Method for measuring and controlling high-yield fucoxanthin and EPA (eicosapentaenoic acid) through diatom light fermentation by adding organic acid coupling spectrum and application of method

Abstract

The invention discloses a method for measuring and controlling high-yield fucoxanthin and EPA (eicosapentaenoic acid) through diatom light fermentation by adding an organic acid coupling spectrum and application of the method. The method comprises the following steps: S1, inoculating diatom into a seed solution culture medium, and performing illumination culture to a logarithmic phase to obtain a diatom mixotrophic seed solution; and S2, inoculating the diatom mixotrophic seed solution into a light fermentation tank filled with a fermentation culture medium containing lactic acid, glycerol and a nitrogen source for light fermentation culture, and performing real-time monitoring and feedback control on intermittent material supplementation by using an online near infrared spectrum analyzer so as to improve the biomass yield of the diatom and the yield of high-value natural products. The method for on-line measurement and control of diatom light fermentation provided by the invention can be used for producing fucoxanthin and eicosapentaenoic acid.

Description

Technical Field

[0001] The invention belongs to industrial biotechnology, and particularly relates to a method for adding organic acid coupled with spectroscopy to measure and control diatom photofermentation to produce high yields of fucoxanthin and EPA, and an application thereof. Background Art

[0002] Diatoms contain a wealth of bioactive substances and high-value products, such as fucoxanthin (Fx), eicosapentaenoic acid (EPA), and β-1,3 glucan. These ingredients are widely used in functional foods, pharmaceutical development, and aquaculture. In aquaculture, Phaeodactylum tricornutum is often used as a starter feed for larvae (shrimp and crab) and as a lifelong feed for shellfish, promoting the growth and development of both larvae and adults, improving disease resistance, survival rates, and yields. In the field of human health, fucoxanthin, a carotenoid, contains active groups such as allene, epoxy, and conjugated carboxyl groups, which can effectively prevent chronic diseases such as cancer, diabetes, obesity, and fatty liver disease. Eicosapentaenoic acid, an ω-3 polyunsaturated fatty acid (PUFA), plays an effective role in improving cardiovascular function, anti-tumor effects, and anti-aging. However, how to increase the production of diatom fucoxanthin and EPA is the main bottleneck for its industrial application.

[0003] The main microalgae sources of fucoxanthin and EPA are diatoms, golden algae, and yellow algae. Fucoxanthin and EPA, as products of photosynthesis, are primarily stored intracellularly. The marine diatom P. tricornutum, due to its ability to be cultivated mixotrophically (also known as "photofermentation" or polyculture) and photosynthetically autotrophically, effectively accumulates these high-value natural products (fucoxanthin and EPA) at levels significantly higher than other microalgae, making it considered the optimal species for producing these products. Fucoxanthin and EPA are both oil-soluble components, allowing for synergistic accumulation in diatoms. Current research on high-value natural products in P. tricornutum focuses primarily on increasing their intracellular content, but has neglected efforts to increase diatom biomass yield. In existing cultivation strategies, when targeting a single product across different diatom species, fucoxanthin content ranges from 0.8% to 6% of dry weight, and EPA content ranges from 1.9% to 11% of dry weight. However, the current diatom biomass yield is generally low, mostly between 0.2 and 6.52 g / L, and rarely exceeds 10 g / L. This ultimately leads to a low total yield of high-value products per unit volume, which cannot meet the multiple requirements of commercial production for target product content and yield.

[0004] Lactic acid, also known as 2-hydroxypropionic acid (C3H6O3), is an organic acid that is a colorless or slightly yellowish viscous liquid and is virtually odorless. Under anaerobic conditions, pyruvate produced during cellular glycolysis is converted to lactic acid by lactate dehydrogenase. Anaerobic fermentation is commonly used in the fermentation industry to produce lactic acid, such as in the production of foods like cheese and yogurt. In biochemical processes, lactate serves as a substrate in metabolic pathways for energy production and as a multifunctional signaling molecule in the cAMP pathway, regulating protein function in various cell types.

[0005] Furthermore, traditional fermentation control methods rely on intermittent offline sampling and testing, such as optical density (OD) and HPLC analysis. These methods exhibit significant feedback lags, making real-time monitoring and control of the production process difficult. Furthermore, manual operations are cumbersome, severely impacting production efficiency. Near-infrared spectroscopy, with its rapid, non-destructive, accurate, multi-indicator simultaneous detection, the absence of chemical reagents, and environmental friendliness, can effectively overcome these challenges. However, no studies have yet applied near-infrared spectroscopy to online monitoring and feedback control of diatom photofermentation, let alone to the measurement and control of diatom synthesis of fucoxanthin and EPA. The near-infrared spectrum, covering the 780nm to 2500nm range, captures the characteristic harmonic and harmonic frequency characteristics of hydrogen-containing groups (such as CH, NH, and OH) in molecules. These hydrogen-containing groups are commonly found in organic molecules such as proteins, sugars, and lipids. During fermentation, microorganisms, substrates, and products often also contain these hydrogen-containing groups. Therefore, near-infrared spectroscopy is particularly suitable for analyzing components directly or indirectly associated with hydrogen-containing groups in biofermentation processes. Currently, in practical applications, online near-infrared detection technology still has the following problems: (1) The actual fermentation system is complex, and there are a large number of outliers between the obtained near-infrared spectral variables and the substrate and target product, which leads to data fluctuations and economic losses. (2) Laboratory equipment is not compatible with production equipment, which leads to mismatches between measured near-infrared spectra and offline analytical values during fermentation scale-up.

[0006] There are currently no reports on the use of lactic acid to promote the high production of fucoxanthin and EPA by photofermentation of P. tricornutum. Photofermentation of P. tricornutum still faces technical bottlenecks such as low algal cell density, biomass yield, and insufficient accumulation of target products. There is also a lack of means for non-contact online measurement and control of fermentation. Summary of the Invention

[0007] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for adding organic acid coupled with spectroscopy to measure and control the high production of fucoxanthin and EPA by diatom photofermentation.

[0008] Another object of the present invention is to provide an application of the method for adding organic acid coupled with spectroscopy to measure and control diatom photofermentation to produce high yields of fucoxanthin and EPA.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A method for adding organic acid coupled with spectroscopy to measure and control diatom photofermentation to produce high yields of fucoxanthin and EPA comprises the following steps:

[0011] S1. Seed liquid culture: inoculating diatoms into seed liquid culture medium, culturing under light until the logarithmic growth phase, and obtaining diatom mixotrophic seed liquid;

[0012] S2. Photofermentation culture (mixotrophic culture): The mixotrophic diatom seed solution is inoculated into a photofermentation tank filled with a fermentation medium containing lactic acid, glycerol and a nitrogen source for photofermentation culture, and an online near-infrared spectrometer is used for real-time monitoring and feedback control of intermittent feeding to increase the biomass yield and the yield of high-value natural products in the diatoms; wherein the high-value natural product is at least one of fucoxanthin (Fx) and eicosapentaenoic acid (EPA).

[0013] The diatom described in step S1 is preferably P. tricornutum.

[0014] The seed liquid culture medium described in step S1 is a modified f / 2 culture medium; preferably, it is a modified f / 2 culture medium containing an organic carbon source and a nitrogen source; further preferably, it is a modified f / 2 culture medium with a total carbon concentration of 300 mmol / L and a nitrogen content of 0.005-0.02 mmol / L; further preferably, it is a modified f / 2 culture medium containing 0-30 mmol / L lactic acid, 20-100 mmol / L glycerol and a nitrogen content of 0.005-0.02 mmol / L; further preferably, it is a modified f / 2 culture medium containing 20 mmol / L lactic acid (1.8 g / L), 40 mmol / L glycerol (3.68 g / L), tryptose with a nitrogen content of 0.01 mol / L (i.e., tryptose 1.17 g / L) and urea with a nitrogen content of 0.01 mol / L (i.e., urea 0.3 g / L).

[0015] The nitrogen source is at least one of tryptone, urea and ammonium sulfate; preferably, it is an organic nitrogen source obtained by mixing tryptone and urea; more preferably, it is an organic nitrogen source obtained by mixing tryptone and urea, wherein the nitrogen content of tryptone is 1 to 2:1 to 2 (molar ratio); further preferably, it is an organic nitrogen source obtained by mixing tryptone and urea, wherein the nitrogen content of tryptone is 1 to 1 (molar ratio) to the nitrogen content of urea.

[0016] The improved f / 2 medium contains the following components: 20 g / L sea salt, 30 mg / L Na2SiO3·5H2O, 4.36 mg / L Na2EDTA·2H2O, 9.8 μg / L CuSO4·5H2O, 6.3 μg / L Na2MoO4·2H2O, 22.0 μg / L ZnSO4·7H2O, 3.15 mg / L FeCl3·6H2O, 10.0 μg / L CoCl2·6H2O, 180.0 μg / L MnCl2·4H2O, and 10.0 mg / L NaH2PO4, with a pH of 7.95 to 8.05 (preferably 8.0).

[0017] The intensity of the light culture in step S1 is 10 to 30 μmol / m 2 / s; preferably 20±2μmol / m 2 / s.

[0018] The light culture time in step S1 is 4 to 12 days, preferably 8 to 12 days, and more preferably 10 to 12 days.

[0019] The temperature of the light culture in step S1 is preferably 20±1°C.

[0020] The culture in step S1 is carried out on a light-controlled constant temperature shaker at a rotation speed of 100 to 160 rpm (preferably 160 rpm).

[0021] The light source used for the culture in step S1 is LED natural white light (its spectral composition is red: green: blue (R:G:B) = 18.9:76.1:5.1, and the LED natural white light is provided by an LED natural white light bar).

[0022] The fermentation medium in step S2 is a modified f / 2 medium.

[0023] The improved f / 2 medium contains the following components: 20 g / L sea salt, 30 mg / L Na2SiO3·5H2O, 4.36 mg / L Na2EDTA·2H2O, 9.8 μg / L CuSO4·5H2O, 6.3 μg / L Na2MoO4·2H2O, 22.0 μg / L ZnSO4·7H2O, 3.15 mg / L FeCl3·6H2O, 10.0 μg / L CoCl2·6H2O, 180.0 μg / L MnCl2·4H2O, and 10.0 mg / L NaH2PO4, with a pH of 7.95 to 8.05 (preferably 8.0).

[0024] The concentration of lactic acid in the fermentation medium in step S2 is 0 to 30 mmol / L (excluding 0); preferably 10 to 20 mmol / L; more preferably 20 mmol / L (1.8 g / L).

[0025] The concentration of glycerol in the fermentation medium in step S2 is 20 to 100 mmol / L, preferably 20 to 60 mmol / L, and more preferably 40 mmol / L (3.68 g / L).

[0026] The nitrogen source (type) in the fermentation medium described in step S2 is at least one of trypsin, urea and ammonium sulfate; preferably, it is an organic nitrogen source obtained by mixing trypsin and urea; more preferably, it is an organic nitrogen source obtained by mixing trypsin and urea, wherein the nitrogen content of trypsin and urea is 1 to 2:1 to 2 (molar ratio); further preferably, it is an organic nitrogen source obtained by mixing trypsin and urea, wherein the nitrogen content of trypsin and urea is 1:1 (molar ratio).

[0027] The nitrogen sources in the fermentation medium in step S2 are tryptone with a nitrogen content of 0.01 mol / L (ie, tryptone 1.17 g / L) and urea with a nitrogen content of 0.01 mol / L (ie, urea 0.3 g / L).

[0028] The inoculation density of the diatom mixotrophic seed solution in step S2 is based on the final density of the diatom mixotrophic seed solution in the fermentation system of 1×10 6 ~2×10 7 cells / mL addition calculation; preferably based on the final density of 5×10 6 ~1×10 7 cells / mL addition calculation; more preferably, the final density in the fermentation system is 1×10 7 cells / mL was added for calculation.

[0029] The initial pH value of the fermentation culture in step S2 is 7.95 to 8.05 (without pH regulation during this period); preferably 8.0.

[0030] The fermentation culture in step S2 is preferably carried out in a light fermentation tank; more preferably, it is carried out in a light fermentation tank of 5 L or larger.

[0031] The light source used for the fermentation described in step S2 is LED red and blue mixed light (the ratio of red light to blue light R:B = 6:1, and its spectral composition is red: green: blue (R:G:B) = 85:0.3:14.7, where the red light is provided by an LED red light bar and the blue light is provided by an LED blue light bar).

[0032] The fermentation conditions in step S2 are: the culture temperature is preferably 20±1°C, the light intensity is 100-300 μmol / m 2 / s (preferably 200 μmol / m 2 / s), aeration volume of 1200-3000 mL / min (preferably 1200 mL / min), rotation speed of 100-300 rpm (preferably 100 rpm), pH of 7.0-9.0 (preferably 8.0±0.1), and culture time of 16-24 days (preferably 16 days).

[0033] The intermittent feeding method described in step S2 is to add lactic acid in batches to adjust the pH to 7.0-9.0 (preferably 8.0±0.1); at the same time, the glycerol level is controlled at 3.6±0.5 g / L. When the glycerol concentration in the fermentation system is less than 3.68 g / L, glycerol is added (high-concentration glycerol mother liquor is added). When the glycerol concentration is higher than 3.68 g / L, the addition of glycerol is stopped; starting from the 14th day, the addition of glycerol is stopped, and the fermentation is terminated when the glycerol is completely consumed.

[0034] The supplementation can be achieved by dropwise addition (addition dropwise during the fermentation process) or stream addition (continuous addition of raw materials at a small flow rate during the fermentation process).

[0035] The concentration of the glycerol mother solution is preferably 80-100 g / L.

[0036] The intermittent feeding described in step S2 also includes adding appropriate amounts of sodium lactate, urea and phosphate according to nutrient consumption. Sodium lactate, urea and phosphate can be added directly or high concentration sodium lactate, urea and phosphate mother liquor can be added.

[0037] The added sodium lactate is a sodium lactate mother liquor with a concentration of 50-150 g / L (preferably 100 g / L), and the sodium lactate mother liquor is added once every two days until the final concentration in the fermentation system is 1.8 g / L. The addition of sodium lactate is stopped from the 14th day.

[0038] The added urea is a urea mother liquor with a concentration of 200-400 g / L (preferably 300 g / L), and the urea mother liquor is added every two days until the final concentration in the fermentation system is 0.3 g / L; and the addition of urea is stopped from the 14th day.

[0039] The added phosphate is a sodium dihydrogen phosphate mother liquor with a concentration of 80-100 g / L (preferably 80 g / L), and the sodium dihydrogen phosphate mother liquor is added once every two days until the final concentration in the fermentation system is 80 mg / L; and the addition of sodium dihydrogen phosphate mother liquor is stopped from the 14th day.

[0040] The real-time monitoring and feedback control of intermittent feeding by the online near-infrared spectrometer in step S2 can be controlled by referring to the method described in Chinese patent application (publication number: CN118190867A, entitled: A method for guiding high-density fermentation of microalgae using online near-infrared technology and its application), and the measured process parameters and cell content, wherein the process parameters include at least one of glycerol concentration, biomass yield, and cell density; the cell content is fucoxanthin; preferably, this is achieved by the following steps:

[0041] (1) Establishment of near-infrared spectral model: diatom seed liquid is inoculated into a fermentation tank containing a fermentation medium containing lactic acid, glycerol and nitrogen source for photofermentation, and near-infrared spectra at more than 60 different time points are continuously collected by an online near-infrared spectrometer. Then, the collected near-infrared spectral data are preprocessed by removing abnormal spectra using algorithms and chemometric methods to obtain preprocessed near-infrared spectral data. At the same time, samples are taken at the collection time point of each spectrum, and process parameters and cell content are measured offline to obtain physical and chemical test data. Finally, linear fitting is performed based on the preprocessed near-infrared spectral data and physical and chemical test data to establish a prediction model (also known as a quantitative prediction model, a joint calibration model or a fermentation liquid model). The process parameters include at least one of glycerol concentration, biomass yield and cell density. The cell content is fucoxanthin.

[0042] (2) Using a near-infrared model to feedback control the fermentation process in real time: using an online near-infrared analysis device to continuously collect near-infrared spectra in the fermentation broth online, and then using algorithms and chemometric methods to remove abnormal spectra from the collected near-infrared spectral data and perform data preprocessing to obtain preprocessed near-infrared spectral data, and then inputting the preprocessed near-infrared spectral data into the prediction model established in step (1) to calculate the process parameters and cell content in the fermentation system, and controlling the peristaltic pump for intermittent feeding according to the process parameters; wherein the process parameters include at least one of glycerol concentration, biomass yield and cell density; and the cell content is fucoxanthin.

[0043] In steps (1) and (2), the online near-infrared analysis equipment includes a near-infrared spectrometer and an industrial computer; wherein the near-infrared spectrometer is connected to the industrial computer via a dedicated USB interface, and is connected to the PLC via an RS232 port, and the PLC is also connected to the industrial computer via an RS232 port to achieve mutual communication.

[0044] In steps (1) and (2), the instrument parameters of the online near-infrared analysis equipment are as follows: a scanning range of 900 nm to 1700 nm, using the built-in background of the instrument as a reference, an electrically cooled indium gallium arsenide diode 512 array detector, a sampling interval of 1 nm, full-wavelength scanning by continuous grating, and a scanning number of more than 100 times; and a spot diameter of the near-infrared spectrum of 100 mm.

[0045] In step (1), near-infrared spectra are collected using online near-infrared analysis equipment, and as many samples as possible are taken at different time points in different batches to ensure the stability and accuracy of the model. The sampling amount is preferably 60 to 150; more preferably 80 to 140; and more preferably 100 to 140.

[0046] The removal of abnormal spectra in steps (1) and (2) is to remove abnormal spectra based on Mahalanobis distance.

[0047] The prediction model building described in step (2) includes the following steps:

[0048] (i) Import the near-infrared spectrum into the software The Unscrambler X and define the spectral variables and chemical value variables;

[0049] (ii) The Unscrambler X software was used to preprocess the near-infrared spectra and screen the characteristic bands;

[0050] (iii) Based on the concentration of each component in the sample, the calibration set and the test set are divided into two groups by taking one out of every three samples;

[0051] (iv) using the near-infrared spectrum collected by online detection as the independent variable and the reference value of the near-infrared instrument analysis of the number of samples measured as the dependent variable, selecting the 950-1350 nm near-infrared band, and correlating the chemical values of the physical and chemical indicators measured in the fermentation broth to establish a prediction model; wherein the physical and chemical indicators include at least one of glycerol concentration, biomass yield and cell density, and fucoxanthin content;

[0052] (v) According to the root mean square error of cross validation (RMSECV), the coefficient of determination of cross validation (R 2 ) and the root mean square error of prediction (RMSEP) to judge the quality of the model, thereby screening out the best near-infrared (quantitative) prediction model.

[0053] The method of adding organic acid coupled with spectroscopy to measure and control diatom photofermentation to produce high yields of fucoxanthin and EPA is applied in diatom culture.

[0054] The diatom is preferably P. tricornutum.

[0055] The application of the method of adding organic acid coupled with spectroscopy to measure and control the high yield of fucoxanthin and EPA by diatom photofermentation in the production of high-value natural products; wherein,

[0056] The high-value natural product is at least one of fucoxanthin and eicosapentaenoic acid (preferably fucoxanthin and eicosapentaenoic acid).

[0057] The present invention has the following advantages and effects compared to the prior art:

[0058] (1) The present invention provides a method for promoting diatom photofermentation to increase biomass yield and the yield of high-value natural products. Diatom mixotrophic seed liquid cultured to the logarithmic phase is inoculated into a fermentation medium containing lactic acid, glycerol and a mixed nitrogen source for mixotrophic culture. At the same time, a model is established for the physical and chemical indicators of the fermentation process of P. tricornutum using online near-infrared technology. By collecting spectral data and combining the model prediction results, feedback control of the culture medium components in the photofermentation tank is achieved, thereby improving the photofermentation capacity of diatoms and thereby increasing the yield of high-value natural products.

[0059] (2) The present invention increases cell density, specific growth rate, biomass yield, fucoxanthin yield, and EPA yield by adding different concentrations (0-30 mmol / L) of lactic acid to a shake flask system. When the lactic acid concentration was 20 mmol / L, the cell density, biomass yield, specific growth rate, fucoxanthin yield, and EPA yield reached their maximum values, significantly increasing by 52.99%, 42.17%, 24.52%, 45.02%, and 53.22% compared to the control group (0 mmol / L).

[0060] (3) In the present invention, under the premise of adding 20 mmol / L lactic acid (1.8 g / L) in a shake flask system, different concentrations of glycerol (1.84-9.2 g / L) were used as the main carbon source to simultaneously improve cell density, specific growth rate, biomass yield, fucoxanthin yield, and EPA yield. When the glycerol concentration was 3.68 g / L, the cell density reached the maximum, increasing by 5.9% compared with the 7.36 g / L group; at the same time, the highest fucoxanthin yield was obtained, increasing by 13.59% compared with the 7.36 g / L group.

[0061] (4) The present invention increases cell density, specific growth rate, biomass yield, fucoxanthin yield, and EPA yield by using different nitrogen sources (tryptone + urea, urea, and ammonium sulfate) in a shake flask system under the premise of adding 1.8 g / L lactic acid and 3.68 g / L glycerol. The maximum cell density, biomass yield, fucoxanthin yield, and EPA yield were obtained when tryptone + urea was added as a mixed nitrogen source. The cell density, biomass yield, and EPA yield were significantly increased by 37.22%, 34.66%, and 22.81% compared with the control group, and the fucoxanthin yield was not significantly different from that of the control group.

[0062] (5) The present invention increases cell density, specific growth rate, biomass yield, and fucoxanthin and EPA production in a 5-L photofermenter system by batch-feeding lactic acid. Under the conditions of batch-feeding lactic acid, fucoxanthin and EPA production reached 148.92 mg / L and 345.276 mg / L, respectively, representing significant increases of 12.82% and 40.68% compared to the control group.

[0063] (6) The present invention increases cell density and biomass yield by adjusting pH by feeding lactic acid in a 5L fermenter. Under the condition of feeding lactic acid, a one-time addition of 9.2 g / L of glycerol can obtain a higher biomass yield (10.38 g / L) and the yields of fucoxanthin and EPA (82.56 mg / L and 336.6 mg / L), which are significantly increased by 26.59%, 12.98% and 36.69% compared with the batch addition of glycerol.

[0064] (7) By establishing a near-infrared online quantitative model, the present invention enables online monitoring and feed-feed feedback control of diatom photofermentation parameters (glycerol concentration, biomass yield, cell density) and intracellular content (fucoxanthin). After 16 days of culture with the addition of lactic acid, glycerol, and an organic nitrogen source, multiple parameters were simultaneously, rapidly, and nondestructively measured. At the end of the culture, the biomass yield was 9.95 g / L, and the yields of fucoxanthin and EPA were 95.97 mg / L and 237.55 mg / L, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 The graph shows the changes in biomass yield, fucoxanthin and EPA content, fucoxanthin and EPA production and yield, and glycerol concentration when Phaeodactylum tricornutum is cultured mixotrophically in the shake flask system of Example 1 with the addition of different concentrations of lactic acid (0-30 mmol / L); wherein A represents biomass yield; B represents fucoxanthin and EPA content; C represents fucoxanthin and EPA production and yield; and D represents glycerol concentration.

[0066] Figure 2This is a graph showing the changes in biomass yield, cell density, glycerol concentration, and fucoxanthin and EPA when P. tricornutum was cultured mixotrophically in the shake flask system of Example 1 with the addition of 20 mmol / L lactic acid and different glycerol concentrations (1.84-9.2 g / L); wherein A is biomass yield; B is cell density; C is glycerol concentration; and D is fucoxanthin and EPA content.

[0067] Figure 3 This graph shows the changes in biomass yield, cell density, glycerol concentration, and fucoxanthin and EPA when P. tricornutum was cultured mixotrophically in the shake flask system of Example 1 with the addition of 20 mmol / L lactic acid and different nitrogen sources (tryptone + urea, urea, and ammonium sulfate); wherein A represents biomass yield; B represents cell density; C represents glycerol concentration; and D represents fucoxanthin and EPA contents.

[0068] Figure 4 This is a graph showing changes in nutrient components in the culture medium under the condition of batch lactic acid supplementation in the 5 L fermentor system of Example 2; wherein A is the glycerol concentration; B is the urea nitrogen concentration; C is the phosphate concentration; and D is the lactic acid concentration.

[0069] Figure 5 This is a graph showing changes in cell density and biomass yield of Phaeodactylum tricornutum under mixotrophic culture conditions of batch lactic acid supplementation in the 5 L fermentor system of Example 2; wherein A represents cell density; and B represents biomass yield.

[0070] Figure 6 This is a graph showing changes in fucoxanthin and EPA content during mixotrophic cultivation of Phaeodactylum tricornutum under batch lactic acid supplementation conditions in the 5 L fermentor system of Example 2.

[0071] Figure 7 This is a graph showing changes in nutrient components in the culture medium under conditions of adding lactic acid and regulating pH in the 5 L fermentation tank system of Example 2; wherein A represents the change in glycerol concentration; B represents the change in urea nitrogen concentration; C represents the change in phosphate concentration; and D represents the change in lactic acid concentration.

[0072] Figure 8 This is a graph showing the changes in biomass yield and cell density of Phaeodactylum tricornutum under mixotrophic culture conditions of lactic acid addition and pH control in the 5 L fermentor system of Example 2; wherein A represents the change in biomass yield; and B represents the change in cell density.

[0073] Figure 9This is a graph showing changes in fucoxanthin and EPA content and yield when Phaeodactylum tricornutum is cultured mixotrophically in the 5 L fermentor system of Example 2 under conditions of flow-feeding lactic acid and pH control; wherein A represents the fucoxanthin content and yield in the fermentation system when glycerol is added in a single batch; B represents the EPA content and yield in the fermentation system when glycerol is added in a single batch; C represents the fucoxanthin content and yield in the fermentation system when glycerol is added in batches; and D represents the EPA content and yield in the fermentation system when glycerol is added in batches.

[0074] Figure 10 This is the original near-infrared spectrum of the fermentation liquid of Phaeodactylum tricornutum in Example 3.

[0075] Figure 11 These are the near-infrared model diagrams of the components in the 5 L photofermentation system of Phaeodactylum tricornutum in Example 3; wherein, A is the near-infrared model diagram of glycerol concentration; B is the near-infrared model diagram of biomass yield; C is the near-infrared model diagram of cell density; and D is the near-infrared model diagram of intracellular fucoxanthin.

[0076] Figure 12 This is a graph showing changes in urea nitrogen concentration, phosphate concentration, and cell density during 5 L photofermentation of Phaeodactylum tricornutum guided by the near-infrared model in Example 3; wherein A represents urea nitrogen concentration; B represents phosphate concentration; and C represents cell density.

[0077] Figure 13 This is a graph showing changes in lactic acid concentration, glycerol concentration, biomass yield, fucoxanthin and EPA yields during 5 L photofermentation of Phaeodactylum tricornutum guided by the near-infrared model in Example 3; wherein A is the lactic acid concentration; B is the glycerol concentration; C is the biomass yield; and D is the fucoxanthin and EPA yields.

[0078] Figure 14 This is a graph showing the changes in fucoxanthin and EPA content and yield during 5 L photofermentation of Phaeodactylum tricornutum guided by the near-infrared model in Example 3. DETAILED DESCRIPTION

[0079] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. It should be noted that if there are any processes not particularly described in detail below, they can be implemented or understood by those skilled in the art with reference to the prior art. If the manufacturer of the reagents, instruments and equipment used is not indicated, it is considered to be a conventional product that can be purchased commercially.

[0080] The detection method adopted in the embodiment of the present invention can be referred to as follows:

[0081] (1) Determination of cell density of P. tricornutum and calculation of specific growth rate

[0082] The cell density was determined by flow cytometry. 2 mL of algae solution was taken into a 2 mL centrifuge tube and centrifuged at 4500 × g for 3 min. The supernatant was discarded and the cells were resuspended in ultrapure water to a certain multiple so that the cell density was 1 × 10 5 ~1×10 6 Cell density was determined by flow cytometry. Three replicates were set for each sample group, and the mean and standard deviation were calculated.

[0083] The specific growth rate calculation formula is:

[0084]

[0085] Where N2 and N1 are the cell densities (cells / mL) measured at time points t2 and t1, respectively.

[0086] (2) Determination of biomass yield of Phaeodactylum triangularis

[0087] The biomass yield (g / L) was determined by the drying differential weight method. The specific method is as follows: take a 2mL centrifuge tube that has been dried to a constant weight and weigh it on an analytical balance, draw 2mL of algae liquid into a pre-weighed centrifuge tube, centrifuge it at 4500×g for 3 minutes, and wash it twice with ultrapure water. The obtained algae mud is placed in a constant temperature blast drying oven at 60°C for drying until constant weight, and then use an analytical balance to weigh the mass of the centrifuge tube and calculate the difference between it and the value before drying. Three parallels are set for each group of samples, and the average and standard deviation are finally calculated. The biomass yield is calculated according to the following formula:

[0088]

[0089] Where: M1 and M2 are the weights of the 2 mL centrifuge tube before and after drying (g), and 0.002 is the volume of the sample (L).

[0090] (3) Determination of the components of the culture medium of Phaeodactylum tricornutum

[0091] 1. Determination of glycerol concentration in culture medium

[0092] The glycerol concentration (g / L) of the culture medium was measured using the M-100 bioanalytical sensor. The specific method is as follows: A glycerol enzyme membrane (purchased from Shenzhen Silman Technology Co., Ltd., catalog number G1008) was installed on the corresponding electrode of the M-100. Glycerol was selected as the detection item. The instrument was first calibrated using a 1.0 g / L glycerol standard solution. After calibration, sample measurement was performed. 2 mL of algae solution was pipetted into a 2 mL centrifuge tube and centrifuged at 4500 × g for 3 minutes. The supernatant was removed and diluted to within the instrument's detection range (0.0-1.0 g / L). The supernatant was then placed in the instrument's test tray for testing. The reading was multiplied by the dilution factor to obtain the sample glycerol concentration. Three replicates were set up for each group, and the average and standard deviation were calculated.

[0093] 2. Determination of urea nitrogen concentration in culture medium

[0094] Urea nitrogen concentration was determined using the urease method. Nanjing Jiancheng Urea Nitrogen (BUN) Kit (Urease Method) was used. A 10 mmol / L urea standard was prepared according to the instructions. A certain volume of sample was centrifuged at 8000 rpm for 3 minutes. 20 μL of the supernatant was collected and the corresponding reagents were added according to the instructions for reaction. The absorbance was measured at 640 nm. Three parallels were set for each sample. Urea nitrogen concentration was calculated according to the following formula:

[0095]

[0096] Where: A 样品 、A 标品 、A 空白 Represent the absorbance values of sample, standard and blank reaction solution at 640nm, C 标品 Represents the concentration of standard (mmol / L).

[0097] 3. Determination of lactic acid concentration in culture medium

[0098] Using an AP-100 online biochemical analyzer, a lactase membrane (purchased from Shenzhen Silman Technology Co., Ltd., catalog number L1002) was installed on the corresponding electrode of the instrument. The instrument automatically calibrated, sampled, diluted, and tested the sample, and the test results were read on the instrument. Each sample was measured three times and the average value was calculated.

[0099] 4. Determination of ammonium ion concentration in culture medium

[0100] An AP-100 online biochemical analyzer equipped with an ammonium ion selective electrode was used. The instrument automatically performed calibration, sampling, dilution, and testing, and the test results were read on the instrument. Each sample was measured three times and the average value was calculated.

[0101] 5. Determination of phosphate concentration in culture medium

[0102] A certain volume of sample was centrifuged at 8000 rpm for 3 minutes. The supernatant was collected and diluted to a measurable range (0.00-30.00 mg / L) to a final volume of 10 mL. Phosphate concentration was measured using an Italian HANNA HI83200 multi-parameter water quality analyzer. The readings were recorded and repeated three times for each sample, averaging the results. The readings were converted using a prepared standard curve. The actual phosphate concentration of the sample was obtained by multiplying the conversion result by the dilution factor.

[0103] (IV) Determination of the content and / or yield of high-value products in the cells of Phaeodactylum tricornutum

[0104] 1. Determination of intracellular fucoxanthin content in Phaeodactylum tricornutum

[0105] High performance liquid chromatography (HPLC) testing was performed, and the specific steps were as follows:

[0106] (1) Accurately weigh 10 mg of freeze-dried algae powder into a 2 mL cryovial and add an appropriate amount of zirconium oxide magnetic beads with a particle size of 0.1-0.6 mm. Then add 1 mL of extraction agent (methanol:acetone = 1:1, v:v, containing 0.1% (w / v) butylated hydroxytoluene), place the tube in liquid nitrogen and quickly freeze it, then place it in a cell disruptor, set the frequency to 70 Hz, and shake it for 60 seconds. After the disruption is completed, quickly freeze the sample in liquid nitrogen, then centrifuge at 4500 × g for 3 minutes, and collect the supernatant into a 10 mL centrifuge tube. Repeat the extraction until the algae powder turns white. Combine all the supernatants, blow dry with nitrogen, and add a constant volume agent (methanol:methyl tert-butyl ether = 1:1, v:v, containing 0.1% (w / v) butylated hydroxytoluene) to make the volume 1 mL. Use a 0.22 μm organic microporous filter membrane to filter it into a 1.5 mL brown injection bottle for subsequent detection and analysis. The entire process of pigment extraction should be carried out in a lightless or low-light environment as much as possible.

[0107] (2) Fucoxanthin standard curve: Accurately weigh 10 mg of fucoxanthin standard sample and dilute to 10 mL with the above-mentioned constant to prepare a standard solution with a concentration of 1000 μg / mL. Then dilute with constant to prepare a gradient of concentrations of 60, 120, 150, 180, and 240 μg / mL. Determine by HPLC and draw a standard curve.

[0108] (3) Fucoxanthin content (mg / g) was determined by high performance liquid chromatography. The instrument was equipped with an Agilent 1260 quaternary pump, a DAD detector, and a YMC Carotenoid column C30 (4.6×150 mm, 3 μm). Specific method parameters were as follows: mobile phase flow rate of 0.8 mL / min, column temperature of 25°C, injection volume of 20 μL, and detection wavelength of 440 nm. The mobile phase consisted of methanol (A) and methyl tert-butyl ether (B), with a gradient elution program as follows: 0-6 min, A: 95% to 80%, B: 5% to 20%; 6-12 min, A: 80% to 60%, B: 20% to 40%; 12-19 min, A: 60% to 55%, B: 40% to 45%; 19-20 min, A: 55% to 95%, B: 45% to 5%; 20-23 min, A: 95%, B: 5%. The peak area was substituted into the standard curve to determine the fucoxanthin content. Three replicates were set for each group, and the average and standard deviation were calculated.

[0109] 2. Determination of fatty acid content in P. tricornutum cells

[0110] (1) Extraction of eicosapentaenoic acid: Add 500 μL of 2 mg / mL nonadecanoic acid-dichloromethane solution to a 15 mL screw-cap glass tube, blow dry with nitrogen, and then accurately weigh 20 mg of freeze-dried algae powder and add it. Then add 2 mL of 0.5 mol / L sodium hydroxide-methanol solution and place the glass tube in an 85°C water bath and heat for 30 min to perform a methylation reaction. After heating, remove the sample and cool it to room temperature. Then add 2 mL of boron trifluoride-methanol solution (15%, v / v) and heat it in a 75°C water bath for 15 min to perform a saponification reaction. After heating, remove the sample and cool it to room temperature. Add 500 μL of saturated sodium chloride solution and 2 mL of n-hexane, shake thoroughly, and centrifuge at 4500 × g for 3 min to separate the layers. Aspirate the upper n-hexane phase with a syringe and filter it through a 0.22 μm organic microporous filter membrane into a 1.5 mL brown injection bottle for subsequent analysis and testing.

[0111] (2) Eicosapentaenoic acid content (mg / g) was determined by GC-MS. The instrument configuration was an Agilent 5975 mass detector and a DB-23 high-efficiency capillary column (30 mm × 0.25 mm, 0.25 μm). The specific method parameters were: helium carrier gas 1 mL / min, injection volume 1 μL, gradient temperature program: initial column temperature 130 °C for 3 min, then temperature increased to 200 °C at a rate of 5 °C / min and maintained for 3 min. The mass spectrometry scanning range was 33 to 400 amu. The NIST05a spectral library and internal standard method (C19:0) were used for qualitative and quantitative analysis of fatty acids. Three replicates were set for each group, and the average and standard deviation were finally calculated.

[0112] The eicosapentaenoic acid (EPA) yield (mg / L / d) was calculated according to the following formula:

[0113]

[0114] Among them, C EPA1 、C EPA2 are the fucoxanthin contents (mg / g) at t1 and t2(d), respectively. B1 and B2 are the biomass yields (g / L) at t1 and t2(d), respectively.

[0115] 3. Calculation of high-value product yield

[0116] High-value product yield (mg / L / d) = (high-value product content (mg / g) × biomass yield (g / L)) / culture days (d).

[0117] Example 1 Optimization of carbon source concentration and nitrogen source type of Phaeodactylum tricornutum in shake flask system

[0118] 1.1 Algae seed activation and seed solution preparation

[0119] P. tricornutum was transferred to the modified f / 2 medium (Table 1) and cultured on the slant surface at 20°C, pH 8.0, and light intensity of 20 μmol / m 2 / s, and observe the growth of triangular phytodactyls. Then pick triangular phytodactyls from the slope and place it in the modified f / 2 medium with a volume of 100 mL, a salinity of 20‰, a pH value of 8.0, and place it under a light intensity of 20 μmol / m 2 The culture was performed in a constant temperature, light-shaking incubator (light source: full-spectrum LED light with a spectral composition of red:green:blue (R:G:B) = 18.9:76.1:5.1, provided by a natural white LED light strip) at 160 rpm. The culture was performed for 10-12 days to prepare the seed solution. The modified f / 2 medium is shown in Table 1.

[0120] Table 1 Improved f / 2 artificial seawater culture medium formula (pH 8.0)

[0121]

[0122]

[0123] Note: EDTA is ethylenediaminetetraacetic acid.

[0124] 1.2 Lactic acid partially replaces glycerol to promote the growth of Phaeodactylum tricornutum cells in a shake flask system

[0125] The seed liquid of Phaeodactylum tricornutum was centrifuged at 4500×g for 3 min, washed twice with sterile and carbon-free modified f / 2 medium, and resuspended to prepare a cell suspension. The cell suspension was transferred to 100 mL of sterile and carbon-free modified f / 2 liquid medium (250 mL Erlenmeyer flask). Lactic acid (C3H6O3, abbreviated as LA) was used to partially replace glycerol (C3H8O3, abbreviated as gly) in the culture medium, while maintaining the total carbon concentration at 300 mmol / L. The lactic acid concentration (0, 5, 10, 20, 30 mmol / L) and glycerol concentration (100, 95, 90, 80, 70 mmol / L) were set to control the initial cell density to 1.0×10 7 cells / mL (the total volume of algae and culture medium after inoculation is 100 mL, and the volume of the shake flask is 250 mL). Each gradient is repeated three times, and the shake flask is placed at a temperature of 20±1℃ and a light intensity of 20±2μmol / m 2 The cells were cultured in a constant temperature and illumination shaker at a speed of 160 r / min and 12 days, and the light source was a full-spectrum LED lamp (the spectrum composition was red: green: blue (R:G:B) = 18.9:76.1:5.1, provided by a natural white LED light strip).

[0126] 1.3 Adding lactic acid and optimizing glycerol concentration in shake flask system

[0127] The seed liquid of Phaeodactylum tricornutum was centrifuged at 4500×g for 3 min, washed twice with sterile and carbon-free modified f / 2 medium, and then resuspended to prepare a cell suspension. The cell suspension was transferred to 100 mL of sterile and carbon-free modified f / 2 liquid medium (250 mL Erlenmeyer flask). Under the optimal lactic acid addition amount, different glycerol concentrations (9.2 g / L, 7.36 g / L, 5.52 g / L, 3.68 g / L, and 1.84 g / L) were set to control the initial cell density to 1.0×10 7 cells / mL (the total volume of algae and culture medium after inoculation is 100 mL, and the volume of the shake flask is 250 mL). Each gradient is repeated three times, and the shake flask is placed at a temperature of 20±1℃ and a light intensity of 20±2μmol / m 2 The cells were cultured in a constant temperature and illumination shaker at a speed of 160 r / min and 12 days, and the light source was a full-spectrum LED lamp (the spectrum composition was red: green: blue (R:G:B) = 18.9:76.1:5.1, provided by a natural white LED light strip).

[0128] 1.4 Effects of lactic acid and different nitrogen sources on the growth of P. tricornutum in shake flasks

[0129] The seed liquid of Phaeodactylum tricornutum was centrifuged at 4500 × g for 3 min, washed twice with sterile, carbon-free modified f / 2 medium, and resuspended to prepare a cell suspension. The cell suspension was then transferred to 100 mL of sterile, nitrogen-free modified f / 2 liquid medium (250 mL Erlenmeyer flask). At the optimal lactic acid dosage, different nitrogen sources (tryptone, urea, and ammonium sulfate) were added at a nitrogen source concentration of 20 mmol / L. These groups were divided into control, treatment group 1, treatment group 2, treatment group 3, and treatment group 4 (see Table 2 for the detailed experimental plan). The initial pH of the treatment groups was controlled at 7.5 using 50 mM PIPES buffer (15.2 g piperazine-1,4-diethanesulfonic acid (PIPES) dissolved in 1000 mL water and adjusted to pH 7.5 with sodium hydroxide). The pH of the control group was adjusted to 7.5 during medium preparation (no PIPES buffer adjustment). The initial cell density was controlled at 1.0 × 10 7 cells / mL (the total volume of algae and culture medium after inoculation is 100 mL, and the volume of the shake flask is 250 mL). Each gradient is repeated three times, and the shake flask is placed at a temperature of 20±1℃ and a light intensity of 20±2μmol / m 2 The cells were cultured in a constant temperature and illumination shaker at a speed of 160 r / min and 12 days, and the light source was a full-spectrum LED lamp (the spectrum composition was red: green: blue (R:G:B) = 18.9:76.1:5.1, provided by a natural white LED light strip).

[0130] Table 2 Experimental plan

[0131] Group Experimental plan control group Carbon source: 9.2 g / L glycerol; Nitrogen source: 1.17 g / L tryptone + 0.3 g / L urea Treatment group 1 Carbon source: 9.2 g / L glycerol; Nitrogen source: 1.17 g / L tryptone + 0.3 g / L urea; PIPES buffer system Treatment group 2 Carbon source: 3.68 g / L glycerol + 1.8 g / L lactic acid; Nitrogen source: 1.17 g / L tryptone + 0.3 g / L urea; PIPES buffer system Treatment group 3 Carbon source: 3.68 g / L glycerol + 1.8 g / L lactic acid; Nitrogen source: 0.6 g / L urea; PIPES buffer system Treatment group 4 Carbon source: 3.68 g / L glycerol + 1.8 g / L lactic acid; Nitrogen source: 1.32 g / L ammonium sulfate; PIPES buffer system

[0132] 1.4 Analysis and testing

[0133] During the culture process, 2 mL samples were collected every two days to measure cell density, biomass yield, glycerol concentration, and pH. After all experiments, the remaining algal liquid was collected, washed, and the algal mud was vacuum-freeze-dried to obtain algal powder. The algal powder was stored at -20°C for subsequent analysis to determine the content of high-value products and calculate yield.

[0134] Origin 2021 and SPSS software were used to process and statistically analyze the data. One-way analysis of variance and paired data t-test were used for significance test analysis. Different letters indicate significant differences between groups (non-significant P>0.05; significant P<0.05; extremely significant P<0.01).

[0135] 1.5 Capacity Analysis Results

[0136] 1.5.1 Lactic acid partially replaces glycerol to promote the growth of Phaeodactylum tricornutum cells in a shake flask system

[0137] The biomass yield, fucoxanthin and EPA content, fucoxanthin and EPA production and yield, and glycerol concentration of P. tricornutum cultured under the conditions of adding different concentrations of lactic acid (0-30 mmol / L) were analyzed. Figure 1 The production performance is shown in Table 3. Figure 1 As shown in Table 3, within the range of 0-20 mmol / L lactic acid concentration, as the proportion of lactic acid replacing glycerol increased, the biomass yield, average specific growth rate and glycerol consumption rate of P. tricornutum all showed a significant upward trend (p<0.05), reaching their maximum values at 20 mmol / L lactic acid, which were 3.54 g / L, 0.193 and 0.219 g / L / d, respectively, which were 42.17%, 24.52% and 33.54% higher than those of the control group (100 mmol / L glycerol), respectively. Figure 1 As can be seen from B, increasing lactic acid concentration has no significant effect on fucoxanthin content, but EPA content gradually increases. At 10mmol / L lactic acid, it is significantly different from the control group. At 20mmol / L lactic acid, the fucoxanthin and EPA contents reach their maximum values, which are 12.96mg / g and 31.47mg / g, respectively. The EPA content is significantly increased by 7.74% compared with the control group (p < 0.05). Figure 1 As shown in Figure C, when 20 mmol / L lactic acid was added, the production of fucoxanthin and EPA in P. tricornutum was significantly higher than that in the control group, reaching 46.27 mg / L and 111.55 mg / L, respectively. This represents a significant increase of 45% and 53.21% compared to the control group. Therefore, adding 20 mmol / L lactic acid instead of glycerol significantly increased the cell density and biomass yield of P. tricornutum, thereby increasing the yield of fucoxanthin and EPA.

[0138] Table 3 Production performance of Phaeodactylum triangularis under the combination of lactic acid and glycerol concentrations

[0139]

[0140] 1.5.2 Adding lactic acid and optimizing glycerol concentration in shake flask system

[0141] The biomass yield, cell density, glycerol concentration, and fucoxanthin and EPA contents of P. tricornutum cultured under the conditions of adding 20 mmol / L lactic acid and different concentrations of glycerol (1.84-9.2 g / L) were as follows: Figure 2 The production performance is shown in Table 4. Figure 2As can be seen from A and Table 4, the highest biomass yield (5.04 g / L) and productivity (0.333 g / L / d) were achieved when lactic acid was 20 mmol / L + 9.2 g / L glycerol; when lactic acid was 20 mmol / L + 3.68 g / L glycerol, the biomass yield was 4.88 g / L and the biomass productivity was 0.32 g / L / d, second only to the 9.2 g / L group, and there was no significant difference between the two groups. Figure 2 As can be seen in B, the highest cell density (1.333×10 8 cells / mL). Figure 2 As shown in Figure D, the highest fucoxanthin content (14.84 mg / g) was achieved when lactate was added to 20 mmol / L and 1.84 g / L glycerol, significantly higher than the groups supplemented with 9.2, 7.36, and 5.52 g / L (p < 0.05). This may be due to glycerol depletion on day 10, when the cells transitioned to an autotrophic state, promoting fucoxanthin accumulation. EPA content also reached its highest level (32.47 mg / g), but this did not differ significantly from the other groups. As shown in Table 4, the maximum fucoxanthin yield (5.918 mg / L / d) was achieved when lactate was added to 20 mmol / L and 3.68 g / L glycerol, significantly different from the groups supplemented with 7.36, 5.52, and 1.84 g / L (p < 0.05), but not significantly different from the group supplemented with 9.2 g / L. Taking both fucoxanthin yield and cost-effectiveness into account, the optimal combination was 20 mmol / L lactate and 3.68 g / L glycerol.

[0142] Table 4 Production performance of Phaeodactylum triangularis under the combination of 20 mmol / L lactic acid and different glycerol concentrations

[0143] Production performance 9.2g / L 7.36g / L 5.52g / L 3.68g / L 1.84g / L Specific growth rate ( / d) <![CDATA[0.223±0.04 b ]]> <![CDATA[0.232±0.003 ab ]]> <![CDATA[0.235±0.004 ab ]]> <![CDATA[0.235±0.007 a ]]> <![CDATA[0.237±0.005 a ]]> Biomass yield (g / L / d) <![CDATA[0.333±0.04 a ]]> <![CDATA[0.297±0.026 ab ]]> <![CDATA[0.282±0.021 ab ]]> <![CDATA[0.320±0.019 ab ]]> <![CDATA[0.273±0.022 b ]]> Glycerol consumption rate (g / L / d) <![CDATA[0.286±0.04 a ]]> <![CDATA[0.261±0.021 ab ]]> <![CDATA[0.267±0.031 ab ]]> <![CDATA[0.239±0.017 bc ]]> <![CDATA[0.197±0.000 c ]]> Fucoxanthin yield (mg / L / d) <![CDATA[5.552±0.04 ab ]]> <![CDATA[5.210±0.303 bc ]]> <![CDATA[4.839±0.004 c ]]> <![CDATA[5.918±0.128 a ]]> <![CDATA[5.286±0.005 b ]]> EPA yield (mg / L / d) <![CDATA[12.238±0.107 a ]]> <![CDATA[10.839±0.408 a ]]> <![CDATA[10.981±0.996 a ]]> <![CDATA[11.849±0.587 a ]]> <![CDATA[11.561±0.169 a ]]>

[0144] 1.5.3 Effects of lactic acid and different nitrogen sources on the growth of Phaeodactylum tricornutum in shake flasks

[0145] The biomass yield, cell density, glycerol concentration, and fucoxanthin and EPA contents of P. tricornutum cultured under the conditions of adding 20 mmol / L lactic acid and different nitrogen sources (tryptone, urea, and ammonium sulfate) were analyzed. Figure 3 The production performance is shown in Table 5. As can be seen from Table 5, there is no significant difference in the specific growth rate, biomass yield, glycerol consumption rate, fucoxanthin yield and EPA yield between the control group and treatment group 1, indicating that 50mM PIPES buffer has no effect on the growth of P. tricornutum. Figure 3 It can be seen that the biomass yield and cell density of treatment group 2 reached the highest, which were 5.37 g / L and 11.85×10 7cells / mL. Compared with the control group, it increased by 37.22% and 34.66%. As shown in Table 5, the specific growth rate, biomass yield and glycerol consumption rate of treatment group 2 were significantly different from those of other groups, and increased by 7.77%, 54.39% and 30.77% respectively compared with the control group. Figure 3 As shown in Figure D, the control group had the highest fucoxanthin content, at 12.44 mg / g, which was significantly different from treatment groups 2, 3, and 4. The control group also had the highest EPA content, at 36.54 mg / g, which was significantly different from treatment groups 1 and 3. However, in terms of fucoxanthin and EPA yields, treatment group 2 had the highest fucoxanthin yield (4.517 mg / L / d), which was significantly different from treatment groups 3 and 4. The highest EPA yield (14.63 mg / L / d) was significantly different from the other groups and increased by 22.81% compared to the control group. Tryptone and urea were the optimal nitrogen sources as a mixed nitrogen source.

[0146] Table 5 Production performance of Phaeodactylum triangularis under the combination of 20 mmol / L lactic acid and different nitrogen sources

[0147] Production performance control group Treatment group 1 Treatment group 2 Treatment group 3 Treatment group 4 Specific growth rate ( / d) <![CDATA[0.206±0.003 b ]]> <![CDATA[0.205±0.003 b ]]> <![CDATA[0.222±0.006 a ]]> <![CDATA[0.140±0.004 d ]]> <![CDATA[0.172±0.005 c ]]> Biomass yield (g / L / d) <![CDATA[0.228±0.006 b ]]> <![CDATA[0.249±0.020 b ]]> <![CDATA[0.352±0.009 a ]]> <![CDATA[0.167±0.014 c ]]> <![CDATA[0.241±0.004 b ]]> Glycerol consumption rate (g / L / d) <![CDATA[0.208±0.014 b ]]> <![CDATA[0.217±0.007 b ]]> <![CDATA[0.272±0.008 a ]]> <![CDATA[0.203±0.016 b ]]> <![CDATA[0.219±0.012 b ]]> Fucoxanthin yield (mg / L / d) <![CDATA[4.057±0.294 a ]]> <![CDATA[4.033±0.153 a ]]> <![CDATA[4.517±0.130 a ]]> <![CDATA[1.940±0.334 c ]]> <![CDATA[2.577±0.136 b ]]> EPA yield (mg / L / d) <![CDATA[11.915±0.568 b ]]> <![CDATA[11.122±0.676 b ]]> <![CDATA[14.633±1.074 a ]]> <![CDATA[8.170±0.444 c ]]> <![CDATA[11.219±0.564 b ]]>

[0148] Example 2 Effects of different feeding methods on the growth and high-value product content and / or yield of Phaeodactylum tricornutum in a 5 L light fermenter

[0149] 2.1 Algae seed activation and seed solution preparation

[0150] P. tricornutum was subcultured according to the method described in 1.1 and cultured for 10 to 12 days for use as seed solution.

[0151] Control strategy of batch addition of lactic acid in 2.25L photofermentation

[0152] The 5L fermenter containing the mixotrophic culture medium was sterilized in a high pressure steam sterilizer at 121°C for 15 minutes. After the culture medium cooled to room temperature, the secondary seed solution cultured for 10 days was transferred to the 5L fermenter, and the initial cell density was controlled to be 1.0×10 7 cells / mL, the total volume of algae and culture medium after inoculation was 4 L (the total volume of the fermenter was 5 L), and the initial light intensity was set to 100 μmol / m 2 / s, red and blue light (R:G:B=85:0.3:14.7), rotation speed of 100 r / min, ventilation volume of 1200 mL / min, culture temperature of 20±1°C, and pH 8.0±0.1 controlled with 0.4 mol / L hydrochloric acid solution and sodium hydroxide solution throughout the culture process. 4 mL each of 300 g / L urea stock solution and 80 g / L sodium dihydrogen phosphate stock solution were added every two days. Lactic acid was added on the second day of culture (first batch, batch 1), and sodium lactate was added to the initial level (1.8 g / L) on the 14th, 16th, and 18th days of culture (second batch, batch 2). 40 mL of samples were collected every 12 hours to determine cell density, biomass yield, and glycerol concentration. The system automatically recorded pH, dissolved oxygen (DO), and temperature throughout the culture process.

[0153] 2.3 In-situ carbon supplementation and pH control strategy of lactic acid in a 5L photofermenter

[0154] Preliminary culture medium sterilization, seed inoculation, and initial culture conditions were the same as in 2.2. Two 5-L photofermenters were divided into two groups: one with an initial glycerol concentration of 9.2 g / L and 100 g / L lactic acid instead of hydrochloric acid for pH adjustment starting on the second day of culture; the other with an initial glycerol concentration of 3.68 g / L and 100 g / L lactic acid instead of hydrochloric acid for pH adjustment. After the first batch of glycerol was consumed, the lactic acid was replaced with 0.4 mol / L hydrochloric acid for pH adjustment, while glycerol was replenished to the initial level (3.68 g / L). The nitrogen source for both groups was 1.17 g / L tryptone plus 0.3 g / L urea. Every two days, 4 mL each of a 300 g / L urea stock solution and an 80 g / L sodium dihydrogen phosphate stock solution were added. Culture was terminated when glycerol was consumed. 40 mL of samples were collected every 12 hours to determine cell density, biomass yield, and glycerol concentration. The system automatically recorded pH, dissolved oxygen (DO), and temperature throughout the culture.

[0155] 2.4 The detection and analysis methods are the same as 1.4.

[0156] 2.5 Capacity analysis results:

[0157] 2.5.1 Control strategy for batch addition of lactic acid in 5 L photofermentation

[0158] The concentrations of glycerol, urea nitrogen, phosphate and lactic acid during the batch addition of lactic acid were as follows: Figure 4 As shown in Figure 2, the cell density and biomass yield of P. tricornutum are as follows: Figure 5 As shown, the fucoxanthin and EPA contents are as Figure 6 The production performance is shown in Table 6. Since the direct addition of lactic acid during the fermentation process will cause drastic pH fluctuations, sodium lactate was used to supplement the pH in batch 2. Figure 5 It can be seen that at the end of batch 2, the cell density and biomass yield of the lactate supplemented group were 24.47×107 cells / mL and 12.93g / L, which were 20.78% and 21.07% higher than those in the control group, respectively. Figure 6 As can be seen, at the end of batch 1, the fucoxanthin and EPA contents in the control group were significantly higher than those in the lactic acid-supplemented group. However, at the end of batch 2, there was no significant difference in fucoxanthin content between the two groups. However, the EPA content in the lactic acid-supplemented group was 26.71 mg / g, a significant increase of 16.18% over the control group. As shown in Table 6, at the end of batch 2, the biomass, fucoxanthin, and EPA yields in the lactic acid-supplemented group were 775 mg / L / d, 12.41 mg / L / d, and 28.77 mg / L / d, respectively, significantly increasing by 25.67%, 12.82%, and 40.68% over the control group (p < 0.05). The fucoxanthin and EPA production rates were 148.92 mg / L and 345.276 mg / L, respectively, significantly increasing by 12.82% and 40.68% over the control group (p < 0.05). Therefore, batch addition of lactic acid in a 5-L fermenter can promote cell division, increase cell density and biomass yield, and significantly enhance the production of fucoxanthin and EPA.

[0159] Table 6 Growth performance of P. tricornutum in 5 L photofermenter with batch addition of lactic acid

[0160]

[0161]

[0162] 2.5.2 In-situ Carbon Replenishment and pH Control Strategy for Lactic Acid Dosing in a 5-L Photofermenter

[0163] The concentrations of glycerol, urea nitrogen, phosphate and lactic acid in the process of pH control by adding lactic acid are as follows: Figure 7 As shown, biomass yield and cell density are Figure 8 As shown in Figure 2, the content and yield of fucoxanthin and EPA are as follows: Figure 9 The production performance is shown in Table 7. Figure 8 It can be seen that the cell density of one batch of glycerol at the end of culture was 30.09×10 7 cells / mL, the biomass was 10.38 g / L, and the cell density at the end of the culture by batch glycerol addition was 22.05×10 7cells / mL, with a biomass of 8.2 g / L. Compared with batch glycerol supplementation, the single-batch glycerol group contained more organic carbon, which was more conducive to the accumulation of biomass of P. tricornutum. In terms of production performance, the specific growth rate, biomass yield, and glycerol consumption rate of the single-batch glycerol group were significantly increased by 10.55%, 21.59%, and 19.41% compared with the batch glycerol group, respectively. The fucoxanthin yield and EPA yield of the single-batch glycerol group were 6.88 mg / L / d and 28.05 mg / L / d, respectively, which were significantly increased by 12.98% and 36.69% compared with the batch glycerol group (6.09 mg / L / d and 20.52 mg / L / d), respectively (p < 0.05). The fucoxanthin and EPA yields of the single-batch glycerol group were 82.56 mg / L and 336.6 mg / L, which were increased by 12.98% and 36.69% compared with the batch glycerol group (p < 0.05). More organic carbon is more conducive to cell growth and biomass accumulation, thereby increasing the production and yield of fucoxanthin and EPA.

[0164] Table 7 Production performance of Phaeodactylum triangularis under in-situ carbon supplementation with lactic acid and pH adjustment

[0165] Production performance A batch of glycerin Batch glycerol supplementation Specific growth rate ( / d) <![CDATA[0.22±0.00 a ]]> <![CDATA[0.20±0.00 b ]]> Biomass yield (mg / L / d) <![CDATA[0.70±0.00 a ]]> <![CDATA[0.58±0.00 b ]]> Glycerol consumption rate (mg / L / d) <![CDATA[0.76±0.00 a ]]> <![CDATA[0.64±0.01 b <!-- 14 -->]]> Fucoxanthin yield (mg / L / d) <![CDATA[6.88±0.07 a ]]> <![CDATA[6.09±0.20 b ]]> EPA yield (mg / L / d) <![CDATA[28.05±0.23 a ]]> <![CDATA[20.52±1.26 b ]]>

[0166] Example 3 Establishment and application of near-infrared model of 5L photofermentation system Phaeodactylum triangularis

[0167] 3.1 Algae seed activation and seed solution preparation

[0168] P. tricornutum was subcultured according to the method described in 1.1 and cultured for 10 to 12 days for use as seed solution.

[0169] 3.2 Sample near-infrared spectrum collection

[0170] During the fermentation processes of 2.2 and 2.3, a total of 132 fermentation broth samples were collected at different time periods (including all periods and states of microalgae growth). The cell density and biomass yield of the collected samples, as well as the glycerol concentration in the supernatant, were tested. The samples were freeze-dried into algae powder and then tested for fucoxanthin content. The DA-7G near-infrared spectrometer of Ruipu Analytical Instruments (Tianjin) Co., Ltd. was used for detection. Near-infrared spectral data within the spectral range of 900nm to 1700nm were collected. The built-in background of the instrument was used as a reference. The detector was an electrically cooled indium gallium arsenide diode 512 array with a sampling interval of 1nm. The full wavelength was scanned by continuous grating, with the number of scans exceeding 100 times. The spot diameter of the near-infrared spectrum was 100mm, and the near-infrared spectra at different time points were obtained. The time points of near-infrared spectrum collection must correspond one to one with the time points of sample collection.

[0171] 3.3 Using spectral data to build near-infrared models

[0172] The software The Unscrambler X was used to preprocess the near-infrared spectra and screen the characteristic bands. The near-infrared spectra collected by online detection were used as the independent variable, and the reference value of the near-infrared instrument analysis of the number of samples measured was used as the dependent variable. The 950-1350nm near-infrared segment was selected to correlate the chemical values of glycerol concentration, biomass yield, cell density, and fucoxanthin content in the fermentation broth. The partial least squares method was used to establish a quantitative prediction model. The cross-validation root mean square error (RMSECV) and cross-validation determination coefficient (R) of the models with different spectral preprocessing methods were compared. 2 ) and the root mean square error of prediction (RMSEP) showed that the prediction model obtained after smoothing derivative and SNV processing was the optimal one.

[0173] 3.4 Using near-infrared model to guide 5 L photofermentation of Phaeodactylum tricornutum

[0174] The initial culture medium sterilization, seed liquid inoculation and initial culture conditions are the same as 2.2. The initial culture conditions are 3.68g / L glycerol + 1.8g / L lactic acid as the carbon source, 1.17g / L tryptone + 0.3g / L urea as the nitrogen source, and the phosphate concentration is 80mg / L. Other culture conditions are the same as 3.1.2.2. Every two days, 4mL of 300g / L urea mother solution and 80g / L sodium dihydrogen phosphate mother solution are added to the initial levels of 0.3g / L and 80mg / L. Every two days, 20mL of 100g / L sodium lactate mother solution is added to the initial level of 1.8g / L. Stop adding glycerol, sodium lactate, urea and phosphate from the 14th day. Associate the glycerol near-infrared model with the central control system of the 5L fermenter, and set the target glycerol concentration to 3.68g / L. When the model detects that the glycerol concentration in the system is less than 3.68 g / L, a peristaltic pump is used to add high-concentration glycerol mother liquor (100 g / L) into the fermentation tank. When the glycerol concentration is higher than 3.68 g / L, glycerol addition is stopped. Glycerol addition is stopped from the 14th day, and fermentation is terminated when the glycerol is completely consumed. In addition, the near-infrared model of cell number and biomass yield is only used as a detection method, and samples are taken for offline testing to verify the accuracy of the prediction results.

[0175] 3.5 Detection and analysis methods are the same as 1.4

[0176] 3.6 Capacity Analysis Results

[0177] 3.6.1 Sample near-infrared spectrum collection

[0178] A total of 132 fermentation broth samples at different time points were collected for model establishment. The original spectra are shown in Figure 2. Figure 10 The glycerol content ranged from 0.1 to 9.5 g / L, the biomass dry weight ranged from 0.7 to 12.93 g / L, and the cell density ranged from 0.39×10 7~29.9×10 7 cells / mL, and the range of fucoxanthin was 5.48-15.53 mg / g. The above parameter concentration ranges cover the parameters of different fermentation periods and can be used to establish the model (Table 8).

[0179] 3.6.2 Using spectral data to build near-infrared models

[0180] The near-infrared model of glycerol concentration, biomass yield, cell density and fucoxanthin is shown in Figure 2. Figure 11 As shown in Figure 2, two important parameters for evaluating the effect of near-infrared models are RMSE (prediction standard deviation) and cross-validation R 2 (Determination coefficient). The smaller the RMSE, the higher the detection accuracy of the near infrared model and the smaller the detection error. 2 The larger the value, the stronger the ability of the near-infrared model to distinguish the high and low values of Y (Table 8). The results show that after removing 16 abnormal spectra, 116 spectral data were used to establish the model. The spectral models of glycerol concentration, biomass yield, and cell density were better and can be used for subsequent online detection. The number of spectra that can be used for fucoxanthin modeling is 85. After removing the abnormal spectra, 65 spectra were used to establish the model. 2 Reached 0.82, when R 2 When it is greater than 0.8, it indicates that the model is relatively reliable and can be used for industrial applications. However, to achieve accurate predictions, it is necessary to continue collecting spectra and optimizing the model.

[0181] Table 8 Correlation between the number of fermentation samples and the measured values of online near-infrared spectrometer

[0182] Element Number of samples Calibration range Standard Deviation <![CDATA[R 2 ]]> Glycerol concentration 116 0.1~9.5g / L 0.5133904 0.9740321 Biomass yield 116 0.7~12.93g / L 0.6534065 0.9523894 Cell density 116 <![CDATA[0.39×10 7 ~29.9×10 7 cells / mL]]> 1.8127029 0.9461187 Fucoxanthin content 65 5.48~15.53mg / g 1.0393838 0.822892

[0183] 3.6.3 Using Near-Infrared Modeling to Guide 5-L Photofermentation of Phaeodactylum tricornutum

[0184] Urea nitrogen concentration, phosphate concentration and cell density during fermentation Figure 12 As shown in Figure 2, the lactic acid concentration, glycerol concentration, biomass yield, fucoxanthin and EPA yields were Figure 13 As shown in Figure 2, the content and yield of fucoxanthin and EPA are as follows: Figure 14 The growth performance of P. tricornutum is shown in Table 9. The cell density and biomass yield were low and the spectral signal was weak in the first two days of fermentation. From the second day on, the fermentation process was monitored and fed using a near infrared model. Figure 13As can be seen from the figure, linking the glycerol near-infrared model with the fermenter's central control system can stably control the glycerol concentration in the fermentation broth at 3.68 ± 0.5 g / L. Glycerol addition was stopped on day 14, and after two days, the glycerol was depleted. After a one-day acclimatization period, the triangular algae began to divide rapidly, and the cell density increased, reaching a maximum on day 10, and then entered a stable period. At the end of the culture on day 16, the cell density was 20.68 × 10 7 cells / mL, biomass yield was 9.95 g / L, fucoxanthin content, yield, and productivity were 9.65 mg / g, 95.97 mg / L, and 6.00 mg / L / d, respectively. EPA content, yield, and productivity were 23.87 mg / g, 237.55 mg / L, and 15.85 mg / L / d, respectively. Furthermore, the root mean square error of prediction (RMSEP) measures the model's prediction accuracy for unknown data using external data. When the RMSEP / RMSE value ranges from 0.8 to 1.2, the model's overfitting and underfitting capabilities are both within acceptable limits, indicating that the model has good predictive performance (Table 9).

[0185] Table 9 Near infrared model verification results

[0186]

[0187]

[0188] 3.7 To evaluate the production levels of fucoxanthin and EPA in the present invention, the yields and yields of fucoxanthin and EPA obtained in the experiment were compared with data from existing literature. The specific data comparison is shown in Table 10 (Note: Table 10 uses yield (in mg / L) and yield (in mg / L / d), that is, the product expression levels per unit volume or unit time, to eliminate the influence of differences in culture cycles on the results).

[0189] In terms of fucoxanthin production and yield, using a near-infrared model to guide the photofermentation of Phaeodactylum tricornutum in a 5-L process, the fucoxanthin production and yield achieved were 95.97 mg / L and 6.00 mg / L / d, respectively, comparable to the highest levels reported in these literatures. Regarding EPA production, the EPA production and yield achieved by the present invention also rank among the highest in these literatures. The present method represents a breakthrough in the production of fucoxanthin and EPA.

[0190] Table 10 Comparison of the yield and yield of fucoxanthin and EPA produced by the present invention and existing literature using Phaeodactylum triangularis

[0191]

[0192] Note: The reference information in Table 10 is as follows:

[0193] [1]BUTLER T O,PADMAPERUMA G,LIZZUL AM,et al.Towards a Phaeodactylumtricornutum biorefinery in an outdoor UK environment[J].BioresourceTechnology,2022,344:126320.

[0194] [2]KIM J,LEE J,VOO AY H,et al.Okara waste as a substrate for themicroalgae Phaeodactylum tricornutum enhances the production of algalbiomass,fucoxanthin,and polyunsaturated fatty acids[J].Fermentation,2022,9(1):31.

[0195] [3]YUAN X,LIANG L,LIU K,et al.Spent yeast as an efficient mediumsupplement for fucoxanthin and eicosapentaenoic acid(EPA)production byPhaeodactylum tricornutum[J].Journal of Applied Phycology,2020,32:59-69.

[0196] [4]WANG H,ZHANG Y,CHEN L,et al.Combined production of fucoxanthin andEPAfrom two diatom strains Phaeodactylum tricornutum and Cylindrothecafusiformis cultures[J].Bioprocess and Biosystems Engineering,2018,41:1061-1071.

[0197] [5]GAO B, CHEN A, ZHANG W, et al. Co-production of lipids, eicosapentaenoic acid, fucoxanthin, and chrysolaminarin by Phaeodactylumtricornutum cultured in a flat-plate photobioreactor under varying nitrogenconditions[J]. Journal of Ocean University of China, 2017, 16: 916-924.

[0198] [6] Yang Zexiong, Yang Runqing, Wei Dong. Effect of wood calcium addition on the yield of high-value products of Phaeodactylum tricornutum[J]. Journal of Food and Biotechnology, 2024, 43(06): 128-134.

[0199] [7]YANG R,WEID.Improving fucoxanthin production in mixotrophicculture of marine diatom Phaeodactylum tricornutum by LED light shift andnitrogen supplementation[J].Frontiers in Bioengineering and Biotechnology,2020,8:820.

[0200] [8]YONGMANITCHAI W,WARD O P.Growth of and omega-3fatty acidproduction by Phaeodactylum tricornutum under different culture conditions[J].Applied and Environmental Microbiology,1991,57(2):419-425.

[0201] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for adding organic acid coupled with spectroscopy to measure and control diatom photofermentation to produce high yields of fucoxanthin and EPA, characterized in that: The steps include: S1. Seed liquid culture: inoculating diatoms into seed liquid culture medium, culturing under light until the logarithmic growth phase, and obtaining diatom mixotrophic seed liquid; S2. Photofermentation culture: The diatom mixotrophic seed liquid is inoculated into a photofermentation tank filled with a fermentation medium containing lactic acid, glycerol, and a nitrogen source for photofermentation culture, and an online near-infrared spectrometer is used for real-time monitoring and feedback control of intermittent feeding to increase the biomass yield of the diatoms and the yield of high-value natural products; wherein the high-value natural product is at least one of fucoxanthin and eicosapentaenoic acid.

2. The method according to claim 1, wherein: The diatom described in step S1 is P. tricornutum; The concentration of lactic acid in the fermentation medium in step S2 is 0 to 30 mmol / L, excluding 0; The concentration of glycerol in the fermentation medium in step S2 is 20 to 100 mmol / L; The nitrogen source in the fermentation medium in step S2 is at least one of trypsin, urea and ammonium sulfate.

3. The method according to claim 2, wherein: The concentration of lactic acid in the fermentation medium in step S2 is 10 to 20 mmol / L; The concentration of glycerol in the fermentation medium in step S2 is 20 to 60 mmol / L; The nitrogen source in the fermentation medium in step S2 is an organic nitrogen source obtained by mixing trypsin and urea, wherein the nitrogen content of trypsin and the nitrogen content of urea are 1-2:1-2.

4. The method according to claim 3, wherein: The concentration of lactic acid in the fermentation medium in step S2 is 20 mmol / L; The concentration of glycerol in the fermentation medium in step S2 is 40 mmol / L; The nitrogen sources in the fermentation medium in step S2 are tryptone with a nitrogen content of 0.01 mol / L and urea with a nitrogen content of 0.01 mol / L.

5. The method according to claim 1, wherein: The intermittent feeding method described in step S2 is to add lactic acid in batches to adjust the pH to 7.0-9.0; at the same time, the glycerol level is controlled at 3.6±0.5 g / L. When the glycerol concentration in the fermentation system is less than 3.68 g / L, glycerol is added. When the glycerol concentration is higher than 3.68 g / L, glycerol addition is stopped. Glycerol addition is stopped from the 14th day, and fermentation is terminated when the glycerol is completely consumed. The intermittent feeding in step S2 further includes adding sodium lactate, urea and phosphate according to nutrient consumption; The sodium lactate supplementation is a sodium lactate mother solution with a concentration of 50 to 150 g / L, and the sodium lactate mother solution is supplemented every two days to a final concentration of 1.8 g / L. The addition of sodium lactate is stopped from the 14th day; The added urea is a urea mother solution with a concentration of 200-400 g / L, and the urea mother solution is added every two days to a final concentration of 0.3 g / L. The addition of urea is stopped from the 14th day; The added phosphate is sodium dihydrogen phosphate mother solution with a concentration of 80-100 g / L. The sodium dihydrogen phosphate mother solution is added every two days to a final concentration of 80 mg / L, and the addition of sodium dihydrogen phosphate mother solution is stopped from the 14th day.

6. The method according to claim 1, wherein: The online near-infrared spectrometer real-time monitoring and feedback control of intermittent feeding described in step S2 are achieved by the following steps: (1) Establishment of near-infrared spectroscopy model: diatom seed liquid is inoculated into a fermentation tank containing fermentation medium containing lactic acid, glycerol and nitrogen source for photofermentation, and near-infrared spectra at more than 60 different time points are continuously collected using an online near-infrared spectrometer. Then, the collected near-infrared spectral data are preprocessed by removing abnormal spectra using algorithms and chemometric methods to obtain preprocessed near-infrared spectral data. At the same time, samples are taken at the collection time point of each spectrum, and process parameters and cell content are measured offline to obtain physical and chemical test data. Finally, linear fitting is performed based on the preprocessed near-infrared spectral data and physical and chemical test data to establish a prediction model. The process parameters include at least one of glycerol concentration, biomass yield and cell density. The cell content is fucoxanthin. (2) Using a near-infrared model to feedback control the fermentation process in real time: using an online near-infrared analysis device to continuously collect near-infrared spectra in the fermentation broth online, and then using algorithms and chemometric methods to remove abnormal spectra from the collected near-infrared spectral data and perform data preprocessing to obtain preprocessed near-infrared spectral data, and then inputting the preprocessed near-infrared spectral data into the prediction model established in step (1) to calculate the process parameters and cell content in the fermentation system, and controlling the peristaltic pump for intermittent feeding according to the process parameters; wherein the process parameters include at least one of glycerol concentration, biomass yield and cell density; and the cell content is fucoxanthin.

7. The method according to claim 1, wherein: The inoculation density of the diatom mixotrophic seed solution in step S2 is based on the final density of the diatom mixotrophic seed solution in the fermentation system of 1×10 6 ~2×10 7 cells / mL added for calculation; The fermentation medium in step S2 is a modified f / 2 medium; The improved f / 2 medium contains the following components: 20 g / L sea salt, 30 mg / L Na2SiO3·5H2O, 4.36 mg / L Na2EDTA·2H2O, 9.8 μg / L CuSO4·5H2O, 6.3 μg / L Na2MoO4·2H2O, 22.0 μg / L ZnSO4·7H2O, 3.15 mg / L FeCl3·6H2O, 10.0 μg / L CoCl2·6H2O, 180.0 μg / L MnCl2·4H2O, 10.0 mg / L NaH2PO4, and a pH of 7.95-8.05; The light source used for the fermentation in step S2 is LED red and blue mixed light, wherein the ratio of red light to blue light is 6:1; The fermentation conditions in step S2 are: culture temperature of 20±1°C, light intensity of 100-300 μmol / m 2 / s, ventilation volume is 1200-3000 mL / min, rotation speed is 100-300 rpm, pH is 7.0-9.0, and culture time is 16-24 days.

8. The method according to claim 1, wherein: The seed liquid culture medium in step S1 is a modified f / 2 culture medium; The improved f / 2 medium contains the following components: 20 g / L sea salt, 30 mg / L Na2SiO3·5H2O, 4.36 mg / L Na2EDTA·2H2O, 9.8 μg / L CuSO4·5H2O, 6.3 μg / L Na2MoO4·2H2O, 22.0 μg / L ZnSO4·7H2O, 3.15 mg / L FeCl3·6H2O, 10.0 μg / L CoCl2·6H2O, 180.0 μg / L MnCl2·4H2O, 10.0 mg / L NaH2PO4, and a pH of 7.95-8.05; The intensity of the light culture in step S1 is 10 to 30 μmol / m 2 / s; The light culture time in step S1 is 4 to 12 days; The temperature of the light culture described in step S1 is 20±1°C; The culture in step S1 is carried out on a light-controlled constant temperature shaker at a rotation speed of 100 to 160 rpm; The light source used for the culture in step S1 is LED natural white light.

9. Application of the method of adding organic acid coupled with spectroscopy to measure and control diatom photofermentation for high yield of fucoxanthin and EPA according to any one of claims 1 to 8 in diatom culture, characterized in that: The diatom is P.tricornutum.

10. Use of the method of adding organic acid coupled with spectroscopy to measure and control diatom photofermentation to produce high-yield fucoxanthin and EPA according to any one of claims 1 to 8 in the production of high-value natural products, characterized in that: The high-value natural product is at least one of fucoxanthin and eicosapentaenoic acid.

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