Sealing bag for breaking cyanobacteriota cell wall and method of cyanobacteriota extraction

Abstract

Provided involves a sealing bag for breaking Cyanobacteriota cell wall and a method of Cyanobacteriota extraction, wherein the sealing bag comprises a square bag body, the square bag body comprises an opening and a heat press part, and the heat press part is located below the opening; when the heat press part is heat-pressed, the opening is sealed to form a sealed space, and the sealed space is located inside the bag body to contain a Cyanobacteriota. The method of Cyanobacteriota extraction comprises inserting the Cyanobacteriota into the bag body of the sealing bag, withdrawing gas from the bag body, and heat pressing the heat press part of the bag body to form a sealed bag; placing the sealed bag under a light to let the Cyanobacteriota perform photosynthesis, and obtaining a cell-broken Cyanobacteriota; opening the sealed bag, and extracting the cell-broken Cyanobacteriota with water to obtain an extraction solution.

Description

Technical Field

[0001] This invention relates to a sealing bag for breaking down the cell walls of cyanobacteria, and a method for physically breaking down and extracting cyanobacteria using the aforementioned sealing bag. Prior Technology

[0002] Phycocyanin is a natural carotenoid with powerful antioxidant and anti-inflammatory effects. It can reduce the risk of cell damage and aging, and alleviate symptoms of chronic diseases such as arthritis and cardiovascular disease. Polyphenols, derived from plants, also have excellent antioxidant properties and can help maintain cardiovascular function and the nervous system. Both phycocyanin and polyphenols offer positive health benefits and are currently used as natural additives in food and cosmetics, while also showing great potential for drug development.

[0003] Phycocyanin and polyphenols, among other active ingredients, can be extracted from cyanobacteria (Cyanobacteriota). Before extraction, the cell walls of the cyanobacteria must be broken. Methods for cell wall breaking include chemical or microbial methods using glycoenzymes, proteases, and repeated freeze-thaw cycles (which are prone to contamination). However, current techniques for physically breaking down and extracting cyanobacteria require cumbersome and time-consuming procedures and are relatively expensive. Furthermore, some extraction methods use organic solvents, high temperatures, high pressures, or grinding, which may alter the molecular structure of phycocyanin or polyphenols and release heavy metals, causing contamination. For example, algal oil is easily extracted by mechanically grinding and breaking down cell walls, leading to the extraction of heavy metals. If chemical reagents are used for cell wall breaking extraction, residues of chemical reagents or organic solvents may remain, resulting in the degradation or loss of active ingredients. If microbial methods are used, contamination of the extracted product by microorganisms must be avoided, and the equipment requirements are also high. Therefore, there is still room for improvement. There is still a need for an easy-to-use and low-cost tool and extraction method for breaking down cyanobacteria cells, so as to extract them efficiently and quickly and retain the active substances in cyanobacteria cells to the greatest extent. Summary of the Invention

[0004] In view of the aforementioned shortcomings of the prior art, one of the objectives of this invention is to provide a resealable bag that can be used to rupture the cell walls of cyanobacteria. This bag is made of a transparent material and can be sealed by heat pressing, allowing the cyanobacteria contained within to perform photosynthesis when exposed to a light source. The resealable bag exhibits excellent sealing performance after sealing and can withstand a certain amount of internal pressure after heat pressing, ensuring that liquids and gases are contained within the sealed bag without leakage.

[0005] Another objective of this invention is to provide a method for extracting active substances from cyanobacteria using the aforementioned sealed bag. This method utilizes the gas produced by cyanobacteria's photosynthesis and the pressure of its own escaping water vapor to force the cyanobacteria's cell walls to rupture, facilitating the subsequent extraction of active substances such as phycocyanin and polyphenols from the cells. Furthermore, it effectively preserves the antioxidant activity of the extract, resulting in a higher-quality extraction solution (less susceptible to contamination by heavy metals, chemical reagents, and microorganisms). The overall extraction effect of this invention for cyanobacteria surpasses existing technologies.

[0006] To achieve the above objectives, this invention provides a sealing bag for breaking down cyanobacteria, comprising a square bag body with an opening and a heat-sealing part located below the opening. The heat-sealing part seals the opening with heat to form a sealed containing space within the bag body for containing cyanobacteria. The material of the sealing bag is selected from the group consisting of polypropylene (PP), polyethylene (PE), polyethylene terephthalate (PET), polycaprolactone (PCL) and polylactic acid (PLA). The light transmittance of the sealing bag material is 90% to 100%, and the thickness of the sealing bag is 80 micrometers (μm) to 100 micrometers.

[0007] By using a heat-sealing process to create a storage space and employing a highly translucent material, this sealed bag can contain and clearly display cyanobacteria, allowing them to receive sufficient light for photosynthesis. Furthermore, the bag offers excellent sealing and can withstand a certain amount of internal pressure after heat sealing, preventing leakage of liquids or gases after the opening is sealed. This effectively preserves the substances released by the cyanobacteria after cell wall disruption.

[0008] In the sealed bag of this creation, the square is either a square or a rectangle.

[0009] In some embodiments, the length of the bag is 10 centimeters (cm) to 3 meters (m), and the width is 10 centimeters to 3 meters. In some embodiments, the length of the bag is 10 centimeters to 2.5 meters, and the width is 10 centimeters to 2.5 meters.

[0010] In some specific embodiments, the heat-sealed portion is located 2 to 5 centimeters below the opening.

[0011] In some specific embodiments, the sealing bag is preferably made of polyethylene.

[0012] In some specific embodiments, the light transmittance of the material of the sealing bag may be 92% to 100%, or 95% to 100%, or 97% to 100%, or 99% to 100%.

[0013] In some specific embodiments, the thickness of the sealing bag may be 85 micrometers to 95 micrometers, or 90 micrometers to 95 micrometers.

[0014] In the resealable bag of this invention, the bag body is composed of a first bag surface and a second bag surface that are oppositely arranged and of the same size, and the ratio of the weight of the cyanobacteria to the area of ​​the first bag surface is from 1 kg: 80 cm² to 1 kg: 2000 cm². In some specific embodiments, the first bag surface and the second bag surface are connected to each other. In some specific embodiments, the bag body is a flat bag body. In some specific embodiments, the area of ​​the first bag surface is half of the surface area of ​​the bag body. In some specific embodiments, the ratio of the weight of the cyanobacteria to the area of ​​the first bag surface can be from 1 kg: 100 cm² to 1 kg: 1500 cm², or from 1 kg: 500 cm² to 1 kg: 1000 cm².

[0015] In some specific embodiments, the blue-green algae may be Spirulina spp. or Nostoc commune, but is not limited to these.

[0016] In some specific embodiments, the sealing bag can withstand temperatures from 0°C to 100°C; more specifically, the sealing bag can withstand temperatures from 4°C to 90°C, or from 4°C to 80°C, or from 4°C to 70°C.

[0017] In some specific embodiments, the sealing bag can withstand an internal pressure of 2 atmospheres (atm) or more after being heat-sealed; specifically, the internal pressure that the sealing bag can withstand after being heat-sealed can be 2 to 5 atmospheres, or 2 to 4.5 atmospheres, or 2 to 3.5 atmospheres, or 2.5 to 3.5 atmospheres.

[0018] To achieve the aforementioned objective, this invention also provides a method for extracting cyanobacteria, comprising: (1) Provide a resealable bag as described above, wherein the bag body is composed of a first bag face and a second bag face that are oppositely arranged and of the same size; (2) Blue-green algae are placed inside the bag body of the sealing bag, the gas inside the bag is extracted until the pressure inside the bag is 0.1 atm to 1 atm, and the hot-press sealing part of the bag body is hot-pressed to seal the opening of the bag body, forming a sealed sealing bag; wherein the ratio of the weight of the blue-green algae to the area of ​​the first bag surface is 1 kg: 80 square centimeters to 1 kg: 2000 square centimeters; (3) Place the sealed bag under a light source at 4°C to 80°C for 8 to 72 hours to allow the cyanobacteria to photosynthesize, and the pressure inside the bag reaches 2 to 4.5 atmospheres. The light intensity of the light source is 1500 to 8000 lux, and the cyanobacteria with broken cell walls are obtained. (4) Open the sealed bag and extract the broken-cell blue-green algae with water to obtain an extract solution.

[0019] This extraction method utilizes the oxygen produced by cyanobacteria through photosynthesis and the evaporated water vapor to create gas pressure within a sealed bag, causing physical cell wall disruption and releasing the substances contained in the cyanobacteria. This facilitates the extraction of active substances such as phycocyanin and polyphenols from the cells. This extraction method is low-cost, simple to operate, and convenient for relocation; since it only uses water, it is also extremely environmentally friendly and protects the health of operators.

[0020] In the extraction method of this invention, the light source can be sunlight or fluorescent lamp. In some specific embodiments, the illuminance of the light source can be 1,500 lux to 5,000 lux, or 3,000 lux to 5,000 lux, or 4,000 lux to 8,000 lux.

[0021] In some specific embodiments, the temperature at which the sealed bag is placed under light can be 4°C to 70°C, or 20°C to 60°C, or 25°C to 50°C, or 25°C to 40°C.

[0022] In some specific embodiments, the sealed bag may be placed under light for 8 to 24 hours, or 24 to 60 hours, or 36 to 48 hours, or 48 to 72 hours.

[0023] In some specific embodiments, the sealed bag is opened to obtain the mixture inside when the internal pressure reaches 2 to 3.5 atmospheres, or 3 to 4.5 atmospheres; wherein the mixture contains phycocyanin and polyphenols, but is not limited to these.

[0024] In some specific embodiments, the extraction temperature is 4°C to 70°C; more specifically, the extraction temperature may be 10°C to 35°C, or 15°C to 30°C.

[0025] In some specific embodiments, the extraction step may involve placing the cell-wall-broken cyanobacteria in ultrapure water at room temperature of 25°C for 1 hour, stirring, and then filtering to obtain an extraction solution; wherein the ratio of the cell-wall-broken cyanobacteria to ultrapure water is 1 kg: 1.5 liters to 1 kg: 4 liters.

[0026] In some specific embodiments, the extraction solution can be further freeze-dried to obtain the aforementioned active substance powder.

[0027] The sealing bag and extraction method for breaking down cyanobacteria provided in this invention allow cyanobacteria to perform photosynthesis while sealed in the bag, and achieve a physical cell wall breaking effect through the gas pressure generated. At the same time, the substances released after cell wall breaking retain better activity, so as to obtain a cyanobacteria extract with excellent components. Simple Explanation of the Diagram

[0028] Figure 1A is a frontal view of the sealing bag used for breaking down the cell walls of cyanobacteria in this invention. Figure 1B is a side view of the sealed bag used for breaking down the cell walls of cyanobacteria in this invention. Figure 2A is a frontal view of the sealed bag used for breaking down cyanobacteria in this creation after the cyanobacteria are placed inside and the bag is sealed. Figure 2B is a frontal view of the sealed bag used for breaking down cyanobacteria in this creation after the cyanobacteria are placed inside, the bag is sealed, and the bag is exposed to light. Figure 3 is a flowchart of the blue-green algae extraction process in this work. Figure 4A shows the FT-IR detection results of algae in Comparative Example 1 (before cell disruption). Figure 4B shows the FT-IR detection results of algae in Example 14 (after cell wall disruption). Implementation

[0029] This invention will further illustrate the structure of the sealing bag for breaking down cyanobacteria with illustrations, and will provide several examples to illustrate the cyanobacteria extraction method of this invention. Comparative examples are also provided for comparison. Those skilled in the art can easily understand the advantages and effects of this invention through the following examples and comparative examples. It should be understood that the examples listed in this specification are merely illustrative of the implementation of this invention and are not intended to limit the scope of this invention. Those skilled in the art can make various modifications and changes based on their ordinary knowledge without departing from the spirit of this invention to implement or apply the content of this invention.

[0030] Please refer to Figures 1A, 1B, 2A, and 2B for the sealing bag used for breaking down cyanobacteria in this invention. As shown in Figures 1A to 2B, the sealing bag 1 comprises a square bag body 10, which includes an opening 11, a closed bottom 12, and a heat-sealed portion 13. The heat-sealed portion 13 is located 2 to 5 centimeters below the opening 11. The heat-sealed portion 13 seals the opening 11 by heat-sealing, forming a sealed containing space 14 within the bag body 10 to contain cyanobacteria 20. The material of the sealing bag 1 is selected from the group consisting of polypropylene, polyethylene, polyethylene terephthalate, polycaprolactone, and polylactic acid, with polyethylene being the most preferred material. The thickness of the sealing bag 1 is 80 to 100 micrometers; the length of the bag body 10 is 10 centimeters to 2.5 meters, and the width is 10 centimeters to 2.5 meters. As shown in Figure 1B, the bag body 10 is composed of a first bag surface 15 and a second bag surface 16 arranged opposite each other and of the same size. The ratio of the weight of the blue-green algae 20 to the area of ​​the first bag surface 15 is from 1 kg: 80 square centimeters to 1 kg: 2000 square centimeters. The sealing bag 1 can withstand a temperature of 0°C to 100°C, and the sealing bag 1 can withstand an internal pressure of more than 2 atmospheres after being heat-sealed.

[0031] When using the sealing bag of this invention for cyanobacterial extraction, firstly, the sealing bag 1 is provided, cyanobacterial algae 20 is placed in the bag body 10 of the sealing bag 1, and the gas inside the bag body 10 is extracted to a rough vacuum state. At this time, the pressure inside the bag body 10 is 0.1 atmospheres to 1 atmosphere. The hot-pressed sealing part 13 is then pressed to seal the opening 11, forming a sealed containment space 14, thereby forming a sealed sealing bag 1' (as shown in Figure 2A). The sealed bag 1' containing cyanobacteria 20 is placed under a light source for irradiation. After a period of photosynthesis, the sealed bag 1' will fill with gas 21, and the pressure inside the bag caused by the gas 21 can reach 2 to 3.5 atmospheres, resulting in cell wall disruption of the cyanobacteria 20 (as shown in Figure 2B). The sealed bag 1' is then opened, and the disrupted cyanobacteria 20 is placed into a quantitative container containing water and stirred to form a mixture containing the disrupted algae and an extraction solution. Finally, the mixture is filtered to separate the disrupted algae from the extraction solution. Please refer to Figure 3 for the above cyanobacteria extraction process.

[0032] [Comparative Example] [1]

[0033] 1.2 kg of *Gnaphalium affine* (purchased from Qinyuan Enterprise Co., Ltd., No. 249-6, Mudan Rd., Mudan Village, Mudan Township, Pingtung County) was placed in a quantitative water bath filled with 4 liters of ultrapure water at pH 7.0 for 1 hour at room temperature of 25°C. The water bath was stirred for 20 seconds every half hour. The algae of *Gnaphalium affine* were then separated by filtering through a 200-mesh sieve to obtain an extraction solution.

[0034] [Example] [1]

[0035] The sealing bag used in this invention has a transmittance of 95% as measured by a transmittance meter (LS116, Pivot Technology Co., Ltd.), and the area of ​​the first surface of the bag is 900 square centimeters. 1.2 kg of *Phyllostachys edulis* (blue-green algae) is placed inside the bag. The gas inside the bag is then evacuated to a rough vacuum (pressure inside the bag is 0.1 to 1 atm), and the heat-sealed opening of the bag is then sealed to form a sealed bag. Here, the ratio of the weight of the blue-green algae to the area of ​​the first surface is 1 kg : 750 square centimeters. The sealed bag was placed under a 60-watt fluorescent lamp at 25°C for 72 hours, with a light intensity of 1500 lux, to induce photosynthesis in the cyanobacteria. During this time, the sealed bag was filled with gas containing oxygen produced by photosynthesis and water vapor evaporated from the cyanobacteria. The pressure inside the bag gradually increased to 2 atmospheres, which caused the cell walls of the *Gnaphalium affine* to break down. The light intensity of the fluorescent lamp was measured using an illuminance meter (ANA-F9, Tokyo Optoelectronics), and the pressure inside the bag was measured using a metal tire pressure gauge (TA-D026, Taige).

[0036] Open the sealed bag and place the cell-wall-broken Ganoderma lucidum in a quantitative water bath filled with 4 liters of ultrapure water at pH 7.0 at room temperature of 25°C for 1 hour. Stir the water bath for 20 seconds every half hour. Then filter the cell-wall-broken Ganoderma lucidum with a 200-mesh sieve to separate the cell-wall-broken algae and obtain an extraction solution.

[0037] [Example] [2] [to]

[16]

[0038] The blue-green algae extraction methods in Examples 2 to 16 are largely the same as those in Example 1, with the only differences being the light transmittance of the sealed bag, the surface area of ​​the first bag, the weight of *Gnaphalium affine* in the bag, the light intensity of the fluorescent lamp, the photosynthetic temperature and time, the pressure inside the gas-filled bag, and the fact that the *Gnaphalium affine* after cell wall disruption in Examples 8 to 16 was placed in a quantitative water bath containing 8 liters of ultrapure water.

[0039] For ease of explanation, the light transmittance of the sealed bag, the surface area of ​​the first bag, the weight of the mushroom, the light intensity, the photosynthetic temperature, the photosynthetic time, and the pressure inside the bag in Examples 1 to 16 are listed in Table 1 below.

[0040] Table 1: Conditions for the blue-green algae extraction methods in Examples 1 to 16 (E1 to E16) Light transmittance of sealed bag (%) Surface area of ​​the first bag (cm²) Weight of *Agrocybe aegerita* (in kg) Light intensity (lx) Photosynthetic temperature (°C) Photosynthesis time (Hour) Pressure inside the bag (atm) E1 95 900 1.2 1500 25 72 2 E2 95 900 1.2 2000 30 72 2.5 E3 90 900 1.2 3000 30 72 3 E4 95 900 1.2 3000 30 72 4 E5 90 900 1.2 2500 35 48 4 E6 95 900 1.2 2500 35 48 4 E7 90 1800 2.4 3000 35 36 4.5 E8 95 1800 2.4 3000 35 36 4.5 E9 90 1800 2.4 3500 35 36 4.5 E10 95 1800 2.4 3500 35 36 4.5 E11 90 1800 2.4 4000 37 24 4.5 E12 95 1800 2.4 4000 37 24 4.5 E13 90 1800 2.4 6000 37 12 4.5 E14 95 1800 2.4 6000 40 8 4.5 E15 95 1800 2.4 6000 50 8 4.5 E16 95 1800 2.4 6000 60 8 4.5

[0041] [Experimental Example] [1] [Measuring phycocyanin content]

[0042] This experimental example used the extract solutions from Comparative Example 1 and Examples 1 to 16 as samples. The phycocyanin content in these samples was detected using the standard analytical method CNS 14721 from the National Standard Analytical Methods (CNS) of the Republic of China. The measurement was conducted by the Ai-Wei Testing and Analysis Center to determine the phycocyanin content (mg) per kilogram of extract solution. Besides the CNS analytical method described above, those skilled in the art may also use other analytical methods to analyze the phycocyanin content in the extract solution.

[0043] The phycocyanin content of Comparative Example 1 and Examples 1 to 16 after extraction, as well as the phycocyanin increase rate of Examples 1 to 16 relative to Comparative Example 1 (phycocyanin content of each example / phycocyanin content of the comparative example), are listed in Table 2 below.

[0044] Table 2: Phycocyanin content and phycocyanin increase rate after extraction in Comparative Example 1 (C1) and Examples 1 to 16 (E1 to E16) Phycocyanin content (mg / kg) Phycocyanin increase rate (times) C1 4.0 1.0 E1 6.0 1.5 E2 14.8 3.7 E3 17.5 4.375 E4 17.9 4.475 E5 19.8 4.95 E6 24.4 6.1 E7 26.7 6.675 E8 28.9 7.225 E9 33.0 8.25 E10 33.4 8.35 E11 38.0 9.5 E12 38.8 9.7 E13 115.4 28.85 E14 141.9 35.475 E15 70.5 17.625 E16 46.9 11.725

[0045] As shown in Table 2, compared to Comparative Example 1, the phycocyanin content obtained after extraction was higher when the cyanobacteria cell wall breaking sealing bag of this invention was used in Examples 1 to 16. Under the same photosynthetic time, the phycocyanin content increased significantly when the light intensity was greater and the gas pressure inside the bag was greater after photosynthesis. Examples 14 to 16 showed that when the photosynthetic temperature of cyanobacteria was above 40°C, the interaction between phycocyanin and other substances became more intense with increasing temperature, which may lead to the formation of more polymers (water-insoluble substances), resulting in a lower content of phycocyanin extracted. Therefore, using the cyanobacteria cell wall breaking sealing bag of this invention under the conditions of Example 14 has the best effect on the photosynthesis of the cyanobacteria contained in the bag, which is beneficial for breaking the cell wall of the cyanobacteria and extracting the phycocyanin contained therein.

[0046] [Experimental Example] [2] [:] [FT-IR]

[0047] This experiment used algae from Comparative Example 1 and Example 14 as samples. Fourier transform infrared spectroscopy (FT-IR) was used for qualitative analysis of these samples, and the measurements were performed by the National Taiwan University Emerging Materials and Advanced Components Technology Research Center. The algae were dried, ground into a fine powder, mixed with potassium bromide powder, and pressed into thin slices. The absorption spectra of these thin slices in the range of 500 cm⁻¹ to 4000 cm⁻¹ were measured using an FT-IR spectrometer (FT / IR-4X, JASCO) with a transmission-type method. The detection results for Comparative Example 1 (before cell disruption) and Example 14 (after cell disruption) are shown in Figures 4A and 4B, respectively. Structural changes in the algae of Comparative Example 1 and Example 14 can be observed at wavenumbers of 1200 cm⁻¹ to 1700 cm⁻¹ and 700 cm⁻¹ to 900 cm⁻¹ in the FT-IR spectra, showing the difference in the degree of cell disruption.

[0048] [Experimental Example] [3] [Measuring polyphenol content]

[0049] This experimental example used the extraction solutions from Comparative Example 1 and Example 6 as samples. The polyphenols in these samples were detected using a colorimetric method, and the results were measured by the Ai-Wei Testing and Analysis Center to determine the polyphenol content (mg) per 100g of extraction solution. The extraction solution was mixed thoroughly with sodium carbonate, and then Folin-Ciocalteu reagent (1.09001, Merck) was added to prepare the sample. Gallic acid was mixed thoroughly with sodium carbonate, and then Folin-Ciocalteu reagent was added to prepare the standard. The absorbance at 750 nm was measured using a spectrophotometer (UV-1700, SHIMADZU). A quantitative check line was plotted using the absorbance of the standard, and the polyphenol content was calculated from the absorbance of the sample based on this check line.

[0050] [Experimental Example] [4] [Testing oxygen free radical absorption capacity]

[0051] This test used the extraction solutions from Comparative Example 1 and Example 6 as samples. The oxygen radical absorbance capacity (ORAC) in the samples was detected by fluorescence attenuation quantification method and the measurement was entrusted to the Ai-Wei Testing and Analysis Center. The extraction solution was mixed with 75 mM NaH 2PO 4 buffer to prepare the sample, and Trolox solutions were prepared as standards at concentrations of 100, 50, 25, 12.5, 6.25, 5.0, 3.0, and 1.0 μM. The sample was ultrasonically disrupted for 5 minutes, then centrifuged at 10000g for 10 minutes at 4°C, and the supernatant was collected. The determination was performed using a fluorescence microplate analyzer (FLUOstar Omega, BMG Labtech), with an excitation wavelength of 485 nm and a scattered wavelength of 520 nm. A quantitative calibrator was plotted using the absorbance of the standard, and the ORAC value was calculated from the absorbance of the sample supernatant based on the quantitative calibrator.

[0052] [Experimental Example] [5] [:test] [DPPH] [Free radical scavenging ability]

[0053] This test used the extraction solutions from Comparative Example 1 and Example 6 as samples to test the DPPH (2,2-Diphenyl-1-picrylhydrazyl) free radical scavenging ability in these samples, which was measured by the Ai-Wei Testing and Analysis Center. The extraction solution was mixed with a DPPH + methanol solution at a concentration of 25 mg / L to prepare the sample, and Trolox solutions ranging from 40 μmol / L to 200 μmol / L were prepared as standards. The absorbance at 517 nm was measured using a spectrophotometer. A quantitative check line was plotted using the absorbance of the standards, and the DPPH free radical scavenging ability was calculated from the absorbance of the samples based on this quantitative check line.

[0054] [Experimental Example] [6] [Testing superoxide dismutase activity]

[0055] This experimental example used the extraction solutions from Comparative Example 1 and Example 6 as samples. The superoxide dismutase (SOD)-like activity in these samples was detected using ultra-weak chemiluminescence (uwCL) and measured by the Ai-Wei Testing and Analysis Center. Hypoxanthine and xanthine oxidase were mixed and reacted, then the extraction solution was added before testing. N,N'-dimethyl-9,9'-diacrylamide dinitrate (M8010, Sigma-Aldrich) was used as a probe, and detection was performed using an ultra-weak chemiluminescence analyzer (CLA-FSI, Tohoku). The SOD-like activity per milliliter of extraction solution was calculated.

[0056] The results of Test Examples 3 to 6 are presented together in Table 3 below, where the oxygen free radical absorption capacity and DPPH free radical scavenging capacity are expressed as Trolox equivalents (TE) antioxidant capacity. In addition, when the measured value is below the detection limit, it is marked "ND" (Not detected) in Table 3 below.

[0057] Table 3: Comparison of polyphenol content, oxygen free radical absorption capacity, DPPH free radical scavenging capacity and superoxide dismutase-like activity test results between Comparative Example 1 (C1) and Example 6 (E6) C1 E6 Polyphenol content (mg / 100g) ND 8.21 Oxygen free radical absorption capacity (μM TE / 100 ml) ND 41.52 DPPH free radical scavenging capacity (μM TE / mL) ND ND Superoxide dismutase-like activity (units / mL) 1995.09 3520.08

[0058] As shown in Table 3, the blue-green algae extraction method of this invention can effectively extract polyphenols from blue-green algae and improve oxygen free radical absorption capacity and superoxide dismutase-like activity, indicating that the antioxidant capacity of the components in the extraction solution is increased.

[0059] In summary, when cyanobacteria are placed and sealed in the cyanobacteria cell-wall breaking bag of this invention, they can perform photosynthesis and generate gas pressure within the bag, thus achieving the cell-wall breaking effect. Based on the results of measuring phycocyanin and polyphenol content, and testing oxygen free radical absorption capacity, DPPH free radical scavenging capacity, and superoxide dismutase-like activity, it is known that the cyanobacteria extraction method of this invention can effectively release the active substances in cyanobacteria after cell-wall breaking, thereby obtaining a cyanobacteria extract containing excellent components.

[0060] 1: Sealed bag 1': Sealed bag 10: Bag body 11: Opening 12: Bottom 13: Hot-press sealing section 14: Sealed accommodating space 15: First bag of flour 16: Second bag of flour 20: Blue-green algae 21: Gas

Claims

1. A sealing bag for breaking down cyanobacteria, comprising a square bag body, the bag body including an opening and a heat-sealing portion, the heat-sealing portion being located below the opening, the opening being sealed by heat-sealing to form a sealed containing space, the sealed containing space being located inside the bag body for containing cyanobacteria, wherein the cyanobacteria is *Gnaphalium affine*; wherein the sealing bag can withstand an internal pressure of 4.5 atm to 5 atm after heat-sealing, the material of the sealing bag is selected from the group consisting of polypropylene, polyethylene, polyethylene terephthalate, polycaprolactone and polylactic acid, the light transmittance of the material of the sealing bag is 90% to 100%, and the thickness of the sealing bag is 80 micrometers to 100 micrometers.

2. The resealable bag as described in claim 1, wherein the length of the bag is 10 cm to 3 meters and the width is 10 cm to 3 meters.

3. The resealable bag as described in claim 1, wherein the bag body is composed of a first bag surface and a second bag surface that are arranged opposite each other and of the same size, and the ratio of the weight of the blue-green algae to the area of ​​the first bag surface is from 1 kg: 80 square centimeters to 1 kg: 2000 square centimeters.

4. The sealing bag as described in claim 1, wherein the sealing bag can withstand temperatures from 0°C to 100°C.

5. A method for extracting cyanobacteria, comprising: (1) providing a resealable bag as described in any one of claims 1 to 4, wherein the bag body is composed of a first bag body and a second bag body disposed opposite to each other and of the same size; (2) placing cyanobacteria inside the bag body, extracting gas from the bag body to a pressure of 0.1 atm to 1 atm inside the bag body, and hot-pressing the hot-pressed sealing portion of the bag body to seal the opening of the bag body, thereby forming a sealed resealable bag; wherein the weight ratio of the cyanobacteria to the area of ​​the first bag body is from 1 kg: 80 square centimeters to 1 kg: 2000 square centimeters, and the cyanobacteria is *Gnaphalium affine*; (3) The sealed bag is placed under a light source at 4°C to 80°C for 8 to 12 hours to allow the blue-green algae to photosynthesize, and the pressure inside the bag reaches 2 to 4.5 atmospheres, where the light intensity of the light source is 6000 to 8000 lux, to obtain the blue-green algae after cell wall disruption; (4) The sealed bag is opened, and the blue-green algae after cell wall disruption is extracted with water to obtain an extract solution.

6. The extraction method as described in claim 5, wherein the extraction temperature is between 4°C and 70°C.

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