A method for restoring protein content in the process of producing glycerol glucoside

Abstract

The present invention discloses a method for restoring protein content during the production of glycerol glucoside. The method comprises the following steps: (1) an initial accumulation stage of algal cells and proteins: the medium used in this stage does not contain a stressor used for salt stress; (2) a glycerol glucoside synthesis stage of algal cells: after the first stage, a stressor required for salt stress at a corresponding concentration is added to the medium, and the algal cells begin to synthesize GG; (3) an algal cell protein recovery stage: after the second stage, the algal cells are harvested and GG is separated, and the remaining algal cells are cultured in the same fresh medium as in the first stage, and then the algal cells are collected to obtain algal powder. Using this process, not only can a high yield of glycerol glucoside be obtained, but 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 invention relates to a method for recovering protein content in the process of producing glycerol glucoside, and belongs to the technical field of microalgae cell cultivation. Background Art

[0002] Spirulina, also known as blue algae, has been shown to mitigate the toxic side effects of cancer radiotherapy and chemotherapy, enhance immune function, and lower blood lipids. Rich in vitamins, protein, unsaturated fatty acids, and trace elements, protein is its primary nutrient. Currently, spirulina has become a highly sought-after protein source in the food industry, recognized as the algae with the greatest potential for producing single-cell protein.

[0003] In recent decades, research has been conducted both domestically and internationally on the separation, extraction, and application of Spirulina algal cell protein, including the enzymatic hydrolysis of algal protein into peptides for use in health supplements and food additives. General literature reports indicate that algal cell protein content is approximately 60-70%. However, in large-scale industrial production, the protein content of most products is only 50-60%. Furthermore, the inventors of this application have discovered that the protein content can be very low during the production of Spirulina. Protein content is a key indicator of algal products, and the national standard for algal powder for food is ≥55%. Therefore, increasing algal protein content is crucial for improving the quality of algal products, developing new products, and utilizing algal protein resources.

[0004] At present, there are many studies on increasing the protein content of algal cells when simply producing Spirulina protein. For example, Li Bosheng et al. studied the method of promoting protein accumulation in Spirulina cells, and Chen Tianfeng et al. studied the effect of the fractional selenium addition method on the cultivation of high-selenium-rich Spirulina and its effect on the protein content of the algae. However, there are fewer studies on increasing the protein content when producing glycerol glucoside (GG), and it is difficult to increase both at the same time.

[0005] Therefore, there is an urgent need to find a suitable cultivation method to restore the protein content when producing GG, so as to meet the industrial application of glycerol glucoside algae cell cultivation and meet the national standard requirement of protein content ≥ 55%. Summary of the Invention

[0006] In response to the above prior art, the inventors discovered during their research into the production of GG from Spirulina that the protein content in the algal cells dropped significantly, to only about 40%, far below the national standard of ≥55% for protein content. This means that the Spirulina powder obtained from conventional GG production processes cannot be sold commercially and can only become a byproduct of glycerol glucoside production. Based on this discovery, the present invention proposes a method for restoring protein content during the production of glycerol glucoside. To this end, the inventors improved the culture process, without affecting algal cell growth and glycerol glucoside synthesis, to increase the protein content of the algal powder to meet the national standard of ≥55%. This allows both the algal powder and GG obtained under this process to be sold as products.

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

[0008] In a first aspect of the present invention, a method for recovering protein content in a glycerol glucoside production process is provided, the method comprising the steps of:

[0009] (1) Initial accumulation stage of algal cells and proteins: The culture medium used in this stage does not contain any additional salt stress substances;

[0010] (2) Algal cell synthesis of glycerol glucoside stage: After the first stage, the corresponding concentration of salt stress required by the stressor is added to the culture medium, and at this stage the algal cells begin to synthesize GG;

[0011] (3) Algal cell protein recovery stage: After the second stage, the algal cells are harvested and GG is separated. The remaining algal cells are cultured in the same fresh culture medium as in the first stage, and then the algal cells are collected to obtain algal powder.

[0012] In a second aspect of the present invention, a method for preparing glycerol glucoside and algal protein is provided, which includes a step of restoring protein content during the production of glycerol glucoside.

[0013] In the third aspect of the present invention, the glycerol glucoside and / or protein product prepared by the above method is also protected.

[0014] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0015] The present invention provides a process for increasing the protein content of algal cells during large-scale culturing of algal cells for glycerol glucoside synthesis. This process not only produces a high yield of glycerol glucoside, but also ensures that the protein content of the resulting algal powder exceeds 60% (w / w), meeting the national standard requirement of ≥55%. DETAILED DESCRIPTION

[0016] It should be noted that the following detailed descriptions are exemplary and 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 skilled in the art to which the present invention belongs.

[0017] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations and / or combinations thereof.

[0018] Explanation of terms:

[0019] Microalgae: generally refers to algae that are so tiny that they can only be identified under a microscope.

[0020] As described in the background art, the prior art algal cell cultivation process has certain deficiencies, resulting in a large amount of microalgal protein loss during the GG production process, which cannot meet the national standard requirement of a protein content of ≥55%. In order to solve the above technical problems, the present invention proposes a method for restoring protein content during the production of glycerol glucoside, which comprises the following steps:

[0021] (1) Initial accumulation of algal cells and proteins: The medium used in this stage does not contain any additional salt stressors, and the algal cells are cultured for 3 to 7 days.

[0022] (2) Algal cell synthesis of glycerol glucoside stage: After the first stage, the corresponding concentration of salt stress required by the culture medium is added. In this stage, the algal cells begin to synthesize GG. The culture time in this stage is more than 3 days, preferably 5-7 days;

[0023] (3) Algal cell protein recovery stage: After the second stage, the algal cells are harvested and GG is separated. The remaining algal cells are cultured in the same fresh culture medium as the first stage for 3-10 days, and then the algal cells are collected to obtain algal powder.

[0024] Among them, step (1) is the process of producing a large number of algal cells and accumulating proteins, step (2) is the process of inducing and accumulating GG, and step (3) is the process of restoring the protein content.

[0025] The reactor used in steps (1) to (3) is not limited and can be a closed reactor or an open raceway tank.

[0026] In the present invention, the algal cell species is green algae, diatom, haematococcus, Dunaliella, Chlorella or Spirulina, etc., and the species is not limited. In the most preferred embodiment of the present invention, the algal cell species is Spirulina.

[0027] In a preferred embodiment of the present invention, the stressor is a substance that can change the osmotic pressure of cells, and the substance can be one or more combinations of inorganic salts and / or organic salts.

[0028] Furthermore, 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.

[0029] In a preferred embodiment of the present invention, the culture medium used in each culturing stage is a freshwater culture medium containing nitrogen, phosphorus, iron, magnesium, sodium, potassium, and trace elements required for microalgae growth, such as commonly used BG-11 medium or Zarrouk's medium (see Tables 1 and 2 for details). This freshwater culture medium enables the proliferation of algal cells, facilitating the synthesis and accumulation of large amounts of GG and protein. The present disclosure is not limited to the freshwater culture media listed above.

[0030] The "additional" in step (1) means that inorganic salts and / or organic salts in the culture medium are not included.

[0031] In a specific embodiment of the present invention, in steps (1) and (3), Zarrouk medium is used, and in step (2), a stressor at a concentration required for salt stress is added to the Zarrouk medium.

[0032] Table 1 Zarrouk medium formula

[0033]

[0034]

[0035] Table 2 Mother liquor formula

[0036]

[0037] The present invention selects appropriate wavelengths of light and carbon sources for photosynthesis. In a preferred embodiment of the present invention, the wavelength of light at each stage is in the range of 400-700 nm. Experiments have found that exposure to light within a specific wavelength range can increase the GG and protein content of microalgae cells.

[0038] As for the carbon source, in a preferred embodiment of the present invention, a mixed air containing carbon dioxide is selected, and the carbon dioxide concentration is within 10% (v / v), preferably, the carbon dioxide concentration is 1-5% (v / v), or an inorganic carbonate is selected, or a mixed air containing carbon dioxide and an inorganic carbonate are selected at the same time.

[0039] In addition to the aforementioned light energy within the wavelength range required for photosynthesis, continuous illumination can also promote GG accumulation. However, intermittent illumination is more effective than continuous illumination, and temperature differences also promote GG accumulation. During the dark reaction under intermittent illumination, reducing the oxygen concentration in the carbon dioxide mixture further promotes GG accumulation.

[0040] Therefore, in a preferred embodiment of the present invention, the illumination conditions of each culture stage adopt intermittent illumination, the light-dark ratio is (1-2): (1-2), the illumination time is 6-18 hours, the dark time is 6-18 hours, and the light intensity is 500-3000 μE·m -2 ·s -2 .

[0041] Furthermore, during the dark reaction under intermittent illumination, the oxygen concentration was reduced to 1-2% (v / v). After a large number of experiments, it was verified that during the dark reaction under intermittent illumination, reducing the oxygen concentration in the introduced carbon dioxide mixture could further promote the accumulation of GG.

[0042] In a preferred embodiment of the present invention, the culture temperature at each stage is: 15-40°C, preferably in the range of 20-40°C, the temperature during darkness is set at 15-25°C, and the temperature during light is set at 25-40°C.

[0043] During the algal cell glycerol glucoside synthesis stage, the culture medium used for cell culture must ensure both the growth of the algal cells and the synthesis of GG within the algal cells. In addition to the nutrients required for the growth of the microalgae in step (1), it is also necessary to add substances that create stress conditions for the cells and induce the GG synthesis reaction. Usually, substances that can change the cell osmotic pressure are selected, such as sodium chloride or potassium chloride at a concentration of 300-1500mM, but are not limited to the above substances. Any substance that can induce the GG synthesis reaction can be used. In a preferred embodiment of the present invention, during the salt stress culture stage of step (2), the concentration of the stressor in the culture medium is 500-1000mmol / L.

[0044] In a preferred embodiment of the present invention, in step (3), the method for separating GG is a hypotonic extraction separation method. The separation liquid used in the hypotonic extraction separation method is a hypotonic solution. When a certain concentration difference occurs between the solutions on both sides of the cell membrane, an osmotic pressure is generated, and osmosis occurs. A solution with a lower osmotic pressure than the intracellular osmotic pressure is called a hypotonic solution, such as distilled water.

[0045] The inventors have found that different culture methods and culture conditions have a great influence on the protein content in algal cells. The present invention obtains algal powder with a higher protein content through a specific culture method (including a day and night combination method, high salinity cultivation after culturing algal species, high light conditions, etc.) without affecting algal cell growth and glycerol glucoside synthesis.

[0046] In order 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 with reference to specific embodiments.

[0047] In a specific embodiment of the present invention, the following method is used to detect the GG content in algae cells, which includes an extraction method and a detection method:

[0048] The extraction method comprises the following steps:

[0049] (1) Take 2 mL of algae solution and centrifuge at 10,000 rpm / min for 30 min to separate the precipitate from the supernatant.

[0050] (2) Add 200 μL of water to the cell pellet and mix well. Then add 800 μL of anhydrous ethanol and mix again. Incubate in a 65°C water bath for 4 h.

[0051] (3) Centrifuge at 10,000 rpm / min for 10 min, discard the precipitate, blow dry the supernatant with N2, add appropriate ddH2O for dilution and then measure.

[0052] The GG detection method includes the following steps:

[0053] (1) Dilute the sample appropriately.

[0054] (2) The diluted solution was filtered using a 0.22 μm filter to obtain the sample before ion chromatography detection.

[0055] (3) Using ion chromatograph ICS-5000 + (Thermo Fisher) for sample determination, the detector is a matching electrochemical detector, and the column is a Dinex column with an inner diameter of 4×250 mm TM CarboPac TM PA10 chromatographic column. Before use, the column was equilibrated with 25 mM NaOH at 1.0 mL / min. Sample measurement was started after the baseline of the electrochemical detector was stable.

[0056] In the specific embodiment of the present invention, the conventional Kjeldahl method is used to detect the protein content.

[0057] Example 1

[0058] A method for recovering protein content in a glycerol glucoside production process, the method comprising the following steps:

[0059] (1) Initial accumulation stage of algal cells and proteins:

[0060] The algal cells were inoculated into the freshwater culture medium Zarrouk (see Table 1 and Table 2 for details) with an initial inoculation concentration of 0.2 g / L. The temperature of the freshwater culture medium for culturing the algal cells was controlled to be 25°C, and the illumination program was continuous illumination with an illumination intensity of 500 μE·m -2 ·s -2 The light wavelength range is 400-700 nm, the ventilation volume is 0.3 VVM, and the gas introduced is a mixed air containing carbon dioxide, and the carbon dioxide concentration is 5% (v / v).

[0061] The culture time at this stage is 5-7 days.

[0062] (2) Algal cells synthesize glycerol glucoside:

[0063] The algae cells are selected from the cells grown after culturing in the above step (1). The culture conditions are the same as those described in step (1), except that sodium chloride is added to the fresh water culture medium in step (1) to make its concentration in the fresh water culture medium 800mmol / L.

[0064] The culture time at this stage is 7 days.

[0065] (3) Algal cell protein recovery stage:

[0066] After the second stage, the algal cells were harvested and GG was separated by hypotonic extraction. The remaining algal cells were cultured in fresh culture medium consistent with the first stage for 5 days. The algal cells were then collected to obtain Spirulina powder. The protein content of the final harvested algal powder was greater than 60%, and the GG yield collected accounted for 16% of the cell dry weight.

[0067] Example 2

[0068] A method for recovering protein content in a glycerol glucoside production process, the method comprising the following steps:

[0069] (1) Initial accumulation stage of algal cells and proteins:

[0070] The algal cells were inoculated into the freshwater culture medium Zarrouk (see Tables 1 and 2 for details) with an initial inoculation concentration of 0.2 g / L. The temperature of the freshwater culture medium for culturing the algal cells was controlled to be 28°C, the light program was: a light-dark ratio of 1:1, a light-dark duration of 12 h and a dark duration of 12 h, respectively, and a light intensity of 650 μE·m -2 ·s -2 The light wavelength range is 400-700 nm, the ventilation volume is 0.3 VVM, and the gas introduced is a mixed air containing carbon dioxide, and the carbon dioxide concentration is 5% (v / v).

[0071] The culture time at this stage is 5 days.

[0072] (2) Algal cells synthesize glycerol glucoside:

[0073] The algae cells are selected from the cells grown after culturing in the above step (1). The culture conditions are the same as those described in step (1), except that sodium chloride is added to the fresh water culture medium in step (1) to make its concentration in the fresh water culture medium 900 mmol / L.

[0074] The culture time at this stage is 7 days.

[0075] (3) Algal cell protein recovery stage:

[0076] After the second stage, the algal cells were harvested and GG was separated by hypotonic extraction. The remaining algal cells were cultured in fresh culture medium consistent with the first stage for 5 days. The algal cells were then collected to obtain Spirulina powder. The protein content of the final harvested algal powder was greater than 60%, and the GG yield collected accounted for 17% of the cell dry weight.

[0077] Example 3

[0078] Spirulina was inoculated into the freshwater Zarrouk medium (see Tables 1 and 2 for details). On the third day of culture, a 500 mL sample was taken and then subjected to salt stress with the addition of sodium chloride, bringing the sodium chloride concentration in the freshwater medium to 900 mM. This sample was harvested, washed, and freeze-dried to obtain sample S1. On the seventh day of salt stress culture, another sample was harvested, washed, and freeze-dried to obtain sample S2 (freeze-dried, S2). The remaining algal cells were harvested, washed (glycerol glucoside extraction), and the entire algal sludge was re-inoculated into the freshwater Zarrouk medium and cultured for three days. The resulting freeze-dried sample was designated S3. Table 3 shows that after three days of salt-free culture (stage 1) followed by salt stress (stage 2), protein levels did not decrease but increased significantly. In the subsequent third stage of recovery culture, protein content continued to increase, but not significantly. Therefore, in the entire process, the first stage of salt-free culture has a significant impact on the final protein content.

[0079] Note: Other culture conditions are as shown in Example 2.

[0080] Table 3 Protein content of algal cells at different culture stages

[0081]

[0082] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for recovering protein content in a glycerol glucoside production process, the method comprising the following steps: (1) Initial accumulation stage of algal cells and proteins: the culture medium used in this stage does not contain any additional stressors used for salt stress; the culture time for the initial accumulation stage of algal cells and proteins is 3 days; (2) Glycerol glucoside synthesis stage of algal cells: After the first stage, the stressor required for salt stress of corresponding concentration is added to the culture medium, and the algal cells begin to synthesize GG at this stage; the culture time of the algal cells in the glycerol glucoside synthesis stage is 5-7 days; the concentration of the stressor in the culture medium is 300-1500 mmol / L; (3) Algal cell protein recovery stage: After the second stage, the algal cells are harvested and GG is separated. The remaining algal cells are cultured in the same fresh culture medium as in the first stage, and then the algal cells are collected to obtain algal powder. The culture time of the algal cell protein recovery stage is 3-10 days. Lighting conditions for each stage of cultivation: the wavelength range of light was 400–700 nm; As the carbon source, a mixed air containing carbon dioxide was selected, and the carbon dioxide concentration was within 10% (v / v); The lighting conditions used were intermittent lighting with a light-dark ratio of 1:1, 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 ; The type of algae cells is Spirulina.

2. The method according to claim 1, wherein: During the dark reaction with intermittent lighting, reduce the oxygen concentration to 1~2% (v / v).

3. The method according to claim 2, wherein: The concentration of carbon dioxide is 1~5% (v / v).

4. The method according to claim 1, wherein: The culture temperature at each stage is: 15-40°C, the temperature is set at 15-25°C during the dark period, and the temperature is set at 25-40°C during the light period.

5. The method according to claim 1, wherein: The stressor is a substance that can change the osmotic pressure of cells, and the substance is one or more combinations of inorganic salts and / or organic salts.

6. The method according to claim 5, wherein: 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.

7. The method according to claim 1, wherein: In step (3), the method for separating GG is a hypotonic extraction separation method.

8. A method for preparing glycerol glucoside and algal protein, the method comprising the step of restoring protein content in the process of producing glycerol glucoside according to any one of claims 1 to 7.

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