Preparation method of chlorella pyrenoidosa fermented beverage

Abstract

The application discloses a preparation method of a chlorella pyrenoidosa fermentation beverage and belongs to the technical field of microorganisms. The preparation method comprises the following steps: S1, uniformly mixing chlorella pyrenoidosa powder with pure water, rehydrating, and obtaining rehydrated algae liquid; S2, adding an enzyme into the rehydrated algae liquid; after enzymolysis, the enzyme is inactivated, the filtrate is filtered and collected, and an enzymolysis liquid is obtained; S3, inoculating the activated strain into the enzymolysis liquid, oscillating and culturing, filtering and collecting the filtrate, and obtaining the chlorella pyrenoidosa fermentation beverage. The method combining neutral protease enzymolysis and lactobacillus plantarum fermentation is adopted, the bitterness of the chlorella pyrenoidosa beverage can be effectively improved without adding a chemical masking agent, and meanwhile, the nutritional value and functional characteristics of the chlorella pyrenoidosa beverage are well reserved.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a method for preparing a fermented beverage made from Chlorella proteoglycans. Background Technology

[0002] With increasing attention paid to functional foods, plant-based raw materials rich in high biological value have become a research hotspot in the food industry. Chlorella proteoglycans, as one of the microalgae with high nutritional value, has received widespread attention in recent years. Chlorella is rich in protein, with a protein content reaching over 60% of its cell dry weight, and contains various essential amino acids, minerals, and natural pigments. Its fatty acids are mainly unsaturated fatty acids, possessing high physiological functional value. However, currently, the microalgae raw materials used on a large scale in the food industry are mostly Spirulina (a type of cyanobacteria), and the research and development of Chlorella proteoglycans-related products are still in the stage of gradual enrichment and improvement.

[0003] In the research and development of Chlorella, protein hydrolysates have attracted widespread attention due to their excellent nutritional value and potential physiological functions. However, many studies have shown that the hydrolysis of algal proteins often produces bitter peptides, which severely affect its sensory quality, thereby reducing consumer acceptance and becoming a major obstacle limiting its application in the food industry. Therefore, how to improve the sensory quality of Chlorella fermented beverages without using masking agents has become an urgent problem to be solved. Summary of the Invention

[0004] To address the aforementioned problems in existing technologies, this invention provides a method for preparing a fermented beverage made from Chlorella proteoglycans. This invention employs a process combining enzymatic hydrolysis and fermentation, effectively improving the bitterness of the Chlorella proteoglycans beverage without the need for chemical masking agents.

[0005] The technical solution of the present invention is as follows: The first aspect of this invention protects a method for preparing a fermented beverage from Chlorella vulgaris, comprising the following steps: S1. Mix Chlorella pyrenoidosa powder with pure water until homogeneous, then rehydrate to obtain rehydrated algal solution; S2. Add enzyme to the rehydrated algae solution; after enzymatic hydrolysis, inactivate the enzyme, filter and collect the filtrate to obtain the enzymatic hydrolysate; S3. Inoculate the activated strain into the enzymatic hydrolysate, shake and culture, filter and collect the filtrate to obtain Chlorella proteoglycans fermented beverage.

[0006] Preferably, in step S1, the ratio of the protein-nucleated Chlorella powder to pure water is 1:(10~30).

[0007] Preferably, in step S2, an enzyme is added to the rehydrated algae solution at 35~39°C, with an enzyme-to-solvent ratio of 4~5%.

[0008] Preferably, in step S2, the enzyme is a neutral protease.

[0009] Preferably, in step S2, the enzymatic hydrolysis temperature is 35~39℃, the pH is 6.5~7.0, and the time is 4~6h.

[0010] Preferably, in step S2, the enzyme inactivation temperature is 95~115℃ and the time is 5~15min.

[0011] Preferably, in step S3, the strain is Lactobacillus plantarum L2, with accession number CGMCC No.37217, which was deposited at the China General Microbiological Culture Collection Center on December 26, 2025.

[0012] Preferably, in step S3, the inoculation amount is 4-5% (w / v).

[0013] Preferably, in step S3, the temperature of the oscillation culture is 35~39℃ and the time is 24~48h.

[0014] The second aspect of this invention protects a fermented beverage of Chlorella vulgaris prepared by the method described in the first aspect.

[0015] The beneficial technical effects of this invention are as follows: This invention employs a method combining neutral protease hydrolysis and Lactobacillus plantarum fermentation. Without adding chemical masking agents, it effectively improves the bitterness of Chlorella proteoglycans beverages, resulting in a more harmonious flavor and a smoother taste. Furthermore, while improving flavor, this invention effectively preserves the nutritional value and functional properties of Chlorella proteoglycans beverages, leading to superior performance in antioxidant activity and other aspects of the fermented beverage.

[0016] The *Lactobacillus plantarum* CGMCC No. 37217 used in this invention exhibits excellent growth and acid-producing characteristics, which are beneficial for the formation of flavor compounds and the stability of product quality during fermentation. Furthermore, it can serve as a safe strain for food fermentation; the entire preparation process requires no additional chemical substances, ensuring safety, health, and a rational process with promising application prospects. Attached Figure Description

[0017] Figure 1 A is the SEM image of the rehydrated algal solution in Comparative Example 27. Figure 1 B is a SEM image of the enzymatic hydrolysate prepared in Comparative Example 28. Figure 1 C is a SEM image of the Chlorella proteoglycans fermented beverage prepared in Comparative Example 26. Figure 1 D is a SEM image of the Chlorella proteoglycans fermented beverage prepared in Example 1.

[0018] Figure 2 The bitter peptide release content is for Examples 1, 26, 27 and 28.

[0019] Figure 3 Fourier transform infrared spectra of Example 1, Comparative Examples 26, 27 and 28.

[0020] Figure 4 The bar chart shows the differential metabolites in comparative examples 27 and 28.

[0021] Figure 5 The bar chart shows the differences in metabolites between Example 1 and Comparative Example 27.

[0022] Figure 6 The bar chart shows the differences in metabolites between Example 1 and Comparative Example 28.

[0023] Figure 7 The bar chart shows the difference in metabolites between Example 1 and Comparative Example 26.

[0024] Figure 8 The pH values ​​for Example 1, Comparative Examples 26, 27, and 28 are given.

[0025] Figure 9 The polyphenol content is for Example 1, Comparative Examples 26, 27 and 28.

[0026] Figure 10 The DPPH removal rates are for Examples 1, 26, 27, and 28. Detailed Implementation

[0027] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0028] In some implementations, the activity of the neutral protease is 5-35 U / g.

[0029] The L2 strain of *Lactobacillus plantarum* in this invention is classified as: *Lactobacillus plantarum* Lactobacillus plantarum It was deposited on December 26, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37217, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0030] The strains used in the specific embodiments of this invention are *Lactobacillus plantarum* L1 and *Lactobacillus salivarius* (…). Ligilactobacillus salivarius ), fermenting lactobacillus ( Limosilactobacillus fermentum All of these were obtained from the laboratory of Ningbo University, and were isolated and purified from fermented vegetables. The specific steps are as follows: After removing yellow and rotten leaves and muddy parts from the roots of fresh vegetables, they were sun-dried for one day. Then, in a large plastic bucket, 15% salt was added according to the weight of the raw materials for dehydration for two days, with occasional turning over until the overall internal and external osmotic pressure reached equilibrium. The dehydrated vegetables were then divided into small plastic buckets, compacted, covered with film, and water-sealed. They were then naturally fermented at approximately 25°C for 25 days. After fermentation, samples were collected and frozen at -80°C for later use. For separation and purification, 10g (or 10mL) of the fermented vegetable sample was added to 90mL of sterile physiological saline and mixed thoroughly to prepare 10... -1 The initial suspension was then serially diluted 10-fold to 10. -6 ~10 -8 MRS medium was used as the isolation medium. 1 mL of appropriately diluted bacterial suspension was added to a sterile Petri dish, and MRS agar medium at approximately 45°C was poured in. The dish was quickly shaken and allowed to solidify. The Petri dishes were then anaerobically incubated at 37°C for 48–72 h. After incubation, the plates were observed, and typical colonies that were milky white or grayish-white, round, with neat edges and smooth surfaces were selected. Purification and identification were then performed. Single colonies were picked up with an inoculation loop and streaked onto fresh MRS plates. This streaking purification was repeated 2–3 times until the colony morphology was consistent. After confirming the acquisition of a pure culture, Gram staining and microscopic examination were performed. Lactic acid bacteria should be Gram-positive bacilli or cocci. A catalase test was also performed; adding 3% hydrogen peroxide solution to the colonies resulted in a negative result if no bubbles were produced. A sugar fermentation test was also performed to detect its metabolic characteristics for glucose, lactose, etc. Finally, the bacterial suspension was mixed with 30% glycerol at a 1:1 ratio and stored at -80°C.

[0031] The MRS culture medium comprises: 10.0 g peptone, 10.0 g beef extract, 5.0 g yeast extract, 20.0 g glucose, 2.0 g dipotassium hydrogen phosphate, 2.0 g triammonium citrate, 5.0 g sodium acetate, 0.2 g magnesium sulfate (MgSO4·7H2O), 0.05 g manganese sulfate (MnSO4·4H2O), and 1.0 g Tween 80, with the pH adjusted to 6.2±0.2. During preparation, each component is dissolved in distilled water, heated to boiling to dissolve, the pH adjusted, dispensed, and autoclaved at 121°C for 15 minutes before use. If a solid culture medium is required, an additional 15.0 g / L of agar powder should be added.

[0032] In the specific embodiments of the present invention: the neutral protease used can be obtained by purchase, namely Shanghai Yuanye Biotechnology Co., Ltd. S10013; the acidic protease used can be obtained by purchase, namely Shanghai Yuanye Biotechnology Co., Ltd. S10012; the alkaline protease used can be obtained by purchase, namely Shanghai Yuanye Biotechnology Co., Ltd. S10154; the papain used can be obtained by purchase, namely Shanghai Yuanye Biotechnology Co., Ltd. S10011; the pepsin used can be obtained by purchase, namely Shanghai Yuanye Biotechnology Co., Ltd. S10027; and the trypsin used can be obtained by purchase, namely Shanghai Yuanye Biotechnology Co., Ltd. S10032.

[0033] Example 1 A method for preparing a fermented beverage from Chlorella vulgaris includes the following steps: S1. Weigh 1 g of Chlorella pyrenoidosa powder, mix the Chlorella pyrenoidosa powder with pure water at a liquid-to-solid ratio (g / ml) of 1:10, let it stand overnight to rehydrate, and obtain the rehydrated algae solution.

[0034] S2. At 37℃, add neutral protease to the rehydrated algae solution at an enzyme-to-base ratio of 4%; after shaking and hydrolyzing for 5 h at 37℃ and pH 7, inactivate the enzyme at 100℃ for 10 min, filter and collect the filtrate to obtain the hydrolysate.

[0035] S3 and activated Lactobacillus plantarum L2 were activated and cultured in MRS medium for 24 h to the logarithmic phase. They were then transferred to the enzyme hydrolysate at a 5% (w / v) inoculation rate and cultured at 37℃ with shaking for 48 h. The filtrate was filtered and collected to obtain the Chlorella proteoglycans fermented beverage.

[0036] Example 2 A method for preparing a fermented beverage of Chlorella proteoglycans is basically the same as that in Example 1, except that: in step S1, the liquid-to-solid ratio (g / ml) is 1:20; in step S3, the inoculum amount is 4% (w / v), and the culture is carried out at 37°C with shaking for 24 hours.

[0037] Example 3 A method for preparing a fermented beverage of Chlorella proteoglycans is basically the same as that in Example 1, except that: in step S1, the liquid-to-solid ratio (g / ml) is 1:30; in step S3, the inoculum amount is 4% (w / v), and the culture is carried out at 37°C with shaking for 24 hours.

[0038] Example 4 A method for preparing a fermented beverage of Chlorella proteoglycans is basically the same as that in Example 1, except that: in step S1, the liquid-to-solid ratio (g / ml) is 1:20; and in step S3, the inoculum amount is 4% (w / v).

[0039] Example 5 A method for preparing a fermented beverage of Chlorella proteoglycans is basically the same as that in Example 1, except that: in step S1, the liquid-to-solid ratio (g / ml) is 1:20; and in step S3, the beverage is cultured at 37°C with shaking for 24 hours.

[0040] Example 6 A method for preparing a fermented beverage of Chlorella proteinensis is basically the same as that in Example 1, except that in step S1, the liquid-to-solid ratio (g / ml) is 1:20.

[0041] Comparative Example 1 A method for preparing a fermented beverage of Chlorella proteoglycans is basically the same as that in Example 1, except that: in step S2, the enzyme used is acidic protease; and in step S3, the strain used is Lactobacillus plantarum L1.

[0042] Comparative Example 2 A method for preparing a fermented beverage of Chlorella proteinensis is basically the same as that in Example 1, except that the enzyme used in step S2 is an acidic protease.

[0043] Comparative Example 3 A method for preparing a fermented beverage of Chlorella proteinensis is basically the same as that in Example 1, except that: in step S2, the enzyme used is acidic protease; and in step S3, the strain used is Lactobacillus salivarius.

[0044] Comparative Example 4 A method for preparing a fermented beverage of Chlorella proteinensis is basically the same as that in Example 1, except that: in step S2, the enzyme used is acidic protease; and in step S3, the strain used is Lactobacillus fermentum.

[0045] Comparative Example 5 A method for preparing a fermented beverage of Chlorella proteinensis is basically the same as that in Example 1, except that in step S3, the strain used is Lactobacillus plantarum L1.

[0046] Comparative Example 6 A method for preparing a fermented beverage of Chlorella proteinensis is basically the same as that in Example 1, except that the strain used in step S3 is Lactobacillus salivarius.

[0047] Comparative Example 7 A method for preparing a fermented beverage of Chlorella proteinensis is basically the same as that in Example 1, except that in step S3, the strain used is Lactobacillus fermentans.

[0048] Comparative Example 8 A method for preparing a fermented beverage of Chlorella proteinensis is basically the same as that in Example 1, except that: in step S2, the enzyme used is alkaline protease; and in step S3, the strain used is Lactobacillus plantarum L1.

[0049] Comparative Example 9 A method for preparing a fermented beverage of Chlorella proteinensis is basically the same as that in Example 1, except that the enzyme used in step S2 is alkaline protease.

[0050] Comparative Example 10 A method for preparing a fermented beverage of Chlorella proteinensis is basically the same as that in Example 1, except that: in step S2, the enzyme used is alkaline protease; and in step S3, the strain used is Lactobacillus salivarius.

[0051] Comparative Example 11 A method for preparing a fermented beverage of Chlorella proteinensis is basically the same as that in Example 1, except that: in step S2, the enzyme used is alkaline protease; and in step S3, the strain used is Lactobacillus fermentum.

[0052] Comparative Example 12 A method for preparing a fermented beverage of Chlorella proteinensis is basically the same as that in Example 1, except that: in step S2, the enzyme used is papain; and in step S3, the strain used is Lactobacillus plantarum L1.

[0053] Comparative Example 13 A method for preparing a fermented beverage of Chlorella proteinensis is basically the same as that in Example 1, except that the enzyme used in step S2 is papain.

[0054] Comparative Example 14 A method for preparing a fermented beverage of Chlorella proteinensis is basically the same as that in Example 1, except that: in step S2, the enzyme used is papain; and in step S3, the strain used is Lactobacillus salivarius.

[0055] Comparative Example 15 A method for preparing a fermented beverage of Chlorella proteinensis is basically the same as that in Example 1, except that: in step S2, the enzyme used is papain; and in step S3, the strain used is Lactobacillus fermentum.

[0056] Comparative Example 16 A method for preparing a fermented beverage of Chlorella proteinensis is basically the same as that in Example 1, except that: in step S2, the enzyme used is pepsin; and in step S3, the strain used is Lactobacillus plantarum L1.

[0057] Comparative Example 17 A method for preparing a fermented beverage of Chlorella proteinensis is basically the same as that in Example 1, except that the enzyme used in step S2 is pepsin.

[0058] Comparative Example 18 A method for preparing a fermented beverage of Chlorella proteinensis is basically the same as that in Example 1, except that: in step S2, the enzyme used is pepsin; and in step S3, the strain used is Lactobacillus salivarius.

[0059] Comparative Example 19 A method for preparing a fermented beverage of Chlorella proteinensis is basically the same as that in Example 1, except that: in step S2, the enzyme used is pepsin; and in step S3, the strain used is Lactobacillus fermentum.

[0060] Comparative Example 20 A method for preparing a fermented beverage of Chlorella proteoglycans is basically the same as that in Example 1, except that: in step S2, the enzyme used is trypsin; and in step S3, the strain used is Lactobacillus plantarum L1.

[0061] Comparative Example 21 A method for preparing a fermented beverage of Chlorella proteinensis is basically the same as that in Example 1, except that the enzyme used in step S2 is trypsin.

[0062] Comparative Example 22 A method for preparing a fermented beverage of Chlorella proteinensis is basically the same as that in Example 1, except that: in step S2, the enzyme used is trypsin; and in step S3, the strain used is Lactobacillus salivarius.

[0063] Comparative Example 23 A method for preparing a fermented beverage of Chlorella proteinensis is basically the same as that in Example 1, except that: in step S2, the enzyme used is trypsin; and in step S3, the strain used is Lactobacillus fermentum.

[0064] Comparative Example 24 A method for preparing a fermented beverage of Chlorella proteoglycans is basically the same as that in Example 1, except that: in step S1, the liquid-to-solid ratio (g / ml) is 1:20; in step S3, the inoculum amount is 4% (w / v), and the culture is carried out at 37°C with shaking for 72 hours.

[0065] Comparative Example 25 A method for preparing a fermented beverage of Chlorella proteoglycans is basically the same as that in Example 1, except that: in step S1, the liquid-to-solid ratio (g / ml) is 1:20; in step S3, the inoculum amount is 3% (w / v), and the culture is carried out at 37°C with shaking for 24 hours.

[0066] Comparative Example 26 A method for preparing a fermented beverage from Chlorella vulgaris includes the following steps: S1. Weigh 1 g of Chlorella pyrenoidosa powder, mix the Chlorella pyrenoidosa powder with pure water at a liquid-to-solid ratio (g / ml) of 1:10, let it stand overnight to rehydrate, and obtain the rehydrated algae solution.

[0067] S2 and activated Lactobacillus plantarum L1 were activated and cultured in MRS medium for 24 h to the logarithmic phase. They were then transferred to the rehydrated algal solution at a 5% (w / v) inoculation rate and cultured at 37℃ with shaking for 48 h. The solution was filtered and the filtrate was collected to obtain the Chlorella proteoglycans fermented beverage.

[0068] Comparative Example 27 Weigh 1 g of Chlorella pyrenoidosa powder, mix the Chlorella pyrenoidosa powder with pure water at a liquid-to-solid ratio (g / ml) of 1:10, let it stand overnight to rehydrate, and obtain the rehydrated algae solution.

[0069] Comparative Example 28 Weigh 1 g of Chlorella pyrenoidosa powder and mix it with pure water at a liquid-to-solid ratio (g / ml) of 1:10. Let it stand overnight to rehydrate and obtain a rehydrated algal solution. Add neutral protease to the rehydrated algal solution at 37℃ with an enzyme-to-solid ratio of 4%. After enzymatic hydrolysis at 37℃ and pH 7 for 5 h with shaking, inactivate the enzyme at 100℃ for 10 min, filter and collect the filtrate to obtain the enzymatic hydrolysate.

[0070] Test case Test Example 1 The peptide content, protein hydrolysis degree, surface hydrophobicity, enzyme production analysis, and viable bacterial count in Example 1 and Comparative Examples 1-23 were determined using the following methods.

[0071] The peptide content refers to the peptides contained in Chlorella vulgaris beverages. Hydrolyzing long-chain proteins into short-chain peptides disrupts their structure and surface hydrophobic groups, thus helping to reduce bitterness. Studies have shown that short peptides with a molecular weight less than 1000 Da have relatively lower bitterness. Therefore, detecting the peptide content in a sample can determine whether the protein has been degraded into short-chain peptides, and thus evaluate the effectiveness of the debittering treatment.

[0072] The degree of protein hydrolysis refers to the amino acid content in Chlorella protein-rich beverages. After hydrolysis, proteins are gradually converted from large protein molecules into smaller peptides and amino acids. Amino acids have lower or no bitterness and can improve the flavor of the product to some extent. Therefore, measuring the free amino acid content can help determine the degree of protein hydrolysis and the effectiveness of debittering.

[0073] Surface hydrophobicity refers to the content and distribution of nonpolar groups (such as hydrophobic amino acid residues) exposed on the surface of a protein molecule in a solvent (usually water). These nonpolar groups mainly include hydrophobic amino acid residues. In Chlorella protein-based beverages, protein surface hydrophobicity can be used to characterize the degree of exposure of hydrophobic groups in the protein molecule. Generally, higher surface hydrophobicity indicates more exposed hydrophobic amino acid residues on the protein molecule's surface, corresponding to a more pronounced bitter taste.

[0074] Enzyme production analysis refers to the enzymatic activity of endogenous enzymes produced by the microbial strain in hydrolyzing proteins, used to characterize the strain's ability to enzymatically degrade proteins. In this invention, the endogenous enzymes produced by the microbial strain can act on protein substrates, causing the hydrolysis of large protein molecules into small peptides or free amino acids, thereby reducing the bitterness caused by proteins and their hydrophobic fragments. Therefore, by detecting the enzyme production capacity of the microbial strain, it can be determined whether the strain can effectively degrade proteins, and its debittering effect can be evaluated accordingly.

[0075] The viable count refers to the growth of a bacterial strain within the same culture time. In this invention, lactic acid bacteria not only produce acid during their growth but also secrete proteases to hydrolyze proteins. Generally, a higher viable count within a shorter culture time indicates more vigorous bacterial growth and metabolism, and stronger acid production and protease secretion capabilities, thereby promoting protein degradation, improving beverage flavor, and reducing bitterness.

[0076] 1. The method for determining polypeptide content is as follows: (1) Preparation of biuret reagent Weigh 0.75 g of copper acetate crystals and dissolve them in 100 mL of ultrapure water. Add 2.25 g of potassium sodium tartrate and 1.25 g of potassium iodide. Stir until completely dissolved, then add 25 mL of 6 mol / L NaOH solution while stirring. Make up the volume to 250 mL with ultrapure water and store in a sealed container.

[0077] (2) Drawing the standard curve Accurately weigh 0.5 g of bovine serum albumin (BSAL) and dissolve it in 0.05 mol / L potassium hydroxide solution, stirring constantly until dissolved. Then, bring the volume to 100 mL to prepare a protein standard solution (5.0 mg / mL). Add the solutions in the order listed in Table 1. Use the tube without BSAL standard as a blank tube. Measure the absorbance at 540 nm. Repeat each sample three times and take the average. Plot a curve with absorbance on the ordinate and protein concentration (mg / mL) on the abscissa.

[0078] Table 1. Biuret Standard Protein Curve

[0079] (3) Determination of peptide content in samples Mix 3 mL of the test sample (fermented Chlorella proteoglycans beverage) with 2 mL of water, add 15 mL of biuret reagent, mix thoroughly, and then react under the same conditions as those used to plot the standard curve. Measure the absorbance at 540 nm. Each sample was measured three times, and the average value was taken. The peptide content in the sample was then calculated based on the standard curve.

[0080] 2. The method for detecting the degree of protein hydrolysis is as follows: A standard curve was plotted using glycine as the standard sample. Glycine standard solutions with concentrations of 0 μmol / L, 40 μmol / L, and 80 μmol / L were prepared. 2 mL of each standard solution was pipetted into a clean test tube, 1 mL of ninhydrin colorimetric reagent was added, and the mixture was thoroughly mixed. The tubes were then incubated in a boiling water bath for 15 min, cooled with cold water, and 5 mL of 40% ethanol was added to each tube. The mixture was thoroughly mixed and allowed to stand for 15 min. The absorbance was measured at 570 nm. A 0 μmol / mL glycine solution was used as a blank control. The absorbance was measured using OD... 570 A standard curve of glycine concentration is obtained by plotting glycine concentration (μmol / L) on the x-axis, with the y-axis as the vertical axis.

[0081] When determining the degree of protein hydrolysis in the sample, the sample to be tested (fermented Chlorella proteinensis beverage) was diluted 1:5, and 2 mL was placed in a test tube. Subsequent procedures were the same as described above. The content of free amino acids in the sample was calculated based on the glycine standard curve and converted according to the sample dilution factor.

[0082] 3. The surface hydrophobicity testing method is as follows: Chlorella beverage was dissolved in water to prepare a 20% (w / v) solution. Then, anhydrous ethanol was slowly added to the chlorella beverage to bring the ethanol concentration in the solution to 40%. The mixture was stirred with a magnetic stirrer for 15 min to ensure thorough mixing. After standing for 10 min, the mixture was centrifuged at 5000 r / min for 15 min to remove the precipitate. Anhydrous ethanol was then added to the supernatant to bring the ethanol concentration in the system to 60% and 80% sequentially. The hydrophobic bitter peptides in the enzymatic hydrolysate were extracted in stages. Finally, the ethanol extract was rotary evaporated, and the resulting ethanol solution was freeze-dried to obtain the bitter peptide sample.

[0083] Weigh 0.2 g of bitter peptide sample and dissolve it in 50 mL of phosphate buffer. Centrifuge at 10000 r / min for 20 min to remove the precipitate. The protein content of the sample solution is determined by the biuret colorimetric method. Then, use phosphate buffer to serially dilute the protein concentration of the above solution to 0.0005, 0.005, 0.05, and 0.5 mg / L. Take 4 mL of each diluted sample solution and add 40 μL of freshly prepared ANS solution (8.0 mmol / L). After shaking and mixing, quickly measure the fluorescence intensity of the solution. Plot a polynomial curve with the fluorescence intensity of the sample as the ordinate and the protein concentration as the abscissa to obtain the surface hydrophobicity value of the protein.

[0084] 4. The enzyme production analysis and detection methods are as follows: (1) Plotting the L-tyrosine standard curve Triple replicates were performed for each tube. Various solutions were added as shown in Table 2, and the mixture was shaken to mix thoroughly. The tubes were then incubated in a constant temperature water bath at (40.0±0.2)℃ for 20 min for color development. After the reaction was complete, a 10 mm cuvette was used, and the solution was adjusted to 0 using a blank tube containing 0 μg / mL tyrosine-free solution. The OD value was then measured. 680 The absorbance of L-tyrosine was measured three times, and the average value was taken. A linear regression equation was constructed based on the data obtained from the spectrophotometer. The L-tyrosine content (µg) corresponding to an absorbance of 1 is the value of the absorbance constant K.

[0085] Table 2. Plotting the standard curve for L-tyrosine.

[0086] (2) In vitro protease activity assay Sample tubes (3 replicates): Preheat 1 mL of bacterial growth medium at 40℃ for 5 min. Preheat 1 mL of 1% casein solution at 40℃ for 5 min. Mix the two and incubate in a 40℃ water bath for 10 min. Accurately and quickly add 2 mL of 0.4 mol / L trichloroacetic acid (TCA) and mix well. Blank tubes (3 replicates): Add 2 mL of 0.4 mol / L TCA first, and follow the same steps as for the sample tubes. After removing the sample and blank tubes, centrifuge at 4000 r / min for 5 min. Take 1 mL of the supernatant, add 0.4 mol / L sodium carbonate solution and mix well, then add 1 mL of Folin-phenol solution and mix well. Incubate at 40℃ for 20 min for color development. Zero the instrument with deionized water and measure the absorbance (OD) of the sample and blank tubes using a 10 mm cuvette. 680 .

[0087] Under conditions of 37°C and pH 7, the amount of enzyme required to hydrolyze the substrate casein in bacterial growth medium to produce 1 μg of tyrosine product within 1 minute is defined as one unit of enzyme activity (1U = 1 μg tyrosine / min).

[0088] X=ΔOD 680 ×K×V / T×n In the formula: X is the enzyme activity (U / mL); ΔOD 680 Σ is the difference between the absorbance of the sample tube and the blank tube; K is the absorbance constant of the tyrosine standard curve; V is the reaction volume (mL); T is the reaction time (min); n is the dilution factor.

[0089] 5. The methods for detecting the growth capacity of microbial strains are as follows: The activated test strains were inoculated into MRS liquid medium at an inoculum rate of 3% and incubated at 37°C for 48 h. The absorbance (OD) of the bacterial culture was measured every 4 h during this period. 600 The viable count was calculated using the dilution plating method.

[0090] The test results are shown in Table 3.

[0091] Table 3: Debittering Effect of Example 1 and Comparative Examples 1-23

[0092] As can be seen from Table 3, the protein hydrolysis degree and peptide content of Example 1 are moderate, and the enzymatic hydrolysis is mild and effective; the high number of viable bacteria indicates that its fermentation activity is high, which can produce a large amount of lactic acid and endogenous enzymes to hydrolyze Chlorella protein, thus masking the bitterness; and the low surface hydrophobicity indicates that fewer small molecule proteins or hydrophobic proteins at the peptide ends are produced, thus producing less bitter substances.

[0093] In Comparative Examples 1-4, acidic protease was used for enzymatic hydrolysis in step S2. As can be seen from Table 3, the polypeptide content was relatively high. This may be because acidic protease can effectively release peptides from Chlorella nucleatum.

[0094] However, the surface hydrophobicity of Comparative Example 1 is as high as 1167, which indicates that the content of hydrophobic amino acids at the ends of the peptides produced is high, which may lead to a strong bitter taste in the beverage, indicating poor debittering effect.

[0095] The protein hydrolysis rate in Comparative Example 2 was too low, indicating that the protein failed to be effectively degraded into amino acids. At the same time, its enzyme activity and bacterial growth were also low, indicating that the strain did not grow in a suitable environment and could not further decompose the enzymatic fragments into shorter peptides and amino acids, nor could it effectively produce acid to improve the bitterness.

[0096] In Comparative Example 3, the protein hydrolysis degree was low and the surface hydrophobicity was high, indicating that the protein was not completely hydrolyzed and the debittering effect was poor.

[0097] Comparative Example 4 showed higher peptide content and protein hydrolysis degree, indicating that the protein hydrolysis effect was relatively good and the peptides cleaved by acidic protease could be utilized by endogenous enzymes produced by fermenting Lactobacillus mucilaginosus; however, the surface hydrophobicity was high, resulting in poor debittering effect.

[0098] Neutral protease was used in step S2 in Comparative Examples 5 to 7. As can be seen from Table 3, the degree of protein hydrolysis and enzyme activity were higher in Comparative Example 5, indicating that Lactobacillus plantarum L1 can utilize small molecule proteins or peptides produced by neutral protease. However, its surface hydrophobicity was also high, resulting in poor debittering effect.

[0099] In Comparative Example 6, the peptide content, protein hydrolysis degree, enzyme activity, and viable bacteria count were all low, indicating a poor debittering effect.

[0100] In Comparative Example 7, the peptide content and protein hydrolysis degree were low, resulting in a poor debittering effect.

[0101] In Comparative Examples 8-11, alkaline protease was used in step S2. As shown in Table 3, although Comparative Examples 8 and 10 exhibited high degrees of protein hydrolysis, their surface hydrophobicity was excessively high, resulting in poor debittering effects. The high degree of protein hydrolysis may be due to the alkaline protease's effective cleavage of Chlorella proteins, leading to the production of a large amount of protein in the samples. However, the alkaline growth environment created after alkaline protease hydrolysis is not suitable for lactic acid bacteria growth, resulting in a large amount of unused free amino acids remaining in the samples.

[0102] Papain was used in Comparative Examples 12-15. As shown in Table 3, Comparative Examples 12 and 14 had excessively low polypeptide content and excessively high surface hydrophobicity, resulting in poor debittering effects. Comparative Examples 13 and 15 also had low polypeptide content and low protein hydrolysis, leading to poor debittering effects.

[0103] Pepsin was used in Comparative Examples 16-19. As shown in Table 3, Comparative Examples 16 and 17 had excessively low polypeptide content and excessively high surface hydrophobicity, resulting in poor debittering effects. This may be because the pepsin cleavage site mainly acts on amino acids such as phenylalanine, valine, and tyrosine, and deep hydrolysis continuously exposes the hydrophobic regions hidden in the folded protein.

[0104] Although Comparative Example 18 showed good enzyme activity and surface hydrophobicity, its peptide content was relatively low, possibly because the peptides were broken down into amino acids.

[0105] Comparative Example 19 had lower peptide content, protein hydrolysis degree, and enzyme activity, resulting in a poor debittering effect.

[0106] One possibility is that the strains used in Comparative Examples 18 and 19 produce different endogenous enzymes with different cleavage sites. This could mean that the peptides that pepsin breaks down might be utilized by the endogenous enzymes of Lactobacillus saliva and Lactobacillus fermentum. Their endogenous enzymes might not produce peptides with hydrophobic amino acid ends. Therefore, although both used pepsin, the hydrophobicity of these two comparative examples was relatively low.

[0107] Trypsin was used in Comparative Examples 20-23. As can be seen from Table 3, although the enzyme activity and surface hydrophobicity were good in Comparative Examples 20 and 23, their polypeptide content was relatively low.

[0108] Comparative Example 21 had relatively low peptide content and protein hydrolysis degree, high surface hydrophobicity, and poor debittering effect.

[0109] Comparative Example 22 showed lower peptide content, protein hydrolysis degree, and enzyme activity, higher surface hydrophobicity, and poorer debittering effect.

[0110] Therefore, Example 1 was selected, Comparative Examples 4 and 5 were selected with relatively high peptide content, protein hydrolysis degree and enzyme activity, and Comparative Examples 18, 20 and 23 were selected with relatively good enzyme activity, viable cell count and surface hydrophobicity for subsequent testing.

[0111] Test Example 2 The sensory evaluation of the Chlorella fermented beverages prepared in Examples 1-6 and Comparative Examples 4, 5, 18, 20, and 23-27 was conducted according to the following criteria: The evaluation was conducted primarily based on five aspects: color, taste, aroma, mouthfeel, and bitterness. A review panel of 10 uniformly trained sensory evaluators evaluated the fermented Chlorella proteinifera beverages prepared sequentially, according to the sensory evaluation standards shown in Table 4. The sensory evaluation results are shown in Table 5.

[0112] Table 4: Sensory Evaluation Standards for Chlorella Protein-Breed Fermented Beverages

[0113] Table 5: Sensory Evaluation Results

[0114] Comparing Example 1, Comparative Examples 4 and 5, 18, 20 and 23, and referring to Table 5, it can be seen that Example 1, which first uses neutral protease and then ferments with Lactobacillus plantarum L2, has the highest sensory score of 84, which is also significantly higher than the other comparative examples (P<0.05).

[0115] Comparing Example 1, Comparative Example 26, and Comparative Example 27, and referring to Table 5, it can be seen that although the Chlorella beverages prepared in Example 1 and Comparative Example 26 show no significant difference in color, the beverage prepared in Example 1 is significantly superior to Comparative Example 26 in terms of bitterness, mouthfeel, and aroma, and far superior to the rehydrated algae liquid in Comparative Example 27. This may be because: before fermentation begins, neutral proteases first hydrolyze large protein molecules into a large number of free amino acids and small peptides, which serve as key precursors for the production of various aroma components in subsequent fermentation. At the same time, microorganisms utilize the enzymatic hydrolysis products to more efficiently generate complex and richer flavor compounds through their own metabolic pathways; and microbial fermentation can further coordinate the enzymatic hydrolysis products to form a more stable and pleasant taste. In addition, the enzymatic hydrolysis performed by neutral proteases has already hydrolyzed most of the bitter peptides into non-bitter small peptides or amino acids, which can significantly reduce bitterness from the source. Microbial fermentation can not only utilize its rich peptide spectrum for further enzymatic hydrolysis, but also utilize the resulting sourness, sweetness, umami, and rich aroma to effectively mask residual bitterness, making the flavor more balanced.

[0116] Comparing Examples 1-6 with Comparative Examples 24 and 25, and referring to Table 5, it can be seen that in step S3, when the inoculum amount is too low or the shaking culture time is too long, the sensory score of the prepared Chlorella beverage is reduced.

[0117] Test Example 3 The fermented Chlorella vulgaris beverage prepared in Example 1, the fermented Chlorella vulgaris beverage prepared in Comparative Example 26, the rehydrated algae liquid in Comparative Example 27, and the enzymatic hydrolysate prepared in Comparative Example 28 were selected and characterized by SEM to evaluate their debittering effect from the perspective of microstructural changes.

[0118] Figure 1 A is a SEM image of the rehydrated algal solution in Comparative Example 27, which shows that macromolecules and proteins stacked together to form a relatively complete spherical structure.

[0119] Figure 1 B is the SEM image of the enzymatic hydrolysate prepared in Comparative Example 28. Its structure shows obvious changes, with the formation of more pores and surface wrinkles, indicating that the enzymatic hydrolysis has affected the original conformation of the protein.

[0120] Figure 1 C is the SEM image of the Chlorella fermented beverage prepared in Comparative Example 26. Compared with the previous two, the pore structure is further increased and more significant, indicating that the fermentation process has a stronger effect on changing the internal material structure of Chlorella.

[0121] Figure 1D is a SEM image of the Chlorella proteoglycans fermented beverage prepared in Example 1. Its spherical structure has essentially disappeared, and its original morphology has been completely destroyed, presenting an overall loose, porous, and filamentous morphology. This indicates that the method of the present invention can achieve the purpose of debittering by gradually and thoroughly destroying the original structure of proteins in Chlorella proteoglycans, degrading them into small molecules.

[0122] Test Example 4 In the preparation of Chlorella protein-rich beverages, the partial degradation of proteins still produces bitter peptides. Different preparation methods yield bitter peptides with their own bitterness thresholds. The total content of these bitter peptides and their corresponding bitterness thresholds jointly regulate the bitterness intensity of the enzymatic hydrolysis products. Based on the principle of "like dissolves like," ethanol fractionation precipitation and macroporous resin adsorption techniques were used to extract and enrich hydrophobic bitter peptides from the samples. The content of bitter peptides in the enzymatic hydrolysis products was determined based on the Kjeldahl nitrogen determination results.

[0123] The following samples were selected for testing: the Chlorella proteoglycans fermented beverage prepared in Example 1, the Chlorella proteoglycans fermented beverage prepared in Comparative Example 26, the rehydrated algal liquid in Comparative Example 27, and the enzymatic hydrolysate prepared in Comparative Example 28.

[0124] The results are as follows Figure 2 As shown, the Chlorella protein-nucleated beverage prepared in Example 1 of this invention has the lowest bitter peptide release content, at 27.94%, indicating that it has the best debittering effect.

[0125] Test Example 5 Fourier transform infrared spectroscopy was performed on samples from the Chlorella fermented beverage prepared in Example 1, the Chlorella fermented beverage prepared in Comparative Example 26, the rehydrated algae solution in Comparative Example 27, and the enzymatic hydrolysate prepared in Comparative Example 28. The results are as follows: Figure 3 As shown.

[0126] according to Figure 3 It can be seen that the absorption peak of the protein-rich Chlorella beverage is mainly distributed at 3200 cm⁻¹. -1 1600cm -1 1400cm -1 1100cm -1The absorption peaks are for OH hydrogen bond stretching vibration, C=C double bond, ester, and ether, respectively. Compared with the rehydrated algae liquid in Comparative Example 27, the Chlorella beverage in Example 1 showed a significant leftward shift in the OH hydrogen bond stretching vibration, and the peak height was reduced. This may be due to the unfolding of peptide bonds during enzymatic hydrolysis, exposing more internal OH / NH groups, which may form new, heterogeneous hydrogen bonds with water or adjacent molecules, leading to peak broadening and red shift. This indicates that the specific spatial conformation of the bitter peptide may be disrupted, or that specific hydrophobic clusters may be reduced. At the same time, the C=C double bond absorption peak was also reduced to some extent, indicating that some substances containing this group were selectively transformed or degraded during the preparation process.

[0127] Test Example 6 Amino acids in food are mainly composed of two parts: non-free amino acids that form the basic framework of proteins, and free amino acids. During consumption, non-free amino acids are not easily hydrolyzed immediately and contribute relatively little to the flavor of food. Therefore, determining the composition and content of free amino acids is particularly important. Based on their flavor characteristics, free amino acids can be divided into the following categories: umami amino acids (such as aspartic acid (Asp) and glutamic acid (Glu)), sweet amino acids (such as glycine (Gly), alanine (Ala), threonine (Thr), serine (Ser), and proline (Pro)), bitter amino acids (such as histidine (His), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine (Phe), and arginine (Arg)), and tasteless amino acids (such as cysteine ​​(Cys), tyrosine (Tyr), and lysine (Lys). These free amino acids collectively constitute the basis of the flavor of Chlorella protein-rich beverages.

[0128] The following samples were selected for the determination of free amino acids: the Chlorella proteoglycans fermented beverage prepared in Example 1, the Chlorella proteoglycans fermented beverage prepared in Comparative Example 26, the rehydrated algae liquid in Comparative Example 27, and the enzymatic hydrolysate prepared in Comparative Example 28. The results are shown in Table 6.

[0129] The methods for amino acid determination are as follows: A certain amount of sample was placed in a hydrolysis tube, and 10 mL of 6 mol / L hydrochloric acid solution and 3-4 drops of phenol were added. Nitrogen gas was introduced for 2 min, and the mixture was hydrolyzed at 110℃ for 24 h. After cooling, the mixture was filtered, and sufficient water was added to reach the standard volume. 1 mL of the filtrate was taken and evaporated to dryness in a concentrator. The residue was dissolved in 1 mL of water and evaporated to dryness again. An appropriate amount of sample diluent was added, and the mixture was sonicated for 1 min. The solution was then filtered through a 0.22 μm needle into a sample vial. The amino acid composition of the peptides in the extract was determined using an amino acid analyzer based on retention time for qualitative analysis and external standard method for quantification by peak area.

[0130] Table 6: Types and contents of free amino acids in the above samples

[0131] As shown in Table 6, the Chlorella protein-nucleated beverage prepared in Example 1 had the highest free amino acid content, reaching 4.082 mg / mL, which was 4.615 times higher than the rehydrated algae solution in Comparative Example 27. This indicates that during enzymatic hydrolysis and fermentation, the proteins and peptides in the sample were effectively hydrolyzed, generating a large number of small peptides and free amino acids, thus significantly increasing the free amino acid content. Further analysis of the amino acid composition revealed that the method of the present invention also altered the proportions of various flavor amino acids. Compared with the rehydrated algae solution in Comparative Example 27, the content of tasteless amino acids such as tyrosine and lysine in the Chlorella protein-nucleated beverage prepared in Example 1 increased to varying degrees, with tyrosine content increasing by 6.51 times. Simultaneously, sweet amino acids such as alanine and glycine also increased significantly. Conversely, the bitter amino acid arginine showed a decreasing trend. These changes demonstrate that the preparation method of the present invention not only increased the total amount of free amino acids in the Chlorella beverage but also adjusted its compositional structure, thereby directionally altering the flavor characteristics of the beverage.

[0132] Table 7: Classification and content of flavor amino acids in samples

[0133] As can be seen from Tables 6 and 7, compared to Comparative Example 27, the proportion of bitter amino acids in the total amino acids of Comparative Example 26 and Example 1 is reduced, with Comparative Example 26 and Example 1 showing reductions of 0.3% and 1.9%, respectively. Furthermore, compared to Comparative Example 27, the total content of umami and sweet amino acids in Example 1 is increased to varying degrees. This enhancement of umami and sweet amino acids likely has an effective flavor masking and harmonizing effect on the residual bitter amino acid intensity in the system through taste interaction. Therefore, the debittering treatment of this invention not only changes the absolute content of amino acids but also, by adjusting their relative composition and flavor profile, directionally shapes the final flavor profile of the Chlorella proteinaceae beverage.

[0134] Table 8: Flavor Threshold and TAV Value of Free Amino Acids in Samples

[0135] Free amino acids, as key non-volatile flavor compounds, have a significant impact on food flavor formation due to their low flavor threshold and high flavor intensity. Evaluation using Taste Activity Value (TAV) can more accurately reflect the actual contribution of each flavor amino acid in the complex system. As shown in Table 8, the TAV values ​​of all free amino acids (sweet, umami, and bitter) in the above examples and comparative examples are not zero, indicating that they collectively participate in the overall flavor composition of the beverage.

[0136] Further analysis revealed significant differences in the TAV values ​​of bitter amino acids among the four samples, at 0.0753, 0.3722, 0.3223, and 0.2265, respectively. Notably, the TAV value of bitter amino acids in Example 1 was lower than that in Comparative Examples 26 and 28, indicating that the method of this invention can weaken the flavor intensity dominated by bitter amino acids compared to simple fermentation or enzymatic hydrolysis. In contrast, the TAV values ​​of sweet amino acids in Comparative Example 26 and Example 1 reached 0.5686 and 0.6161, respectively, forming a clear flavor advantage. Based on the flavor interaction mechanism, this significantly increased sweet signal likely improves flavor perception through two pathways: firstly, by directly masking bitterness; and secondly, by integrating and harmonizing flavors at the central nervous system level, thereby reducing the overall prominence of bitterness and achieving the goal of eliminating bitterness. Therefore, the method of this invention can directionally regulate the flavor spectrum, balancing and masking bitterness by enhancing pleasant flavors such as sweetness.

[0137] The fermented Chlorella vulgaris beverage prepared in Example 1, the fermented Chlorella vulgaris beverage prepared in Comparative Example 26, the rehydrated algae solution in Comparative Example 27, and the enzymatic hydrolysate prepared in Comparative Example 28 were selected as samples for comparison. The differentially expressed metabolites were sorted by p-value from smallest to largest, and the top 20 differentially expressed metabolites were displayed in a bar chart to clarify the content of metabolites in different sample groups. The results are as follows: Figures 4-7 As shown.

[0138] Compared to the rehydrated algae solution of Comparative Example 27, the glycine-phenylalanine content in the enzymatic hydrolysate of Comparative Example 28 was significantly increased. Glycine-phenylalanine is a dipeptide that is predicted to bind to sweet taste receptors and is a hallmark metabolite of tea flavor, which is positively correlated with its mellowness and freshness. This may be one of the taste mechanisms that reduced the bitterness of this group of products.

[0139] Compared to the rehydrated algae solution in Comparative Example 27, the ornithine content in Example 1 was significantly increased. Ornithine, as an important flavor precursor, can be degraded in food systems via Strecker degradation or undergo Maillard reactions with reducing sugars to generate volatile flavor compounds such as pyrazines, which possess roasted and nutty aromas. In fermented foods, ornithine is often a key intermediate in forming signature flavors, and its accumulation helps enhance the product's body, roasted flavor, and overall complexity. Furthermore, ornithine can synergistically enhance flavors with umami amino acids such as glutamic acid, further enriching the taste experience.

[0140] Compared to the enzymatic hydrolysate in Comparative Example 28, the Chlorella beverage prepared in Example 1 showed a significant increase in the content of tasteless amino acids such as tyrosine, which is likely related to the overall improvement in flavor of the final fermented beverage. Compared to the Chlorella beverage in Comparative Example 26, the Chlorella beverage prepared in Example 1 showed the most significant increase in L-lysine content. This indicates that enzymatic hydrolysis and microbial fermentation not only enhance the nutritional value and flavor characteristics of Chlorella proteoglycans but also alter the amino acid ratio to adjust the flavor balance.

[0141] Test Example 7 The following samples were selected: the Chlorella proteoglycans fermented beverage prepared in Example 1, the Chlorella proteoglycans fermented beverage prepared in Comparative Example 26, the rehydrated algae liquid in Comparative Example 27, and the enzymatic hydrolysate prepared in Comparative Example 28. The following physicochemical properties were tested.

[0142] (1) pH measurement The pH value of food was determined according to GB 5009.237-2016, "National Food Safety Standard - Determination of pH Value in Food". The results are as follows: Figure 8 As shown, Example 1 had the lowest pH value, which was 21% lower than that of the rehydrated algae solution in Comparative Example 27.

[0143] (2) Determination of polyphenol content The detection method is as follows: Gallic acid standard stock solution (concentration 1 mg / mL) was prepared in advance. 0.3, 0.6, 0.9, 1.2, 1.5, and 1.8 mL of the stock solution were sequentially pipetted into 10 mL volumetric flasks, and water was added to the mark to prepare test solutions with concentrations of 0.03, 0.06, 0.09, 0.12, 0.15, and 0.18 mg / mL, respectively. 0.2 mL each of the above series of standard solutions, the supernatant of the beverage sample, and the blank solution (distilled water) were taken, and 1 mL of Folin-Ciocalteu reagent was added. The mixture was vortexed and stirred for 5 min. Then, 3 mL of 7.5% Na₂CO₃ solution was added, and the mixture was incubated at room temperature in the dark for 1 h. The absorbance was measured at 765 nm. A gallic acid standard curve was plotted with gallic acid concentration as the X-axis and absorbance as the Y-axis, and the total phenol content of the sample was further calculated. The results are as follows: Figure 9 As shown, the Chlorella beverage prepared in Example 1 of this invention has the highest polyphenol content, which is 0.991 mg / mL.

[0144] (3) DPPH scavenging rate determination Take 10 mg of 2,2-diphenyl-1-trinitrophenylhydrazine (DPPH) standard (purity ≥ 98%) and dilute to 100 mL in a brown volumetric flask with pure methanol to obtain a standard solution with a concentration of 100 μg / mL. Then dilute to prepare standard solutions with concentrations of 0.5, 1.25, 2.5, 5, 10, 20, and 25 μg / mL. Measure the UV absorbance of each concentration at 517 nm at room temperature, using pure methanol as a parallel blank. Plot absorbance as the ordinate (Y) and concentration as the abscissa (X) (μg / mL).

[0145] Add 0.1 mL of different crude extracts to 3.9 mL of DPPH with a mass concentration of 20 μg / mL. For the blank control, add 0.1 mL of the solvent used for the test solution (2 tubes). React in a 30 °C water bath in the dark for 30 min. Centrifuge at 2500 rpm for 10 min. Take out and measure the absorbance at 517 nm. Substitute the measured value into the DPPH regression equation to calculate the remaining concentration of DPPH. Calculate the DPPH free radical scavenging rate according to the following formula.

[0146]

[0147] A0: DPPH concentration of blank sample, at t=0; A: Remaining DPPH concentration of sample, at t=45 min.

[0148] The results are as follows Figure 10 As shown, Example 1 showed the best performance in terms of antioxidant capacity, with a scavenging rate of 70.05%, which is 2.2 times higher than that of Comparative Example 27.

[0149] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and variations that are directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. A method for preparing a fermented beverage made from Chlorella proteinifera, characterized in that, Includes the following steps: S1. Mix Chlorella pyrenoidosa powder with pure water until homogeneous, then rehydrate to obtain rehydrated algal solution; S2. Add enzyme to the rehydrated algae solution; after enzymatic hydrolysis, inactivate the enzyme, filter and collect the filtrate to obtain the enzymatic hydrolysate; S3. Inoculate the activated strain into the enzymatic hydrolysate, shake and culture, filter and collect the filtrate to obtain Chlorella proteoglycans fermented beverage.

2. The preparation method according to claim 1, characterized in that, In step S1, the ratio of the protein-nucleated Chlorella powder to pure water is 1:(10~30).

3. The preparation method according to claim 1, characterized in that, In step S2, at 35~39℃, an enzyme is added to the rehydrated algae solution, with an enzyme-to-bottom ratio of 4~5%.

4. The preparation method according to claim 1, characterized in that, In step S2, the enzyme is a neutral protease.

5. The preparation method according to claim 1, characterized in that, In step S2, the enzymatic hydrolysis temperature is 35~39℃, the pH is 6.5~7.0, and the time is 4~6h.

6. The preparation method according to claim 1, characterized in that, In step S2, the enzyme inactivation temperature is 95~115℃ and the time is 5~15min.

7. The preparation method according to claim 1, characterized in that, In step S3, The strain is Lactobacillus plantarum L2, with accession number CGMCC No.37217, which was deposited at the China General Microbiological Culture Collection Center on December 26, 2025.

8. The preparation method according to claim 1, characterized in that, In step S3, the inoculation amount is 4-5% (w / v).

9. The preparation method according to claim 1, characterized in that, In step S3, the temperature of the oscillation culture is 35~39℃, and the time is 24~48h.

10. A fermented beverage of Chlorella proteinensis prepared by the method according to any one of claims 1 to 9.

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