A method for preparing salt-induced yeast nano-selenium

Abstract

The invention discloses a method for preparing salt-induced yeast nano-selenium, comprising the following steps: preparing a culture medium; preparing an activated seed solution; inoculating the activated seed solution into the culture medium for fermentation, adding a salt solution into the culture medium for continued fermentation, and then adding sodium selenite solution for fermentation and culture; collecting the fermentation liquid, centrifuging, washing, drying and crushing to obtain dry selenium yeast powder; mixing the selenium yeast aqueous solution with the wall-breaking enzyme solution and then incubating, adding an extractant and mixing and standing, collecting the intermediate aqueous phase, centrifuging, washing, and collecting the precipitate as yeast nano-selenium. The invention uses Saccharomyces boulardii as a seed liquid fermentation bacterium, screens out the optimal fermentation conditions and the addition amount of sodium selenite, and the prepared nano-selenium has good stability and high conversion rate. When the concentration of sodium selenite is 400 μg / mL and the concentration of sodium chloride solution is 0.6 mol / L, the conversion rate of yeast nano-selenium is as high as 90%.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation technology and nano-selenium preparation, and in particular to a method for preparing salt-induced yeast nano-selenium. Background Art

[0002] Selenium is an essential trace element for the human body. In recent years, the role of nano-selenium particles in numerous physiological processes has attracted much attention. Compared with inorganic selenium and organic selenium, nano-selenium is less toxic and easier to absorb. Therefore, how to solve the problem of improving the bioavailability of nano-selenium is urgent. Nano-selenium particles have antioxidant and antibacterial effects and are widely used in the biomedical and pharmaceutical fields. The use of microorganisms to synthesize biogenic nano-selenium is considered to be the most promising green and renewable biosynthetic route. In the prior art, people use the addition of a reducing agent (such as ascorbic acid) to yeast fermentation medium to reduce sodium selenite to nano-selenium, and the process does not involve the participation of yeast cells. Therefore, the reduced nano-selenium part in this method remains in the fermentation medium, and the conversion rate of inorganic selenium is relatively low.

[0003] Saccharomyces boulardii has unique physiological and metabolic characteristics. The use of Saccharomyces boulardii to produce nano-selenium particles is considered a green technology. During the process of selenium enrichment, Saccharomyces boulardii will be subjected to various stresses, such as high osmotic pressure, high sulfite dosage, acid stress and increasing ethanol content. In order to avoid the damage caused by environmental stress, microorganisms have evolved stress mechanisms to cope with environmental induction. Among these stresses, oxidative stress and osmotic stress are receiving more and more attention. In the prior art, nano-selenium is prepared by using microorganisms, mainly by adding selenium salts to the culture medium. When selenium salts are added to the culture medium, the osmotic pressure of the cells will increase, and the cells will lose water and die. Summary of the invention

[0004] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a method for preparing yeast nano-selenium, by setting different concentrations of sodium chloride solution to increase its salt adaptability, select the best sodium chloride concentration, to increase the enrichment of sodium selenite salt, thereby increasing the yield of nano-selenium.

[0005] Technical solution: In order to achieve the above purpose, the method for preparing salt-induced yeast nano-selenium of the present invention comprises the following steps:

[0006] (1) preparing culture medium;

[0007] (2) preparing activated seed solution;

[0008] (3) inoculating the activated seed liquid into the culture medium for fermentation, adding a salt solution to the culture medium for further fermentation, and then adding sodium selenite for fermentation and culture;

[0009] (4) collecting the fermentation liquid, centrifuging, washing, drying and crushing to obtain dry selenium yeast powder;

[0010] (5) The selenium yeast aqueous solution and the cell wall breaking enzyme solution are mixed and incubated, and the extractant is added and mixed and allowed to stand, and the intermediate aqueous phase is collected, centrifuged, washed, and the precipitate is collected as yeast nanoparticles.

[0011] Furthermore, the culture medium in step (1) includes YPD solid culture medium, YPD liquid culture medium, and inorganic salt fermentation culture medium.

[0012] Furthermore, the YPD solid culture medium is: tryptone 20-40g / L, yeast powder 10-20g / L, glucose 40-50g / L, agar powder 20-30g / L; the YPD liquid culture medium is: tryptone 20-40g / L, yeast powder 10-20g / L, glucose 40-50g / L; the inorganic salt fermentation medium is: glucose 80-120g / L, urea 3-5g / L, potassium dihydrogen phosphate 2-4g / L, potassium chloride 2-3g / L, magnesium sulfate 1-3g / L, calcium chloride 0.2-0.6g / L and trace elements and vitamins.

[0013] Furthermore, in step (3), a salt solution with a concentration of 0 to 0.8 mol / L is added at 0 to 24 h, wherein the salt solution is a sodium chloride solution. Preferably, a salt solution with a concentration of 0.6 mol / L is added at 4 h of fermentation.

[0014] Furthermore, in step (3), a sodium selenite solution with a concentration of 200-500 μg / mL is added at 8-10 hours of fermentation. Preferably, a sodium selenite solution with a concentration of 400 μg / mL is added at 9 hours of fermentation.

[0015] Furthermore, the culturing conditions of the activated seed solution in step (2) are: 30-40°C, 250-400rpm, and 24-35h.

[0016] Furthermore, the fermentation conditions of step (3) are: 30-40°C, 250-400rpm, and culture for 48-58h.

[0017] Furthermore, the seed liquid in step (3) is Saccharomyces boulardii seed liquid.

[0018] Furthermore, the extractant in step (5) is n-hexane.

[0019] Furthermore, the pH value of the selenium yeast aqueous solution in step (5) is 4-5.

[0020] Principle of the invention: By adding different concentrations of sodium chloride to the culture medium, the osmotic pressure of the cells is changed, allowing the cells to adapt to the high osmotic pressure environment, and the cells will not lose water and die. The addition of sodium chloride can not only serve as a nutrient element for cell growth, but also stimulate cells to adapt to the high osmotic pressure environment, thereby promoting the conversion of nano-selenium.

[0021] The present invention utilizes salt to induce yeast to synthesize nano-selenium, and the salt induces protein to reduce inorganic selenium, so that most of the nano-selenium is inside the yeast cells. Not only the conversion efficiency of sodium selenite is improved, but also the outer layer of the nano-selenium formed in the yeast cells is wrapped with a layer of protein film, which is a non-toxic, repeatable and green production method. In the present invention, sodium chloride must be added first and then sodium selenite. The order is very important. If sodium selenite is added first, the bacteria will be significantly inhibited; and when sodium chloride and sodium selenite are mixed and fermented in one step at the same time, the effect is obviously not as good as the step-by-step fermentation (adding sodium chloride first and then sodium selenite). The synergistic effect, so the present invention adopts a specific step-by-step fermentation method.

[0022] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0023] (1) The present invention uses Saccharomyces boulardii as a starting strain for fermentation, screens out the optimal fermentation conditions and the amount of sodium selenite added, and the prepared nano-selenium has good stability and high conversion rate.

[0024] (2) When a certain concentration of sodium chloride solution and sodium selenite solution are added to the fermentation medium, the conversion rate of yeast nano-selenium can reach over 90%. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is an operation flow chart of the present invention;

[0026] Figure 2 This is a scanning electron microscope of yeast cells;

[0027] Figure 3 It is a radio electron microscope of nanoselenium in yeast cells;

[0028] Figure 4 It is a nano-selenide radio microscope;

[0029] Figure 5 The effect of sodium chloride concentration on the conversion rate of nano-selenium in yeast;

[0030] Figure 6 The effect of sodium chloride addition time on the conversion rate of nano-selenium in yeast;

[0031] Figure 7 This is the effect of sodium selenite concentration on the conversion rate of yeast nano-selenium. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0033] Drugs and reagents: Saccharomyces boulardii was provided by Huaiyin Institute of Technology or purchased directly or the wild type could be used.

[0034] Example 1

[0035] like Figure 1 As shown, the preparation method of salt-induced nano-selenium specifically comprises the following steps:

[0036] (1) Prepare YPD solid medium: 20 g / L tryptone, 10 g / L yeast powder, 40 g / L glucose, and 20 g / L agar powder;

[0037] Prepare YPD liquid medium: 20 g / L tryptone, 10 g / L yeast powder, 40 g / L glucose;

[0038] Prepare mother solution: 5 g sodium selenite in 100 mL deionized water;

[0039] Prepare inorganic salt fermentation medium: glucose 100g / L, urea 4g / L, potassium dihydrogen phosphate 2.5g / L, potassium chloride 2g / L, magnesium sulfate 1.5g / L, calcium chloride 0.32g / L, add trace elements and vitamins;

[0040] Among them, trace elements in the fermentation medium mg / L: zinc sulfate 5.4mg / L, ferrous sulfate 7mg / L, copper sulfate 0.22mg / L, manganese sulfate 0.38mg / L, sodium molybdate 0.38mg / L, potassium iodide 0.2mg / L, nickel chloride 0.32mg / L;

[0041] Vitamins in fermentation medium mg / L: biotin 0.024 mg / L, B5 4.4 mg / L, B1 3.1 mg / L, B8 26.8 mg / L, B6 0.7 mg / L, B3 0.62 mg / L, B2 0.05 mg / L; the above culture medium mother liquor is sterilized.

[0042] (2) Activation of the strain: Take out the Saccharomyces boulardii bacterial tube from -80°C, inoculate it into the YPD solid medium prepared in step (1), streak it on a plate, and then place it in a 30°C constant temperature incubator for 2 days. Pick a single colony with good growth in 50 / 250mL YPD liquid medium, culture it at 30°C, 250rpm for 24 hours, and prepare an activated seed solution for use.

[0043] (3) The seed liquid activated in step (2) is inoculated into the inorganic salt fermentation medium prepared in step (1) at an inoculum amount of 8% by volume. Initially (0 h), sterilized sodium chloride solutions with different final concentrations are added to the inorganic salt fermentation medium, and the concentration gradient (mol / L) is: 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, to obtain a salt-induced medium for fermentation. When the fermentation is 9 h, a sodium selenite solution with a final concentration of 300 μg / mL is added to continue the fermentation culture. The entire fermentation conditions are: 30° C., 250 rpm, and culture for 48 h.

[0044] (4) After fermentation, the fermentation liquid was collected and centrifuged at 10,000 rpm for 15 min to obtain yeast paste, which was washed three times with deionized water to remove the surface fermentation liquid, and then dried in an oven at 60°C for 30 h, dried and crushed to obtain dry selenium yeast powder.

[0045] (5) Weigh 2 g of cell wall breaking enzyme (Nanning Pangbo Bioengineering Co., Ltd.) and dissolve it in 50 mL of deionized water. Weigh 2 g of dry selenium yeast powder prepared in step (4), dilute to 25 mL with deionized water, and adjust the pH to 4.5 with 1 mol / L NaOH and 1 mol / L HCl. Take 4 mL of cell wall breaking enzyme solution and add it to the selenium yeast aqueous solution. Incubate in a constant temperature water bath shaker at 50°C and 170 rpm for 10 h to obtain cell wall breaking solution. Transfer the cell wall breaking solution to a separatory funnel, add 20 mL of extractant n-hexane at the same time, mix well, and let stand for 24 h.

[0046] (6) Remove the broken yeast fragments in the lower layer of the separatory funnel in step (5) and collect the intermediate aqueous phase. Centrifuge at 10,000 rpm for 40 min to obtain a nano-selenium solution. Wash with deionized water three times to remove excess extractant and other impurities, collect the precipitate, which is nano-selenium, and resuspend the selenium nanoparticles in deionized water and store at 4°C.

[0047] The nano-selenium obtained by the preparation method described in this embodiment is a biologically active nano-selenium wrapped by protein. Figure 2 The morphology of yeast cells is shown. Figure 3 Shown is a successfully prepared nano-selenium cell slice, with the nano-selenium particle size being 50-200nm.

[0048] At the same time, the color of the fermentation liquid changes from yellow to orange to red and then to dark red when the present invention is cultured under different concentrations of sodium chloride. The darker the color, the higher the content of nano-selenium. The conversion rate of nano-selenium without salt induction is only 56.7%. Figure 5 As shown, when the sodium chloride concentration is 0-0.6 mol / L, the conversion rate of induced yeast nano-selenium shows an upward trend. When it is 0.6 mol / L, the conversion rate of induced yeast nano-selenium can reach 80%. When the concentration is greater than this, the conversion rate shows a downward trend.

[0049] Example 2

[0050] (1) The method of Example 1 is adopted.

[0051] (2) The method of Example 1 is adopted.

[0052] (3) The seed liquid activated in step (2) is inoculated into the inorganic salt fermentation medium prepared in step (1) at an inoculum amount of 8% by volume, and a sterilized sodium chloride solution with a final concentration of 0.6 mol / L is added to the inorganic salt fermentation medium at 0, 4, 8, 12, 16, 20, and 24 hours of fermentation to obtain a salt-induced medium, and a sodium selenite solution with a final concentration of 300 μg / mL is added at 9 hours of fermentation to continue fermentation. The entire fermentation culture conditions are 30° C. and 250 rpm for 48 hours.

[0053] (4) The method of Example 1 is adopted.

[0054] (5) The method of Example 1 is adopted.

[0055] (6) The method of Example 1 is adopted.

[0056] The nano-selenium obtained by the preparation method described in this example is a biologically active nano-selenium wrapped by protein, and the particle size is between 50-200nm. The experimental results show that the conversion rate of nano-selenium without salt induction is only 56.7%. Figure 6 As shown, the conversion rate of yeast nano-selenium induced by adding 0.6 mol / L sodium chloride during 0-4 h showed an upward trend. The conversion rate of yeast nano-selenium induced by adding 0.6 mol / L sodium chloride after 4 h can reach 87%. After more than 4 h, the conversion rate of yeast nano-selenium showed a downward trend.

[0057] Example 3

[0058] (1) The method of Example 1 is adopted.

[0059] (2) The method of Example 1 is adopted.

[0060] (3) The seed liquid activated in step (2) is inoculated into the inorganic salt fermentation medium prepared in step (1) at an inoculum amount of 8% by volume, and at 4 hours, a sterilized sodium chloride solution with a final concentration of 0.6 mol / L is added to the inorganic salt fermentation medium to obtain a salt-induced medium for fermentation. At 9 hours of fermentation, sodium selenite solutions with different final concentrations are added to continue fermentation, namely: 200 μg / mL, 250 μg / mL, 300 μg / mL, 400 μg / mL, and 500 μg / mL, and the entire fermentation culture conditions are: 30° C., 250 rpm, and culture for 48 hours.

[0061] (4) The method of Example 1 is adopted.

[0062] (5) The method of Example 1 is adopted.

[0063] (6) The method of Example 1 is adopted.

[0064] The nano-selenium obtained by the preparation method described in this embodiment is a biologically active nano-selenium wrapped by protein, such as Figure 4 The figure shows the successful preparation of nano-selenium TEM. The experimental results show that the conversion rate of nano-selenium without salt induction is only 62.7%. Figure 7 As shown, when the concentration of sodium selenite added is 200μg / mL-400μg / mL, the conversion rate of yeast nano-selenium shows an upward trend. When the concentration of sodium selenite added is 400μg / mL, the conversion rate of yeast nano-selenium can reach more than 90%. When the concentration is greater than this, the conversion rate shows a downward trend.

Claims

1. A method for preparing salt-induced yeast nano-selenium, characterized in that: The following steps are involved: (1) preparing a culture medium, wherein the culture medium includes a YPD solid culture medium, a YPD liquid culture medium, and an inorganic salt fermentation culture medium; (2) preparing an activated seed solution, wherein the seed solution is a Saccharomyces boulardii seed solution; (3) inoculating the activated seed liquid into the culture medium for fermentation, adding a salt solution to the culture medium for further fermentation, and then adding sodium selenite for fermentation culture; (4) collecting the fermentation liquid, centrifuging, washing, drying and crushing to obtain dry selenium yeast powder; (5) mixing the selenium yeast aqueous solution and the cell wall breaking enzyme solution, incubating, adding an extractant, collecting the intermediate aqueous phase, centrifuging, washing, and collecting the precipitate as yeast nano-selenium; In step (3), at 4 hours, a salt solution with a concentration of 0.6 mol / L is added, wherein the salt solution is a sodium chloride solution; at 9 hours of fermentation, a sodium selenite solution with a concentration of 400 μg / mL is added; The fermentation conditions of step (3) are: 30° C., 250 rpm, and 48 h.

2. The method for preparing salt-induced yeast nano-selenium according to claim 1, characterized in that: The YPD solid culture medium in step (1) is: 20-40 g / L tryptone, 10-20 g / L yeast powder, 40-50 g / L glucose, and 20-30 g / L agar powder; the YPD liquid culture medium is: 20-40 g / L tryptone, 10-20 g / L yeast powder, and 40-50 g / L glucose; the inorganic salt fermentation culture medium is: 80-120 g / L glucose, 3-5 g / L urea, 2-4 g / L potassium dihydrogen phosphate, 2-3 g / L potassium chloride, 1-3 g / L magnesium sulfate, 0.2-0.6 g / L calcium chloride, and trace elements and vitamins.

3. The method for preparing salt-induced yeast nano-selenium according to claim 1, characterized in that: The activation seed solution culture conditions in step (2) are: 30-40°C, 250-400rpm, 24-35h.

4. The method for preparing salt-induced yeast nano-selenium according to claim 1, characterized in that: The extractant in step (5) is n-hexane.

5. The method for preparing salt-induced yeast nano-selenium according to claim 1, characterized in that: The pH value of the selenium yeast aqueous solution in step (5) is 4-5.

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