A microalgae active ingredient extraction method based on solid-state fermentation enzyme phase state regulation

Abstract

This invention belongs to the field of biomanufacturing and high-value utilization of natural products, specifically relating to a method for extracting active ingredients from microalgae based on phase regulation of solid-state fermentation enzyme systems. The solid-state fermentation product of microalgae is reconstituted at a material-to-liquid ratio of approximately 1:10, a suitable pH is established, and the mixture is incubated at this temperature. The endogenous enzyme system and residual bacterial cells in the fermentation product are used for enzymatic hydrolysis and metabolism, significantly improving the cell wall disruption efficiency of the fermentation substrate. This promotes the efficient release of intracellular active ingredients from the microalgae and further degrades unused carbon sources and macromolecules into small peptides and other active metabolites. An auxiliary method is used: after incubation, the pH of the system is adjusted to induce a phase transition, promoting the flocculation and sedimentation of impurity polysaccharides and cell fragments. This invention not only significantly improves the overall yield of various active ingredients but also achieves extremely simple solid-liquid separation of the extract, possessing high industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of biomanufacturing and high-value utilization of natural products, specifically relating to a method for extracting active ingredients from microalgae based on phase regulation of solid-state fermentation enzyme systems. Background Technology

[0002] Solid-state fermentation technology for microalgae (such as spirulina) has attracted much attention in recent years, as it can effectively enrich bioactive substances such as small peptides, microalgal polysaccharides, fatty acids, and phycocyanin. It has been proven to possess various physiological activities, including antioxidant, anti-inflammatory, and immunomodulatory effects. However, current industrial extraction processes generally suffer from technical bottlenecks such as insufficient substrate utilization and loss of active ingredients.

[0003] Traditional extraction methods typically involve adding a large amount of water and heating the solution to above 80°C after solid-state fermentation for inactivation and extraction. This process of inactivation followed by extraction has several technical drawbacks: First, solid-state fermentation is limited by mass transfer resistance, and the substrate often contains microalgal cells that have not been completely destroyed and carbon sources that have not been fully consumed. Direct high-temperature inactivation causes the microorganisms and enzymes that could have continued to function to instantly lose their activity, resulting in extremely low efficiency in the breakdown and utilization of the fermentation substrate. This not only hinders the dissolution of active ingredients within the microalgal cells but also wastes substrate macromolecules that cannot be further enzymatically broken down into smaller peptides. Second, traditional aqueous extracts contain a large amount of incompletely degraded extracellular polysaccharides and cell wall fragments, resulting in extremely high viscosity. Subsequent solid-liquid separation is extremely difficult, and traditional centrifugation or filtration is prone to clogging, limiting the efficiency of industrial production.

[0004] Therefore, breaking through existing technological bottlenecks and developing a new green extraction method that can avoid enzyme inactivation at high temperatures, reduce the viscosity of the extract from the source, and achieve efficient and synergistic recovery of the target product has significant industrial importance and market value. Summary of the Invention

[0005] To address the above problems, this invention provides a method for extracting active ingredients from microalgae based on phase regulation of solid-state fermentation enzyme systems, comprising the following steps:

[0006] (1) Resolution and phase expansion: The solid fermentation product of microalgae is mixed with the aqueous medium at a material-liquid ratio of 1:8 to 1:12 to obtain a liquid suspension system;

[0007] (2) Incubation and deep utilization: The liquid suspension system described in step (1) is incubated at a constant temperature of 45℃~55℃ for 40~90 minutes;

[0008] (3) pH adjustment and self-flocculation: After incubation, an acidic regulator was added to the system to adjust the pH value of the system to 4.2~4.8, and the system was allowed to stand to obtain flocculated precipitate;

[0009] (4) Solid-liquid separation and collection: Separate and remove the flocculated precipitate generated in step (3) and collect the supernatant rich in active ingredients.

[0010] Furthermore, the material-to-liquid ratio in step (1) is 1:10.

[0011] Furthermore, the microalgae solid fermentation product mentioned in step (1) is obtained by fermenting microalgae biomass and excipients with probiotics; the probiotics include at least one of Bacillus, Lactobacillus or Saccharomyces.

[0012] Furthermore, the settling time in step (3) is 30 to 60 minutes.

[0013] The present invention also provides a microalgae active ingredient, which is obtained by any of the above extraction methods.

[0014] The present invention also provides the application of the above-mentioned microalgae active ingredients in the preparation of pharmaceuticals that enhance immunity, have antioxidant or anti-inflammatory effects.

[0015] The present invention has the following beneficial effects:

[0016] This invention achieves the following through phase regulation of solid-state fermentation enzyme systems:

[0017] The dissolution rate of microalgae's inherent active substances (such as phycocyanin) is significantly improved, while the further degradation of macromolecular proteins increases the extraction rate of small peptides by more than 16.3% and reduces the total sugar content by more than 94%. The auxiliary pH phase control method induces self-flocculation, reducing the original turbid extract with OD750>0.7 to below 0.3, which can improve the filter membrane flux and thus significantly reduce the downstream purification cost.

[0018] Based on the above findings, this invention proposes the following applications for solid-state fermentation phase-controlled enzyme systems:

[0019] (1) Application in the preparation of microalgae active extracts rich in small peptides;

[0020] (2) Application in the preparation of low-viscosity, easily separable microalgal fermentation extracts;

[0021] (3) Application in simultaneously improving the release rate of intracellular proteins in microalgae and the clarity of the extract. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The process principle and overall flow diagram of this invention.

[0024] Figure 2 : Phycocyanin content before enzyme inactivation in each example.

[0025] Figure 3 Comparison of physical photos of the extracts obtained in each embodiment.

[0026] Figure 4 : Analysis chart of total sugar content in the supernatant of each example.

[0027] Figure 5 : Analysis chart of small peptide content in supernatant of each example. Detailed Implementation

[0028] Various exemplary embodiments of the present invention are now described in detail. Unless otherwise specified, the methods used in the embodiments are conventional methods, and the reagents used are commercially available reagents or reagents prepared using conventional methods. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and embodiments of the present invention.

[0029] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0031] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0032] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0033] 1. Experimental materials

[0034] Spirulina powder, starch, Bacillus subtilis (100 billion CFU / g), Lactobacillus plantarum (10 billion CFU / g)

[0035] 2. Experimental Methods

[0036] Example 1: 32.32 g of spirulina powder, 30.166 g of starch, and 37.5 mL of water were mixed. 0.1 g of Bacillus subtilis and 0.1 g of Lactobacillus plantarum were inoculated, and solid-state fermentation was carried out at 37°C for 12 hours. 10.0 g of the solid-state fermentation material was weighed and placed in a 250 mL Erlenmeyer flask. 100 mL of deionized water was added, and the mixture was stirred evenly with a magnetic stirrer. The pH of the system was adjusted to 5.7 with 1M HCl or 1M NaOH, while the system temperature was raised to 50°C in a water bath. The mixture was stirred at 50°C and pH 5.7 for 60 minutes. The pH of the mixture was adjusted to 4.5 with 1M HCl, and stirring was continued for 5 minutes. Then, stirring was stopped, and the mixture was allowed to stand for 1 hour. The flocculation and sedimentation were observed and recorded. The supernatant after standing was heated in a 90°C water bath for 10 minutes, and then rapidly cooled to room temperature. The enzyme-inactivated supernatant was filtered to obtain a clear extract for subsequent determination.

[0037] Example 2: Weigh 10.0 g of the same batch of solid-state fermentation material and place it in a 250 mL Erlenmeyer flask. Follow the same extraction process as described in this invention (i.e., 50°C, pH 5.7, recovery time 60 minutes). Then, without adjusting the pH, filter directly and collect the supernatant.

[0038] Comparative Example 1: Weigh 10.0 g of the same batch of solid-state fermentation material and place it in a 250 mL Erlenmeyer flask. Add 100 mL of deionized water, stir well, adjust the pH to 5.7, and place directly in a 90℃ water bath for immediate high-temperature enzyme inactivation for 10 min. Then, adjust the pH to 4.5, stir for 5 minutes, and let stand for 1 hour. Filter the extract as described in the same steps for analysis.

[0039] Phycocyanin content determination: Spectrophotometry was used. In each example, the absorbance of the supernatant at 620 nm and 652 nm was measured before enzyme inactivation, and the absorbance was calculated according to the following formula.

[0040] Phycocyanin (mg / mL) = 0.187A 620 -0.08A 652 (1)

[0041] Allophycocyanin (mg / mL) = 0.196A 652 -0.041A 620 (2)

[0042] Determination of small peptide content: Dissolve 3 mL of enzyme-inactivated supernatant in 7.5 mL of 15% trichloroacetic acid solution, let stand for 5 min, centrifuge (4000 rpm, 10 min), take all supernatant, and then use the BCA method to determine the content of soluble nitrogen (i.e. soluble small peptides) in the solution with bovine serum albumin as standard.

[0043] Total sugar content determination: Using glucose as the standard, the total sugar content in the supernatant after enzyme inactivation was determined by the phenol-sulfuric acid method. The glucose standard curve was y = 0.01078x + 0.05594 (R²). 2 =0.994).

[0044] Turbidity determination: Take 40 mL of solutions from Examples 1 and 3 before enzyme inactivation, and 40 mL of solution from Control Example 1 before filtration, and measure the OD values ​​of each group at 0 min, 30 min, and 60 min. 750 The value.

[0045] 3. Experimental Results

[0046] 3.1 Effect of different extraction processes on phycocyanin content

[0047] By comparing the phycocyanin content of the three groups, this invention shows that, as Figure 2 As shown, the phycocyanin content in Examples 1 and 2 reached 1.50 mg / mL and 1.71 mg / mL, respectively, significantly higher than the 0.82 mg / mL in Control Example 1. Control Example 1 represents the initial release state after reconstitution of the solid-state fermentation product without any heat treatment; its protein content is only the portion naturally released by the broken cells in the fermentation product. In Example 2, after incubation at 50°C for 60 min, the phycocyanin content increased to 2.08 times that of Control Example 1, and the allophycocyanin content also increased from 0.27 mg / mL to 0.53 mg / mL. This result fully demonstrates that the 50°C incubation stage described in this invention can effectively activate the residual endogenous enzyme system in the fermentation product, further breaking down the microalgal cell wall structure through enzymatic hydrolysis, thereby significantly promoting the release of intracellular proteins.

[0048] 3.2 Effects of different extraction processes on the solid-liquid separation efficiency of microalgal fermentation products

[0049] This invention measured the optical density (OD) of the extract at 750 nm under three treatment conditions. 750 As shown in Table 2, the turbidity data show that the three OD values ​​in the initial state are... 750Similar to each other (approximately 1.5–1.6), significant differences were observed after standing for 60 min. The turbidity of Example 2 (heat incubation only, no pH adjustment) remained as high as 0.781, indicating that although 50°C incubation promoted protein release, a large amount of unsettled cell debris and impurity polysaccharides remained in the system, preventing spontaneous flocculation. The turbidity of Control Example 1 (enzyme inactivation + pH adjustment) after standing was 0.333, significantly lower than Example 2, confirming that pH adjustment to 4.5 effectively induced the isoelectric point precipitation of cell wall debris and large impurity molecules. The turbidity of Example 1 (heat incubation + pH adjustment) further decreased to 0.287, lower than Control Example 1, indicating that after 50°C incubation, large molecules in the fermentation product were partially degraded into smaller fragments. These fragments formed denser flocs under pH induction, resulting in better sedimentation. In Comparative Example 1, the reduction in turbidity mainly stemmed from the combined effect of thermal denaturation and aggregation of macromolecules after inactivation and pH flocculation. However, due to the lack of an enzymatic hydrolysis step, the macromolecules were not fully degraded, resulting in a slightly less effective flocculation than in Example 1. Analysis of the phycocyanin data revealed that the flocculation condition at pH 4.5 may have caused some phycocyanin to co-precipitate with impurities, while the bulk phycocyanin remained. This selective precipitation effect helps improve the relative purity of the target active ingredient, small peptides, in the final product.

[0050]

[0051] 3.3 Effect of different extraction processes on total sugar content

[0052] The total sugar content of the supernatant from each treatment was determined using the phenol-sulfuric acid method, such as... Figure 4 As shown, the total sugar content of Examples 1 and 2 was 11.5 mg / mL and 10.9 mg / mL, respectively, while that of Control Example 1 was 22.9 mg / mL and 22.4 mg / mL after pH adjustment. This indicates that the 50℃ incubation stage effectively activated the polysaccharide-degrading enzyme system in the fermentation product, further degrading the carbon source and cell wall polysaccharides remaining from solid-state fermentation into small-molecule oligosaccharides or monosaccharides. Some of these may be metabolized and utilized by the surviving bacteria, while others exist in the system as soluble small molecules. In contrast, Control Example 1 suffered from polysaccharide-degrading enzyme inactivation due to high-temperature inactivation, and the large-molecule polysaccharides were not effectively degraded, resulting in a significantly higher total sugar content than the previous two groups. This result fully demonstrates that the enzymatic hydrolysis stage described in this invention not only promotes protein release but also achieves deep degradation of polysaccharides, laying the foundation for subsequent reduction of extract viscosity and improvement of flocculation effect.

[0053] 3.4 Effect of different extraction processes on small peptide content

[0054] The content of small peptides in the supernatant of each treatment was determined by trichloroacetic acid precipitation combined with the BCA method. The content of small peptides in Example 1 reached 606.57 μg / mL, which was significantly higher than that in Control Example 1 before and after pH adjustment (521.40 μg / mL and 526.49 μg / mL, respectively), representing an increase of 16.3%. Figure 5 This indicates that incubation at 50°C not only promotes the release of intracellular proteins, but more importantly, it further enzymatically degrades the released proteins and residual macromolecular proteins in the fermentation product into highly bioactive small peptides. This process is a key step in achieving high-value utilization of microalgal proteins in this invention. In Control Example 1, the high-temperature inactivation led to protease inactivation, and the macromolecular proteins could not be deeply degraded, resulting in a significantly lower amount of small peptides generated compared to Example 1.

[0055] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for extracting active ingredients from microalgae based on phase regulation of solid-state fermentation enzyme systems, characterized in that, Includes the following steps: (1) Resolution and phase expansion: The solid fermentation product of microalgae is mixed with the aqueous medium at a material-liquid ratio of 1:8 to 1:12 to obtain a liquid suspension system; (2) Incubation and deep utilization: The liquid suspension system described in step (1) is incubated at a constant temperature of 45℃~55℃ for 40~90 minutes; (3) pH adjustment and self-flocculation: After incubation, an acidic regulator was added to the system to adjust the pH value of the system to 4.2~4.8, and the system was allowed to stand to obtain flocculated precipitate; (4) Solid-liquid separation and collection: Separate and remove the flocculated precipitate generated in step (3) and collect the supernatant rich in active ingredients.

2. The extraction method according to claim 1, characterized in that, The material-liquid ratio in step (1) is 1:

10.

3. The extraction method according to claim 1, characterized in that, The microalgae solid fermentation product mentioned in step (1) is obtained by fermenting microalgae biomass and excipients with probiotics; the probiotics include at least one of Bacillus, Lactobacillus or Saccharomyces.

4. The extraction method according to claim 1, characterized in that, The settling time mentioned in step (3) is 30 to 60 minutes.

5. A microalgae active ingredient, characterized in that, It is prepared by the extraction method described in any one of claims 1-4.

6. The use of the microalgae active ingredient as described in claim 5 in the preparation of pharmaceuticals that enhance immunity, have antioxidant or anti-inflammatory effects.

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