Method for producing high-quality protein raw material microalgae

Abstract

The invention discloses a method for producing high-quality protein raw material microalgae. By combining a reduction product formic acid driven by carbon dioxide clean energy with edible microalgae culture, microalgae growth and protein synthesis are enhanced. The method specifically comprises the following steps: carrying out adaptive evolution of microalgae by using 20-50mM of low-concentration formate, culturing under an illumination condition by using 50-300mM of formate as an auxiliary carbon source, and finally harvesting the microalgae biomass of which the protein content exceeds 30% and the amino acid composition meets the high-quality protein requirements of international grain and agricultural tissues, high-quality protein raw materials of food and feed can be used. The method has a remarkable application prospect in the field of green non-grain high-quality protein raw material production.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and green biomanufacturing, and involves combining liquid sunlight technology with traditional microalgae cultivation and production. It uses the energetic carbon dioxide reduction product obtained by liquid oxidation technology as a carrier to break through the bottleneck of low energy and carbon source transfer efficiency in traditional photosynthetic culture of microalgae, and successfully obtain high-quality protein raw materials. Background Art

[0002] As a green carbon source alternative, small-molecule organic compounds such as formic acid and acetic acid have attracted people's interest as alternative carbon sources and are an important goal for achieving clean and green production. This type of small-molecule organic molecules can be obtained through solar-driven CO2 reduction and is a class of energy-containing compounds known as solar fuels. With the successful industrial demonstration of solar-driven CO2 reduction to methanol by Li Can's team, solar fuels have become a research hotspot that takes into account both carbon dioxide emission reduction and the further high-value conversion of low-carbon molecules. The bioconversion of solar fuels has received additional attention due to the complexity and edibility of its products.

[0003] Microalgae theoretically have high photosynthetic efficiency and high protein content, so they have attracted attention as a source of high-level protein. However, due to the low efficiency of photoautotrophic culture in traditional culture models, which is limited by carbon transfer efficiency and light utilization efficiency, the industry has developed slowly. Over the past few decades, efficiency has increased slowly, and the annual output is only tens of thousands of tons. Compared with the inefficient transfer of gaseous CO2, liquid carbon-containing compounds including bicarbonate, formic acid, acetic acid, etc. have a high solubility in water, and the carbon flux is much higher than CO2 transfer. Imitating C4 plants to separate the fixation of carbon dioxide from the light capture and oxygen release process, and using organic acids as carriers to enhance carbon transport,

[0004] Various microalgae have been approved as common protein food ingredients in countries including the United States, EU countries, Singapore, and China. For example, Chlamydomonas reinhardtii, a green alga, has achieved commercialization by converting acetic acid through fermentation technology. However, the high energy consumption and relatively stringent environmental requirements of traditional fermentation have limited its larger-scale application. It is possible to use microalgae to convert and utilize energetic liquid small molecule organic substances such as formic acid. People have achieved acetic acid fermentation, co-cultivation of formic acid and acetic acid, and growth of formic acid as a single carbon source. Although formic acid utilization in microalgae is feasible, there is a problem that limits its large-scale application, namely the low concentration of formic acid in the culture, poor economic feasibility, and the need for significant technological advancement.

[0005] One of the main reasons for the low formate addition concentration is that formate can bind to the key proteins for quinone conversion in the photoreaction center and photosynthetic electron transport chain of the photosynthetic system, forming competitive inhibition with the original bicarbonate. When the formate concentration is low, this competitive inhibition can be alleviated by changing the bicarbonate concentration, but when the formate concentration is too high, photosynthetic electron transport will be significantly and irreversibly inhibited. According to literature reports, the inhibition of natural photosynthetic systems to no more than 25mM formate can be relieved by adding 50mM bicarbonate, but the inhibition caused by higher concentrations of formate cannot be reversibly relieved. By gradually increasing the formate concentration in the culture environment, people have achieved cultivation in Chlamydomonas reinhardtii with a certain amount of formate as an enhanced carbon source. At present, the formate concentration in the formate and acetic acid co-culture can reach 20mM, and the formate carbon source alone can achieve a maximum cultivation under 50-60mM formic acid conditions. However, the low formate addition concentration leads to a low overall spatiotemporal conversion rate of formate (≤0.4mMh -1 ).

[0006] By analyzing the mechanism and characteristics of formic acid's inhibition of photosynthetic activity, it was found that the above-mentioned competitive inhibition should have a certain dose-effect relationship between microalgae biomass and formic acid concentration. That is, for a unit biomass of microalgae, since its own photosynthetic activity requires maintaining a certain bicarbonate concentration, a certain amount of formate is required to produce effective inhibition. Conversely, if the ratio of microalgae biomass to formic acid concentration is controlled below a certain level, it should be possible to achieve effective growth of microalgae under conditions of higher formic acid concentrations, thereby improving the spatiotemporal conversion rate of formic acid.

[0007] Therefore, in view of the shortcomings of existing formate culture, the present invention explores the stable culture technology of formate with a concentration of 100mM or above on the basis of rational analysis and the early low-concentration formate culture, and proposes an optimized culture strategy, which can achieve a spatiotemporal conversion rate of formate of 0.5mM / h. -1 The results show that the biomass yield reaches 0.6g biomass / g formate. Combined with the analysis of the amino acid content of the obtained algal powder, the present invention successfully uses formate as a carbon-enhanced raw material for large-scale photosynthetic cultivation aimed at producing high-quality protein food raw materials. Summary of the Invention

[0008] This method aims to provide a strategy for enhancing the growth of microalgae photosynthetic cultures by using formate as a carbon source. By combining adaptive evolution with culture optimization, high spatiotemporal formate yield and biomass conversion rates were achieved in photosynthetic cultures. Using Chlamydomonas reinhardtii as an example, the amino acid content of the biomass obtained met the requirements of the Food and Agriculture Organization (FAO) and the World Health Organization (WHO) for high-quality protein food ingredients.

[0009] To achieve the above objectives, the present invention provides a method for producing high-quality protein raw material microalgae, comprising the following steps:

[0010] 1. Under photosynthetic culture conditions, gradually add formate, and while maintaining the pH of the culture medium unchanged, increase the formate concentration to above 50-60 mM. Use the chlorophyll fluorescence kinetic parameter, the maximum quantum conversion efficiency of photosystem II (Fv / Fm), as an indicator to monitor the culture. The obtained algae species will serve as the basis for subsequent scale-up culture.

[0011] 2. Transfer the above algae into a culture medium containing 100-300 mM formate and grow at an initial OD 750 Inoculate between 4.0 and 8.0, or contain 2 to 4 g / L by weight -1 The dry weight of microalgae was inoculated.

[0012] 3. Use air as the gas source, or add 0-5% CO2 to the air for methyl-enriched enhanced photosynthetic culture.

[0013] 4. Monitoring the chlorophyll fluorescence kinetic parameter Fv / Fm of microalgae during the cultivation process is used as a process control for methyl-enriched enhanced photosynthetic cultivation.

[0014] The present invention combines adaptive evolution with microalgae cultivation guided by chlorophyll fluorescence kinetic parameters, successfully achieving photosynthetic cultivation of microalgae under conditions of high formate concentration. Taking Chlamydomonas reinhardtii as an example, its amino acid content was determined to meet the FAO / WHO requirements for high-quality protein food ingredients.

[0015] The present invention includes the following contents:

[0016] Based on the photosynthetic characteristics of microalgae cultivation, the adaptive evolution process was monitored using photosynthetic activity as a guide, resulting in microalgae with a certain degree of formate tolerance, which was then used as a seed solution. Starting from the aforementioned microalgae species, the ratio of formate to inoculum in the culture medium was controlled to achieve photosynthetic growth of the microalgae under high formate concentrations, and chlorophyll fluorescence kinetic parameters were used as monitoring indicators for cultivation.

[0017] The technical solutions of the present invention are as follows:

[0018] (1) Obtaining microalgae seed solution suitable for high formate concentration cultivation

[0019] Under optimal light and temperature conditions for the microalgae, gradually increase the formate content in the culture environment during subculture to above 50-60 mM. To determine suitability for further subculture, select cells where the difference in the highest chlorophyll fluorescence kinetic parameter after increasing formate concentration is no more than 10% compared to the value obtained under formate-free culture.

[0020] When the microalgae can finally grow under the condition of 50-60 mM or more formate, the formate concentration is maintained unchanged and the culture is expanded.

[0021] (2) High formate concentration culture

[0022] Take the microalgae seed liquid in the exponential growth phase and calculate the initial OD 750 Inoculate between 4.0 and 8.0, or according to the weight containing 2 to 4 g L -1 The microalgae were inoculated with a dry weight of 100-300 mM formate in fresh culture medium. After inoculation, the microalgae were cultured under suitable light conditions. Chlorophyll fluorescence kinetics were used as an indicator to determine the end point of the culture.

[0023] (3) Evaluation of nutritional quality of biomass protein

[0024] The amino acid relative content evaluation algorithm AAS recommended by FAO / WHO was used for scoring.

[0025] The beneficial effects of the present invention are:

[0026] Compared with the prior art, the present invention has the following advantages and effects:

[0027] 1. The present invention successfully achieved high formate concentration photosynthetic culture far exceeding the previously reported level, and its spatiotemporal conversion rate of formate was greater than 0.5 mMh -1 , and the conversion rate of formate to biomass reached more than 0.6g microalgae dry weight / g formate.

[0028] 2. The microalgae biomass obtained by the present invention has a total protein content of more than 30% dry weight, and its amino acid composition meets the FAO / WHO standards for high-quality edible protein raw materials.

[0029] 3. Overall, by introducing formate as a medium for carbon and energy transfer, while ensuring the quality of microalgae, a green and efficient CO2-driven microalgae production technology route driven by clean energy is provided. Compared with heterotrophic culture, it is easier to achieve large-scale sustainable production. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a diagram for verifying the growth changes of formate adaptive culture;

[0031] Figure 2 The inoculation diagram is different initial OD values ​​from 100 to 300 mM;

[0032] Figure 3 The inoculum diagram is different for initial dry weights from 100 to 300 mM;

[0033] Figure 4The graph shows the protein content in the culture medium with 100-300 mM formic acid. DETAILED DESCRIPTION

[0034] Because during the culture process, formic acid or formate is in a dissociated state, that is, it exists in the form of formate and cations. For microalgae culture, the formate is utilized. Therefore, in the description of the embodiments, for ease of description, the formic acid culture mentioned actually refers to the culture with a certain amount of formate ions in the solution, rather than formic acid molecules.

[0035] Example 1

[0036] Culture medium preparation

[0037] (1) The formula of TAP (Tris-Acetate-Phosphate) medium is as follows:

[0038] To prepare 1L of TAP medium, add the substances listed in Table 1 below, where Tris is 2-amino-2-hydroxymethyl-1,3-propanediol (Tris(hydroxymethyl)aminoethane).

[0039] Table 1 TAP Medium formula

[0040]

[0041] Mother solution 1: add double distilled water to 500mL

[0042] NH4Cl 20.0g

[0043] MgSO4·7H2O 5.0g

[0044] CaCl2·2H2O 2.5g

[0045] Mother solution 2: add double distilled water to 100mL of phosphate solution

[0046] K2HPO4 10.8g

[0047] KH2PO4 5.6g

[0048] Mother solution 3: Hutner's trace metal salt solution

[0049]

[0050] Preparation process of mother solution 3

[0051] a) Prepare the salt solutions listed in the table above separately. Dissolve EDTA disodium salt in boiling water, and dissolve ferrous sulfate last to prevent oxidation.

[0052] b) Combine all solutions except EDTA disodium salt, bring to a boil, and then add EDTA disodium salt (dissolve in boiling water bath).

[0053] c) The mixture turns green and, after all components have dissolved, is cooled to 70°C while maintaining the water temperature at 70°C. 85 ml of a 70°C, 20% KOH solution (20 g / 100 ml) is added. (An iron-brown precipitate forms with a pH around 13. Adjust the pH to 6.8 with concentrated HCl.)

[0054] d) After cooling to room temperature, dilute the solution to 1 L in a volumetric flask. The solution should initially be clear green, but will turn dark red or purple after 3-7 days, with the formation of an iron-brown precipitate. If no precipitate forms, or the solution remains green, check the pH, which should be around 6.7. Plug the flask with a cotton ball to allow for gas exchange. Shake the flask daily and wait 1-2 weeks before use.

[0055] e) Filter through two layers of Whatman #1 filter paper, and filter again if necessary, until the solution is clear.

[0056] f) The prepared solution will eventually be wine red in color. Even a slight change in pH will change the color of the solution.

[0057] g) Aliquot and store in the freezer.

[0058] (2) TP / TFP (Tris-Formate-Phosphate) (liquid) culture medium

[0059] Except for the organic carbon sources of formic acid and acetic acid, respectively, the remaining components of TFP and TAP (liquid) media are identical. 1 mL / L of glacial acetic acid in TAP medium is replaced with formic acid or sodium formate at final concentrations of 5 mM, 10 mM, 20 mM, 35 mM, 40 mM, 50 mM, 60 mM, 80 mM, 100 mM, 150 mM, 200 mM, and 300 mM, respectively. The pH can be adjusted to approximately 7.5 with approximately 10% hydrochloric acid or 1 M NaOH (TAP medium does not require pH adjustment because its pH is approximately 7.5). Liquid media such as TP, TFP5, TFP10, TFP20, TFP35, TFP40, TFP50, TFP60, TFP80, TFP100, TFP150, TFP200, and TFP300 are obtained, respectively. TP refers to the culture after removing acetic acid and without adding formate, and the number after TFP is the corresponding formate concentration.

[0060] The prepared culture medium needs to be sterilized at 121°C for 20 minutes as soon as possible and cooled to room temperature before use.

[0061] (3) Preparation of solid culture medium plates

[0062] Agar powder was added to the liquid culture medium (comprising TAP and TFP, respectively) at a mass volume ratio of 1%-1.5% (here 1.5%), sterilized at 121°C for 20 minutes, cooled to 60°C, and introduced into a 10 cm inner diameter culture dish to prepare the corresponding solid plate (culture medium thickness of about 5 mm). TAP solid medium, TFP10 (10 mM formate) solid medium, TFP20 (20 mM formate) solid medium, TFP35 (35 mM formate) solid medium, TFP40 (40 mM formate) solid medium, TFP50 (50 mM formate) solid medium, TFP60 (60 mM formate) solid medium, TFP80 (80 mM formate) solid medium, TFP100 (100 mM formate) solid medium, and TFP150 (150 mM formate) solid medium were obtained respectively.

[0063] Example 2

[0064] Adaptive evolution culture of microalgae formate roots based on solid culture medium plates

[0065] Take 10 μL of Chlamydomonas reinhardtii algae solution grown in TAP liquid medium to the middle of the exponential growth phase (about OD 750 =0.8), and evenly spread the solid plate prepared in Example 1 using a coating rod (or glass beads) for solid plate culture. Then, the clones were streaked and cultured using an inoculation loop.

[0066] The specific operations are:

[0067] a. The TAP-grown microalgae were inoculated into 10 mM formate solid medium and cultured for 7 days to form distinct algal colonies of 1 mm in diameter or greater. The culture temperature was 25-28°C.

[0068] b. The microalgae obtained in step a were inoculated with an inoculating loop and streaked into 20 mM formate solid medium and cultured for 7 days to form a distinct algal colony of 1 mm in diameter or more at a temperature of 25 to 28 ° C.

[0069] c. The microalgae obtained in step b were inoculated with an inoculation loop into 35 mM formate solid medium and cultured for 10 days to form clear algae colonies with a diameter of 1 mm or more at a temperature of 25 to 28 ° C.

[0070] d. The microalgae obtained in step c were inoculated with an inoculation loop into 50 mM formate solid medium and cultured for 10 days to form a clear algae colony with a diameter of 1 mm or more, and the culture temperature was 25 to 28 ° C;

[0071] e. The microalgae obtained in step d were inoculated with an inoculation loop into 60 mM formate solid medium and cultured for 10 days to form a clear algae colony with a diameter of 1 mm or more, and the culture temperature was 25 to 28 ° C (group e);

[0072] f. The microalgae obtained in step e were inoculated with an inoculating loop and streaked into 100 mM formate solid medium and cultured for 15 days to form a distinct algal colony of 1 mm in diameter or more at a temperature of 25 to 28 ° C.

[0073] g. The microalgae obtained in step f were inoculated with an inoculating loop into 150mM formate solid medium and cultured for 15 days to form a distinct algae colony of 1mm in diameter and above at a culture temperature of 25 to 28°C.

[0074] During the culture process, the light intensity was controlled at 30-100 μmol m -2 s -1 .

[0075] Evaluation of culture effect based on chlorophyll fluorescence kinetic parameters

[0076] Due to individual differences in microalgae, it is necessary to evaluate the culture effect to determine whether to further carry out culture with increased formate concentration and to select the timing for subculture inoculation. The chlorophyll fluorescence kinetic parameters (Fv / Fm) of the microalgae grown in TAP liquid culture medium and the microalgae cultured in steps af of Example 2 were measured using a water-PAM chlorophyll fluorometer. The chlorophyll fluorescence kinetic parameters after culture were measured and compared (the chlorophyll fluorescence values ​​of the cultured in steps af were divided by the chlorophyll fluorescence values ​​of the TAP culture respectively). The group that initially showed less than 90% and whose chlorophyll fluorescence maximum value reached 90% (greater than or equal to 90%) under the corresponding TAP conditions was the best choice and could be used for the next culture operation. Therefore, the microalgae after culture in group e (step e) were selected as the microalgae species for adaptive culture.

[0077]

[0078] Subsequently, the algae grown in the TAP liquid medium and the microalgae cultured in step e were inoculated into the TFP60 formate liquid medium prepared in Example 1. The inoculation ratio was 1 algae colony not less than 1 mm was inoculated into 50 mL of liquid medium. The culture was carried out at 25-28°C and a light intensity of 30-100 μmol m -2 s -1 After 10 days of cultivation, it was found that the algae inoculated in group e grew to OD 7501.0, with an Fv / Fm value of 0.72. However, the algae grown in the original TAP liquid medium directly transferred to the 60 mM liquid medium could not directly adapt to the high concentration of formate. 750 Less than 0.01, the Fv / Fm value of the algae liquid is 0.08.

[0079] The microalgae species cultured in step e were further inoculated into the TAP, TFP35, TFP50, TFP100 and TFP150 liquid culture media prepared in Example 1 above at a concentration of OD 750 0.1 was inoculated, the culture volume was 500 mL, the temperature was 25-28 °C, and the light intensity was 30-100 μmol m -2 s -1 The growth results were as follows: Figure 1 As shown in the results, it was found that its growth at 100 and 150 mM formate conditions was significantly weaker than that at 35 mM and 50 mM formate conditions, which was consistent with the plate screening results.

[0080] After that, the microalgae grown in the TAP liquid medium in the previous step after the culture in step e were inoculated as seed liquid, and the initial concentration OD 750 0.1 was re-inoculated into the TFP60 liquid culture medium prepared in Example 1 above, with a culture volume of 250 mL, at 25-28°C, and a light intensity of 30-100 μmol m -2 s -1 After 10 days of cultivation, it was found that the original TAP algae could not adapt to growth, and OD 750 It dropped to 0.08, and the Fv / Fm value of the algae solution was 0.35, indicating that its adaptability disappeared after being cultured in formate-free medium.

[0081] Example 3

[0082] Cultivation and evaluation of microalgae with high formate content in group e

[0083] The algae liquid after the algae culture of group e in Example 2 was used as algae seed, and after centrifugation and concentration, it was inoculated into the TFP100, TFP200 and TFP300 liquid culture media prepared in Example 1. The inoculation amount corresponded to OD 750 =6.0, 7.0 and 7.0, culture volume 700mL, culture temperature 28-30℃. Compared with the optimized concentration of group e (60mM), the concentration increased to about 1.5, 3 and 5 times. Figure 2 The growth changes show that it can maintain a certain growth under the conditions of 100-300mM formate. Even under the condition of 300mM formate, the biomass decreases at the beginning and then gradually increases steadily, and growth is still achieved.

[0084] By measuring the changes in Fv / Fm during the culture process, it was found that the Fv / Fm in TFP100 and TFP200 culture medium remained above 0.4, except that it began to drop to 0.28 on the 4th day, which was 50% lower than the highest value of 0.60.

[0085] Therefore, 100mM to 200mM is preferred.

[0086] The inoculation concentration in Example 3 was increased by 60-70 times compared to that in Example 2. By comparing the performance of Group e in Examples 2 and 3, increasing the inoculation concentration facilitates the growth of microalgae under conditions of higher formic acid concentrations.

[0087] The protein content of the microalgae inoculated in TFP100 and TFP200 after harvest in group e of Example 3 was analyzed, accounting for 31%, 33% and 29% of the dry weight, respectively. At the same time, the protein in TFP100-TFP300 was scored according to the FAO / WHO recommended algorithm (for the calculation process, see Yang, F.; Huang, X.; Zhang, C.; Zhang, M.; Huang, C.; Yang, H., Amino acid composition and nutritional value evaluation of Chinese chestnut (Castanea mollissima Blume) and its protein subunit. RSC Advances 2018, 8(5), 2653-2659), as shown in Table 1. The results show that the overall amino acid composition meets the requirements of high-quality protein (EAA greater than 100), is close to that of pork, beef and mutton, and slightly lower than eggs.

[0088] Example 4

[0089] Liquid-based adaptive evolution culture of microalgae formate

[0090] According to the inoculation ratio of 0.05 mg / mL based on the inoculation dry weight, the Chlamydomonas reinhardtii algae solution grown to the middle of the exponential growth phase in TAP liquid medium (approximately 0.4 mg / mL dry weight) was inoculated into 100 mL of fresh formate culture medium, and the formate concentration in the liquid culture medium was gradually increased with the passage.

[0091] The specific operations are:

[0092] h. Chlamydomonas reinhardtii algae solution (approximately 0.4 mg / mL dry weight) grown in TAP liquid medium to the mid-exponential growth phase was inoculated into 5 mM formate liquid medium (100 mL) at 0.05 mg / mL and cultured for 7 days at a temperature of 28 to 30°C.

[0093] i. The microalgae obtained in h were inoculated into 10mM formate liquid culture medium at 0.05mg / mL, 100mL, culture temperature 28-30°C, cultured for 7 days, and the dry weight exceeded 0.5

[0094] mg / mL;

[0095] j. The microalgae obtained in i were inoculated into 25mM formate liquid medium at 0.1mg / mL, 100mL, culture temperature 28-30°C, and cultured for 7 days until the dry weight exceeded 0.5mg / mL;

[0096] k. The microalgae obtained in j were inoculated into 40mM formate liquid medium at 0.1mg / mL, 100mL, culture temperature 28-30℃, cultured for 7 days, and the dry weight exceeded 0.5mg / mL; l. The microalgae obtained in h were inoculated into 60mM formate liquid medium at 0.1mg / mL, 100mL, culture temperature 28-30℃, cultured for 7 days, and the dry weight exceeded 0.5mg / mL; m. The microalgae obtained in l were inoculated into 80mM formate liquid medium at 0.1mg / mL, 100mL, culture temperature 28-30℃, cultured for 7 days, and the dry weight exceeded 0.5mg / mL; n. The microalgae obtained in m were inoculated into 100mM formate liquid medium at 0.1mg / mL, 100mL, culture temperature 28-30℃, cultured for 7 days, and the dry weight exceeded 0.5

[0097] mg / mL.

[0098] During the culture process, the light intensity was controlled at 30-100 μmol m -2 s -1 .

[0099] Evaluation of the Cultivation Effect of Chlorophyll Fluorescence Kinetic Parameters in Liquid Formate Seed Solution

[0100] Taking the HN process as an example, using the Water-PAM chlorophyll fluorescence kinetics analyzer, samples were taken daily to measure the maximum value of the chlorophyll fluorescence kinetic parameter Fv / Fm during the culture process. For comparison, a maximum chlorophyll fluorescence value reaching 90% of that under TAP culture conditions was considered optimal and suitable for the next step. The Fv / Fm value of the seed solution from liquid culture is generally higher than that from plate culture.

[0101]

[0102] Subsequently, the L group and the TAP group were inoculated into TFP60 liquid formate culture medium at 0.1 mg / mL, with a volume of 100 mL, and cultured for 7 days at a temperature of 28-30°C. During the culture process, the light intensity was controlled at 30-100 μmol m -2 s -1 The results showed that when the algae in Group 1 were grown to 1 mg / mL, their Fv / Fm value was 0.70. However, the algae directly transferred to 60 mM TAP could not adapt to the high formate concentration, with a biomass of approximately 0.03 mg / mL and a Fv / Fm value of 0.05. When the algae in Group 1 and TAP were cultured separately in TAP at an inoculum size of 0.1 mg / mL and under the same culture conditions, no significant differences were observed between the two groups.

[0103] When the algae from group 1 were again inoculated with TFP60 at a concentration of 0.1 mg / mL in TAP, using the same culture conditions as above, after seven days of cultivation, the biomass dropped to 0.04 mg / mL, and the Fv / Fm ratio dropped to 0.1, indicating ineffective growth.

[0104] Therefore, group 1 seed solution in TFP60 medium was selected for subsequent high formic acid culture.

[0105] Example 5

[0106] Cultivation and evaluation of microalgae with high formate content based on liquid formate seed solution

[0107] The preferred algae species of group 1 obtained in Example 4 were expanded to the exponential growth phase, with a dry weight of about 0.7 mg / mL. After centrifugation and concentration, they were directly inoculated into 700 mL of the corresponding TFP100, TFP200, and TFP300 formate liquid culture medium at a dry weight concentration of 2 mg / mL, 2.5 mg / mL, and 3.5 mg / mL, respectively. The cultures were cultured for 7 days at a temperature of 28-30°C. During the culture process, the light intensity was controlled at 30-100 μmol m -2 s -1 The formic acid concentration used in the culture was increased to about 1.5, 3 and 5 times the formic acid concentration of 60 mM in Group 1 of Example 4. Figure 3 From the growth results, it can be seen that in TFP300 medium, it first decreased significantly, then gradually maintained a slight increase, while in TFP100 and TFP200, it showed a certain upward trend. In TFP100 culture, the maximum dry weight of biomass could reach 3.6 mg / mL, an increase of 1.6 mg / mL.

[0108] At the same time, the protein content during the culture process was monitored, such as Figure 4As shown, except for TFP300, where the protein content dropped to 20% of the dry weight, the protein content in TFP100 and TFP200 also exceeded 30% of the dry weight. Considering the changes in biomass and protein content, 100-200 mM formate culture medium is preferred.

[0109] The amino acid content was determined and scored according to the FAO / WHO recommended algorithm (for the calculation process, see Yang, F.; Huang, X.; Zhang, C.; Zhang, M.; Huang, C.; Yang, H., Amino acid composition and nutritional value evaluation of Chinese chestnut (Castanea mollissima Blume) and its protein subunit. RSC Advances 2018, 8(5), 2653-2659), as shown in Table 1. The overall amino acid composition of chestnut (Castanea mollissima Blume) was in line with the requirements of high-quality protein (EAA greater than 100 as defined by the Food and Agriculture Organization (FAO) and the World Health Organization (WHO), close to that of pork, beef, and mutton, and slightly lower than that of eggs).

[0110] Table 1 Amino acid nutrition score table

[0111]

[0112] The present invention discloses a method for producing high-quality protein raw materials from microalgae. By combining formic acid, a reduction product driven by clean energy from carbon dioxide, with the cultivation of edible microalgae, the growth of microalgae and protein synthesis are enhanced. Specifically, the method comprises the following steps: adaptive evolution of microalgae using a low concentration of formate (20-50 mM), followed by cultivation under light conditions using 50-300 mM formate as an auxiliary carbon source, and ultimately harvesting microalgae biomass having a protein content exceeding 30% and an amino acid composition that meets the FAO's high-quality protein requirements. The microalgae biomass can be used as a high-quality protein raw material for food and feed. The present invention has significant application prospects in the field of green non-grain high-quality protein raw material production.

Claims

1. A method for producing high-quality protein raw material microalgae, characterized in that: Here are the steps: 1) Seed solution preparation: Formate is added to the microalgae culture medium and the formate concentration in the culture medium is gradually increased to perform adaptive evolution culture. The changes in photosynthetic activity are used as a guide to determine the microalgae's tolerance to formate inhibition, which is used as the seed solution; 2) High formate culture: The seed solution is inoculated with a high cell concentration, and photosynthetic culture is carried out in a high formate culture medium. The photosynthetic activity is monitored as a judgment of the end of the culture process.

2. The method according to claim 1, wherein: The method uses formate as an auxiliary carbon source, adds one or more of formic acid or formates (preferably sodium formate) to the microalgae culture medium, and adjusts the pH to maintain the pH value corresponding to the original culture medium, with the concentration range of 0 to 300 mM.

3. The method according to claim 1 or 2, wherein: The microalgae adaptive evolution cultivation process uses microalgae cultured in a medium without formic acid as a starting algae species and a control, and uses a medium containing 5-10 mM formate as an initial culture medium to obtain algae species for culturing the next group of higher formate concentration groups. The incremental step length of the formate concentration in the two adjacent groups of culture media is 10-20 mM formate, and the formate concentration in the culture medium is gradually increased to obtain high formate tolerance seeds.

4. The method according to claim 1 or 3, wherein: The adaptive evolutionary culture is guided by changes in photosynthetic activity, and refers to measuring the maximum quantum conversion efficiency (Fv / Fm) of the chlorophyll fluorescence kinetic parameter of the photosystem II to ensure that this kinetic parameter of the selected maximum tolerance group culture does not decrease by more than 10% after the formate concentration in the culture medium is increased compared with the culture medium without formate, that is, the Fv / Fm after culture in formate medium / Fv / Fm after culture in formate medium without formate is greater than or equal to 90%, and the Fv / Fm of the next group after culture in formate medium / Fv / Fm after culture in formate medium without formate is less than 90%.

5. The method according to claim 1, wherein: During the seed solution preparation process, the inoculation concentration of the microalgae seeds in the liquid culture is 0.05 to 0.2 OD value, or the inoculation amount calculated by dry weight is 25 to 100 mg / L. -1 .

6. The method according to claim 1, wherein: The high formic acid culture has a formate concentration range of 1.5 to 5 times, preferably 1.5 to 3.5 times, the formate concentration in the corresponding culture medium obtained from the seed solution.

7. The method according to claim 1 or 6, wherein: The high formic acid culture is carried out by inoculating the seed solution with a higher cell concentration, which means that the cell inoculation concentration is 4.0 to 8.0 calculated by OD value, or the cell inoculation amount is 2 to 4 g / L calculated by dry weight. -1 .

8. The method according to claim 1, wherein: The microalgae include green algae and / or blue algae, the green algae include one or more of Chlamydomonas reinhardtii, Chlorella vulgaris, and Dunaliella; the blue algae include Spirulina.

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