CN115895922B - Rhodotorula graminea for high-yield carotenoid and application thereof - Google Patents
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Abstract
The invention discloses a novel rhodotorula graminea with high carotenoid yield and application thereof, and belongs to the technical field of microorganisms. The rhodotorula graminea strain is named as rhodotorula graminea (Rhodotorula graminis) YM2777 and is preserved in China general microbiological culture collection center (CGMCC) of China general microbiological culture Collection center (CGMCC) for 10-21 of 2022, and the preservation number is CGMCC No.25943. The rhodotorula graminea strain with high carotenoid yield provided by the invention has the carotenoid yield as high as 9.71+/-0.26 mg/L. The molasses is industrial and agricultural waste, the fermentation temperature is as low as 12 ℃, and the carotenoid is produced in a low-temperature season or in a low-temperature environment, so that the 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 rhodotorula graminea with high carotenoid yield and application thereof.
Background
Carotenoids are a class of fat-soluble tetraterpene compounds of well-defined structure, almost 750, which are critical for the survival of numerous photosynthetic and non-photosynthetic organisms due to their diverse biological properties of antioxidant, antitumor, immunomodulating, etc. At present, carotenoids play an important role in various fields such as cosmetic industry, food industry and aquaculture industry. With the increasing human living standard and the emphasis on health problems, the global market for carotenoids is increasing, from 20 billion dollars in 2022 to 27 billion dollars in 2027, with a annual compound growth rate of up to 5.7%.
Carotenoids on the market can be extracted from plants and microorganisms with carotenoid activity, but in practice are mostly synthesized chemically. However, the natural carotenoid is extracted by microbial fermentation, so that the problems that the planted plants occupy a large amount of land and are limited by geography and climate can be solved, hidden danger of quality and safety in chemical synthesis can be eliminated, and more importantly, the cost is low, the purity is high and the production period is short. The carotenoid-producing yeasts grow and propagate rapidly and are easy to culture, and the carotenoid-producing yeasts have obvious economic advantages when being used as materials for producing carotenoids. Optimizing fermentation conditions is an important means to increase fermentation production levels. Over the years, different optimization methods have been developed, wherein the 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 of carotenoid production has been developed, the biggest obstacle is the cost problem. The substrate chosen for the production of natural carotenoids is usually a high-valence compound, so how to reduce the production costs while increasing the yield of their carotenoids is of great importance for the future mass production of carotenoids.
Disclosure of Invention
The invention aims to provide rhodotorula graminifolia capable of producing carotenoids in high yield and application thereof, which are used for solving the technical problem of high production cost caused by high-valence compounds as substrates used in the existing carotenoid production.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
rhodotorula graminea with high carotenoid yield has a collection number of CGMCC No.25943.
The strain is preserved in China general microbiological culture Collection center, address: the Beijing city, the Chaoyang district, north Chen Xili No. 1, 3, is classified and named: rhodotorula graminea (Rhodotorula graminis) YM2777.
The use of rhodotorula graminearum with high carotenoid yield as a fermenting microorganism in the microbial fermentation production of carotenoids.
More preferably, the microbial fermentation process is as follows: inoculating seed liquid of rhodotorula graminearum with high yield of carotenoid into a 250mL triangular flask containing 50mL of liquid fermentation medium according to the inoculation amount with the volume fraction of 10%, and fermenting, wherein the liquid culture medium comprises the following components: 80.45g/L molasses as independent carbon source, 10g/L yeast extract and 3.86g/L urea as mixed nitrogen source, inorganic salt as ZnSO 0.5g/L 4 And 0.5g/L K 2 HPO 4 The method comprises the steps of carrying out a first treatment on the surface of the 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 is 180-220 rpm during fermentation.
Compared with the prior art, the invention has the following beneficial effects:
the rhodotorula graminea strain with high carotenoid yield provided by the invention has the carotenoid yield as high as 9.71+/-0.26 mg/L. Molasses which is 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 ℃, and the carotenoid can be produced in low-temperature seasons or low-temperature environments, so that the method has the advantages of high yield, environmental friendliness, low production cost and the like.
Drawings
FIG. 1 is a colony morphology and microscopic morphology diagram of the high carotenoid strain rhodotorula graminis (Rhodotorula graminis) YM 2777;
FIG. 2 is a bar graph of carotenoid production at various concentrations of medium components;
FIG. 3 is a bar graph of carotenoid production under fermentation conditions at different gradients;
FIG. 4 is a ranking chart of importance of factors affecting carotenoid production;
fig. 5 is a response surface curvature map and a contour map.
Detailed Description
The invention will be further illustrated with reference to the following examples, which are included by way of illustration, but not limitation.
Example 1
The rhodotorula graminea (Rhodotorula graminis) YM2777 with high carotenoid yield is separated from surface water of the Dian pond.
1. Bacterial identification process
1. Morphological identification:
colony morphology was observed using 5% malt agar medium (g/L): malt extract powder 50.0g, agar 20.0g, natural pH. Streaking in 5% malt agar medium, culturing at 25℃for 6d, the results are shown in FIG. 1A, characterized in that: coral red, smooth surface, glossy, mucilaginous, irregular to complete edges.
Microscopic morphological observations were made using 5% malt agar medium (g/L): malt extract powder 50.0g, agar 20.0g, natural pH. The culture was carried out at 25℃for 6 days in a 5% malt agar medium. The water slide was prepared and observed under a 400-fold optical microscope, and the results are shown in fig. 1B, and are characterized in that: the cells are spherical, oval, egg-shaped or oblong.
2. Molecular biological identification
The two gene fragments of strain YM2777 were PCR amplified using universal primers (NLI and NL 4) for the 26S rRNA D1/D2 region sequences and universal primers (ITS 1 and ITS 4) for the ITS1-5.8S-ITS2 fragment sequences, respectively, and the amplified products were sequenced, and the resulting sequences were subjected to a search alignment with known sequences disclosed in GenBank of U.S. The National Center for Biotechnology Information (NCBI) by Blastn (http:// blast. NCBI. Lm. Nih. Gov), and the strain YM2777 was identified as Rhodotorula graminea (Rhodotorula graminis) with 100% and 99.6% similarity to the 26S rRNA D1/D2 fragment sequences (accession No. AF 070431) and ITS1-5.8S-ITS2 fragment sequences (accession No. AF 444505) of model strain Rhodotorula graminis CBS 2826, respectively.
2. Method for extracting and measuring carotenoid
1. Determination of cell dry weight: weighing 5mL clean centrifuge tubes as A1; taking 3mL of fermentation liquor which is uniformly mixed, centrifuging for 5min at 10000r/min, discarding supernatant, washing a precipitation part with deionized water, centrifuging again, and discarding supernatant; and (3) placing the centrifuge tube with the precipitated cell mud in a blast drying oven at 65 ℃ for baking for more than 24 hours to constant weight, taking out and weighing to obtain 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 by acid heating and then carotenoid was extracted with Acetone (AR) (this method was derived from Yang Wen, ji Chunming. A simple cell wall disruption method [ J ]. Microbiological bulletins, 1995,22 (1): 58-59). Using acetone as a blank, measuring the absorbance of the sample at 475nm by using an ultraviolet-visible spectrophotometer, and calculating the carotenoid content according to the following formula: carotenoid content (μg/g) =aλmaxdv/0.16DCW. Wherein Aλmax is absorbance at a maximum absorption wavelength of 475nm, V is total volume of acetone (mL) used for extraction, D is dilution of leaching solution, DCW is dry cell weight (g), and 0.16 is extinction coefficient of carotenoid. Carotenoid yield (mg/L) = (biomass×carotenoid content)/1000.
3. Fermentation process of strain
Rhodotorula graminea (Rhodotorula graminis) YM2777 with high carotenoid yield is used for fermentation.
1. The fermentation process is explored by adopting a single factor experiment: inoculating small amount of thallus on YPD culture medium (yeast extract 5.0g/L, peptone 10.0g/L, glucose 20.0g/L, agar 20.0g/L, natural pH value), inoculating thallus activated for 24 hr into seed fermentation culture medium (glucose 40.0g/L, yeast extract 10.0g/L, peptone 10.0g/L, mgSO) of 50mL/250mL 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 medium according to 10% (V/V) inoculum size to perform fermentation. The basic fermentation method comprises the following steps: 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, 28.+ -. 2C, 200rpm for 120h, the carotenoid yield obtained under this method was 5.88.+ -. 0.49mg/L. The different liquid media in the single factor experiment were as follows:
1) Based on the basic fermentation method, under the condition that other factors are unchanged, glucose is changed into molasses, and the concentration of the molasses is changed into 20g/L, 30g/L, 40g/L, 50g/L, 60g/L, 70g/L and 80g/L respectively, and the result is shown in FIG. 2A;
2) Taking the basic fermentation method as a center, under the condition that other factors are unchanged, replacing peptone with urea, and changing the concentration of the peptone 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, wherein the result is shown in FIG. 2B;
3) Based on basic fermentation method, under the condition of other factors being unchanged, mgSO is used as the main material 4 Conversion to ZnSO 4 And the concentrations thereof 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, and the results are shown in FIG. 2C;
4) Based on the basic fermentation method, under the condition that other factors are unchanged, the initial pH values are respectively 4.0, 5.0, 6.0, 7.0, 7.5, 8.0 and 9.0, and the results are shown in FIG. 3A;
5) The fermentation temperature is changed to be 12 ℃, 16 ℃, 20 ℃, 24 ℃, 28 ℃, 32 ℃ and 36 ℃ respectively under the condition of taking the basic fermentation method as the center and keeping other factors unchanged, and the result is shown in figure 3B;
6) Based on the basic fermentation method, under the condition that other factors are unchanged, the fermentation time is changed to be 12h, 24h, 36h, 48h, 72h, 96h, 120h, 144h, 168h and 192h respectively, and the result is shown in figure 3C;
7) The shaking table oscillation rates were changed to 100rpm, 120rpm, 140rpm, 160rpm, 180rpm, 200rpm, 220rpm, respectively, with the basic fermentation method as a center, and the results are shown in FIG. 3D, with other factors unchanged.
2. Response surface optimization is carried out on the fermentation process:
(1) Plackett-Burman experimental design screens important factors: two levels were selected for each factor over the range of peaks that occur in the single factor experiment: low level "-1" and high level "+1", the above 7 factors examined as variables, and carotenoid production as response values (Y). The results are shown in Table 1 and FIG. 4, the main factors that have a significant impact on the yield of carotenoids of the strain rhodotorula graminis (Rhodotorula graminis) YM2777 are, in turn, the concentration of A molasses, the D initial pH and the G fermentation time (A: P=0.0126; D: P=0.0281; G: P=0.0310), with higher yields of carotenoids being obtained for high levels of molasses than for low levels of molasses, and conversely, higher yields of carotenoids being obtained for low levels of initial pH and fermentation time. The effects of other factors (urea concentration B, zinc sulfate concentration C, shaking rate of the E shaker, F fermentation temperature) were insignificant (P > 0.05), where B and E were positive effects and C and F were negative effects, which were taken as high and low values, respectively, in subsequent experiments. Based on the above conclusion, 3 factors of molasses concentration, initial pH and fermentation time will be further examined.
TABLE 1 principal effect analysis of factors of Plackett-Burman experimental design
(2) The steepest climbing experiment determines the center point of the response surface and the Box-Benhnken design to optimize the response surface
Molasses is a positive effect, increasing in sequence from a minimum level value "-" to an intermediate value "0"; the pH and fermentation time are negative effects, decreasing in sequence from a highest level value "+" to an intermediate value "0". Other factors respectively take the highest and lowest level values 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. The highest carotenoid yield is obtained when the molasses concentration is 80g/L, the pH is 6.5 and the fermentation time is 124h in the steepest climbing experiment, so that the highest carotenoid yield is used as a central point to perform response surface optimization by using a Box-Benhnken design.
Performing a quadratic polynomial regression fit on the experimental results of the Box-Benhnken design to obtain a ternary quadratic regression equation (Y=9.23+0.21A+0.02D-0.05G-0.01AD+0.14AG-0.06 DG-0.46A) with carotenoid yield (Y) as a response value, molasses concentration (A), pH (D), fermentation time (G) as independent variables 2 －0.30D 2 －0.52G 2 ). The variance analysis is carried out on the fitting model, the result is shown in the table 2, the P value (0.00005) of the regression model is less than 0.05, the fitting model is obvious, the model is reasonable to select, and the experimental result can be fitted well; the P value (0.08906) of the model mismatching item is more than 0.05, the mismatching item is not obvious, and the experimental result can be analyzed by using the model instead of experimental true points. Complex correlation coefficient R of model 2 = 0.9789, demonstrating good correlation between individual terms in the model; the CV value (1.14%) of the variation coefficient is less than 10%, which indicates that the accuracy and the credibility of the model are good; the Precision Adeq Precision value (18.22) > 4.0 indicates that the model is reasonable. The response surface profile and contour plot of FIG. 5 further intuitively reflect the concentration of molasses, initial pH and fermentation time versus Rhodotorula graminea (Rhodotorula graminis)) Influence of YM2777 carotenoid production. The contour map can directly reflect the interaction strength between two factors, and the tighter the ellipse arrangement is, the larger the influence of factor change on the result is. FIGS. 5A, 5B are graphs showing the effect of molasses concentration and pH on carotenoid production, with increasing amounts of the same, with increasing amounts of carotenoid production followed by decreasing amounts slightly, with molasses concentration at 79-81g/L and pH at 6.4-6.6, with the highest carotenoid production, and with Table 2, the interaction between the two was not significant and the oval arrangement was sparse, with no significant effect; FIGS. 5C, 5D are effects of molasses concentration and fermentation time on carotenoid production, with increasing carotenoid production increasing and decreasing, with molasses concentration at 79-81g/L and fermentation time at 122-126h, carotenoid production reaching highest, and with Table 2, interactions between the two have a significant effect on carotenoid production, and the tighter the ellipses, the greater the effect; FIGS. 5E, 5F are effects of pH and fermentation time on carotenoid production, with increasing amounts of carotenoids production increasing and decreasing, with pH 6.4-6.6 and fermentation time 122-126h, with highest carotenoid production, with insignificant effect of interaction between the two on carotenoid production, sparse oval arrangement, and no significant effect, as can be obtained in conjunction with Table 2.
TABLE 2Box-Benhnken test results analysis of variance table
(3) Verification of response surface experiments
And predicting the optimal fermentation condition by using Design Expert 11 software with the maximum carotenoid yield as a target value, namely: molasses concentration is 80.45g/L, pH is 6.51, and maximum carotenoid yield of 9.25mg/L is achieved at a fermentation time of 123.93 h. Under the optimal fermentation condition, the strain rhodotorula graminea (Rhodotorula graminis) YM2777 is subjected to fermentation culture, and the yield of the finally obtained carotenoid 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, by using industrial and agricultural waste molasses as the sole carbon source and through single factor experiments and response surface optimization, compared with a basic fermentation method, the carotenoid yield is improved by 65.14%, so that the carotenoid yield of the strain rhodotorula graminis (Rhodotorula graminis) YM2777 is improved while the production cost is reduced, the fermentation temperature is as low as 12 ℃, and the carotenoid production activity is suitable for low-temperature seasons and low-temperature environments. Through verification, the model has a certain practical value, and the reliability of the optimal preparation condition of the yield of the obtained carotenoid is high.
The above 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 all the insubstantial modifications or color changes made in the main design concept and spirit of the present invention are still consistent with the present invention, and all the technical problems to be solved are included in the scope of the present invention.
Claims (3)
1. Rhodotorula graminea producing carotenoidRhodotorula graminis) The microbial strain is preserved in China general microbiological culture Collection center (CGMCC) at the month 10 and 21 of 2022, and the preservation number is CGMCC No.25943.
2. Use of the carotenoid-producing rhodotorula graminearum of claim 1 as a fermenting microorganism in the fermentative production of carotenoids by microorganisms.
3. The use according to claim 2, wherein the microbial fermentation process is: inoculating seed liquid of rhodotorula graminearum for producing carotenoid into a 250mL triangular flask containing 50mL of liquid fermentation medium for fermentation according to the inoculation amount with the volume fraction of 10%, wherein the liquid culture medium comprises the following components: 80.45g/L molasses as independent carbon source, 10. 10g/L yeast extract and 3.86g/L urea as mixed nitrogen source, inorganic salt as ZnSO of 0.5. 0.5g/L 4 And K of 0.5g/L 2 HPO 4 The method comprises the steps of carrying out a first treatment on the surface of the The fermentation conditions are as follows: initial fermentation pH is 6-7, fermentation temperature is 10-18 ℃, fermentation time is 120-140 h, and shaking table is used during fermentationThe oscillation rate is 180-220 rpm.
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