CN111793569A

CN111793569A - High-yield fermentation method and application of carotenoid high-yield spore-casting yeast strain

Info

Publication number: CN111793569A
Application number: CN202010776701.XA
Authority: CN
Inventors: 李朝霞; 陈慧; 李丁; 杨成飞; 仇明; 梁慧星
Original assignee: Yancheng Institute of Technology
Current assignee: Yancheng Institute of Technology; Yancheng Institute of Technology Technology Transfer Center Co Ltd
Priority date: 2020-08-05
Filing date: 2020-08-05
Publication date: 2020-10-20

Abstract

A high-yield fermentation method of a carotenoid high-yield spore-casting yeast strain and application thereof belong to the technical field of microbial fermentation and food processing. The preservation number of the strain is CGMCC No.17429, and the preservation name isSporobolomyces reseuGCJM 3-6. Through single factor test and orthogonal test optimization, the proper fermentation conditions of the strain are determined as follows: 5% (V/V) inoculation amount, 180r/min oscillation, high yield spore throwing yeast strain seed liquor bacterial content of 6.0 x 10⁸~8.0×10⁸The fermentation broth is characterized by comprising the following components of (by volume)/mL, a liquid filling ratio of 5-10% (V/V), an initial fermentation pH of 6.0, a fermentation temperature of 16-18 ℃, a fermentation time of 60-78 h, and a liquid fermentation culture medium, wherein the components of the liquid fermentation culture medium are 20-22 g/L of sucrose, 12-14 g/L of peptone, 10-12 g/L of yeast extract, 0.4-0.6 mg/L of lycopene and 0.2-0.6 mg/L of riboflavin. This optimizationThe fermentation conditions obviously improve the yield of the carotenoid, the average yield is 822.6-898.4 mug/g (dry thallus), and the fermentation conditions of the high-yield spore-throwing yeast have the advantages of simple culture medium, mild reaction, high yield, low production cost, environmental friendliness and the like.

Description

High-yield fermentation method and application of carotenoid high-yield spore-casting yeast strain

Technical Field

The invention belongs to the technical field of microbial fermentation and food processing, and particularly relates to a high-yield fermentation method of a carotenoid high-yield sporobolomyces strain and application thereof.

Background

Carotenoids, represented by beta-carotene, lycopene, rhodotorula, anthocyanidin, etc., are yellow, orange-red or red polyene compounds, have biological activity of converting into vitamin A, and also have various physiological functions of preventing cancers, cardiovascular diseases, etc. At present, carotenoids are recognized as a class a nutritional pigment by international organizations such as FAO and WHO, and are approved as a food additive with dual functions of nutrition and coloring in more than 50 countries and regions, and are widely used in health food and pharmaceutical and cosmetic industries. The carotenoid extracted from natural fruits and vegetables has the defects of high cost, high price, complex process, poor coloring power and the like.

The carotenoid in Sporobolomyces mainly includes beta-carotene, lycopene, rhodotorula, purpurin and xanthophyll, and riboflavin (VB)₂) And lycopene may be a precursor or growth-promoting factor for beta-carotene. Although the production of carotenoid by the spore-casting yeast is limited by pigment yield at present, the method has the advantages of simple nutrient requirement, short growth period, rich thallus nutrition and the like, and has great practical value and development prospect.

CN102115716B locked yeast strain and application thereof provide a method for producing carotenoid by using Sporidiobolus pararosaeus JD-2CCTCC M2010326, glucose is used as an optimal carbon source, the optimal concentration is 40g/L, corn steep liquor is used as an optimal nitrogen source for enriching beta-carotene, and the optimal concentration is 20 g/L; the yeast extract is used as the optimal nitrogen source for enriching the rhodotorula, the optimal concentration is 20g/L, and the fermentation is carried out for more than 16h under the conditions of pH 5-9 and temperature of 25-28 ℃. Finally, the total yield of the carotenoid is 500-700 mu g/g and the yield of the beta-carotene is 350-400 mu g/g through high liquid chromatography and mass spectrometry. But the method still has the defects of large consumption of nutrient substrates, complex separation and identification method, difficult industrial production and the like.

Therefore, the key problem of realizing the industrial production is how to breed the high-yield sporogenous yeast strain of the carotenoid and properly optimize the nutritional factors, the growth promoting factors and various fermentation conditions, reduce the production cost as much as possible and improve the yield of the carotenoid produced by the sporogenous yeast.

Disclosure of Invention

The technical problem to be solved is as follows: aiming at the technical problems, the invention provides a high-yield fermentation method of a high-yield sporogenous yeast carotenoid strain and application thereof, and solves the technical problems of properly optimizing nutritional factors, growth promoting factors and various fermentation conditions and improving the yield of the carotenoid produced by a sporogenous yeast high-yield mutant strain as much as possible.

The technical scheme is as follows: the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation number is CGMCC No.17429, the preservation name is Sporobolomyces reseu GCJM3-6, and the preservation date is 2019, 3 months and 22 days.

The application of the high-yield sporogenous yeast strain of the carotenoid is to produce the carotenoid.

The fermentation method of the carotenoid high-yield spore-casting yeast strain comprises the following steps: respectively inoculating the seed liquid of the high-yield spore-casting yeast strain into 1000mL triangular flasks containing a certain volume of liquid fermentation medium according to the inoculation amount of 5% (V/V), wherein the bacterium content of the seed liquid of the high-yield spore-casting yeast strain is about 2.0 multiplied by 10⁸～10.0×10⁸The fermentation broth is prepared from (by volume/mL), a liquid loading ratio of a liquid fermentation medium is 5-25% (V/V), an initial fermentation pH is 4.5-7.0, a fermentation temperature is 14-34 ℃, and the liquid fermentation medium is subjected to oscillation fermentation for 48-84 hours in a constant temperature oscillator at a rotating speed of 180r/min, wherein the liquid fermentation medium comprises 16-24 g/L sucrose, 6-16 g/L peptone, 6-16 g/L yeast extract, 0.2-1.0 mg/L lycopene, 0.2-1.0 mg/L riboflavinAnd (4) element.

Preferably, the bacterium content of the seed liquid of the high-yield spore-casting yeast strain is 6.0 multiplied by 10⁸～8.0×10⁸one/mL.

Preferably, the liquid filling ratio is 5% to 10% (V/V).

Preferably, the initial fermentation pH of the liquid fermentation medium is 6.0.

Preferably, the fermentation temperature is 16-18 ℃.

Preferably, the time of the shaking fermentation is 60-78 h.

Preferably, the liquid fermentation medium comprises 20-22 g/L of sucrose, 12-14 g/L of peptone, 10-12 g/L of yeast extract, 0.4-0.6 mg/L of lycopene and 0.2-0.6 mg/L of riboflavin.

Preferably, the fermentation process is scaled up for laboratory pilot and actual production.

Has the advantages that: the optimized culture conditions of the high-yield sporogenous yeast carotenoid strain can obviously improve the yield of the carotenoid, and the average yield of the carotenoid is 822.6-898.4 mu g/g (dry thallus), and meanwhile, the fermentation conditions of the high-yield sporogenous yeast strain have the advantages of simple culture medium, mild reaction, high yield, low production cost, environmental friendliness and the like.

Drawings

FIG. 1 shows the change in carotenoid content for different seed liquid contents;

FIG. 2 shows the change of carotenoid content in different liquid loading ratios;

FIG. 3 change of carotenoid content at different initial fermentation pH;

FIG. 4 the carotenoid content variation at different fermentation temperatures;

FIG. 5 the carotenoid content variation at different fermentation times;

FIG. 6 the carotenoid content changes at different sucrose concentrations;

FIG. 7 variation of carotenoid content for different peptone concentrations;

FIG. 8 the carotenoid content variation for different yeast extract concentrations;

FIG. 9 the carotenoid content variation at different lycopene concentrations;

FIG. 10 different VB₂The carotenoid content at concentration varies.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below with reference to the specific contents of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.

Breeding of high-yield carotenoid spore-throwing yeast strain

1 experimental strain: sporobolomyces deposited in laboratory.

2 culture Medium

Solid seed culture medium: 20g/L of sucrose, 20g/L of peptone, 10g/L of yeast extract, 17g/L of agar powder and pH 6.0;

liquid seed culture medium: 20g/L sucrose, 20g/L peptone, 10g/L yeast extract, pH 6.0.

Solid fermentation medium: 20g/L of sucrose, 10g/L of peptone, 10g/L of yeast extract, 17g/L of agar powder and pH 6.0;

liquid fermentation medium: 20g/L sucrose, 10g/L peptone, 10g/L yeast extract, 0.4mg/L lycopene, 0.4mg/L riboflavin, pH 6.0.

3 operating method

3.1 preparation of seed solutions

Continuously selecting 3-4 rings of sporobolomyces activated for 3d on a solid seed culture medium into a 5mL plastic centrifuge tube containing 2mL of sterile water, uniformly mixing, respectively transferring 0.2mL of sporobolomyces into 1 bottle of 250mL conical flask containing 50mL of liquid seed culture medium, culturing at 16 ℃ for 36-48 h at the rotating speed of about 180r/min, and adding sterile water to obtain seed liquid. The bacteria content of the seed liquid is measured and controlled to be 3.0 × 10 by using a microscopic counting method⁸～4.0×10⁸And (4) putting the rice seeds/mL into a refrigerator at 4 ℃ for storage for later use.

3.2 ultraviolet mutagenesis of Sporobolomyces strains

Adding 5mL of prepared seed liquid into a sterile culture dish with the diameter of 6cm, placing the sterile culture dish on a magnetic stirrer at a distance of 30 w/254 nm ultraviolet lamp for 30-60 s, and taking the seed liquid which is not irradiated by ultraviolet light as a control group. After the irradiation is finished, respectively taking 0.2mL of bacterial suspension under the dark light condition, coating the bacterial suspension on a solid fermentation medium flat plate, and then culturing at the constant temperature of 16 ℃ for 48-72 h. And selecting the most red and/or largest growth circle on the solid fermentation medium plate as the starting strain for the next ultraviolet mutagenesis. Repeating the steps for 3-5 times of ultraviolet mutagenesis, and finally screening 4-6 strains of the high-yield sporogenous yeast of carotenoid for primary screening.

3.3 screening of Carotenoid high-producing Strain

Using carotenoid yield as measure index, the bacteria content is about 3.0 × 10⁸～4.0×10⁸Inoculating the same amount of primary screening strains per mL into a 1000mL conical flask filled with 100mL of liquid fermentation medium, culturing for 2-3 days in a constant temperature oscillator at 16 ℃ for secondary screening, and finally obtaining the high-yield carotenoid spore-throwing yeast strain.

The high-yield carotenoid spore-casting yeast strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 17429.

The extraction and yield determination of the carotenoids was as follows (this process was derived from the patent application with application number 201910344401.1):

wet cell harvest: putting the fermentation liquor into a centrifuge tube, centrifuging for 15min at 3000r/min and 4 ℃, pouring out supernatant, adding a little sterile water for washing, freezing and centrifuging again, pouring out supernatant, combining a proper amount of centrifuged wet cells into another clean centrifuge tube with constant weight, inverting and drying the water on the tube wall and the tube cover of the centrifuge tube until the weight is constant, and obtaining the wet cells with constant weight;

acid heat crushing: taking 1.0-5.0 g of wet cells with constant weight, transferring the wet cells into a clean glass test tube, performing acid-heat crushing under the conditions of 4.5mol/L hydrochloric acid concentration, 3.6mL/g of hydrochloric acid dosage of wet cells, 30min hydrochloric acid soaking time and 4min boiling water bath time, then rapidly cooling, taking out the test tube, pouring cell mud into a centrifuge tube, centrifuging at 4000r/min and 4 ℃ for 15min, discarding supernatant fluid to obtain cell fragment sediment, adding water to wash, centrifuging again, and discarding the supernatant fluid. Repeating the steps until the precipitate is white, and obtaining the crushed cells;

extraction of carotenoids: adding 10ml acetone aqueous solution into the crushed cells for extraction, oscillating by a vortex oscillator, centrifuging, and pouring the supernatant into another clean test tube to obtain acetone carotenoid extracting solution;

quantitative determination of carotenoids: the carotenoid content was determined spectrophotometrically at 450 nm. The carotenoid content was calculated according to the following formula: x is A_450nm XRXV 1 X1000/(0.16 XV 2)/W. Wherein X is carotenoid content (μ g/g), A_450nmThe absorbance measured at 450nm, R is the dilution factor, V1 is the volume (mL) of the acetone extract, V2 is the volume (mL) of the fermentation broth taken, 0.16 is the extinction coefficient, and W is the dry bacterial weight (g/L).

Secondly, fermentation conditions of the high-yield sporobolomyces throwing yeast strain of carotenoid

1 Single factor experiment

Respectively inoculating 5% (V/V) inoculum sizes of high-yield spore-casting yeast strain seed liquids with different bacterial contents into 1000mL conical flasks containing a certain volume of liquid fermentation culture medium, carrying out shake culture in a constant-temperature oscillator at 180r/min, and exploring suitable conditions for single-factor fermentation of high-yield carotenoid. Wherein the central values of the bacteria content, liquid loading ratio, fermentation pH, fermentation temperature and fermentation time of the seed liquid are respectively 6.0 × 10⁸each/mL, 10% (V/V), pH6.0, 16 ℃ and 60h, the central value of each component in the liquid fermentation medium is: 20g/L of sucrose, 10g/L of peptone, 10g/L of yeast extract, 0.4mg/L of lycopene and 0.4mg/L of riboflavin.

1) Under the condition of no change of other single-factor central values, the bacterium content of seed liquor of high-yield spore-casting yeast strain is changed to 2.0 x 10 respectively⁸、4.0×10⁸、6.0×10⁸、8.0×10⁸And 10.0X 10⁸one/mL, the results are shown in FIG. 1;

2) in the case where the other single-factor center values were not changed, the liquid-filling ratios were changed to 5%, 10%, 15%, 20% and 25% (V/V), respectively, and the results are shown in fig. 2;

3) in the case of other single-factor central values being unchanged, initial fermentation pH was changed to 4.5, 5.0, 5.5, 6.0, 6.5 and 7.0, respectively, and the results are shown in fig. 3;

4) under the condition that the central value of other single factors is not changed, the fermentation temperatures are respectively changed to 14 ℃, 16 ℃, 18 ℃, 20 ℃, 22 ℃, 24 ℃, 26 ℃, 28 ℃, 30 ℃, 32 ℃ and 34 ℃, and the result is shown in figure 4;

5) under the condition that the central value of other single factors is not changed, the fermentation time is respectively changed into 48h, 54h, 60h, 66h, 72h, 78h and 84h, and the result is shown in FIG. 5;

6) under the condition that the central value of other single factors is not changed, the sucrose contents are respectively changed to 16g/L, 18g/L, 20g/L, 22g/L and 24g/L, and the result is shown in FIG. 6;

7) under the condition that other single-factor central values are not changed, the peptone contents are respectively changed to 6g/L, 8g/L, 10g/L, 12g/L, 14g/L and 16g/L, and the results are shown in FIG. 7;

8) under the condition that the central value of other single factors is not changed, the content of yeast extract is respectively changed to 6g/L, 8g/L, 10g/L, 12g/L, 14g/L and 16g/L, and the result is shown in figure 8;

9) under the condition that the central value of other single factors is not changed, the lycopene content is respectively changed to be 0mg/L, 0.2mg/L, 0.4mg/L, 0.6mg/L, 0.8mg/L and 1.0mg/L, and the result is shown in figure 9;

10) in the case where the other single-factor central values were not changed, the riboflavin contents were changed to 0mg/L, 0.2mg/L, 0.4mg/L, 0.6mg/L, 0.8mg/L, and 1.0mg/L, respectively, and the results are shown in FIG. 10.

2 orthogonal experiments

Selecting three factors of initial fermentation pH, sucrose concentration and fermentation temperature which have the most obvious influence on the yield of the carotenoid in a single-factor experiment, selecting appropriate range levels of the three factors, designing an orthogonal experiment, and finishing the orthogonal experiment by controlling different factors of each experiment, wherein the results are shown in table 1.

TABLE 1 orthogonal test Table and results

As can be seen from Table 1, the three main fermentation factors affecting carotenoid production are sucrose concentration>Initial fermentation pH>The fermentation temperature, in which the sucrose concentration of 18g/L, the pH of the initial fermentation of 6.0 and the culture temperature of 16 ℃ are the optimum conditions, is just the test No. 4 in Table 1, and the carotenoid yield is indeed the highest in the table, reaching 898.4 mug/g. The best fermentation conditions of the high-yield sporogenous yeast strain of carotenoid are obtained by combining the results of the single-factor test as follows: the content of the seed liquid inoculated into 1000mL of fermentation medium according to the inoculation amount of 5% (V/V) is 6.0 multiplied by 10⁸～8.0×10⁸The concentration per mL, the liquid filling ratio is 5%, the fermentation pH is 6.0, the fermentation temperature is 16 ℃, the fermentation time is 66-72 hours, the sucrose concentration is 18g/L, the peptone concentration is 12-14 g/L, the yeast extract concentration is 10-12 g/L, the lycopene concentration is 0.4-0.6 mg/L, the riboflavin concentration is 0.2-0.6 mg/L, and the rotating speed is 180 r/min. The carotenoid yield under the optimized condition is as high as 822.6 mu g/g (dry bacterial cells) to 898.4 mu g/g (dry bacterial cells) on average.

Comparative example 1

A wild type Sporobolomyces NGR, the fermentation condition is: the cells were inoculated at 1% inoculum size into a 250mL Erlenmeyer flask containing 50mL YPD liquid medium and cultured at 28 ℃ for 72 hours with shaking at 200 r/min. The liquid culture medium is 20g/L glucose, 20g/L peptone, 10g/L yeast extract and pH6.0. The total carotenoid content in the fermentatively cultured wild-type Sporobolomyces NGR was determined by spectrophotometric assay to be 1238.04. mu.g/g (wet cells), which corresponded to 309.51. mu.g/g (dry cells) since an average of 1g of dry cells corresponded to 4g of wet cells.

Comparative example 2

The rhodotorula mucilaginosa K-1 has the fermentation conditions that: the medium was inoculated at 5% inoculum size into a 250mL Erlenmeyer flask containing 50mL of liquid medium and cultured at 25 ℃ for 5d with shaking at 150 r/min. The liquid culture medium is glucose20g/L、(NH₄)₂SO₄20g/L of anhydrous CaCl₂2g/L, trisodium citrate 5g/L, pH 4.0.0. The total carotenoid content in the fermentation-cultured Rhodotorula mucilaginosa K-1 was 221.28. mu.g/g (dry cell) as determined by spectrophotometer.

The above examples are only for illustrating the technical idea and features of the present invention, and the purpose of the present invention is to enable those skilled in the art to understand the content of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

1. The high-yield carotenoid spore-casting yeast strain is characterized in that the strain is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.17429 and the preservation name of CGMCC No.17429Sporobolomyces reseuGCJM3-6 with a preservation date of 2019, 3 months and 22 days.

2. The use of a carotenoid high yielding sporobolomyces strain as claimed in claim 1 for the production of carotenoids.

3. The fermentation process of a carotenoid high yielding sporobolomyces strain of claim 1, wherein said process is: according to 5%, (V/V) Respectively inoculating the seed liquid of the high-yield spore-casting yeast strain into 1000mL triangular flasks containing a certain volume of liquid fermentation medium, wherein the bacterium content of the seed liquid of the high-yield spore-casting yeast strain is about 2.0 multiplied by 10⁸~10.0×10⁸Per mL, the liquid loading ratio of the liquid fermentation medium is 5-25% ((V/V) The pH value of initial fermentation is 4.5-7.0, the fermentation temperature is 14-34 ℃, and the fermentation is carried out in a constant temperature oscillator at a rotating speed of 180r/min for 48-84 h, wherein the liquid fermentation medium comprises 16 g/L-24 g/L of sucrose, 6 g/L-16 g/L of peptone, 6 g/L-16 g/L of yeast extract, 0.2 mg/L-1.0 mg/L of lycopene and 0.2 mg/L-1.0 mg/L of riboflavin.

4. The fermentation method of a carotenoid highly productive sporobolomyces strain as claimed in claim 3, wherein the bacterial content of the seed liquid of said highly productive sporomyces bolomyces strain is 6.0 x 10⁸~8.0×10⁸one/mL.

5. The fermentation method of a carotenoid high-yielding sporobolomyces sp strain as claimed in claim 3, wherein the liquid loading ratio is 5-10% (S) ((S))V/V）。

6. A fermentation process of a carotenoid high yielding spore-casting yeast strain as claimed in claim 3 wherein the initial fermentation pH of the liquid fermentation medium is 6.0.

7. The fermentation method of a carotenoid high-yielding sporobolomyces sp strain as claimed in claim 3, wherein the fermentation temperature is 16-18 ℃.

8. The fermentation method of a carotenoid high-yielding sporobolomyces sp strain as claimed in claim 3, wherein the time of shaking fermentation is 60-78 h.

9. The fermentation method of a carotenoid high-yield spore-casting yeast strain as claimed in claim 3, wherein the composition of the liquid fermentation medium is 20g/L to 22g/L of sucrose, 12g/L to 14g/L of peptone, 10g/L to 12g/L of yeast extract, 0.4mg/L to 0.6mg/L of lycopene, and 0.2mg/L to 0.6mg/L of riboflavin.

10. A fermentation process of a carotenoid highly productive sporobolomyces strain as claimed in claim 3, wherein said fermentation process is scaled up for laboratory pilot and actual production.