Spirulina alpha-glucan containing soluble dietary fibers and having immunomodulatory activity and preparation method thereof

Abstract

The invention discloses spirulina alpha-glucan containing soluble dietary fibers and having immunomodulatory activity and a preparation method thereof, spirulina is subjected to continuous illumination culture under nutrient salt deficiency or low temperature or combined regulation and control of low temperature and nutrient salt deficiency; a large amount of alpha-glucan which contains more soluble dietary fibers and has immunoregulatory activity is accumulated in the spirulina, and the spirulina alpha-glucan is prepared by adopting a method of hot water extraction, trichloroacetic acid deproteinization and ethanol precipitation. The prepared spirulina alpha-glucan contains 10%-26% of dietary fibers, the content of the dietary fibers is 9-27 times higher than the content of the dietary fibers of maltodextrin, the spirulina alpha-glucan can induce secretion of NO, TNF-alpha and IL-6 of macrophages RAW264.7, the activation index of the NO reaches 1.5-3.6, the activation index of the TNF-alpha reaches 2.0-4.1, the activation index of the IL-6 reaches 1.6-3.0, and the spirulina alpha-glucan has a remarkable immunoregulation effect. Technical support and product cases are provided for development of spirulina glucan type high-added-value functional products.

Description

Technical Field

[0001] The invention belongs to the technical field of development of algae active natural products, and specifically relates to spirulina alpha-glucan containing soluble dietary fiber and having immunomodulatory activity and a preparation method thereof. Background Art

[0002] Dietary fiber is known as the seventh nutrient for the human body. It is a carbohydrate polymer with 10 or more units that cannot be hydrolyzed by endogenous enzymes in the human small intestine. Dietary fiber is divided into soluble and insoluble according to its solubility in the intestine. Soluble dietary fiber mainly includes oligosaccharides, glucans, fructans, pectin, etc. It can reach the colon and is fermented and utilized by intestinal microbial communities to form functional active factors such as short-chain fatty acids. It has the functions of regulating blood lipids, blood sugar and intestinal flora, and is an important component of functional prebiotic carbohydrates. At present, soluble dietary fiber is mainly extracted from plants by physical, chemical, enzymatic and membrane separation methods, or produced by further fermentation and transformation of microorganisms. The source of raw materials is uncontrollable and the process is complicated.

[0003] Immunomodulators are active substances that can activate the body's immune system, improve the level of innate immunity and adaptive immunity, enhance the body's ability to resist the invasion of pathogenic microorganisms, and improve human health. Spirulina, also known as Arthrospira, belongs to the phylum Cyanobacteria, class Cyanobacteria, order Oscillatoria, family Oscillatoria, and genus Arthrospira. It is a type of basophilic photosynthetic autotrophic cyanobacteria. Spirulina is the world's largest commercially produced edible algae, especially in China, where the annual output of spirulina cultivation has reached more than 8,000 tons, accounting for more than 57% of the global output. Spirulina is rich in phycocyanin, carotenoids, vitamins and trace elements, has high nutritional value, and has biological activities such as antioxidant, anti-radiation, antiviral, antibacterial, anti-obesity and immune regulation. It is praised by the World Health Organization as a "super food" and has broad application prospects in food, medicine, health products, feed and cosmetics. However, at present, the spirulina industry mainly uses primary raw material algae powder as the market application form, with low added value and limited industrial development; the development and deep processing technology of its high value-added products need to be developed, and related products are lacking. The growth of Spirulina is affected by multiple environmental factors, such as light intensity, temperature, salinity, pH, and nutrient supply. Among them, nutrient deficiency (nitrogen, phosphorus, sulfur) or low temperature will lead to a decrease in cell photosynthetic efficiency, slow growth, and change cell metabolism from protein synthesis required for cell division to storage sugar (such as glycogen) synthesis. The optimum temperature of Spirulina is usually 28-35°C, while the production of Spirulina in high-latitude areas (such as Ordos, Inner Mongolia) is usually affected by low temperatures (5-15°C), and the quality of Spirulina with high protein content cannot be guaranteed, so the production cycle is limited to May to October. How to improve the value of Spirulina biomass in low-temperature environments and expand the production cycle is an urgent problem to be solved. Spirulina polysaccharides, as one of its active ingredients, have received widespread attention. The main component of Spirulina polysaccharides reported so far is acidic heteropolysaccharides (sulfated), which show good biological activity in immunomodulation, anti-inflammatory, hypoglycemic and antioxidant aspects. However, the content of Spirulina polysaccharides reported so far is usually not higher than 10%, the yield is low, and the extraction and separation are difficult, resulting in high production costs. In addition, the content and activity of Spirulina polysaccharides are also affected by outdoor culture conditions, and the yield and quality stability need to be improved. Spirulina polysaccharides obtained under different culture conditions may have different structures and their activities may also differ. Obtaining a large amount of storage sugars in Spirulina by regulating environmental factors such as nutritional stress and low temperature is an important way to solve problems such as low polysaccharide content, poor product stability, and low product added value at low temperatures. However, the structure and function of Spirulina glucan under different culture conditions are currently unclear, and whether it has the potential to be developed into functional products such as dietary fiber or immunomodulators remains to be studied.

[0004] In view of this, a spirulina α-glucan and a preparation method thereof that can effectively achieve immune regulation and at the same time take into account functionalities such as prebiotics are urgently needed in the industry. Summary of the invention

[0005] The technical problem to be solved by the present invention is: how to achieve efficient and controllable production of functional glucan containing soluble dietary fiber and having immunomodulatory activity by regulating the spirulina culture process in combination with downstream extraction and separation processes.

[0006] The present invention is achieved through the following technical solutions:

[0007] The present invention firstly discloses a method for preparing spirulina alpha-glucan containing soluble dietary fiber and having immunomodulatory activity, comprising:

[0008] Spirulina is cultured under special conditions with continuous illumination to allow Spirulina to accumulate a large amount of α-glucan, and Spirulina α-glucan is prepared by a method of hot water extraction-trichloroacetic acid protein removal-ethanol precipitation; wherein:

[0009] The special conditions are selected from any one or two of the following:

[0010] Lack of nutrients and low temperature.

[0011] Furthermore, the nutrient deficiency is nitrogen deficiency or phosphorus deficiency; and the low temperature is 10-20°C.

[0012] Furthermore, the nitrogen-deficient culture medium is composed of: each 1L of nutrient agent includes: NaHCO3 10-25g, K2HPO4 0.2-1.0g, NaCl 1.0-4.0g, K2SO4 0.5-2.0g, MgSO4·7H2O 0.1-0.3g, CaCl2 20-40mg, FeSO4·7H2O 0.005-0.02g, H3BO3 1.5-3.5mg, MnCl2·4H2O 1.0-2.5mg, ZnSO4·7H2O 0.1-0.3mg, Na2MoO4·2H2O 0.2-0.5mg, CuSO4·5H2O 0.05-0.1mg, and Na2EDTA 60-100mg.

[0013] Furthermore, the phosphorus-deficient culture medium is composed of: each 1L of nutrient agent includes: NaHCO3 10-25g, NaNO3 1-4g, NaCl 0.5-2.5g, K2SO4 0.5-2.0g, MgSO4·7H2O 0.1-0.3g, CaCl2 20-40mg, FeSO4·7H2O 0.005-0.02g, H3BO3 1.5-3.5mg, MnCl2·4H2O 1.0-2.5mg, ZnSO4·7H2O 0.1-0.3mg, Na2MoO4·2H2O 0.2-0.5mg, CuSO4·5H2O 0.05-0.1mg, and Na2EDTA 60-100mg.

[0014] Furthermore, the continuous illumination culture time is 2 to 8 days, and the cells are harvested before the cells enter the decline phase; the illumination intensity is 50 to 200 μE / (m 2 ·s).

[0015] Furthermore, the initial inoculation density of Spirulina is OD 560 =0.5~1.0.

[0016] Furthermore, the material-liquid ratio of the hot water extraction is 1:20-1:40 (g / mL); the mass concentration of the added trichloroacetic acid is 5%-10% (w / w); and the mass concentration of the ethanol is 70%-100%.

[0017] The present invention also discloses spirulina alpha-glucan prepared according to any one of the above preparation methods.

[0018] Furthermore, the Spirulina α-glucan has an α-(1→4) glycosidic bond as the main chain, an α-(1→6) glycosidic bond as a branch, or α-(1→6) and α-(1→2) glycosidic bonds together form a branch, and the total branching degree reaches 2% to 13%;

[0019] The spirulina alpha-glucan contains 10% to 26% of dietary fiber.

[0020] Furthermore, the Spirulina α-glucan can induce the secretion of NO, TNF-α and IL-6 in macrophages RAW264.7, with the NO activation index reaching 1.5 to 3.6, the TNF-α activation index reaching 2.0 to 4.1, and the IL-6 activation index reaching 1.6 to 3.0.

[0021] The invention also discloses a use of the spirulina alpha-glucan in preparing a prebiotic or an immunomodulator.

[0022] After the regulation of the above environmental factors, the α-glucan yield of Spirulina reaches 0.4-1g / L, which is 15-38 times higher than that of unregulated culture; the α-glucan content reaches 30%-51% of the cell dry weight, which is 14-37 times higher than that of unregulated culture. The soluble α-glucan obtained after hot water extraction-trichloroacetic acid protein removal-ethanol precipitation has a new structure with α-(1→4) glycosidic bond as the main chain, α-(1→6) glycosidic bond as the branch or α-(1→6) and α-(1→2) glycosidic bonds as the branch, and the total branching degree reaches 2%-13%. The obtained soluble α-glucan contains 10%-26% dietary fiber, which is 9-27 times higher than the dietary fiber content of maltodextrin. The obtained soluble α-glucan can induce the secretion of NO, TNF-α and IL-6 in macrophages RAW264.7, with the NO activation index reaching 1.5-3.6, the TNF-α activation index reaching 2.0-4.1, and the IL-6 activation index reaching 1.6-3.0, having a significant immunomodulatory effect.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The present invention proposes a strategy for controlling the production of active natural products from Spirulina by using environmental factors. Compared with the traditional production of active polysaccharides using unregulated Spirulina, the present invention has the advantages of high yield / content of active α-glucan (more than 14 times higher than before regulation) and stable raw material sources; at the same time, the regulation method of the present invention is simple and controllable, easy to scale up, and the α-glucan extraction method is simple, which has good commercial application prospects.

[0025] 2. The present invention obtains multiple new α-glucans containing soluble dietary fiber and immunomodulatory activity through the cultivation and regulation of Spirulina, which provides technical support and product cases for the deep processing of Spirulina biomass and the development of high-value-added functional glucan products.

[0026] 3. The present invention proposes a production strategy for Spirulina to accumulate α-glucan containing soluble dietary fiber and having immune activity under low temperature environment, which can improve the added value of Spirulina biomass produced under low temperature environment, broaden the application field of Spirulina biomass products, and is conducive to expanding the production cycle of Spirulina in high-latitude and low-temperature areas, significantly increasing the annual output of Spirulina, and promoting the development of Spirulina-related industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The changes of glucan and soluble sugar contents over time in the experimental group and the control group under the regulation of nitrogen deficiency in Spirulina in Example 1 of the present invention;

[0028] Figure 2The changes in the yields of glucan and soluble sugars over time in the experimental group and the control group under the regulation of nitrogen deficiency in Spirulina in Example 1 of the present invention;

[0029] Figure 3 The glycosidic bond types of Spirulina glucan under nitrogen deficiency regulation in Example 1 of the present invention, wherein the left side is the original hydrogen nuclear magnetic resonance spectrum, and the right side is the local enlarged view of 4.4-6.0ppm;

[0030] Figure 4 Comparison of the immunomodulatory activity of Spirulina glucan under nitrogen deficiency control and that of the control in Example 1 of the present invention;

[0031] Figure 5 The changes of the glucan and soluble sugar contents in the experimental group and the control group under the regulation of Spirulina phosphorus deficiency over time in Example 2 of the present invention;

[0032] Figure 6 The changes in the yields of glucan and soluble sugars over time in the experimental group and the control group under the regulation of Spirulina phosphorus deficiency in Example 2 of the present invention;

[0033] Figure 7 The glycosidic bond types of Spirulina glucan under phosphorus deficiency control in Example 2 of the present invention, wherein the left side is the original H NMR spectrum, and the right side is the local enlarged view of 4.4-6.0 ppm;

[0034] Figure 8 Comparison of the immunomodulatory activity of Spirulina glucan under phosphorus deficiency control and that of the control in Example 2 of the present invention;

[0035] Fig. 9 The changes of the glucan content of the experimental group and the control group under the control of low temperature alone and low temperature combined with nitrogen deficiency in Example 3 of the present invention over time;

[0036] Fig.10 The changes in the glucan production of Spirulina in the experimental group and the control group over time under the control of low temperature alone and low temperature combined with nitrogen deficiency in Example 3 of the present invention;

[0037] Fig.11 The glycosidic bond types of Spirulina glucan under the regulation of low temperature and nitrogen deficiency in Example 3 of the present invention, wherein the left side is the original hydrogen nuclear magnetic resonance spectrum, and the right side is the local enlarged view of 4.4-6.0ppm;

[0038] Fig.12 The immunomodulatory activity of Spirulina glucan under low temperature combined with nitrogen deficiency and the control is compared in Example 3 of the present invention;

[0039] Fig.13 The changes of the glucan content of the experimental group and the control group under the control of low temperature alone and low temperature combined with phosphorus deficiency in Example 4 of the present invention over time;

[0040] Fig.14 The changes in the glucan production of Spirulina in the experimental group and the control group over time under the control of low temperature alone and low temperature combined with phosphorus deficiency in Example 4 of the present invention;

[0041] Fig.15 The glycosidic bond types of Spirulina glucan under the control of low temperature alone and low temperature combined with phosphorus deficiency in Example 4 of the present invention, wherein the left side is the original hydrogen nuclear magnetic resonance spectrum, and the right side is the local enlarged view of 4.4-6.0ppm;

[0042] Fig.16 This is a comparison of the immunomodulatory activity of Spirulina glucan and the control under the control of low temperature alone and low temperature combined with phosphorus deficiency in Example 4 of the present invention. DETAILED DESCRIPTION

[0043] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0044] The present invention uses Arthrospira platensis as the experimental material, and adopts the cultivation methods of nutrient salt stress, low temperature and nutrient salt stress combined with low temperature in a photobiological reaction device, and continuous illumination to make the spirulina accumulate glucan. The gas source is an air compressor, and its flow rate is controlled by a gas pressure reducing valve and a gas flow meter arranged on the pipeline.

[0045] Storage solution configuration:

[0046] A5 stock solution (100 mL): 0.286 g H3BO3, 39 mg Na2MoO4·2H2O, 0.18 g MnCl2·4H2O, 8 mg CuSO4.5H2O, 22 mg ZnSO4.7H2O.

[0047] Stock solution 1 (1 L): 1 g FeSO4·7H2O, 8 g Na2EDTA.

[0048] Storage solution 2 (100 mL): 3 g CaCl2, sterilized at 121°C for later use

[0049] The meanings of the numbers in the tables and figures in the embodiments are as follows:

[0050] -N represents nitrogen deficiency; -P represents phosphorus deficiency; NT represents 25℃; LT represents 15℃; MD represents maltodextrin; G-N2 represents Spirulina glucan on the second day under nitrogen deficiency; G-P6 represents Spirulina glucan on the sixth day under phosphorus deficiency; G-P8 represents Spirulina glucan on the eighth day under phosphorus deficiency; GLT-N2 represents Spirulina glucan on the second day under nitrogen deficiency combined with low temperature; GLT6 represents Spirulina glucan on the sixth day under low temperature; GLT-P6 represents Spirulina glucan on the sixth day under phosphorus deficiency combined with low temperature; *, ** and *** indicate significant differences compared with the control group without addition at p<0.05, p<0.01 and p<0.001; + and ++ indicate significant differences between the two glucans at the same concentration at p<0.05 and p<0.01; different letters indicate significant differences compared with the same concentration of MD at p<0.05.

[0051] Example 1

[0052] Using nitrogen deficiency stress to regulate Spirulina to produce a new type of α-glucan containing soluble dietary fiber and immunomodulatory activity.

[0053] (1) Obtaining algal cell seed solution

[0054] Preparation of complete culture medium (1L): NaHCO3 16.8 g, K2HPO4 0.5 g, NaNO3 2.5 g, NaCl 1.0 g, K2SO4 1.0 g, MgSO4·7H2O 0.2 g, storage solution 110 mL, A5 storage solution 1 mL, sterilize at 121℃ for 20 min, add 1 mL of sterilized storage solution 2 after cooling, and set aside.

[0055] Cultivation of algal cell seed solution: Mix the Spirulina mother solution in the shake flask with fresh complete culture medium at a ratio of 1:1, inoculate into the sterilized photoreactor with a working volume of 500 mL and culture conditions: temperature 25°C, continuous light intensity 80 μE / (m 2 ·s), and after 2 days of aeration culture, the above operation was repeated once.

[0056] (2) Photobioreactor culture

[0057] Two culture conditions, nitrogen stress and full nutrition, were set up, and three replicates were set up for each condition. To achieve nitrogen-deficient culture conditions, the full culture was replaced with nitrogen-deficient medium.

[0058] Preparation of nitrogen-deficient culture medium: Replace NaNO3 with an equimolar amount of NaCl. The remaining steps are the same as those for the preparation of complete culture medium.

[0059] The composition of nitrogen-deficient culture medium is (1L): NaHCO3 16.8 g, K2HPO4 0.5 g, NaCl 2.72 g, K2SO4 1.0 g, MgSO4·7H2O 0.2 g, CaCl2 30 mg, FeSO4·7H2O 0.01 g, H3BO3 2.86 mg, MnCl2·4H2O 1.86 mg, ZnSO4·7H2O 0.22 mg, Na2MoO4·2H2O 0.39 mg, CuSO4·5H2O 0.08 mg, and Na2EDTA 80 mg.

[0060] Cultivation of algal cells: The algal cell seed solution was filtered through a 1000 nylon cloth to collect the algal mud, and then washed once or twice with nitrogen-deficient medium. The algal mud was resuspended in nitrogen-deficient medium and then inoculated into a sterilized photoreactor and inoculated with an initial OD 560 The temperature was controlled between 0.7 and 0.8, and the cells were placed on a light reaction device for culture. The culture conditions were: temperature 25°C, continuous light intensity 80 μE / (m 2 ·s), ventilation rate 0.4vvm.

[0061] (3) Determination of glucan

[0062] Weigh about 2 mg of algae powder and record it, add 0.25 mL of 30% KOH solution, maintain at 95 ° C for 90 minutes to extract α-glucan in algae mud; after the solution cools, add 0.75 mL of anhydrous ethanol and precipitate at 0 ° C for 2 hours; then centrifuge at 8000 r / min for 5 minutes to collect the precipitate. The precipitate uses the sulfuric acid-anthrone method to determine the glucose concentration. The principle is that sugars are dehydrated to produce furfural or hydroxymethylfurfural under the action of concentrated sulfuric acid, and then anthrone will undergo dehydration condensation with furfural or hydroxymethylfurfural to produce blue-green furfural derivatives. The glucose concentration can be determined according to the absorbance at 621 nm.

[0063] (4) Extraction of glucan

[0064] Algal powder and deionized water were mixed in a ratio of 1:30, and the mixture was stirred and extracted at 80°C for 4 hours. After cooling, the mixture was centrifuged at 8000 rpm, and the supernatant was collected. Trichloroacetic acid (TCA) was added to the supernatant at a concentration of 8%, and the mixture was allowed to stand at 4°C for 3 hours. The mixture was then centrifuged at 13000 rpm and 4°C for 10 minutes. The supernatant was filtered with a 0.22 μm filter, and 4 times the volume of anhydrous ethanol was added, and the mixture was allowed to stand at 4°C for 12 hours. Finally, the mixture was centrifuged at 8000 r / min at 4°C for 5 minutes, and the precipitate was collected and freeze-dried.

[0065] (5) H NMR spectroscopy

[0066] About 5 mg of the sample was dissolved in 0.55 mL of D2O and tested using a 400 MHz nuclear magnetic resonance spectrometer (Brukeravance 400) at room temperature.

[0067] (6) Determination of dietary fiber content

[0068] Weigh about 7 mg of sample, dissolve in 200 μL 50 mM sodium maleate (pH = 6.0), take out 5 μL of solution as the control at time 0, then add a magnetic stirrer and 79 μL enzyme working solution, keep warm at 37 ° C for 4 h, take out 5 μL of solution at 20, 40, 80, 120, 180, and 240 min respectively, add 5 μL of solution to 200 μL 66% ethanol solution, centrifuge at 8000 rpm for 5 min to remove undigested glucan, and use the GOPOD kit to determine the glucose concentration of the supernatant.

[0069] Enzyme working solution: accurately weigh 123 mg of porcine pancreatic α-amylase (PAA) powder and dissolve it in 1 mL of 50 mM sodium maleate (pH 6.0), place it on a suspension mixer and shake for 5 minutes, then centrifuge it at more than 1500g for 5 minutes, aspirate 955 μL of supernatant, mix it with 45 μL of amyloglucosidase (AMG) mother solution, shake well and set aside.

[0070] (7) Immunoactivity assay

[0071] Macrophages RAW264.7 were treated with samples and controls at different concentrations. The inhibitory effect of dextran on macrophages was detected by MTT method, the secretion expression of macrophage tumor necrosis factor (TNF-α) and interleukin 6 (IL-6) was detected by enzyme-linked immunosorbent assay, and the release of macrophage NO was detected by Griess method. The activation index was calculated according to the following formula:

[0072]

[0073] from Figure 1 and Figure 2 It can be seen that nitrogen deficiency stress (-N) significantly increased the content and yield of Spirulina glucan. The glucan content under -N reached a peak of 50.37% on the second day, which was 14 times higher than that of the +N group on the second day. The glucan yield reached a maximum of 0.72g / L on the third day, which was 36 times higher than that of the +N group (0.02g / L) in the same period. The soluble total sugar content and yield of -N both reached a maximum of 62.77% and 0.91g / L on the third day. Figure 3It can be seen that the spirulina glucan (G-N2) regulated by -N is a glucan with α-1,4 glycosidic bonds as the main chain and α-1,6 branches, and the branching degree is 5.50% (Table 1). The soluble dietary fiber content of G-N2 is 10.34%, which is 9.8 times higher than the control maltodextrin (MD) (Table 1). Figure 4 As shown in the figure, G-N2 did not significantly inhibit the growth of macrophages at any of the three concentrations (p>0.05), indicating that it was not cytotoxic. At a concentration of 1000 mg / L, G-N2 significantly promoted the secretion of NO, TNF-α, and IL-6 (p<0.05), with activation factors of 1.72, 2.03, and 2.32, respectively, which were comparable to the in vitro immune activity of MD; G-N2 significantly promoted the secretion of NO and TNF-α at 500 mg / L (p<0.05), and there was no significant difference in activation index with that at 1000 mg / L (p>0.05). At 100 mg / L, the activation level of G-N2 for NO was comparable to that at 1000 mg / L.

[0074] Table 1 Soluble dietary fiber content, glycosidic bond type and branching degree of Spirulina glucan and control

[0075]

[0076]

[0077] Example 2

[0078] Using phosphorus deficiency stress to regulate Spirulina to produce a new type of α-glucan containing soluble dietary fiber and immunomodulatory activity.

[0079] The nitrogen-deficient culture medium in Example 1 is replaced with a phosphorus-deficient culture medium, and the remaining steps are the same as in Example 1.

[0080] Preparation of phosphorus-deficient medium: replace K2HPO4 with an equimolar amount of NaCl, and the remaining steps are the same as the preparation of full medium.

[0081] The composition of phosphorus-deficient culture medium is (1L): NaHCO3 16.8 g, NaNO3 2.5 g, NaCl 1.17 g, K2SO4 1.0 g, MgSO4·7H2O 0.2 g, CaCl2 30 mg, FeSO4·7H2O 0.01 g, H3BO3 2.86 mg, MnCl2·4H2O 1.86 mg, ZnSO4·7H2O 0.22 mg, Na2MoO4·2H2O 0.39 mg, CuSO4·5H2O 0.08 mg, and Na2EDTA 80 mg.

[0082] like Figure 5 and Figure 6As shown in the figure, phosphorus deficiency stress (-P) significantly increased the content and yield of glucan. The glucan content under -P conditions continued to increase from 10.15% on the first day to 38.86% on the eighth day. However, due to sufficient nutrition, +P began to degrade glucan from the first day to provide energy for cell division, and its content continued to decrease from 13.56% to 2.05% on the eighth day. At the same time, the glucan yield of -P continued to increase, reaching 0.99g / L on the eighth day, which was 23 times higher than that of the control group during the same period. Figure 7 It can be seen that the glucans obtained on the 6th and 8th days of phosphorus deficiency regulation (G-P6 and G-P8, respectively) are glucans with α-1,4 glycosidic bonds as the main chain and α-1,6 branches, and the branching degree is 5.82% and 6.33%, respectively (Table 1). As can be seen from Table 1, the soluble dietary fiber content of G-P6 and G-P8 reached 25.30% and 12.24%, respectively, which is 12 to 26 times higher than MD. Figure 8 It can be seen that G-P6 had no significant effect on cell viability in the concentration range of 100-500 mg / L (p>0.05); when the concentration increased to 1000 mg / L, it slightly inhibited cell activity, with an inhibition rate of 6.60% (p<0.01); G-P8 inhibited 5.40% of cell activity at 500 mg / L (p<0.05), but there was no difference with G-P6 at high concentration (1000 mg / L), with an inhibition rate of 6.83%. Compared with high concentration MD (1000 mg / L) (inhibition rate 3.00%), there was no significant difference in cytotoxicity between G-P6 and G-P8 (p>0.05), indicating that their toxicity was low. G-P6 has a promoting effect on the secretion of NO, TNF-α and IL-6, and shows a certain concentration dependence. The activation index reached 3.17, 2.46 and 1.70 respectively at a concentration of 1000 mg / L. G-P8 also had a significant improvement effect on the above indicators. The activation index of IL-6 reached 1.93 at 1000 mg / L, which was not significantly different from G-P6 and MD (p>0.05), while the activation index of NO and TNF-α reached 1.85 and 2.07, which were significantly lower than those of G-P6 under the same conditions (p<0.05).

[0083] Example 3

[0084] Low temperature combined with nitrogen deficiency stress was used to regulate Spirulina to produce a new type of α-glucan containing soluble dietary fiber and immunomodulatory activity.

[0085] (1) Obtaining algal cell seed solution: the same as in Example 1.

[0086] (2) Photobioreactor culture

[0087] The culture conditions were set to be 25°C with nitrogen (NT+N), 15°C with nitrogen (LT+N) and 15°C without nitrogen (LT-N). The culture condition of 15°C was achieved by connecting the jacket outside the photoreactor to circulating condensed water. The rest of the operation was exactly the same as in Example 1.

[0088] from Fig. 9 and Fig.10 It can be seen that low temperature combined with nitrogen deficiency (LT-N) can increase the glucan content and yield of Spirulina. The glucan content of the LT-N group reached 39.13% on the first day, which was higher than the LT+N and NT+N groups in the same period (28.32% and 3.34%), and then further increased to about 45%, which was also higher than the LT+N and NT+N groups in the same period, indicating that nitrogen deficiency stress can further increase the glucan content of low-temperature Spirulina. Correspondingly, the glucan yield of the LT-N group reached a peak of 0.45g / L on the second day, an increase of 68.9% over the LT+N group in the same period, indicating that nitrogen deficiency stress will further increase the glucan yield of low-temperature Spirulina. Compared with the NT+N group, low temperature combined with nitrogen deficiency regulation (LT-N) increased the glucan content and yield of Spirulina by 14 times and 15 times, respectively. Fig.11 It can be seen that the Spirulina glucan (GLT-N2) obtained on the second day of low temperature combined with nitrogen deficiency regulation is a glucan with α-1,4 glycosidic bonds as the main chain and α-1,6 branches. It also contains a small amount of α-1,2 branches, and the branching degree is 2.19% (Table 1). The soluble dietary fiber content of GLT-N2 reaches 23.68% (Table 1), which is 24 times higher than that of MD. Fig.12 As shown in the results, GLT-N2 had no significant inhibitory effect on the growth of macrophages (p>0.05), but could significantly promote the secretion of NO, TNF-α and IL-6 at 1000 mg / L (p<0.01), with the corresponding activation indexes of 1.73, 2.53 and 2.92, respectively; at 500 mg / L, it significantly activated the secretion of NO and TNF-α. In addition, the activation index of GLT-N2 for TNF-α was significantly higher than that of MD at the same concentration (p<0.01), while the in vitro immune activity of G-N2 was comparable to that of MD, indicating that Spirulina glucan has higher in vitro immune regulation activity under low temperature combined with nitrogen deficiency stress.

[0089] Example 4

[0090] Low temperature and low temperature combined with phosphorus deficiency stress were used to regulate Spirulina to produce a new type of α-glucan containing soluble dietary fiber and immunomodulatory activity.

[0091] The nitrogen-deficient culture medium in Example 3 was replaced with a phosphorus-deficient culture medium, wherein the phosphorus-deficient culture medium was exactly the same as that in Example 2, and the rest of the process was exactly the same as that in Example 3.

[0092] from Fig.13 and Fig.14 It can be seen that low temperature (LT+P) can increase the glucan content and yield of Spirulina. The glucan content of the LT+P group remained between 26.20% and 30.32% from 1 to 6 days, while under normal temperature culture (NT+P), it dropped from 10.15% on the first day to below 5%; the glucan yield of the LT+P group gradually increased from 0.16g / L on the first day to 0.47g / L on the sixth day, while the NT+P group dropped from 0.09g / L to below 0.05g / L. Compared with NT+P, the glucan content and yield of Spirulina under low temperature control (LT+P) increased by 27 times and 24 times, respectively.

[0093] Under low temperature combined with phosphorus deficiency (LT-P), the glucan content of Spirulina gradually increased to a peak of 41.49% on the 6th day, and the glucan yield also reached a peak of 0.72 g / L on the 6th day, an increase of 53.19% over the LT+P group in the same period, indicating that phosphorus deficiency will further increase the glucan yield of Spirulina under low temperature. Compared with the NT+P group, low temperature combined with phosphorus deficiency (LT-P) increased the glucan content and yield of Spirulina by 37 times and 38 times, respectively.

[0094] Depend on Fig.15 It can be seen that the Spirulina glucan (GLT6 and GLT-P6, respectively) obtained on the 6th day of low temperature and low temperature combined with phosphorus deficiency regulation are both glucans with α-1,4 glycosidic bonds as the main chain and α-1,6 branches, with branching degrees of 7.27% and 12.11%, respectively. The soluble dietary fiber content of GLT6 and GLT-P6 reached 24.09% and 24.22%, respectively (Table 1), which is 24 times higher than MD.

[0095] like Fig.16As shown in the results, GLT 6 and GLT-P6 had almost no cytotoxicity to macrophages. GLT6 significantly promoted the secretion of NO at 200 mg / L (p<0.05), and with the increase of concentration, the activation index gradually increased to 3.54, which was significantly higher than MD (p<0.05); GLT-P6 only significantly promoted the secretion of NO at 500 mg / L (p<0.05), and the activation index reached a maximum of 1.57 at 1000 mg / L, and there was no significant difference with MD (p>0.05); in addition, the NO activation index of GLT6 at 500 and 1000 mg / L was significantly higher than that of GLT-P6 at the same concentration (p<0.01). GLT6 can significantly promote the secretion of TNF-α, and it shows concentration dependence, with the activation index up to 4.01, which is significantly higher than MD (p<0.05); GLT-P6 also shows a certain concentration dependence on the activation of TNF-α, with the maximum activation index of 2.00, which is equivalent to MD (p<0.05); and at 500 and 1000 mg / L concentrations, the activation index of GLT6 for TNF-α is significantly higher than that of GLT-P6 (p<0.01). GLT6 and GLT-P6 only have a significant promoting effect on the release of IL-6 at 1000 mg / L (p<0.05), and there is no significant difference with MD (p>0.05). In summary, both GLT6 and GLT-P6 have immune immunomodulatory activity, and the effect of GLT6 is better.

[0096] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions only describe the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention. These changes and improvements fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for preparing spirulina α-glucan containing soluble dietary fiber and having immunomodulatory activity, comprising: Spirulina is cultured under special conditions with continuous illumination, so that Spirulina accumulates a large amount of α-glucan, and Spirulina α-glucan is prepared by a method of hot water extraction-trichloroacetic acid protein removal-ethanol precipitation; wherein: The special conditions are selected from any one or two of the following: Lack of nutrients and low temperature.

2. The preparation method according to claim 1, wherein: The nutrient deficiency is nitrogen deficiency or phosphorus deficiency; The low temperature is 10-20°C.

3. The preparation method according to claim 2, wherein: The nitrogen-deficient culture medium is composed of: each 1L of nutrient agent includes: NaHCO3 10-25g, K2HPO4 0.2-1.0g, NaCl 1.0-4.0g, K2SO4 0.5-2.0g, MgSO4·7H2O 0.1-0.3g, CaCl2 20-40mg, FeSO4·7H2O 0.005-0.02g, H3BO3 1.5-3.5mg, MnCl2·4H2O 1.0-2.5mg, ZnSO4·7H2O 0.1-0.3mg, Na2MoO4·2H2O 0.2-0.5mg, CuSO4·5H2O 0.05-0.1mg, Na2EDTA 60-100mg; The phosphorus-deficient culture medium is composed of: each 1L of nutrient agent includes: NaHCO3 10-25g, NaNO3 1-4g, NaCl 0.5-2.5g, K2SO4 0.5-2.0g, MgSO4·7H2O 0.1-0.3g, CaCl2 20-40mg, FeSO4·7H2O 0.005-0.02g, H3BO3 1.5-3.5mg, MnCl2·4H2O 1.0-2.5mg, ZnSO4·7H2O 0.1-0.3mg, Na2MoO4·2H2O 0.2-0.5mg, CuSO4·5H2O 0.05-0.1mg, and Na2EDTA 60-100mg.

4. The preparation method according to claim 1, wherein: The continuous illumination culture time is 2 to 8 days; The light intensity is 50 to 200 μE / (m 2 ·s).

5. The preparation method according to claim 1, wherein: The initial inoculation density of Spirulina is OD 560 =0.5~1.

0.

6. The preparation method according to claim 1, wherein: The solid-liquid ratio of the hot water extraction is 1:20 to 1:40 (g / mL); The mass concentration of trichloroacetic acid added is 5% to 10% (w / w); The mass concentration of the ethanol is 70% to 100%.

7. Spirulina α-glucan obtained according to the preparation method according to any one of claims 1 to 6.

8. The Spirulina α-glucan according to claim 7, wherein: The spirulina α-glucan has an α-(1→4) glycosidic bond as the main chain, an α-(1→6) glycosidic bond as the branch, or α-(1→6) and α-(1→2) glycosidic bonds as the branch, and the total branching degree reaches 2% to 13%. The spirulina alpha-glucan contains 10% to 26% of dietary fiber.

9. The Spirulina α-glucan according to claim 7, wherein: The spirulina alpha-glucan can induce the secretion of NO, TNF-alpha and IL-6 of macrophage RAW264.7; the NO activation index reaches 1.5-3.6, the TNF-alpha activation index reaches 2.0-4.1, and the IL-6 activation index reaches 1.6-3.

0.

10. Use of the Spirulina α-glucan according to claim 7 in the preparation of prebiotics or immunomodulators.

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