CN115895922A - Rhodotorula benthica for high yield of carotenoid and application thereof - Google Patents
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Abstract
The invention discloses a new high-yield carotenoid-producing rhodotorula graminis and application thereof, belonging to the technical field of microorganisms. The Rhodotorula graminis strain is named as Rhodotorula graminis YM2777, and is preserved in China general microbiological culture Collection center (CGMCC) at 10 and 21 months in 2022 with the preservation number of CGMCC No.25943. The high-yield carotenoid strain of the rhodotorula graminis provided by the invention has the carotenoid yield of 9.71 +/-0.26 mg/L. The molasses in the fermentation method is industrial and agricultural waste, the fermentation temperature is as low as 12 ℃, carotenoid production is carried out in low-temperature seasons or low-temperature environments, and the fermentation method has the advantages of high yield, environmental friendliness, low production cost and the like.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a high-carotenoid-yield rhodotorula benthica and application thereof.
Background
Carotenoids are fat-soluble tetraterpenes, almost 750 kinds of structures of which are well-defined, and are vital to the survival of a plurality of photosynthetic organisms or non-photosynthetic organisms due to various biological properties such as oxidation resistance, tumor resistance, immunoregulation and the like. At present, carotenoids play an important role in many fields such as the cosmetic industry, the food industry and the breeding industry. With the increasing levels of human living and the health concerns, the global market for carotenoids is increasing, with annual compound growth rates as high as 5.7% from 20 billion dollars in 2022 to 27 billion dollars in 2027.
Carotenoids on the market can be extracted from plants and microorganisms having carotenoid activity, but are in fact mostly synthesized chemically. However, the extraction of natural carotenoids by microbial fermentation not only solves the problems that the planted plants occupy a large amount of land and are limited by geography and climate, but also eliminates the hidden troubles of quality and safety in chemical synthesis, and more importantly, the cost is low, the purity is high and the production period is short. The yeast for producing carotenoid grows and breeds quickly and is easy to culture, and the yeast has obvious economic advantages when being used as a material for producing carotenoid. Optimizing fermentation conditions is an important means to improve the level of fermentation production. Over the years, different optimization methods have been developed, where a response surface method is used to determine the optimal settings of experimental factors and to account for the interaction effects between factors during fermentation.
However, although the technology for carotenoid production has been well developed, the greatest obstacle is the cost problem. The substrate chosen for natural carotenoid production is usually a high-priced compound, and therefore how to reduce the production cost while increasing the carotenoid yield is crucial for future mass production of carotenoids.
Disclosure of Invention
The invention aims to provide a rhodotorula graminis with high carotenoid yield and application thereof, which are used for solving the technical problem of high production cost caused by high-valence compounds as substrates in the existing carotenoid production.
In order to realize the purpose, the technical scheme adopted by the invention is as follows:
a strain of high-yield carotenoid-producing rhodotorula benthica has a preservation number of CGMCC No.25943.
The strain is preserved in China general microbiological culture Collection center (CGMCC) at 10 months and 21 days in 2022, and the address is as follows: the Xilu No. 1 Hospital No. 3, beijing, chaoyang, is classified and named as: rhodotorula glutinis (Rhodotorula graminis) YM2777.
The application of the rhodotorula benthica with high carotenoid yield as a fermentation microorganism in the production of the carotenoid by microbial fermentation.
More preferably, the process of microbial fermentation is as follows: inoculating the seed liquid of the high-yield carotenoid-producing rhodotorula graminis into a 250mL triangular flask containing 50mL of liquid fermentation medium according to the inoculation amount with the volume fraction of 10% for fermentation, wherein the liquid fermentation medium comprises the following components: 80.45g/L of molasses used as an independent carbon source, 10g/L of yeast extract and 3.86g/L of urea used as mixed nitrogen source, and 0.5g/L of ZnSO as inorganic salt 4 And 0.5g/L of K 2 HPO 4 (ii) a The fermentation conditions are as follows: the initial fermentation pH is 6-7, the fermentation temperature is 10-18 ℃, the fermentation time is 120-140 h, and the shaking table oscillation speed during fermentation is 180-220 rpm.
Compared with the prior art, the invention has the following beneficial effects:
the high-yield carotenoid strain of the rhodotorula graminis provided by the invention has the carotenoid yield of 9.71 +/-0.26 mg/L. The molasses used as the only carbon source in the fermentation method is an industrial and agricultural byproduct, and the cost is extremely low; the fermentation temperature is as low as 12 ℃, the carotenoid production can be carried out in low-temperature seasons or low-temperature environments, and the method has the advantages of high yield, environmental friendliness, low production cost and the like.
Drawings
FIG. 1 is a colony morphology and a microscopic morphology of a high-carotenoid-producing strain Rhodotorula glutinis YM 2777;
FIG. 2 is a bar graph showing the change in carotenoid production at different concentrations of the media components;
FIG. 3 is a bar graph of carotenoid production changes under different gradients of fermentation conditions;
FIG. 4 is a graph of the ranking of importance of the factors affecting carotenoid production;
FIG. 5 is a response surface plot and a contour plot.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.
Example 1
The Rhodotorula glutinis YM2777 with high carotenoid yield is separated from the surface water of Dian lake.
1. Strain identification process
1. And (3) morphological identification:
colony morphology observation, using 5% malt agar medium (g/L): malt extract powder 50.0g, agar 20.0g, natural pH. Streaking on 5% malt agar medium and incubating at 25 ℃ for 6 days resulted in FIG. 1A, which is characterized by: coral red, smooth surface, lustrous, mucus-like, irregular to complete edge.
Microscopic morphology observation, using 5% malt agar medium (g/L): malt extract powder 50.0g, agar 20.0g, natural pH. Cultured in 5% malt agar medium at 25 ℃ for 6 days. A water slide was prepared and observed under an optical microscope of 400 times, and the result is shown in FIG. 1B, which is characterized in that: the cells are spherical, ovoid, or long ovoid.
2. Molecular biological identification
These two gene fragments of strain YM2777 were PCR-amplified and The amplified products were sequenced using universal primers for The 26S rRNA D1/D2 region sequence (NLI and NL 4) and universal primers for The ITS1-5.8S-ITS2 fragment sequence (ITS 1 and ITS 4), respectively, and The resulting sequences were search-aligned with known sequences disclosed in The National Center for Biotechnology Information (NCBI) GenBank of The United states via Blastn (http:// blast.nc. N.n.n.nih.gov), and The similarity of strain YM2777 with The 26S D1/D2 region sequence (accession No. AF 070431) and ITS1-5.8S-ITS2 fragment sequence (accession No. AF 444505) of The model strain Rhodotorula grandis CBS 2826 reached 100% and 99.6%, respectively, and thus The strain Rhodotorula rubra was identified as Rhodotorula rubra strain 2777 (Rhodotorula rubra).
2. Method for extracting and measuring carotenoid
1. Determination of the dry cell weight: weighing 5mL of a clean centrifuge tube as A1; centrifuging the uniformly mixed fermentation liquor 3mL at 10000r/min for 5min, discarding the supernatant, washing the precipitate with deionized water, centrifuging again, and discarding the supernatant; and placing the centrifuge tube with the precipitated cell mud in a forced air drying oven at 65 ℃ for baking for more than 24 hours to constant weight, taking out the centrifuge tube, and weighing the centrifuge tube as A2. Cell dry weight (DCW) (g) = (A2-A1), biomass (g/L) = (DCW × 1000)/3.
2. Cell disruption, carotenoid extraction and assay: the fermentation broth was subjected to wall-breaking treatment by acid-thermal method, and then carotenoid was extracted with Acetone (AR) (this method was derived from Yangyun, gichumin. A simple method for cell wall disruption [ J ] microbiological report, 1995,22 (1): 58-59). Taking acetone as a blank control, measuring the absorbance of a sample at 475nm by using an ultraviolet-visible spectrophotometer, and calculating the carotenoid content according to the following formula: carotenoid content (μ g/g) = a λ maxDV/0.16DCW. Where A λ max is the absorbance at maximum absorption wavelength of 475nm, V is the total volume of acetone used for extraction (mL), D is the dilution factor of the leach liquor, DCW is the dry cell weight (g), and 0.16 is the extinction coefficient of the carotenoid. Carotenoid production (mg/L) = (biomass × carotenoid content)/1000.
3. Fermentation process of strain
The Rhodotorula glutinis YM2777 with high carotenoid yield is used for fermentation.
1. The fermentation process is explored by adopting a single-factor experiment: inoculating a small amount of thallus on YPD medium (yeast extract 5.0g/L, peptone 10.0g/L, glucose 20.0g/L, agar 20.0g/L, natural pH), activating thallus for 24 hr, inoculating 50mL/250mL seed fermentation medium (glucose 40.0g/L, yeast extract 10.0g/L, peptone 10.0g/L, mgSO 4 1.0g/L，K 2 HPO 4 0.5g/L, pH 6.0) to obtain seed solutions, and respectively inoculating the seed solutions into 250mL triangular flasks containing 50mL of liquid fermentation culture medium according to the inoculation amount of 10% (V/V) for fermentation. The basic fermentation method comprises the following steps: 40.0g/L glucose, 10.0g/L yeast extract, 10.0g/L peptone and MgSO 4 1.0g/L，K 2 HPO 4 0.5g/L, pH 6.0, 28. + -. 2C, cultured at 200rpm for 120h, the carotenoid yield obtained in this way is 5.88. + -. 0.49mg/L. The different liquid media in the one-factor experiment are as follows:
1) Taking a basic fermentation method as a center, under the condition that other factors are not changed, replacing glucose with molasses, and changing the concentrations of the molasses to be 20g/L, 30g/L, 40g/L, 50g/L, 60g/L, 70g/L and 80g/L respectively, wherein the results are shown in a figure 2A;
2) Taking the basic fermentation method as the center, under the condition that other factors are not changed, the peptone is changed into urea, and the concentrations of the peptone are changed to be 0.43g/L, 1.29g/L, 2.14g/L, 3.00g/L, 3.86g/L, 4.71g/L and 5.57g/L respectively, and the result is shown in figure 2B;
3) Taking basic fermentation method as the center, under the condition that other factors are not changed, mgSO 4 By conversion to ZnSO 4 And the concentrations were changed to 0.25g/L, 0.50g/L, 0.75g/L, 1.00g/L, 1.25g/L, 1.50g/L, 1.75g/L, respectively, the results are shown in FIG. 2C;
4) With the basic fermentation method as the center and other factors unchanged, the initial pH values were changed to 4.0, 5.0, 6.0, 7.0, 7.5, 8.0, and 9.0, respectively, and the results are shown in FIG. 3A;
5) The basic fermentation method is used as the center, and the fermentation temperatures are respectively changed to 12 ℃, 16 ℃, 20 ℃, 24 ℃, 28 ℃, 32 ℃ and 36 ℃ under the condition that other factors are not changed, and the results are shown in figure 3B;
6) Taking the basic fermentation method as the center, under the condition that other factors are not changed, the fermentation time is changed to 12h, 24h, 36h, 48h, 72h, 96h, 120h, 144h, 168h and 192h respectively, and the result is shown in FIG. 3C;
7) The results are shown in FIG. 3D, which is based on the basic fermentation process, except that the shaking table oscillation rates are changed to 100rpm, 120rpm, 140rpm, 160rpm, 180rpm, 200rpm, and 220rpm, respectively, without changing other factors.
2. Response surface optimization is carried out on the fermentation process:
(1) Plackett-Burman experiment design screening important factors: two levels were chosen for each factor in the range where the peak occurred in the single factor experiment: low level "-1" and high level "+1", with the above 7 factors examined as variables and carotenoid yields as response values (Y). As a result, as shown in table 1 and fig. 4, the main factors having significant influence on the yield of carotenoids of strain Rhodotorula glutinis (Rhodotorula glutinis) YM2777 are, in order, a molasses concentration, D initial pH, and G fermentation time (a: P = 0.0126D, P = 0.0281G. The influence of other factors (urea concentration B, zinc sulfate concentration C, shaking rate of shaker E, fermentation temperature F) was not significant (P > 0.05), where B and E are positive effects and C and F are negative effects, and in subsequent experiments, high and low levels were taken, respectively. Based on the above conclusions, further consideration will be made on the 3 factors of molasses concentration, initial pH and fermentation time.
TABLE 1 analysis of the Main Effect of the factors of the Plackett-Burman Experimental design
(2) Response surface optimization is carried out by determining response surface central point and Box-Benhnken design through steepest climbing experiment
Molasses is a positive effect, increasing from the lowest level value "-" to the intermediate value "0" in sequence; the pH and fermentation time are negative effects, decreasing from the highest level "+" to the middle "0". Other factors are respectively selected according to positive and negative effects, namely 3.86g/L of urea, 0.5g/L of zinc sulfate, 220rpm of shaking table oscillation speed and 12 ℃ of fermentation temperature. In the steepest climbing experiment, the highest carotenoid yield is obtained when the concentration of molasses is 80g/L, the pH value is 6.5 and the fermentation time is 124h, so that the highest carotenoid yield is obtained by taking the molasses as a central point and carrying out response surface optimization by using a Box-Benhnken design.
The experimental result designed by Box-Benhnken is subjected to secondary multiple regression fitting to obtain a ternary secondary regression equation (Y =9.23+0.21A +0.02D-0.05G-0.01AD +0.14AG-0.06 DG-0.46A) with the carotenoid yield (Y) as a response value and the molasses concentration (A), the pH (D) and the fermentation time (G) as independent variables 2 －0.30D 2 －0.52G 2 ). Performing variance analysis on the fitting model, wherein the result is shown in table 2, and the P value (0.00005) of the regression model is less than 0.05, which shows that the fitting model is remarkable, and shows that the model is reasonable in selection and can better fit the experimental result; the P value (0.08906) of the mismatching item of the model is more than 0.05, and the mismatching item is not significant, which indicates that the model can be used for analyzing the experimental result instead of the experimental real point. Complex correlation coefficient R of model 2 =0.9789, indicating good correlation between the individual terms in the model; the coefficient of variation CV value (1.14%) is less than 10%, which shows that the accuracy and the reliability of the model are good; precision Adeq Precision value (18.22) > 4.0, indicating a reasonable model. The response surface plot and contour plot of FIG. 5 further visually reflect the effect of molasses concentration, initial pH and fermentation time on the yield of carotenoids in Rhodotorula glutinis (Rhodotorula grandis) YM2777. The contour map can directly reflect the strength of interaction between two factors, and the tighter the ellipse arrangement is, the greater the influence of factor change on the result is. FIGS. 5A and 5B show molassesThe concentration and pH influence on the yield of the carotenoid, along with the increase of the carotenoid, the yield of the carotenoid is slightly reduced after the increase, the concentration of molasses is 79-81g/L, and when the pH is 6.4-6.6, the yield of the carotenoid reaches the highest value, the carotenoid yield is obtained by combining the table 2, the interaction between the two has no obvious influence on the yield of the carotenoid, and the ellipse arrangement is sparse without obvious influence; FIGS. 5C and 5D are graphs showing the effect of molasses concentration and fermentation time on carotenoid yield, increasing and then decreasing with increasing carotenoid yield, with molasses concentration at 79-81g/L and fermentation time at 122-126h, the highest carotenoid yield was achieved, as can be seen from Table 2, the interaction between the two had a significant effect on carotenoid yield, and the tighter the elliptical arrangement, the greater the effect; FIGS. 5E and 5F show the effect of pH and fermentation time on carotenoid yields, increasing and decreasing with increasing, with pH between 6.4 and 6.6 and fermentation time between 122 and 126h, the carotenoid yields were highest, as can be seen from Table 2, and the interaction between the two had no significant effect on carotenoid yields, with sparse elliptical arrangement and no significant effect.
TABLE 2Box-Benhnken test results ANOVA TABLE
(3) Verification of response surface experiments
With the maximum carotenoid yield as a target value, predicting the optimal fermentation conditions by using Design Expert 11 software, namely: the concentration of molasses is 80.45g/L, pH is 6.51, and the maximum carotenoid yield is 9.25mg/L when the fermentation time is 123.93 h. Under the optimal fermentation condition, the strain Rhodotorula (Rhodotorula gracilis) YM2777 is subjected to fermentation culture, the finally obtained carotenoid yield is 9.71 +/-0.26 mg/L, and the relative deviation from the theoretical predicted value of the model is only 0.46mg/L.
In conclusion, industrial and agricultural waste molasses is used as a unique carbon source, and compared with a basic fermentation method, the yield of the carotenoid is improved by 65.14% through a single-factor experiment and response surface optimization, so that the production cost is reduced, the yield of the carotenoid of a strain Rhodotorula glutinis (Rhodotorula graminis) YM2777 is improved, the fermentation temperature is as low as 12 ℃, and the carotenoid production activity is suitable for low-temperature seasons and low-temperature environments. The model has certain practical value through verification, and the reliability of the optimal preparation condition of the obtained carotenoid output is higher.
The above-mentioned embodiment is only one of the preferred embodiments of the present invention, and should not be used to limit the scope of the present invention, but any insubstantial modifications or changes made in the spirit and the spirit of the main design of the present invention, which still solves the technical problems consistent with the present invention, should be included in the scope of the present invention.
Claims (3)
1. A strain of high-yield carotenoid-producing rhodotorula benthica has a preservation number of CGMCC No.25943.
2. The application of the high-yield carotenoid rhodotorula graminis as a fermentation microorganism in producing carotenoid by microbial fermentation.
3. Use according to claim 2, wherein the microbial fermentation is carried out by: inoculating the seed liquid of the high-yield carotenoid rhodotorula graminis into a 250mL triangular flask containing 50mL of liquid fermentation medium according to the inoculation amount with the volume fraction of 10% for fermentation, wherein the liquid fermentation medium comprises the following components: 80.45g/L of molasses is used as an independent carbon source, 10g/L of yeast extract and 3.86g/L of urea are used as mixed nitrogen sources, and inorganic salt is 0.5g/L of ZnSO 4 And 0.5g/L of K 2 HPO 4 (ii) a The fermentation conditions were: the initial fermentation pH is 6-7, the fermentation temperature is 10-18 ℃, the fermentation time is 120-140 h, and the shaking table oscillation speed during fermentation is 180-220 rpm.
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