A method for efficiently producing fucoxanthin through fermentation-illumination coupling of chrysophytes

Abstract

The present application relates to a kind of by chrysophyte fermentation-light coupling high-efficiency production of fucoxanthin method, comprising: chrysophyte is inoculated into fermenter and is carried out heterotrophic culture, while the algal liquid is continuously or intermittently lighted;Culture medium includes basic medium and feed medium, C / N is 4:1-40:1, nitrogen source uses organic nitrogen source or inorganic ammonium nitrogen;The glucose concentration in basic medium is 5-15g / L, temperature is 25-32℃, dissolved oxygen is controlled at 5-80%, pH is 4-8, light intensity 5-600 μmol m ‑2 s ‑1 ;Glucose concentration is monitored in time during the cultivation process, when glucose concentration is reduced to 0.5-3g / L, using peristaltic pump to flow feed medium is added to flow culture, control the glucose concentration in whole flow culture process in 1-10g / L;After 100h of cultivation, every 1-5h is sampled to detect the biomass dry weight contained in the culture medium in fermenter, when the detection value is unchanged or begins to drop, fermentation is finished, harvest, and the harvested product is used for extracting fucoxanthin.The present application establishes the feasibility basis of fermentation-light coupling chrysophyte cultivation to scale production of fucoxanthin.

Description

Technical Field

[0001] This invention relates to the field of microalgae cultivation technology, specifically to a method for producing fucoxanthin through high-density fermentation culture coupled with light and heterotrophic processes. Background Technology

[0002] Fucoxanthin is a propadiene carotenoid, unlike common carotenoids (such as β-carotene and astaxanthin). Fucoxanthin molecules possess a polyene skeleton, containing not only unique conjugated double bonds but also mono-epoxy, carbonyl, and hydroxyl groups. This unique molecular structure endows fucoxant with various biological activities, including antioxidant, anti-obesity, anti-tumor, blood pressure regulation, hypoglycemic, and lipid-lowering effects. Therefore, fucoxanthin, as a functional ingredient, can be widely used in cosmetics, health products, pharmaceuticals, aquaculture, and livestock industries. Currently, commercially available fucoxanthin is mainly extracted from macroalgae (kelp, wakame, nori, etc.). However, due to the low fucoxanthin content, seasonal growth, limited resources, and long cultivation cycles of these macroalgae, large-scale production of fucoxanthin using these natural resources is not only costly but also unsustainable, making it difficult to meet market demand. Compared to large brown algae, many single-celled microalgae (such as diatoms) have advantages such as high fucoxanthin content, rapid growth rate, low extraction cost, and short culture cycle, and are considered important alternative sources for the commercial production of fucoxanthin. Although microalgal cells contain higher levels of fucoxanthin, most fucoxanthin-producing algal strains currently employ photoautotrophic culture. In this culture mode, microalgal cell growth is limited by light, resulting in low microalgal biomass concentration and fucoxanthin yield. Therefore, existing technologies propose using marine diatoms to produce fucoxanthin. For example, Xue Lu, Han Sun, Weiyang Zhao, and others proposed using a heterotrophic marine diatom strain (specifically, a smooth rhomboid alga, *Nitzschialaevis*) to produce fucoxanthin using a two-stage heterotrophic-photoautotrophic method. The first stage involves high-density culture of the smooth rhomboid alga in a fermenter to obtain a large number of algal cells.

[0003] However, this method utilizes heterotrophic culture of marine diatoms, requiring the use of sea salt, which can corrode stainless steel fermenters during large-scale cultivation. Furthermore, transferring the smooth nigrum cells cultured in the fermenter to a photobioreactor system for photoautotrophic culture poses a risk of microbial contamination (especially during scale-up cultivation), making it difficult to ensure the quality and stable production of fucoxanthin powder. In addition, because smooth nigrum cells contain cell walls, to improve fucoxanthin extraction efficiency, pretreatment of the microalgae cells using mechanical, chemical, or biological methods is generally required to disrupt the cell walls before extraction, significantly increasing the extraction cost of fucoxanthin. Summary of the Invention

[0004] (a) Technical problems to be solved

[0005] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a method for efficient production of fucoxanthin through fermentation-light coupling of golden algae. The method uses freshwater golden algae rich in fucoxanthin that can be heterotrophically cultured as a carrier for fucoxanthin accumulation. The golden algae is placed in a fermenter for high-density culture and light-coupled culture. In the fermenter, the growth of golden algal cells and the accumulation of fucoxanthin are promoted by regulating the nitrogen source supply and light conditions.

[0006] This invention addresses two main issues: firstly, the low fucoxanthin content, high extraction costs, limited resources, and unsustainable raw material supply of existing commercially available fucoxanthin sources (such as large brown algae like kelp, wakame, and nori). Secondly, it solves problems related to the susceptibility to contamination by other microorganisms in the two-stage heterotrophic-autotrophic culture of marine diatoms, the instability of scale-up culture, and the high extraction costs due to the cell walls of diatoms. This invention lays the foundation for the feasible large-scale production of fucoxanthin from golden algae cultivated using fermentation-photocoagulation coupling technology, providing a new technological direction for fucoxanthin production.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0009] In a first aspect, the present invention provides a method for the efficient production of fucoxanthin via golden algae fermentation-light coupling, the method comprising:

[0010] Heterotrophic freshwater golden algae were inoculated into a fermenter for heterotrophic culture. During the culture, the algal solution was subjected to continuous or intermittent light. The culture media used for heterotrophic culture included basal medium and supplemented medium, with a C / N ratio of 4:1-40:1. The carbon source was glucose, and the nitrogen source was either organic nitrogen or inorganic ammonium nitrogen. The culture conditions were as follows:

[0011] The glucose concentration in the basal culture medium is 5-15 g / L, the culture temperature is 25-32℃, and the stirring speed and dissolved oxygen are coupled and controlled during the culture process, with dissolved oxygen maintained at 5-80%. A pH adjuster is used to maintain the pH in the fermenter at 4-8; the light intensity is 5-600 μmol / m². -2 s -1 During the cultivation process, the glucose concentration in the fermenter should be monitored and measured in a timely manner. When the glucose concentration drops to 0.5-3 g / L, the feed medium should be fed in a fed culture using a peristaltic pump. The glucose concentration should be controlled at 1-10 g / L throughout the entire fed culture process.

[0012] After 100 hours of cultivation, samples were taken every 1-5 hours to measure the dry weight of biomass in the culture medium in the fermenter. When the measured value remained unchanged or began to decrease, the fermentation was terminated and the product was harvested. The harvested product was used to extract fucoxanthin.

[0013] According to a preferred embodiment of the present invention, the inoculation method is to first prepare a shake flask seed solution, and then inoculate the shake flask seed solution into a fermenter at an inoculation amount of 5-10% (v / v) for heterotrophic culture.

[0014] The preparation method of the shake flask seed culture is as follows: Under sterile conditions, the activated golden algae cells are inoculated into a 250mL shake flask (100mL liquid volume) at an inoculation rate of 2-10% (v / v) and cultured in the dark with shaking at a temperature of 25-32℃ and a rotation speed of 120-220rpm for 2-5 days.

[0015] According to a preferred embodiment of the present invention, the heterotrophic freshwater golden algae is *Poterioochromonas malhamensis* CMBB-01, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.1162, deposited on December 2, 2015, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0016] CMBB-01 is a heterotrophic golden algae strain rich in fucoxanthin, which can achieve high-density growth and efficient accumulation of fucoxanthin under fermentation-light coupling conditions.

[0017] According to a preferred embodiment of the present invention, the nitrogen source is one or more selected from urea, NH4Cl, yeast powder, liver extract powder, and peptone. According to a preferred embodiment of the present invention, the nitrogen source is a mixture of inorganic and organic nitrogen sources; preferably, the nitrogen content ratio of the inorganic and organic nitrogen sources is 1:1.

[0018] According to a preferred embodiment of the present invention, the C / N ratio of the basal culture medium and the supplemental culture medium is 16:1-24:1, more preferably 16:1.

[0019] According to a preferred embodiment of the present invention, the pH adjuster is a 3M NaOH solution or a 1M HCl solution.

[0020] According to a preferred embodiment of the present invention, the culture conditions during the cultivation process are as follows: dissolved oxygen controlled at 20-40%, pH at 5.5-6.5; and light intensity at 50-150 μmol / m². -2 s -1 .

[0021] According to a preferred embodiment of the present invention, the illumination is an artificial light source, such as an LED lamp, which is installed inside the fermentation tank or located outside the fermentation tank with a light-transmitting window.

[0022] According to a preferred embodiment of the present invention, the light intensity / color of the artificial light source is adjustable, and the light color of the light source is white light or monochromatic light; preferably white light. The monochromatic light is one or a mixture of two or more of blue light, red light, and green light.

[0023] Experimental results show that when the carbon-to-nitrogen ratio in the basal and supplemental culture media is 4:1–24:1, the pH is 5.5–6.5, the dissolved oxygen is 20–40%, and white light is used with an average light intensity of 50–150 μmol m², the optimal conditions for successful treatment are achieved. -2 s -1 When the nitrogen source in the basal medium and supplemental medium is a mixture of organic and inorganic nitrogen sources, it is more conducive to improving the total yield of fucoxanthin than inorganic or organic nitrogen sources alone; especially when the carbon-nitrogen ratio in the basal medium and supplemental medium meets the condition of 16-24:1, it is easiest to obtain a high yield of fucoxanthin.

[0024] (III) Beneficial Effects

[0025] The main technical effects of this invention include the following:

[0026] (1) This invention utilizes a heterotrophic golden algae obtained from previous screening (most of the currently reported fucoxanthin-rich microalgae cannot be heterotrophically cultured), and uses high-density fermentation-light coupling to efficiently produce fucoxanthin, thereby solving the problems of low fucoxanthin content, high extraction cost, limited resources and unsustainable raw material supply in existing commercial fucoxanthin sources (such as kelp, wakame, nori and other large brown algae).

[0027] (2) Compared with the existing technology of producing fucoxanthin through two-stage culture of *Rhizophora smoothifolia*, this invention uses cell-wall-free freshwater golden algae as the biological carrier for accumulating fucoxanthin, enabling high-density culture and accumulation of fucoxanthin in cells within a fermenter. During fucoxanthin extraction, since golden algae lack cell walls, no pretreatment such as cell wall disruption is required, resulting in higher extraction efficiency and lower cost. Furthermore, the freshwater algae culture process does not require the use of sea salt, the culture medium is non-corrosive, and scale-up culture can be achieved using stainless steel fermenters / liquid transport pipeline components, ensuring a longer service life for the fermentation equipment.

[0028] (3) This invention produces fucoxanthin through high-density fermentation coupled with light in a sterile fermenter. The reactor is not transferred during the cultivation process, and the cultivation process is controllable and stable. This solves the technical defects of low microalgal biomass concentration, susceptibility to contamination by other bacteria, and unstable scale-up cultivation when using microalgae for photoautotrophic cultivation or two-stage heterotrophic-autotrophic cultivation. Attached Figure Description

[0029] Figure 1 The changes in golden algae biomass concentration and fucoxanthin content over time under four different experimental conditions in Examples 1-2 and Control Examples 1-2 are shown.

[0030] Figure 2 The changes in golden algae biomass concentration and fucoxanthin content over time are shown under two different experimental conditions, Example 3 and Control Example 3. Detailed Implementation

[0031] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0032] Preparation of experimental materials

[0033] Algal strain: *Poterioochromonas malhamensis* CMBB-01, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.1162, deposited on December 2, 2015, at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0034] Shake flask culture medium: glucose 10 g / L; yeast powder 1 g / L; liver extract powder 1 g / L; KH2PO4 0.4 g / L; MgSO4·7H2O 0.4 g / L; adjust pH to 4-8.

[0035] Basic culture medium: glucose 5-15 g / L; yeast extract 0-5 g / L; liver extract 0-5 g / L; peptone 0.1-5 g / L; KH2PO4 0.1-0.5 g / L; NH4Cl 0-2 g / L; urea 0-2 g / L; MgSO4·7H2O 0.1-0.5 g / L; FeCl3+EDTA stock solution 0.1-1 mL / L; CaCl2·2H2O stock solution 0.1-1 mL / L; trace element stock solution 0.1-1 mL / L; VB1 0.1-5 mg / L; VB12 0.1-5 mg / L; adjust pH to 4-8.

[0036] Supplemented culture medium: glucose 200-500 g / L; yeast extract 0-50 g / L; liver extract 0-50 g / L; peptone 0-50 g / L; KH2PO4 5-50 g / L; NH4Cl 0-50 g / L; urea 0-50 g / L; MgSO4·7H2O 5-50 g / L; FeCl3+EDTA stock solution 10-50 mL / L; CaCl2·2H2O stock solution: 10-50 mL / L; trace element stock solution: 10-50 mL / L; VB1 0.1-50 mg / L; VB12 0.1-50 mg / L.

[0037] The composition of the mother liquor in the above culture medium is shown in Table 1 below:

[0038] Table 1: Composition of the stock solution in the culture medium

[0039]

[0040] The cultivation method is as follows:

[0041] (1) Shake flask seed culture

[0042] Under sterile conditions, activated golden algae cells were inoculated at a 10% inoculum (v / v) in a 250 mL shake flask (100 mL of liquid) and cultured in the dark with shaking at a temperature of 25-32℃ and a rotation speed of 120-220 rpm for 2-5 days.

[0043] (2) Fermentation-light coupled culture

[0044] Culture conditions: The cultured shake-flask seed culture is inoculated into a 1L fermenter at an inoculum rate of 5-10% (v / v). The culture temperature is 25-32℃, and the aeration ratio is 0.5-2:1 (vvm). During culture, the stirring speed and dissolved oxygen are coupled and controlled, with dissolved oxygen maintained at 5-80% (preferably 20-40%). During fermentation, the pH is controlled at 4-8 (preferably pH 5.5-6.5) using 3M NaOH or 1M HCl. Adjustable LED lights with light intensity / color are installed outside or inside the fermenter. During fermentation, the LED lights are simultaneously turned on for supplemental lighting-heterotrophic fermentation coupled culture, with a light intensity of 5-600 μmol m². -2 s -1 The preferred light intensity is 50-150 μmol m. -2 s -1 The LED light color is white light or monochromatic light; preferably, the monochromatic light is one or a mixture of two or more of blue light, red light, and green light.

[0045] Culture medium addition control: The carbon source in both basal and fed-batch media is glucose, and the nitrogen source is one or more of urea, NH4Cl, yeast extract, liver extract, and peptone. The C / N ratio in the medium is 4:1-40:1, preferably 16:1-24:1, and the initial glucose concentration is 5-15 g / L. When the glucose concentration drops to 0.5-3 g / L during culture, fed-batch culture is initiated using an adjustable-rate peristaltic pump. The glucose concentration is monitored and measured regularly during culture, and the feeding rate is adjusted accordingly to maintain the glucose concentration within 1-10 g / L throughout the entire fed-batch culture process.

[0046] After 100 hours of cultivation, samples were taken every 1-5 hours to measure the dry weight of biomass in the culture medium in the fermenter. When the measured value remained unchanged or began to decrease, the fermentation was terminated and the product was harvested. The harvested product was used to extract fucoxanthin.

[0047] The following are embodiments and comparative examples of the present invention.

[0048] Example 1

[0049] The method for cultivating golden algae in this embodiment is as follows:

[0050] Under sterile conditions, activated golden algae cells were inoculated at a 10% inoculum (v / v) into 250 mL shake flasks (100 mL of liquid) and cultured in the dark with shaking at 30 °C and 220 rpm for 4 days to obtain the shake flask seed culture.

[0051] The cultured shake-flask seed culture was inoculated into a 1L fermenter at a 10% (v / v) inoculation rate. The culture temperature was 28-30℃, and the stirring speed and dissolved oxygen were coupled and controlled during cultivation, with dissolved oxygen maintained at 20-40%. The pH was maintained at 5.5-6.5 during fermentation. In this embodiment, ammonium chloride was used as the inorganic nitrogen source for both the basal and fed-batch media. The glucose concentration in the basal medium was 10 g / L, and the glucose concentration in the fed-batch media was 240 g / L, with a carbon-to-nitrogen ratio of 16:1 in both media. Other components of the basal and fed-batch media were taken as intermediate values. White LED lights were installed inside the fermenter, with an average light intensity of 50-150 μmol / m² during fermentation. -2 s -1 Throughout the experiment, the glucose concentration in the fermenter was maintained at the same level (1-5 g / L).

[0052] Compare with Example 1

[0053] This example modifies the culture conditions of Example 1, specifically by omitting LED lights and culturing the golden algae in the dark in a fermenter. All other conditions remain the same as in Example 1.

[0054] Example 2

[0055] This embodiment modifies the culture conditions based on Example 1. The basal and supplemental culture media use organic nitrogen sources, yeast extract and liver extract, as nitrogen sources. The glucose concentration in the basal culture medium is 10 g / L, and the glucose concentration in the supplemental culture medium is 240 g / L. All other conditions are the same as in Example 1.

[0056] Compare with Example 2

[0057] This example modifies the cultivation conditions of Example 2, specifically by omitting LED lights and culturing the golden algae in the dark in a fermenter. All other conditions remain the same as in Example 2.

[0058] During the experiment, samples were taken every 24 hours to measure the biomass concentration (g / L) and fucoxanthin content (mg / g - cell dry weight) of the algal broth in the fermenter, and curves were plotted to compare the changes in biomass concentration and fucoxanthin content of golden algae over time under four different experimental conditions: Example 1 and Control Example 1, and Example 2 and Control Example 2. The experimental results are as follows: Figure 1 As shown.

[0059] like Figure 1 As shown in Figure A, with the increase of fermentation culture time, the cell biomass concentration of Control Example 1 reached its highest value after 144 hours of culture, at which point the biomass concentration of Example 1 also reached its peak. However, the highest biomass concentration of Control Example 1 was slightly higher than that of Example 1. This indicates that under the same conditions, fermentation-dark culture is more likely to obtain higher biomass than fermentation-light coupled culture mode. During the 0-192 hours of culture, the cell biomass concentration of Control Example 2 and Example 2 continued to increase. Starting from 144 hours of culture, the biomass concentration of Control Example 2 began to be slightly higher than that of Example 2, and the highest biomass concentration of Control Example 2 was slightly higher than that of Example 2. This is consistent with the experimental conclusions of Example 1 and Control Example 1.

[0060] The above experimental results indicate that, in terms of biomass concentration alone, heterotrophic fermentation-dark culture is more conducive to obtaining higher biomass concentrations than heterotrophic fermentation-light coupled culture; and compared to using inorganic ammonium nitrogen sources, organic nitrogen culture is more conducive to obtaining higher biomass concentrations. Although fermentation-dark culture and the use of organic nitrogen sources have an effect on increasing biomass concentration, the increase is not significant. In other words, while using fermentation-light coupled culture or inorganic nitrogen sources is not conducive to obtaining high biomass concentrations, the negative impact on biomass concentration is not significant, and inorganic nitrogen sources are very beneficial in promoting the accumulation of fucoxanthin in golden algal cells.

[0061] See Figure 1As shown in Figure B, in Control Examples 1-2, the fucoxanthin content accumulated in cells cultured under both organic and inorganic nitrogen source conditions was almost the same and remained at a low level. However, the fucoxanthin content in the golden algae cells cultured in Example 1 (fermentation-light coupling + inorganic nitrogen source) and Example 2 (fermentation-light coupling + organic nitrogen source) was significantly higher than that in Control Examples 1-2, especially in Example 1 where an inorganic nitrogen source was used, the fucoxanthin content in the golden algae cells was significantly higher than that in Example 2. This indicates that light coupling and inorganic nitrogen sources are highly beneficial for the accumulation of fucoxanthin in golden algae cells.

[0062] Example 3

[0063] The method for cultivating golden algae in this embodiment is as follows:

[0064] Under sterile conditions, activated golden algae cells were inoculated at a 10% inoculum (v / v) into 250 mL shake flasks (100 mL of liquid) and cultured in the dark with shaking at 30 °C and 220 rpm for 4 days to obtain the shake flask seed culture.

[0065] The cultured shake-flask seed culture was inoculated into a 1L fermenter at a 10% (v / v) inoculation rate. The culture temperature was 28-30℃, and the stirring speed and dissolved oxygen were coupled and controlled during cultivation, with dissolved oxygen maintained at 20-40%. The pH was maintained at 5.5-6.5 during fermentation. In this embodiment, the basal and fed-batch media used a mixed nitrogen source (inorganic nitrogen source ammonium chloride and organic nitrogen source yeast extract + liver extract, with a nitrogen ratio of 1:1). The glucose concentration in the basal medium was 10 g / L, and the glucose concentration in the fed-batch media was 240 g / L, with a carbon-to-nitrogen ratio of 16:1 in both media. Other components of the basal and fed-batch media were taken as intermediate values. White LED lights were installed inside the fermenter, with an average light intensity of 50-150 μmol m² during fermentation. -2 s -1 Throughout the experiment, the glucose concentration in the fermenter was maintained at the same level (1-5 g / L).

[0066] Compare with Example 3

[0067] This example modifies the cultivation conditions of Example 3 by omitting LED lights and culturing the golden algae in the dark in a fermenter. All other conditions remain the same as in Example 3.

[0068] During the experiment, samples were taken every 24 hours to measure the biomass concentration (g / L) and fucoxanthin content (mg / g - cell dry weight) of the algal liquid in the fermenter, and curves were plotted to compare the changes in the biomass concentration and fucoxanthin content of golden algae over time under two different experimental conditions, Example 3 and Control Example 3. The experimental results are as follows: Figure 2 As shown.

[0069] like Figure 2 As shown in Figure A, with the increase of fermentation culture time, the cell biomass concentration of Control Example 3 reached its highest value after 168 hours of culture. At this time, the biomass concentration of Example 3 also reached its peak. However, the highest biomass concentration of Golden Algae cells cultured in Control Example 3 (fermentation-dark culture + mixed nitrogen source) was slightly higher than that in Example 3 (fermentation-light coupling + mixed nitrogen source). However, as... Figure 2 As shown in Figure B, compared to the heterotrophic fermentation-dark culture mode, the fucoxanthin content accumulated in single cells of *Gynostemma pentaphyllum* obtained by heterotrophic fermentation-light coupling (Example 3) was significantly higher than that obtained by fermentation-dark culture (Control Example 3). The highest fucoxanthin content in *Gynostemma pentaphyllum* cells in Example 3 was approximately 10-13 times that in Control Example 3, and this trend was already evident at 48 hours of culture, reaching its peak at 168 hours. This demonstrates that a mixed nitrogen source and a fermentation-light coupling culture mode are more effective in promoting fucoxanthin accumulation in single cells. This is consistent with the previous experimental conclusions.

[0070] The highest biomass concentration, highest fucoxanthin content, highest fucoxanthin yield, and highest fucoxanthin production rate of fermented golden algae from Examples 1-3 and Control Examples 1-3 are compared as follows (Table 2):

[0071] Table 2:

[0072]

[0073] Combined with Table 2 and Figure 1-2 It was found that under the three conditions of inorganic nitrogen source (ammonium chloride), organic nitrogen source, and mixed nitrogen source in the basal culture medium and supplemented culture medium, although heterotrophic fermentation-dark culture could obtain a slightly higher biomass concentration, the heterotrophic fermentation-light coupling mode could obtain significantly higher fucoxanthin content under all three nitrogen source conditions, thus achieving higher total fucoxanthin yield and productivity. Therefore, from the perspective of large-scale cultivation of golden algae to produce fucoxanthin, heterotrophic fermentation-light coupling is superior to heterotrophic fermentation-dark culture, and inorganic nitrogen source is superior to organic nitrogen source.

[0074] Examples 4-6

[0075] Examples 4-6 all involved heterotrophic fermentation coupled with light, with glucose concentration maintained at a consistent level (1-5 g / L), pH 5.5-6.5, and dissolved oxygen 20-40% throughout the entire culture process. In Examples 4-6, the basal and fed media used a mixed nitrogen source (inorganic nitrogen source ammonium chloride and organic nitrogen source yeast extract + liver extract, with a nitrogen ratio of 1:1). The glucose concentration in the basal medium was 10 g / L, and the glucose concentration in the fed media was 240 g / L. In Example 4, the carbon-to-nitrogen ratio in both the basal and fed media was 24:1. In Example 5, the carbon-to-nitrogen ratio in both the basal and fed media was 4:1. In Example 6, the carbon-to-nitrogen ratio in both the basal and fed media was 40:1. Other components of the culture media in each example were taken as intermediate values. White LED lights were installed inside the fermenter, with an average light intensity of 50-150 μmol / m² during fermentation. -2 s -1 The light quality is LED white light.

[0076] Example 7

[0077] Example 7 is based on Example 3, but with increased light intensity, and the average light intensity is adjusted to 400-600 μmol m. - 2 s -1 The light quality was white LED light. All other experimental conditions were exactly the same as in Example 3.

[0078] Example 8

[0079] Example 8 is based on Example 3, but with reduced light intensity, adjusting the average light intensity to 5-30 μmol m. -2 s -1 The light quality was white LED light. All other experimental conditions were exactly the same as in Example 3.

[0080] Example 9

[0081] Example 9 is based on Example 3, but with a reduced dissolved oxygen concentration, controlled at 5-15%. All other experimental conditions are exactly the same as in Example 3.

[0082] Example 10

[0083] Example 10 is based on Example 3, but with an increased dissolved oxygen concentration, controlled at 60-80%. All other experimental conditions are exactly the same as in Example 3.

[0084] Example 11

[0085] Example 11 is based on Example 3, but the pH of the fermentation broth was adjusted to 4-4.5 throughout the entire culture process. All other experimental conditions were exactly the same as in Example 3.

[0086] Example 12

[0087] Example 12 is based on Example 3, but the pH of the fermentation broth was adjusted to 7.5-8 throughout the entire culture process. All other experimental conditions were exactly the same as in Example 3.

[0088] Example 13

[0089] Example 13 maintains the same average illumination intensity as Example 3, but replaces the white LED with a monochromatic blue LED. All other experimental conditions are exactly the same as in Example 3.

[0090] Example 14

[0091] Example 14 maintains the same average illumination intensity as Example 3, but replaces the white LED with a monochromatic red LED. All other experimental conditions are exactly the same as in Example 3.

[0092] The highest biomass concentration, highest fucoxanthin content, highest fucoxanthin yield, and highest fucoxanthin production rate of fermented golden algae from Examples 1-14 are compared as follows (Table 3):

[0093] Table 3:

[0094]

[0095] Compared to Example 6, the highest fucoxanthin content and yield were significantly higher in the fermented golden algae cells of Examples 1-5. This indicates that the fucoxanthin yield from golden algae fermentation is higher when the carbon-to-nitrogen ratio in the basal and supplemental media is 4:1–24:1 than under the 40:1 carbon-to-nitrogen ratio condition. A comparison of Examples 3-6 shows that a higher fucoxanthin yield was obtained when the carbon-to-nitrogen ratio in the basal and supplemental media was 16:1, and when both media used a mixed nitrogen source.

[0096] Compared to the inorganic nitrogen source used in Example 2 and the organic nitrogen source used in Example 1, the highest fucoxanthin content and yield were significantly higher in the fermented golden algae cells of Examples 3-5. This indicates that using inorganic nitrogen alone is not conducive to obtaining a high fucoxanthin yield. From the perspective of obtaining a high fucoxanthin yield, mixed nitrogen sources are superior to inorganic nitrogen sources, and inorganic nitrogen sources are superior to organic nitrogen sources. Organic nitrogen sources are mainly beneficial for obtaining high biomass concentrations, while inorganic nitrogen sources better promote fucoxanthin synthesis and accumulation.

[0097] Example 3 and Example 7 (average light intensity 400-600 μmol m) were compared. -2 s -1 ) and Example 8 (average light intensity 5-30 μmol m -2 s-1 Comparison shows that excessively high or low average light intensity is detrimental to obtaining higher fucoxanthin yields. The optimal average light intensity is 50-150 μmol / m². -2 s -1 .

[0098] Comparing Example 3 with Example 9 (dissolved oxygen 5-15%) and Example 10 (dissolved oxygen 60-80%), it can be seen that too high or too low dissolved oxygen concentrations are not conducive to obtaining higher fucoxanthin yields. The optimal dissolved oxygen concentration is 20-40%, but overall, the fucoxanthin yield obtained under the 5-15% dissolved oxygen concentration condition is higher than that under the 60-80% dissolved oxygen concentration condition.

[0099] Comparing Example 3 with Examples 11 (pH 4-4.5) and 12 (pH 7.5-8), it can be seen that excessively high or low pH is not conducive to obtaining higher fucoxanthin yields, and the optimal pH range is 4-8. Furthermore, overall, the fucoxanthin yield obtained at a weakly acidic pH of 4-4.5 is higher than that at a weakly alkaline pH of 7.5-8.

[0100] Comparing Example 3 with Examples 13 (blue light) and 14 (red light), it is evident that using a single monochromatic light source is not conducive to obtaining a higher fucoxanthin yield compared to white light; white light is the preferred light source. Furthermore, overall, the biomass concentration is higher when irradiated with red light, but the fucoxanthin yield obtained from irradiation with blue light is higher than that obtained from irradiation with red light.

[0101] The above experimental results show that when the carbon-to-nitrogen ratio in the basal and supplemental culture media is 4:1 to 24:1, the pH is 5.5-6.5, the dissolved oxygen is 20-40%, and white light is used with an average light intensity of 50-150 μmol / m², the optimal conditions for successful cultivation are achieved. -2 s -1 When the nitrogen source in the basal medium and the supplemental medium is a mixed nitrogen source, it is beneficial to improve the yield of fucoxanthin, especially when the carbon-nitrogen ratio in the basal medium and the supplemental medium is 16-24:1, the yield of fucoxanthin is the highest.

[0102] In summary, this invention provides a method for high-density heterotrophic fermentation-light coupling of golden algae to efficiently produce fucoxanthin. During the cultivation of golden algae in a heterotrophic fermenter, continuous or intermittent light is simultaneously provided to the algal solution. By changing the type of nitrogen source and C / N ratio in the culture medium, optimizing the dissolved oxygen concentration, adjusting the pH range, adjusting the light intensity, and selecting the light color, the aim is to increase the cell density (biomass concentration) of golden algae and the total yield of fucoxanthin.

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for efficient production of fucoxanthin via golden algae fermentation-light coupling, characterized in that, The method includes: Heterotrophic freshwater golden algae were inoculated into a fermenter for heterotrophic culture. During the culture process, the algal solution was subjected to continuous or intermittent light. The culture medium used for heterotrophic culture included a basal medium and a supplemental medium, with a C / N ratio of 4:1-40:

1. The carbon source was glucose, and the nitrogen source was either organic nitrogen or inorganic ammonium nitrogen. The heterotrophic freshwater golden algae was *Gynostemma pentaphyllum*. Poterioochromonas malhamensis CMBB-01, deposited at the China General Microbiological Culture Collection Center, accession number: CGMCC NO.1162, deposit date: December 2, 2015, deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; The cultivation conditions were as follows: glucose concentration in the basal medium was 5-15 g / L; cultivation temperature was 25-32℃; stirring speed and dissolved oxygen were coupled and controlled during cultivation, with dissolved oxygen maintained at 5-80%; pH in the fermenter was controlled at 4-8 using a pH adjuster; and the light intensity was 5-600 μmol / m². -2 s -1 During the cultivation process, the glucose concentration in the fermenter should be monitored and measured in a timely manner. When the glucose concentration drops to 0.5-3 g / L, the feed medium should be fed in a fed culture using a peristaltic pump. The glucose concentration should be controlled at 1-10 g / L throughout the entire fed culture process. After 100 hours of cultivation, samples were taken every 1-5 hours to measure the dry weight of biomass in the culture medium in the fermenter. When the measured value remained unchanged or began to decrease, the fermentation was terminated and the product was harvested. The harvested product was used to extract fucoxanthin.

2. The method according to claim 1, characterized in that, The inoculation method involves first preparing a shake-flask seed culture, then inoculating the shake-flask seed culture into a fermenter at a volume of 5-10% for heterotrophic culture. The shake-flask seed culture is prepared as follows: under sterile conditions, activated golden algae cells are inoculated into a 250 mL shake flask at a volume of 2-10% for shaking and dark culture at a temperature of 25-32℃ and a rotation speed of 120-220 rpm for 2-5 days.

3. The method according to claim 1 or 2, characterized in that, The nitrogen source is one or more of urea, NH4Cl, yeast powder, liver extract powder, and peptone.

4. The method according to claim 3, characterized in that, The nitrogen source is a mixture of inorganic and organic nitrogen sources.

5. The method according to claim 1, characterized in that, The C / N ratio of the basal culture medium and the supplemental culture medium is 16:1-24:

1.

6. The method according to claim 1, characterized in that, The cultivation conditions were as follows: dissolved oxygen controlled at 20-40%, pH at 5.5-6.5; and light intensity at 50-150 μmol / m². -2 s -1 .

7. The method according to claim 1, characterized in that, The illumination uses an artificial light source, which is installed inside the fermentation tank or located outside the fermentation tank with a light-transmitting window.

8. The method according to claim 7, characterized in that, The intensity / color of the artificial light source is adjustable, and the light color of the light source is white light or mixed light; the mixed light is one or more monochromatic lights selected from blue light, red light, and green light.

9. The method according to claim 1, characterized in that, The basal and supplemental culture media have a carbon-to-nitrogen ratio of 4:1-24:1, pH 5.5-6.5, dissolved oxygen 20-40%, and are irradiated with white light at an average intensity of 50-150 μmol / m². -2 s -1 The nitrogen source in the basal culture medium and the supplemental culture medium is a mixture of organic and inorganic nitrogen sources.

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