KR100390546B1 - Saccharomyces cerevisiae strain having high immune-boosting activity and preparation thereof - Google Patents

Abstract

The present invention relates to a yeast mutant strain with improved immune activity and a method for producing the same, wherein the mutations are NTG (N-methyl-N'-nitro-N-nitrosogu anidine), EMS (ethylmethane sulfonate) and UV and cell wall degrading enzyme Saccharomyces cerevisiae IS 2 (KCTC 0959BP), IS 9 (KCTC 0960BP), a novel strain from Saccharomyces, a mutant yeast strain developed using glass beads and glass beads. , IB 54 (KCTC 0961BP) and IB 56 (KCTC 0962BP) showed excellent immunostimulating activity.

Description

Saccharomyces cerevisiae strain having high immune-boosting activity and preparation etc.

The present invention relates to a Saccharomyces cerevisiae strain with improved immune activity promoting function and a preparation method thereof. More specifically, the method of preparing yeast mutants with improved immune activity by changing the components of glucan, which is a cell wall component of yeast, and Saccharomyces tax, which can be used for the production of yeast products that enhance immune function. Saccharomyces cerevisiae relates to IS 2, IS 9, IB 54, IB 56.

Yeast has a solid cell wall, which consists of anoproteins, glucans, and small amounts of chitin and fat. The cell wall of yeast is layered with three components, the inner side of the cell is composed of beta 1,3 and 1,6 glucan as the main components, a small amount of chitin and agate protein, and the outside is the mannan linked to the protein. Agate protein (Fleet, GH 1991. Cells Walls. In: Rose AH, Harrison Js (eds) The yeasts, vol. 4, edn.Academic Press, London, pp. 199-277 and Klis, FM 1994. Review: Cell Wall assembly in yeast.Yeast 10: 851-869). These cell wall polysaccharides are divided into water-soluble manganese, alkali-soluble glucans, alkali insoluble glucans, and chitin. (Fleet, GH and DJ Manners, 1977. The enzymatic degradation of an alkali-soluble glucan from the cell wall of Saccharomyces cerevisiae. J. Gen. Microbiol. 94: 315-327)

Yeast is a microorganism that is used in various industries including the food sector. In the livestock sector, it is added to livestock feed to promote the secretion of digestive enzymes necessary for livestock growth, stabilize the intestinal bacteria, and prevent metabolic diseases. It is widely used as a probiotics that is effective in improving livestock productivity. Recently, glucan, a major component of the yeast cell wall, has been reported as a substance that enhances the immune function of animals, and when it is fed to animals, it is known to have an effect of increasing the disease resistance of animals. It is currently attracting attention as an alternative to solve livestock residue and tolerance problems (James, S., J. Easson, D. Davidson and GR Ostroff, 1996. Use of aqueous soluble glucan preparation to stimulate platelet production.US patent 5,532,223 and Williams, DL, HA Pretus, RB McNamee, EL Jones, HE Ensley and IW Borwder, 1992. Development of a water-soluble, sulfated (1-> 3) -β-D-glucan biological response modifier derived from Saccharomyces cerevisiae. Carbohydr.Res. 235: 247-257). In addition to animal feed products, glucans are used in various industries as substances that lower blood cholesterol levels, as anticancer agents, cosmetics, surfactants, or as food additives. In the related art related to the present invention, a mutant selected through a mutation of Saccharomyces cerevis is used to exhibit resistance to hydrolysis by glucanase and exhibits excellent particle size between 0.2 and 10 microns. U.S. Pat.No. 5,506,124 describing how to make glucans and selecting mutants (Jamas, Spiros, Rha, ChoKyun, Sinskey, J. Anthony, 1996. Process for preparing glucanase resistant yeast mutants.US patent 5,506,124), By inducing mutations in the Candida genus isolated from the soil, mutants with 70% less mannan, the main component of the cell wall, were produced and produced microbial protein components used in the production of food, livestock and fish feed. U.S. Patent No. 3,855,063 describing the method of selection and selection of mutant strains (Nagasawa, T., J. Ono, T. Kudo, Y. Harada 1974. Process for p roducing a micbial proteinous substance.US patent 3,855,063).

However, as a result of the selection of yeast mutant strains with enhanced immune activity-induced function induced by the change of glucan component by the method of US Pat. No. 5,506,124, beta-1,3-glucanase (β-1, Using only one 3-glucanase did not facilitate selection of the desired mutant strain. For this reason, when the mutated yeast is treated with beta-1,3-glucanase, a cell wall degrading enzyme, sporeoplasts are formed due to the characteristics of the yeast, and then the cell walls are restored when cultured in liquid medium and plate medium. Due to the phenomenon of regeneration), the beta-1,3-glucanase treatment alone changed the composition of the cell wall, resulting in little selection of the mutant strains resistant to beta-1,3-glucanase. In view of the above, the present invention introduces a method using glass beads capable of mechanically stressing mutant yeasts firstly selected with cell wall degrading enzymes for optimization of selection factors. In addition, in order to avoid selection of strains derived from other metabolic mutations that may occur during the mutation process, the present invention was used for selection of mutant strains intended for molasses medium.

Therefore, an object of the present invention is to produce a yeast strain Saccharomyces cerevisiae IS 2, IS 9, IB 54, IB 56 with improved immune activity promoting function.

Another object of the present invention is to provide a method for producing the yeast strain Saccharomyces cerevisiae ( Saccharomyces cerevisiae ).

The object of the invention is to use the mutations NTG (N-methyl-N'-nitro-N-nit rosoguanidine), EMS (ethylmethane sulfonate) EMS and UV and cell wall degrading enzymes and glass beads (glass bead) to make the glucan composition This was achieved by making yeast mutant strains IS 2, IS 9 and IB 54, IB 56 and confirming that the immune activity promoting function was improved over the existing yeast.

Hereinafter, the configuration of the present invention will be described.

The present invention is inoculated with Saccharomyces cerevisiae (Saccharomyces cerevisiae) KCTC 7911 in liquid YPD medium and cultured and harvesting the yeast by centrifugation; Diluting the number of yeasts to 10 ⁸ per mL, washing three times with sterile distilled water, and mutating them to a survival rate of 0.1% using NTG as a mutagen; Harvesting the mutated yeast and incubating for 6 hours at 30 ° C. in a liquid YPD medium to which 600 μg / mL of zymolase was added, followed by harvesting by centrifugation; Washing with sterile distilled water and then adding the same amount of glass beads and shaking vigorously for 30 seconds; Take the liquid portion and re-yield the yeast, incubate for 3 hours at 30 ° C in liquid YPD medium containing 600μg / mL zymolase, harvest it, wash it with sterile distilled water, and then wash the yeast strain Saccharomyces at room temperature. Leaving Saccharomyces cerevisiae IS and IB strains for 10 minutes; The above-described process was repeated two more times, and the harvested yeast was molasses medium (molasses 30 mL / L, urea 5 g / L. Phosphoric acid 2.5 mL / L, ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) 7.5 g / L, pH 6.5) and selecting colonies growing by storage at 30 ℃ as a mutant strain resistant to zymolase; Culturing Saccharomyces cerevisiae IS 2, IS 9 and IB 54, IB 56 strains; Extracting yeast glucan from Saccharomyces cerevisiae IS 2, IS 9 and IB 54, IB 56 cells; Saccharomyces cerevisiae (Saccharomyces cerevisiae) IS 2, IS 9 and IB 54, IB 56, using the glucan glucans of the NO production capacity (nitric oxide) and macrophages activation and phagocytic activity consists of the step of identifying.

Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) used in the present invention was used for sale in a microbial deposit center in the Biotechnology Research Institute, the accession number is KCTC 7911.

The inventors of the present invention, strains Saccharomyces cerevisiae IS 2, IS 9, and IB 54, IB 56, which have been selected to enhance the function of promoting immune activity, are transferred to IS 2 (KCTC 0959BP) at the microbial deposit center of the Biotechnology Research Institute. ), IS 9 (KCTC 0960BP) and IB 54 (KCTC 0961BP), IB 56 (KCTC 0962BP).

In this step, 100-1000 μg / mL of the cell wall lyase is added, and preferably 600 μg / mL.

In this step, Saccharomyces cerevisiae ( Saccharomyces cerevisiae) KCTC 7911 was mutated and cultured in liquid YPD medium (2% glucose, 1% yeast extract, 2% peptone) to which cell wall lyase was added. In the middle of repeating the process of washing the harvested mutants with sterile distilled water, the addition of glass beads to the strong shaking process, the relative growth, the resistance to the cell wall degrading enzyme zymolase In the experiments measured in%), about 5% of the colonies in molasses medium showed better resistance than the parent strain. These figures showed about 20 times higher selection ability than the conventional method for treating only one glucanase (Table 1), and the resistance to the selected mutant zymolase was also selected by the method used in the present invention. Mutant strains showed higher levels.

Hereinafter, the specific configuration and operation of the present invention will be described with reference to Examples, but the scope of the present invention is not limited only to the following Examples.

Example 1 yeast mutant strain selection method

A colony of Saccharomyces cerevisiae KCTC 7911 was inoculated in liquid YPD medium and cultured until mid-log phase. The yeast was harvested by centrifugation, and the number of yeasts was diluted to 10 ⁸ per mL, washed three times with sterile distilled water, and then mutated to a survival rate of 0.1% using NTG. The mutated yeast was harvested and washed three times with sterile distilled water, followed by incubation for 6 hours at 30 ° C. in a liquid YPD medium containing 600 μg / mL of zymolase and harvested by centrifugation. The harvested yeast was washed with sterile distilled water, then the same amount of glass beads was added and vigorously shaken for 30 seconds (Vortex GENIE 2, Scientific Instrument). The liquid portion was taken and the yeast was re-harvested, and again harvested after incubation at 30 ° C. for 3 hours in a liquid YPD medium to which 600 μg / mL of zymolase was added. Again washed with sterile distilled water and left for 10 minutes at room temperature. Repeat the above procedure two more times and harvest the yeast harvested molasses medium (molasses 30mL / L, urea 5g / L. phosphoric acid 2.5mL / L, ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) 7.5g / L, pH 6.5) and colonies growing by storage at 30 ℃ was selected as a mutant strain resistant to zymolase.

The yeast mutated by the above method was incubated in a liquid YPD medium to which zymolase was added, and then plated on a molasses plate medium at an appropriate dilution ratio and left at 30 ° C. As shown in Table 1, only 705 colonies of fast growing colonies in molasses medium were selected and inoculated in liquid YPD medium to which 600 μg / mL of zymolase was added. After 6 hours of incubation, the cell growth was measured by O.D (optical density). As a control, the parent strain, KCTC 7911, was used to select mutant strains resistant to Zymolase. As the selection conditions, only beta-1,3-gluganase was selected for the selection of two species, and combinations of xymolase and glass beads were selected for 36 species. The first resistance test for cell wall degrading enzyme was repeated three times.

Number of Cell Wall Degrading Resistant Yeast Mutants Primary Selected from Molasses Plate Medium After Mutation Treatment and Cell Wall Degrading Enzyme Treatment Selection method Selection using only beta-1,3-glucanase Selection of Zymolase and Glass Beads in Combination Number of yeast colonies showing rapid growth in molasses platelets after mutation and cell wall digestase treatment 705 705 Number of mutant yeast colonies resistant to cell wall lyase (600 μg / mL zymolase) finally selected from 705 colonies 2 36

Example 2 Resistance Test by Zymolase Concentration of Saccharomyces cerevisiae IS, IB Strain

In Example 1, the mutant strains showing a cell growth of 55% or more even at a concentration of 800 μg / mL of zymolase were finally selected by performing the resistance test against zymolase of 36 mutant strains selected first. 36 yeast mutants were inoculated in YPD medium containing 0 μg / mL to 800 μg / mL zymolase and incubated for 6 hours in a shaker incubator. Superior Saccharomyces cerevisiae IS 2, IS 9, IB 54, IB 56 strains were selected. The resistance test results of the final selected yeast mutant strains of zymolase concentration are shown in Table 2 below. As a control, Saccharomyces cerevisiae KCTC 7911, a parent strain, was used, and cell growth concentration and 400-800 μg / mL of Zymolase were grown in the condition that Zymolase was not added after 3 replicates. It is the average value of the ratio of cell growth concentration grown in the normalized condition.

Results of Tolerance Tests by Zymolase Concentration in Yeast Variants Strain zymolase concentration (μg / mL) Control (KCTC 7911%) IS 2 (%) IS 9 (%) IB 54 (%) IB 56 (%) 0 100 100 100 100 100 400 35.4 81.6 100 76.1 77.2 600 23 74.6 93.6 65 75.4 800 17.7 64 91.5 57.3 64.9

The inventors of the present invention, strains Saccharomyces cerevisiae IS 2, IS 9, IB 54, and IB 56, which have improved immune activity-promoting function, were selected for the development of IS 2 (KCTC 0959BP) in a microbial deposit center of the Biotechnology Institute. ), IS 9 (KCTC 0960BP) and IB 54 (KCTC 0961BP), IB 56 (KCTC 0962BP).

Example 3 Culture of Saccharomyces cerevisiae IS 2, IS 9, IB 54, IB 56 Strains

The initial medium was 100g / L molasses, 3g / L yeast extract, 5g / L peptone. Cultivation of inoculum for inoculation used liquid YPD medium. Growth medium composition is molasses 150-200g / L, yeast extract 3g / L, urea 2.5g / L, potassium phosphate 0.5g / L, magnesium sulfate 0.11g / L, calcium chloride 0.06g / L. Sterilize 2L of growth medium in 5L fermenter and inoculate 100mL of Saccharomyces cerevisiae IS and IB mutant strains (seed), rotation speed 300rpm, aeration rate 1vvm, culture temperature 30 ℃, pH 5.5 After culturing in a batch at and obtained yeast cells corresponding to 44-60g / L through the incubation of supplying growth medium.

Example 4 Extraction of Yeast Glucan from Saccharomyces cerevisiae IS 2, IS 9, IB 54, IB 56 Cells

The cell wall was harvested by autolysis of the yeast cells obtained in Example 3. To promote self-extinguishing, 5% ethanol, 1% salt (NaCl) and 0.5% papain were added to the yeast cells and reacted at 50 ° C. for 24 hours while stirring well. In order to separate the glucan from the cell wall obtained after self-extinguishing, 30 g of the cell wall was dispersed in 1 L of 0.25 M sodium hydroxide (NaOH) and left at 100 ° C. for 24 hours. The sodium hydroxide solution and the solid component were separated by centrifugation, washed three times with sterile distilled water, and then treated with 0.5 M acetic acid for 24 hours at 80 ° C., and the solid component was washed three times with sterile distilled water after centrifugation. The obtained solid component was dispersed in Tris-EDTA-mercaptoethanol buffer (0.1M, EDTA 5mM, mercaptoethanol 100mM, pH 6.8) containing 2% sodium dodecylsulfate. Heated for 1 hour 30 minutes. Again washed three times with sterile distilled water and centrifuged to harvest the solid components, and then lyophilized to obtain yeast glucan. The amount and yield of yeast glucan extracted from the finally selected yeast mutant strain are shown in Table 3 below.

Yield and Yield of Yeast Glucan Extracted from Finally Selected Yeast Variants Yeast strain Control (KCTC 7911) IS 2 IS 9 IB 54 IB 56 Amount of yeast used to harvest glucans (g, wet) 50 53 44 50 60 Amount of Glucan Harvested (g) 0.87 1.74 0.86 1.21 1.23 Glucan Extraction Yield (%) 1.74 3.28 1.95 2.42 2.05

Example 5 NO Production Capacity Test Using Glucan of Saccharomyces cerevisiae IS 2, IS 9, IB 54, IB 56 Strains

Glucan extracted in Example 4 was dissolved in dimethyl sulfoxide (DMSO), and then PBs (PBS, 2.56 g / L NaH ₂ PO ₄ .H ₂ O, 22.5 g / L Na ₂ HPO ₄ .7H ₂ O , 87.9g / L NaCl, pH 7.2), diluted to a concentration of 5mg / mL and intraperitoneally injected into 7-week-old mice (balb / c) 0.2mL each. Cold Hanks' solution 3 days after injection

(Hank's solution, 0.185 g / L Calcium Chloride. ₂ H ₂ O, 0.09767 g / L MgSO ₄ (anhydrous), 0.4 g / L Potassium Chloride, 0.06 g / L Potassium Phosphate Monobasic (anhydrous), 0.8 g / L Sodium Chloride, 0.04788g / L Sodium Phosphate Dibasic (anhydrous), 1.0g / L D-Glucose, 0.011g / L Phenol Red.Na, 0.35g / L Sodium Bicarbonate, pH 7.0) 5 × 10 ⁶ cells After diluting to / mL, each 100μL was dispensed into a microtiter plate, and the glucan sample was added to 100μL by 1mg / mL. After 20 hours of incubation at 37 ° C, 100 μL of the culture supernatant was transferred to another well, followed by grease (grease, 10g / L Sulfanilamide, 1g / L N- (1-Naphthyl) ethylenediamine -dihydrochloride, 2.5g / L Phosphoric acid) 100 μL of reagent was added and mixed. After standing at room temperature for 10 minutes, the absorbance was measured at 550 nm with a microplate reader.

As shown in Table 4, the glucan sample of Saccharomyces cerevisiae KCTC 7911, a control result of nitric oxide production, showed an OD value of 0.052, and the glucan of IS 2, IS 9 mutant strains. Samples showed 0.088 and 0.138, respectively. In IB 54 and IB 56 mutant strains, 0.092 and 0.101, respectively, showed a significant increase in NO production of 1.69-2.65 times compared to the control glucan sample. This suggests that physiological activity of celiac cells is induced by glucan samples of mutant strains.

Measurement of NO (nitric oxide) production capacity of celiac immune cells DMSO PBS KCTC7911 IS 2 IS 9 IB 54 IB 56 O.D (550 nm) 0.052 0.052 0.052 0.088 0.138 0.092 0.101

Example 6 Measurement of Activation and Phagocytosis of Macrophages Using Collected Peritoneal Immune Cells

Activation and phagocytosis of macrophages were measured using celiac immune cells harvested in Example 5. Cell activation was determined by cell size (FSC) and shape (SSC), showing the percentage of activated macrophages out of total macrophages in%. Phagocytosis was measured by measuring the fluorescence intensity by the phenotypic fluorescent latex beads (latex beads) and expressed the ratio of the population with a strong fluorescence intensity among all macrophages in%. The peritoneal cells collected in Example 5 were diluted to 1 × 10 ⁶ cells / mL, and 1.125 mL of diluted beads (fluorescent latex beads) and 0.3 mL of cells were mixed in a 15 mL tube. It was kept at 37 ° C. for 1-hour while shaking. After centrifugation, cells were washed three times with cold PBS and suspended in 5 mL of 2.5% formaldehyde / PBS. After standing on ice for 20 minutes, the phagocytes were measured by a Fluorescence-Activated Cell Sorter (FACS). The activity of macrophages was highest in IB 56 mutants with 62.61% in control, 74.09% in IS 2 mutant, 66.29% in IS 9 mutant, 69.8% in IB 54 mutant, and 76.53% in IB 56 mutant. The glucan sample of the mutant strain showed about 6-22% higher activity than the glucan sample of the control. The phagocytosis was 35.3% in the control and 50.45, 37.68, 51.67 and 47.9% in the IS 2, IS 9, IB 54 and IB 56 mutants, respectively. According to the experimental results, as shown in Table 5, the phagocytic activity was increased in proportion to the activity of the macrophages.

Measurement of Macrophage Activity and Phagocytosis Using Peritoneal Immune Cells cell PBS KCTC7911 IS 2 IS 9 IB 54 IB 56 Activity (%) 10.03 28.98 62.61 74.09 66.29 69.80 76.53 Phagocytosis (%) 0 17.61 35.3 50.45 37.68 51.67 47.90

As described above, the Saccharomyces cerevisiae yeast mutant of the present invention can be applied to the preparation of various functional yeast products by changing the composition of glucan, which is a cell wall component, and thus promoting immune activity. It is a very useful invention in the industrial and biomedical industry.

Claims (6)

(a) inoculating Saccharomyces cerevisiae in liquid YPD medium, culturing, and mutating the cultured yeast with 0.1% survival with NTG; (b) culturing the mutated yeast of step (a) in a liquid YPD medium to which 100-1000 μg / mL of zymolase is added; (c) harvesting the cultured yeast of step (b) and vigorously shaking with glass beads; (d) Re-harvesting the yeast in step (c), incubating in a liquid medium to which 100-1000 μg / mL zymolase is added, harvesting and rinsing with sterile water twice, and then molasses plate medium A method for producing a yeast mutant strain selected by mutant strains that show resistance to zymolase by smearing on. delete The yeast mutant strain (Accession No. KCTC 0959BP) according to claim 2, wherein the yeast mutant strain is Saccharomyces cerevisiae IS 2. The yeast mutant strain (Accession No. KCTC 0960BP) according to claim 2, wherein the yeast mutant strain is Saccharomyces cerevisiae IS 9. The yeast mutant strain (Accession No. KCTC 0961BP) according to claim 2, wherein the yeast mutant strain is Saccharomyces cerevisiae IB 54. The yeast mutant strain (Accession No. KCTC 0962BP) according to claim 2, wherein the yeast mutant strain is Saccharomyces cerevisiae IB 56.