Microbial fermentation-based phycocyanin crude protein extraction method and application of obtained product

Abstract

The invention relates to the technical field of microbial fermentation, in particular to an extraction method of phycocyanin crude protein based on microbial fermentation and application of an obtained product. The method comprises the following steps: S1, dissolving spirulina powder in a phosphate buffer solution, and adding glucose to obtain an algae powder suspension; s2, inoculating saccharomycetes into the algae powder suspension, and culturing for 12-24 hours to obtain a first-stage fermentation product; s3, inoculating the first-stage fermentation product with plant lactobacillus, and continuing fermentation culture for 24-48 hours to reduce the pH value of the system; s4, after fermentation is finished, supernate is taken and concentrated through an ultrafiltration membrane with the molecular weight cut-off of 50 kDa, dialysis or nanofiltration desalination is carried out, and phycocyanin crude protein is obtained. The method for extracting the phycocyanin crude protein by utilizing the microorganisms to cooperatively ferment the spirulina powder is low in energy consumption, free of organic solvent and capable of being amplified, has remarkable technical effect improvement and process comprehensive advantages, and provides technical support for deep application in the fields of food and health.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and in particular to a method for extracting crude phycocyanin based on microbial fermentation and the application of the resulting product. Background Technology

[0002] Spirulina is a microalgae resource rich in protein, phycocyanin, phycobilichrome, and various active ingredients. Phycocyanin (PC), as a natural water-soluble pigment, is widely used in the food, health product, and cosmetic industries. However, existing phycocyanin extraction methods mostly rely on physical (freeze-thaw, ultrasound, microwave) or chemical (buffer extraction, salting out) methods. Specifically, while physical methods can improve cell wall disruption efficiency, they are energy-intensive and can easily lead to partial denaturation and color value reduction of phycocyanin. Chemical methods have relatively high extraction efficiency, but the introduction of exogenous chemical reagents poses food safety risks and environmental pressures. Enzymatic methods require the addition of cellulase and protease to break down algal cell walls. However, enzyme preparations are expensive and the processes are complex, making widespread industrial application difficult. These methods suffer from high energy consumption, high risk of protein denaturation, and unstable purity and yield. In recent years, some studies have attempted to introduce microbial fermentation to improve phycocyanin extraction efficiency, but most of these studies involve the application of single strains (such as lactic acid bacteria or Bacillus subtilis) and have the following shortcomings: 1. If the acidity or enzymatic hydrolysis environment is too uniform during fermentation, it can easily lead to the degradation of phycocyanin; 2. The enzyme production spectrum of a single microbial strain is insufficient, making it difficult to achieve the synergistic release of Spirulina cell wall and intracellular proteins; 3. Lack of systematic process optimization makes it difficult to balance yield and stability.

[0003] Therefore, how to release phycocyanin efficiently in a mild and controllable manner while ensuring its activity and structural integrity, and at the same time maintaining its functional activity and structural stability, is a key technical problem that urgently needs to be solved.

[0004] Existing methods for extracting phycocyanin, including Chinese patents CN120248096A (a method for extracting phycocyanin from Spirulina), CN112812176A (a method for extracting phycocyanin from Spirulina via low-salt flocculation), CN112851800A (a method for preparing phycocyanin from Spirulina), and CN109456406A (a method for extracting phycocyanin from Spirulina), mostly employ traditional steps such as suspension with phosphate buffer, freeze-thaw cycles, centrifugation, and salting-out precipitation to obtain phycocyanin. While these methods have some effectiveness, they generally suffer from high energy consumption, chemical reagent residues, or the risk of protein denaturation. Existing technologies, such as Chinese patent CN105524165A (a method for extracting phycocyanin by coupling an expanded bed and an adsorption resin fixed bed), CN118420749A (a method for preparing high-purity phycocyanin), CN113583111A (a high-pressure carbon dioxide non-thermal extraction method for high-quality phycocyanin pigment), and CN112480241A (a method for extracting phycocyanin from fresh spirulina harvested in a high-salt environment), still emphasize obtaining phycocyanin through physical methods (adsorption, ultrasound, expanded bed) and chemical cascade extraction.

[0005] While the methods mentioned above emphasize industrialization, they still fall under the category of physical and chemical methods. There are few publicly available technologies that utilize the synergistic effect of microorganisms to ferment spirulina for the extraction of phycobiliproteins. There is a lack of a method that achieves mild, efficient, and green extraction of phycocyanin through the synergistic effect of multiple bacterial species. Summary of the Invention

[0006] In view of this, the present invention aims to provide a method for extracting crude phycocyanin from spirulina powder by utilizing the synergistic fermentation of spirulina powder by microorganisms. Through the metabolic complementarity and enzymatic hydrolysis synergistic effect of different types of microorganisms, the method achieves gentle cell wall disruption of spirulina and efficient release of crude phycocyanin, providing a green, safe, simple and scalable method for extracting crude phycocyanin.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The first aspect of this invention provides a method for extracting crude phycocyanin based on microbial fermentation, comprising the following steps: S1. Dissolve spirulina powder in phosphate buffer, add glucose to obtain an algae powder suspension; S2. Inoculate yeast into the algae powder suspension and culture for 12-24 hours to obtain the first stage fermentation product; S3. Inoculate the first-stage fermentation product with Lactobacillus plantarum and continue fermentation for 24-48 hours to lower the pH of the system. S4. After fermentation, solid-liquid separation is performed to remove the cells and residues. The supernatant is concentrated using an ultrafiltration membrane with a molecular weight cutoff of 50 kDa. The supernatant is then desalted by dialysis or nanofiltration to obtain crude phycocyanin.

[0008] In some preferred embodiments, the ratio of spirulina powder to phosphate buffer is 1 g: (20-50) mL.

[0009] In some preferred embodiments, the concentration of glucose in the algal powder suspension is 30-50 g / L.

[0010] In some preferred embodiments, in step S2, the fermentation temperature of the yeast is 30-37 °C, and the dissolved oxygen is controlled to be not less than 1.5 mg / L.

[0011] In some preferred embodiments, the inoculum size of the yeast is 1-3 × 10⁻⁶. 6 CFU / mL In some preferred embodiments, in step S3, the fermentation temperature of *Lactobacillus plantarum* is 30-37°C, and the dissolved oxygen is controlled to be no less than 1.5 mg / L.

[0012] In some preferred embodiments, the inoculum size of *Lactobacillus plantarum* is 0.5-1.5 × 10⁻⁶. 6 CFU / mL In some preferred embodiments, in step S4, the pH of the system is reduced to 5.0-6.0.

[0013] In some preferred embodiments, the yeast is Kluyveromyces marxianus or Saccharomyces cerevisiae At least one of the following, wherein the *Lactobacillus plantarum* is Lactiplantibacillus plantarum At least one of them.

[0014] A second aspect of the present invention provides the application of the crude phycocyanin obtained by the above extraction method in the preparation of food or cosmetics.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The two-step synergistic fermentation method of "yeast pretreatment + mild acidification by *Lactobacillus plantarum*" proposed in this invention is a novel approach not found in existing technologies. Therefore, based on the above, this invention develops a method for extracting crude phycocyanin from spirulina powder using microbial synergistic fermentation. This method is low-energy, solvent-free, scalable, and possesses significant technological improvements and comprehensive process advantages, providing technical support for its in-depth application in the food and health fields.

[0016] 2. This invention provides a method for extracting crude phycocyanin from spirulina powder using microbial co-fermentation. Specifically, it utilizes yeast-secreted polysaccharide enzymes and proteases to partially degrade the cell wall structure and organic acids produced by *Lactobacillus plantarum* to regulate the microenvironment, achieving gentle cell wall disruption of spirulina and efficient dissolution of crude phycocyanin. The advantages of this crude phycocyanin extraction method are: efficient cell wall disruption under gentle conditions, maintaining the natural conformation and biological activity of crude phycocyanin; synergistic effect of yeast and *Lactobacillus plantarum* to enhance the dissolution rate of crude phycocyanin; a green and environmentally friendly fermentation system with no chemical solvent residue; and low energy consumption, simple process, and wide applicability. Attached Figure Description

[0017] Figure 1 Images of crude phycocyanin solutions from Example 1 and Comparative Examples 1-14. Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Spirulina powder is commercially available.

[0020] The yeast used in Example 1 is Kluyveromyces marxianus From the China Center for Type Culture Collection, accession number CCTCC SY2022081. *Lactobacillus plantarum* is... Lactiplantibacillus plantarum It comes from the China Center for Type Culture Collection, accession number CCTCC AB 206133.

[0021] Example 1 This embodiment provides a method for extracting crude phycocyanin based on microbial fermentation, including the following steps: S1. Dissolve 10g of spirulina powder in 300mL of 0.1 mol / L phosphate buffer (pH=7.5), add 15g of glucose, and stir magnetically for 20min at 28℃ and 300rpm to obtain an algae powder suspension; S2. Inoculate yeast into the algal powder suspension at an inoculum size of 2 × 10⁻⁶. 6 The dissolved oxygen was controlled at 1.5 mg / L at 30℃ under aerobic conditions for 24 hours to obtain the first stage fermentation product. S3. Inoculate the first-stage ferment with *Lactobacillus plantarum* at a rate of 1 × 10⁻⁶. 6The CFU / mL concentration was further fermented at 36℃ under conditions where dissolved oxygen was controlled to be no less than 1.5 mg / L for 24 hours, allowing the pH of the system to slowly decrease to 5.5. S4. After fermentation, centrifuge at 8000 rpm, 25℃, and 15 min to separate solids and liquids, remove bacteria and residues, and concentrate the supernatant using an ultrafiltration membrane with a molecular weight cutoff of 50 kDa. Then, desalinate the solution by dialysis or nanofiltration to obtain crude phycocyanin.

[0022] Comparative Example 1 The specific implementation method of this comparative example is the same as that of Example 1, except that the culture time in S2 is 10 hours and the culture time in S2 is 38 hours. Comparative Example 2 The specific implementation method of this comparative example is the same as that of Example 1, except that the culture time in S2 is 36 hours and the culture time in S2 is 12 hours.

[0023] Comparative Example 3 The specific implementation method of this comparative example is the same as that of Example 1, except that it does not include step S3, which separates the first-stage fermentation product into solid and liquid components. Comparative Example 4 The specific implementation method of this comparative example is the same as that of Example 1, except that it does not include step S2, inoculating the algal powder suspension with Lactobacillus plantarum.

[0024] Comparative Example 5 This comparative example provides a method for extracting crude phycocyanin based on microbial fermentation, including the following steps: S1. Dissolve 10g of spirulina powder in 300mL of 0.1 mol / L phosphate buffer (pH=7.5), add 15g of glucose, and stir magnetically for 20min at 28℃ and 300rpm to obtain an algae powder suspension; S2. Place the algal powder suspension in an ultrasonic disruptor, set the power to 300W, the working time to 3s, the interval time to 5s, the total ultrasonic treatment time to 20min, and the temperature to 25℃ to complete cell disruption; S4. After sonication, the algal powder suspension is centrifuged at 8000 rpm, 25°C, and 15 min to separate solids and liquids, remove bacteria and residues, and concentrate the supernatant using an ultrafiltration membrane with a molecular weight cutoff of 50 kDa. The concentrate is then desalted by dialysis or nanofiltration to obtain crude phycocyanin protein.

[0025] Comparative Example 6 This comparative example provides a method for extracting crude phycocyanin based on microbial fermentation, including the following steps: S1. Dissolve 10g of spirulina powder in 300mL of 0.1 mol / L phosphate buffer (pH=7.5), add 15g of glucose, and stir magnetically for 20min at 28℃ and 300rpm to obtain an algae powder suspension; S2. Inoculate the algal powder suspension with *Lactobacillus plantarum* at a concentration of 1 × 10⁻⁶. 6 The fermentation was carried out at CFU / mL for 24 hours at 36℃ with dissolved oxygen controlled at no less than 1.5 mg / L, so that the pH of the system slowly decreased to 5.5, and the first stage fermentation product was obtained. S3. Inoculate the first-stage fermentation product with yeast at an inoculum size of 2 × 10⁻⁶. 6 CFU / mL, under aerobic conditions at 30℃, dissolved oxygen is controlled to be no less than 1.5 mg / L, and cultured for 24 hours; S4. After fermentation, centrifuge at 8000 rpm, 25℃, and 15 min to separate solids and liquids, remove bacteria and residues, and concentrate the supernatant using an ultrafiltration membrane with a molecular weight cutoff of 50 kDa. Then, desalinate the solution by dialysis or nanofiltration to obtain crude phycocyanin.

[0026] Comparative Example 7 The specific implementation method of this comparative example is the same as that of comparative example 3, except that the yeast is... Saccharomyces cerevisiae It comes from the China Center for Type Culture Collection, accession number CCTCC CY 20081195. Comparative Example 8 The specific implementation method of this comparative example is the same as that of Example 1, except that the yeast is Saccharomycescerevisiae var. ellipsoideus, which comes from the China Agricultural Microbial Culture Collection Center, with accession number ACCC20163.

[0027] Comparative Example 9 The specific implementation method of this comparative example is the same as that of Comparative Example 4, except that *Lactobacillus plantarum* is used. Streptococcus thermophilus It comes from the China Industrial Microbial Culture Collection Center, with accession number CICC20174.

[0028] Comparative Example 10 The specific implementation method of this comparative example is the same as that of Comparative Example 4, except that *Lactobacillus plantarum* is used. Lacticaseibacillus casei It comes from the China General Microbiological Culture Collection Center, with accession number CGMCC1.29.

[0029] Comparative Example 11 The specific implementation method of this comparative example is the same as that of comparative example 4, except that the plant lactobacillus is Lactiplantibacillus plantarum subsp. plantarum, which comes from the American Technical Reference Center (ATCC) with accession number ATCC 14917.

[0030] Comparative Example 12 The specific implementation method of this comparative example is the same as that of Example 1, except that the yeast is replaced with Saccharomyces bayanus, which is from the China General Microbiological Culture Collection Center, with accession number CGMCC2.1885.

[0031] Comparative Example 13 The specific implementation method of this comparative example is the same as that of Example 1, except that Lactobacillus plantarum is replaced with Lactobacillus acidophilus, which is from the China Center for Type Culture Collection, accession number CCTCC CB20081799.

[0032] Comparative Example 14 The specific implementation method of this comparative example is the same as that of Example 1, except that yeast and Lactobacillus plantarum are inoculated at the same time, and the two bacteria ferment together for 48 hours.

[0033] Performance testing In this embodiment and the comparative example, all experiments were repeated three times, and the data are expressed as the mean ± standard deviation of the three replicates. The data were analyzed for significance using one-way ANOVA and Duncan's test in SPSS. p< 0.05 ).

[0034] Method for determining crude protein content of phycocyanin: Refer to Chinese National Standard SN / T 1113 Method for determining the phycocyanin content in Spirulina 2002.

[0035] The formula for calculating the extraction rate is: Extraction rate =

[0036] The formula for calculating the recovery rate is: Recovery rate =

[0037] Purity calculation method: The absorbance of the crude phycocyanin sample solution at 620 nm and 280 nm was measured using a UV-Vis spectrophotometer, and the values ​​were recorded. Then, the purity was calculated using the formula: Purity = calculate.

[0038] The test data above are shown in Table 1.

[0039] Table 1

[0040] (1) Extraction rate of crude phycocyanin The extraction rates of crude phycocyanin in Example 1 and Comparative Examples 1-14 are shown in Table 1. As can be seen from the figure, Example 1 achieved the highest extraction rate. This is because yeast fermentation for 24 hours fully decomposes the polysaccharides in the Spirulina cell wall, loosening the cell structure; subsequent fermentation by *Lactobacillus plantarum* for another 24 hours produces metabolites (organic acids) that assist in cell wall rupture, allowing for the full release of crude phycocyanin and achieving the highest extraction rate. Comparative Example 6, although a co-fermentation, had a reversed fermentation sequence, resulting in a slightly lower extraction rate compared to Example 1. Compared to sequential fermentation with two bacteria, Comparative Example 14's co-fermentation lacked a clear division of labor, leading to competition for dissolved oxygen and carbon sources, thus reducing the extraction rate. However, these three examples validated the advantages of co-fermentation combining yeast's physical disruption of cell structure with *Lactobacillus plantarum*'s chemical cell structure modification. Comparative Examples 1-2 showed poor co-fermentation effects due to uneven fermentation times. In Comparative Example 1, the yeast fermented for only 12 hours, resulting in insufficient decomposition of cell wall polysaccharides and incomplete destruction of cell structure. Although the fermentation time of *Lactobacillus plantarum* was extended to 36 hours, its acid production intensity decreased due to cell structure obstruction, limiting the release of phycocyanin crude protein, and the extraction rate decreased by approximately 9.8% compared to Example 1. In Comparative Example 2, yeast fermentation for 36 hours excessively consumed nutrients and even decomposed some of the released phycocyanin crude protein, and increased metabolic byproducts; *Lactobacillus plantarum* fermented for only 12 hours, resulting in insufficient acid production and cell disruption. These two factors combined led to a significant decrease in the extraction rate. Comparative Examples 3-4, 7, and 9-10 were all single-strain fermentations, with effects far lower than synergistic fermentation. Comparative Examples 3 and 7 involved only yeast single-strain fermentation, without *Lactobacillus plantarum* to assist in cell disruption, resulting in limited cell rupture. Comparative Examples 4 and 9-10 involved only *Lactobacillus plantarum* single-strain fermentation, which could only slightly damage the cell surface because it could not decompose the rigid cell wall polysaccharides of *Spirulina*. The release of phycocyanin crude protein was low, resulting in a low extraction rate. The bacterial strains used in Comparative Examples 8 and 11 were inferior to those in Comparative Examples 3 and 7, resulting in poor extraction of phycocyanin. The bacterial communities in Comparative Examples 12-13 showed poor compatibility, leading to the lowest extraction efficiency among bacterial fermentation methods for phycocyanin extraction. The ultrasonic disruption extraction method in Comparative Example 5 relied solely on mechanical force to disrupt Spirulina cells. While it could directly destroy the structure, it lacked the selective decomposition ability of cell wall polysaccharides, and the localized high temperatures easily caused denaturation of crude phycocyanin (some proteins could not be released or were inactivated), resulting in a significantly lower extraction rate than the microbial fermentation extraction method.

[0041] (2) Recovery rate of crude protein from phycocyanin The extraction rates of crude phycocyanin in Example 1 and Comparative Examples 1-14 are shown in Table 1. In Example 1, the balanced fermentation time distribution of the two bacteria resulted in sufficient polysaccharide breakdown by yeast in the first stage and sufficient acid production by *Lactobacillus plantarum* in the second stage, stabilizing the pH at 5.5. This metabolic environment was optimal for the stability of crude phycocyanin, achieving a recovery rate as high as 94%, close to the upper limit. In Comparative Example 6, the reversed fermentation sequence led to insufficient initial cell disruption, resulting in some protein not being released. In Comparative Example 14, bacterial competition caused metabolic disturbances, with a small number of contaminating bacteria consuming the protein, reducing the recovery rate. In Comparative Example 1, the yeast fermentation time was too short, resulting in insufficient polysaccharide breakdown and limiting the release of some crude phycocyanin. The *Lactobacillus plantarum* fermentation time was too long, leading to increased metabolic byproducts due to insufficient nutrients in the later stages of acid production, which damaged the protein structure and reduced the crude phycocyanin recovery rate. In Comparative Example 2, the yeast fermentation time was too long, excessively consuming nutrients and even decomposing some of the released crude phycocyanin. Furthermore, the *Lactobacillus plantarum* fermentation time was too short, resulting in insufficient acid production and insufficient inhibition of contaminating bacteria growth. The protein was either consumed by contaminating bacteria or denatured due to an unsuitable environment, leading to a decreased recovery rate. Comparative Example 5 showed the lowest recovery rate of crude phycocyanin protein, at only 50%. This was because the ultrasonic cavitation effect in Comparative Example 5 directly disrupted cells and damaged the structure of crude phycocyanin protein, while failing to decompose cell wall polysaccharides, resulting in cell debris mixing with protein. Furthermore, the mechanical force and localized high temperature of ultrasound directly denatured the crude phycocyanin protein, rendering nearly half of the original protein unrecoverable, leading to significant waste. The remaining comparative examples involved single-strain fermentation or co-fermentation with non-specified strains, which affected protein stability: single-strain fermentation lacked acidification assistance, resulting in an unsuitable pH and some protein denaturation; substrain or non-specified strain fermentation increased metabolic byproducts, damaging protein structure, and the recovery rate was 5%-10% lower than the specified strain group.

[0042] (3) Purity of crude phycocyanin extraction Purity (A) 620 / A 280The purity of crude phycocyanin extracted from Examples 1 and Comparative Examples 1-14 depends on the amount of residual impurities, which is directly related to the thoroughness of cell destruction (reducing cell debris) and the antibacterial effect (reducing metabolic waste from bacteria). Table 1 shows the purity results of crude phycocyanin extraction from Examples 1 and 1-14. During co-fermentation, yeast decomposes polysaccharides to reduce impurities, while *Lactobacillus plantarum* produces acid to inhibit bacterial growth. In Example 1, the fermentation time of yeast and *Lactobacillus plantarum* was balanced and sufficient, resulting in good polysaccharide decomposition and acid production for antibacterial effects, achieving a purity of ≥1.8%. In Comparative Example 6, the reverse fermentation sequence led to insufficient polysaccharide decomposition, resulting in a small amount of residual polysaccharides; in Comparative Example 14, bacterial competition led to the proliferation of bacteria, resulting in a small amount of residual metabolites; the purity of both was slightly lower than that of Example 1. In Comparative Example 1, insufficient yeast pretreatment resulted in a large amount of undecomposed polysaccharides remaining; although the fermentation time of *Lactobacillus plantarum* was extended, it could not degrade the residual polysaccharides, and impurities interfered with the purity reduction. The lower purity of Comparative Example 2 was due to the excessive fermentation of yeast producing small-molecule proteins and other metabolic byproducts, and the short fermentation time of *Lactobacillus plantarum* resulting in insufficient acid production and antibacterial effect, leading to increased cell debris and residual microbial metabolites. The purity of Comparative Examples 3-4, 7, 9-11 and Comparative Examples 12-13, which involved mixed-strain fermentation during single-strain fermentation, was significantly lower than in the examples, indicating that single-strain fermentation and non-specified-strain fermentation resulted in more residual cell debris and a larger amount of microbial metabolites. Comparative Example 5, due to non-selective cell disruption, resulted in a mixture of cell debris, denatured proteins, and crude phycocyanin, with impurities exceeding 40%, achieving a purity only reaching the low-value industrial standard (<1.5%). This purity was not only far lower than the microbial co-fermentation extraction of the examples but also showed no advantage compared to the fermentation extraction of the remaining comparative examples.

[0043] (4) Colony count Colony count reflects the antimicrobial activity of the process. The colony count results for Example 1 and Comparative Examples 1-14 are shown in Table 1. During the co-fermentation process, yeast consumes carbon sources from other microorganisms, while *Lactobacillus plantarum* creates an unsuitable acidic environment for other microorganisms by producing acid to lower the pH, thus achieving dual antimicrobial activity. In Example 1, the balanced fermentation time of the first yeast fermentation and the subsequent fermentation time of *Lactobacillus plantarum* allowed the carbon source consumption and acid production to fully exert their antimicrobial effects, resulting in the lowest colony count and the best antimicrobial effect. In Comparative Examples 1-2, the imbalance between the two fermentation times led to a mismatch between the carbon source consumption / acid production rhythm and the proliferation rhythm of other microorganisms, resulting in insufficient capacity to eliminate already multiplied microorganisms, leading to a higher colony count and poorer antimicrobial effect. In Comparative Example 14, due to competition among microorganisms during co-fermentation, the target microorganism's metabolism was disrupted, its antimicrobial activity decreased, and other microorganisms proliferated, resulting in a higher colony count than the stepwise co-fermentation group. Compared with the single-strain fermentation systems of Examples 3-4, 7, and 9-11, the simple microbial community structure and lack of synergistic antimicrobial mechanisms (yeast rapidly consumes oxygen and nutrients, and *Lactobacillus plantarum* rapidly lowers pH through acid production) result in limited ability to inhibit environmental contaminants. Although the total colony count at the experimental endpoint may be low due to issues such as the activity of the initial inoculated strain, the microenvironmental stability of these fermentation systems is poor, making them more susceptible to contamination by other microorganisms. This is evidenced by the significantly lower and more stable colony count in the synergistic fermentation system of Example 1, which demonstrates the significant advantages of the synergistic fermentation mode of this invention in inhibiting contaminants and ensuring fermentation purity.

[0044] (5) Sugar consumption Sugar consumption is a direct indicator of microbial metabolic intensity. Table 1 directly reflects the sugar consumption results of Comparative Examples 1-2 and Examples 1-3. In co-fermentation, yeast decomposes Spirulina cell wall polysaccharides, a difficult-to-use carbon source, releasing small-molecule sugars, an easily usable carbon source, for use by *Lactobacillus plantarum*. This forms a metabolic coupling from yeast destroying cells to provide carbon sources to *Lactobacillus plantarum* enhancing acid production. Higher sugar consumption indicates more efficient coupling. This sugar consumption process can be verified in the example groups. In the first stage of fermentation in Example 1, yeast efficiently decomposes Spirulina cell wall polysaccharides and exogenous glucose to release small-molecule sugars, which are fully utilized by *Lactobacillus plantarum* in the second stage of fermentation. The metabolic coupling is the most efficient, and the sugar consumption is the highest, corresponding to excellent extraction rate and purity. Short yeast fermentation time leads to insufficient decomposition of cell wall polysaccharides, thus reducing the efficiency of converting difficult-to-use carbon sources into easily usable carbon sources. In addition, the available carbon sources for *Lactobacillus plantarum* are limited, resulting in insufficient metabolic coupling and thus lower sugar consumption in Comparative Example 1 compared to Example 1. The co-fermentation in Comparative Example 2 also had many problems: excessive yeast fermentation consumed some non-carbon source nutrients such as phycocyanin crude protein, and increased metabolic byproducts; the fermentation time of *Lactobacillus plantarum* was too short, resulting in insufficient utilization of readily available carbon sources. Although sugar consumption was high, the proportion of ineffective consumption was large, leading to a failure to increase extraction rate and purity in sync with sugar consumption. Comparative Example 6, with its reversed fermentation sequence, resulted in insufficient initial carbon source decomposition and slightly lower sugar consumption; Comparative Example 14, with its microbial community competition, led to carbon source waste, and sugar consumption was 0.8 g / L lower than Comparative Example 6. Comparative Examples 3-4, 7, and 9-11 all relied solely on single-strain fermentation, resulting in insufficient sugar source decomposition, extremely weak metabolic activity, and extremely low sugar consumption, showing no advantage in either cell disruption or antibacterial activity. Comparative Examples 12-13 involved co-fermentation with non-specified strains, resulting in poor microbial community compatibility, insufficient metabolic coupling, low carbon source utilization efficiency, and sugar consumption lower than in Example 1.

[0045] Examples 1 and Comparative Examples 1-2, 6, and 14 demonstrate the triple advantages of metabolic coupling through the synergistic fermentation of yeast and Lactobacillus plantarum, which complement each other in cell disruption and acid production, create a synergistic environment through nutrient competition and acidification inhibition, and achieve mutual carbon source supply. This results in a comprehensive process with high extraction rate, high purity, excellent safety, and high resource utilization of phycocyanin crude protein, significantly superior to the single-strain fermentation mode of Comparative Examples 3-4. It is an efficient, high-quality, and safe technical approach for extracting phycocyanin crude protein.

[0046] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for extracting crude phycocyanin based on microbial fermentation, characterized in that, Includes the following steps: S1. Dissolve spirulina powder in phosphate buffer, add glucose to obtain an algae powder suspension; S2. Inoculate yeast into the algae powder suspension and culture for 12-24 hours to obtain the first stage fermentation product; S3. Inoculate the first-stage fermentation product with Lactobacillus plantarum and continue fermentation for 24-48 hours to lower the pH of the system. S4. After fermentation, solid-liquid separation is performed to remove the cells and residues. The supernatant is concentrated using an ultrafiltration membrane with a molecular weight cutoff of 50 kDa. The supernatant is then desalted by dialysis or nanofiltration to obtain crude phycocyanin.

2. The extraction method according to claim 1, characterized in that, The ratio of spirulina powder to phosphate buffer is 1g:(20-50)mL.

3. The extraction method according to claim 1, characterized in that, The concentration of glucose in the algal powder suspension is 30-50 g / L.

4. The extraction method according to claim 1, characterized in that, In S2, the fermentation temperature of the yeast is 30-37℃, and the dissolved oxygen is controlled to be no less than 1.5 mg / L.

5. The extraction method according to claim 1, characterized in that, The inoculation amount of the yeast is 1-3 × 10⁻⁶. 6 CFU / mL.

6. The extraction method according to claim 1, characterized in that, In S3, the fermentation temperature of Lactobacillus plantarum is 30-37℃, and the dissolved oxygen is controlled to be no less than 1.5 mg / L.

7. The extraction method according to claim 1, characterized in that, The inoculum size of *Lactobacillus plantarum* is 0.5-1.5 × 10⁻⁶. 6 CFU / mL.

8. The extraction method according to claim 1, characterized in that, In step S4, the pH value of the system is reduced to 5.0-6.

0.

9. The extraction method according to claim 1, characterized in that, The yeast is Kluyveromyces marxianus or Saccharomyces cerevisiae At least one of the following, wherein the *Lactobacillus plantarum* is Lactiplantibacillus plantarum At least one of them.

10. The application of the crude phycocyanin obtained by the extraction method according to any one of claims 1-8, characterized in that, It is used in the preparation of food or cosmetics.

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