Chlorella pyrenoidosa heterotrophic high-density proliferation method

Abstract

The invention relates to the technical field of microalgae heterotrophic culture and biomass proliferation, in particular to a chlorella pyrenoidosa heterotrophic high-density proliferation method. The specific technical scheme is as follows: the heterotrophic high-density proliferation method of chlorella pyrenoidosa comprises the following steps: pre-culture and heterotrophic propagation of chlorella pyrenoidosa: inoculating a chlorella pyrenoidosa solution into a BG11 culture medium, and culturing for 3-4 days in a constant-temperature shaking table with the temperature of 25 + / -1 DEG C, the rotating speed of 200rpm and the light-dark ratio of 0h: 24h to obtain a primary culture chlorella solution; inoculating the primary culture algae liquid into a pre-sterilized and optimized heterotrophic culture medium, and carrying out expanding culture under the same culture condition for 3-4 days to obtain a secondary expanding culture algae liquid; (2) a heterotrophic high-density culture stage: regularly supplementing a nutrient culture medium containing high-concentration glucose, nitrate and phosphate by adopting an index fed-batch feeding strategy; other conditions are kept consistent during culture; and (3) an in-situ protein accumulation induction stage: when the chlorella pyrenoidosa reaches an exponential growth end stage, adding paclobutrazol into the system, and keeping other conditions consistent while keeping exponential fed-batch culture. According to the method disclosed by the invention, the biomass of the chlorella pyrenoidosa is increased by about 128.6% compared with that of the traditional batch culture, the protein content can reach 49.14%, meanwhile, the configuration of essential amino acids is improved, and the requirements of high-end biomass resource application are met. The method has the advantages of simplicity and convenience in operation and high production efficiency, and is suitable for the fields of high-quality protein extraction and related biomass production.

Description

Technical Field

[0001] The present invention relates to the technical field of heterotrophic cultivation of microalgae and biomass value-added, and particularly relates to a method for heterotrophic high-density proliferation of Chlorella pyrenoidosa. Background Art

[0002] The scientific community has devoted a great deal of effort to developing new protein sources to meet the growing demand. Microalgae can synthesize all essential amino acids, and their average protein content is generally higher than that of traditional plant protein sources, making them a promising alternative protein source. In addition, since the production of microalgae protein is technically feasible and has shown application potential in the food and feed industries, this new protein source is expected to play an important role in alleviating the global protein supply shortage.

[0003] As typical photoautotrophic organisms, microalgae are known as "multifunctional cell factories" and show potential application values in the fields of food, medicine, and feed. Currently, photoautotrophic cultivation is still the main method for industrial production of microalgae protein. For example, open raceway ponds and closed tubular photobioreactors have been widely used for the cultivation of Chlorella and Spirulina. In this mode, the protein content of microalgae biomass can reach 50-70% of the dry weight and can be used for human food and animal feed. However, the productivity of photoautotrophic cultivation is limited by the low microalgae biomass, and high-density cultivation is easily affected by microbial contamination, thus reducing the efficiency of protein production. To overcome these problems, researchers have proposed a heterotrophic cultivation strategy, that is, using organic carbon sources (such as glucose) instead of light as the energy source to enable microalgae to grow under dark conditions. Under heterotrophic cultivation conditions, microalgae can achieve higher growth rates and accumulate a large amount of biomass. Therefore, compared with traditional photoautotrophic cultivation, the heterotrophic cultivation mode shows great potential in improving the production efficiency of microalgae protein.

[0004] Although heterotrophic cultivation can achieve a higher cell density than photoautotrophic cultivation, the core challenge it faces is the low protein content, usually less than 40%, which is significantly lower than that under photoautotrophic cultivation conditions. To address this issue, researchers have explored various strategies. For example, a sequential heterotrophic-autotrophic cultivation mode is adopted. After the heterotrophic stage ends, light is introduced and carbon dioxide is supplied to the culture medium to increase the protein content of Chlorella vulgaris to 60.1%. Another method is to establish a continuous heterotrophic-dilution-photoinduced cultivation system, that is, after heterotrophic cultivation reaches a high cell density, the algal solution is diluted to an appropriate concentration and transferred to the light condition to promote protein synthesis. However, these methods still face many challenges in practical applications: (1) The heterotrophic-autotrophic or mixed cultivation modes that are easy to operate at the laboratory scale are difficult to ensure that all algal cells can effectively receive light during large-scale industrial production, resulting in limited autotrophic growth; in addition, the high cell density under heterotrophic / mixed cultivation will significantly weaken the light penetration ability, further limiting the photosynthetic efficiency. (2) In the heterotrophic-dilution-photoinduced process, the photoautotrophic cultivation system is an essential component, but this process prolongs the cultivation cycle, increases the operation complexity, and raises the risk of culture contamination.

[0005] Recently, researchers have attempted to develop a new method for promoting microalgae protein synthesis based on the "compensatory growth" theory. In the heterotrophic cultivation mode, microalgae first grow under nitrogen-sufficient conditions to accumulate biomass, then undergo a short-term nitrogen starvation stage, and then a large amount of nitrogen source is supplemented to induce the "compensatory absorption" of nitrogen by microalgae, thereby promoting protein synthesis. However, this method only increases the protein content of Chlorella vulgaris to 44.3%. Previous studies have shown that there are significant differences in the protein synthesis ability of different microalgae species, and the currently reported methods for increasing protein content still have limitations and are difficult to achieve economically viable protein accumulation in industrial production. Therefore, in the future, it is still necessary to further explore more efficient and low-cost heterotrophic cultivation strategies to achieve the sustainable production of microalgae protein. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the present invention provides a method for heterotrophic high-density proliferation of Chlorella pyrenoidosa. During the heterotrophic cultivation of Chlorella pyrenoidosa, an exponential fed-batch feeding strategy is adopted to maintain the optimal concentration of nutrients in the culture medium, and paclobutrazol is added when cell proliferation reaches a certain level to induce in-situ protein accumulation in cells. It solves the problems of low microalgae biomass accumulation and low protein synthesis efficiency under traditional heterotrophic cultivation, and at the same time realizes the efficient production of heterotrophic high density and high-quality protein.

[0008] To achieve the above objectives, the present invention is realized through the following technical solutions:

[0009] The present invention discloses a method for heterotrophic high-density proliferation of Chlorella pyrenoidosa, comprising the following steps:

[0010] (1) Pre-cultivation and heterotrophic expansion culture of Chlorella pyrenoidosa:

[0011] Inoculate the Chlorella pyrenoidosa liquid into the BG11 medium containing glucose, and culture it in a constant-temperature shaking incubator at a temperature of 25 ± 1°C, a rotation speed of 200 rpm, and a light-dark ratio of 0 h:24 h for 3 - 4 days to obtain the primary culture algal liquid; inoculate the primary culture algal liquid into the optimized heterotrophic medium that has been pre-sterilized, and expand the culture for 3 - 4 days under the same culture conditions to obtain the secondary expanded culture algal liquid;

[0012] (2) Heterotrophic high-density culture stage: Adopt an exponential fed-batch feeding strategy, and regularly supplement the feeding medium containing high concentrations of glucose, nitrate, and phosphate; the culture conditions during the culture are the same as those in step (1);

[0013] (3) In-situ protein accumulation induction stage: When Chlorella pyrenoidosa reaches the end of the exponential growth phase, add paclobutrazol to the system, and while maintaining the exponential fed-batch culture, the culture conditions are the same as those in step (1).

[0014] Preferably, in steps (1) and (2), the composition of the optimized heterotrophic medium that has been pre-sterilized is: 1.7 g / L BG11 medium, 2.5 - 40 g / L glucose, 0.3 - 4 g / L potassium nitrate, 25 - 150 mg / L potassium dihydrogen phosphate.

[0015] Preferably, during the process of adopting the exponential fed-batch feeding strategy, it is achieved by establishing a heterotrophic exponential fed-batch model of Chlorella pyrenoidosa. The fed-batch model is shown in equation (a):

[0016]

[0017] where S and X respectively represent the nutrient concentration and biomass, F S is the concentration of the fed-batch substrate, and Y XS is the ratio of biomass to substrate concentration.

[0018] Preferably, when the nutrient concentration in the feeding medium remains constant, equation (a) can be converted to:

[0019]

[0020] where X 0 is the initial biomass, μ is the specific growth rate, and t is the time.

[0021] Preferably, further introduce a fed-batch control parameter based on volume to convert equation (b) to:

[0022]

[0023] Among them, V FS represents the flow acceleration rate (mL / h), and V 0 is the initial culture volume (mL), and C FS represents the concentration of nutrients in the feeding medium.

[0024] Preferably, in step (3), the concentration of paclobutrazol is 10 - 50 mg / L.

[0025] The present invention has the following beneficial effects:

[0026] 1. In order to achieve the sustainable production of microalgal protein, the present invention adopts a strategy of directly promoting the in-situ accumulation of protein under heterotrophic culture conditions, avoiding additional culture stages and complex conversion processes. By optimizing the carbon source supply through the exponential feeding strategy, the metabolic state of microalgae can be precisely regulated in a heterotrophic environment, improving biomass accumulation while promoting protein synthesis. In addition, the application of plant growth regulators such as paclobutrazol has shown the potential to increase the microalgal protein content, which may enhance the nitrogen assimilation ability by regulating the carbon-nitrogen metabolic balance, thereby improving the protein accumulation efficiency. These methods not only overcome the limitations of the traditional heterotrophic-autotrophic conversion mode but also provide a feasible technical path for large-scale and efficient production of high-protein microalgal biomass.

[0027] 2. The present invention provides a method for heterotrophic high-density proliferation of Chlorella pyrenoidosa, specifically using two-stage culture regulated by exponential feeding and paclobutrazol to achieve heterotrophic high-density growth and in-situ protein accumulation of Chlorella pyrenoidosa. Based on the heterotrophic growth characteristics of Chlorella pyrenoidosa and combined with the exponential feeding strategy, the carbon source supply is precisely controlled to maintain a stable growth rate. At the same time, paclobutrazol regulation is introduced to optimize the carbon metabolic pathway and improve the protein accumulation efficiency. This method can effectively promote the high-density growth of Chlorella pyrenoidosa and achieve in-situ protein accumulation during the culture process, improving the biomass utilization rate. Compared with the traditional culture method, the present invention significantly improves the protein yield, reduces the production cost, and provides an efficient and feasible technical approach for the industrial development of microalgal protein, with broad application prospects. Description of the Drawings

[0028] Figure 1 shows the effects of different glucose (A), nitrate (B), and phosphate (C) concentrations on the biomass and specific growth rate of Chlorella pyrenoidosa in Example 1;

[0029] Figure 2 shows the exponential growth of the biomass of Chlorella pyrenoidosa within a certain culture time (A) and the relationship between the biomass and glucose, nitrate, and phosphate in the exponential phase (B) in Example 2;

[0030] Figure 3 In Example 2, comparison of the biomass (A) and protein content (B) of Chlorella pyrenoidosa in exponential fed-batch culture and batch culture;

[0031] Figure 4 In Example 2, effects of different concentrations of paclobutrazol on the biomass (A) and protein content (B) of Chlorella pyrenoidosa;

[0032] Figure 5 In Example 3, during exponential fed-batch culture with the addition of 20 mg / L paclobutrazol, i.e., changes in the biomass (A) and protein content (B) of Chlorella pyrenoidosa over time during the two-stage culture process. Detailed implementation manner

[0033] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention.

[0034] Unless otherwise specified, the technical means used in the implementation examples are conventional means well known to those skilled in the art.

[0035] The present invention discloses a method for heterotrophic high-density proliferation of Chlorella pyrenoidosa, comprising the following steps:

[0036] (1) Pre-culture and heterotrophic expansion of Chlorella pyrenoidosa: Add BG11 medium to a conical culture flask, and inoculate 10% (v / v) of Chlorella pyrenoidosa liquid into the BG11 medium containing 10 g / L glucose under sterile conditions. After mixing evenly, place the conical culture flask in a constant temperature shaking incubator at a temperature of 25 ± 1 °C, a rotation speed of 200 rpm, and a light-dark ratio of 0 h:24 h for 3 - 4 days to obtain the primary culture algal liquid; then, inoculate the primary culture algal liquid into the pre-sterilized optimized heterotrophic medium at an inoculation ratio of 10% (v / v), and expand the culture for 3 - 4 days under the same culture conditions to obtain the secondary expanded culture algal liquid, realizing the preliminary proliferation of Chlorella pyrenoidosa;

[0037] Among them, the composition of the pre-sterilized optimized heterotrophic medium is: 1.7 g / L BG11 medium, 2.5 - 40 g / L glucose, 0.3 - 4 g / L potassium nitrate, 25 - 150 mg / L potassium dihydrogen phosphate.

[0038] (2) Heterotrophic high-density cultivation stage: An exponential fed-batch strategy is adopted to regularly supplement the fed-batch medium containing high concentrations of glucose, nitrate, and phosphate to maintain the key nutrients in the culture system at the optimal growth level, thereby promoting high-density cell growth; other conditions during the cultivation are kept consistent;

[0039] Among them, during the process of adopting the exponential fed-batch strategy, it is achieved by establishing a heterotrophic exponential fed-batch model for Chlorella pyrenoidosa. The fed-batch model is shown in Equation (a):

[0040]

[0041] Among them, S and X represent the nutrient concentration and biomass respectively, and F S is the concentration of the fed substrate, and Y XS is the ratio of biomass to substrate concentration.

[0042] When the nutrient concentration in the fed-batch medium remains constant, Equation (a) can be converted to:

[0043]

[0044] Among them, X 0 is the initial biomass, μ is the specific growth rate, and t is the time.

[0045] Further introducing the fed-batch control parameter based on volume, Equation (b) is converted to:

[0046]

[0047] Among them, V FS represents the feeding rate (mL / h), V 0 is the initial culture volume (mL), and C FS represents the concentration of nutrients in the fed-batch medium.

[0048] Taking glucose as the main fed substance, according to the linear relationship between biomass accumulation and nutrient consumption, Y XS = 0.4699 g / L can be calculated. According to Equation (c), the exponential fed-batch equation can be established as V FS = 1.299×10 -2 ×exp(0.037t).

[0049] (3) In-situ protein accumulation induction stage: When Chlorella pyrenoidosa reaches the end of the exponential growth phase, a certain concentration of paclobutrazol is added to the system. While maintaining the exponential fed-batch culture, other conditions are kept consistent, and the cultivation is continued for a period of time to induce the in-situ synthesis and accumulation of proteins in the cells;

[0050] Among them, during the exponential fed-batch culture process, 10-50 mg / L of paclobutrazol was added and the culture was continued.

[0051] (4) Analyze the protein yield, amino acid composition, and essential amino acid index (EAAI) of Chlorella pyrenoidosa after two-stage culture to evaluate the nutritional value of the obtained protein.

[0052] Among them, the protein content of Chlorella pyrenoidosa after two-stage culture was determined using a BCA kit, and its amino acid nutritional components were evaluated using an automatic amino acid analyzer (Hitachi-L8900).

[0053] The present invention will be further elaborated below in conjunction with specific embodiments.

[0054] Example 1

[0055] 1. Pre-culture and heterotrophic expansion of Chlorella pyrenoidosa were carried out, including the following steps:

[0056] (1) Add BG11 medium (100 mL) to a 250 mL conical culture flask, sterilize it at 115 °C for 30 min, and then add 10% (v / v) of Chlorella pyrenoidosa liquid to the BG11 medium containing 10 g / L glucose under sterile conditions. After mixing evenly, place the conical culture flask in a constant temperature shaking incubator at a temperature of 25 ± 1 °C, a rotation speed of 140 rpm, and a light-dark ratio of the light cycle of 0 h:24 h for 3-4 days to obtain the primary culture algal liquid;

[0057] (2) Subsequently, the primary culture algal liquid that had grown to the exponential phase was inoculated into BG11 media containing different concentrations of glucose (2.5, 5, 10, 15, 20, 30, 40 g / L) at the same inoculation ratio, with other conditions remaining unchanged. By comparing the biomass and specific growth rate, the optimal glucose concentration for heterotrophic culture was determined. On this basis, the optimal KNO 3 concentration (0.3, 0.75, 1.5, 2.5, 3.5, 4 g / L) and KH 2 PO 4 concentration (25, 50, 75, 100, 125, 150 mg / L) were further determined. The optimal heterotrophic medium for Chlorella pyrenoidosa was obtained, and it was expanded for 3-4 days to obtain the secondary expanded algal liquid, achieving the preliminary proliferation of Chlorella pyrenoidosa;

[0058] Methods for measuring biomass and specific growth rate: Take a certain volume of the sample, centrifuge it at 5000 rpm for 5 min, and discard the supernatant. Repeat this process twice to reduce the influence of residual nutrients on cell biomass. The obtained precipitate was dried at 60 °C for at least 6 h until constant weight. The specific growth rate (μ) was calculated according to the following formula:

[0059] μ = (lnN t - lnN 0 ) / t

[0060] where μ is the specific growth rate (d - -1), Nt is the biomass concentration (g / L) at day t, and N 0 is the initial biomass concentration (g / L).

[0061] During the microalgae cultivation process of this step, nutrients have been proven to affect the flux of cell metabolism, play a crucial role in cell growth, and optimizing it can significantly increase the microalgae biomass. As Figure 1 shown in A, under batch cultivation, the biomass of Chlorella pyrenoidosa increases with the increase of glucose concentration. However, the maximum specific growth rate is obtained when the glucose concentration is 10 g / L. This is mainly because too high glucose concentration increases the biomass by extending the cultivation time, but it will greatly limit the rapid growth of cells. The effect of nitrate concentration on the specific growth rate of Chlorella pyrenoidosa first increases and then decreases, and the highest biomass and specific growth rate are both obtained when the nitrate concentration is 2.5 g / L (as Figure 1 shown in B). When the phosphorus source concentration is 50 mg / L, the biomass and specific growth rate of Chlorella pyrenoidosa are the highest (as Figure 1 shown in C). Optimizing the concentrations of C, N, and P during the heterotrophic cultivation of Chlorella pyrenoidosa lays a good foundation for the subsequent exponential feeding.

[0062] Among them, batch cultivation is ordinary cultivation, which is the cultivation process of the above steps (1)-(2), that is, the process of obtaining the secondary expanded culture algal solution.

[0063] 2. Method for establishing a heterotrophic exponential feeding model of Chlorella pyrenoidosa, including the following steps:

[0064] To maintain the optimal level of nutrient concentration during the heterotrophic cultivation process, the present invention adopts an exponential feeding strategy to supplement the nutrients consumed by microalgae growth. This method is based on the following assumptions: (1) Intracellular nutrients are mainly used for growth, and the consumption for cell maintenance is small; (2) The concentration of extracellular metabolic by-products is low. This example focuses on studying the consumption of key nutrients such as glucose, nitrate, and phosphate during the microalgae growth process, while other nutrient components are supplemented according to the proportion of the initial medium (heterotrophic medium). The feeding model can be established by the following equation:

[0065]

[0066] where S and X represent the nutrient concentration and biomass respectively, F S is the feeding substrate concentration, and Y XS is the ratio of biomass to substrate concentration. When the nutrient concentration in the heterotrophic medium remains constant, equation (a) can be converted to:

[0067]

[0068] where X 0 is the initial biomass, μ is the specific growth rate, and t is the time.

[0069] For ease of fed-batch operation, a fed-batch control parameter based on volume is further introduced to transform Equation (b) into:

[0070]

[0071] where V FS represents the feeding rate (mL / h), V 0 is the initial culture volume (mL), and C FS represents the concentration of nutrients in the fed-batch medium. Specifically, C FS includes C FG , C FN and C FP , where C FG , C FN and C FP correspond to the concentrations of glucose, nitrate, and phosphate in the fed-batch medium, respectively. These parameters are used to regulate the addition rate of nutrients during the exponential fed-batch process to maintain continuously optimized growth conditions.

[0072] Determination of residual glucose: Centrifuge the algal solution at 10,000 rpm, take 1 mL of the supernatant into a 15-mL graduated test tube, add 2 mL of DNS reagent, heat in a boiling water bath for 2 min, cool, and make up the volume to the mark with distilled water. Measure the absorbance at 540 nm and establish a standard curve.

[0073] Determination of residual nitrogen content: After centrifuging the algal solution at 10,000 rpm, take 2 mL of the supernatant into a 25-mL colorimetric tube, make up the volume to 10 mL with deionized water, add 5 mL of alkaline persulfate solution, and digest at 120 °C for 30 min. After cooling, add 1 mL of hydrochloric acid (hydrochloric acid: water = 1:9, v / v), and measure the absorbance at wavelengths of 220 nm and 275 nm.

[0074] Determination of residual phosphorus content: After centrifuging the algal solution at 10,000 rpm, take 2 mL of the supernatant into a 25-mL colorimetric tube, make up the volume to 10 mL with deionized water, add 4 mL of 50 g / L potassium persulfate solution, and digest at 120 °C for 30 min. After cooling, add 1 mL of 100 g / L ascorbic acid solution, mix well for 30 s, then add 2 mL of ammonium molybdate solution, let stand at room temperature for 15 min, and measure the absorbance at 700 nm.

[0075] An exponential fed-batch model is established during the processing of this step. The fed-batch formula (c) is a function of the fed-batch volume and time, and multiple parameters involved have been determined, where X0 = 0.6 g / L, V 0 = 110 mL, C FS = 400 g / L (taking glucose as an example). Figure 2 A shows that the biomass of Chlorella pyrenoidosa increases exponentially during this cultivation time. As the biomass increases, the nutrient substrates in the culture medium are gradually consumed. As Figure 2 shown in B, during the exponential growth period, the consumption of glucose, nitrate, and phosphate in the culture medium is linearly related to the increase in biomass. The relationship between glucose and biomass is y = -11.834x + 67.069 (R 2 = 0.98), that is, for every 1 g increase in biomass, 11.834 mmol of glucose needs to be consumed. Similarly, the relationships between nitrate and phosphate and biomass are y = -5.4296x + 30.09 (R 2 = 0.98) and y = -0.5043x + 2.7781 (R 2 = 0.90), respectively. Therefore, the molar ratio of glucose, nitrate, and phosphate in the feeding medium is 23:10:1. The glucose concentration in the feeding medium is set to 400 g / L, and the corresponding concentrations of potassium nitrate and potassium dihydrogen phosphate are 97.6 g / L and 13.1 g / L, respectively. It should be noted that: the feeding medium only includes glucose, potassium nitrate, and potassium dihydrogen phosphate.

[0076] Taking glucose as the main feeding substance, its Y can be calculated according to the linear relationship between biomass accumulation and nutrient consumption XS = 0.4699 g / L. According to equation (c), the exponential feeding equation can be established as V FS = 1.299×10 -2 ×exp(0.037t) (shake flask level). According to this method, at the shake flask culture level, exponential feeding cultivation of heterotrophic Chlorella pyrenoidosa was carried out. By accurately and timely supplementing the nutrients consumed during the cultivation process, the biomass of Chlorella pyrenoidosa was increased to 8.0 ± 0.2 g / L, while the maximum biomass of batch cultivation could only reach 3.5 ± 0.1 g / L (as Figure 3 shown). It confirmed the superiority of the exponential feeding strategy in promoting cell growth and biomass accumulation.

[0077] 3. A method for in-situ protein accumulation using paclobutrazol, comprising the following steps:

[0078] When Chlorella pyrenoidosa reaches the end of the exponential growth phase, add 1 mL of paclobutrazol solutions at different concentrations (0, 10, 20, 30, 40, 50 mg / L), and determine the optimal paclobutrazol concentration based on the protein content. Then inoculate the algal cells grown to the exponential growth phase into the optimized heterotrophic medium (the same as step (2) in step 1 above), and add 1 mL of the optimal paclobutrazol concentration on the 4th day of fed-batch culture. The culture lasts for 6 days, and the biomass is monitored every day, and the protein content is measured from the 4th day to the 6th day to evaluate the effect of paclobutrazol on protein accumulation.

[0079] During this treatment process, when Chlorella pyrenoidosa reaches the end of the exponential growth phase, add 1 mL of paclobutrazol at 0, 10, 20, 30, 40, 50 mg / L, and the results are as Figure 4 shown in A. After adding paclobutrazol on the 2nd day, the biomass of Chlorella pyrenoidosa showed a downward trend, and the decline amplitude was positively correlated with the addition amount of paclobutrazol to a certain extent. Figure 4 B shows the changes in the protein content of Chlorella at different time points (the 4th day and the 6th day) without adding paclobutrazol (the 2nd day) and after adding. The results show that the addition of paclobutrazol can increase the protein content to a certain extent. Especially on the 4th day of culture (i.e., the 2nd day after adding paclobutrazol), the treatment with 20 mg / L of paclobutrazol had the most significant effect on the increase of protein content, and the protein content reached 48.79 ± 1.54%, and the protein yield increased to 1.62 ± 0.05 g / L.

[0080] Combined with the above experimental results, two-stage heterotrophic culture is carried out to achieve in-situ accumulation of high-quality protein. The first stage is exponential fed-batch culture, which is cultured until the end of the exponential growth phase (the 4th day), and then add 1 mL of 20 mg / L of paclobutrazol to the medium to enter the second stage of culture. As Figure 5 shown in A, the biomass of Chlorella pyrenoidosa can reach 7.03 g / L on the 4th day of shake flask culture. Subsequently, add 20 mg / L of paclobutrazol and continue to culture for 2 days, and the protein content increases to 49.14% ( Figure 5 B), and the protein yield reaches 3.56 g / L, while that of the control group is only 2.59 g / L, which is 2.04 times higher than that of ordinary batch culture.

[0081] 4. Select the algal cells after two-stage culture in step 3, measure their protein content with a BCA kit, and measure the types and contents of their amino acids by acid hydrolysis method. The specific methods are as follows:

[0082] To determine the protein content, the microalgae cells were freeze-dried. A certain amount of algal powder was weighed, then broken by liquid nitrogen. 200 μL of 1 M sodium hydroxide was added for hydrolysis, incubated at 80 °C for 10 min, 1.8 mL of deionized water was added, and centrifuged at 13000 rpm for 30 min. The extraction operation was repeated 2 more times, the supernatants were combined, and the protein content was determined using a BCA kit (Beyotime, Guangzhou, China).

[0083] The sample was weighed and placed in an ampoule bottle. 10 mL of 6 mol / L hydrochloric acid was added and hydrolyzed at 110 °C for 22 hours. After hydrolysis, it was taken out and cooled, and made up to 50 mL with 0.02 mol / L hydrochloric acid. 1 mL of the hydrolyzate was taken into a rotary evaporation flask, and ultrapure water was added for evaporation to remove acid. This process was repeated three times until the solution was completely dry. Finally, the residue was dissolved in 2 mL of 0.02 mol / L hydrochloric acid. The resulting solution was filtered through a 0.22 μm water-based filter membrane and transferred to a liquid phase bottle for subsequent analysis. The amino acid composition of the algal powder was qualitatively and quantitatively analyzed using an automatic amino acid analyzer (Hitachi - L8900).

[0084] Amino acid score (AAS) represents the ratio of the content of each amino acid in the sample to the amino acid pattern of the ideal protein. Generally, the pattern protein scoring standard proposed by the Food and Agriculture Organization of the United Nations / World Health Organization (FAO / WHO, 1973) is adopted, and this scoring pattern is also used in this study. The calculation formula is as follows:

[0085] AAS (%) = content of amino acid in the sample to be measured / content of amino acid in the FAO / WHO standard pattern protein × 100%

[0086] The availability of protein amino acids was evaluated according to the essential amino acid index (EAAI), which is a method for evaluating protein feeds:

[0087]

[0088] a, … j: represent the percentage of essential amino acids in the test sample in the total amount of essential amino acids;

[0089] A, … J: represent the percentage of this essential amino acid in the reference protein in the total amount of essential amino acids;

[0090] n: the number of essential amino acids.

[0091] During this processing, the amino acid nutritional components of Chlorella pyrenoidosa after two-stage cultivation were evaluated, and the results are shown in Table 1-2. As shown in Table 1, under two-stage cultivation, the contents of multiple amino acids increased compared with ordinary cultivation, especially amino acids such as Glu, Ala, and Leu. This indicates that the addition of paclobutrazol not only optimizes the efficiency of nitrogen metabolism to a certain extent but also helps to increase the amino acid accumulation in microalgae. The EAA / TAA of two-stage cultivation was 35%, which was higher than that of traditional feeds such as soybean meal (12.43%) and fish meal (23.97%).

[0092] In addition, as shown in Table 2, all AASs in two-stage cultivation were better than those in ordinary cultivation. The EAAI value was 1.36. The higher the EAAI value, the closer the essential amino acid configuration of the sample is to the ideal ratio required by humans or other animals, and thus it has higher nutritional value. The experimental results show that two-stage cultivation not only improves the synthesis and accumulation ability of essential amino acids in microalgae but also may optimize the amino acid configuration of microalgae. This change provides support for subsequent protein synthesis and nutritional value improvement, and further enhances the application potential of microalgae as a biomass resource in food or feed.

[0093] Table 1 Amino acid composition and content of Chlorella pyrenoidosa

[0094] Amino acid Two-stage cultivation (mg / g dry weight) Normal cultivation (mg / g dry weight) Lys* 30.418 26.588 Phe* 22.576 19.452 Tyr* 17.462 14.704 Glu 83.07 76.492 Asp 51.544 48.136 Ser 21.99 19.256 Thr* 22.158 19.236 Ala 40.962 36.36 Val* 20.04 17.202 Gly 25.792 22.29 Arg 62.076 63.204 Met* 7.948 6.816 Ile* 12.592 10.466 His 11.328 9.884 TAA 505.546 453.722 EAA 176.404 152.028 EAA / TAA 0.349 0.3351

[0095] "*" represents essential amino acids; TAA: total content of amino acids; EAA: essential amino acids; EAA / TAA: ratio of essential amino acids to total amino acids.

[0096] Table 2 AAS and EAAI of essential amino acids of Chlorella pyrenoidosa

[0097]

[0098]

[0099] The embodiments described above are only descriptions of the preferred modes of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for heterotrophic high-density proliferation of Chlorella pyrenoidosa, characterized in that: The following steps are involved: (1) Pre-culture and heterotrophic expansion of Chlorella pyrenoidosa: The protein nucleus Chlorella liquid was inoculated into the BG11 medium containing glucose, and cultured in a constant temperature shaking incubator at a temperature of 25±1°C, a rotation speed of 200 rpm, and a light-dark ratio of 0h:24h for 3-4 days to obtain a primary culture algae liquid; the primary culture algae liquid was inoculated into a pre-sterilized optimized heterotrophic medium, and expanded for 3-4 days under the same culture conditions to obtain a secondary expansion algae liquid; (2) Heterotrophic high-density culture stage: an exponential feeding strategy is adopted to regularly supplement the feed medium containing high concentrations of glucose, nitrate and phosphate; the culture conditions during the culture period are the same as those in step (1); (3) In situ protein accumulation induction stage: When Chlorella pyrenoidosa reaches the end of exponential growth, paclobutrazol is added to the system, and the culture conditions are the same as step (1) while maintaining exponential fed-batch culture.

2. The method for heterotrophic high-density proliferation of Chlorella pyrenoidosa according to claim 1, characterized in that: In steps (1) and (2), the composition of the pre-sterilized optimized heterotrophic culture medium is: 1.7 g / L BG11 culture medium, 2.5-40 g / L glucose, 0.3-4 g / L potassium nitrate, and 25-150 mg / L potassium dihydrogen phosphate.

3. The method for heterotrophic high-density proliferation of Chlorella pyrenoidosa according to claim 1, characterized in that: In the process of adopting the exponential feeding strategy, the heterotrophic exponential feeding model of Chlorella pyrenoidosa is established, and the feeding model is shown in equation (a): Where S and X represent nutrient concentration and biomass, respectively, and F S is the feed matrix concentration, Y XS is the ratio of biomass to substrate concentration.

4. The method for heterotrophic high-density proliferation of Chlorella pyrenoidosa according to claim 3, characterized in that: When the nutrient concentration in the feed medium is kept constant, equation (a) can be converted to: Among them, X0 is the initial biomass, μ is the specific growth rate, and t is the time.

5. The method for heterotrophic high-density proliferation of Chlorella pyrenoidosa according to claim 4, characterized in that: Further introducing the volume-based feed control parameter, equation (b) is transformed into: Among them, V FS represents the flow rate (mL / h), V0 is the initial culture volume (mL), C FS Indicates the concentration of nutrients in the feed medium.

6. The method for heterotrophic high-density proliferation of Chlorella pyrenoidosa according to claim 1, characterized in that: In step (3), the concentration of paclobutrazol is 10-50 mg / L.

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