MD1853Z - Method for culturing pigmented yeast Rhodotorula mucilaginosa CNMN-Ys-10 - Google Patents

Abstract

The invention relates to biotechnology, in particular to a method for cultivating pigmented yeast Rhodotorula mucilaginosa CNMN-Ys-10 for the purpose of increasing biomass productivity, carotenogenesis and catalase activity of the yeast strain. The method for cultivating pigmented yeast Rhodotorula mucilaginosa CNMN-Ys-10, according to the invention, provides for submerged cultivation of the strain in Erlenmeyer flasks, on a shaker with a rotation speed of 200 rpm, at a temperature of +27°C - +28°C for 120 hours, on natural liquid nutrient media obtained from agro-food plant waste: carrot and celery peelings.

Description

[0001] The invention relates to biotechnology, in particular to a process for cultivating the yeast strain Rhodotorula mucilaginosa and can be applied to obtain yeast biomass with an increased carotene content.

[0002] Currently, various processes are known for cultivating R. mucilaginosa on different nutrient media, including natural ones, obtained from different agri-food waste: peels, seeds, bagasse.

[0003] The closest solution involves cultivating the yeast strain R. mucilaginosa CCT 3892 on a nutrient medium based on sisal bagasse hydrolysate (Agave sisalana) supplemented with yeast extract and various mineral salts, which allows a significant increase, by about 50%, of the strain's biomass productivity from 8.42 to 12.17-12.87 g/L BAU, but with a low carotenoid content, of a maximum of 92.44 µg/g, which allows the production of 1.13 mg/L of carotenoids. At the same time, in the variant with a maximum carotenoid content of 179.66 µg/g, the biomass productivity decreases significantly to 4.69 g/L BAU, which allows the production of only 0.84 mg/L of carotenoids [1].

[0004] The disadvantage of this process is the low amount of stem biomass in the variant with maximum carotenoid content and low carotenoid content in the variant with maximum biomass productivity.

The problem solved by the invention consists in increasing the biomass productivity of the stem, the carotenoid content in the biomass, the carotenoid production and the catalase (CAT) activity therein.

[0006] The problem is solved by the process of cultivating the yeast strain Rhodotorula mucilaginosa CNMN-Ys-10, which consists in obtaining the yeast suspension Rhodotorula mucilaginosa CNMN-Ys-10 by cultivating on YPD medium for 48 hours, inoculating the suspension in a concentration of 5% in the nutrient medium, which contains carrot peel extract or carrot peel extract and celery peel extract in a ratio of 1:1, submerged cultivation of the strain at a temperature of +27-28°C with continuous stirring at 200 rpm for 120 hours, at the same time the carrot peel and celery peel extracts are obtained by grinding the peels, mixing with distilled water in a ratio of 1:1, autoclaving at a temperature of 115°C, a pressure of 0.5 atm, for 30 min, separation of the supernatant by centrifugation and its sterilization at a temperature of 115°C, a pressure of 0.5 atm, for 30 min.

[0007] The technical result of the invention consists in increasing the amount of biomass by 6.9-68.1%, the content of carotenoids in biomass by 145.7-226.9%, the production of carotenoids by 3-5.5 times, the CAT activity by 29.8-43.0% compared to the control (table) and increasing the content of carotenoids in biomass by 17.9-56.8% and the production of carotenoids by about 3 times compared to the closest solution.

[0008] The result of the invention is conditioned by the use of natural nutrient media, obtained based on extracts from carrot and celery peels with a valuable biochemical composition as stimulators of the productivity of the R. mucilaginosa strain CNMN-Ys-10, the biosynthesis of carotenoids and CAT activity.

[0009] Embodiments of the invention

Example 1

[0011] Carrot peel extract (EM) is prepared as follows: carrot peels are washed repeatedly to remove impurities; they are ground to a particle size of 0.5-1.0 cm; then, 1 volume of peels (750 g of wet mass with 10.12±0.48% S.U.) is mixed with 1 volume of distilled water (750 mL) and autoclaved for 30 min at a temperature of 115°C, a pressure of 0.5 atm, after cooling, the supernatant (extract) is separated from the sediment (carrot peels) by centrifugation; then the supernatant (extract) is sterilized in an autoclave for 30 min at a temperature of 115°C, a pressure of 0.5 atm. The extract obtained has a sugar content of 5.0°Blg and pH-5.6.

[0012] Celery peel extract (EP) is prepared as follows: celery peels are washed repeatedly to remove impurities; they are ground to a particle size of 0.5-1.5 cm; then, 1 volume of peels (750 g of wet mass with 9.35±0.06% S.U.) is mixed with 1 volume of distilled water (750 mL) and autoclaved for 30 min at a temperature of 115°C, a pressure of 0.5 atm, after cooling, the supernatant (extract) is separated from the sediment (celery peels) by centrifugation; then the supernatant (extract) is sterilized in an autoclave for 30 min at a temperature of 115°C, a pressure of 0.5 atm. The extract obtained has a sugar content of 4.5°Blg and pH-5.5.

[0013] The nutrient medium is prepared by combining (mixing) EM and EŢ extracts in a ratio of 1:1 and sterilized in an autoclave for 30 min at a temperature of 115°C, a pressure of 0.5 atm. The nutrient medium obtained is used as a sole source of carbon and nitrogen for submerged cultivation of the yeast strain. The seed material is obtained by cultivating the yeast strain on YPD nutrient medium for 48 hours, on a shaker (200 rpm), at a temperature of +25-26°C. The inoculum constitutes 5% of the volume of the nutrient medium, and submerged cultivation is carried out in Erlenmeyer flasks, on a shaker with a rotation speed of 200 rpm, at a temperature of +27-28°C for 120 hours.

[0014] As a result, the productivity of the strain is 7.7±0.3 g/L BAU, the carotenoid content is 211.8±11.2 µg/g, the carotenoid production is 1.63 mg/L and the CAT activity is 1108.9±114.0 mmol/min.mg protein.

Example 2

[0016] Prepare the carrot peel extract (EM) as follows: wash the carrot peels repeatedly to remove impurities; chop them to a particle size of 0.5-1.0 cm; then, 1 volume of peels (750 g of wet mass with 10.12±0.48% S.U.) is mixed with 1 volume of distilled water (750 mL) and autoclaved for 30 min at a temperature of 115°C, a pressure of 0.5 atm; after cooling, separate the supernatant (extract) from the sediment (carrot peels) by centrifugation; then, sterilize the supernatant (extract) in an autoclave for 30 min at a temperature of 115°C, a pressure of 0.5 atm. The obtained extract, with a sugar content of 5.0°Blg and pH-5.6, is used as a nutrient medium, as a sole source of carbon and nitrogen for submerged cultivation of the yeast strain. The seed material is obtained by cultivating the yeast strain on YPD nutrient medium, for 48 hours, on a shaker (200 rpm), at a temperature of +25-26°C. The inoculum constitutes 5% of the volume of the nutrient medium, and submerged cultivation is carried out in Erlenmeyer flasks, on a shaker with a rotation speed of 200 rpm, at a temperature of +27-28°C for 120 hours.

[0017] As a result, the productivity of the strain is 12.1±0.6 g/L BAU, the carotenoid content is 281.8±11.7 µg/g, the carotenoid production is 3.41 mg/L and the CAT activity is 1222.1±133.7 mmol/min.mg protein.

[0018] Table

[0019] Productivity, content, carotenoid production and CAT activity

[0020] of the R. mucilaginosa strain CNMN-Ys-10 on various nutrient media

Nutrient medium

Productivity, g/L BAU

Productivity, % of control

Carotenoids, µg/g

Carotenoids, % of control

Carotenoid productivity, mg/L

CAT activity, mmol/min.mg protein

CAT activity, % of control

YPD (Witness)

7.2±0.3

100

86.2±9.4

100

0.62

854.5±64.2

100

EM:ET (1:1)

7.7±0.3

106.9

211.8±11.2*

245.7

1.63

1108.9±114.0*

129.8

EM

12.1±0.6*

168.1

281.8±11.7*

326.9

3.41

1222.1±133.7*

143.0

[0022] * statistically significant for p≤0.05

[0023] Research to achieve the invention was carried out within the project 020101 InBioS - Innovative biotechnological solutions for agriculture, medicine and environment, financed by the Ministry of Education and Research of the Republic of Moldova.

1. Josevan da Silva, Flávio Luiz Honorato da Silva, José Evangelista Santos Ribeiro, Débora Jamila Nóbrega de Melo, Felipe Augusto Santos, Lorena Lucena de Medeiros. Effect of supplementation, temperature and pH on carotenoids and lipids production by Rhodotorula mucilaginosa on sisal bagasse hydrolyzate. Biocatalysis and Agricultural Biotechnology, 2020, v. 30, 101847, found on the Internet, URL: <https://doi.org/10.1016/j.bcab.2020.101847>

Claims (1)

1. Process for cultivating the yeast strain Rhodotorula mucilaginosa CNMN-Ys-10, which consists in obtaining the yeast suspension Rhodotorula mucilaginosa CNMN-Ys-10 by cultivating on YPD medium for 48 hours, inoculating the suspension at a concentration of 5% in the nutrient medium, which contains carrot peel extract or carrot peel and celery peel extracts in a ratio of 1:1, submerged cultivation of strains at a temperature of +27-28°C with continuous stirring at 200 rpm for 120 hours, at the same time, carrot peel extract and celery peel extract are obtained by grinding the peels, mixing with distilled water in a 1:1 ratio, autoclaving at a temperature of 115°C, a pressure of 0.5 atm, for 30 min, separating the supernatant by centrifugation and sterilizing it at a temperature of 115°C, a pressure of 0.5 atm, for 30 min.