Method for extracting and preparing phycocyanin by means of autolysis of arthrospira platensis

Abstract

The present invention relates to a method for extracting, preparing, and producing phycocyanin by freezing and thawing Arthrospira platensis and using the autolysis reaction. When phycocyanin is extracted by using the method of the present invention, the extraction concentration, yield, and purity of phycocyanin are significantly increased.

Description

Method for extracting and producing phycocyanin through autolysis of spirulina

[0001] The present invention relates to a method for extracting and producing high-concentration and high-purity phycocyanin by autolyzing spirulina cultured using a medium containing bicarbonate.

[0002] Phycocyanin (PC), found in cyanobacteria, is a water-soluble, light-absorbing protein used in the cosmetics and pharmaceutical industries and for fluorescent labeling. Spirulina (Arthrospira platensis) is a widely used, abundant, and inexpensive natural source of PC pigment. Extracting PC from cyanobacteria is challenging due to its highly resistant, multilayered cell wall. Therefore, developing a simple and effective extraction process for PC from Spirulina is crucial for its industrial applications.

[0003] Phycocyanin is an industrially useful substance known to effectively remove harmful reactive oxygen species, preventing lipid peroxidation and suppressing inflammatory responses. Furthermore, phycocyanin has anti-inflammatory effects by reducing the activity of cycloxygenase-2, an enzyme that produces inflammatory substances, and suppressing the release of histamine from mast cells. Furthermore, it is known to suppress carbon tetrachloride-induced liver toxicity through lipid peroxidation and inhibition of cycloxygenase-2.

[0004] Various techniques have been used to extract phycocyanin (PC), including freeze-thawing, chemical (organic and inorganic acid) treatment, lysozyme treatment, ultrasonic treatment, pulsed electric field treatment, and mechanical homogenization. Meanwhile, the freeze-thawing method is known to be used to extract phycocyanin (PC) from wet biomass of Spirulina. However, when extracting phycocyanin (PC) from Spirulina using the conventional freeze-thawing method, there was a problem in that it required multiple freeze-thawing cycles, which was time-consuming, and the use of extraction salts such as sodium phosphate or ammonium chloride, which incurred additional process costs.

[0005] Accordingly, the inventors of the present invention developed an efficient extraction method for phycocyanin (PC) and completed the present invention.

[0006] The purpose of the present invention is to provide a method for culturing spirulina (Arthrospira platensis) in a medium containing bicarbonate;

[0007] A step of producing the above culture into wet biomass; and

[0008] A method for producing phycocyanin is provided, comprising the steps of extracting phycocyanin by freezing and thawing.

[0009] Another object of the present invention is to provide phycocyanin manufactured by the above manufacturing method.

[0010] Another object of the present invention is to provide a food composition comprising phycocyanin manufactured by the above manufacturing method.

[0011] Another object of the present invention is to provide a health functional food composition comprising phycocyanin manufactured by the above manufacturing method.

[0012] Another object of the present invention is to provide a feed composition comprising phycocyanin manufactured by the above manufacturing method.

[0013] In order to achieve the above purpose, the present invention comprises the steps of culturing Spirulina (Arthrospira platensis) in a medium containing bicarbonate;

[0014] A step of producing the above culture into wet biomass; and

[0015] A method for producing phycocyanin is provided, comprising the steps of extracting phycocyanin by freezing and thawing.

[0016] In addition, the present invention provides phycocyanin manufactured by the above manufacturing method.

[0017] In addition, the present invention provides a food composition comprising phycocyanin manufactured by the above manufacturing method.

[0018] In addition, the present invention provides a health functional food composition comprising phycocyanin manufactured by the above manufacturing method.

[0019] In addition, the present invention provides a feed composition comprising phycocyanin manufactured by the above manufacturing method.

[0020] The present invention relates to a method for extracting, manufacturing, and producing phycocyanin by freezing and thawing spirulina. When phycocyanin is extracted using the method of the present invention, phycocyanin can be extracted quickly and easily at a high concentration, high yield, and high purity without having to repeat the freeze-thaw cycle several times.

[0021] Figure 1a shows the extraction concentration of phycocyanin at a storage temperature of 25°C by the autolysis method of Spirulina (Arthrospira platensis).

[0022] Figure 1b shows the extraction concentration of phycocyanin at a storage temperature of 37°C by the autolysis method of Spirulina (Arthrospira platensis).

[0023] Figure 1c shows the extraction concentration of phycocyanin at a storage temperature of 4°C by the autolysis method of Spirulina (Arthrospira platensis).

[0024] Figure 2 shows the change in shape according to temperature conditions observed under a microscope during the wet biomass autolysis process of Spirulina (Arthrospira platensis).

[0025] Figure 3a shows the growth curve of the biomass of Spirulina according to the influence of the initial concentration of bicarbonate (16.8 g / L or 8.4 g / L) of the growth medium in the autolysis method of the present invention.

[0026] Figure 3b shows the concentration of phycocyanin (PC) in wet biomass of Spirulina according to the influence of the initial bicarbonate concentration (16.8 g / L or 8.4 g / L) of the growth medium by the autolysis method of the present invention.

[0027] Figure 3c shows the purity of phycocyanin (PC) of wet biomass of Spirulina according to the influence of the initial bicarbonate concentration (16.8 g / L or 8.4 g / L) of the growth medium by the autolysis method of the present invention.

[0028] Figure 4a shows the effect of initial wet biomass concentration in Spirulina on the phycocyanin (PC) extraction yield by autolysis method.

[0029] Figure 4b shows the effect of wet biomass concentration in initial Spirulina on phycocyanin (PC) purity by autolysis method.

[0030] The present invention is described in more detail through the following preparation examples and examples.

[0031] However, the examples are merely examples intended to easily illustrate the content and scope of the technical concept of the present invention and are not intended to limit or alter the technical scope of the present invention. Furthermore, those skilled in the art will readily recognize that various modifications and variations within the scope of the technical concept of the present invention are possible based on these examples.

[0032] Preparation Example 1. Cultivation of Spirulina

[0033] Spirulina (Arthrospira platensis) KCTC AG40101 was obtained from the Korea Center for Biological Resources (KCTC). Spirulina was cultured in a transparent vertical column photobioreactor (height 45 cm, diameter 12 cm, custom-made) on Zarrouk medium (Hirano et al., 1990) containing 8.4 g / L of NaHCO₃ (bicarbonate of soda). The mixture was mixed by constant bubbling air and aeration through a 0.2 μm polytetrafluoroethylene (PTFE) filter at the bottom of the reactor.

[0034] The above Zarrouk medium contains 8.4 g / L of NaHCO3, 0.5 g / L of K2HPO4, 2.5 g / L of NaNO3, 1.0 g / L of K2SO4, 1.0 g / L of NaCl, 0.2 g / L of MgSO4·7H2O, 0.04 g / L of CaCl2·2H2O, 0.01 g / L of FeSO4·7H2O, 0.08 g / L of ethylenediaminetetraacetic acid (EDTA), and 1 mL of a micronutrient solution, which contains 2.86 g / L of H3BO3, 1.81 g / L of MnCl2·4H2O, 0.222 g / L of ZnSO4·4H2O, 0.0177 g / L of Na2MoO4, and 1 mL of a micronutrient solution. Contains 0.079 g / L.

[0035] All cultures were maintained in transparent vertical column reactors (45 cm high, 12 cm diameter) at 25°C and aerated with constant bubbling air through a 0.2 μm polytetrafluoroethylene (PTFE) filter at the bottom of the reactor.

[0036] The light source was a white light-emitting diode (LED), and the standard incident photon flux density at the front of the photobioreactor was 100 μmol photons / m during cultivation. 2 / s continuous light illumination was used. Cell growth was measured daily using a spectrophotometer (JASCO-V-730 Spectrophotometer, Tokyo, Japan) at a wavelength of 680 nm.

[0037] Meanwhile, for phycocyanin extraction, Spirulina cells were cultured until the spectrophotometric value at a wavelength of 680 nm reached 2.

[0038] The dry cell weight of Spirulina was measured after filtering through a pre-dried and pre-weighed 0.45 μm cellulose nitrate membrane (Whatman, USA) and drying the cells in an oven at 80°C for 24 h.

[0039]

[0040] Preparation Example 2. Extraction of phycocyanin through autolysis using freeze-thaw method

[0041] Spirulina wet biomass was autolyzed by freeze-thawing to disrupt the cell membrane and extract phycocyanin contained in the cytoplasm using the following method.

[0042] To determine the difference in the effect of the type of salt solution on the extraction yield of phycocyanin (PC) in the freezing-thawing method, phycocyanin was extracted using the freezing-thawing method of Manirafasha et al. (2017).

[0043] Specifically, Spirulina cells cultured in the same manner as in Preparation Example 1 were harvested using a standard sieve with a mesh size of 0.045 mm, filtered cells (250 mg) were obtained, and the cells were washed three times with distilled water to obtain 100% Spirulina wet biomass (hereinafter referred to as wet biomass), which was diluted to a defined concentration using distilled water (for example, 75% wet biomass is composed of 7.5 mL of the 100% wet biomass and 2.5 mL of distilled water). The prepared wet biomass was frozen once at -20°C for 24 hours and then thawed at 25°C for 1 hour. The subsequent autolysis process was performed by reacting the wet biomass at various concentrations in a chamber at 4, 25, or 37°C for 0, 3, 6, 9, 12, or 24 hours. Morphological changes in cells during the autolysis process were observed microscopically (Fig. 2). After incubation, the cell lysate was centrifuged at 8960 × g for 15 min at 4°C, and the blue supernatant containing phycocyanin (hereinafter referred to as the crude extract) was collected.

[0044]

[0045] Preparation Example 3. Measurement of phycocyanin purity and concentration

[0046] To measure the purity and extraction yield of the extracted phycocyanin, the absorption spectrum of the crude extract (blue supernatant) of Preparation Example 2 was confirmed in the wavelength range of 280 to 750 nm using a spectrophotometer (JASCO-V-730 Spectrophotometer, Tokyo, Japan). The purity of the extracted phycocyanin was expressed as the absorbance ratio A 620 / A 280 The concentration and extraction yield of phycocyanin (PC) were determined using the following equation.

[0047] Phycocyanin concentration (mg / mL) = (A 615 - 0.474 × A 652 ) / 5.34

[0048] Extraction yield (mg / g) = (phycocyanin (PC) concentration × solvent volume) / dry cell weight

[0049]

[0050] Example 1. Extraction of phycocyanin (PC) through autolysis

[0051] In order to confirm whether the extraction efficiency of phycocyanin (PC) increases depending on the reaction temperature and time during the autolysis process of the present invention, 75% wet biomass prepared as in Preparation Example 2 was frozen at -20°C for 24 hours and then thawed at 25°C for 1 hour. The cells were reacted under three temperature conditions of 4°C, 25°C, or 37°C, and the absorbance spectrum of the crude extract supernatant sample was measured in the same manner as in Preparation Example 3 (Fig. 1).

[0052] As a result, as shown in Fig. 1, it was confirmed that the peak intensity at 620 nm increased as the reaction time increased at 25°C and 37°C. Phycocyanin (PC) was measured based on the characteristic of a maximal absorption peak appearing at 620 nm.

[0053] When reacted at 25°C, a significant amount of phycocyanin was extracted after 9 hours (confirmed by the 620 nm absorption peak), but when reacted at 37°C, a 91% maximum absorption peak was observed after 3 hours of reaction (Fig. 1a, Fig. 1b). On the other hand, when reacted at 4°C, it was confirmed that phycocyanin (PC) was not extracted through autolysis (Fig. 1c).

[0054] Furthermore, microscopic observation of the autolysis process revealed dramatic changes in the morphology of the wet biomass of Spirulina (Arthrospira platensis) (Fig. 2). Spirulina cells, which had been intact, completely disappeared after 3 hours of autolysis at 37°C. However, when the cells were autolyzed at 4°C, no changes in their coiled shape were observed (Fig. 2).

[0055] Using the methods of Preparation Examples 2 and 3, the concentration, extraction yield, and purity of phycocyanin (PC) in the crude extract according to each autolysis reaction time were calculated and are shown in Table 1. When the reaction was performed at 37℃, the purity index of phycocyanin exceeded 1 after 3 hours of reaction, and the value increased as the reaction time elapsed (Table 1). Table 1 shows the results of analyzing the phycocyanin concentration, extraction yield, and purity using one-way ANOVA test and Duncan test (p<0.05).

[0056] Item Temperature 0 h3 h6 h9 h12 h24 h Phycocyanin Concentration (mg / mL) 25℃ 0.042 h 0.080 h 0.0127 gh 0.885 f 2.973 e 5.155 cd 4℃0.042 h 0.065 h 0.092 gh 0.103 gh 0.105 gh 0.133 gh 37℃0.259 g 5.030 d 5.522 a 5.303 bc 5.334 b 5.202 bc Extraction yield (mg / g) 25℃ 0.779 h 1.495 h 2.372 gh 16.467 f 55.320 e 95.919cd 4℃0.780 h 1.206 h 1.716 gh 1.923 gh 1.960 gh 2.470 gh 37℃4.813 g 93.603 d 102.751 a 98.677 bc 99.252 b 96.807 bc Purity 25℃ 0.070 h 0.106 ghi 0.145 g 0.589 e 1.045 c 0.947 d 4℃0.078 hi 0.108 ghi 0.140 gh 0.146 g 0.143 g 0.155 g 37℃0.257 f 1.194 b 1.168 b 1.261 a 1.271 a 1.318 a

[0057]

[0058] Example 2. Extraction of phycocyanin by optimized autolysis of the present invention

[0059] The following experiments were conducted to determine the effects of the initial bicarbonate concentration of Spirulina growth medium and the concentration of wet biomass on the phycocyanin (PC) extraction yield according to the autolysis method.

[0060] Example 2-1. Initial bicarbonate concentration of growth medium

[0061] The following experiments were conducted to determine whether the initial bicarbonate concentration of the Spirulina growth medium affects the growth, extraction yield, and purity of phycocyanin in Spirulina.

[0062] Specifically, Spirulina growth medium was cultured with an initial bicarbonate (NaHCO3) concentration of 8.4 g / L (Zarrouk medium of Preparation Example 1) or 16.8 g / L (standard Zarrouk medium) in the same manner as Example 1, and 75% Spirulina wet biomass prepared in the same manner as Example 1 was frozen once at -20°C for 24 hours, thawed at 25°C for 1 hour, and then reacted at 37°C, and the yield of phycocyanin (PC) extraction by autolysis was compared with the phycocyanin content extracted from Spirulina cells grown in a medium containing 16.8 g / L of bicarbonate (NaHCO3) and Spirulina cells grown in a medium containing 8.4 g / L of bicarbonate (NaHCO3) (Fig. 3).

[0063] As a result, it was confirmed that there was no difference in the growth of Spirulina cells when grown in a medium containing 16.8 g / L sodium bicarbonate (NaHCO3) or a medium containing 8.4 g / L sodium bicarbonate (NaHCO3) (Fig. 3a). On the other hand, when Spirulina cells were grown in a medium containing 16.8 g / L sodium bicarbonate (NaHCO3), no significant extraction of phycocyanin was observed within 24 hours, but when Spirulina cells were grown in a medium containing 8.4 g / L sodium bicarbonate (NaHCO3), it was confirmed that the phycocyanin (PC) concentration increased by approximately 90% when the autolysis reaction was performed at 37°C for 3 hours (Fig. 3b). In addition, when Spirulina cells were cultured in a medium containing 16.8 g / L of sodium bicarbonate (NaHCO3), the phycocyanin (PC) purity was only 0.1 (A620nm / A280nm) after the initial 3-hour autolysis reaction, but when Spirulina cells were cultured in a medium containing 8.4 g / L of sodium bicarbonate (NaHCO3), the phycocyanin (PC) purity was confirmed to quickly reach 1.2 (A620nm / A280nm) after the initial 3-hour autolysis reaction (Fig. 3c).

[0064] According to the above results, it was confirmed that the concentration of bicarbonate (NaHCO3) contained in the Spirulina cell medium affects the concentration and extraction purity of phycocyanin from Spirulina cells.

[0065] Example 2-2. Concentration of wet biomass

[0066] To determine whether the dilution rate of spirulina wet biomass affects the extraction yield and purity of phycocyanin from spirulina, the following experiments were conducted.

[0067] Specifically, in order to confirm the effective concentration of wet biomass for optimal phycocyanin (PC) extraction by autolysis, Spirulina was cultured in a growth medium with a bicarbonate (NaHCO3) concentration of 8.4 g / L in the same manner as Example 1, mixed with distilled water to have different wet biomass dilution rates of 100%, 75%, or 50% in the same manner as Preparation Example 2, frozen once at -20°C for 24 hours, thawed at 25°C for 1 hour, and then reacted sequentially at 37°C, and the phycocyanin purity and concentration were measured in the same manner as Preparation Example 3.

[0068] According to the above results, it was confirmed that the extraction yield reached the maximum when 100% wet biomass was subjected to autolysis reaction at 37°C for 12 hours, and it was confirmed that 50% biomass did not reach the maximum extraction yield within 24 hours at 37°C, but 75% wet biomass confirmed the maximum extraction yield of phycocyanin (PC) within 3 hours when subjected to autolysis reaction at 37°C (Fig. 4a).

[0069] In addition, in the case of 100% wet biomass, it was confirmed that there was no change in phycocyanin purity even if the reaction time increased after 10 hours of autolysis reaction, and in the case of 50% wet biomass, it was confirmed that the phycocyanin purity increased as the reaction time increased when observed for 24 hours. In addition, in the case of 75% wet biomass, it was confirmed that there was no change in phycocyanin purity even if the reaction time increased after 3 hours of reaction, reaching the maximum purity in only 3 hours (Fig. 4b).

[0070] Hereinafter, the present invention will be described in detail.

[0071] Those skilled in the art will appreciate that variations and modifications can be made without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered illustrative rather than restrictive. The scope of the present invention is defined by the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as encompassed by the present invention.

[0072] Furthermore, the terminology used in this specification is intended to appropriately express preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the customs of the field to which the present invention pertains. Therefore, the definitions of these terms should be determined based on the contents throughout this specification. Throughout this specification, when a part is said to "include" a certain component, this does not mean that other components are excluded, but rather that other components may be included, unless otherwise specifically stated.

[0073] In the present invention, any one of the following methods for extracting phycocyanin from spirulina may be selected and used: hot water extraction, cold immersion extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, dissolution, and pressing. Furthermore, the desired extract may additionally undergo a conventional fractionation process, and may be purified using a conventional purification method. There is no limitation on the method for producing the extract of the present invention, and any known method may be used.

[0074] In the present invention, "spirulina biomass" may refer to the organic matter mass produced by culturing the blue-green algae known as spirulina. Spirulina contains various nutrients, including protein, vitamins, minerals, omega-3 fatty acids, and vegetarian vitamin B12, and is an environmentally friendly food ingredient with numerous health benefits and industrial applications, but is not limited thereto.

[0075] In the present invention, “spirulina wet biomass” may mean a product collected by washing a spirulina culture with distilled water. Specifically, the wet biomass may mean a product collected by washing the product obtained by filtering a spirulina culture with a standard mesh size of 0.045 mm with distilled water, and specifically, the product obtained by filtering with the standard mesh is referred to as 100% wet biomass, and a product obtained by diluting 100% wet biomass by adding distilled water to the product by 75% may be referred to as 75% wet biomass, and a product obtained by diluting 50% may be referred to as 50% wet biomass.

[0076] In the present invention, "autolysis" refers to the phenomenon, or self-digestion, in which cellular components are broken down by endogenous enzymes when a living organism dies or is destroyed. This process can liberate enzymes within the cell. Spirulina autolysis can be performed through various processes that kill cells, such as freeze-thawing, and through this process, phycocyanin, a useful substance produced by Spirulina, can be extracted and manufactured.

[0077] Meanwhile, when spirulina wet biomass is autolyzed using the method of the present invention, phycocyanin can be extracted, manufactured, and produced with high concentration, yield, and purity.

[0078] Hereinafter, the present invention will be described in detail with reference to the attached drawings as embodiments of the present invention. However, the following embodiments are presented as examples of the present invention, and if it is determined that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

[0079] One aspect of the present invention comprises the steps of culturing Spirulina (Arthrospira platensis) in a medium containing bicarbonate;

[0080] A step of producing the above culture into wet biomass; and

[0081] A method for producing phycocyanin, comprising the steps of extracting spirulina by freezing and thawing.

[0082] According to one embodiment of the present invention, in the step of culturing in the medium containing bicarbonate, the bicarbonate may be characterized in that it includes at least one selected from the group consisting of sodium bicarbonate (NaHCO3), potassium bicarbonate (KHCO3), and calcium bicarbonate (Ca(HCO3)2), and preferably, in the step of culturing in the medium containing bicarbonate, the bicarbonate may be sodium bicarbonate (NaHCO3), but is not limited thereto.

[0083] According to one embodiment of the present invention, in the step of culturing in the medium containing bicarbonate, the bicarbonate concentration of the medium containing bicarbonate is characterized in that it is 3 g / L to 14 g / L, preferably 6.4 g / L to 12.6 g / L, and even more preferably 8.4 g / L. If the concentration of sodium bicarbonate added in the step of culturing Spirulina (Arthrospira platensis) in the medium containing bicarbonate is less than 3 g / L, there may be a problem that the growth of Spirulina is inhibited, and if the concentration of sodium bicarbonate added exceeds 14 g / L, the cell membrane carbonate may become hard due to adhesion, so that the extraction yield and purity of phycocyanin may be significantly reduced.

[0084] According to one embodiment of the present invention, the step of manufacturing the culture into wet biomass may mean that the step of culturing Spirulina (Arthrospira platensis) in a medium containing bicarbonate is performed through a process of collecting the product obtained by filtering the Spirulina culture through a standard sieve having a mesh size of 0.045 mm and washing the product with distilled water. Specifically, the product obtained by filtering through the standard sieve is referred to as 100% wet biomass, and the product obtained by diluting 100% wet biomass by adding distilled water to the product by 75% is referred to as 75% wet biomass, and the product obtained by diluting 50% is referred to as 50% wet biomass.

[0085] In the present invention, the dilution ratio based on the 100% wet biomass is called the wet degree.

[0086] The above spirulina wet biomass (hereinafter referred to as wet biomass) may be characterized by a wetness of 60 to 100%, preferably 70 to 100%, and most preferably 75%.

[0087] In the step of manufacturing the above culture into wet biomass, if spirulina wet biomass with a wetness of less than 60% is manufactured and autolyzed by freezing and thawing, the leaked autolytic enzyme may be too diluted, which may lower the phycocyanin extraction efficiency. In addition, if spirulina wet biomass with a wetness of more than 100% is manufactured and autolyzed by freezing and thawing, the movement of the leaked autolytic enzyme may be hindered due to insufficient surrounding moisture, which may lower the phycocyanin extraction efficiency.

[0088] The step of extracting phycocyanin by freezing and thawing may refer to extracting and manufacturing phycocyanin by freezing and thawing wet biomass. Meanwhile, the step of extracting phycocyanin by freezing and thawing may additionally include a step of subjecting the wet biomass to an autolysis reaction after freezing and thawing.

[0089] According to one embodiment of the present invention, the step of extracting phycocyanin by freezing and thawing may mean a step of producing the culture into wet biomass, in which the produced wet biomass is frozen once at -20°C for 24 hours, thawed at 25°C, and then subjected to an autolysis reaction at a specific temperature.

[0090] According to one embodiment of the present invention, the step of extracting spirulina by freezing and thawing may mean freezing the wet biomass once at -20°C for 24 hours, then thawing it at 25°C for 1 hour, and then subjecting the wet biomass to an autolysis reaction at 18°C ​​to 40°C, but may preferably be subjected to a reaction at 25°C to 38°C, and more preferably may be subjected to a reaction at 37°C, but is not limited thereto.

[0091] If the above wet biomass is reacted at a temperature lower than 18°C, the activity of the autolytic enzyme of the wet biomass may decrease, resulting in a slow culture rate and low spirulina extraction efficiency. If the above wet biomass is reacted at a temperature higher than 40°C, the activity or structure of the enzyme of the wet biomass may be altered, resulting in problems with the production of spirulina itself.

[0092] In the step of extracting spirulina by freezing and thawing, the autolysis reaction is characterized in that it proceeds for 1.5 to 7.5 hours, but preferably it may proceed for 2 to 6 hours, more preferably it may proceed for 2.5 to 5 hours, and even more preferably it may proceed for 3 hours. If the autolysis reaction is performed for less than 1.5 hours in the step of extracting spirulina by freezing and thawing, the time itself for extracting phycocyanin by autolysis may be insufficient, and if it is performed for more than 7.5 hours, denaturation of the phycocyanin extracted by autolysis may occur.

[0093] According to one embodiment of the present invention, when the wet biomass was subjected to an autolysis reaction at 37°C after freezing and then sequentially, the phycocyanin concentration, extraction yield, and purity rapidly increased when phycocyanin was measured after about 3 hours of reaction. That is, when the autolysis reaction was performed for 3 hours, the phycocyanin concentration, extraction yield, and purity rapidly increased, and when the autolysis reaction was performed for 1.5 to 7.5 hours, the phycocyanin concentration, extraction yield, and purity gradually increased as the reaction time elapsed.

[0094] According to one embodiment of the present invention, the phycocyanin extracted in the step of extracting spirulina by freezing and thawing can have a significantly increased phycocyanin concentration, extraction yield, and purity compared to when phycocyanin is extracted using a conventional freezing and thawing method, when phycocyanin is extracted without freezing and thawing, when phycocyanin is extracted after culturing spirulina in a medium not containing bicarbonate, or when phycocyanin is extracted after culturing spirulina under conditions different from those used in the present invention.

[0095] According to one embodiment of the present invention, when spirulina was cultured in a medium containing bicarbonate, wet biomass was prepared, and this was freeze-thawed and subjected to an autolysis reaction, the phycocyanin concentration, extraction yield, and purity were significantly increased. Specifically, when spirulina cells were cultured in a growth medium containing 8.4 g / L bicarbonate (NaHCO3), and a 75% wet biomass of the cultured spirulina diluted with distilled water was subjected to an autolysis reaction for 3 hours, it was confirmed that high-concentration and high-purity phycocyanin (PC) was extracted.

[0096]

[0097] The specific details of the method for extracting phycocyanin in the present invention can be applied identically or similarly to the extent that they do not deviate from the essential details of the use of phycocyanin produced by the method of the present invention. Specifically, the specific details of the method for extracting phycocyanin in the present invention can be applied identically or similarly to phycocyanin produced by using the method of the present invention, a food composition comprising phycocyanin produced by using the method of the present invention, a health functional food composition comprising phycocyanin produced by using the method of the present invention, and a feed composition comprising phycocyanin produced by using the method of the present invention.

[0098]

[0099] In another aspect, the present invention relates to phycocyanin produced by the above production method.

[0100] In another aspect, the present invention relates to a food composition comprising phycocyanin produced by the above production method.

[0101] The food composition of the present invention may contain, in addition to containing the extract as an effective ingredient, various flavoring agents or natural carbohydrates as additional ingredients, like a conventional food composition.

[0102] Examples of the natural carbohydrates mentioned above include monosaccharides such as hops, fructose, etc.; disaccharides such as maltose, sucrose, etc.; and polysaccharides such as dextrin, cyclodextrin, etc., common sugars, and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As the flavoring agent mentioned above, natural flavoring agent (thaumatin), stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agent (saccharin, aspartame, etc.) can be advantageously used. The food composition of the present invention can be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candies, ice cream, alcoholic beverages, vitamin complexes, and health supplements.

[0103] In addition, the food composition may contain, in addition to the extract as an active ingredient, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain fruit pulp for producing natural fruit juice, fruit juice drinks, and vegetable drinks.

[0104] In another aspect, the present invention relates to a health functional food composition comprising phycocyanin manufactured by the above manufacturing method.

[0105] The functional food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. In the present invention, the term "health functional food composition" refers to a food manufactured and processed using raw materials or ingredients having functionality useful to the human body according to Act No. 6727 on Health Functional Foods, and means to be consumed for the purpose of obtaining a useful effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological effects. The health functional food of the present invention may include conventional food additives, and whether it is suitable as a food additive is determined by the specifications and standards for the relevant item according to the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety, unless otherwise specified. Items listed in the above "Food Additives Codex" include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, sulphate pigment, and guar gum; Examples thereof include mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline agents, preservative preparations, and tar color preparations. For example, health functional foods in tablet form can be prepared by mixing the active ingredient of the present invention with excipients, binders, disintegrants, and other additives, granulating the mixture using a conventional method, and then adding a lubricant, etc. to perform compression molding, or directly compressing the mixture. In addition, the health functional foods in tablet form can contain a maturing agent, etc., if necessary. Among health functional foods in capsule form, hard capsules can be prepared by filling a mixture of the active ingredient of the present invention with additives such as excipients into a conventional hard capsule, and soft capsules can be prepared by filling a mixture of the active ingredient of the present invention with additives such as excipients into a capsule base such as gelatin. The soft capsules can contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, etc., if necessary.The ring-shaped health functional food can be prepared by molding a mixture of the active ingredient of the present invention with excipients, binders, disintegrants, etc. using a conventionally known method, and, if necessary, can be coated with white sugar or other coating agents, or the surface can be coated with a substance such as starch or talc. The granular health functional food can be manufactured into a granular form using a mixture of the active ingredient of the present invention with excipients, binders, disintegrants, etc. using a conventionally known method, and, if necessary, can contain a flavoring agent, a flavoring agent, etc.

[0106] In another aspect, the present invention relates to a feed composition comprising phycocyanin manufactured by the above manufacturing method.

[0107] The feed composition of the present invention can be expected to have the effect of replacing existing anti-inflammatory agents and inhibiting the growth of harmful food pathogenic bacteria, thereby improving the health of animals, improving weight gain and meat quality of livestock, and increasing milk production and immunity. The feed composition of the present invention can be manufactured in the form of fermented feed, compound feed, pellets, silage, etc. The fermented feed can be manufactured by fermenting organic matter by adding various microbial groups or enzymes other than the peptide of the present invention, and the compound feed can be manufactured by mixing various types of general feed with the peptide of the present invention. The pellet-type feed can be manufactured by applying heat and pressure to the compound feed, etc. in a pelletizer, and the silage can be manufactured by fermenting green feed with the microorganism according to the present invention. Wet fermented feed can be manufactured by collecting and transporting organic matter such as food waste, mixing it with a sterilizing process and excipients for moisture control at a certain ratio, and then fermenting it at an appropriate temperature for fermentation for more than 24 hours to adjust the moisture content to approximately 70%. Fermented dried feed can be manufactured by adding a drying process to wet fermented feed to adjust the moisture content to approximately 30% to 40%.

[0108] The feed composition of the present invention may further include ingredients added to conventional feed. Examples of such ingredients added to the feed may include grain powder, meat powder, and legumes. The grain powder may be at least one selected from rice flour, wheat flour, barley flour, and corn flour. The meat powder may be a powdered meat powder obtained by pulverizing at least one selected from chicken, beef, pork, and other meats. The legumes may be at least one selected from soybeans, kidney beans, peas, and black beans.

[0109] The feed composition of the present invention may, in addition to the grain powder, meat powder, and legumes that are ingredients added to the conventional feed mentioned above, add at least one selected from among nutrients and minerals to increase the nutritional value of the feed, and may include at least one selected from among antifungal agents, antioxidants, anticoagulants, emulsifiers, and binders to prevent deterioration of feed quality.

[0110]

[0111] Since phycocyanin (PC) is a substance widely used in the pharmaceutical industry, etc., utilizing the technology of extracting phycocyanin (PC) from spirulina of the present invention has industrial utility.

Claims

1. A step of culturing Spirulina (Arthrospira platensis) in a medium containing bicarbonate; A step of producing the above culture into wet biomass; and A method for producing phycocyanin, comprising the steps of extracting phycocyanin by freezing and thawing.

2. In paragraph 1, A method for producing phycocyanin, characterized in that in the step of culturing in a medium containing bicarbonate, the bicarbonate comprises at least one selected from the group consisting of sodium bicarbonate (NaHCO3), potassium bicarbonate (KHCO3), and calcium bicarbonate (Ca(HCO3)2).

3. In paragraph 2, A method for producing phycocyanin, characterized in that in the step of culturing in a medium containing bicarbonate, the bicarbonate is sodium bicarbonate (NaHCO3).

4. In paragraph 1, A method for producing phycocyanin, characterized in that the bicarbonate concentration of the medium containing bicarbonate in the step of culturing in the medium containing bicarbonate is 3 g / L to 14 g / L.

5. In paragraph 1, A method for producing phycocyanin, characterized in that the step of producing the above culture into wet biomass has a wetness of 60 to 100%.

6. In paragraph 1, A method for producing phycocyanin, characterized in that the step of extracting spirulina by freezing and thawing comprises autolysis of wet biomass at 18°C ​​to 40°C after freezing.

7. In paragraph 6, A method for producing phycocyanin, characterized in that the above autolysis reaction proceeds for 1.5 to 7.5 hours.

8. In paragraph 1, A method for producing phycocyanin, characterized in that the amount of phycocyanin extracted in the step of extracting spirulina by freezing and thawing is increased.

9. Phycocyanin manufactured by the manufacturing method of paragraph 1.

10. A food composition comprising phycocyanin manufactured by the manufacturing method of paragraph 1.

11. In paragraph 10, A food composition containing phycocyanin, characterized in that in the step of culturing in a medium containing bicarbonate, the bicarbonate is sodium bicarbonate (NaHCO3).

12. A health functional food composition containing phycocyanin manufactured by the manufacturing method of paragraph 1.

13. In paragraph 12, A health functional food composition containing phycocyanin, characterized in that in the step of culturing in a medium containing bicarbonate, the bicarbonate is sodium bicarbonate (NaHCO3).

14. A feed composition comprising phycocyanin manufactured by the manufacturing method of paragraph 1.

15. In paragraph 14, A feed composition containing phycocyanin, characterized in that in the step of culturing in a medium containing bicarbonate, the bicarbonate is sodium bicarbonate (NaHCO3).

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