Monascus ruber strain with high extracellular yellow pigment yields, method for breeding monascus ruber strain and application thereof

A technology of Monascus and Monascus red, which is applied in the field of microbial strain breeding, can solve the problems of complicated production process, limited use, unfavorable water-soluble occasions, etc., and achieve the effect of simple operation, mild cultivation conditions and less investment

Active Publication Date: 2015-11-18

SOUTH CHINA UNIV OF TECH

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Problems solved by technology

However, at present, most of the red yeast yellow pigments are intracellular fat-soluble yellow pigments, the production process is relatively complicated, and it is not conducive to use in water-soluble occasions

Chemical transformation methods a...

Abstract

The invention discloses monascus ruber strain with high extracellular yellow pigment yields, a method for breeding the monascus ruber strain and application thereof. The monascus ruber strain is named as monascus ruber WQ15 and is preserved in the China General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms of the institute of microbiology of the Chinese Academy of Science in the No.3 yard on the No.2 Beichen West road of the Chaoyang district of Beijing on 2 July, 2015, and a preservation number of the monascus ruber strain is CGMCC No.10910. The monascus ruber strain with the high extracellular yellow pigment yields is obtained from riginal strains by means of ultraviolet mutation breeding. The monascus ruber strain with the high extracellular yellow pigment yields, the method and the application have the advantages that color level of extracellular yellow pigment of the monascus ruber strain is upgraded by 72% as compared with original strains by means of conventional liquid fermentation; the color level of the extracellular yellow pigment of the monascus ruber strain can be upgraded by 7.04 times as compared with the original strains if the monascus ruber strain is refilled with 300g/L of glucose during fermentation, and accordingly the monascus ruber strain can be used for producing the extracellular yellow pigment.

Application Domain

FungiMutant preparation +4

Technology Topic

Laboratory cultureYard (length) +5

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Examples

Experimental program(4)

Example Embodiment

[0050] Example 1 Obtaining of Monascus strains with high production of extracellular yellow pigment

[0051] (1) Preparation of spore suspension: Inoculate the original red yeast strain (MonascusruberGIM3.240, purchased from Guangdong Province Microbial Culture Collection) on the PDA slope, and after incubating at 30°C for 3 days, rinse the bacterial slope with 5 mL of normal saline. Disperse by glass beads, filter out the hyphae with 4 layers of sterile lens cleaning paper, and make 1×10 spores 5 Pieces/mL suspension.

[0052] (2) Ultraviolet mutagenesis: Each petri dish with a diameter of 7 cm is filled with 3 mL of the suspension prepared in step (1); the petri dish is placed under a 15W ultraviolet lamp at a vertical distance of 30 cm, and irradiated for 5 min under magnetic stirring. UV radiation mutagenesis.

[0053] (3) Plate separation and screening: Take 0.5 mL of the mutagenized bacterial solution, add 4.5 mL of sterile water to dilute, and then apply an appropriate amount to the PDA plate (0.06g potato powder, 0.4g glucose, 0.3g agar powder, Distilled water to 20mL, pH natural), 30℃ constant temperature and dark inverted culture for 48h, after the growth of bacteria, select the bacterial colony morphology (including colony size, color, aerial hyphae) changed strains, and single The colony was separated by streaking, and after repeated separation and purification, a pure mutagenic strain was obtained, which was stored at 4°C.

[0054] (4) Fermentation and breeding: Take the isolated and purified mutagenic strains in step (3), and conduct fermentation experiments, that is, the mutagenized strains are inoculated into the seed medium for culture and propagation, and each 50mL seed medium (1g glucose, 0.15 yeast extract powder) g, fish meal peptone 0.5g, KCl 0.025g, KH 2 PO 4 0.2g, FeSO 4 ·7H 2 O0.5mg, distilled water to 50mL, pH natural)) inoculate 5 single colonies cultured for 6 days, placed on a shaker controlled 180rpm culture for 30h, so that the seed medium has mature spores to obtain seed liquid; Inoculate 25mL fermentation medium (glucose 1.25g, (NH 4 ) 2 SO 4 0.125g, KH 2 PO 4 0.125g, MgSO 4 ·7H 2 O0.0125g, KCl0.0125g, FeSO 4 ·7H 2 O0.25mg, ZnSO 4 ·7H 2 O0.25mg, MnSO 4 ·H 2 O0.75mg, distilled water to a constant volume of 25mL, pH natural), controlled 180rpm and 30℃ under a shaker condition. After fermentation and culture for 6 days, the fermentation broth was centrifuged and suction filtered to obtain a supernatant containing extracellular monascus pigment. Liquid, by detecting the color value and hue of the extracellular pigments, the high-yielding bacteria are verified by multiple subcultures, and the Monascus strains that can produce extracellular yellow pigments are selected.

[0055] After selection and breeding, a Monascus strain with high production of extracellular yellow pigment was obtained. It has the following characteristics: cultivated on a PDA plate for 7 days, the colony is larger, with a diameter of 30.8-31.8mm, the colony is yellow-brown, and the back is orange-yellow , The edge of the colony is intact and round, the center is raised into a hilly shape, no radiation cracks, white fluffy aerial hyphae grow, and the surrounding is smooth and shiny, without oil drop-like substances, such as figure 1 Shown in (a); hyphae are smooth, transparent and colorless, with septa; conidia are spherical or inverted, solitary or chained; closed cyst shell is larger, spherical, spores in the cyst are visible, oval, figure 1 (b) Shown. It was named Monascusruber WQ15, and it was deposited on July 2, 2015 at the General Microbiology Center of the Chinese Academy of Sciences Institute of Microbiology, No. 3 Beichen West Road, Chaoyang District, Beijing , The deposit number is CGMCCNo.10910.

Example Embodiment

[0056] Example 2 Preparation of Monascus yellow pigment by conventional method using Monascus strain with high production of extracellular yellow pigment

[0057] (1) Preparation of seed solution: the original strain (MonascusruberGIM3.240) and mutant strain (MonascusruberWQ15) plate seeds streaked on the PDA plate were respectively inoculated into sterilized 50mL seed medium (1g glucose, 0.15g yeast extract powder). , Fish meal peptone 0.5g, KCl 0.025g, KH 2 PO 4 0.2g, FeSO 4 ·7H 2 O0.5mg, distilled water to 50mL, pH natural) for culture and propagation, the inoculum is 5 single colonies cultured for 6 days, placed on a shaker controlled 180rpm and cultivated for 30h, so that the seed medium has mature spores. Seed liquid.

[0058] (2) Fermentation culture: inoculate the seed liquid into 25mL basic fermentation culture liquid (glucose 1.25g, (NH 4 ) 2 SO 4 0.125g, KH 2 PO 4 0.125g, MgSO 4 ·7H 2 O0.0125g, KCl0.0125g, FeSO 4 ·7H 2 O0.25mg, ZnSO 4 ·7H 2 O0.25mg, MnSO 4 ·H 2 O0.75mg, distilled water to a constant volume of 25mL, pH natural), controlled 180rpm and 30℃ under a shaker to ferment and culture for 6 days, then centrifuge and filter the fermentation broth to obtain a supernatant containing extracellular monascus pigment The color value and hue of the extracellular fermentation broth were determined.

[0059] (3) Method for determining color value and hue: Take 25 mL of fermentation broth and centrifuge for 10 min at 8000 rpm, take the supernatant and filter it, and use a spectrophotometer to detect the color value of the yellow, orange and red pigments of the filtrate ( That is, the absorbance at 350, 470, and 510nm at the absorption wavelength) and the hue of extracellular yellow pigment (AU 350 /AU 510 ).

[0060] The result is figure 2 As shown, the absorbance at 350, 470, and 510nm of the extracellular fluid of the mutant strain fermented with UV spectrophotometer was 25.20, 2.12, 0.56AU/mL, respectively, and the yellow tone of extracellular red yeast rice was 45.00 (AU 350 /AU 510 ); The absorbance at 350, 470, and 510nm measured by the extracellular liquid of the original strain fermented with UV spectrophotometer was 14.62, 1.35, 0.47AU/mL, respectively, and the yellow tone of extracellular red yeast rice was 31.11 (AU 350 /AU 510 ). It can be seen that the extracellular yellow pigment of the mutant strain is 1.72 times that of the original strain, and the yellow tone of extracellular red yeast rice is increased by 0.45 times.

Example Embodiment

[0061] Example 3 Preparation of Monascus Yellow Pigment by Feeding Method Using Monascus Strains with High Yield of Extracellular Yellow Pigment

[0062] The Monascusruber WQ15 and the original strain MonascusruberGIM3.240 were fermented and cultured according to the method of Example 2. The difference from Example 2 was that after the third day of fermentation, 1.25g of glucose was supplemented, resulting in fermentation culture. The glucose concentration in the base was increased to about 60 g/L, and the fermentation was continued for 6 days, and the color value and hue of the extracellular fermentation broth were measured according to the method of Example 2.

[0063] The result is image 3 As shown, the absorbance at 350, 470, and 510 nm of the extracellular liquid of the mutant strain fermented by an ultraviolet spectrophotometer was 80.40, 4.14, and 1.00 AU/mL, respectively, and the yellow tone of the fermented extracellular red yeast rice was 80.40 (AU 350 /AU 510 ); the extracellular liquid of the original strain was used to measure the absorbance at 350, 470, and 510nm with an ultraviolet spectrophotometer to be 16.8, 1.93, and 0.64 AU/mL, respectively, and the yellow tone of extracellular red yeast rice was 26.25 (AU 350 /AU 510 ). It can be seen that the extracellular yellow tint of the mutant strain is 4.79 times that of the original strain, and the yellow tint of extracellular red yeast rice is increased by 2.06 times.

PUM

Property	Measurement	Unit
Absorbance	0.56 ~ 25.2
Absorbance	1.0 ~ 80.4
Absorbance	0.64

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Monascus ruber strain with high extracellular yellow pigment yields, method for breeding monascus ruber strain and application thereof

AI-Extracted Technical Summary

Problems solved by technology

Abstract

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Examples

Example Embodiment

Example Embodiment

Example Embodiment

PUM

Description & Claims & Application Information

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