CN113151019A - Yeast separation method - Google Patents
Yeast separation method Download PDFInfo
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- CN113151019A CN113151019A CN202110319668.2A CN202110319668A CN113151019A CN 113151019 A CN113151019 A CN 113151019A CN 202110319668 A CN202110319668 A CN 202110319668A CN 113151019 A CN113151019 A CN 113151019A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
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Abstract
The invention provides a yeast separation method, which comprises the following steps: step one, preparing organic selenium mother liquor: adding 0.2-0.5mg of sodium selenite, 3-6mg of nicotinic acid and 1-2mg of glycine into each liter of water, stirring and uniformly mixing, and adjusting the pH value to 5-6 to obtain organic selenium mother liquor; step two, incubation: putting the yeast fermentation liquor and the organic selenium mother liquor in a volume ratio of 80-100:1 on a blending machine for room temperature incubation; step three, separating a magnetic rack; and step four, washing with 0.1% PBST and resuspending with PBS buffer solution. The invention separates the yeast rich in selenium by a reasonable method, and has simple method and low cost.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a yeast separation method.
Background
Selenium (Selenium, Se) is a necessary trace element for human and animal bodies, and has important effects on immunity, growth, breeding, aging resistance and the like of animals. Se deficiency can lead to various diseases in humans and animals, such as hypothyroidism and hypoimmunity in animals, keshan disease (KSD) and Kaschin-Beck disease in humans, exudative diathesis in birds, pancreatic necrosis, and decreased conception and hatchability of hatching eggs. In the prior art, people mainly add selenium into food or health care products in the form of inorganic matters or organic matters, but the problems of poor absorption and unsatisfactory comprehensive effect generally exist.
The selenium-enriched yeast is a selenium-enriched preparation prepared by conveying inorganic selenium into cells in various ways in the fermentation process of yeast to convert the inorganic selenium into organic selenium, centrifuging, drying and the like, wherein the content of the organic selenium is more than 90%, and the organic selenium mainly comprises selenomethionine and selenocysteine.
Therefore, it is important to establish an efficient and fast separation method to separate and obtain the selenium-enriched yeast.
Disclosure of Invention
The invention aims to solve the technical problems in the prior art. Therefore, the invention provides a yeast separation method, aiming at facilitating separation of selenium-enriched yeast.
Based on the above purpose, the present invention provides a yeast separation method, comprising the following steps:
step one, preparing organic selenium mother liquor: adding 0.2-0.5mg of sodium selenite, 3-6mg of nicotinic acid and 1-2mg of glycine into each liter of water, stirring and uniformly mixing, and adjusting the pH value to 5-6 to obtain organic selenium mother liquor;
step two, incubation: putting the yeast fermentation liquor and the organic selenium mother liquor in a volume ratio of 80-100:1 on a blending machine for room temperature incubation;
step three, separating a magnetic rack;
and step four, washing with 0.1% PBST and resuspending with PBS buffer solution.
The stirring and uniformly mixing in the step one are carried out at the temperature of 60-80 ℃.
And in the second step, the incubation time at room temperature is 20-30min, and the rotating speed is 25-30 r/min.
The separation time of the magnetic frame in the third step is 3-5 min.
And step two, adding a pretreated multi-arm meteor polymer, wherein the mass volume ratio of the pretreated multi-arm meteor polymer to the yeast fermentation liquor is 1-2:80-100mg/mL, and the preparation method of the pretreated multi-arm meteor polymer is that 1mg of the multi-arm meteor polymer is suspended in 3-4mL of PBS phosphate buffer, then stirring and dropwise adding 25% glutaraldehyde aqueous solution to make the final concentration of glutaraldehyde be 1-2%, and then carrying out table shaking reaction and reduced pressure spin-drying of the solvent to obtain the product.
The concentration of the PBS phosphate buffer solution is 0.01mol/L, the pH value is 8, the rotation speed of a shaking table is 100-120r/min, and the reaction time is 3-4 h.
The multi-arm wellstar polymer is multi-arm wellstar polyamide-amine with the molecular weight of 70000 Da.
The separation method also comprises the steps of drying, crushing and sieving the yeast after the PBS buffer solution is mixed and resuspended.
The invention has the beneficial effects that:
1. the invention separates the yeast rich in selenium by a reasonable method, and has simple method and low cost.
2. The invention can realize the separation of the yeast under lower magnetic field intensity by means of the cascade amplification effect of the multi-arm meteor polymer, and has stronger separation capability in the same time compared with the conventional immunomagnetic bead separation method. Meanwhile, the multi-arm meteor polymer is adopted, so that the reaction solution is more stable and is not easy to precipitate.
3. The selenium-enriched yeast separated by the invention has the characteristics of easy culture and preservation, and can be used as a raw material for preparing a selenium-containing nutritional health food.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to specific embodiments below.
It is to be noted that technical terms or scientific terms used in the embodiments of the present invention should have the ordinary meanings as understood by those having ordinary skill in the art to which the present disclosure belongs, unless otherwise defined. The word "comprising" or "comprises", and the like, means that the element or item listed before the word covers the element or item listed after the word and its equivalents, but does not exclude other elements or items.
Example 1
A method for separating yeast comprises the following steps:
step one, preparing organic selenium mother liquor: weighing 0.3mg of sodium selenite, 3mg of nicotinic acid and 1mg of glycine, dissolving in 1 liter of water, heating to 80 ℃ while stirring, uniformly stirring, and adjusting the pH value to 5.5 to obtain organic selenium mother liquor for later use;
step two, incubation: placing the yeast fermentation liquor and the organic selenium mother liquor on a blending machine according to the volume ratio of 80:1, and incubating at the room temperature for 20min at the rotating speed of 25 r/min; wherein, the yeast fermentation liquor is obtained by the following method: inoculating yeast in 5% of the inoculation amount into a yeast fermentation culture medium, and fermenting for 20h at 30 ℃. During the culture process, ammonia water and hydrochloric acid are adopted to adjust the pH of the culture medium, and the pH is adjusted to 5.5.
Per liter of fermentation medium contains:
40g of mixture of maltose and glucose, 8g of mixture of soybean meal and soybean cake powder, 2g of yeast powder, 25g of 10% bran extract, 1g of sodium chloride, 2g of monopotassium phosphate, 1.5g of sweet potato powder and 0.8g of betaine. The pH was adjusted to 5.5.
Separating for 4min by adopting a conventional magnetic frame;
and step four, after magnetic separation, washing the mixture twice by using 0.1% PBST, and then resuspending the mixture by using PBS buffer solution to obtain the compound.
Preparation method of 0.1% PBST: 8.0g NaCl, 0.2g KCl, 0.24gKH2PO4、1.44gNa2HPO4Dissolving in 800mL of distilled water, adjusting pH to 7.3 with 5M NaOH, and diluting to 1000mL to obtain 0.01 MPBS. Tween20 was added at a volume ratio of 1/1000(V/V) to obtain 0.1% PBST.
Example 2
A method for separating yeast comprises the following steps:
step one, preparing organic selenium mother liquor: weighing 0.5mg of sodium selenite, 6mg of nicotinic acid and 2mg of glycine, dissolving in 1 liter of water, heating to 85 ℃ while stirring, uniformly stirring, and adjusting the pH value to 5.8 to obtain organic selenium mother liquor for later use;
step two, incubation: placing yeast fermentation liquor and organic selenium mother liquor on a blending machine according to the volume ratio of 100:1, and incubating for 20min at the room temperature at the rotating speed of 30 r/min; wherein, the yeast fermentation liquor is obtained by the following method: inoculating yeast in 5% of the inoculation amount into a yeast fermentation culture medium, and fermenting for 20h at 30 ℃. During the culture process, ammonia water and hydrochloric acid are adopted to adjust the pH of the culture medium, and the pH is adjusted to 5.8.
Per liter of fermentation medium contains:
40g of mixture of maltose and glucose, 8g of mixture of soybean meal and soybean cake powder, 2g of yeast powder, 25g of 10% bran extract, 1g of sodium chloride, 2g of monopotassium phosphate, 1.5g of sweet potato powder and 0.8g of betaine. The pH was adjusted to 5.8.
Separating for 5min by adopting a conventional magnetic frame;
and step four, after magnetic separation, washing the mixture twice by using 0.1% PBST, and then resuspending the mixture by using PBS buffer solution to obtain the compound.
Preparation method of 0.1% PBST: 8.0g NaCl, 0.2g KCl, 0.24gKH2PO4、1.44gNa2HPO4Dissolving in 800mL of distilled water, adjusting pH to 7.3 with 5M NaOH, and diluting to 1000mL to obtain 0.01 MPBS. Tween20 was added at a volume ratio of 1/1000(V/V) to obtain 0.1% PBST.
Example 3
A method for separating yeast comprises the following steps:
step one, preparing organic selenium mother liquor: weighing 0.4mg of sodium selenite, 5mg of nicotinic acid and 1mg of glycine, dissolving in 1 liter of water, heating to 80 ℃ while stirring, uniformly stirring, and adjusting the pH value to 5.5 to obtain organic selenium mother liquor for later use;
step two, incubation: putting the yeast fermentation liquor, the pretreated multi-arm Jingxing polymer and the organic selenium mother liquor on a blending instrument in a volume ratio of 80:1:1, and incubating at the rotating speed of 25r/min for 20min at room temperature; wherein, the yeast fermentation liquor is obtained by the following method: inoculating yeast in 5% of the inoculation amount into a yeast fermentation culture medium, and fermenting for 20h at 30 ℃. During the culture process, ammonia water and hydrochloric acid are adopted to adjust the pH of the culture medium, and the pH is adjusted to 5.5.
Per liter of fermentation medium contains:
40g of mixture of maltose and glucose, 8g of mixture of soybean meal and soybean cake powder, 2g of yeast powder, 25g of 10% bran extract, 1g of sodium chloride, 2g of monopotassium phosphate, 1.5g of sweet potato powder and 0.8g of betaine. The pH was adjusted to 5.5.
The preparation method of the pretreated multi-arm Weeking polymer comprises the steps of suspending 1mg of multi-arm Weeking polymer multi-arm Weeking polyamide-amine in 3mL of PBS phosphate buffer solution (the concentration is 0.01mol/L, and the pH value is 8.0), then stirring and dropwise adding 25% glutaraldehyde aqueous solution to enable the final concentration of glutaraldehyde to be 1%, then reacting for 3h at the rotating speed of 100r/min of a shaking table, and finally carrying out reduced pressure spin drying on the solvent to obtain the multi-arm Weeking polymer.
Separating for 3min by adopting a conventional magnetic frame;
and step four, after magnetic separation, washing the mixture twice by using 0.1% PBST, then resuspending the mixture by using PBS buffer solution, drying the mixture by using hot air at 50 ℃, and crushing and sieving the dried mixture by using a conventional method to obtain the compound.
The preparation method of the 0.1% PBST comprises the following steps: 8.0g NaCl, 0.2g KCl, 0.24gKH2PO4、1.44g Na2HPO4Dissolving in 800mL of distilled water, adjusting pH to 7.3 with 5M NaOH, and diluting to 1000mL to obtain 0.01 MPBS. Tween20 was added at a volume ratio of 1/1000(V/V) to obtain 0.1% PBST.
Example 4
A method for separating yeast comprises the following steps:
step one, preparing organic selenium mother liquor: weighing 0.4mg of sodium selenite, 5mg of nicotinic acid and 1mg of glycine, dissolving in 1 liter of water, heating to 80 ℃ while stirring, uniformly stirring, and adjusting the pH value to 5.5 to obtain organic selenium mother liquor for later use;
step two, incubation: putting the yeast fermentation liquor, the pretreated multi-arm meteoric polymer and the organic selenium mother liquor on a blending instrument according to the volume ratio of 100:2:1, and incubating at the rotating speed of 30r/min for 20min at room temperature; wherein, the yeast fermentation liquor is obtained by the following method: inoculating yeast in 5% of the inoculation amount into a yeast fermentation culture medium, and fermenting for 20h at 30 ℃. During the culture process, ammonia water and hydrochloric acid are adopted to adjust the pH of the culture medium, and the pH is adjusted to 5.5.
Per liter of fermentation medium contains:
40g of mixture of maltose and glucose, 8g of mixture of soybean meal and soybean cake powder, 2g of yeast powder, 25g of 10% bran extract, 1g of sodium chloride, 2g of monopotassium phosphate, 1.5g of sweet potato powder and 0.8g of betaine. The pH was adjusted to 5.5.
The preparation method of the pretreated multi-arm Weeking polymer comprises the steps of suspending 1mg of multi-arm Weeking polymer multi-arm Weeking polyamide-amine in 3mL of PBS phosphate buffer solution (the concentration is 0.01mol/L, and the pH value is 8.0), then stirring and dropwise adding 25% glutaraldehyde aqueous solution to enable the final concentration of glutaraldehyde to be 2%, then reacting for 3h at the rotating speed of 120r/min of a shaking table, and finally carrying out reduced pressure spin drying on the solvent to obtain the multi-arm Weeking polymer.
Separating for 3min by adopting a conventional magnetic frame;
and step four, after magnetic separation, washing the mixture twice by using 0.1% PBST, then resuspending the mixture by using PBS buffer solution, drying the mixture by using hot air at 50 ℃, and crushing and sieving the dried mixture by using a conventional method to obtain the compound.
The preparation method of the 0.1% PBST comprises the following steps: 8.0g NaCl, 0.2g KCl, 0.24gKH2PO4、1.44g Na2HPO4Dissolving in 800mL of distilled water, adjusting pH to 7.3 with 5M NaOH, and diluting to 1000mL to obtain 0.01 MPBS. Tween20 was added at a volume ratio of 1/1000(V/V) to obtain 0.1% PBST.
The yeast separated in each example was placed in a triangular flask, and was dispersed with distilled water by ultrasonic dispersion for 20min, and after standing for 15min, the yeast slurry was transferred to a centrifuge tube, centrifuged at 5000r/min for 15min, and the supernatant was taken out and the selenium content was measured by atomic absorption spectrometry. The test results show that the yeasts obtained in examples 1 to 4 all contain selenium.
The yeast separation results of the above examples show that examples 3 and 4 can achieve better separation of yeast, reduce separation time, and improve separation efficiency.
Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to these examples; within the idea of the invention, also features in the above embodiments or in different embodiments may be combined, steps may be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.
The embodiments of the invention are intended to embrace all such alternatives, modifications and variances that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, substitutions, improvements and the like that may be made without departing from the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (8)
1. A method for separating yeast, which is characterized by comprising the following steps:
step one, preparing organic selenium mother liquor: adding 0.2-0.5mg of sodium selenite, 3-6mg of nicotinic acid and 1-2mg of glycine into each liter of water, stirring and uniformly mixing, and adjusting the pH value to 5-6 to obtain organic selenium mother liquor;
step two, incubation: putting the yeast fermentation liquor and the organic selenium mother liquor in a volume ratio of 80-100:1 on a blending machine for room temperature incubation;
step three, separating a magnetic rack;
and step four, washing with 0.1% PBST and resuspending with PBS buffer solution.
2. The method for separating yeast according to claim 1, wherein the stirring and mixing in the first step are performed at 60 to 80 ℃.
3. The method for separating yeast according to claim 1, wherein the incubation time at room temperature in the second step is 20-30min, and the rotation speed is 25-30 r/min.
4. The method for separating yeast according to claim 1, wherein the time for separating the magnetic frame in the third step is 3-5 min.
5. The yeast separation method according to claim 1, wherein a pre-treated multi-armed Venus polymer is further added in the second step, the mass-to-volume ratio of the pre-treated multi-armed Venus polymer to the yeast fermentation broth is 1-2:80-100mg/mL, the pre-treated multi-armed Venus polymer is prepared by suspending 1mg of the multi-armed Venus polymer in 3-4mL of PBS phosphate buffer, then stirring and dropwise adding 25% glutaraldehyde aqueous solution to make the final concentration of glutaraldehyde be 1-2%, and then carrying out table shaking reaction and reduced pressure rotary drying on the solvent to obtain the product.
6. The yeast separation method of claim 5, wherein the concentration of the PBS phosphate buffer solution is 0.01mol/L, the pH is 8, the rotation speed of the shaking table is 100-120r/min, and the reaction time is 3-4 h.
7. The method for isolating yeast according to claim 5, wherein the multiarm meteor polymer is multiarm meteor polyamidoamine with molecular weight of 70000 Da.
8. The method for separating yeast according to claim 1, further comprising the steps of drying, pulverizing and sieving the yeast after the PBS buffer mixed with the resuspended yeast.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1762207A (en) * | 2005-11-11 | 2006-04-26 | 浙江省天然药用植物研究中心 | Method for cultivating selenium-enriched officinal dendrobium stem |
| CN103275879A (en) * | 2013-06-05 | 2013-09-04 | 南昌大学 | Novel method for enriching and separating Candida albicans |
| CN106906206A (en) * | 2017-04-26 | 2017-06-30 | 中国科学院合肥物质科学研究院 | A kind of Se-enriched yeast, preparation method and application |
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- 2021-03-25 CN CN202110319668.2A patent/CN113151019A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1762207A (en) * | 2005-11-11 | 2006-04-26 | 浙江省天然药用植物研究中心 | Method for cultivating selenium-enriched officinal dendrobium stem |
| CN103275879A (en) * | 2013-06-05 | 2013-09-04 | 南昌大学 | Novel method for enriching and separating Candida albicans |
| CN106906206A (en) * | 2017-04-26 | 2017-06-30 | 中国科学院合肥物质科学研究院 | A kind of Se-enriched yeast, preparation method and application |
Non-Patent Citations (2)
| Title |
|---|
| 王涛: "高生物量富硒酵母的选育研究", 《电子制作》, pages 229 - 230 * |
| 陈妍等: "硒浓度梯度驯化结合紫外诱变法 选育富硒酵母菌研究", 《粮食与饲料工业》, no. 4, pages 55 - 59 * |
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