A method for restoring protein content during the production of glycerol glucoside

Abstract

The present invention discloses a method for restoring protein content during the production of glycerol glucoside, which method comprises the following steps: obtaining the stage of cultured algal cells; and including: GG boosting stage one: the culture medium in this stage contains a substance capable of changing the cell osmotic pressure, and the concentration of this substance in the culture medium is 300 < C1 ≤ 800 mmol / L; and / or, GG boosting stage two: the culture medium in this stage contains a substance capable of changing the cell osmotic pressure, and the concentration of this substance in the culture medium is 800 < C2 ≤ 1500 mmol / L; during the culture process of any of the above stages, 20% - 50% of the nitrogen source is supplemented into the culture medium every 3 - 5 days. By adopting this process, not only can a relatively high yield of glycerol glucoside be obtained, but also the protein content of the finally harvested algal powder is greater than 60% (w / w), meeting the national standard requirement of ≥ 55%.

Description

Technical Field

[0001] The present invention relates to a method for restoring protein content during the production of glucosylglycerol, belonging to the technical field of microalgae cell cultivation. Background Art

[0002] Spirulina, also known as blue-green algae, has the effects of reducing the toxic and side effects of cancer radiotherapy and chemotherapy, enhancing immune function, and lowering blood lipid. It is rich in vitamins, proteins, unsaturated fatty acids, and trace elements. In particular, the protein it contains is the main nutrient of Spirulina. Currently, Spirulina has become a protein source that has attracted much attention in the food industry and is considered the most potential alga for producing single-cell protein.

[0003] In recent decades, research on the separation, extraction, and application of Spirulina alga cell protein at home and abroad has been carried out, or the alga protein has been enzymatically hydrolyzed into polypeptides for use in health products and food additives. Generally reported in the literature, the protein content of alga cells is about 60 - 70%. However, in large-scale industrial production by enterprises, the protein content of most products is 50 - 60%. In addition, the inventors of the present application found that the protein content is very low when preparing and producing GG. The protein content is one of the important indicators of alga products. The national standard for food alga powder requires a protein content of ≥55%. Therefore, increasing the alga protein content is of great significance for improving the quality of alga products, developing new products, and utilizing alga protein resources.

[0004] Currently, when simply producing Spirulina alga protein, there are relatively many studies on increasing the protein content of alga cells. For example, the method for promoting the accumulation of protein in Spirulina cells studied by Li Bosheng et al., and the study by Chen Tianfeng et al. on the cultivation of high-selenium Spirulina by the method of adding selenium in batches and its effect on the protein content of the alga body. However, for the production of glucosylglycerol (GG), there are few studies on increasing the protein content, and it is rather difficult to increase both simultaneously.

[0005] Therefore, there is an urgent need to find a suitable cultivation method to restore the protein content during the production of GG, so as to meet the industrial application of the cultivation of glucosylglycerol alga cells and the requirement of the national standard for a protein content of ≥55%. Summary of the Invention

[0006] In view of the above prior art, in the process of researching the production of GG from Spirulina, the present inventors found that the protein content in algal cells decreased significantly, only about 40%, far lower than the national standard of protein content ≥ 55%. That is, the Spirulina powder obtained after producing GG by the conventional process cannot be used for commercial sales and can only become a by-product of the production of glyceroglucoside. Based on this discovery, the present invention proposes a method for restoring the protein content during the production of glyceroglucoside. Therefore, without affecting the growth of algal cells and the synthesis of glyceroglucoside, the present inventors improved the culture process to increase the protein content of the algal powder to meet the requirement of the national standard ≥ 55%, so that the algal powder and GG obtained under this process condition can be sold as products.

[0007] Based on this, the present invention specifically adopts the following technical solutions:

[0008] In the first aspect of the present invention, a method for restoring the protein content during the production of glyceroglucoside is provided. The method includes the following steps:

[0009] Obtaining the stage of cultured algal cells; and including:

[0010] GG elevation stage 1: The culture medium in this stage contains a substance that can change the cell osmotic pressure, and the concentration of this substance in the culture medium is 300 < C1 ≤ 800 mmol / L; and / or,

[0011] GG elevation stage 2: The culture medium in this stage contains a substance that can change the cell osmotic pressure, and the concentration of this substance in the culture medium is 800 < C2 ≤ 1500 mmol / L;

[0012] During the culture process of any of the above stages, 20%-50% of the nitrogen source is supplemented into the culture medium every 3-5 days.

[0013] Further, after the step of obtaining the stage of cultured algal cells, a GG generation stage is further included: The culture medium in this stage contains a substance that can change the cell osmotic pressure, and the concentration of this substance in the culture medium is 100-300 mmol / L.

[0014] In the second aspect of the present invention, a preparation method of glyceroglucoside and algal protein is provided. The method includes the steps of the method for restoring the protein content during the production of glyceroglucoside as described above.

[0015] In the third aspect of the present invention, glyceroglucoside and / or protein products prepared by the above method are also protected.

[0016] Compared with the prior art, the beneficial effects of the technical solution of the present invention are:

[0017] (1) The present invention first proposes the technical problem that the algal protein content decreases significantly during the production of GG from Spirulina, and provides a process for restoring the protein content of algal cells during the large-scale cultivation of algal cells to synthesize glucosylglycerol. By using this process, not only can a relatively high yield of glucosylglycerol be obtained, but also the protein content of the finally harvested algal powder is greater than 60% (w / w), meeting the national standard requirement of ≥55%.

[0018] (2) Through step-by-step regulation of cultivation, the present invention can obtain algal cells with a relatively high content of GG. After the first stage of GG and protein elevation, the GG content reaches more than 15% (w / w), and after the second stage of GG and protein elevation, the GG content reaches more than 30%, enabling the large-scale industrial production of GG.

[0019] (3) Through four stages of specific culture conditions, the present invention can obtain algal cells with a high content of GG, reducing the separation and extraction cost of GG. Detailed implementation mode

[0020] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0021] It should be noted that the terms used herein are only for describing specific implementation modes and are not intended to limit the exemplary implementation modes according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0022] Term explanation:

[0023] Microalgae: Generally refer to those algae that are microscopic and can only be distinguished in terms of morphology.

[0024] As introduced in the background art, there are certain deficiencies in the existing algal cell cultivation process, resulting in a large loss of microalgal protein during the production of GG and being unable to meet the national standard requirement of protein content ≥55%. To solve the above technical problems, the present invention proposes a method for restoring the protein content during the production of glucosylglycerol, which includes the following steps:

[0025] The stage of obtaining cultured algal cells; and including:

[0026] GG elevation stage 1: The culture medium in this stage contains a substance that can change the cell osmotic pressure, and the concentration of this substance in the culture medium is 300 < C1 ≤ 800 mmol / L; and / or,

[0027] GG elevation stage 2: The culture medium in this stage contains a substance that can change the cell osmotic pressure, and the concentration of this substance in the culture medium is 800 < C2 ≤ 1500 mmol / L;

[0028] During the cultivation process of any of the above stages, 20%-50% of the nitrogen source is supplemented into the culture medium every 3-5 days (that is, 20-50% of the total mass of the nitrogen source in the original culture medium is added). The nitrogen source includes but is not limited to one or more of urea, ammonium bicarbonate, sodium nitrate, monoammonium phosphate, diammonium phosphate, and ammonium nitrate. The protein content of the algal powder harvested by this method is greater than 60%, and it does not affect the growth of Spirulina and the synthesis of GG.

[0029] In the present invention, the types of the algal cells are Chlorella, diatom, Haematococcus pluvialis, Dunaliella, Chlorella vulgaris or Spirulina, etc., and the types are not limited. In the most preferred specific embodiment of the present invention, the type of the algal cells is Spirulina.

[0030] As an alternative embodiment, after the step of obtaining the cultured algal cells, a GG generation stage is further included: The culture medium in this stage contains a substance that can change the cell osmotic pressure, and the concentration of this substance in the culture medium is 100-300 mmol / L.

[0031] In a more preferred embodiment of the present invention, a method for restoring the protein content during the production of glycerol glucoside is provided, and this method includes the following steps:

[0032] The stage of obtaining the cultured algal cells;

[0033] GG elevation stage 1: Inoculate the algal cells in the above stage into a culture medium containing a substance that can change the cell osmotic pressure for cultivation, wherein the concentration of this substance in the culture medium is 300 < C1 ≤ 800 mmol / L;

[0034] GG elevation stage 2: Inoculate the algal cells in GG elevation stage 1 into a culture medium containing a substance that can change the cell osmotic pressure for cultivation, wherein the concentration of this substance in the culture medium is 800 < C2 ≤ 1500 mmol / L;

[0035] During the cultivation process of any of the above stages, 20%-50% of the nitrogen source is supplemented into the culture medium every 3-5 days.

[0036] In the most preferred embodiment of the present invention, a method for restoring the protein content during the production of glycerol glucoside is provided, and this method includes the following steps:

[0037] Obtaining the stage of cultured algal cells;

[0038] GG generation stage: inoculating the algal cells in the above stage into a culture medium containing a substance capable of changing the cell osmotic pressure for culture, wherein the concentration of the substance in the culture medium is 100 - 300 mmol / L;

[0039] GG elevation stage 1: inoculating the algal cells in the GG generation stage into a culture medium containing a substance capable of changing the cell osmotic pressure for culture, wherein the concentration of the substance in the culture medium is 300 < C1 ≤ 800 mmol / L;

[0040] GG elevation stage 2: inoculating the algal cells in the GG elevation stage 1 into a culture medium containing a substance capable of changing the cell osmotic pressure for culture, wherein the concentration of the substance in the culture medium is 800 < C2 ≤ 1500 mmol / L;

[0041] During the culture process of any of the above stages, 20% - 50% of the nitrogen source is supplemented into the culture medium every 3 - 5 days.

[0042] In a more preferred embodiment of the present invention, during the culture process of the last stage, 20% - 50% of the nitrogen source is supplemented into the culture medium every 3 - 5 days.

[0043] In the most preferred embodiment of the present invention, during the culture process of all stages, 20% - 50% of the nitrogen source is supplemented into the culture medium every 3 - 5 days.

[0044] In the present invention, the substance capable of changing the cell osmotic pressure is one or more combinations of inorganic salts and / or organic salts.

[0045] Preferably, the inorganic salt is one or more of sodium chloride, sodium sulfate, potassium chloride or other inorganic salts; the organic salt is one or more of sodium formate, ammonium acetate or other organic salts.

[0046] In the present invention, natural light or variable light can be used for the light in each culture stage. To make the culture effect optimal, in the preferred embodiment of the present invention, the light wavelength range in each culture stage is 400 - 700 nm.

[0047] In the most preferred embodiment, the GG generation stage, the GG elevation stage 1 and the GG elevation stage 2 are processes of inducing GG, accumulating GG and preventing the synthesized GG from being consumed by other environmental conditions, and are processes of gradually increasing the GG content step by step.

[0048] In the stage of obtaining the cultured algal cells:

[0049] There are various ways or methods to obtain cultured algal cells, and there is no special limitation here. However, in order to quickly and effectively produce a large number of algal cells, the present invention proposes a preferred method for culturing algal species, which includes: inoculating algal cells in a low-salt fresh water medium for culture, where the salt concentration is less than 100 mmol / L.

[0050] Preferably, the culture time at this stage is 3 - 20 days.

[0051] Preferably, the culture temperature at this stage is: 15 - 40 °C, and further preferably, the culture temperature at this stage is 20 - 40 °C.

[0052] Preferably, the low-salt fresh water medium contains nitrogen, phosphorus, iron, magnesium, sodium, potassium, and trace elements required for the growth of microalgae.

[0053] Preferably, the formula of the low-salt fresh water medium is Zarrouk medium, and the detailed components are shown in Table 1 and Table 2. This low-salt fresh water medium can enable the algal cells to multiply in large numbers, laying a foundation for the large-scale synthesis and accumulation of GG.

[0054] Table 1 Formula of Z medium

[0055]

[0056] Table 2 Formula of mother liquor

[0057]

[0058] In this stage, light of an appropriate wavelength and a carbon source are selected for photosynthesis.

[0059] For the carbon source, under autotrophic conditions, a mixed air containing carbon dioxide is selected, with the carbon dioxide concentration within 10% (v / v), preferably, the carbon dioxide concentration is 1 - 5% (v / v), or an inorganic carbonate is selected, or both a mixed air containing carbon dioxide and an inorganic carbonate are selected; under heterotrophic conditions, glucose, maltose, glycerol, acetic acid, etc. are additionally added to the low-salt fresh water medium.

[0060] In the GG generation stage:

[0061] The algal cells selected are the cells grown after being cultured in the above-mentioned stage of obtaining cultured algal cells. Under normal circumstances, before being inoculated into the medium for the GG generation stage, the cells do not contain GG components inside, or even if they do, the amount is very small.

[0062] The culture medium used for cell culture should not only ensure the growth of algal cells but also enable the synthesis of GG within the algal cells. In addition to obtaining the nutrient elements required for the growth of microalgae in the cultured algal cell stage described above, substances that create stress conditions for the cells and induce the GG synthesis reaction need to be added. Usually, substances that can change the cell osmotic pressure are selected. For example, substances with a concentration of 100 - 300 mmol / L that can change the cell osmotic pressure can be added. This substance can be sodium chloride, potassium chloride, etc., and is not limited to the aforementioned substances, as long as it can induce the GG synthesis reaction.

[0063] In addition to the light energy within the wavelength range required for photosynthesis described above, although continuous light illumination can also promote the accumulation of GG, compared with continuous light illumination, intermittent light illumination can promote the accumulation of GG more effectively, and temperature difference can also promote the accumulation of GG more effectively. During the dark reaction process of intermittent light illumination, reducing the oxygen concentration in the introduced carbon dioxide mixture can promote the accumulation of GG more effectively.

[0064] Based on this, the culture conditions at this stage are optimized in order to obtain algal cells containing GG, providing a good foundation for the boosting stage.

[0065] Preferably, the culture time at this stage is 3 - 20 days, and more preferably, the culture time at this stage is 5 - 10 days.

[0066] Preferably, the culture temperature is 15 - 40 °C, and more preferably, the culture temperature is 20 - 40 °C.

[0067] Preferably, intermittent light illumination is selected at this stage, with a light - dark ratio of 1:1, and the light and dark durations are 6 - 18 h and 6 - 18 h respectively, and the light intensity is 500 - 3000 μE·m -2 ·s -2 。

[0068] More preferably, during the dark reaction process of intermittent light illumination, the oxygen concentration is reduced to 1 - 2% (v / v). After a large number of experimental verifications, reducing the oxygen concentration in the introduced carbon dioxide mixture during the dark reaction process of intermittent light illumination can promote the accumulation of GG more effectively.

[0069] In the first GG boosting stage:

[0070] In the most preferred implementation, the algal cells are characterized by already containing a certain amount of GG after being cultured in the GG generation stage. During long - term continuous culture in the GG generation stage, the cells will secrete some substances that hinder cell growth, resulting in a decrease in cell metabolic activity and a decline in the GG synthesis ability within the cells. However, when the cells containing a certain amount of GG after culture are inoculated into the culture medium of the first GG boosting stage, the cells can recover their activity and continue to grow, providing a driving force for the GG synthesis reaction and improving the efficiency of GG accumulation.

[0071] The culture medium conditions in the first stage of GG elevation are basically the same as those in the GG generation stage. The culture medium used for cell culture should ensure the growth of algal cells while allowing the synthesis of GG in the algal cells. In addition to the nutrient elements required for the growth of microalgae, substances that can create stress conditions for cells and induce the GG synthesis reaction need to be added. The addition amount of this GG inducer should be at least maintained at the same level as in the GG generation stage. Increasing the addition amount of the GG inducer can more effectively promote the GG synthesis reaction. For example, adding sodium chloride or potassium chloride at a concentration of 300 < C1 ≤ 800 mmol / L, and it is not limited to the aforementioned substances, as long as they can induce the GG synthesis reaction.

[0072] In addition to the light energy within the wavelength range required for photosynthesis mentioned above, continuous light can also promote the accumulation of GG. However, compared with continuous light, intermittent light can promote the accumulation of GG more effectively, and temperature difference can also promote the accumulation of GG more effectively. During the dark reaction process of intermittent light, reducing the oxygen concentration in the mixed carbon dioxide gas introduced can promote the accumulation of GG more effectively.

[0073] Based on this, the culture conditions in the first stage of GG elevation are optimized to obtain algal cells with a high content of GG.

[0074] Preferably, the culture time is greater than 2 days, and the preferred time is 3 - 5 days.

[0075] Preferably, the culture temperature is 15 - 40°C; more preferably, the temperature during the dark period is set at 15 - 25°C, and the temperature during the light period is set at 25 - 40°C.

[0076] Preferably, intermittent light is selected at this stage, the light - dark ratio is 1:1, the light and dark durations are 6 - 18 h and 6 - 18 h respectively, and the light intensity is 500 - 3000 μE·m -2 ·s -2 。

[0077] More preferably, during the dark reaction process of intermittent light, the oxygen concentration is reduced to 1 - 2% (v / v). After a large number of experimental verifications, during the dark reaction process of intermittent light, the mixed gas introduced can not only reduce the oxygen concentration but also promote the accumulation of GG.

[0078] In the most preferred embodiment, after culturing in the first stage of GG elevation, algal cells with a GG content of 10% (w / w) or more can be obtained.

[0079] In the second stage of GG elevation:

[0080] For autotrophic microalgae, the same culture conditions as in the first stage of GG enhancement are selected, with the difference that the concentration of the substance capable of changing the cell osmotic pressure in the culture medium is 800 < C2 ≤ 1500 mmol / L.

[0081] For heterotrophic microalgae or microalgae cells with enhanced small molecule permeability of the cell wall after genetic engineering modification, in addition to making the concentration of the substance capable of changing the cell osmotic pressure 800 < C2 ≤ 1500 in this culture medium, reaction substrates for synthesizing GG need to be added, such as glycerol or available sugars, such as glucose and maltose, but not limited to the above two sugars, which can further increase the GG content in microalgae cells. Preferably, the glycerol or glucose is maintained at a concentration of 0.5 - 2 g / L for glycerol and 0.5 - 5 g / L for glucose by means of feeding.

[0082] Preferably, the culture time is greater than 2 days, and the preferred time is 3 - 5 days.

[0083] The reactor used in each step of the present invention is not limited and can be a closed reactor or an open raceway pond. For heterotrophic microalgae cells, using a closed reactor can more effectively prevent contamination by miscellaneous bacteria.

[0084] In a specific embodiment of the present invention, the following method is used to detect the GG content in microalgae cells, and this method includes an extraction method and a detection method:

[0085] Among them, the extraction method includes the following steps:

[0086] (1) Take 2 mL of the algal solution and centrifuge it at 10,000 rpm / min for 30 min to separate the precipitate from the supernatant.

[0087] (2) Add 200 μL of water to the cell precipitate and mix well, then add 800 μL of absolute ethanol and mix again, and incubate in a water bath at 65 °C for 4 h.

[0088] (3) Centrifuge at 10,000 rpm / min for 10 min, discard the precipitate, dry the supernatant with N2, and dilute it with appropriate ddH2O for determination.

[0089] The GG detection method includes the following steps:

[0090] (1) Dilute the sample appropriately.

[0091] (2) Filter the dilution using a 0.22 μm filter to obtain the sample before ion chromatography detection.

[0092] (3) Use ion chromatography ICS - 5000 +(Thermo Fisher) performs sample determination, with the detector being a supporting electrochemical detector and the column being a Dinex CarboPac PA10 column with an inner diameter of 4×250 mm. Before use, the column is equilibrated with 25 mM NaOH at a flow rate of 1.0 mL / min. After the baseline of the electrochemical detector is stable, sample determination begins. TM CarboPac TM PA10 column. Before use, the column is equilibrated with 25 mM NaOH at a flow rate of 1.0 mL / min. After the baseline of the electrochemical detector is stable, sample determination begins.

[0093] To enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0094] Example 1

[0095] A method for recovering protein content during the production of glycerol glucoside, which specifically includes the following steps:

[0096] (1) Obtaining the stage of cultured algal cells:

[0097] The algal cells are inoculated into a low-salt fresh water medium, Zarrouk medium (see Tables 1 and 2 for details), with an initial inoculation concentration of 0.2 g / L. The temperature program of the fresh water medium for culturing the algal cells is controlled as follows: constant temperature of 25°C, light program: continuous light, light intensity of 500 μE·m -2 ·s -2 , light wavelength range of 400 - 700 nm, aeration rate of: 0.3 VVM, and the gas introduced is a mixed air containing carbon dioxide, with a carbon dioxide concentration of 5% (v / v).

[0098] The culturing time at this stage is 5 - 7 days.

[0099] (2) GG elevation stage:

[0100] The algal cells selected are the cells grown after culturing in the above step (1). The temperature program of the fresh water medium for culturing the algal cells is controlled as follows: temperature of 32°C during light, temperature of 22°C during darkness, light program: light-dark ratio of 1:1, light and dark durations are 12 hours and 12 hours respectively, light intensity of 1000 μE·m -2 ·s -2 , light wavelength range of 400 - 700 nm, aeration rate of: 0.3 VVM, and the gas introduced is a mixed air containing carbon dioxide, with a carbon dioxide concentration of 5% (v / v).

[0101] Among them, the fresh water medium is the fresh water medium in step (1) with sodium chloride added so that its concentration in the fresh water medium is 400 mmol / L.

[0102] The culturing time at this stage is 3 - 5 days.

[0103] On the 3rd day at the beginning of the boosting stage in the above cultivation process, add 20% of the nitrogen source to the culture medium, and the nitrogen source is urea.

[0104] After culturing through the above two steps and detecting, algal cells with a GG content of 17% and a protein content of 61% (w / w) or more can be obtained.

[0105] Example 2

[0106] A method for restoring protein content during the production of glycerol glucoside, the method comprising the following steps:

[0107] (1) Obtaining the stage of cultured algal cells:

[0108] Inoculate the algal cells into the low-salt fresh water medium Zarrouk medium (see Tables 1 and 2 for details), with an initial inoculation concentration of 0.2 g / L, and control the temperature program of the fresh water medium for culturing the algal cells as follows: keep the temperature constant at 25 °C, and the light program is: continuous light, with a light intensity of 500 μE·m -2 ·s -2 , the light wavelength range is 400 - 700 nm, the aeration rate is: 0.3 VVM, and the gas introduced is a mixed air containing carbon dioxide, with a carbon dioxide concentration of 5% (v / v).

[0109] The culturing time in this stage is 5 - 7 days.

[0110] (2) GG boosting stage:

[0111] Select the algal cells grown after culturing in the above step (1), and control the temperature program of the fresh water medium for culturing the algal cells as follows: the temperature is 32 °C during light, 22 °C during darkness, the light program is: the light-dark ratio is 1:1, the light and dark durations are 12 hours and 12 hours respectively, and the light intensity is 1000 μE·m -2 ·s -2 , the light wavelength range is 400 - 700 nm, the aeration rate is: 0.3 VVM, and the gas introduced is a mixed air containing carbon dioxide, with a carbon dioxide concentration of 5% (v / v).

[0112] Among them, the fresh water medium is the fresh water medium in step (1) with sodium chloride added so that its concentration in the fresh water medium is 900 mmol / L.

[0113] The culturing time in this stage is 3 - 5 days.

[0114] During the whole process of the above cultivation, add 20% of the nitrogen source to the culture medium every 4 days, and the nitrogen source is ammonium bicarbonate and sodium nitrate.

[0115] After culturing through the above two steps, it can be detected that algal cells with a GG content of 31% and a protein content of 61% (w / w) or more can be obtained.

[0116] Example 3

[0117] A method for restoring protein content during the production of glycerol glucoside, the method comprising the following steps:

[0118] (1) Obtaining the stage of cultured algal cells:

[0119] Inoculate the algal cells into the low-salt fresh water medium Zarrouk medium (see Tables 1 and 2 for details), with an initial inoculation concentration of 0.2 g / L. Control the temperature program of the fresh water medium for culturing the algal cells as follows: constant temperature of 25 °C, and the light program: continuous light, with a light intensity of 500 μE·m -2 ·s -2 , the light wavelength range is 400 - 700 nm, the aeration rate is: 0.3 VVM, and the gas introduced is a mixed air containing carbon dioxide, with a carbon dioxide concentration of 5% (v / v).

[0120] The culturing time at this stage is 5 - 7 days.

[0121] Among them, the specific formula of the fresh water medium is Zarrouk medium (see Tables 1 and 2 for details).

[0122] (2) The first stage of GG elevation:

[0123] Select the algal cells grown after culturing in the above step (1). Control the temperature program of the fresh water medium for culturing the algal cells as follows: the temperature is 32 °C during light and 22 °C during darkness, and the light program: the light-dark ratio is 1:1, with the light and dark durations being 12 hours and 12 hours respectively, and the light intensity is 1000 μE·m -2 ·s -2 , the light wavelength range is 400 - 700 nm, the aeration rate is: 0.3 VVM, and the gas introduced is a mixed air containing carbon dioxide, with a carbon dioxide concentration of 5% (v / v).

[0124] Among them, the fresh water medium is the fresh water medium in step (1) with sodium chloride added so that its concentration in the fresh water medium is 400 mmol / L.

[0125] The culturing time at this stage is 5 - 7 days.

[0126] (3) The second stage of GG elevation:

[0127] The algal cells are the cells grown after culturing in the above step (2). The culturing conditions at this stage are basically the same as those in step (2), with the difference being that sodium chloride is further added to the fresh water medium in step (2) to make its concentration in the fresh water medium 900 mmol / L.

[0128] The culturing time at this stage is 5 days.

[0129] During the whole process of the above culturing, a 40% nitrogen source is supplemented to the medium every 5 days. The nitrogen source is diammonium phosphate.

[0130] After culturing through the above three steps and through detection, algal cells with a GG content of 30% and a protein content of 61% (w / w) or more can be obtained.

[0131] Example 4

[0132] A method for restoring the protein content during the production of glycerol glucoside, the method comprising the following steps:

[0133] (1) Stage of obtaining cultured algal cells:

[0134] Inoculate the algal cells into a low-salt fresh water medium, Zarrouk medium (see Tables 1 and 2 for details), with an initial inoculation concentration of 0.2 g / L. Control the temperature program of the fresh water medium for culturing the algal cells as follows: keep the temperature constant at 25 °C, and the light program as follows: continuous light, with a light intensity of 500 μE·m -2 ·s -2 , with the light wavelength range of 400 - 700 nm, and the aeration rate of: 0.3 VVM. The gas introduced is mixed air containing carbon dioxide, and the carbon dioxide concentration is 5% (v / v).

[0135] The culturing time at this stage is 5 - 7 days.

[0136] Among them, the specific formula of the fresh water medium is Zarrouk medium (see Tables 1 and 2 for details). (2) GG generation stage:

[0137] Select the algal cells grown after culturing in the above step (1). Control the temperature program of the fresh water medium for culturing the algal cells as follows: the temperature is 32 °C during light and 22 °C during darkness, and the light program as follows: the light-dark ratio is 1:1, with the light and dark durations being 12 hours and 12 hours respectively, and the light intensity of 1000 μE·m -2 ·s -2 , with the light wavelength range of 400 - 700 nm, and the aeration rate of: 0.3 VVM. The gas introduced is mixed air containing carbon dioxide, and the carbon dioxide concentration is 5% (v / v).

[0138] Among them, the fresh water culture medium is the fresh water culture medium in step (1) added with sodium chloride so that its concentration in the fresh water culture medium is 200 mmol / L.

[0139] The culture time at this stage is 5 - 7 days.

[0140] (3) GG boosting stage one:

[0141] The characteristics of the algal cells at this stage are that they already contain a certain amount of GG after being cultured in step (2). The culture conditions at this stage are basically the same as those in step (2), with the difference being that: the fresh water culture medium is the fresh water culture medium in step (2) further added with sodium chloride so that its concentration in the fresh water culture medium is 400 mmol / L.

[0142] The culture time at this stage is 3 - 5 days.

[0143] After being cultured in step (3) and detected, algal cells with a GG content of more than 11% (w / w) can be obtained.

[0144] (4) GG boosting stage two:

[0145] For autotrophic microalgae - Spirulina, the same culture conditions as in step (3) are selected, with the difference being that: the microalgal cells obtained in step (3) are inoculated into the fresh culture medium in step (4), and the fresh culture medium is Zarrouk medium containing 900 mmol / L sodium chloride.

[0146] The culture time at this stage is 5 days.

[0147] During the whole process of the above culture process, 20% of the nitrogen source is supplemented in the culture medium every 5 days, and the nitrogen source is ammonium bicarbonate and sodium nitrate.

[0148] After being cultured through the above four steps and detected, algal cells with a GG content of 32% and a protein content of more than 64% (w / w) can be obtained.

[0149] Example 5

[0150] A method for restoring the protein content during the production of glycerol glucoside, the method comprising the following steps:

[0151] (1) Obtaining the cultured algal cell stage:

[0152] The algal cells are inoculated into a fresh water culture medium, with an initial inoculation concentration of 0.15 g / L, and the temperature program of the fresh water culture medium for culturing the algal cells is controlled as: constant temperature of 28 °C, and the light program is: continuous light, with a light intensity of 200 μE·m -2 ·s -2, the light wavelength range is 400 - 700 nm, the aeration rate is 0.2 VVM, the gas introduced is mixed air containing carbon dioxide, the carbon dioxide concentration is 2% (v / v), and 100 mmol of NaHCO3 is added to the culture medium.

[0153] The culture time for this stage is 3 - 5 days.

[0154] Among them, the specific formula of the fresh water culture medium is: Zarrouk medium.

[0155] (2) GG generation stage:

[0156] The algal cells selected are the cells grown after culturing in the above step (1). The temperature program of the fresh water culture medium for culturing the algal cells is controlled as follows: the temperature is 32 °C during illumination and 23 °C during darkness. The illumination program is: the light - dark ratio is 1:1, the light and dark durations are 12 hours and 12 hours respectively, and the light intensity is 300 μE·m -2 ·s -2 , the light wavelength range is 400 - 700 nm, the aeration rate is 0.2 VVM, the gas introduced is mixed air containing carbon dioxide, and the carbon dioxide concentration is 5% (v / v).

[0157] Among them, the fresh water culture medium is the fresh water culture medium in step (1) with sodium chloride added so that its concentration in the fresh water culture medium is 150 mmol / L.

[0158] The culture time for this stage is 3 - 5 days.

[0159] (3) GG elevation stage one:

[0160] The characteristics of the algal cells in this stage are that they already contain a certain amount of GG after culturing in step (2). The culture conditions for this stage are basically the same as those in step (2), with the difference being that: the fresh water culture medium is the fresh water culture medium in step (2) with sodium chloride continued to be added so that its concentration in the fresh water culture medium is 500 mmol / L.

[0161] The culture time for this stage is 3 days.

[0162] After culturing through step (3) and detection, algal cells with a GG content of more than 10% (w / w) can be obtained.

[0163] (4) GG elevation stage two:

[0164] For autotrophic microalgae - Spirulina, the same culture conditions as in step (3) are selected, with the difference being that: the microalgae cells obtained in step (3) are inoculated into the fresh culture medium in step (4), and the fresh culture medium is Zarrouk medium containing 900 mmol of sodium chloride.

[0165] The cultivation time for this stage is 3 days.

[0166] During the above cultivation process, at the beginning of the GG boosting stage, 20% of the nitrogen source, which is diammonium phosphate, is supplemented to the culture medium every 4 days.

[0167] After cultivation through the above four steps and detection, algal cells with a GG content of 31% and a protein content of 63% (w / w) or more can be obtained.

[0168] Example 6

[0169] A method for restoring the protein content during the production of glucosylglycerol, the method comprising the following steps:

[0170] (1) Obtaining the stage of cultivated algal cells:

[0171] Inoculate the algal cells into a freshwater culture medium with an initial inoculation concentration of 0.1 g / L, and control the temperature program of the freshwater culture medium for cultivating the algal cells as follows: keep the temperature constant at 30 °C, and the light program is: continuous light, with a light intensity of 200 μE·m -2 ·s -2 , the light wavelength range is 400 - 700 nm, the aeration rate is: 0.2 VVM, and the gas introduced is a mixed air containing carbon dioxide, with a carbon dioxide concentration of 2% (v / v).

[0172] The cultivation time for this stage is 5 - 7 days.

[0173] Among them, the specific formula of the freshwater culture medium is: Zarrouk medium.

[0174] (2) GG generation stage:

[0175] Select the algal cells grown after cultivation in the above step (1), and control the temperature program of the freshwater culture medium for cultivating the algal cells as follows: the temperature is 32 °C during light and 25 °C during darkness, the light program is: the light - dark ratio is 1:1, the light and dark durations are 12 hours and 12 hours respectively, and the light intensity is 250 μE·m -2 ·s -2 , the light wavelength range is 400 - 700 nm, the aeration rate is: 0.2 VVM, and the gas introduced is a mixed air containing carbon dioxide, with a carbon dioxide concentration of 5% (v / v).

[0176] Among them, the freshwater culture medium is the freshwater culture medium in step (1) with sodium chloride added so that its concentration in the freshwater culture medium is 200 mmol / L.

[0177] The cultivation time for this stage is 3 - 5 days.

[0178] (3) GG boosting stage one:

[0179] The characteristics of the algal cells at this stage are that they already contain a certain amount of GG after being cultured in step (2). The culture conditions at this stage are basically the same as those in step (2), with the difference being that sodium chloride is continuously added to the fresh water medium in step (2) to make its concentration in the fresh water medium 500 mmol / L.

[0180] The culture time at this stage is 3 days.

[0181] After being cultured in step (3) and detected, algal cells with a GG content of more than 10% (w / w) can be obtained.

[0182] (4) GG Enhancement Stage Two:

[0183] For autotrophic microalgae - Spirulina, the same culture conditions as in step (3) are selected, with the difference being that the microalgae cells obtained in step (3) are inoculated into the fresh medium in step (4), and the fresh medium is Zarrouk medium containing 950 mmol / L sodium chloride.

[0184] The culture time at this stage is 3 - 5 days.

[0185] On the 4th day of the culture in the second enhancement stage during the above - mentioned culture process, 50% of the nitrogen source, which is diammonium phosphate, is supplemented to the medium.

[0186] After being cultured through the above four steps and detected, algal cells with a GG content of 33% and a protein content of more than 60.5% (w / w) can be obtained.

[0187] The above - mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above - mentioned embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for restoring protein content during the production of glycerol glucoside, characterized in that The method comprises the following steps: Obtaining the stage of cultured Spirulina platensis algal cells; the culture time is 3 - 20 days; And including GG boosting stage two, or, GG boosting stage one and GG boosting stage two, wherein, GG boosting stage one: The culture medium in this stage contains a substance capable of changing the cell osmotic pressure, and the concentration of this substance in the culture medium is 300 < C1 ≤ 800 mmol / L; the culture time is 3 - 20 days; the culture temperature is: 20 - 40 °C; Intermittent light is selected at this stage, with a light duration of 6 - 18 hours, a dark duration of 6 - 18 hours, and a light intensity of 500 - 3000 μE·m -2 ·s -2 ; During the dark reaction of intermittent light, the oxygen concentration is reduced to 1 - 2% (v / v); GG boosting stage two: The culture medium in this stage contains a substance capable of changing the cell osmotic pressure, and the concentration of this substance in the culture medium is 800 < C2 ≤ 1500 mmol / L; the culture time is 3 - 5 days; Intermittent light is selected at this stage, with a light duration of 6 - 18 hours, a dark duration of 6 - 18 hours, and a light intensity of 500 - 3000 μE·m -2 ·s -2 ; During the dark reaction of intermittent light, the oxygen concentration is reduced to 1 - 2% (v / v); During the culture process of any of the above stages, 20% - 50% of the nitrogen source is added to the culture medium every 3 - 5 days, and any of the above stages refers to GG boosting stage two, or, GG boosting stage one and GG boosting stage two; After the step of obtaining the stage of cultured Spirulina platensis algal cells, it further includes the GG generation stage: The culture medium in this stage contains a substance capable of changing the cell osmotic pressure, and the concentration of this substance in the culture medium is 100 - 300 mmol / L; the culture time is 3 - 20 days; the culture temperature is: 20 - 40 °C; Intermittent light is selected at this stage, with a light duration of 6 - 18 hours, a dark duration of 6 - 18 hours, and a light intensity of 500 - 3000 μE·m -2 ·s -2 ; During the dark reaction of intermittent light, the oxygen concentration is reduced to 1 - 2% (v / v); The substance capable of changing the cell osmotic pressure is one or more combinations of inorganic salts and / or organic salts; The light wavelength range of the light in each culture stage is 400 - 700 nm; For the carbon source, under autotrophic conditions, a mixed air containing carbon dioxide is selected, and the concentration of carbon dioxide is 1 - 5% (v / v), or an inorganic carbonate is selected, or a mixed air containing carbon dioxide and an inorganic carbonate are selected simultaneously; under heterotrophic conditions, one or two of glucose, maltose, glycerol, and acetic acid are selected.

2. The method according to claim 1, wherein: The inorganic salts are one or more of sodium chloride, sodium sulfate, potassium chloride, sodium bicarbonate, sodium carbonate, or other inorganic salts; the organic salts are one or more of sodium formate, ammonium acetate, or other organic salts.

3. A method for preparing glyceroglucoside and algal protein, the method comprising the steps of the method for restoring the protein content in the production of glyceroglucoside according to any one of claims 1 - 2.

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