تاثیر غلظت‌های مختلف روی آلی و معدنی محیط کشت بر رشد و محتوای روی در مخمر نانوایی (Saccharomyces cerevisiae)

Introduction

Development of the aquaculture industry is not possible without the use of live foods, especially those used in the early stages of larvae life, such as algae, yeasts, Artemia, rotifers and daphnia (1). Yeasts are mono-cellular fungi, without rashes and are seen in spherical and elliptic forms. Different types of yeasts, such as Saccharomyces cerevisiae, Candida, Rhodotorula, Dipodascus capitatus, and carotenoid yeast (Phaffia rhodozyma) are used to feed a variety of aquatic species, mass culture and enrichment the live food, such as rotifer and Artemia (2). Yeasts are the most important and mostly-used microorganisms in the food industry for production of yeast extracts (3). Typically, yeasts are used alive for the feeding of live food or used as an additive food in the aquaculture industry (4).

Saccharomyces cerevisiae due to the proper cell diameter (4-5µm), desirable nutritional value (a suitable source of proteins, vitamins, nucleotides and polysaccharides), cellular resistance, lack of leakage the organic soluble materials in the medium, easy preparation and affordable prices for aquaculture, especially live food, has been widely used in research. Also in certain physicochemical environments, yeasts are able to accumulate various minerals and form organic bonds at concentrations higher than normal (5). Baker's yeast is used as an interface to deliver nutrients to Artemia and other live foods. Adding minerals to baker's yeast makes it possible to produce live food containing sufficient amounts of minerals (6).

Zinc accumulation in yeast occurs in two steps. The first stage of bioavailability or inactive adsorption is related to accumulation on the cell wall surface. The second stage is the active accumulation or absorption stage, which is associated with intracellular metabolism and intracellular penetration of ions by cell membrane and cellular circulation (7).

Although only a small amount of minerals is present in the body, zinc is known as an essential ingredient in aquatic nutrition and acts as a cofactor in many active enzyme systems. Therefore, the activity of aldolase, peptidases, and phosphatases that interfere with digestion is dependent on zinc (8). Zinc is a significant ingredient in larvae, because it is necessary for the growth, development and maintenance bone structure and its deficiency causes bones deformity. The zinc requirements of fish are 15 to 40 mg kg^-1 of zinc in diets (9). This amount is not available in live foods for larvae (10). Live foods such as zinc-enriched Artemia and rotifer can improve growth and prevent stress in nourished fish (11). Based on the need for minerals in live foods, yeast can be used as an interface for maximizing these materials. Zinc is also an essential element for the growth and metabolism of Saccharomyces cerevisiae. Zinc deficiency in the yolk formation stage prevents growth and causes the cells to stop in the G1 phase and in this case, Zn recruitments of yeast cannot be compensated by other metal ions. Zinc in metabolism plays a role in the production and operation of many enzymes, proteins, nucleic acids, gene expression and cellular immune development (12). Therefore, in the present study, the effect of different concentrations of organic (Zinc-threonine) and inorganic (ZnSO₄.7H₂O) zinc on the growth and zinc content of yeast (Saccharomyces cerevisiae) was investigated.

Materials and methods

Preparation of the materials: Saccharomyces cerevisiae purchased from Iranian Biological Research Center. Yeast extract, Glucose, inorganic zinc (ZnSO₄.7H₂O) and K₂HPO₄ were obtained from Jahan Kimia Company in Urmia. Threonine amino acid was purchased from Aryan Roshd Company.

Zinc-threonine preparation (organic form of zinc): Zinc-threonine was obtained by mixing the threonine amino acid with zinc sulfate. The solution of ZnSO₄.7H₂O (50.6 mg zinc sulfate in 6 ml of 50% ethanol) was added as a dropper to a threonine solution (5 ml ethanol 50%, final concentration of 0.016 mol.l^-1). Then, 100 μl of water was added to it, stirred continuously for an hour at room temperature, and finally at 8000 rpm the solution was centrifuged for 15 minutes (13).

Enrichment of Yeast: The yeast culture medium (YEPD) was papered with yeast extract (2%), glucose (5%) and K₂HPO₄ (1%) and after adjusting the pH to 5.5, the culture medium was autoclaved at 121°C for 15 minutes (13). Organic and inorganic zinc forms were used to enrich yeast in YEPD medium. In summary, 10 g of yeast was grown in 1000 ml of YEPD culture medium at pH 8.5 and then incubated in an incubator shaker at 160 rpm at 27.4 ° C for 24 hours. After 24 hours of yeast incubation, two organic and inorganic zinc forms with 3 concentrations (2.5, 5 and 7.5 g L^-1) were added to the sterile culture medium of yeast and the incubation for another 24 hours continued with the same conditions. Then, the sterile medium was centrifuged with 3000 rpm for 15 minutes and washed twice with sterile physiological serum to remove the excess. The enriched yeast was then stored at -20°C (14).

Experimental design: In the present study, the effect of organic zinc threonine (C₈H₂₀N₂O₈Zn) and inorganic zinc sulfate (ZnSO₄.7H₂O) on the growth and yeast enrichment in the 7 treatments including 1) control (yeast without adding zinc source), 2) yeast + 2.5 g L^-1 zinc sulfate, 3) yeast + 5 g L^-1 of zinc sulfate, 4) yeast + 7.5 g L^-1 of zinc sulfate, 5) yeast + 2.5 g L^-1 zinc threonine 6) yeast + 5 g L^-1 zinc threonine and 7) yeast + 7.5 g L^-1 zinc threonine on growth and enrichment of yeast were investigated. 

Evaluation the yeast cells: After the preparation of the YEPD culture medium, the yeast was added. The number of yeast cells was immediately counted by hemocytometer lam. Also, 24 hours after adding zinc, the number of cells in the samples was counted (13).

Analysis procedure: 0.1 gram of Zn-enriched yeast was poured into a 200 ml flask and then 3 ml of concentrated nitric acid was added until the steam evolved (indicating the digestion of the sample), then nitric acid added to 10 ml. The amount of absorbed zinc in the yeast cell was measured by atomic absorption (15).

Statistical analyses: Data were analyzed by SPSS statistical software (version 21) using 2-way ANOVA. Levene's test were used for checking the homogeneity of variances (P≤0.05). Significance level between treatments was tested by Tukey's honestly (HSD) post-hoc test. Differences among means were considered at P≤0.05 and the data were shown as mean ± SD.

Results

Dtermination of yeast growth in YEPD medium before adding zinc: The number of yeast cells at the first 24 hours after culture is shown in Figure 1. Based on t-test, the number of yeast cells increased 24 h after the culture compared to the beginning, and a significant difference was observed (P <0.05).

Growth of yeast cells after adding zinc threonine and zinc sulfate: Results showed that the zinc source and zinc concentration significantly affected the yeast cell count (Table 1). Also there was a significant interaction between zinc source and zinc concentration on yeast cell count (Table 1).

The number of yeast cells 24 hours after adding zinc threonine and zinc sulfate were shown in Figure 2. In treated groups, the most growth inhibitory of yeast was observed in treatment 2.5 g L^-1 of zinc threonine (P<0.05). By increasing the concentration of zinc threonine, the amount of inhibition decreased. Also in this group, the highest number of yeast cell has been shown in the treatment of 5 g L^-1 of zinc sulfate and significant differences were observed among treatments (P <0.05). In the treatment of 7.5 g L^-1 of zinc sulfate, the number of  yeast cells decreased significantly compared to the control group (P <0.05).

Figure 1- The number of yeast cells (mean ±SD) for 24 hours before adding zinc

Figure 2- The effect of zinc source and zinc concentration on the yeast cell count, the number in horizontal axis indicates the amount of zinc sulfate and zinc threonine (g L^-1). Different letters have a significant difference (P <0.05). The data were shown as mean ± SD.

Table 1- Two-way ANOVA of the effect of different Zn source and concentration on Zn content and cell count in a growing population of yeast, Saccharomyces cerevisiae

Explanation: ∗∗ P < 0.001

Evaluation of zinc content in enriched yeast: Based on Table 1, the zinc source and zinc concentration significantly affected the zinc content of yeast. Also, the results showed that there was a significant interaction between zinc source and zinc concentration on the zinc content of yeast (Table 1). Figure 3 shows the zinc content of yeast on the measured atomic absorption in different treatments. This amount in three concentrations significantly was higher in zinc sulfate compared to zinc threonine groups (P <0.05). The maximum Zn content (115.67 ± 4.65 mg g^-1) significantly was observed at high concentration of zinc sulfate (P <0.05). Also with increasing the zinc sulfate, the amount of zinc content of yeast was increased (Figure 3). In Zn-threonine treatments, the maximum amount (23.07 ± 1.14mg g^-1) of Zn content of yeast was obtained in 5 g L^-1of zinc threonine (P <0.05).

Figure 3- The effect of zinc source and zinc concentration on the zinc content of yeast, the number on horizontal axis indicate the amount of zinc sulfate and zinc threonine (g L-1). Different letters have a significant difference (P <0.05). The data were shown as mean ± SD.