Phycobiliprotein and / or red algae oligosaccharide extracted based on biological soluble wall and preparation method of phycobiliprotein and / or red algae oligosaccharide

Abstract

The invention discloses phycobiliprotein and / or red algae oligosaccharide extracted based on a biological soluble wall and a preparation method of the phycobiliprotein and / or the red algae oligosaccharide, and belongs to the technical field of biological functional component extraction. The invention provides a preparation method for simultaneously extracting phycobiliprotein and phycobiliprotein of red algae, which is high in efficiency, low in loss rate and suitable for process amplification, specifically, cell wall polysaccharide in the red algae is matched with a compound enzyme in a matching manner, and multiple enzymes are cooperated, so that the contradiction problem between high-temperature dissolution of the cell wall polysaccharide of the red algae and heat sensitivity of phycobiliprotein is effectively solved, and the yield of the phycobiliprotein and the oligosaccharide of the red algae is improved. The wall breaking efficiency is improved, and cell wall breaking is promoted to form porous and macroporous cell wall structures; the outward migration of intracellular substances such as phycobiliprotein is promoted, so that higher extraction efficiency is obtained. According to the preparation method provided by the invention, the extraction amount of phycobiliprotein reaches 224 g / kg after production conversion, and the dissociation loss of phycobiliprotein polymers is effectively reduced. In addition, the phycobiliprotein is efficiently extracted, meanwhile, the red algae oligosaccharide product is obtained, and the process economic benefit and the raw material utilization rate are improved. The preparation method provided by the invention does not need special equipment, is simple and convenient in process, green, environment-friendly, low in cost and beneficial to industrial production.

Description

Technical Field

[0001] This invention belongs to the field of biofunctional component extraction technology, specifically relating to a phycobiliprotein and / or red algal oligosaccharide extracted from bio-walled surfaces and their preparation method. Background Technology

[0002] Phycobiliproteins are water-soluble pigment proteins unique to algae, typically found within the cells of cyanobacteria (blue-green algae), red algae, and cryptophytes. They have practical and / or potential biotechnological applications in nutritional supplements, pharmaceuticals, food, cosmetics, biomedical research, and clinical diagnostics. Currently, most phycobiliproteins on the market are derived from microalgae such as Spirulina, but this is insufficient to meet the continuously growing market demand. my country possesses abundant red algae resources, providing ample resources for the production and application of phycobiliproteins. Effective release of phycobiliproteins requires cell wall disruption during extraction. However, the unique marine environment results in the tough and dense cell walls of red algae, posing a challenge to traditional cell wall disruption techniques. To date, no effective large-scale extraction and purification technology has been developed, hindering the development and application of red algal phycobiliprotein resources. High prices and inefficient preparation methods remain the biggest constraints on the development of the phycobiliprotein industry.

[0003] The key to phycobiliprotein extraction is the disruption of algal cells; the higher the proportion of disrupted algal cells, the higher the yield of phycobiliproteins. To address this issue, various technical solutions have been proposed in the existing technology, including traditional extraction methods such as immersion and freeze-thaw, as well as novel extraction technologies such as ultrasound-assisted extraction, microwave-assisted extraction, high-pressure homogenization, and pulsed electric field extraction, and enzyme (lysozyme) treatment and its combinations. Commonly used immersion methods, such as ultrafine powder combined with alcohol-ketone extraction and water extraction, achieve high phycobiliprotein purity, but the extraction cycle is as long as 14 hours and introduces organic substances such as methanol, ethanol, and acetone, which is detrimental to applications in the food industry. Existing technologies utilize fusion swelling and wet ultrafine pulverization processes to extract phycobiliproteins from *Porphyra tenera*, but the extraction cycle is also long, exceeding 18 hours, and the phycobiliprotein extraction rate is less than 4.8%. Existing technologies use salt solvents to extract phycobiliproteins from *Spirulina platensis*, consistent with other immersion methods, but the extraction cycle is as long as 30 hours. Repeated freeze-thaw cycles are another common method in existing technologies for extracting phycobiliproteins from microalgae. However, the extraction cycle can take several days and is less efficient.

[0004] To improve extraction efficiency, emerging technologies such as ultrasound-assisted extraction, microwave-assisted extraction, high-pressure homogenization, and pulsed electric field extraction have been applied. For example, combining wet pulverization with ultrasound to extract phycobiliproteins from laver shortens the extraction cycle to within 2 hours, achieving an actual extraction rate of 26.6% for crude phycobiliproteins, although the purity is only 0.56%. Furthermore, existing technologies have documented significant differences in the effectiveness of these technologies for different algae. Different methods, such as maceration, freeze-thaw, ultrasound, and high-pressure assisted extraction, have been evaluated for phycobiliprotein extraction from Gracilaria. Maceration was found to be the most effective method, achieving an extraction rate of 0.58%. Existing technologies have also evaluated the effectiveness of ultrasound, ultrasound + maceration, and ultrasound + enzymatic hydrolysis in extracting phycobiliproteins from Gelidium pusillum and Gratierupia turuturu. Due to the limitations of ultrasound in disrupting the phycobiliprotein structure, although good cell wall disruption can be achieved, the phycobiliprotein extraction rate is only 0.27-0.43%.

[0005] Furthermore, while the aforementioned emerging technologies offer advantages such as non-toxicity, good raw material adaptability, and ease of scaling up production, issues like heat generation and high-pressure control during the process can cause the disintegration or denaturation and inactivation of phycobiliprotein aggregates. Chemical methods are also suitable for large-scale production, but their low stability, toxicity, and the introduction of chemical pollution do not align with current industrial development trends. Enzyme-mediated hydrolysis-assisted extraction offers advantages such as mild processing conditions, non-toxicity, and high efficiency, making it suitable for large-scale production. With the price of industrial enzymes gradually decreasing, it is the most promising method for industrial application.

[0006] However, enzyme-assisted extraction has not been very effective in practical applications. For example, existing techniques using β-agarase and cellulase to extract phycobiliproteins from marine red algae yield phycobiliproteins with a purity of 1.2%, but the extraction rate is only 0.83%. Another method uses xylanase to extract phycobiliproteins from palmate red algae; the optimized scheme achieves an extraction rate of 1.24% and a purity of 0.4%. Although these techniques have made some progress, they are only laboratory-scale, kilogram-level preparation techniques and have not yet solved the problem of process scale-up. On the other hand, the techniques and processes for disrupting algal cell walls depend on factors such as biological composition, stability, and cell wall resistance. The challenge lies in releasing phycobiliproteins from cells while maintaining their structure and function. Existing techniques do not address this aspect.

[0007] In addition, polysaccharides are another major active component of red algae, and their cross-linking forms the dense structure of the red algal cell wall. After cell wall disruption, red algal oligosaccharides are formed. If these can be recycled, the added value of the process and the utilization rate of raw materials can be improved. However, current technical solutions rarely address this. Therefore, there is an urgent need to explore a mild and green method that can simultaneously and efficiently prepare phycobiliproteins and red algal oligosaccharides, which would have significant economic benefits and broad market prospects. Summary of the Invention

[0008] Based on technical needs, the purpose of this invention is to provide a method for extracting phycobiliproteins and / or red algae oligosaccharides from bio-dissolution and their preparation. This invention provides a method for simultaneously extracting phycobiliproteins and red algae oligosaccharides from red algae with high efficiency, low loss rate, and suitability for process scale-up. The preparation method provided by this invention has the advantage of high extraction rate, improves the utilization rate of red algae raw materials, brings significant economic benefits, and has important reference significance for its large-scale production.

[0009] To achieve the above-mentioned objectives, the present invention employs the following technical solution:

[0010] This invention provides a method for preparing phycobiliproteins and / or red algal oligosaccharides based on bio-dissolution extraction, the preparation method comprising the following steps:

[0011] (1) Pretreatment: Mix red algae with water thoroughly, rehydrate, and grind into a homogenous slurry to obtain red algae slurry;

[0012] (2) Bio-dissolving: The red algae slurry is transferred into a reaction vessel, the pH and temperature of the red algae slurry are adjusted, and a compound enzyme is added. The mixture is stirred continuously to fully react and obtain red algae extract.

[0013] (3) Solid-liquid separation: The red algae extract is coarsely filtered and centrifuged to remove insoluble matter, and the supernatant is collected;

[0014] (4) Liquid-liquid separation: The centrifuged clear liquid is pumped into a separation tank, and polyethylene glycol and inorganic salt are added to form a two-phase aqueous separation system. The system is stirred rapidly, and after the phases come into contact, it is allowed to stand. After the two phases are separated, the top phase and the bottom phase are collected respectively.

[0015] (5) Collection of phycobiliproteins: The top phase is separated by ultrafiltration to remove impurities, and washed with water. The concentrated liquid is collected to obtain the phycobiliprotein solution.

[0016] (6) Collection of red algae oligosaccharides: The substrate is separated by ultrafiltration to remove impurities, washed with water, and the filtrate is collected. Then, impurities are removed again by nanofiltration, and the concentrate is collected to obtain the red algae oligosaccharide solution.

[0017] (7) Drying: After drying the phycobiliprotein solution and the red algae oligosaccharide solution respectively, phycobiliprotein powder and red algae oligosaccharide powder are obtained.

[0018] Furthermore, the rehydration time is 1-2 hours, and the red algae is either dried or fresh. The dried red algae is thoroughly mixed with water and then rehydrated; or fresh red algae is mixed with water and then homogenized using a colloid mill to obtain a red algae slurry.

[0019] Furthermore, in step (2), the pH of the red algae slurry is adjusted to 4.5–7.0, and the temperature is raised to 35–45°C and kept warm; the complex enzyme is a complex enzyme composed of α-galactosidase, cellulase, and pectinase, with an enzyme activity ratio of 0.5–2: 6–12: 0.7–1.7; the amount of the complex enzyme added is 1‰–15‰, based on the dry weight of the red algae.

[0020] Furthermore, the complex enzyme is a complex enzyme composed of α-galactosidase, cellulase and pectinase, with an enzyme activity ratio of 1.1:10:1.3.

[0021] Furthermore, the centrifugation speed in step (3) is 6000 to 18000 rpm.

[0022] Furthermore, in step (4), the molecular weight of ethylene glycol is 2000-20000 Da, and the inorganic salt is ammonium sulfate or phosphate.

[0023] Furthermore, the aqueous two-phase separation system specifically has the following characteristics: TLL of 18%–27%, Vr of 0.26–0.77, and pH of 5.2–7.0.

[0024] Furthermore, the molecular weight of polyethylene glycol (PEG) is 2000–6000 kDa, which is beneficial for increasing the phase separation rate.

[0025] Furthermore, the ultrafiltration membrane used for the top phase in step (5) has a molecular weight cutoff of 8 to 100 kDa; the ultrafiltration membrane used for the bottom phase in step (6) has a molecular weight cutoff of 0.8 to 2 kDa.

[0026] Furthermore, in step (5), the ultrafiltration membrane has a molecular weight cutoff of 10 kDa. This molecular weight cutoff can efficiently remove impurities such as polyethylene glycol and inorganic salts, avoid the loss of phycobiliproteins in different specific forms, and achieve the purpose of rapid purification and concentration.

[0027] Furthermore, in step (6), the ultrafiltration membrane has a molecular weight cutoff of 1 kDa, which can efficiently remove larger molecular impurities such as polyethylene glycol and red algae peptides, thereby achieving the purpose of rapid purification of oligosaccharides.

[0028] Furthermore, the nanofiltration membrane used in step (6) has a molecular weight cutoff of 100-200 Da. The purpose of nanofiltration is to rapidly remove small molecule impurities such as inorganic salts, thereby achieving rapid purification of oligosaccharides.

[0029] Furthermore, the red algae mentioned are at least one of Porphyra, Gracilaria, Porphyra yezoensis, Porphyra yezoensis, and Polytubula.

[0030] The present invention also provides phycobiliprotein powder and / or red algae oligosaccharide powder prepared by the method.

[0031] Furthermore, the phycobiliprotein powder has a protein content of 50% to 60%, an average particle size of 0.5 to 1.5 mm, a pore volume of 4.5 to 5.5 mm³ / g, an average pore size of 30 to 40 nm, and an effective porosity of 50% to 60%.

[0032] Furthermore, the extract of the red algae oligosaccharide powder has a viscosity of 30–40 mPa·s and a molecular weight of 0.5–1.5 kDa.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] 1. This invention provides a method for extracting phycobiliproteins based on bio-dissolution. It fully utilizes the structure and characteristics of red algae cell wall polysaccharides, employing a compatible enzyme combination ratio and synergistic enzyme cleavage sites to rapidly disrupt the cell wall polysaccharide structure, forming a porous cell wall, increasing the macropore ratio, and promoting the dissolution of phycobiliproteins. The preparation method provided by this invention achieves a phycobiliprotein extraction yield of 224 g / kg after conversion, with a purity of 1.38 (A562 / A280) and a protein content of 54%.

[0035] 2. The method for extracting phycobiliproteins based on bio-wall melting provided by this invention shortens the phycobiliprotein extraction cycle from tens of hours to several hours compared to traditional methods such as freeze-thaw and swelling. Furthermore, this method yields a higher extraction yield of phycobiliproteins, increasing it by 5.4 to 7.1 times compared to traditional methods; the purity of phycobiliproteins in the extract is increased by 1.6 to 2.4 times.

[0036] 3. The method for extracting phycobiliproteins based on bio-wall melting provided by this invention has a higher extraction rate than emerging technologies such as ultrasound and high-pressure homogenization, with a relative improvement of 1.5 to 1.8 times. The purity of phycobiliproteins in the extract is increased by 1.8 times. Moreover, the conditions are mild, and the dissociation rate of phycobiliprotein aggregates is only 19.4%, while the dissociation rates of phycobiliprotein aggregates extracted by ultrasound and high-pressure homogenization reach 34.5% and 42%, respectively, effectively avoiding the loss of phycobiliprotein aggregates due to dissociation.

[0037] 4. The method for extracting phycobiliproteins based on biological wall dissolution provided by this invention has a higher extraction rate than the enzyme extraction method in the prior art, which is 3 to 5 times higher, and the purity of phycobiliproteins in the extract is increased by 1.4 times.

[0038] 5. This invention efficiently extracts phycobiliproteins while simultaneously converting red algae cell wall polysaccharides into red algae oligosaccharides, thereby improving the economic benefits of the process and the utilization rate of raw materials.

[0039] 6. The method for comprehensive and efficient extraction of phycobiliproteins and preparation of red algal oligosaccharides based on bio-wall dissolution provided by this invention is simple, green, environmentally friendly, and low in cost, which is conducive to industrial production. Attached Figure Description

[0040] Figure 1 These are fluorescence micrographs of algal residues before and after treatment in Examples 1-2 and Comparative Examples 1-8.

[0041] Figure 2 These are scanning electron microscope (SEM) and transmission electron microscope (TEM) images of changes in the cell walls of algae before and after treatment in Examples 1 and 2.

[0042] Figure 3 This is a distribution diagram of the average pore size of algal cell walls before and after treatment in Example 1 and Comparative Examples 1-4. Detailed Implementation

[0043] The present invention will now be described more completely and clearly with reference to the accompanying drawings and specific embodiments. The described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0044] Example 1: Preparation of phycobiliproteins and red algal oligosaccharides based on bio-dissolution extraction

[0045] This embodiment provides a comprehensive and efficient method for extracting phycobiliproteins and preparing red algal oligosaccharides based on bio-wall melting. The preparation method includes the following steps:

[0046] (1) Pretreatment: Add 1000 kg of water to the reactor and stir slowly; add 100 kg of dried seaweed powder, then add 1000 kg of water, stir quickly and mix well, rehydrate for 1 h and then grind into a homogenate using a colloid mill to obtain red algae slurry.

[0047] (2) Bio-dissolving: Adjust the pH of the red algae slurry to 5.5, heat it to 43℃ and keep it warm. Take 220g of 10,000 U / g α-galactosidase, 200g of 200,000 U / g cellulase and 260g of 10,000 U / g pectinase respectively, dissolve them in water and add them to the enzymatic hydrolysis tank. Mix them thoroughly, stir and hydrolyze for 5 hours to obtain red algae extract.

[0048] (3) Solid-liquid separation: The red algae extract obtained in step (2) is coarsely filtered through a plate centrifuge with a 100-mesh filter bag; then the coarse filtrate is centrifuged at 14,000 rpm to remove insoluble matter and the supernatant is collected.

[0049] (4) Liquid-liquid separation: The supernatant obtained in step (3) is pumped into a separation tank with an effective volume of 2500L. Polyethylene glycol with a molecular weight of 2000 and phosphate are added respectively. After stirring and dissolving, a two-phase aqueous separation system with a TLL (ti-line length) of 21.88%, Vr (upper-lower phase volume ratio) of 0.57 and pH of 6.5 is formed. After stirring rapidly for 0.5 h, the system is allowed to stand. After the two phases are separated, the top phase and the bottom phase are collected respectively.

[0050] (5) Collection of phycobiliproteins: The top phase collected in step (4) is filtered through an ultrafiltration system with a molecular weight cutoff of 10 kDa to remove impurities including polyethylene glycol and phosphate, and then washed with 3 volumes of water to collect the concentrate, thus obtaining a phycobiliprotein concentrate.

[0051] (6) Collection of red algae oligosaccharides: The substrate collected in step (4) is subjected to ultrafiltration with a membrane with a molecular weight cutoff of 1000 Da to remove impurities such as polyethylene glycol, and is washed with 5 volumes of water. The filtrate is collected, and then nanofiltration with a membrane with a molecular weight cutoff of 100-200 Da is used to remove impurities such as inorganic salts. The concentrate is collected to obtain red algae oligosaccharide concentrate.

[0052] (7) Drying of phycobiliprotein: freeze-dry the phycobiliprotein concentrate collected in step (5) to obtain phycobiliprotein freeze-dried powder, and store it at 2-8℃ for later use.

[0053] (8) Drying of red algae oligosaccharides: Spray dry the red algae oligosaccharide concentrate collected in step (6) with an inlet temperature of 155°C and an outlet temperature between 80°C and 95°C. Collect the red algae oligosaccharide powder and store it at 2-8°C for later use.

[0054] Example 2: Preparation of phycobiliproteins and red algal oligosaccharides based on bio-dissolution extraction

[0055] This embodiment provides a comprehensive and efficient method for extracting phycobiliproteins and preparing red algal oligosaccharides based on bio-wall melting. The preparation method includes the following steps:

[0056] (1) Pretreatment: Take 800 kg of fresh Porphyra tenuifolia, add 1500 kg of water, stir and mix well, and grind into a homogenate using a colloid mill to obtain red algae slurry;

[0057] (2) Bio-dissolving: Adjust the pH of the red algae slurry to 5.3, heat it to 45℃ and keep it warm. Take 264g of 10,000 U / g α-galactosidase, 258g of 200,000 U / g cellulase and 300g of 10,000 U / g pectinase respectively, dissolve them in water and add them to the enzymatic hydrolysis tank. Mix them thoroughly, stir and hydrolyze for 6 h to obtain red algae extract.

[0058] (3) Solid-liquid separation: The red algae extract obtained in step (2) is coarsely filtered through a plate centrifuge with a 100-mesh filter bag; then the coarse filtrate is centrifuged at 14,000 rpm to remove insoluble matter and the supernatant is collected.

[0059] (4) Liquid-liquid separation: The supernatant obtained in step (3) is pumped into a separation tank with an effective volume of 3000L. Polyethylene glycol with a molecular weight of 2000 and ammonium sulfate are added respectively. After stirring and dissolving, a two-phase aqueous separation system with a TLL of 24.98%, Vr of 0.57 and pH of 6.3 is formed. After stirring rapidly for 0.5 h, the system is allowed to stand. After the two phases are separated, the top phase and the bottom phase are collected respectively.

[0060] (5) Collection of phycobiliproteins: The top phase collected in step (4) is filtered through an ultrafiltration system with a molecular weight cutoff of 10 kDa to remove impurities including polyethylene glycol and phosphates. The phase is then washed with water in three volumes and the concentrate is collected to obtain phycobiliprotein concentrate.

[0061] (6) Collection of red algae oligosaccharides: The substrate collected in step (4) is subjected to ultrafiltration with a membrane with a molecular weight cutoff of 1000 Da to remove impurities such as polyethylene glycol, and is washed with 5 volumes of water. The filtrate is collected, and then nanofiltration with a membrane with a molecular weight cutoff of 100-200 Da is used to remove impurities such as inorganic salts. The concentrate is collected to obtain red algae oligosaccharide concentrate.

[0062] (7) Drying of phycobiliprotein: freeze-dry the phycobiliprotein concentrate collected in step (5) to obtain phycobiliprotein freeze-dried powder, and store it at 2-8℃ for later use.

[0063] (8) Drying of red algae oligosaccharides: Spray dry the red algae oligosaccharide concentrate collected in step (6) with an inlet temperature of 155°C and an outlet temperature between 80°C and 95°C. Collect the red algae oligosaccharide powder and store it at 2-8°C for later use.

[0064] Example 3: A preparation method different from Examples 1-2 is provided for the preparation of phycobiliproteins and red algal oligosaccharides.

[0065] This embodiment provides comparative examples 1-8, demonstrating the preparation of phycobiliproteins and red algal oligosaccharides using different processes, as detailed below:

[0066] Comparative Example 1

[0067] This comparative example provides a method for single-enzyme-assisted extraction of phycobiliproteins and preparation of red algal oligosaccharides. The preparation method includes the following steps:

[0068] (1) Weigh 50 kg of dried seaweed powder, add 1000 kg of water, stir and mix well, rehydrate for 1 h, and then grind into a homogenate using a colloid mill to obtain red algae slurry.

[0069] (2) Adjust the pH of the red algae slurry to 5.5, raise the temperature to 43℃ and keep it warm. Take 88g of 10,000 U / g α-galactosidase, dissolve it in water and add it to the enzymatic hydrolysis tank. Mix it thoroughly, stir and hydrolyze for 5h.

[0070] Collect the liquid material and proceed with the same steps (3) to (8) as in Example 1.

[0071] Comparative Example 2

[0072] This comparative example provides a method for single-enzyme-assisted extraction of phycobiliproteins and preparation of red algal oligosaccharides. The preparation method is as follows:

[0073] (1) Weigh 10 kg of dried seaweed powder, add 200 kg of water, stir and mix well, rehydrate for 1 h, and then grind into a homogenate using a colloid mill to obtain red algae slurry.

[0074] (2) Adjust the pH of the red algae slurry to 5.5, raise the temperature to 43℃ and keep it warm. Take 44g of 200,000 U / g cellulase, dissolve it in water and add it to the enzymatic hydrolysis tank. Mix it thoroughly, stir and hydrolyze for 5 hours.

[0075] Collect the liquid material and proceed with the same steps (3) to (8) as in Example 1.

[0076] Comparative Example 3

[0077] This comparative example provides a method for single-enzyme-assisted extraction of phycobiliproteins and preparation of red algal oligosaccharides. The preparation method is as follows:

[0078] (1) Weigh 10 kg of dried seaweed powder, add 200 kg of water, stir and mix well, rehydrate for 1 h, and then grind into a homogenate using a colloid mill to obtain red algae slurry.

[0079] (2) Adjust the pH of the red algae slurry to 5.5, raise the temperature to 43℃ and keep it warm. Take 88 g of 10,000 U / g pectinase, dissolve it in water and add it to the enzymatic hydrolysis tank. Mix it thoroughly, stir and hydrolyze for 5 hours.

[0080] Collect the liquid material and proceed with the same steps (3) to (8) as in Example 1.

[0081] Comparative Example 4

[0082] This comparative example provides a method for extracting phycobiliproteins with the assistance of a compound enzyme and preparing oligosaccharides from red algae. The preparation method is as follows:

[0083] (1) Weigh 10 kg of dried seaweed powder, add 200 kg of water, stir and mix well, rehydrate for 1 h, and then grind into a homogenate using a colloid mill to obtain red algae slurry.

[0084] (2) Adjust the pH of the red algae slurry to 6.8, raise the temperature to 43℃ and keep it warm. Take 104 g of 10,000 U / g agarase and 50 g of 200,000 U / g cellulase respectively, dissolve them in water and add them to the enzymatic hydrolysis tank. Mix them thoroughly, stir and enzymatically hydrolyze for 5 hours.

[0085] Collect the liquid material and proceed with the same steps (3) to (8) as in Example 1.

[0086] Comparative Example 5

[0087] This comparative example provides a method for freeze-thaw extraction of phycobiliproteins, and the preparation method is as follows:

[0088] Weigh 10 kg of dried seaweed powder, add 200 kg of water, stir and mix well, rehydrate for 1 h, freeze at -20℃ for 12 h, thaw at room temperature in the dark, repeat freeze-thaw 5 times; collect the liquid, and the subsequent treatment is the same as steps (3) to (8) in Example 1.

[0089] Comparative Example 6

[0090] This comparative example provides a method for extracting phycobiliproteins using a swelling method, and the preparation method is as follows:

[0091] Weigh 10 kg of dried seaweed powder, add 200 kg of water, stir and mix well, rehydrate for 1 h, add an equal volume of potassium dihydrogen phosphate-sodium hydroxide buffer, place at 38℃ for 10 h to swell, then grind into a homogenate using a colloid mill, collect the liquid, and follow up with the same steps (3) to (8) as in Example 1.

[0092] Comparative Example 7

[0093] This comparative example provides a method for ultrasound-assisted extraction of phycobiliproteins, and the preparation method is as follows:

[0094] Weigh 1 kg of dried seaweed powder, add 20 kg of water, stir and mix well, rehydrate for 1 h, and then sonicate at 100% amplitude and 50 kHz for 30 min, using pulsed ultrasonic cycles of 10 s / 10 s, and control the temperature of the liquid below 15℃. Collect the liquid and proceed with the same steps (3) to (8) as in Example 1.

[0095] Comparative Example 8

[0096] This comparative example provides a method for high-pressure homogenization-assisted extraction of phycobiliproteins, and the preparation method is as follows:

[0097] Weigh 10 kg of dried seaweed powder, add 200 kg of water, stir and mix well, rehydrate for 1 h, then homogenize under 500 bar pressure, control the temperature of the liquid below 15℃, and perform two homogenization cycles at a flow rate of 50 L / h; collect the liquid and process it as in Example 1.

[0098] The following analysis focuses on the phycobiliproteins and red algal oligosaccharides prepared by the different methods described above.

[0099] Example 4: Evaluation of Phycobiliprotein Quality Obtained in Examples and Comparative Examples

[0100] 1. Purity of phycobiliproteins:

[0101] Collect the concentrated solution and measure its absorbance A at 280 nm, 562 nm, 620 nm, and 652 nm using spectrophotometry. Calculate the purity of the phycobiliprotein according to formula (1):

[0102] Formula (1): Purity of phycobiliprotein (PI) = A562 / A280.

[0103] 2. Phycobiliprotein extraction yield:

[0104] Collect the lyophilized phycobiliprotein powder and calculate the extraction yield of phycobiliprotein according to formula (2):

[0105] Formula (2): Extraction amount (EA) = m / m0 (unit g / kg, where m is the weight of phycobiliprotein freeze-dried powder and m0 is the dry weight of red algae).

[0106] 3. Determination of protein content in phycobiliprotein lyophilized powder

[0107] The method specified in Method I of GB 5009.5 shall be followed, and the conversion factor for nitrogen to protein shall be 6.25.

[0108] 4. Determination of polymer dissociation rate

[0109] Phycobiliproteins are hexamers composed of α, β, and γ subunits, with a molecular weight of approximately 240 kDa. They can dissociate into α, β, and γ subunits or αβ monomers under the influence of environmental factors. This dissociation of the polymer affects the optical properties and activity of phycobiliproteins.

[0110] Take 5 g of the phycobiliprotein lyophilized powder prepared in Examples 1-2 and Comparative Examples 1-8 respectively, add distilled water to dissolve it completely, and then perform ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 50 kDa. Collect the concentrate and filtrate respectively, freeze dry them, and calculate the polymer dissociation rate of phycobiliprotein according to formula (3):

[0111] Formula (3): Polymer dissociation rate = M2 / M1×100% (where M2 is the weight of the lyophilized filtrate powder and M1 is the weight of the concentrated filtrate powder).

[0112] 5. Experimental Results and Analysis

[0113] Table 1. Phycobiliprotein quality analysis obtained from the examples and comparative examples.

[0114]

[0115] As shown in Table 1, the purity of phycobiliproteins obtained from Examples 1 and 2 reached 1.3, and the extraction yield reached 224 g / kg, both higher than Comparative Examples 1-8. The purity of phycobiliproteins obtained from Examples 1 and 2 was 40%-47% higher than that of Comparative Examples 1-4, and 60%-144% higher than that of Comparative Examples 5-8. The extraction yield of phycobiliproteins from Examples 1 and 2 was 3-5 times that of Comparative Examples 1-4, and 1.5-7.1 times that of Comparative Examples 5-8. Simultaneously, the protein content of phycobiliproteins obtained from Examples 1 and 2 reached 54%, the highest level among the comparative methods. This indicates that the phycobiliproteins obtained from Examples 1 and 2 contained fewer impurities. The purity of phycobiliproteins in the crude extracts obtained from Examples 1-2 and Comparative Examples 1-4 using the enzyme-assisted extraction method was higher than that of Comparative Examples 5-8 using the physical extraction method. Therefore, enzyme-assisted extraction is beneficial for improving the purity of phycobiliproteins in the crude extract.

[0116] Conventional freeze-thaw (Comparative Example 5) and swelling (Comparative Example 6) extraction methods are simple to operate, require minimal equipment, and offer extraction conditions favorable to phycobiliproteins, effectively preserving their integrity and achieving the lowest polymer dissociation rate. These are currently the main technologies used in production. However, their relatively low extraction yield and purity, along with their long extraction cycle, are bottlenecks restricting their further large-scale application. Ultrasonic and high-pressure homogenization disrupt algal cells through mechanical forces such as cavitation and high-pressure shearing, facilitating the outflow of intracellular substances. However, these mechanical forces also disrupt the three-dimensional structure of phycobiliproteins, resulting in high polymer dissociation rates in the phycobiliproteins extracted in Comparative Examples 7 and 8, thus reducing the effective yield of phycobiliproteins.

[0117] Enzyme-assisted extraction, with its mild conditions, effectively controlled the depolymerization of phycobiliproteins, resulting in low levels of polymer dissociation in Examples 1, 2, and Comparative Examples 1-4, significantly lower than in Comparative Examples 7 and 8. However, single glycosidases have weak degradation capabilities on the red algal cell walls, leading to low phycobiliprotein extraction yields in Comparative Examples 1-3. While the simple combination of glycosidases improved the phycobiliprotein extraction yield in Comparative Example 4, the effect was limited. This invention, tailored to the polysaccharide composition of red algal cell walls, offers mild conditions that avoid polymer dissociation caused by high shear, cavitation, and turbulence, effectively reducing phycobiliprotein loss due to high-temperature dissolution of algal cell wall polysaccharides, thus achieving higher phycobiliprotein extraction yield, purity, and effective recovery rate.

[0118] Example 5: Exploring the morphological microscopic analysis of algae before and after treatment with different methods

[0119] (1) After fixation, the algal body and residual algal tissue after different treatments were observed using optical microscope, fluorescence microscope and electron microscope to observe the morphology of algal tissue.

[0120] The results are as follows Figure 1 As shown, untreated algal cells are tightly packed, forming intact sheets, and emit strong green fluorescence, which is the autofluorescence of phycobiliproteins. Algal residue samples treated with different methods showed varying degrees of coexistence of cell debris and residual tissue, and their autofluorescence also decreased to varying degrees, indicating that phycobiliproteins had leaked out of the cells to varying degrees. The algal residue treated with freeze-thaw (Comparative Example 5) and swelling (Comparative Example 6) contained more residual tissue and almost no cell debris, indicating that these two methods had very limited effect on cell disruption; moreover, their fluorescence intensity was also stronger, indicating that the phycobiliproteins extracted from these two samples were insufficient, consistent with their extraction amounts.

[0121] In contrast, ultrasound-assisted treatment (Comparative Example 7), high-pressure homogenization (Comparative Example 8), and enzyme treatment (Examples 1-2 and Comparative Examples 1-4) showed good effects on algal tissue destruction, leaving only a small amount of residual tissue with loosely arranged, swollen cells. High-pressure homogenization (Comparative Example 8) and combined enzyme treatment (Examples 1-2) resulted in higher cell fragmentation, indicating a higher cell disruption rate. Furthermore, compared to Comparative Examples 5-8, the residual cells after enzyme treatment (Examples 1-2 and Comparative Examples 1-4) exhibited lower fluorescence intensity; particularly, the residual cells in Examples 1 and 2 showed weak fluorescence intensity. This suggests that enzyme treatment, in addition to causing complete cell disruption and releasing phycobiliproteins, also increased cell permeability, leading to the outward migration of phycobiliproteins.

[0122] (2) The cell morphology of untreated algae and algae treated in Examples 1 and 2 was further observed by scanning electron microscopy and transmission electron microscopy.

[0123] like Figure 2 As shown, untreated algal cells have an intact, compact, and smooth surface under a scanning electron microscope. However, a transmission electron microscope reveals a dense and thick cell wall structure, which effectively hinders the release of intracellular macromolecules such as phycobiliproteins and proteins, maintaining the algae's survival in seawater. After treatment in Examples 1 and 2, the algal cells have a rough surface with an irregular porous structure; under a transmission electron microscope, their cell morphology is distorted, and the cell walls are significantly thinned, weakened, and broken, with intracellular substances leaking out in some locations. This indicates that the present invention effectively disrupts the barrier function of the algal cell wall, promoting the release of phycobiliproteins. This is fundamentally different from the physical comparative examples 5-8.

[0124] Example 6: Determination of BET specific surface area and pore structure analysis of algal cells before and after treatment

[0125] 1. Analytical Principles and Methods

[0126] In its natural state, phycobiliprotein exists as a hexamer, with a disc-shaped structure approximately 12 nm in diameter and 85 nm thick. The dissociated dimer (αβ) has a diameter of approximately 5 nm, while the trimer (αβ3) has a disc-shaped structure approximately 12 nm in diameter and 3 nm thick. Therefore, measuring the porosity characteristics of algal cells before and after treatment can reflect the extraction efficiency and principle of phycobiliprotein.

[0127] Algal samples and algal residues after different treatments were collected. A surface and pore area analyzer was used to measure the particle size, specific surface area, pore volume, and pore size of the samples through gas adsorption and desorption. Effective pore size refers to the pores capable of accommodating phycobiliprotein hexamers. The effective pore size ratio reflects the efficiency of the technique in releasing phycobiliproteins from algal cells. The effective pore size ratio is calculated according to formula (4):

[0128] Formula (4): ,

[0129] Where S 12.4 Specific surface area of ​​pores with a diameter ≥ 12.4 nm; S T This represents the total pore surface area.

[0130] 2. Experimental Results and Analysis

[0131] Table 2. Pore characteristics analysis before and after treatment in the examples and comparative examples.

[0132]

[0133] 3. Analysis of the impact of different preparation methods on the degree of algal degradation

[0134] The average particle size of the residual algae more accurately reflects the degree of fragmentation of the algal tissue. As shown in Table 2, the average particle size of the untreated algal powder was approximately 6.09 mm. The treatment in Comparative Example 5 did not significantly improve the particle size of the algae, while other preparation methods reduced the particle size of the algae to varying degrees.

[0135] In the comparative examples, the enzyme treatment schemes (Comparative Examples 1-4) showed better fragmentation effects on algal tissues than Comparative Examples 5-8. The average particle size of algal tissues treated with the composite enzyme scheme in Comparative Example 4 was not significantly different from that treated with the single enzyme schemes in Comparative Examples 1-3. However, the algal particle size treated with the schemes in Examples 1 and 2 was reduced to approximately 1 mm, significantly better than the comparative examples. This further verifies that the preparation methods provided in Examples 1-2 have a higher ability to fragment algal tissues.

[0136] 4. Effects of different preparation methods on the porosity characteristics of algal cells

[0137] Figure 3Table 2 shows that the total pore volume of untreated algal cells is only 1.89 mm³ / g, mainly composed of pores with a diameter less than 8 nm and an average pore diameter of 7.21 nm, which is insufficient to accommodate the release of phycobiliprotein aggregates. The physical cell disruption techniques in Comparative Examples 5 and 6 had little effect on algal porosity; the total pore volume did not change significantly, and the average pore diameter expanded to 11-12.37 nm, only reaching the minimum diameter required for phycobiliprotein aggregates to pass through. Comparative Example 6 treatment increased the average pore diameter to 15.78 nm, allowing phycobiliprotein aggregates to pass through smoothly, but it did not significantly change the pore volume of the sample. Therefore, although Comparative Examples 5–7 increased the pore diameter and promoted the release of phycobiliproteins, the release efficiency was limited due to the pore volume limitation, which is basically consistent with the phycobiliprotein extraction results. The treatment of Comparative Example 8 increased the average pore size to 21.06 nm, further improving the permeability of phycobiliprotein aggregates. Its pore volume also changed significantly, reaching 2.67 mm³ / g, but its effective pore ratio was only 8.76%.

[0138] The pore characteristic analysis results of Comparative Examples 5-8 confirmed that the physical technology for extracting phycobiliproteins relies on the disruption of algal cells and has very limited effect on altering the pore characteristics of the cell wall. After treatment with the enzyme-assisted technology (Comparative Examples 1-4), the average pore size increased to 23.16-28.25 nm. Although the improvement compared to Comparative Example 8 was limited, the effective porosity was significantly increased, reaching 25.02%-45.46%, greatly improving the permeability of phycobiliproteins. In Examples 1 and 2, the average pore size of the algal cell wall reached 34.75 nm, an improvement of 23%-50.8% compared to Comparative Examples 1-4, and 1.65-3.2 times that of Comparative Examples 5-8; the effective porosity reached 56%, 1.2-2.3 times that of Comparative Examples 1-4, and 6.4-12 times that of Comparative Examples 5-8. Therefore, enzyme treatment can not only disrupt cells through cell wall lysis but also efficiently release phycobiliproteins by increasing the pore size of the algal cell wall. This has significant advantages compared to physical methods.

[0139] Figure 3The results show that the cell wall pore size distribution shifted towards macropores after enzyme treatment. Compared to untreated algal cells, the enzyme-treated samples exhibited a wider pore size distribution, ranging from 8.7 nm to over 250 nm. This indicates that enzyme treatment induces porous characteristics in the algal cell wall. Among the enzyme-treated comparative examples, Comparative Example 3 showed relatively better results, with its pore size concentrated in the ranges of 8.7-29.37 nm and above 92.9 nm, and an average pore size of 28.15 nm, significantly better than the 23.35 nm of the enzyme combination-treated sample in Comparative Example 4 (Table 2). However, the proportion of effective pores that could accommodate phycobiliprotein aggregates in the enzyme combination-treated sample in Comparative Example 4 reached 45.46%, significantly higher than the single-enzyme-treated samples in Comparative Examples 1-3, which is consistent with its higher phycobiliprotein extraction yield (Table 1). The samples treated in Examples 1 and 2 of this invention exhibited a wider pore size distribution range (9.7-60 nm and above 250 nm), and the pore volume in the range with a pore size greater than 30 nm was significantly greater than that in Comparative Examples 1-4. Therefore, the present invention significantly improves the porosity of algal cell walls and increases the proportion of macropores, thereby obtaining a higher yield of phycobiliproteins.

[0140] In summary, this invention, through a cell wall-compatible enzyme combination, not only efficiently catalyzes the thinning, weakening, and breakage of red algal cell walls, significantly improving the efficiency of algal tissue disruption and releasing phycobiliproteins; but also induces residual algal cells to form porous and macroporous cell wall structures, significantly increasing the proportion of effective pores, increasing cell permeability, and promoting the outward migration of intracellular substances such as phycobiliproteins, thus achieving higher extraction efficiency. This differs from the principle of physical cell wall disruption extraction of phycobiliproteins, and it has significant advantages in extraction yield, purity, and efficiency; it also exhibits higher cell wall disruption rate and phycobiliprotein extraction efficiency compared to single enzymes and simple enzyme combination schemes.

[0141] Example 7: Evaluation of the effects of different preparation methods on red algae polysaccharides

[0142] Table 3. Polysaccharide analysis of the examples and comparative examples

[0143]

[0144] The unique high-pressure, high-permeability environment of the ocean has resulted in the distinctive composition and compact structure of red algal cell walls. The cell walls of red algae are primarily composed of gel-like polysaccharides. Because these polysaccharides contain a certain amount of sulfate groups, they exhibit weak gelling properties and a certain viscosity.

[0145] As shown in Table 3, the high-pressure homogenization process in Comparative Example 8 dissolves the polysaccharides from red algae, but hardly degrades them. The extracted polysaccharides have a high molecular weight (149-227 kDa), resulting in a high-viscosity liquid (viscosity 1192 mPa·s), which also increases the difficulty of separating phycobiliproteins. The ultrasonic process in Comparative Example 7 increases the yield of oligosaccharides, but their molecular weight remains high (43-64 kDa). Comparative Examples 5 and 6 show very limited polysaccharide extraction, only dissolving a small amount of low-molecular-weight sugars, resulting in low-viscosity extracts. Enzyme treatment effectively promotes the extraction of red algae polysaccharides. However, while single-enzyme treatments in Comparative Examples 1-3 promote polysaccharide extraction, their degradation ability is limited, resulting in high-viscosity extracts with high-molecular-weight oligosaccharides that remain within the oligosaccharide range. Comparative Example 4 shows good degradation of red algae polysaccharides, yielding a low-viscosity extract, a high oligosaccharide yield, and a low molecular weight distribution. The oligosaccharide yields of Examples 1 and 2 reached approximately 570 g / kg, which is 1.5 times that of Comparative Example 4 and 2.4 to 4.5 times that of Comparative Examples 1 to 3. Furthermore, the molecular weight of the oligosaccharides was further reduced to 0.5-1.15 kDa, falling within the oligosaccharide range, resulting in a low viscosity of the extract (35 mPa·s). Therefore, this invention efficiently prepares red algal oligosaccharides while achieving high extraction yield and purity of phycobiliproteins, yielding two high-value active substances.

[0146] In summary, this invention provides a highly efficient, low-loss, and scale-up method for extracting phycobiliproteins from red algae. This invention effectively resolves the conflict between the high-temperature dissolution of red algae cell wall polysaccharides and the heat sensitivity of phycobiliproteins through multi-enzyme synergy, improving cell wall disruption efficiency and promoting the formation of porous and macroporous cell wall structures. It also facilitates the outward migration of intracellular substances such as phycobiliproteins, resulting in higher extraction efficiency. Furthermore, this invention enhances the extraction and conversion of red algae polysaccharides through enzymatic action, yielding red algae oligosaccharide products and improving the utilization rate of red algae raw materials and the economic benefits of the process. The multi-enzyme synergistic method for preparing phycobiliproteins and red algae oligosaccharides provided by this invention requires no special equipment, is simple to implement, and is easily scalable for production.

[0147] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A method for preparing phycobiliproteins and / or red algal oligosaccharides based on bio-wall extraction, characterized in that, The preparation method includes the following steps: (1) Pretreatment: Mix red algae with water thoroughly, rehydrate, and grind into a homogenous slurry to obtain red algae slurry; (2) Bio-dissolving: The red algae slurry is transferred into a reaction vessel, the pH and temperature of the red algae slurry are adjusted, and a compound enzyme is added. The mixture is stirred continuously to fully react and obtain red algae extract. (3) Solid-liquid separation: The red algae extract is coarsely filtered and centrifuged to remove insoluble matter, and the supernatant is collected; (4) Liquid-liquid separation: The centrifuged clear liquid is pumped into a separation tank, and polyethylene glycol and inorganic salt are added to form a two-phase aqueous separation system. The system is stirred rapidly, and after the phases come into contact, it is allowed to stand. After the two phases are separated, the top phase and the bottom phase are collected respectively. (5) Collection of phycobiliproteins: The top phase is separated by ultrafiltration to remove impurities, and washed with water. The concentrated liquid is collected to obtain the phycobiliprotein solution. (6) Collection of red algae oligosaccharides: The substrate is separated by ultrafiltration to remove impurities, washed with water, and the filtrate is collected. Then, impurities are removed again by nanofiltration, and the concentrate is collected to obtain the red algae oligosaccharide solution. (7) Drying: The phycobiliprotein solution and the red algae oligosaccharide solution are dried separately to obtain phycobiliprotein powder and red algae oligosaccharide powder.

2. The preparation method according to claim 1, characterized in that, In step (2), the pH of the red algae slurry is adjusted to 4.5-7.0; the complex enzyme is a complex enzyme composed of α-galactosidase, cellulase and pectinase, with an enzyme activity ratio of 0.5-2: 6-12: 0.7-1.7; the amount of the complex enzyme added is 1‰-15‰, based on the dry weight of the red algae.

3. The preparation method according to claim 1, characterized in that, In step (4), the molecular weight of ethylene glycol is 2000-20000 Da, and the inorganic salt is ammonium sulfate or phosphate.

4. The preparation method according to claim 1, characterized in that, The two-phase aqueous separation system in step (4) is specifically characterized by a system length of 18% to 27%, an upper and lower phase volume ratio of 0.26 to 0.77, and a pH of 5.2 to 7.

0.

5. The preparation method according to claim 1, characterized in that, The ultrafiltration membrane used in step (5) has a molecular weight cutoff of 8 to 100 kDa.

6. The preparation method according to claim 1, characterized in that, The ultrafiltration membrane used in step (6) has a molecular weight cutoff of 0.8 to 2 kDa, and the nanofiltration membrane used in nanofiltration has a molecular weight cutoff of 100 to 200 Da.

7. The preparation method according to claim 1, characterized in that, The red algae mentioned are at least one of the following: Porphyra, Gracilaria, Porphyra yezoensis, Porphyra yezoensis, and Polytubula.

8. Phycobiliprotein powder and / or red algae oligosaccharide powder prepared by the method according to any one of claims 1 to 7.

9. The phycobiliprotein powder and / or red algae oligosaccharide powder according to claim 8, characterized in that, The phycobiliprotein powder has a protein content of 50%–60%, an average particle size of 0.5–1.5 mm, a pore volume of 4.5–5.5 mm³ / g, an average pore size of 30–40 nm, and an effective porosity of 50%–60%.

10. The phycobiliprotein powder and / or red algae oligosaccharide powder according to claim 8, characterized in that, The extract of the red algae oligosaccharide powder has a viscosity of 30–40 mPa·s and a molecular weight of 0.5–1.5 kDa.

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