Comparative Study of Selenium-Enriched Conditions from Ganoderma lucidum and Yeasts

Liu Pei-Li^{1, *}, Wang Da-Yan², Hao Yong-Chun³, Huang Shu-Ming³

¹Department of Chemical and Materials Engineering, Hefei University, Hefei, Anhui, China

²Anhui Star New Materials Technology Co., Ltd., Hefei, Anhui, China

³Department of Biological and Environmental Engineering, Hefei University, Hefei, Anhui, China

Email address:

(Liu Pei-Li)

^*Corresponding author

To cite this article:

LiuPei-Li, Wang Da-Yan, Hao Yong-Chun, Huang Shu-Ming. Comparative Study of Selenium-Enriched Conditions from Ganoderma lucidum and Yeasts. International Journal of Nutrition and Food Sciences. Vol. 5, No. 2, 2016, pp. 124-128. doi: 10.11648/j.ijnfs.20160502.15

Received: January 17, 2016; Accepted: January 28, 2016; Published: March 22, 2016

Abstract: This paper compared the conditions of selenium-enriched from Ganoderma lucidum and yeasts. The optimal conditions for shake flask were carried out by a single factor test and orthogonal test L₉(3⁴), giving a comparative study on the ability of biotransformation of inorganic selenium between Ganoderma lucidum and yeasts. The orthogonal experimental results showed that the optimal conditions for shake flask growth of Ganoderma lucidum were as bellow: culture medium final concentration of Na₂SeO₃ (Se⁴⁺) was 20mg/L, 15% inoculum, incubation time 42h, medium volume 60ml/250ml. The total content of selenium in fermentation of G. lucidum was 1540.34±19.21µg/g, and the growth capacity of mycelium of G. lucidum was 51.56±0.065g/L, respectively. The optimal conditions for shake flask cell growth of yeasts were as bellow: culture medium final concentration of Na₂SeO₃ (Se⁴⁺) was 25mg/L, 10% inoculum, incubation time 48h, medium volume 50ml/250ml. The total content of selenium in fermentation of yeasts was 2454.86±29.82µg/g, and the growth capacity of cell of yeasts was 6.44±0.023g/L, respectively.

Keywords: Ganoderma lucidum (Fr.) Karst, Saccharomyces Cerevisiae, Selenium-Enriched, Single-Factor Test, Orthogonal Experiment

1. Introduction

Selenium is an essential trace element in human body which strengthens immune system, enhances antioxidant and anti-cancer effects, plays a physiological role in the prevention and treatment of Keshan disease, etc. The total accumulation of selenium in the human body is about 14~21mg, which maintains dynamic balance in ingestion and excretion. By microbial fermentation, biotransformation of inorganic selenium can reduce the toxicity of inorganic selenium, improve the absorption and utilization, which can be used as a safe source of selenium. It is an ideal functional food ingredient. This thesis compared optimized conditions for culturing selenium-enriched gloss G. lucidumand Yeast through single-factor test and orthogonal experiments L₉ (3⁴).

2. Instrument and Materials

2.1. Instrument

Tableconcentrator(Sartorius AG). superclean Benches (Shanghai Bo Xun Industrial Co., Ltd. Medical Equipment Factory). Vertical Pressure Steam Autoclave (Shanghai Bo Xun Industrial Co., Ltd. Medical Equipment Factory). Digital stainless steel electric incubator (Shanghai Bo Xun Industrial Co., Ltd. Medical Equipment Factory). 721 spectrophotometer (Shanghai Bo Xun Industrial Co., Ltd. Medical Equipment Factory).

2.2. Materials

Ganoderma lucidum (Fr.) Karst，provided by College of Life Science, Anhui University and identified by Professor Shen Ye-shou). Brewer's yeast (Saccharomyces cerevisiae, preserved in our laboratory). Na₂SeO₃ (purity of 99.99%, Anhui Star New Materials Technology Co., Ltd.). Edetate disodium (EDTA-2Na), 3, 3- diaminobenzidine (DAB) and so were of analytical reagent.

3. Experimental Method

3.1. Digestive Juice, Standard Stock Solution and Standard Working Solution Preparation

Digestive juice preparation: measureaccurately and mix ulfuric acid, hydrogen peroxide, perchlorate solution according to the ration 1: 3: 3, then store the mixture in a brown light-shading bottle at 4°C for backup.

Selenite standard stock solution (1mg/ml) preparation: weigh 0.0500g Na₂SeO₃; after nitric acid solution (nitric: ultrapure water =1: 30, V/V) was dissolved, reserve the volume (50ml volumetric flask) and formulate as a 1mg / ml standard stock solution, then pour into flask, 121°C, and autoclave it for 20min. After cooling, the solution was aseptically transferred to bottle with blue cap to saved up for use at 4°C.

Na₂SeO₃ working standard solution (10μg/ml) formulation: exact selenite stock standard solution Pipette 1ml, with a solution of nitric acid (nitrate: ultrapure water =1: 30, V/V) volume to 100ml; store for use at 4°C.

3.2. Single-Factor Test

3.2.1. Plus Selenium

Liquid volume 90ml/250ml, shaking speed 150r·min^-1, 28°C, culture time 56h. When the shaker has cultured for 8h, aseptic solution was added to Na₂SeO₃ G. lucidum and yeast broth so that the final concentration of the culture medium Na₂SeO₃ was 10mg/L, 15mg/L, 20mg/L, 25mg/L, 30mg/L; then culture it after mixing in the shaker.

3.2.2. Inoculum

Set five inoculum levels at 5%, 10%, 15%, 20% and 25%, respectively remove with a pipette the corresponding bacterial suspension volume of fungus and yeast cell; add it to marked Erlenmeyer flask containing the corresponding culture medium; set the rotation speed at 150r·min^-1, at 28°C; the culture time is 56h; when the shake flask has cultured for 8h, with the aseptic technique the Na₂SeO₃ solution was added to the G. lucidum andyeast broth to end the culture medium at a Na₂SeO₃ concentration of 20mg/L.

3.2.3. Culture Time

Shaking speed 150r·min^-1, 28°C, time and amount of adding selenium is the same as in 2.2.2. when shaking culture time of the G. lucidum and yeast is 30h, 36h, 42h, 48h, 52h and 56h, take out flasks respectively with the corresponding number, put in 5000 r·min^-1 centrifugal for 15min, and discard the supernatant and wash with deionized water; break fast the fermentation product on the mixers; let stand for 12h, then remove the supernatant after centrifugation again; repeat the above operation twice. The resulting fermentation was placed in 60°C oven, drying to a constant weight, and weighed.

3.2.4. Liquid Volume

Setting liquid volume to 50ml/250ml, 60ml/250ml, 70ml/250ml, 80ml/250ml and 90ml/250ml respectively, with a pipett each flask was added the corresponding amount of broth, then add it to the activated G. lucidum mycelia and yeast suspension; shaking speed 150r·min^-1, 28°C; shaking culture for 54h, time and acceleration of adding selenium is the same as in 2.2.2.

3.3. Orthogonal Experiment

The orthogonal test conditions at the phase of shake flask cultures of G. lucidum and yeast were designed on the basis of single factor experiments, select four factors, the amount of selenium, inoculation, incubation time and the medium volume using L₉ (3⁴) orthogonal optimization test. Table head design as shown in Table 1.

Table 1. G. lucidum and yeast orthogonal experiments [L₉ (3⁴)] factor level coding table.

Rotation speed 150r·min^-1, the culture temperature 28°C, added Na₂SeO₃ 2 times, respectively, added after the first shake flask cultures at 8h and 13h.

3.4. Determination of Selenium Content

3.4.1. Selenium (Se⁴⁺) Standard Curve

Exact amount of Na₂SeO₃ standard working solution 0.0ml, 2.0ml, 4.0ml, 6.0ml, 8.0ml, 10.0ml, sequentially apply to six clean separating funnel (125ml); add the appropriate amount of deionized water; adjust to neutral pH with 5% sodium hydroxide solution, plus 3ml formic acid solution (2mol/L), then add water to 35ml (at this point pH of the solution is about 2 to 3); shake, and then add 5ml 0.5% of DAB to each; shake and put in the dark to react for 30mins. After the reaction completed, adjust it to neutral pH with a 5% sodium hydroxide solution; add 10ml toluene, shaking extraction for 2mins; let it stand to be stratified, put the toluene layer filtrate in the cuvette; absorbance was measured at a wavelength of 420nm. Standard selenium (Se⁴⁺) work concentration as abscissa, absorbance ordinate to draw selenium standard curve.

3.4.2. Sample Preparation

I. fermentation separated and dried

G. lucidum and yeast fermentation broth were poured into the appropriate number of centrifuge tube, 5000r·min^-1centrifugal 15min; the supernatant was removed, and the precipitate was broken by the ultra-pure water dip 2-3 times in rapid mixers; the supernatant was removed by centrifugation and dried to a constant weight, then respectively Se Ganoderma mycelium and yeast powder were harvested.

II. sample digest

Accurately weighed mycelia and yeast that were dried to constant weight in 150ml beaker, add a small amount of deionized water wet first sample, and then add the appropriate amount of digestive juices, respectively, shake until the bubbles subside, then stamped surface dish, put in the ventilating kitchen, with electric stove digested to end (ie heating to fuming, until the solution becomes pale yellow, reheat 2~3min, then the solution becomes colorless). If not completely digested, after cooling a bit, add hydrogen peroxide and heating until it digested completely. After the beaker cooled, rinse glass and the beaker wall with 1ml deionized water; plus 2ml 5% EDTA-2Na solution and shake; pH was adjusted to neutral with 40% NaOH in a cold water bath, then set the volume to 100ml, use the same method to make blank solution.

3.4.3. For Sample Preparation and Measurement

Put the precise amount of the after-digestion juice and an equal amount of blank digestion solution in separating 125ml funnels; add water; adjust to neutral pH with 5% NaOH solution; plus 3ml formic acid solution (2mol / L), then add water to 35ml, shake, then plus 5ml 0.5% DAB solution to each, and shake, then set in the dark to react for 30mins, then adjust pH to 6.5~7.0 with 5% NaOH solution, adding precise 10ml toluene, shaking extraction for1min; let it stand to be stratified; put the toluene layer filtrate in the cuvette; absorbance is measured at a wavelength of 420nm. Calculate the selenium content in the sample with the comparison to the standard curve.

4. Results and Discussion

4.1. Selenium (Se⁺⁴) Standard Curve

Linear equation of Selenium (Se⁺⁴) standard curve is y = 0.0094x + 0.0214. R²=0.998. Significant test result shows P<0.0001 (n=6, n is the concentration gradient of the selenium standard sample number). Selenium (Se⁺⁴) standard curve working range is 0μg/ml~100μg/ml.

4.2. Single-Factor Test Results and Analysis

4.2.1. Plus Selenium

Test results of shake flask culture with different concentrations of Na₂SeO₃ are shown in Figure 1. The results show: added amount selenium at 10~15mg/L G. lucidum has higher growth, followed by G. lucidum of lower growth with increasing amount of selenium. When added Selenium is 15~20mg/L, G. lucidum has higher content of selenium; when Selenium is added at 15~20mg/L, yeast cells have higher growth, followed by lower growth of yeast with increasing amount of selenium. When Selenium is added at 20~25mg/L, yeast cells have high content of selenium.

Figure 1. Selenium yeast growth rate on G. lucidum and influence diagrams.

Figure 2. Affect the amount of inoculum of G. lucidum and yeast growth.

4.2.2. Inoculation Amount

Figure 2 is the test results showing how different inoculum affects selenium content and growth of G. lucidum and yeast. The results showed that, when inoculation was 20%, G. lucidum has higher growth, followed by a slight decline, while the growth of yeast has been an upward trend with the increasing inoculation amount. When inoculation amount is at 10% to 15%, the fungus selenium content is at a high level. When inoculation is greater than 15%, the selenium content in fungus begins to decrease. When inoculum concentration is greater than 20%, selenium content in the yeast began to decline.

4.2.3. Culture Time

Figure 3 is the test resultsshowing how different culture time affects selenium content and growth of G. lucidum and yeast. The results showed that when the culture time is 48h~52h, selenium content in both fungus and yeast are of the maximum range; if cultured 52h, selenium content in G. lucidum and yeast declines.

Figure 3. Affect the cultivation time of G. lucidum and yeast growth.

Figure 4. Affect the liquid volume of G. lucidum and yeast growth.

4.2.4. Liquid Volume

Figure 4 is the test results showing how different liquid volume affects selenium content and growth of G. lucidum and yeast. The results show that liquid volume is at 60~70ml/250ml, selenium content volume and growth of G. lucidum appear higher. After the value reaches the highest it begins to decline. When the liquid volume is at 50~60ml/250ml, yeast growth is slightly higher than the other groups. after liquid volume reaching the highest value, with the increase of liquid volume, the selenium content in yeast shows a more obvious downward trend.

4.3. Orthogonal Test Results of the Discussions

Based on the single factor test results, design 4 factors culturing G. lucidum and yeast including selenium amount, inoculation, incubation time and the medium volume and 3 levels orthogonal experiment L₉ (3⁴).

According to the results of the orthogonal experiment, factors that affect the content of selenium in Ganoderma lucidum were as bellow: selenium amount>liquid volume>inoculation>incubation time. Referring to Table 1, the culture conditions of fungus in the test No. 8 (A₃B₂C₁D₂) ie optimized culture conditions of G. lucidum conversion to inorganic selenium (ie, Se optimal conditions): final concentration of G. lucidum broth added Na₂SeO₃ was 20mg/L; inoculation amount was 15%, culture time was 42h; liquid volume was 60ml/250ml; maximum content of selenium of G. lucidum appears at 1540μg/g; culture conditions of G. lucidum in Test No. 3 (A₁B₃C₁D₁), ie conditions for G. lucidum growth of cell culture can be optimized as follows: final concentration of G. lucidum fermentative inorganic selenium Na₂SeO₃ is 10mg/L; inoculation amount was 20%; culture time was 42h; liquid volume was 50ml/250ml; the emergence of the maximum growth of G. lucidum was at 74.12g/L. Factors affecting the content of selenium yeast were: selenium amount>inoculation>culture time>liquid volume. Analysis referring to Table 1, yeast in the test number 7 (A₃B₁C₂D₁) culture conditions, i.e., optimized culture conditions for yeast cell’s biotransformation inorganic selenium (i.e., conditions optimal Se): final concentration of yeast fermentation liquid with added Na₂SeO₃ was at 25mg/L; inoculation amount was 10%; culture time was 48h; liquid volume was 50ml/250ml; yeast cells showed a maximum content of selenium at 2454μg/g; culture conditions of yeast in Test No. 3 (A₁B₃C₁D₁), ie optimized culture conditions of growth of yeast cells; inorganic final concentration of selenium yeast fermentation broth with added Na₂SeO₃ was 15mg/L; inoculation amount was 20%, culture time was 42h; liquid volume was 50ml/250ml; yeast growth of maximum amount occurs at 11.84g/L.

5. Conclusion

The optimal conditions for shake flask were carried out by a single factor test and orthogonal test L₉ (3⁴), giving a comparative study on the ability of biotransformation of inorganic selenium between G. lucidum and yeasts. According to the results of the orthogonal experiment, factors that affect the content of selenium in G. lucidum were: selenium amount>liquid volume>inoculation>incubation time. The orthogonal experimental results showed that the optimal conditions for shake flask growth of G. lucidum were as bellow: culture medium final concentration of Na₂SeO₃ (Se⁴⁺) was 20mg/L, 15% inoculum, incubation time 42h, medium volume 60ml/250ml. The total content of selenium in fermentation of G. lucidum was 1540.34±19.21µg/g, and the growth capacity of mycelium of G. lucidum was 51.56±0.065g/L, respectively. The optimal conditions for shake flask cell growth of yeasts were as bellow: culture medium final concentration of Na₂SeO₃ (Se⁴⁺) was 25mg/L, 10% inoculum, incubation time 48h, medium volume 50ml/250ml. The total content of selenium in fermentation of yeasts was 2454.86±29.82µg/g, and the growth capacity of cell of yeasts was 6.44±0.023g/L, respectively.

Project Source

Anhui Province Department of Education, college Innovation and Entrepreneurship Project in 2015.

Research Direction

Basic chemical analysis and food processing.

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