Method for enriching spirulina alpha-glucan

Abstract

The invention discloses a method for enriching spirulina alpha-glucan, and belongs to the technical field of glucan preparation. The method adopts two-stage culture and is combined with washing transfer, and specifically comprises the following steps: inoculating spirulina into a sterilized Zarrouk culture medium with nitrogen source content of 30mM, and culturing to OD560gt; 3, finishing the pre-culture; collecting algae slurry, and cleaning the algae slurry with deionized water for several times to reduce extracellular nitrogen residues; and inoculating the washed algae slurry into a sterilized Zarrouk culture medium with the nitrogen source content of 1mM according to the inoculation density OD560 of 0.2, and culturing for 96 hours under the condition same as the pre-culture condition. The method disclosed by the invention has the beneficial effects that the spirulina alpha-glucan is remarkably enriched under the industrial operable condition by combining two-stage culture with washing transfer, the method is good in repeatability, and meanwhile, the glycogen content and the volume yield are also considered.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for enriching alpha-glucan of Spirulina, belonging to the technical field of glucan preparation. BACKGROUND

[0002] Spirulina, also known as Arthrospira, belongs to Cyanophyta, Cyanophyceae, Oscillatoriales, Osciallatoriaceae and Arthrospira. It is a kind of alkaliphilic photosynthetic autotrophic cyanobacteria. Spirulina is the largest commercially produced edible algae in the world, which contains rich phycocyanin, carotenoids, vitamins and trace elements, and has high nutritional value. It is praised as superfood by the World Health Organization. It also has biological activities such as antioxidant, anti-radiation, anti-virus, anti-bacteria, anti-obesity and immune regulation, and is widely used in food, health care products, feed and cosmetics industries. The growth of Spirulina is affected by multiple environmental factors, such as light intensity, temperature, salinity, pH, and nutrient salt supply. Among them, the lack of nutrient salts (nitrogen deficiency, phosphorus deficiency, sulfur deficiency) will lead to the decline of cell photosynthetic efficiency, slow down the growth, and make the cell metabolism change from protein synthesis required for cell division to storage carbohydrate (such as glycogen) synthesis.

[0003] Aikawa et al. increased the glycogen production of Spirulina to 1.03 g / L by high light intensity combined with nitrogen stress, reaching the highest level reported so far (Aikawa et al., Synergistic enhancement of glycogen production in Arthrospria platensis by optimization of light intensity and nitrate supply, Bioresource Technology, 108, 211-215).

[0004] Cuellar-Bermudez S.P. et al found that nitrogen limitation increased the intracellular carbohydrate content of Spirulina to 688 mg / g, and the intracellular glycogen content to 564 mg / g (Cuellar-Bermudez S.P. et al, Quantification of extracellular and biomass carbohydrates by Arthrospira under nitrogen starvation at lab-scale, Algal Research-Biomass Biofuels and Bioproducts, 2022.102907).

[0005] Yao Changhong et al. increased the yield of Spirulina alpha-glucan to 0.4-1 g / L and the content of alpha-glucan to 30-51% of cell dry weight by regulating environmental factors (CN119614652A, A kind of soluble dietary fiber containing and having immune regulation activity Spirulina alpha-glucan and its preparation method).

[0006] However, the sharp stress caused by nitrogen limitation greatly reduces the carbon fixation efficiency of Spirulina, and the cells die rapidly within 2-3 days, resulting in low biomass yield, although the glycogen content is high, the yield is ultimately limited.

[0007] In summary, the prior art usually increases the polysaccharide content by nitrogen limitation or culture condition optimization, but these are common sense parameter adjustments, and there are problems such as limited enrichment range of alpha-glucan, poor repeatability, and difficulty in balancing glycogen content and volumetric yield. SUMMARY

[0008] To solve the problems of the prior art, the purpose of the present application is to provide a method for significantly enriching Spirulina alpha-glucan with good repeatability and balancing glycogen content and volumetric yield.

[0009] In order to achieve the above-mentioned goal, the technical scheme adopted by the present application is as follows: A method for enriching Spirulina alpha-glucan, which adopts two-stage culture and combines washing and transfer, specifically comprising the following steps: (1) First stage culture: inoculate Spirulina into sterilized Zarrouk medium with a nitrogen content of 30 mM, and culture until OD 560 >3, and the pre-culture is completed; (2) Washing: collect the algal slurry, and wash the algal slurry several times with deionized water to reduce the residual extracellular nitrogen; (3) Second stage culture: inoculate the algal slurry according to the inoculation density OD 560=0.2 The washed algae slurry was inoculated into sterilized Zarrouk medium with 1 mM nitrogen source and cultured under the same conditions as the first stage culture for 96 h.

[0010] Preferably, in step (1), the Spirulina is Arthrospira platensis or Arthrospira maxima.

[0011] Preferably, in step (1), the inoculation density is OD 560 =0.1.

[0012] Preferably, in steps (1) and (3), the culture conditions are: LED light source, color temperature 5000 K, 150 µmol / m 2 / s, light 14 h:dark 10 h, 30°C, aeration stirring, aeration volume 150 mL / min.

[0013] Preferably, in step (2), after each washing, the algae slurry is diluted with deionized water at a volume ratio of 1:1, and the conductivity of the system is detected. When the conductivity of the system remains basically unchanged after three consecutive washes, the washing is completed.

[0014] The present application has the advantages that: by two-stage culture and combined with washing transfer, significant enrichment of Spirulina alpha-glucan is achieved under industrial operable conditions, and the method has good repeatability, while also taking into account glycogen content and volumetric yield. DETAILED DESCRIPTION

[0015] The present application will be specifically described below in combination with specific examples.

[0016] I. Germplasm screening The candidate algae species are: Arthrospira platensis, Arthrospira maxima, and Spirulina major.

[0017] The alpha-glucan content of the candidate algae species was detected, and the detection method is as follows: weigh about 2 mg of algae powder and record, then add 0.25 mL of 30% (w / v) potassium hydroxide solution, maintain in a 95°C water bath for 90 min (to extract alpha-glucan in the algae), then cool the reaction solution to room temperature, next add 0.75 mL of anhydrous ethanol, and precipitate at 0°C for 2 h, then centrifuge at 8000 r / min for 5 min, collect the precipitate, and finally determine the glucose concentration by the sulfuric acid-anthrone method.

[0018] Through detection, the alpha-glucan content of Arthrospira platensis, Arthrospira maxima, and Spirulina major accounts for 70 wt%, 72 wt%, and 60% of the dry weight of the algae powder, respectively.

[0019] The above test results show that the α-glucan content varies significantly among different algal species. *Spirulina platensis* and *Spirulina maxima* are the preferred algal species for enriching α-glucan.

[0020] II. Enrichment of Spirulina α-glucan The following explanation uses Spirulina platensis as an example.

[0021] 1. First stage of training Prepare Zarrouk medium with a nitrogen source concentration of 30 mM: NaHCO3 16.8g / L, K2HPO30.5g / L, NaNO32.5g / L, NaCl 1.0g / L, MgSO4 0.2g / L, Fe2(SO4)3 0.01g / L, K2SO4 1.0g / L, CaCl2·H2O 0.04 g / L, EDTA 0.08g / L, pH=9.

[0022] According to vaccination density OD 560 =0.1 Spirulina platensis was inoculated into the sterilized Zarrouk medium described above, and the mixture was kept under LED light source, color temperature 5000K, and 150µmol / m 2 Pre-culture was conducted under the following conditions: 14 hours of light followed by 10 hours of darkness, at 30°C, with aeration and stirring, and an aeration rate of 150 mL / min. The OD value of the culture medium was measured daily. 560 When OD 560 When the temperature reaches >3 (approximately 7 days of culture), the pre-culture ends.

[0023] The purpose of pre-culturing is to expand the culture and prepare algal strains for the second stage of culture.

[0024] 2. Washing After the pre-culture was completed, the culture medium was filtered through a 400-mesh sieve and the algal slurry was collected.

[0025] Wash the algal slurry several times with deionized water. After each wash, filter the slurry through a 400-mesh sieve, then dilute it with deionized water at a 1:1 volume ratio. Measure the conductivity of the system. When the conductivity of the system remains basically unchanged after three consecutive washes, it indicates that there is very little extracellular nitrogen residue, and the washing process is complete.

[0026] The purpose of washing algal slurry is to reduce extracellular nitrogen residue, establish a clear two-stage enrichment window, improve reproducibility, and increase volumetric yield.

[0027] 3. Second stage training Prepare five Zarrouk culture media with different nitrogen source contents: (1) Zarrouk medium with a nitrogen source content of 0 mM (medium A) NaHCO3 16.8g / L, K2HPO30.5g / L, NaNO30g / L, NaCl 1.0g / L, MgSO4 0.2g / L, Fe2(SO4)3 0.01g / L, K2SO4 1.0g / L, CaCl2·H2O 0.04 g / L, EDTA 0.08g / L, pH=9.

[0028] (2) Zarrouk medium (medium B) with a nitrogen source content of 1 mM. NaHCO3 16.8g / L, K2HPO30.5g / L, NaNO30.085g / L, NaCl 1.0g / L, MgSO4 0.2g / L, Fe2(SO4)3 0.01g / L, K2SO4 1.0g / L, CaCl2·H2O 0.04 g / L, EDTA 0.08g / L, pH=9.

[0029] (3) Zarrouk medium with a nitrogen source content of 5 mM (medium C) NaHCO3 16.8g / L, K2HPO30.5g / L, NaNO30.425g / L, NaCl 1.0g / L, MgSO4 0.2g / L, Fe2(SO4)3 0.01g / L, K2SO4 1.0g / L, CaCl2·H2O 0.04 g / L, EDTA 0.08g / L, pH=9.

[0030] (4) Zarrouk medium with a nitrogen source content of 10 mM (medium D) NaHCO3 16.8g / L, K2HPO30.5g / L, NaNO30.85g / L, NaCl 1.0g / L, MgSO4 0.2g / L, Fe2(SO4)3 0.01g / L, K2SO4 1.0g / L, CaCl2·H2O 0.04 g / L, EDTA 0.08g / L, pH=9.

[0031] (5) Zarrouk medium (medium E) with a nitrogen source content of 30 mM. NaHCO3 16.8g / L, K2HPO30.5g / L, NaNO32.55g / L, NaCl 1.0g / L, MgSO4 0.2g / L, Fe2(SO4)3 0.01g / L, K2SO4 1.0g / L, CaCl2·H2O 0.04 g / L, EDTA 0.08g / L, pH=9.

[0032] According to different inoculation densities (OD) 560=0.1, 0.2, 0.4, 0.8) The cleaned algal slurry was inoculated into sterilized Zarrouk medium with different nitrogen source contents, and the second stage of culture was carried out under the same conditions as the first stage of culture.

[0033] After inoculation, samples were taken at the start of culture (0h) and at 24h, 48h, and 96h after culture to detect the OD of the culture medium. 560 The test results are shown in Tables 1-1, 1-2, 1-3 and 1-4.

[0034] Table 1-1 OD of Spirulina during the second stage of culture 560 Test Results (I)

[0035] Table 1-2 OD of Spirulina during the second stage of culture 560 Test Results (II)

[0036] Table 1-3 OD of Spirulina during the second stage of culture 560 Test Results (III)

[0037] Table 1-4 OD of Spirulina during the second stage of culture 560 Test Results (IV)

[0038] After cultivation, the biomass of Spirulina in each group, as well as the content, volumetric yield, and production rate of α-glucan, were calculated. The results are shown in Tables 2-1, 2-2, 2-3, and 2-4.

[0039] Table 2-1 Calculation results of Spirulina biomass, α-glucan content, volumetric yield, and production rate (I)

[0040] Table 2-2 Calculation results of Spirulina biomass, α-glucan content, volumetric yield, and production rate (II)

[0041] Table 2-3 Calculation results of Spirulina biomass, α-glucan content, volumetric yield, and production rate (Part III)

[0042] Table 2-4 Calculation results of Spirulina biomass, α-glucan content, volumetric yield, and production rate (IV)

[0043] From Tables 2-1, 2-2, 2-3, and 2-4, we can see that: (1) Inoculation density OD 560 At concentrations of 0.1 and 0.2, the biomass of Spirulina and the volumetric yield of α-glucan both showed a trend of first increasing and then decreasing with the increase of nitrogen source content in the culture medium. This is because nitrogen-free culture limits the increase of Spirulina biomass, while low concentrations of nitrogen can promote the increase of Spirulina biomass and induce the accumulation of α-glucan. (2) Inoculation density OD 560 At concentrations of 0.4 and 0.8, the yield of α-glucan gradually decreased with increasing nitrogen source content, which was due to nitrogen excess.

[0044] The optimal method for enriching spirulina α-glucan was ultimately determined to be: Phase 1 culture: Inoculation density OD 560 =0.1, Zarrouk medium with a nitrogen source concentration of 30 mM, under LED light source, color temperature of 5000 K, and 150 µmol / m 2 Pre-cultured at OD under the following conditions: 14h light exposure followed by 10h darkness, 30℃, aeration and stirring, and aeration rate of 150mL / min until OD. 560 >3; Wash the algal slurry several times with deionized water to remove residual extracellular nitrogen. Second stage culture: Inoculation density OD 560 The medium (medium B) with a nitrogen source content of 1 mM and a concentration of 0.2 was cultured under the same conditions as the first stage for 96 hours.

[0045] Under this optimal method, the volumetric yield of α-glucan is as high as 0.454 g / L, and the production rate is 0.114 g / L / d.

[0046] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. A method for enriching spirulina α-glucan, characterized in that, The two-stage culture method, combined with washing and transfer, specifically includes the following steps: (1) First stage of culture: Spirulina was inoculated into sterilized Zarrouk medium with a nitrogen source content of 30mM and cultured until OD 560 When the temperature reaches >3, the pre-culture is complete; (2) Washing: Collect the algal slurry and wash it several times with deionized water to reduce extracellular nitrogen residue; (3) Second stage culture: according to the inoculation density OD 560 =0.2 The washed algal slurry was inoculated into sterilized Zarrouk medium with a nitrogen source content of 1mM and cultured under the same conditions as the first stage for 96 hours.

2. The method for enriching spirulina α-glucan according to claim 1, characterized in that, In step (1), the spirulina is Spirulina platensis or Spirulina macrophylla.

3. The method for enriching spirulina α-glucan according to claim 1, characterized in that, In step (1), the inoculation density is OD. 560 =0.

1.

4. The method for enriching spirulina α-glucan according to claim 1, characterized in that, In steps (1) and (3), the cultivation conditions are: LED light source, color temperature 5000K, 150µmol / m 2 / s, 14h light exposure: 10h darkness, 30℃, aeration and stirring, aeration rate 150mL / min.

5. The method for enriching spirulina α-glucan according to claim 1, characterized in that, In step (2), after each cleaning, the algal slurry is diluted with deionized water at a volume ratio of 1:1, and the conductivity of the system is detected. When the conductivity of the system remains basically unchanged after three consecutive cleanings, the cleaning is completed.

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