological conditions. To determine the amount of zinc released from the selected yeasts in the simulated medium of the stomach and intestines, 0.2 g of the dried yeast powder was added to 20 mL of the simulated medium of the stomach and placed on a shaker incubator with a speed of 150 rpm and a temperature of 30℃. It was placed for seven hours and sampled in four-hour and seven-hour intervals^21,22.

To investigate the bioavailability of zinc-rich yeast in the intestinal environment, the supernatant was discarded, and the remaining biomass was washed twice with distilled water. Then, the yeast was inoculated into the intestinal simulator’s environment. After five hours of incubation at 30℃ with a speed of 150 rpm, a sample was prepared from the solution. This sample and those prepared from the stomach simulator solution were then sent for ICP-MS measurement.

Examining the toxic effects of zinc-enriched yeast using the MTT assay

Saccharomyces cerevisiae and a selected strain were inoculated in a YPD broth culture medium. The cultures were incubated for 48 h in a shaker incubator set at 150 rpm and a temperature of 30 °C. Following incubation, the cell pellets were dried at 50 °C for 24 h to remove residual moisture. Once dried, the yeasts were ground into a fine powder. A sample of 0.2 g of this powdered yeast was then prepared for the MTT assay to assess cell viability and metabolic activity⁹. The MTT assay was performed according to Kumar’s method²³ on the HUVEC cell line.

Statistical analysis

The results of this study were analyzed statistically using Design Expert (version 22). Data are presented as mean ± SD in the tables, and error bars in the figures indicate the standard deviation. The ANOVA test assessed differences between means, with a significance threshold set at p < 0.05.

Among the 50 yeast strains tested, some yeasts could grow in a culture medium containing zinc metal. Among all, two yeasts, S9 and S46, were selected for their ability to grow in 2000 and 1 g/L ZnCl2 concentrations and because they had the highest amount of biomass.

Bioaccumulation capacity and the ability of strains to absorb zinc

Among the examined strains, S. cerevisiae and the novel strains S46 and S9 showcased varying capacities for zinc absorption. Strain S46 demonstrated a remarkable ability to accumulate approximately 700 ± 60 mg of zinc per gram of dry biomass (700 ± 60 mg/g) of zinc in its cells. Studies on S. Cerevisiae have reported zinc accumulation ranging from 100 to 500 ± 30 mg/g, depending on the strain and growth conditions. For example, a study by Wang et al. demonstrated that engineered S. cerevisiae strains could accumulate up to 500 ± 30 mg/g of zinc under optimized conditions²⁴. While most studies have focused on S. cerevisiae, research on novel yeast strains is limited. However, a study by Li et al. identified a marine yeast strain (Rhodotorula mucilaginosa) capable of accumulating up to 600 mg/g of zinc, highlighting the potential of non-conventional yeasts for bioaccumulation. S46, with an accumulation potential of 700 mg/g, exceeds this amount, underscoring its potential for industrial applications. This absorption level indicates a highly efficient uptake mechanism, positioning S46 as a promising candidate for the production of enriched yeast. The results suggest that yeast’s mechanisms underlying zinc uptake in yeasts are mediated by both high- and low-affinity transport systems. In S. cerevisiae, zinc import across the plasma membrane is facilitated by the ZRT1 and ZRT2 transporters, whereas intracellular sequestration occurs through vacuolar transporters such as Cot1p and Zrc1p. Although the molecular transporters responsible for Candida sp. pz46 have not yet been identified, the enhanced zinc accumulation observed in this strain indicates the likely involvement of highly efficient zinc-binding proteins and vacuolar storage pathways²⁵.

TEM was used to investigate the intracellular accumulation of zinc metal in yeast. In these yeasts, zinc metal is observed within the yeast cell as a point (Fig. 1). Additionally, the dense granules of zinc metal are primarily located in the cytoplasmic region, indicating that zinc metal is in transit. It has entered the cell through the selectively permeable membrane, and the yeast has absorbed it intracellularly. Zinc was transported into the cytoplasm through different layers (peptidoglycan, teichoic acids) of the cell wall and cytoplasmic membrane, and dense granules gradually accumulated inside the cell.

The findings from TEM highlight the intricate processes through which zinc enters yeast cells. Identifying zinc particles indicates successful translocation across the selectively permeable membrane, demonstrating the yeast’s ability to absorb essential minerals effectively. This uptake process involves navigating through the layers of the cell wall, including peptidoglycan and teichoic acids, showcasing the yeast’s complex structural adaptations for nutrient acquisition. The observed accumulation of zinc granules within the cytoplasm suggests that yeast not only absorbs this metal but also stores it for future metabolic needs. Clemens et al. reported that excess zinc is sequestered in vacuoles to prevent toxicity, while cytoplasmic zinc is utilized for metabolic processes²⁶. This observation of cytoplasmic zinc granules supports these findings and suggests that S46 may have efficient mechanisms for both storage and utilization (compare with control cells in Supplementary data, Figure S1).

Molecular identification of the selected strain

The selected S46 strain was identified using specific primers for the ITS1-4 region. The results showed that this strain is 98% similar to Candida viswanathii ATCC 22981. Considering this result, the S46 strain was registered in the database under the name Candida sp. pz46 and the accession number of PP982893.

Growth optimization on the low-cost substrates

The initial investigation focused on biomass production in YPD broth, a standard culture medium for yeasts (Fig. 2). For Candida sp. pz46, the dry weight of biomass produced in YPD broth was measured at 2 g/L. Among various carbon sources, the highest cell biomass, 5.05 g/L, was achieved using sugarcane molasses. The high sugar content and growth-promoting compounds in molasses likely contributed to this enhanced growth, indicating that Candida sp. pz46 has a strong capacity for utilizing complex carbohydrates. In a study by Silva et al., the use of sugarcane molasses for S. cerevisiae cultivation was investigated, and biomass yields of 4.8 g/L were reported, closely aligning with our findings. The study highlighted molasses as a cost-effective and sustainable carbon source due to its rich nutrient profile²⁷.

Regarding nitrogen sources, the culture medium containing corn steep liquor (CSL) yielded the highest biomass production, reaching 3.05 g/L. A comparison of biomass production in environments with different carbon and nitrogen sources, relative to the common YPD broth, revealed significant increases. Specifically, the biomass in the medium with sugarcane molasses increased by 152.5%, while the medium with CSL saw a 52.5% increase. CSL is known for its rich nutrient profile, including amino acids and vitamins essential for yeast growth. The significant improvement in biomass production when using CSL suggests that optimizing nitrogen sources can further enhance the growth performance of Candida sp. pz46. In 2020, Papizadeh et al. demonstrated that sugarcane molasses and CSL could replace synthetic media for yeast cultivation without compromising growth performance. Their results, showing biomass yields of 5.0 to 5.3 g/L, are consistent with the present study’s observations²⁸.

Furthermore, the analysis indicated that Candida sp. pz46 and S. cerevisiae exhibited better growth in sugarcane molasses and CSL than in the standard YPD medium (Fig. 1). These results advocate for a paradigm shift in yeast cultivation practices, emphasizing the potential of utilizing agricultural by-products like sugarcane molasses and CSL.

To further enhance biomass production, a Box–Behnken Design (BBD) was employed, considering three independent variables: carbon source concentration (sugarcane molasses, 30 to 60 g/L), nitrogen source concentration (Corn steep liquor, 20 to 40 g/L), and mineral salts (K₂HPO₄ and MgSO₄, 0.05 to 0.20 g/L each). The experimental design comprised 27 runs, each performed in duplicate, with biomass dry weight as the response variable.

The Design Expert software’s analysis revealed that the best-proposed model, according to the model table, was a linear equation (quadratic) (Table 1).

A p-value < 0.05 at the 95% confidence level indicates the significance of the statistical model. The mean square is high in this case, indicating that this model has covered most of the data. Also, the non-significance of the lack of fit of the model (Lack of fit p-value > 0.05) indicates the appropriateness of the model. In this proposed model, the lack of fit p-value is equal to 0.2321, and the p-value is less than 0.05, which indicates the significance of the model.

The F-value of the model is equal to 20.87, which is appropriate and significant, and there is only a 0.01% chance that this large F-value is caused by noise. P-values ​​less than 0.05 indicate that the model is appropriate and significant. The p-values ​​are generally significant for the model and not significant for the lack of fit, indicating the model’s significance and acceptability. In this case, the p-value is substantial for models A, B, C, CD, and B2 (Supplementary data, Table S1).

Usually, an R2 or R-squared above 0.7 or 70% indicates a relatively good correlation coefficient, and the closer this value is to 1 or 100%, the greater the compatibility of the experimental data and the more accurate the model. In general, R2 determines the quality of fitting the experimental data with the model, which means the model’s correctness can be understood.

Model adequacy diagrams are provided in the supplementary data (Figures S2 to S8).

Sugarcane molasses has the most significant impact on biomass production and directly affects it. As its concentration increases, biomass production reaches its highest concentration among other parameters. A study by Oliveira et al. demonstrated that sugarcane molasses significantly enhanced biomass production in S. cerevisiae, with yields increasing proportionally to molasses concentration up to a certain threshold. This aligns with this study’s observation that molasses concentration directly affects biomass production²⁹.

Besides the carbon source, the nitrogen source has the most significant effect on cell biomass production and directly affects it. K₂HPO₄ salt has a negligible impact on increasing biomass production, and according to the graph, at concentrations above 0.14 g/L, it does not have such an effect on increasing biomass production. MgSO₄ salt has no special effect on increasing cell biomass production. However, with the increase in amount, there was a minimal increase in biomass (Fig. 3). Li et al. (2020) reported that MgSO₄ had a negligible effect on biomass production in S. cerevisiae. The study suggested that MgSO₄ is more critical for maintaining cellular functions than directly promoting growth³⁰.

To achieve the maximum cellular biomass, the optimal conditions involve using a carbon source at 55 g/L and a nitrogen source at 35 g/L. Additionally, salts such as K₂HPO₄ and MgSO₄ should each be used at a concentration of 0.15 g/L. Under these conditions, biomass production can reach 14.49 g/L. This optimal formulation highlights the potential for significant advancements in yeast cultivation practices, especially in utilizing agricultural by-products like sugarcane molasses to enhance biomass yield sustainably. A 2019 study by Zhang et al. optimized carbon and nitrogen concentrations for S. cerevisiae growth and found that 50 to 60 g/L of carbon source (glucose) and 30 to 40 g/L of nitrogen source (yeast extract) yielded biomass concentrations of 12 to14 g/L, which is similar to the findings of this study³¹.

This study highlights the intricate relationships between nutrient sources and biomass production in Candida sp. pz46. The findings advocate for a strategic approach to nutrient optimization, emphasizing the importance of carbon and nitrogen sources while recognizing the limited roles of certain salts. Establishing these optimal conditions can enhance the efficiency of yeast cultivation, paving the way for more sustainable biotechnological applications and improved yields in commercial settings.

Bioavailability of zinc in the digestive environment

Sampling was done four and seven hours after staying in the stomach environment, and the atomic absorption was read. Table 2 shows the results of the atomic absorption of zinc metal at specific time intervals in the simulator environment.

Zinc release from Candida sp. pz46 was rapid and substantial in simulated gastric and intestinal environments. After four hours of introducing the enriched yeasts into a gastric environment (pH 2.0, with pepsin) and comparing them with control samples, it was observed that more than 77 ± 2% of the accumulated zinc in the yeast cells was released during this exposure, compared to only 26 ± 5% in S. cerevisiae. The release continued gradually, reaching over 85 ± 3% after 7 hours. this controlled release reflects a dual-phase mechanism of zinc mobilization in yeast, beginning with the passive release of surface-bound zinc under acidic conditions. This is followed by the enzymatic and pH-dependent degradation of intracellular compartments, which gradually liberates zinc stored in the vacuoles. The release suggests that the enriched yeasts can effectively deliver essential minerals in a form that the body can readily absorb. This prolonged-release mechanism may help maintain adequate zinc levels in the bloodstream over an extended period, reducing the need for frequent supplementation. The controlled release profile of zinc from the Candida sp. pz46 biomass observed in this study presents a potential advantage over conventional inorganic zinc supplements. In contrast to the rapid, burst release typically reported for inorganic zinc salts, such as zinc sulfate, which can lead to a sharp spike in bioavailability and potential gastrointestinal discomfort, our yeast-based zinc demonstrated a more gradual and sustained release. This modulated release pattern is likely due to the intracellular nature of the zinc, which requires enzymatic and pH-dependent degradation of the cell wall and organelles to be liberated.” A 2021 study by Grešáková et al. demonstrated that zinc from yeast-based delivery systems had higher bioavailability than inorganic zinc supplements, with 70 to 85% of zinc absorbed in simulated gastric conditions³². For this reason, yeast-based delivery systems effectively improve zinc bioavailability and have significant potential for nutritional supplementation.

Toxicity analysis

MTT assay was used to investigate the toxicity of yeasts (Table 3). For Candida sp. pz46, the survival rate of the cell line exposed to yeast enriched with this strain at concentrations of 0.12 and 0.25 mg/mL and above is nearly 100%, matching the survival rate of the control group. This indicates that the exposure to the enriched yeast was not toxic to the human endothelial cells. A decline in viability at higher doses (≥ 0.5 mg/mL) is likely attributable to non-specific effects from the high biomass concentration in the in vitro environment, which exceeds projected supplemental intake levels. The key outcome is the demonstrated safety at physiologically relevant doses, supporting the biomass’s potential for further development as a food-grade supplement. Ferrari et al. reported that selenium-enriched yeast showed no cytotoxicity at concentrations up to 0.5 mg/mL, further validating the non-toxic profile of enriched yeast³³. Dadkhodazade et al. also confirmed that yeast-encapsulated nutrients are generally recognized as safe (GRAS) by regulatory agencies, supporting our conclusion³⁴. This indicates that Candida sp. pz46 does not exert harmful effects on the cell line, even at elevated concentrations. This non-toxic profile is crucial for any food supplement intended for consumption. The cell line’s ability to thrive in enriched yeast suggests it is safe for ingestion, alleviating concerns about adverse reactions or toxicity. Given the non-toxic nature of Candida sp. pz46, it could be a candidate for nutritional supplementation.

This non-toxic profile is crucial for any food supplement intended for consumption. The ability of the cell line to thrive in the presence of enriched yeast suggests that it is safe for ingestion, thereby alleviating concerns about adverse reactions or toxicity. Given the non-toxic nature of Candida sp. pz46, it stands as an excellent candidate for nutritional supplementation.

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Authors and Affiliations

1. Department of Microbiology, School of Biology, College of Science, University of Tehran, Tehran, Iran

   Pegah Zare, Minoo Giyahchi, Tabasom Entezari & Hamid Moghimi

Contributions

H.M. designed and supervised the project. P.Z. performed the experiments and analyzed the data, M.G. provided scientific consultation, P.Z. and M.G. wrote the paper, and T.E., M.G., and H.M. edited the manuscript. All the authors read and approved the final manuscript.

Corresponding author

Correspondence to Hamid Moghimi.

Competing interests

The authors declare no competing interests.

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