KR101810567B1 - Method for producing dry yeast containing selenium using fermentation - Google Patents
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Abstract
The present invention relates to a process for the production of selenium-containing dry yeast which reduces the residual amount of inorganic selenium in the medium by serine addition and increases the selenium content in the yeast, and to the selenium-containing dry yeast produced by said process. Using the method for producing selenium-containing dry yeast of the present invention, the residual amount of inorganic selenium can be lowered by adding serine, and the selenium content in the yeast can be increased to maximize the efficiency of producing selenium-containing dry yeast. In addition, it is possible to omit the step of diluting the dried yeast by supplying the selenium salt at a low concentration during the manufacturing process, and excessive selenium may not be discharged to the waste solution during washing. Thus, the cost for treating the residual inorganic selenium can be reduced.
Description
The present invention relates to a process for the production of selenium-containing dry yeast which reduces the residual amount of inorganic selenium in the medium by serine addition and increases the selenium content in the yeast, and to the selenium-containing dry yeast produced by said process.
Selenium, a trace element, is an essential component of selenium-containing proteins, which play an important role in the functions of antioxidant, inflammation, hormone synthesis, gene synthesis and reproduction. In addition, the selenium metabolite in the body reduces the blood supply to the tumor, induces necrosis of cancer cells, and acts as an antioxidant as a component of glutathione peroxidase in the body.
Selenium can be ingested through various plants, but its contents are distributed differently according to geographical factors, and therefore, when sufficient selenium is not supplied from plants depending on the region, growth disorders, pregnancy disorders, liver cirrhosis, muscle disorders, chronic liver dysfunction Of selenium deficiency symptoms. Therefore, the supply of selenium-containing adjuvants is becoming important.
The pathway of selenium metabolism of microorganisms such as plants and yeasts converts selenium absorbed into the body into selenomethionine form and contains the same constituents as methonein. Thus, selenium intake through yeast or microorganisms with increased selenium content is considered to be an effective way to overcome selenium deficiency symptoms.
Most of the methods for producing selenium-containing yeast utilize a fermentation method using strains such as Saccharomyces cerevisiae or Saccharomyces uvarium . When these strains are cultured in a fermentation medium containing selenium so that the selenium can be absorbed well, they are produced as selenium-containing yeast. When a yeast is cultured using a medium containing selenium, yeast accumulates a large amount of selenium in the form of selenium-containing methionine. That is, the inorganic component, sodium selenite, is absorbed by the yeast and converted to selenium methionine, which is an organic selenium accumulated in protein components in the yeast. In this process, inorganic selenium, which is a toxic component, is converted to safe organic selenium and can be used as a nutritional supplement.
In order to mass-produce selenium-containing yeast, various fermentation methods such as batch culture, oil culture, and continuous culture are used, but oil culture is widely used. At this time, the content of inorganic selenium, the concentration of selenium, the selenium supply method, the medium composition, the pH, the temperature, the mixing rate, the oxygen supply amount, and the incubation time affect the absorption rate of selenium into the yeast and the growth rate of the cell. These conditions make it difficult to stably produce organic selenium without the residual amount of inorganic selenium in the actual mass production, so that selenium which is insufficient at the end of the culture is present in a large amount of the culture broth. Residual selenium that has not been absorbed after culturing causes the residual selenium concentration in the culture wastewater to increase after cell collection, resulting in exceeding the permissible selenium limit of wastewater (1 ppm or less).
The cost of wastewater treatment is increased in order to treat a large amount of waste medium containing inorganic selenium which is generated after cultivation, so that the production cost is inevitably increased and the environmental problems are caused by the accumulation of heavy metals in the river and soil during discharge.
Among the conventional methods for producing selenium-containing yeast, Korean Patent Registration No. 0092586 discloses a method for producing selenized yeast, which comprises preparing a yeast containing high concentration of selenium by fermentation of yeast, removing the remaining selenium after incubation using activated carbon And in a method for producing yeast strains containing high concentration of selenium in Korean Patent Registration No. 0142891 and a method for producing the same, the fermentation waste medium is reused to suppress the generation of fermentation waste liquid containing inorganic selenium. However, such a method can not be considered as a fundamental method, and a high-cost wastewater treatment cost is incurred (Patent Documents 1 and 2).
Accordingly, the present inventors have completed the present invention by developing a method for producing selenium-containing dried yeast in which the amount of inorganic selenium in the culture medium is reduced and the content of selenium in the yeast is increased after culturing by adding serine to the yeast culture medium.
The object of the present invention is 1) to inoculate yeast into a YM medium containing 1 to 50 ppm of selenium, ferment at a fermentation temperature of 20 to 35 ° C, aeration amount of 0.5 to 1.5 vvm and a stirring speed of 300 to 1000 rpm for 12 to 20 hours step; 2) culturing the medium with selenium salt, serine and nutrient solution at a culture temperature of 20 to 35 ° C, aeration amount of 0.5 to 1.5 vvm and a stirring speed of 300 to 1000 rpm for 52 to 60 hours; 3) harvesting and washing the cultured yeast; And 4) re-harvesting the washed yeast after sterilization, and drying the dried yeast.
Another object of the present invention is to provide a selenium-containing dry yeast.
In order to achieve the above object,
One embodiment of the present invention provides a method for producing selenium-containing dry yeast,
1) inoculating yeast into YM medium containing 1 ppm or more of selenium and then fermenting;
2) adding the selenium salt (selenium salt), serine and the nutrient solution to the culture medium;
3) harvesting and washing the cultured yeast; And
4) re-harvesting and washing the washed yeast after sterilization,
More specifically
1) inoculating yeast into a YM medium containing 1 to 50 ppm of selenium, fermenting at a fermentation temperature of 20 to 35 ° C, aeration amount of 0.5 to 1.5 vvm and a stirring speed of 300 to 1000 rpm for 12 to 20 hours;
2) culturing the medium with selenium salt, serine and nutrient solution at a culture temperature of 20 to 35 ° C, aeration amount of 0.5 to 1.5 vvm and a stirring speed of 300 to 1000 rpm for 52 to 60 hours;
3) harvesting and washing the cultured yeast; And
4) re-harvesting and drying the washed yeast after sterilization treatment.
As used herein, the term "selenium" is an element of atomic number 34 and the elemental symbol is Se, a trace mineral that is essential for various actions in the body and is an antioxidant. Selenium exists in the form of selenomethionine and selenocysteine in animal and plant tissues and is a protein that contains selenium such as glutathione peroxidase (GSHPx) and selenoprotein P As a component of the plasma. The average recommended selenium requirement for male and female adults is 42 μg / day. The recommended intake is 50 μg / day using the 10% coefficient of variation and the upper limit is 400 μg / day (Korean Nutrition Society, 2005; Expert Group on Vitamins and Minerals, Safe Upper Levels for Vitamins and Minerals, 2003; Selenium can be ingested through yeast or plants that have increased selenium content.
As used herein, the term "dry yeast" means a yeast that has been dried so that the yeast that is susceptible to a large amount of water may be stored without being decomposed.
Thus, the term "selenium-containing dry yeast " as used herein means dry yeast prepared by drying yeast with enhanced selenium content.
In the above step 1), a medium (pH 6.2 ± 0.2) containing 3 g of yeast extract, 3 g of malt extract, 5 g of peptone and 10 g of glucose per 1 L of the medium may be used as the YM medium.
The yeast of the above step 1) may be a known strain, specifically Saccharomyces cerevisiae cerevisiae), Saccharomyces access to my story Honors L (Saccharomyces pastorianus), Cluj Vero My process lactis (Kluyveromyces lactis) and Cluj Vero My process infrastructure jilriseu (Kluyveromyces fragilis ) can be used. For example, saccharomyces cerevisiae ( Saccharomyces cerevisiae) cerevisiae ) may be used, but the present invention is not limited thereto.
The yeast of step 1) may be selected from strains grown in YM medium containing 1 to 50 ppm of selenium before step 1), and yeasts selected as strains resistant to selenium salts may be used have.
The yeast of step 1) can be inoculated in an amount of 5 to 30% (v / v) to the medium, specifically in an amount of 5 to 20% (v / v) The seeds can be inoculated in an amount of 10% (v / v).
The seed culture can be obtained by inoculating cultured yeast strains with the yeast strain colony in the YM medium. For example, seed culture can be used which is cultured at 30 DEG C and 150 rpm for 16 hours with shaking.
In the above step 1), "fermentation" is a phenomenon in which microorganisms decompose organic acids of relatively large molecules, and the energy generated thereby is used as a driving force for living things. For example, saccharomyces cerevisiae among fermenting bacteria undergoes alcohol fermentation.
The yeast is inoculated in a YM medium containing 1 to 50 ppm of selenium at a fermentation temperature of 20 to 35 ° C, aeration amount of 0.5 to 1.5 vvm and a stirring speed of 300 to 1000 rpm for 12 to 20 hours More specifically, after yeast is inoculated into YM medium containing 10 to 40 ppm of selenium, the yeast is cultivated at a fermentation temperature of 28 to 32 DEG C, aeration amount of 0.8 to 1.2 vvm, a stirring speed of 400 to 600 rpm for 14 to 18 hours For example, yeast can be inoculated in a YM medium containing 20 ppm of selenium, followed by fermentation at a temperature of 30 ° C, aeration amount of 1.0 vvm, and a stirring speed of 500 rpm for 16 hours.
The step 1) may be performed to adjust the yeast to selenium at the early stage of fermentation, and to produce a logarithmic growth phase in which metabolism is most active. If the selenium salt is added and fermented from the beginning of the yeast fermentation, the yeast can not grow and die, and the production amount can be reduced. Thus, by performing the above step 1), the yeast is adapted to selenium, and after the increase of the growth, the selenium salt is added in the next step, so that the yield of selenium-containing dry yeast can be increased by using a small amount of yeast .
In the above step 2), "Selenium salt" may be sodium selenite (Na 2 SeO 3 ), and sodium selenite is an inorganic selenium.
The selenium salt of step 2) may be added so that the concentration of selenium is 10-200 ppm based on the medium, specifically, 30-100 ppm of selenium, more specifically, 30-50 ppm of selenium For example, at a concentration of 40 ppm of selenium. In the prior art, a selenium salt is supplied at a high concentration to produce a selenium-containing yeast having a high concentration. In this case, the amount of residual inorganic selenium is increased, and excess selenium is discharged to the waste solution during washing. Therefore, conventionally, attempts have been made to solve this problem by using activated carbon or reusing the medium. However, in the present invention, a selenium salt is supplied at a low concentration, so that a dried yeast having a selenium content of about 500 ppm, which is a selenium content in the yeast, can be prepared, and excessive selenium is not discharged into the waste solution during washing, Can be saved.
In the above step 2), "serine" is a kind of amino acid constituting a protein, and the abbreviation of the residue is Ser, and specifically, L-serine can be used. Serine can be a commercially available known serine. In one embodiment of the present invention, Kyowa Hakko Bio co. ltd. L-serine was purchased and used, but not always limited thereto.
The serine of step 2) may be added at a molar concentration of 0.1 to 10 times the selenium salt, specifically 0.5 to 5 times the molar concentration of the selenium salt. More specifically, Mol, more specifically 0.1 to 7.6 mM, and may be added at a concentration of 0.5 mM, for example. The selenium salt adsorbed into the cells reacts with serine to convert to selenomethionine. By supplying serine from the outside, the conversion of selenium in the cells can be increased. In addition, the addition of serine to the selenium salt at the same molar concentration can increase the reaction rate.
By adding the serine in the step 2), the inorganic selenium residual amount in the medium decreases and the selenium content in the yeast can be increased. Therefore, the cost for removing residual inorganic selenium can be reduced.
The nutrient solution of step 2) may be water containing glucose, specifically water containing 1 to 20% by weight of glucose. More specifically, water containing 1 to 10% by weight of glucose may be used. For example, water containing 2% by weight of glucose can be used. The glucose acts as a nutrient to generate an energy source necessary for the process of the inorganic selenium salt absorbed into the cells and metabolized. The nutrient may further include nutrients necessary for yeast growth.
More specifically, the culture in step 2) can be carried out at a culture temperature of 20 to 35 ° C, aeration amount of 0.5 to 1.5 vvm, and a stirring speed of 300 to 1000 rpm for a period of 52 to 60 hours, Aeration rate of 0.8 to 1.2 vvm and a stirring speed of 300 to 500 rpm for 54 to 58 hours. For example, the culture can be performed at a temperature of 27 to 28 ° C, aeration rate of 1.0 vvm, and a stirring speed of 400 rpm for 56 hours.
In the step 3), the harvesting of the yeast means separating the yeast after the culture is recovered from the fermentation tank. For example, centrifugation, microfiltration or the like may be used.
In step 3), washing of the yeast can be carried out by adding physiological saline to the harvested cells, mixing them, and recovering the cells. For example, physiological saline is added to the harvested cells at a ratio of And then the cells are recovered by repeating the above steps three times or more. The washing can completely remove residual inorganic selenium that may remain in the medium.
In step 4), the yeast may be sterilized to stop the activity of the yeast. Specifically, the yeast may be sterilized at 55 to 90 ° C for 0.5 to 3 hours.
In the step 4), the yeast may be dried using lyophilization, spray drying or hot air drying. Specifically, lyophilization may be used. When the freeze-drying method is used, the sterilized yeast is no longer subjected to heat, so that the amount of the harvested bacteria can be maximally preserved.
The mass production process of selenium-containing dry yeast is generally subjected to a process of culture, washing, pasteurization and drying. At this time, the washing process is for removing the residual inorganic selenium which is not absorbed into the yeast, and the pasteurization heat treatment process is for stopping intracellular metabolism by sterilizing the yeast cells.
Unless the inorganic selenium is completely removed by the washing process, the residual selenium becomes red color inherent to the element type during the heat treatment, resulting in a reddish color of the final product. According to the Korea Food and Drug Administration's amendment to Article 11 of the Korean Pharmacopoeia, each part of the Pharmaceuticals Regulations, Part 1, reports on selenium-containing dry yeast, "This drug is a yeast cultured in a medium containing selenium and organically bound to selenium This drug contains more than 540 ug of selenium per 1 g of the indicated amount when quantified. In addition, the properties of this drug is yellowish brown to brown powder, odor and taste peculiar to the yeast, "he said. In other words, since the remaining amount of inorganic selenium in the manufacturing process ultimately turns the color of the final product red, the quality of the product is not suitable for the medicinal product notification, and the final product can not be manufactured. Since the absorption rate of selenium into the yeast body is the most important factor in production of the product, the present invention intends to maximize the selenium absorption rate during the mass production process of the selenium yeast.
Explaining the conversion of inorganic selenium to organic selenium by yeast, the inorganic selenium absorbed in the form of sodium selenite reacts with glutathione to become selenoglutathione and then to selenite. Selenium hydrogen reacts with serine to produce selenomethionine through selenocysteine. The selenomethionine thus formed is included as a protein component in place of methionine through a protein synthesis process.
In this process, the intermediate selenium (H 2 S) acts as a toxic element in the yeast, which lowers the yeast growth and lowers the yeast selenium absorption rate. In order to overcome this, supplying serine as an external medium component can increase serine concentration in the yeast cells and allow the selenium hydrogen to react with serine to rapidly convert to selenized cysteine. As a result, the efficiency of selenium absorption of the yeast is increased, and thus the yeast containing selenium containing the maximum yield can be produced.
The present invention relates to a mass production method that enables a yeast to absorb selenium as efficiently as possible by modifying a fermentation method for producing selenium-containing yeast. The present invention can minimize the occurrence of residual inorganic selenium, Dry yeast can be mass produced. Selenium-containing yeast By adding serine to the raw material ingredients added during the yeast production process, the absorption rate of selenium in the yeast can be improved, and the amount of residual selenium outside the cell can be dramatically lowered and the selenium-containing dry yeast can be mass-produced.
Another embodiment of the present invention provides a selenium-containing dry yeast produced by the above method.
The selenium-containing dry yeast may have a selenium content in the yeast of 500 to 1500 ppm, specifically 600 to 900 ppm, more specifically 650 to 850 ppm, for example 800 ppm, but not always limited thereto .
The prior art has focused on producing yeast containing selenium as high as possible when producing selenium-containing dry yeast. Thus, a method of preparing a yeast containing high concentration of selenium, and then drying yeast were mixed to lower the total selenium content according to the pharmacopoeial standard. For example, when a high-concentration selenium-containing yeast having a concentration of 10,000 to 20,000 ppm is prepared, it is necessary to use a suitable method such as dry yeast and dilute it 10 to 20 times with a suitable excipient to produce a final product. Or if it is not uniformly mixed according to the physical properties of the excipient, the selenium content in the final product may be shifted to a certain region.
However, since the selenium-containing dry yeast of the present invention has a selenium content close to about 500 ppm, which is the required concentration of selenium in the yeast, it is not necessary to further dilute the dry yeast. Even if it is diluted, the dilution ratio is low, Less.
Using the method for producing selenium-containing dry yeast of the present invention, the residual amount of inorganic selenium can be lowered by adding serine, and the selenium content in the yeast can be increased to maximize the efficiency of producing selenium-containing dry yeast. In addition, it is possible to omit the step of diluting the dried yeast by supplying the selenium salt at a low concentration during the manufacturing process, and excessive selenium may not be discharged to the waste solution during washing. Thus, the cost for treating the residual inorganic selenium can be reduced.
1 is a schematic view showing a method for producing selenium-containing dry yeast according to an embodiment of the present invention.
Fig. 2 is a graph showing selenium concentration in ppm (ppm) and residual selenium concentration (ppm) in the medium during the production of dry yeast containing selenium under serine addition (example) or serine addition (comparative example) conditions.
Hereinafter, the present invention will be described in more detail in the following Examples. It should be noted, however, that the following examples are illustrative only and do not limit or limit the scope of the present invention. It will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Example 1. Preparation of selenium-containing dry yeast using fermentation method with addition of serine
The most important factor in preparing selenium-containing yeast of the present invention is that the absorption rate can be maximized by adding serine to the process of absorbing inorganic selenium into the body of yeast.
Various strains of Saccharomyces cerevisiae were obtained for the production of selenium-containing yeast by fermentation. KCTC7915 strain, KCTC7924 strain, KCTC7924 strain and KCTC27141 strain were distributed from the microbial resource center. KCM11201 strain, KCCM11306 strain, and KCCM11920 strain were distributed from Korean Microorganism Conservation Center. Thereafter, strains growing on YM plate medium (containing 3 g yeast extract, 3 g malt extract, 5 g peptone and 10 g glucose, pH 6.2) containing 20 ppm of selenium were selected to be resistant to selenium salts Were selected. In order to identify strains having a high growth rate, each of the colonies was inoculated into a 250 ml flask containing 25 ml of a liquid medium composed of the same components as the culture medium, and cultured at 30 DEG C and 150 rpm for 24 hours. The strain was selected as a strain with a rapid growth rate. The selected strains were used for the production of selenium containing dry yeast. The strain selected was KCCM11306 strain.
For culturing, 2 L of YM medium (3 g yeast extract, 3 g malt extract, 5 g peptone and 10 g glucose, pH 6.2) containing 20 ppm of selenium was added to a 5 L fermenter. Further, the selected Saccharomyces cerevisiae colony was inoculated into the flask containing the same medium (YM medium containing 20 ppm of selenium) and cultured with shaking at 150 rpm at 30 DEG C for 16 hours. The prepared seeds were inoculated in a volume of 10% (v / v) in 2 L of YM medium and fermented for 16 hours at a culture temperature of 30 ° C, aeration amount of 1 vvm, and a stirring speed of 500 rpm. Nutrient solution containing serine (sodium selenite; Na 2 SeO 3 ) and serine (L-serine from Kyowa Hakko Bio Co., Ltd., Japan were purchased and used) 28 ° C, aeration amount of 1 vvm, and a stirring speed of 500 rpm for 56 hours. Water containing 2 wt% of glucose was used as the nutrient solution, and 173 mg of sodium selenium corresponding to 40 ppm of selenium (Se) was used as the selenium salt based on 2 L of the medium. Also, 52.5 mg of the serine was added and added at the same molar concentration as the molar concentration (0.5 mM) of the selenium salt to the medium. Thereafter, the culture broth was recovered from the fermentation tank, and the cells were harvested using a centrifugal separator. Then, physiological saline solution of one volume of the culture was added, and the cells were centrifuged after the re-mixing, and the cells were washed three times. Then, it was re-harvested after sterilization treatment at 55 to 90 ° C for 0.5 to 3 hours and dried by freeze drying.
A flow chart of the method for producing dried yeast containing selenium is shown in Fig.
Comparative Example 1. Preparation of selenium-containing dry yeast using fermentation method without addition of serine
A selenium-containing dry yeast was prepared by fermentation in the same manner as in Example 1, except that serine was not added to the medium components.
Experimental Example 1. Analysis of residual selenium and selenium content in yeast by addition of serine
The dry yeast of Example 1 and Comparative Example 1 was analyzed to analyze the content of selenium in the residual selenium and yeast in the culture broth according to the addition of serine.
Specifically, in Example 1 and Comparative Example 1, after the completion of the incubation, the supernatant was washed with acid and the dried yeast was subjected to acid hydrolysis to obtain an Inductively Coupled Plasma-Atomic Emission Spectrometer (ICP-AES; Selenium content was measured by the manufacturer: PerkinElmer, model: OPTIMA 5300DV). At this time, in the condition that no serine was added (Comparative Example 1), residual selenium content in the culture solution not absorbed by the yeast was detected to be about 6 to 8 ppm, and selenium content in the yeast was about 600 ppm. On the other hand, in the experiment (Example 1) in which fermentation was carried out with the composition of the medium to which serine was added together, the residual amount of inorganic selenium in the culture solution was 0 to 0.5 ppm, the selenium content in the yeast was about 800 ppm, (Fig. 2).
Therefore, it has been confirmed that when selenium in the medium is added to produce selenium-containing dry yeast, the residual selenium content of the medium can be reduced and the content of selenium in yeast cells can be increased.
Claims (5)
2) culturing the medium with selenium salt and serine at a culture temperature of 20 to 35 ° C, aeration amount of 0.5 to 1.5 vvm and a stirring speed of 300 to 1000 rpm for 52 to 60 hours;
3) harvesting and washing the cultured yeast; And
4) re-harvesting the washed yeast after sterilization, and drying the dried yeast.
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