Porphyridium exopolysaccharide extract as well as extraction method and application thereof

Abstract

According to the method, the porphyridium liquid is homogenized, the cell disruption degree is controlled, the cell polysaccharide extraction rate is increased, the solid-liquid separation problem of the viscous cell-polysaccharide solution is effectively solved, a membrane separation technology is used for replacing a traditional alcohol precipitation technology, operation is easy and convenient, product stability is improved, and the method is suitable for continuous production and low in cost. The porphyridium polysaccharide retains the moisturizing, soothing and other effects of the porphyridium polysaccharide, solves the problem that the traditional extracted polysaccharide interferes with the formula system, is a high-adaptability functional raw material more suitable for industrial application, can be widely applied to moisturizing cream, anti-wrinkle essence and sensitive skin care products, and effectively repairs the skin barrier and delays senescence. In the field of medicines, the compound can be used as an immunopotentiator for assisting in improving the body resistance, and can also be used for repairing skin inflammation, healing wounds and assisting in conditioning chronic diseases; in the field of food, the functional nutritional supplement can be used as a functional nutritional supplement, and can also be used as a natural stabilizer and a thickening agent to improve food texture.

Description

Technical Field

[0001] This invention belongs to the field of microalgae industry, specifically relating to an extracellular polysaccharide extract of *Porphyra yezoensis*, its extraction method, and its application. Background Technology

[0002] Porphyridium is a single-celled red alga widely distributed in marine, brackish, and freshwater environments, belonging to the phylum Rhodophyta, class Rhodophyta, and order Porphyridales. Its cells are typically spherical or oval, exhibiting a characteristic purplish-red or deep red color due to the presence of phycobiliproteins such as phycoerythrin and phycocyanin. Porphyridium exhibits strong adaptability, tolerating a wide range of environmental conditions (such as light, temperature, and salinity), making it easy to cultivate on a large scale under controlled conditions, thus enabling industrial production. Porphyridium polysaccharides are a class of natural macromolecular sulfated heteropolysaccharides secreted extracellularly or present in the cell wall during its growth. They are mainly composed of various monosaccharides (such as xylose, glucose, galactose, and mannose) linked by glycosidic bonds, and their molecular structure contains numerous sulfate ester groups, giving them excellent bioactivities such as antioxidant and immunomodulatory effects. They show broad commercial prospects in the fields of medicine (such as immune adjuvants), food (such as functional additives), and cosmetics (such as moisturizing active ingredients).

[0003] The immense application value of *Porphyra yezoensis* polysaccharides has made their extraction and purification a research hotspot. However, existing technologies still face numerous bottlenecks, severely hindering the industrialization and product quality of *Porphyra yezoensis* polysaccharides. First, traditional photobioreactor cultivation methods suffer from uneven lighting, low dissolved oxygen efficiency, susceptibility to contamination, and high energy consumption, resulting in low algal biomass and polysaccharide yield, leading to high production costs. Second, separation is challenging. Low-concentration polysaccharide culture media require high-energy centrifugation to separate algal cells, widely used in laboratories but with poor separation results in large-scale production. High-concentration polysaccharide culture media, due to their high viscosity, cannot achieve effective separation of algal cells from the culture medium even with high-speed multiple centrifugation or conventional filtration. Third, traditional processes typically use alcohol precipitation to obtain polysaccharides, which suffers from low purity and poor stability. Furthermore, high ethanol consumption necessitates the establishment of explosion-proof workshops, significantly increasing industrial costs and hindering large-scale production. Summary of the Invention

[0004] To address the above problems, this invention provides a method for extracting extracellular polysaccharides from *Porphyra yezoensis*, comprising the following steps:

[0005] S1: The culture of *Porphyra yezoensis* was subjected to limited homogenization at a low cell disruption rate, with the cell disruption rate controlled to be <10%;

[0006] S2: Centrifugation yields a supernatant containing extracellular polysaccharides from *Porphyra yezoensis*.

[0007] S3: Microfilter the supernatant to remove large particulate impurities;

[0008] S4: Desalting and concentration yields an extracellular polysaccharide solution of *Porphyra yezoensis*.

[0009] In one specific implementation, in S1, the *Purpleella pulidonis* culture is homogenized by high-pressure treatment at a pressure of 20 bar.

[0010] In one specific implementation, S2 is centrifuged at 10,000 rpm for 10 min.

[0011] In one specific implementation, in S3, microfiltration is performed using a hollow fiber membrane with a pore size of 0.2-1.0 μm.

[0012] In one specific implementation, in S4, ultrafiltration is performed using a ceramic membrane to achieve desalination and concentration, wherein the ultrafiltration membrane has a pore size of 5-20 nm.

[0013] In one specific implementation, the *Porphyra yezoensis* culture is obtained by inoculating *Porphyra yezoensis* species into a deep-pool reactor with a built-in light source.

[0014] In one specific implementation, the illumination scheme for cultivating *Porphyra yezoensis* is as follows: 30% illumination for 0-7 days, 60% illumination for 7-10 days, and 100% illumination for 10-22 days; or 30% illumination for 0-3 days, 60% illumination for 3-7 days, and 100% illumination for 7-22 days; wherein the illumination power per unit volume at 100% illumination is 0.8-4 kW / m³.

[0015] In one specific embodiment, the *Porphyra yezoensis* culture medium is a nitrogen-enriched f / 2 medium, and during the culture process, 2% carbon dioxide air is introduced and the pH of the culture medium is controlled at 7.5±0.2, and the culture temperature is controlled at 25±2℃.

[0016] The present invention also provides an extracellular polysaccharide extract of *Porphyra yezoensis* prepared by the above method.

[0017] This invention also provides the application of the above-mentioned *Porphyra yezoensis* extracellular polysaccharide extract in the preparation of cosmetics, medical products or food.

[0018] This invention utilizes high-pressure homogenization of algal liquor, which not only improves polysaccharide extraction rate but also effectively solves the solid-liquid separation problem. It replaces the traditional alcohol precipitation process with membrane separation technology, simplifying operation, improving product stability, and making it suitable for continuous production. Furthermore, this invention employs a deep-pond reactor with an integrated light source to cultivate *Porphyra yezoensis*, effectively preventing pollution and achieving efficient light utilization.

[0019] The polysaccharide from *Plasmodium purpureum* of this invention retains the moisturizing and soothing effects of the polysaccharide itself, while solving the problem of interference with the formulation system caused by traditional polysaccharide extraction. It is a "highly adaptable functional raw material" that is more suitable for industrial application and can be widely used in moisturizing creams, anti-wrinkle essences, and sensitive skin care products to effectively repair the skin barrier and delay aging.

[0020] In addition, in the pharmaceutical field, it can be used as an immune enhancer to help improve the body's resistance, and can also be used for skin inflammation repair, wound healing and chronic disease management; in the food field, it can be used as a functional nutritional supplement, as well as a natural stabilizer and thickener to improve food texture. Attached Figure Description

[0021] Figure 1 The biomass / polysaccharide production per unit of lighting energy consumption is compared for different photoreactors. Among them, 1 is an external light source plate reactor; 2 is an internal light source laboratory deep pool reactor; and 3 is an internal light source ton-scale deep pool reactor.

[0022] Figure 2 This represents the biomass / polysaccharide production per unit of lighting energy consumption for different lighting schemes in a deep-pond reactor with an internal light source. Scheme 1 represents full illumination for 0-22 days; Scheme 2 represents 30% illumination for 0-3 days, 60% illumination for 3-7 days, and 100% illumination for 7-22 days; Scheme 3 represents 30% illumination for 0-7 days, 60% illumination for 7-10 days, and 100% illumination for 10-22 days.

[0023] Figure 3 The growth curve (A) and polysaccharide content curve (B) of *Porphyra yezoensis* cultured in a deep-pool reactor with built-in light source using illumination scheme 3 are shown.

[0024] Figure 4 The centrifugation effects of different treatments on *Porphyra yezoensis* cultures are shown. A represents ultrasonic treatment; B represents high-pressure homogenization.

[0025] Figure 5 The cell breakage rate and viscosity changes of *Porphyra yezoensis* culture after high-pressure homogenization.

[0026] Figure 6 This is the state of the polysaccharide from *Plasmodium purpureum* after centrifugation and micro Detailed Implementation

[0027] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0028] 1. Purple Gastrodia elata cultivation - deep-pool built-in light source reactor

[0029] Inoculate with *Porphyra yezoensis*, culture with a mixture of carbon dioxide and air, pH (7.5±0.2) with feedback carbon dioxide supplementation, and temperature (25±2)℃.

[0030] The cultivation effects of different reactors are as follows: Figure 1 As shown, the plate reactor with external light source exhibits lower biomass and polysaccharide yields per unit of lighting energy consumption, with a biomass output of 1.5 g / kWh and a polysaccharide output of 0.5 g / kWh. Reactors with internal light source show higher material yields; in laboratory deep-pool reactors, biomass reaches 6.6 g / kWh and polysaccharide yield reaches 2.7 g / kWh. After scale-up, the material yield per unit of lighting energy consumption in ton-scale deep-pool reactors can reach a high level, with biomass at 5.2 g / kWh and polysaccharide yield at 2.2 g / kWh.

[0031] The influence of different illumination schemes was further investigated using a deep pool reactor with an integrated light source, and the results are as follows: Figure 2 As shown, Scheme 1 involves full illumination for 0-22 days, where the algae concentration is low and the light intensity is high, thus inhibiting the growth of *Porphyra yezoensis*. Scheme 2 involves 30% illumination for 0-3 days, with a light intensity of 0.28 kW / m². 3 Light intensity was 60% for 3-7 days, with a light intensity of 0.56 kW / m². 3 Option 1 uses full illumination from day 7 to 22; Option 2 uses 30% illumination from day 0 to 7, with an illumination rate of 0.28 kW / m². 3 Light intensity was 60% for 7-10 days, with a light intensity of 0.56 kW / m². 3 Full illumination for 10-22 days, with a light intensity of 0.93 kW / m². 3 Both illumination schemes 2 and 3 produce similar final biomass and polysaccharide yields, but scheme 3 yields higher material output per unit of lighting energy consumption, making scheme 3 the preferred option.

[0032] 100 L of algal inoculum was inoculated into a one-ton deep tank and cultured using an air-fuel mixture with 2% carbon dioxide. The carbon dioxide supply was controlled via pH feedback to maintain a stable pH of 7.5 ± 0.2. The culture temperature was maintained at (25 ± 2) °C, following the photobioreactor and illumination protocols defined above. Results are as follows... Figure 3 As shown in the growth curve of *Porphyra yezoensis*, the overall growth is continuous and in good condition, with a biomass of 1.8 g / L after 22 days. Figure 3 A). Changes in polysaccharide content: As biomass increases, polysaccharide content increases. According to the curve trend, in the later stages of growth, a plateau is reached, and polysaccharides continue to accumulate, eventually reaching 0.73 g / L ( Figure 3 B). Therefore, appropriately extending the growth time can significantly increase the polysaccharide content.

[0033] 2. Optimization of solid-liquid separation

[0034] The collected algal solutions were homogenized by ultrasound and high pressure, and then centrifuged at 10,000 rpm for 10 min to observe the centrifugation effect.

[0035] The results are as follows Figure 4 As shown, when ultrasonic treatment was performed for 0 min, 1 min, 2 min, 5 min, 10 min, and 20 min respectively, the centrifugation effect of the treated liquid was not good. Figure 4 A). When high-pressure homogenization was used, with processing pressures of 0, 20 bar, 40 bar, 60 bar, 80 bar, 100 bar, 120 bar, 140 bar, 160 bar, 180 bar, and 200 bar, the results showed that the solid-liquid separation effect was better at pressures of 20 bar and above. Figure 4 B).

[0036] like Figure 5 As shown, the cell disruption rate was less than 10% during treatment at 0-20 bar, with little change in solution viscosity. However, above 20 bar, the cell disruption rate increased significantly, while the solution viscosity decreased. Therefore, high-pressure homogenization at 20 bar, controlling the cell disruption rate to <10%, can both improve polysaccharide yield and achieve better solid-liquid separation.

[0037] In summary, high-pressure homogenization (preferably 20 bar) can achieve limited cell disruption while solving the solid-liquid separation problem.

[0038] 3. Remove large particulate impurities

[0039] Microfiltration of the supernatant was performed using 0.2, 0.45, 1.0, and 5 μm PES hollow fiber membranes to remove residual algal residue and large molecular impurities. When the membrane pore size was 0.2-1.0 μm, the solution was clear and the impurity removal effect was good. Figure 6 As shown.

[0040] 4. Concentration and Desalination

[0041] The microfiltered feed solution was concentrated and desalinated using 0.01 μm, 0.03 μm, and 0.04 μm PVDF hollow fiber membranes or 5 nm, 10 nm, 20 nm, 40 nm, and 50 nm ceramic membranes, respectively.

[0042] The results are shown in Table 1. After treatment with a 50 nm ceramic membrane, the polysaccharide concentration in the concentrate was 2.7 g / L, the polysaccharide recovery rate was 21%, and the polysaccharide permeation rate was 72%. After treatment with a 40 nm ceramic membrane, the polysaccharide concentration in the concentrate was 3 g / L, the polysaccharide recovery rate was 35%, and the polysaccharide permeation rate was 58%. After treatment with a 20 nm ceramic membrane, the polysaccharide concentration in the concentrate was 4.0 g / L, the polysaccharide recovery rate was 72%, and the polysaccharide permeation rate was 23%. After treatment with a 10 nm ceramic membrane, the polysaccharide concentration in the concentrate was 4.5 g / L, the polysaccharide recovery rate was 78%, and the polysaccharide permeation rate was 18%. After treatment with a 5 nm ceramic membrane, the polysaccharide concentration in the concentrate was 5 g / L, the polysaccharide recovery rate was 85%, and the polysaccharide permeation rate was 10%. The polysaccharide recovery rate was higher when the pore size was 5-10 nm. After treatment with a 0.01 μm hollow fiber membrane, the polysaccharide concentration in the concentrate was 2.8 g / L, with a polysaccharide recovery rate of 25% and a polysaccharide permeation rate of 15%. After treatment with a 0.03 μm hollow fiber membrane, the polysaccharide concentration in the concentrate was 1.9 g / L, with a polysaccharide recovery rate of 18% and a polysaccharide permeation rate of 21%. After treatment with a 0.04 μm hollow fiber membrane, the polysaccharide concentration in the concentrate was 1.1 g / L, with a polysaccharide recovery rate of 11% and a polysaccharide permeation rate of 30%. Using PVDF hollow fiber membranes resulted in significant polysaccharide loss due to membrane adsorption, leading to poor recovery. After desalination, the conductivity of the feed solution decreased to below 200 μs / cm, compared to 53 ms / cm before desalination, indicating a good desalination effect. In summary, 5-20 nm ceramic membranes can retain more polysaccharides and achieve better polysaccharide recovery.

[0043] Table 1 Ultrafiltration membrane treatment data

[0044]

[0045] 5. Comparison of polysaccharides extracted by the present invention with those extracted by alcohol precipitation.

[0046] The effects of different *Porphyra yezoensis* samples on the stability of the carbomer system were tested according to the *TGDICST 001-2023 Guidelines for Stability Testing in Cosmetics*. Carbomer U20, after neutralization with triethanolamine (TEA), forms a high-viscosity three-dimensional hydrogel network. Certain raw materials (such as electrolytes, ionic components, specific polysaccharides, or surfactants) can disrupt this network structure, leading to a sharp decrease in viscosity (thinning). This experiment quantitatively evaluated and compared the effects of alcohol-precipitated *Porphyra yezoensis* polysaccharide sample 1 and membrane-technology-based *Porphyra yezoensis* polysaccharide sample 2 on the viscosity (i.e., viscosity reduction effect) of the neutralized carbomer U20 gel at specific addition levels, and assessed its stability.

[0047] The results are shown in Table 2. Experimental group A (sample 1 with 1% purple algae polysaccharide) further reduced the viscosity of the system (4880 mPa.s), and a slight viscosity reduction phenomenon was observed by the naked eye, indicating that the sample has a certain interference effect on the carbomer gel structure.

[0048] Experimental group B (sample 2 with 1% added *Plasmodium purpureum* polysaccharide) showed the opposite trend, with a significant increase in viscosity to 6564 mPa·s and no obvious change in appearance, indicating that this sample enhanced the stability of the carbomer gel to some extent. Therefore, polysaccharides extracted using membrane technology have better stability.

[0049] Table 2 Results of the effect of samples on the viscosity of carbomer gel

[0050]

[0051] Carbomer is the most commonly used thickener in cosmetics (gels, serums, creams, etc.), and the stability of its gel network directly determines the product's texture and shelf life. Polysaccharides extracted using traditional alcohol precipitation methods (Sample 1) interfere with the carbomer gel structure, leading to a decrease in system viscosity and a thinner texture. The polysaccharide of this invention (Sample 2) not only does not damage the gel network but also increases its viscosity to 6564 mPa·s (higher than the control group), with no change in appearance. This means that after addition, cosmetics are less prone to quality problems such as layering and thinning, thus extending shelf life. The *Plasmodium purpureum* polysaccharide of this invention retains the moisturizing and soothing effects of *Plasmodium purpureum* polysaccharide itself while solving the problem of traditional polysaccharide extraction interfering with the formulation system. It is a more suitable "highly adaptable functional raw material" for industrial application and can be widely used in moisturizing creams, anti-wrinkle serums, and sensitive skin care products, effectively repairing the skin barrier and delaying aging.

[0052] In addition, in the pharmaceutical field, it can be used as an immune enhancer to help improve the body's resistance, and can also be used for skin inflammation repair, wound healing and chronic disease management; in the food field, it can be used as a functional nutritional supplement, as well as a natural stabilizer and thickener to improve food texture.

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for extracting extracellular polysaccharides from *Porphyra yezoensis*, characterized in that, Includes the following steps: S1: Perform limited homogenization on the *Porphyra yezoensis* culture with a low cell disruption rate, controlling the cell disruption rate to ≤10%; S2: Centrifugation yields a supernatant containing extracellular polysaccharides from *Porphyra yezoensis*. S3: Microfilter the supernatant to remove large particulate impurities; S4: The microfiltrate was desalted and concentrated to obtain an extracellular polysaccharide solution of *Porphyra yezoensis*.

2. The method according to claim 1, characterized in that, In S1, the *Purpleella pulidonis* culture is homogenized by high-pressure treatment at a pressure of 20 bar.

3. The method according to claim 1, characterized in that, Centrifuge at 10,000 rpm for 10 min in S2.

4. The method according to claim 1, characterized in that, In S3, microfiltration is performed using a hollow fiber membrane, wherein the pore size of the hollow fiber membrane is 0.2-1.0 μm.

5. The method according to claim 1, characterized in that, In S4, ultrafiltration is performed using a ceramic membrane with a pore size of 5-20 nm to achieve desalination and concentration.

6. The method according to any one of claims 1-6, characterized in that, The *Porphyra yezoensis* culture was obtained by inoculating *Porphyra yezoensis* species into a deep-pool reactor with a built-in light source and cultivating it.

7. The method according to claim 7, characterized in that, The illumination scheme for cultivating *Porphyra yezoensis* is as follows: 30% illumination for 0-7 days, 60% illumination for 7-10 days, and 100% illumination for 10-22 days; or 30% illumination for 0-3 days, 60% illumination for 3-7 days, and 100% illumination for 7-22 days; wherein, the unit volume illumination power at 100% illumination is 0.8-4 kw / m³.

8. The method according to claim 7, characterized in that, The culture medium for *Porphyra yezoensis* is an f / 2 medium enriched with nitrogen and phosphorus by 1-5 times. During the culture process, a gas containing 1-100% carbon dioxide is introduced, and the pH is controlled at 7.5±0.2 and the temperature at 25±2℃.

9. An extracellular polysaccharide extract of *Porphyra yezoensis*, characterized in that, It is prepared by the method according to any one of claims 1-8.

10. The use of the *Porphyra yezoensis* extracellular polysaccharide extract according to claim 9 in the preparation of cosmetics, medical products or food.

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