Method for screening rhodotorula and application thereof

Abstract

According to the method for screening the rhodotorula and the application of the method, toluidine blue O is adopted as a reagent for screening the rhodotorula, the rhodotorula with high carotenoid yield can be rapidly and efficiently screened out, and the method has important significance in the field of carotenoid production.

Description

Technical Field

[0001] The invention belongs to the technical field of red yeast screening, and particularly relates to a method for screening red yeast and application thereof. Background Art

[0002] Carotenoids are a type of red, yellow or orange fat-soluble pigment widely found in nature, including β-carotene, astaxanthin, lutein and lycopene. As precursors, carotenoids can generate a variety of derivatives with important physiological functions, such as vitamin A, plant hormones, pigments and aromatic compounds. They play an important role in enhancing immunity, anti-oxidation, anti-radiation, anti-tumor and treating photosensitive diseases, and are widely used in food, cosmetics, medicine and feed industries.

[0003] Industrial production methods of carotenoids mainly include chemical synthesis, plant extraction and biological fermentation. Chemical synthesis is technically complex, and the synthesized carotenoids are far from natural carotenoids, with certain side effects; plant extraction is costly and complex, and is subject to greater restrictions in industrial production and application; the use of microbial fermentation to produce carotenoids is low-cost, simple, safe and efficient, and has become the main method for producing carotenoids.

[0004] The invention provides a red yeast screening method with low cost, convenient operation and high yield, which is of great significance in the field of carotenoid production. Summary of the invention

[0005] The invention provides a method for screening red yeast and application thereof. Toluidine blue O is used to screen the red yeast, which not only improves the screening efficiency but also improves the pigment yield of the red yeast.

[0006] The present invention adopts the following technical solutions: In a first aspect, the present invention provides a reagent for screening red yeast.

[0007] Toluidine blue O is a photosensitizer that produces oxygen anions under strong light and is mainly used for tissue staining. In the present invention, by adding TBO to the culture medium of red yeast, an oxygen stress environment can be created. Only red yeast with high carotenoid production can quickly generate colonies, which significantly improves the screening efficiency of red yeast.

[0008] According to the present invention, the concentration of toluidine blue O is 0.1-5 mmol / mL.

[0009] According to the present invention, the concentration of toluidine blue O can be 0.1 mmol / mL, 0.2 mmol / mL, 0.8 mmol / mL, 1.5 mmol / mL, 2.0 mmol / mL, 2.8 mmol / mL, 3.0 mmol / mL, 3.3 mmol / mL, 4.0 mmol / mL, 4.5 mmol / mL or 5.0 mmol / mL.

[0010] According to the present invention, the concentration of toluidine blue O is preferably 0.5-2 mmol / mL.

[0011] According to the present invention, the concentration of toluidine blue O is further preferably 1-2 mmol / mL.

[0012] In a second aspect, the present invention provides a method for screening red yeast.

[0013] According to the present invention, the screening agent Toluidine Blue O as described in the first aspect is used to screen red yeast.

[0014] According to the present invention, the method for screening red yeast comprises the following steps: (1) Cultivating red yeast using a liquid culture medium supplemented with toluidine blue O; (2) Spreading the red yeast suspension on a solid culture medium and culturing the selected red yeast.

[0015] According to the present invention, the concentration of toluidine blue O is 0.1-5 mmol / mL.

[0016] According to the present invention, the concentration of toluidine blue O can be 0.1 mmol / mL, 0.2 mmol / mL, 0.8 mmol / mL, 1.5 mmol / mL, 2.0 mmol / mL, 2.8 mmol / mL, 3.0 mmol / mL, 3.3 mmol / mL, 4.0 mmol / mL, 4.5 mmol / mL or 5.0 mmol / mL.

[0017] According to the present invention, the concentration of toluidine blue O is preferably 0.5-2 mmol / mL.

[0018] According to the present invention, the concentration of toluidine blue O is further preferably 1-2 mmol / mL.

[0019] According to the present invention, the liquid culture medium in step (1) is a yeast extract peptone glucose culture medium.

[0020] According to the present invention, the culture temperature in step (1) is 25-30°C.

[0021] According to the present invention, the culture temperature may be, for example, 25°C, 26°C, 27°C, 28°C, 29°C or 30°C.

[0022] According to the present invention, the culturing time in step (1) is 22-25 hours.

[0023] According to the present invention, the culturing time may be, for example, 22 h, 23 h, 24 h or 25 h.

[0024] According to the present invention, the rotation speed of the shaking table during the culturing in step (1) is 180-220 rpm.

[0025] According to the present invention, the rotation speed of the shaker during the culturing may be, for example, 180 rpm, 190 rpm, 200 rpm, 210 rpm or 220 rpm.

[0026] According to the present invention, the culturing temperature in step (2) is 25-30°C, for example, 25°C, 26°C, 27°C, 28°C, 29°C or 30°C.

[0027] According to the present invention, the culturing time in step (2) is 2-5 days, for example, 2 days, 3 days, 4 days or 5 days.

[0028] According to the present invention, the steps of primary screening and mutagenesis are further included before step (1).

[0029] According to the present invention, the primary screening and mutagenesis specifically include: (1') inoculating the activated red yeast into a liquid culture medium for culturing to prepare a mutagenesis strain; (2') irradiating the strain prepared for mutation in step (1') with ultraviolet light to obtain mutant red yeast.

[0030] In the present invention, before using toluidine blue O to screen red yeast, ultraviolet irradiation is performed on the red yeast, so as to further improve the ability of the red yeast to produce carotenoids.

[0031] According to the present invention, the power of the ultraviolet lamp in step (2') is 30-50W, for example, 30W, 35W, 40W, 45W or 50W.

[0032] According to the present invention, the ultraviolet light irradiation time in step (2') is 1-10 min, for example, it can be 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min or 10 min.

[0033] As a preferred technical solution, the present invention provides a method for screening red yeast, using the screening reagent as described in the first aspect, comprising the following steps: (1) inoculating activated red yeast into a yeast extract powder peptone glucose liquid medium for culturing to prepare a mutagenesis strain; (2) irradiating the mutagenized strain described in step (1) under an ultraviolet lamp with a power of 30-50 W for 1-10 min to obtain a mutagenized red yeast; (3) using a yeast extract peptone glucose liquid medium supplemented with 0.5-2 mmol / mL toluidine blue O, culturing the induced red yeast described in step (2) at 25-30° C. for 22-25 h, with the rotation speed of the shaker being 180-220 rpm; (4) Spreading the red yeast suspension on a solid culture medium and culturing at 25-30° C. for 2-5 days to obtain the screened red yeast.

[0034] In a third aspect, the present invention provides a red yeast obtained by screening using the method described in the second aspect.

[0035] In the present invention, the pigment yield of the screened red yeast is as high as 5.027 μg / mL, which is 79.9% higher than that of wild red yeast. The screened red yeast is used for industrial fermentation to produce carotenoids, which is of great significance in the field of carotenoid production.

[0036] In a fourth aspect, the present invention provides a red yeast as described in the third aspect for use in fermentation production.

[0037] Preferably, the red yeast is used for fermentation production of carotenoids.

[0038] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention adds toluidine blue O to the culture medium of red yeast to create an oxygen stress environment, thereby being able to quickly and efficiently screen out red yeast with high carotenoid production; (2) The present invention uses ultraviolet mutagenesis and toluidine blue O together. Under the conditions of ultraviolet lamp irradiation time of 5 minutes and toluidine blue O concentration of 1 mmol / mL, the maximum pigment yield of the red yeast screened is 5.027 μg / mL, which is 79.9% higher than that of wild red yeast; (3) The screening method of the present invention has simple process, low cost and high efficiency, and can be applied to the industrial production of carotenoids. Implementation Example 1

[0039] (1) Activating red yeast, inoculating it into a yeast extract peptone dextrose (YPD) liquid medium, and culturing it at 200 rpm at 28° C. for 2 days to prepare a mutagenesis strain; (2) Irradiate the prepared mutagenized strain described in step (1) under an ultraviolet lamp with a power of 40 W for 5 min to obtain mutagenized Rhodotorula; (3) Use a yeast extract peptone dextrose liquid medium supplemented with 1 mmol / mL toluidine blue O and culture the mutagenized Rhodotorula described in step (2) at 28 °C for 24 h, with the shaker rotating at 200 rpm; (4) Spread the Rhodotorula suspension on a solid medium and culture it at 28 °C for 4 days to obtain the screened Rhodotorula. Example 2

[0040] (1) Activate Rhodotorula and inoculate it into a yeast extract peptone dextrose (YPD) liquid medium. Culture it at 28 °C and 200 rpm for 2 days to obtain a prepared mutagenized strain; (2) Irradiate the prepared mutagenized strain described in step (1) under an ultraviolet lamp with a power of 40 W for 3 min to obtain mutagenized Rhodotorula; (3) Use a yeast extract peptone dextrose liquid medium supplemented with 1.5 mmol / mL toluidine blue O and culture the mutagenized Rhodotorula described in step (2) at 28 °C for 24 h, with the shaker rotating at 200 rpm; (4) Spread the Rhodotorula suspension on a solid medium and culture it at 28 °C for 4 days to obtain the screened Rhodotorula. Example 3

[0041] (1) Activate Rhodotorula and inoculate it into a yeast extract peptone dextrose (YPD) liquid medium. Culture it at 28 °C and 200 rpm for 2 days to obtain a prepared mutagenized strain; (2) Irradiate the prepared mutagenized strain described in step (1) under an ultraviolet lamp with a power of 40 W for 7 min to obtain mutagenized Rhodotorula; (3) Use a yeast extract peptone dextrose liquid medium supplemented with 0.5 mmol / mL toluidine blue O and culture the mutagenized Rhodotorula described in step (2) at 28 °C for 24 h, with the shaker rotating at 200 rpm; (4) Spread the Rhodotorula suspension on a solid medium and culture it at 28 °C for 4 days to obtain the screened Rhodotorula. Example 4

[0042] (1) Activate Rhodotorula and inoculate it into a yeast extract peptone dextrose (YPD) liquid medium. Culture it at 28 °C and 200 rpm for 2 days to obtain a prepared mutagenized strain; (2) Irradiate the prepared mutagenized strain described in step (1) under an ultraviolet lamp with a power of 50 W for 1 min to obtain mutagenized Rhodotorula; (3) using a yeast extract peptone glucose liquid medium supplemented with 2 mmol / mL toluidine blue O, culturing the induced red yeast described in step (2) at 28° C. for 24 h, with a shaking speed of 200 rpm; (4) Spreading the red yeast suspension on a solid culture medium and culturing it at 28° C. for 4 days to obtain the screened red yeast. Example 5

[0043] (1) Activating red yeast, inoculating it into a yeast extract peptone dextrose (YPD) liquid medium, and culturing it at 200 rpm at 28° C. for 2 days to prepare a mutagenesis strain; (2) irradiating the prepared mutagenesis strain in step (1) under a 30 W ultraviolet lamp for 10 min to obtain a mutagenized red yeast; (3) using a yeast extract peptone glucose liquid medium supplemented with 0.1 mmol / mL toluidine blue O, culturing the induced red yeast described in step (2) at 25° C. for 25 h, with the rotation speed of the shaker at 220 rpm; (4) Spreading the red yeast suspension on a solid culture medium and culturing it at 25° C. for 5 days to obtain the screened red yeast. Example 6

[0044] (1) Activating red yeast, inoculating it into a yeast extract peptone dextrose (YPD) liquid medium, and culturing it at 200 rpm at 28° C. for 2 days to prepare a mutagenesis strain; (2) using a yeast extract peptone glucose liquid medium supplemented with 5 mmol / mL toluidine blue O, culturing the mutagenized strain described in step (1) at 30° C. for 22 h, with the shaking speed of the incubator at 180 rpm; (3) Spread the red yeast suspension on a solid culture medium and culture it at 30° C. for 2 days to obtain the screened red yeast.

[0045] The red yeast of the embodiment was inoculated on a YPD plate for 10 generations, cultured at 28°C for 4 days, the OD600 value of the culture solution was adjusted with a YPD liquid medium, 10 mL of the bacterial solution was taken, centrifuged at 8000 rpm for 10 min, the supernatant was discarded, 20 mL of concentrated hydrochloric acid with a concentration of 3 mol / L was added, the solution was bathed at 40°C for 60 min, treated in a boiling water bath for 4 min, placed in a -20°C refrigerator and rapidly cooled, washed twice with distilled water after centrifugation, 10 mL of acetone was added, and the solution was shaken in a 40°C water bath for 30 min, the acetone supernatant containing the pigment was taken, the maximum absorbance at 200-600 nm was measured, and the pigment yield was calculated according to the following formula, wherein A represents the maximum absorbance, V represents the volume (mL) of acetone added when extracting the pigment, D represents the dilution multiple of the sample, 0.16 is the carotenoid extinction coefficient, and L represents the volume (mL) of the fermentation solution.

[0046] Pigment yield (μg / mL) = A×V×D / 0.16×L Pigment content = pigment yield / biomass The results are shown in Table 1. Red yeast formed red colonies on the YPD medium.

[0047] Group Pigment yield (μg / mL) Improvement rate (%) Example 1 5.027 79.9 Example 2 4.600 64.6 Example 3 3.845 37.6 Example 4 3.781 35.3 Example 5 3.516 25.8 Example 6 3.123 11.8 Wild red yeast 2.794 —— It can be seen that the pigment yields of the red yeasts of Examples 1-6 are all higher than 3 μg / mL, and the pigment yield is increased by more than 10% compared with the wild red yeast. Among them, Example 1 adopts the optimal ultraviolet lamp irradiation time of 5 min and the optimal toluidine blue O concentration of 1 mmol / mL, and the pigment yield of the screened red yeast is as high as 5.027 μg / mL, which is 79.9% higher than that of the wild red yeast. Compared with other examples, Example 6 is directly screened with toluidine blue O without ultraviolet mutagenesis, and the pigment yield of the obtained red yeast is increased by 11.8% compared with that of the wild red yeast, indicating that toluidine blue O can increase the pigment yield of red yeast, but the red yeast can be further improved by toluidine blue O screening after ultraviolet mutagenesis.

[0048] In summary, the present invention adds toluidine blue O to the culture medium of red yeast to create an oxygen stress environment, and can quickly and efficiently screen out red yeast with high carotenoid production. Ultraviolet mutagenesis and toluidine blue O cooperate with each other. Under the conditions that the ultraviolet lamp irradiation time is 5 minutes and the concentration of toluidine blue O is 1 mmol / mL, the pigment production of the screened red yeast is as high as 5.027 μg / mL, and the pigment production is increased by 79.9% compared with that of wild red yeast.

Claims

1. A method for screening red yeast, It is characterized in that Toluidine blue O was used as a reagent for screening red yeast.

2. The method according to claim 1, It is characterized in that The concentration of the toluidine blue O is 0.1-5 mmol / mL.

3. The method according to claim 1 or 2, It is characterized in that The following steps are involved: (1) Cultivating red yeast using a liquid culture medium supplemented with toluidine blue O; (2) Spreading the red yeast suspension on a solid culture medium and culturing the selected red yeast.

4. The method according to claim 3, It is characterized in that The liquid culture medium in step (1) is a yeast extract peptone glucose culture medium, the culture temperature in step (1) is 25-30° C., the culture time is 22-25 hours, and the rotation speed of the shaking table during the culture is 180-220 rpm.

5. The method according to claim 3 or 4, It is characterized in that The culture temperature in step (2) is 25-30° C. and the culture time is 2-5 days.

6. The method according to any one of claims 4 to 5, It is characterized in that Prior to step (1), the method also includes the steps of preliminary screening and mutagenesis.

7. The method according to claim 6, It is characterized in that The initial screening and mutagenesis specifically include: (1) inoculating the activated red yeast into a liquid culture medium for culturing to prepare a mutagenesis strain; (2) irradiating the strain prepared for mutation in step (1) with ultraviolet light to obtain mutant red yeast.

8. The method according to claim 7, It is characterized in that The power of the ultraviolet lamp in step (2) is 30-50W, and the irradiation time is 1-10min.

9. A red yeast obtained by screening according to the method of any one of claims 1 to 8.

10. Use of the red yeast of claim 9 in fermentation production of carotenoids.