Methods for improving the flavor of Chlorella mutants with chlorophyll synthesis defects

Abstract

This invention belongs to the field of functional food processing technology, specifically relating to a method for improving the flavor of a chlorophyll-deficient Chlorella proteoglycan mutant. The method includes the following steps: S1, obtaining a Chlorella proteoglycan mutant strain; S2, centrifuging the Chlorella proteoglycan mutant strain and collecting the algal sludge, then pulverizing the collected algal sludge and dispersing it in water to obtain an algal powder suspension, then adding a compound enzyme to the algal powder suspension, and inactivating the enzyme after the reaction; S3, cooling the system to room temperature, adding glucose and stirring to dissolve, sterilizing, inoculating with a compound bacterial strain and fermenting, and sterilizing again after fermentation; S4, cooling the fermented system to room temperature, then adding β-cyclodextrin and stirring, and drying the system after stirring to obtain the algal protein product. This invention employs a synergistic process of compound enzymatic hydrolysis, compound bacterial fermentation, and β-cyclodextrin encapsulation to remove the off-flavor of the mutant algae from the source, significantly improving the flavor and enhancing the product's storage stability.

Description

Technical Field

[0001] This invention belongs to the field of functional food processing technology, specifically relating to a method for improving the flavor of Chlorella mutants with chlorophyll synthesis defects. Background Technology

[0002] Due to the limited chlorophyll synthesis, the algal powder produced by the protein-nucleated Chlorella mutant is significantly lighter in color, reducing the reliance on decolorization processes in food applications.

[0003] However, in actual food applications, it has been found that the chlorophyll synthesis defective Chlorella proteoglycan mutant, after being made into algae powder or protein products, has significantly different flavor characteristics from wild-type Chlorella proteoglycan. It is characterized by enhanced bitterness, prominent fishy or off-flavors, which affects the sensory quality of the product and consumer acceptance.

[0004] Existing flavor improvement technologies for Chlorella or algal proteins mainly focus on decolorization, flavor masking, or flavor compounding, without considering the impact of metabolic changes caused by chlorophyll synthesis defects on the composition of flavor substances. Therefore, it is difficult to effectively improve the flavor problems unique to the above mutants. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a method for improving the flavor of Chlorella mutants with chlorophyll synthesis defects. The method employs a synergistic process of compound enzymatic hydrolysis, compound bacterial fermentation, and β-cyclodextrin encapsulation to remove the off-flavor of the mutant algae from the source, significantly improving the flavor and enhancing the product's storage stability.

[0006] The specific technical solution adopted in this invention is as follows: A method for improving the flavor of Chlorella mutants with chlorophyll synthesis defects includes the following steps: S1. Obtain the Chlorella proteoglycan mutant strain; S2. Centrifuge the Chlorella proteoglycans mutant strain and collect the algal mud. Then, crush the collected algal mud and disperse it in water to obtain an algal powder suspension. Add the compound enzyme to the algal powder suspension and react at 35-45℃ for 1-3 hours. After the reaction is completed, inactivate the enzyme at 85-95℃ for 10-15 minutes. S3. Cool the enzyme-inactivated system from step S2 to room temperature, add glucose and stir to dissolve, sterilize, inoculate with compound bacterial strains and ferment for 12-24 hours, sterilize again after fermentation. S4. Cool the fermented system to room temperature, then add β-cyclodextrin and stir. After stirring, dry the system to obtain the algal protein product.

[0007] The Chlorella proteoglycan mutant strain mentioned in step S1 is named RMCP012, and is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 65141, deposit date of September 14, 2024, and deposit address of Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] In step S2, the mass ratio of algae powder to water in the algae powder suspension is 1:10-20.

[0009] The compound enzyme in step S2 includes aldehyde dehydrogenase and alcohol oxidase, with a mass ratio of aldehyde dehydrogenase to alcohol oxidase of 1:1-2, and the amount of compound enzyme added is 0.2%-0.8% of the mass of algae powder.

[0010] In step S3, the sterilization conditions before inoculating the compound strain are 120-122℃ for 15-20 minutes, and the sterilization conditions after fermentation are 75-85℃ for 10-15 minutes.

[0011] The amount of glucose added in step S3 is 0.5%-1% of the mass of the algae powder.

[0012] The compound bacterial strain mentioned in step S3 includes lactic acid bacteria and yeast, with a live count ratio of lactic acid bacteria to yeast of 3-5:1, and an inoculation amount of 1×10⁻⁶. 6 -5×10 6 CFU / mL.

[0013] The amount of β-cyclodextrin added in step S4 is 2%-5% of the mass of algae powder.

[0014] After adding β-cyclodextrin in step S4, stir at 100-200 r / min for 30-60 min at 25-30℃.

[0015] The moisture content of the algal protein product after drying in step S4 is ≤8%.

[0016] The beneficial effects of this invention are: 1. Chlorophyll synthesis defects lead to the accumulation of large amounts of volatile small molecules such as aldehydes and alcohols in mutant algae, which are the main sources of their bitterness and fishy taste. This invention uses a combination of aldehyde dehydrogenase and alcohol oxidase to catalyze the decomposition of residual aldehydes and alcohols in the algae, thus eliminating the off-flavor at its source.

[0017] 2. In this invention, organic acids are produced by the metabolism of lactic acid bacteria to neutralize the alkaline bitter peptides in the algae and adjust the pH value of the system, thereby weakening the bitterness from a sensory perspective. At the same time, yeast is used to further decompose the trace odor molecules remaining after enzymatic hydrolysis and metabolize them to generate natural ester and alcohol flavor substances, which mask the residual fishy smell and give the finished product a mellow aroma. The synergistic effect of the two further improves the flavor of algae-derived protein products.

[0018] Furthermore, sterilization at a low temperature of 75-85℃ after fermentation can both inactivate the bacteria and stop fermentation, while preserving the flavor substances produced during fermentation, thus avoiding the destruction of flavor by high temperature.

[0019] 3. This invention utilizes the hydrophobic cavity structure of β-cyclodextrin, which can encapsulate residual bitter and fishy small molecules in the system to form inclusion complexes, thereby sealing off odors. Detailed Implementation

[0020] The present invention will be further described below with reference to specific embodiments: I. Specific Implementation Methods The method for inducing the chlorophyll synthesis-deficient Chlorella mutant strain in this invention is as follows: S101, Preparation of culture medium for Chlorella proteoglycans The formula for the liquid culture medium is as follows: Each liter of liquid culture medium contains 10-20g of glucose, 5-10g of urea, 0.005-0.01g of citric acid, 0.005-0.01g of ferric ammonium citrate, 0.01-0.05g of K2HPO4·3H2O, 0.05-0.1g of MgSO4·7H2O, 0.02-0.1g of CaCl2·2H2O, 0.03-0.05g of Na2HCO3, and the balance of water. After the raw materials are mixed evenly, they are sterilized at 105-125℃ to obtain the liquid culture medium for Chlorella proteoglycans. Solid culture medium formulation: Solid culture medium is based on liquid culture medium with the addition of 0.8%-2% agar powder; S102. Prepare the mutagenic working solution for Chlorella proteoglycans. Wild-type Chlorella proteoglycans (naturally isolated from Hubei waters) were cultured on plates under aseptic conditions. Single colonies were picked and transferred to the prepared liquid culture medium (50 mL). The culture was carried out in the dark for 3-5 days at 20-32℃ and pH 6-8. During this period, Chlorella proteoglycans were in the logarithmic growth phase. A small amount of the algal solution was taken for microscopic observation of cell morphology and cell counting. The algal solution was then diluted with sterile water to different cell densities: 10-1 5 -10 6 The concentration was set at 1 / mL as the mutagenic working solution.

[0021] S103, mutagenesis of Chlorella proteoglycans Prepare a UV manipulation equipment table, set the UV lamp power to 40-60W, and keep the working solution 30-50cm away from the UV lamp. Treat for different times of 10-40min to determine the time when the UV lethality of cells in the working solution is 85-95%. Then treat for the same time (23min). Dilute the treated algal solution evenly and spread it on Chlorella proteoglycans solid culture medium. Incubate in the dark at 20-32℃ for 7-10 days and observe the results. Screening of S104 and Chlorella proteoglycan mutants After 2-3 rounds of UV mutagenesis and screening for strains with reduced pigment, the color of the strains gradually lightened after repeated mutagenesis and screening. Finally, several treated Chlorella proteoglycan plates were obtained. The initial screening involved picking algal strains that appeared to be lighter or yellower in color from these plates and culturing them in new heterotrophic plates. After more than 8 generations of continuous culture, the Chlorella proteoglycan mutant strain with chlorophyll synthesis deficiency was finally obtained.

[0022] Examples of methods for improving the flavor of Chlorella mutants with chlorophyll synthesis defects are as follows: Example 1 S1. Obtain the Chlorella proteoglycan mutant strain; S2. Centrifuge the Chlorella proteoglycans mutant strain at 8000 r / min for 8 min and collect the algal sludge. After freeze-drying the algal sludge, pulverize it to 100 mesh and disperse it in deionized water at a material-to-liquid ratio of 1:15. Add 0.5% of the algal powder mass of a compound enzyme, which is aldehyde dehydrogenase and alcohol oxidase in a mass ratio of 1:1.5. Adjust the pH to 7.0, enzymatically hydrolyze at 40℃ for 2 h, and then incubate at 90℃ for 12 min to inactivate the enzyme. S3. After cooling the enzyme-inactivated system from step S2 to room temperature, adjust the pH to 6.5, then add 0.8% glucose (by weight of algae powder) and stir to dissolve. Sterilize at 121℃ for 20 minutes. After cooling to room temperature, inoculate with a compound bacterial strain consisting of lactic acid bacteria and yeast in a live-to-live ratio of 4:1. The total inoculation amount is 3 × 10⁻⁶. 6 CFU / mL, then anaerobic fermentation at 35℃ for 18h, and sterilization at 80℃ for 12min after fermentation; S4. Cool the fermented system to room temperature, add β-cyclodextrin at 3% of the algae powder mass, stir at 150 r / min for 45 min at 28℃, and then dry to a moisture content of 6% to obtain the flavor-improved algae protein product.

[0023] Example 2 S1. Obtain the Chlorella proteoglycan mutant strain; S2. Centrifuge the Chlorella mutant strain at 8000 r / min for 8 min and collect the algal sludge. After freeze-drying the algal sludge, pulverize it to 100 mesh and disperse it in deionized water at a material-to-liquid ratio of 1:10. Add 0.2% of the algal powder mass of a compound enzyme, which is aldehyde dehydrogenase and alcohol oxidase in a mass ratio of 1:1. Adjust the pH value to 6.8, enzymatically hydrolyze at 35℃ for 1 h, and then incubate at 85℃ for 10 min to inactivate the enzyme. S3. After cooling the enzyme-inactivated system from step S2 to room temperature, adjust the pH to 6.2, then add 0.5% glucose (by weight of algae powder) and stir to dissolve. Sterilize at 120℃ for 15 minutes. After cooling to room temperature, inoculate with a compound bacterial strain consisting of lactic acid bacteria and yeast in a live-to-live ratio of 3:1. The total inoculation amount is 1×10⁻⁶. 6 CFU / mL, then anaerobic fermentation at 32℃ for 12h, and sterilization at 75℃ for 10min after fermentation; S4. Cool the fermented system to room temperature, add β-cyclodextrin at 2% of the algae powder mass, stir at 100 r / min for 30 min at 25℃, and then dry to a moisture content of 8% to obtain the flavor-improved algae protein product.

[0024] Example 3 S1. Obtain the Chlorella proteoglycan mutant strain; S2. Centrifuge the Chlorella proteoglycans mutant strain at 8000 r / min for 8 min and collect the algal sludge. After freeze-drying the algal sludge, pulverize it to 100 mesh and disperse it in deionized water at a material-to-liquid ratio of 1:20. Add 0.8% of the algal powder mass of a compound enzyme, which is aldehyde dehydrogenase and alcohol oxidase in a mass ratio of 1:2. Adjust the pH value to 7.2, enzymatically hydrolyze at 45℃ for 3 h, and then incubate at 95℃ for 15 min to inactivate the enzyme. S3. After cooling the enzyme-inactivated system from step S2 to room temperature, adjust the pH to 6.8, then add 1.0% glucose (by weight of algae powder) and stir to dissolve. Sterilize at 122℃ for 20 minutes. After cooling to room temperature, inoculate with a compound bacterial strain consisting of lactic acid bacteria and yeast in a live-to-live ratio of 5:1. The total inoculation amount is 5 × 10⁻⁶. 6 CFU / mL, then anaerobic fermentation at 38℃ for 24h, and sterilization at 85℃ for 15min after fermentation; S4. Cool the fermented system to room temperature, add β-cyclodextrin at 5% of the algae powder mass, stir at 200 r / min for 60 min at 30℃, and then dry to a moisture content of 7% to obtain the flavor-improved algae protein product.

[0025] Example 4 S1. Obtain the Chlorella proteoglycan mutant strain; S2. Centrifuge the Chlorella mutant strain at 8000 r / min for 8 min and collect the algal sludge. After freeze-drying the algal sludge, pulverize it to 100 mesh and disperse it in deionized water at a material-to-liquid ratio of 1:18. Add 0.6% of the algal powder mass of a compound enzyme, which is aldehyde dehydrogenase and alcohol oxidase in a mass ratio of 1:1.8. Adjust the pH to 7.0, enzymatically hydrolyze at 42℃ for 2.5 h, and then inactivate the enzyme at 92℃ for 13 min. S3. After cooling the enzyme-inactivated system from step S2 to room temperature, adjust the pH to 6.6, then add 0.7% glucose (by weight of algae powder) and stir to dissolve. Sterilize at 121℃ for 18 minutes. After cooling to room temperature, inoculate with a compound bacterial strain consisting of lactic acid bacteria and yeast in a live-to-live ratio of 4.5:1, with a total inoculation amount of 4 × 10⁻⁶. 6 CFU / mL, then anaerobic fermentation at 36℃ for 20h, and sterilization at 82℃ for 13min after fermentation; S4. Cool the fermented system to room temperature, add β-cyclodextrin at 4% of the algae powder mass, stir at 180 r / min for 50 min at 27℃, and then dry to a moisture content of 6.5% to obtain the flavor-improved algae protein product.

[0026] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the algae mud system is not enzymatically hydrolyzed in Comparative Example 1. The specific steps are as follows: S1. Obtain the Chlorella proteoglycan mutant strain; S2. Centrifuge the Chlorella mutant strain at 8000 r / min for 8 min and collect the algal sludge. After freeze-drying the algal sludge, pulverize it to 100 mesh and disperse it in deionized water at a material-to-liquid ratio of 1:15. S3. Adjust the pH of the system to 6.5, then add 0.8% glucose (by weight of algae powder) and stir to dissolve. Sterilize at 121℃ for 20 minutes. After cooling to room temperature, inoculate with a compound bacterial strain consisting of lactic acid bacteria and yeast in a live-to-live ratio of 4:1. The total inoculation amount is 3 × 10⁻⁶. 6 CFU / mL, then anaerobic fermentation at 35℃ for 18h, and sterilization at 80℃ for 12min after fermentation; S4. Cool the fermented system to room temperature, add β-cyclodextrin at 3% of the algae powder mass, stir at 150 r / min for 45 min at 28℃, and then dry to a moisture content of 6% to obtain the flavor-improved algae protein product.

[0027] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the algae mud system is not fermented in Comparative Example 2. The specific steps are as follows: S1. Obtain the Chlorella proteoglycan mutant strain; S2. Centrifuge the Chlorella proteoglycans mutant strain at 8000 r / min for 8 min and collect the algal sludge. After freeze-drying the algal sludge, pulverize it to 100 mesh and disperse it in deionized water at a material-to-liquid ratio of 1:15. Add 0.5% of the algal powder mass of a compound enzyme, which is aldehyde dehydrogenase and alcohol oxidase in a mass ratio of 1:1.5. Adjust the pH to 7.0, enzymatically hydrolyze at 40℃ for 2 h, and then incubate at 90℃ for 12 min to inactivate the enzyme. S3. Cool the enzyme-inactivated system to room temperature, add β-cyclodextrin at 3% of the algal powder mass, stir at 150 r / min for 45 min at 28℃, and then dry to a moisture content of 6% to obtain the algal protein product.

[0028] II. Performance Testing The algal protein products obtained in Examples 1-4 and Comparative Examples 1-2 were used as samples for performance testing.

[0029] 1. Sensory flavor evaluation Five professionally trained sensory evaluators were selected. The evaluators were required to have no olfactory or gustatory impairments and to refrain from consuming spicy, sweet, sour, or strongly scented foods or drinking stimulating beverages within one hour prior to the evaluation to ensure the objectivity and accuracy of the evaluation results.

[0030] Each group of samples was placed in a container of the same size, numbered, and randomly provided to the evaluators. The evaluators tasted and smelled the samples in turn to judge their bitterness, fishiness, and off-odors, and scored them according to the standards. The average score of the five evaluators for each group of samples was taken as the final sensory score, and one decimal place was retained.

[0031] 1.1 Sensory Flavor Evaluation Criteria (1) Bitterness: 10 points (no bitterness), 7-9 points (slightly bitter, negligible), 4-6 points (moderate bitterness, acceptable), 1-3 points (severe bitterness, unacceptable). (2) Fishy smell: 10 points (no fishy smell), 7-9 points (slightly fishy, ​​negligible), 4-6 points (moderate fishy smell, acceptable), 1-3 points (severe fishy smell, unacceptable). (3) Odor: 10 points (no odor), 7-9 points (slight odor, negligible), 4-6 points (moderate odor, acceptable), 1-3 points (severe odor, unacceptable); (4) Overall acceptability: 10 points (smooth taste, no unpleasant flavor, can be eaten directly), 7-9 points (good taste, weak unpleasant flavor, can be eaten directly), 4-6 points (average taste, obvious unpleasant flavor, needs further improvement), 1-3 points (extremely poor taste, prominent unpleasant flavor, cannot be eaten).

[0032] 1.2 Sensory Flavor Detection Results and Analysis The sensory flavor test results are shown in Table 1.

[0033] Table 1 As shown in Table 1, the sensory flavor scores of the samples in Examples 1-4 were in the high range of 8.9-9.3, indicating a mellow flavor and high acceptability. In Comparative Example 1, due to the lack of aldehyde dehydrogenase and alcohol oxidase to degrade off-odor precursors, the scores for bitterness, fishiness, and off-odor were only 3.8-4.5, with an overall acceptability score of less than 4. This confirms that aldehydes and alcohols enriched in chlorophyll-deficient algae are the core cause of flavor degradation, and that enzymatic hydrolysis is the fundamental key to deodorization. In Comparative Example 2, only enzymatic hydrolysis and cyclodextrin encapsulation were retained. Although the sensory flavor score was higher than that of Comparative Example 1, it was still far lower than that of the examples, indicating that simply degrading off-odor substances cannot neutralize bitter peptides. The lack of the synergistic effect of lactic acid bacteria adjusting pH to suppress bitterness and yeast producing natural aromas significantly reduced the flavor improvement effect.

[0034] 2. Detection of volatile odor substances Hexanal, heptanal, phenylethanol, and isoamyl alcohol were selected as detection indicators. Gas chromatography-mass spectrometry was used to determine the content of the above four substances in the sample. The detection results are shown in Table 2.

[0035] Table 2 As shown in Table 2, the content of the four odor substances in Examples 1-4 was all below 1.5%, which is a low level. Comparative Example 1, which did not undergo enzymatic hydrolysis, had approximately five times the content of the four types of odor substances compared to the Example group, with a large amount of aldehydes and alcohols remaining, indicating that enzymatic hydrolysis is a necessary step in degrading primordial odor substances. Comparative Example 2, which did not undergo fermentation, had approximately four times the content of odor substances compared to the Example group, indicating that enzymatic hydrolysis cannot completely remove trace amounts of residual odor molecules, and that compound microbial fermentation can achieve deep deodorization.

[0036] 3. Product stability Each group of samples was placed in a sealed plastic bag and stored in a cool, dark, and dry environment for 3 months. During this period, the bags were kept dry and protected from moisture.

[0037] After 3 months of storage, the flavor of the samples was tested according to the sensory flavor evaluation method, and the test results are shown in Table 3. At the same time, the contents of four substances, namely hexanal, heptanal, phenylethanol and isoamyl alcohol, were determined according to the volatile odor content detection method. The results were compared with the test results before storage to evaluate the degree of odor residue, and the test results are shown in Table 4.

[0038] Table 3 Table 4 As can be seen from Table 3, the scores of the samples in Examples 1-4 decreased slightly after storage, while the scores of bitterness, fishiness and off-flavor remained above 8.5, and the overall acceptability remained excellent. The products showed strong stability under long-term storage conditions. The average decline in sensory flavor of the samples in Comparative Examples 1 and 2 after storage was relatively large, with severe undesirable flavors, indicating that the improved process in this invention can give the product better stability and effectively solve the problem of flavor deterioration of mutant algal proteins during storage.

[0039] As can be seen from Table 4, the odor substances in the samples of Examples 1-4 increased only slightly after storage, while the odor substances in Comparative Examples 1 and 2 increased significantly. This further demonstrates that the improved process in this invention has a long-lasting effect on odor control.

Claims

1. A method for improving the flavor of a mutant Chlorella vulgaris with a chlorophyll synthesis defect, characterized in that, Includes the following steps: S1. Obtain the Chlorella proteoglycan mutant strain; S2. Centrifuge the Chlorella proteoglycans mutant strain and collect the algal mud. Then, crush the collected algal mud and disperse it in water to obtain an algal powder suspension. Add the compound enzyme to the algal powder suspension and react at 35-45℃ for 1-3 hours. After the reaction is completed, inactivate the enzyme at 85-95℃ for 10-15 minutes. S3. Cool the enzyme-inactivated system from step S2 to room temperature, add glucose and stir to dissolve, sterilize, inoculate with compound bacterial strains and ferment for 12-24 hours, sterilize again after fermentation. S4. Cool the fermented system to room temperature, then add β-cyclodextrin and stir. After stirring, dry the system to obtain the algal protein product.

2. The method for improving the flavor of Chlorella mutants with chlorophyll synthesis defects as described in claim 1, characterized in that, The Chlorella proteoglycan mutant strain mentioned in step S1 is named RMCP012, and is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 65141, deposit date of September 14, 2024, and deposit address of Building 59, No. 100 Xianlie Middle Road, Guangzhou.

3. The method for improving the flavor of Chlorella mutants with chlorophyll synthesis defects as described in claim 1, characterized in that, In step S2, the mass ratio of algae powder to water in the algae powder suspension is 1:10-20.

4. The method for improving the flavor of Chlorella mutants with chlorophyll synthesis defects as described in claim 1, characterized in that, The compound enzyme in step S2 includes aldehyde dehydrogenase and alcohol oxidase, with a mass ratio of aldehyde dehydrogenase to alcohol oxidase of 1:1-2, and the amount of compound enzyme added is 0.2%-0.8% of the mass of algae powder.

5. The method for improving the flavor of Chlorella mutants with chlorophyll synthesis defects as described in claim 1, characterized in that, In step S3, the sterilization conditions before inoculating the compound strain are 120-122℃ for 15-20 minutes, and the sterilization conditions after fermentation are 75-85℃ for 10-15 minutes.

6. The method for improving the flavor of Chlorella mutants with chlorophyll synthesis defects as described in claim 1, characterized in that, The amount of glucose added in step S3 is 0.5%-1% of the mass of the algae powder.

7. The method for improving the flavor of Chlorella mutants with chlorophyll synthesis defects as described in claim 1, characterized in that, The compound bacterial strain mentioned in step S3 includes lactic acid bacteria and yeast, with a live count ratio of lactic acid bacteria to yeast of 3-5:1, and an inoculation amount of 1×10⁻⁶. 6 -5×10 6 CFU / mL.

8. The method for improving the flavor of Chlorella mutants with chlorophyll synthesis defects as described in claim 1, characterized in that, The amount of β-cyclodextrin added in step S4 is 2%-5% of the mass of algae powder.

9. The method for improving the flavor of Chlorella mutants with chlorophyll synthesis defects as described in claim 1, characterized in that, After adding β-cyclodextrin in step S4, stir at 100-200 r / min for 30-60 min at 25-30℃.

10. The method for improving the flavor of Chlorella mutants with chlorophyll synthesis defects as described in claim 1, characterized in that, The moisture content of the algal protein product after drying in step S4 is ≤8%.

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