Method for breaking the cell wall of haematococcus pluvialis by biological fermentation and application thereof

Abstract

The present application belongs to the technical field of microbial processing, and particularly relates to a method for breaking the cell wall of Haematococcus Pluvialis by biological fermentation and application thereof. The method for breaking the cell wall of Haematococcus Pluvialis by biological fermentation provided by the present application comprises: adding bran koji to high-temperature sterilization pretreated Haematococcus Pluvialis or non-pretreated Haematococcus Pluvialis for sealed fermentation treatment, and obtaining a broken cell wall Haematococcus Pluvialis fermentation liquor after the fermentation is completed. The method for breaking the cell wall of Haematococcus Pluvialis by biological fermentation can effectively break the cell wall of Haematococcus Pluvialis, and in the latter case, the fermentation hardly affects the astaxanthin and other carotenoid components.

Description

Technical Field

[0001] This invention belongs to the field of microbial treatment technology, specifically relating to a method for breaking the cell wall of Haematococcus pluvialis using biological fermentation and its application. Background Technology

[0002] Astaxanthin is a red-orange ketone carotenoid and is the most powerful natural antioxidant discovered to date. Haematococcus pluvialis is one of the main microalgal sources of natural astaxanthin. Haematococcus pluvialis cultivation has developed into a mature biotechnology, with large-scale production facilities established worldwide. The cell disruption steps for commercially scalable extraction of astaxanthin from Haematococcus pluvialis involve mechanical processes such as pressing and ball milling. Extraction steps are typically carried out via ethanol extraction or supercritical CO2 extraction, with the final product existing in forms such as soft gels, capsules, creams, energy drinks, oils, extracts, and tablets. However, astaxanthin from this mature production system remains expensive, primarily due to the complexity of the cultivation and purification processes, with cell wall disruption being a major obstacle in the purification step.

[0003] The scientific community has a sufficient understanding of the rigid three-layered cell wall formed by the immobile spores in the life cycle and non-motile phase of Haematococcus pluvialis. Mature cyst cells develop into three distinct cell wall layers, consisting of an outer triple sheath (TLS), a secondary wall, and interstitial spaces. These different layers are primarily composed of alginate biopolymers and amorphous structures of cellulosic polysaccharides, mannose, and cellulose, as well as the amorphous arrangement of mannose and cellulose. This physicochemical structure of the three thick-walled cells exhibits high resistance to general physical, chemical, and enzymatic treatments.

[0004] Traditional and widely used methods, including mechanical disruption, ultrasonic disruption, high-pressure homogenization, and microwave-assisted extraction, can disrupt all types of microbial cells, but are neither economical nor efficient. Researchers have followed two basic approaches to address this problem: the first is to develop new equipment and extraction solvents for cell wall disruption extraction, such as supercritical (subcritical) fluid extraction, accelerated solvents, nanoemulsions, and ionic liquid extraction. These technology-dependent methods remain expensive for large-scale applications. The second approach is inspired by nature, such as germination disruption, multi-enzyme degradation, and interference with thick cell wall formation pathways. These methods are energy-efficient and have potential economic and environmental benefits, warranting further improvement to meet the demands of green industry. In the latter nature-inspired strategy, multi-enzyme co-processing has proven effective in degrading the cell walls of Haematococcus pluvialis. However, the large-scale implementation of multi-enzyme cell lysis processes remains challenging due to slow reaction rates, the cost of large-scale enzyme production, and issues with stability and reusability. Summary of the Invention

[0005] Therefore, the technical problem to be solved by this invention is to provide a method for disrupting the cell walls of Haematococcus pluvialis using bio-fermentation and its application. In this method, the target astaxanthin (Ast) and astaxanthin ester (Ast-E) in Haematococcus pluvialis are not affected or are almost unaffected. Simultaneously, the carbohydrates and proteins in the cell walls of Haematococcus pluvialis can be converted into bio-fermentation products, rather than being used as waste after pigment extraction, as animal feed, or further fermented to produce bioethanol.

[0006] This invention provides a method for breaking the cell wall of Haematococcus pluvialis using biological fermentation, comprising the following steps;

[0007] The bran koji was added to either high-temperature sterilized pretreated Haematococcus Pluvialis or untreated Haematococcus Pluvialis for sealed fermentation. After fermentation, the cell wall-broken Haematococcus Pluvialis fermentation broth was obtained.

[0008] Optionally, when Haematococcus pluvialis is not subjected to high-temperature sterilization, the following steps are included:

[0009] 1) Inoculate the bran koji into a carbon source aqueous solution for pre-activation;

[0010] 2) Add untreated Haematococcus pluvialis to the pre-activated bran solution in step 1) and carry out aerobic coexistence culture;

[0011] 3) After aerobic coexistence culture in step 2), sealed fermentation is carried out, and the cell wall broken Haematococcus pluvialis fermentation broth is obtained after fermentation.

[0012] Optionally, the high-temperature sterilization refers to sterilizing Haematococcus pluvialis at 80℃-160℃ for >0 minutes and ≤120 minutes.

[0013] Optionally, the high-temperature sterilization refers to sterilizing Haematococcus pluvialis at 120°C for 15 minutes.

[0014] The cell-wall broken Haematococcus pluvialis fermentation broth obtained after the fermentation of this invention includes cell-wall broken Haematococcus pluvialis and fermentation broth.

[0015] Optionally, the Haematococcus pluvialis can be selected from fresh, mature Haematococcus pluvialis (dehydrated by plate and frame pressing) or Haematococcus pluvialis powder obtained by freeze drying.

[0016] Preferably, the bran koji in step 1) is at least one of Angel Yeast koji, Gutian red yeast rice, Aspergillus oryzae powder, Mucor koji powder, acetic acid bacteria powder, or Aspergillus niger.

[0017] And / or, the inoculation amount of the bran koji is 1-5 wt% of the total mass of water;

[0018] And / or, the mass concentration of the carbon source in the aqueous solution is 1-5 wt%.

[0019] Optionally, the bran koji mentioned in step 1) is Angel baijiu koji;

[0020] Optionally, the inoculum amount of the bran koji is 2 wt% of the total mass of water;

[0021] Optionally, the mass concentration of the carbon source in the aqueous carbon source solution is 2 wt%.

[0022] Optionally, the present invention does not specifically limit the type of carbon source, as long as it is used to provide the nutrients required for the initial growth of the microbial community. Typically, glucose is used as a carbon source without limitation.

[0023] Preferably, the pre-activation temperature in step 1) is 32-36℃, and the pre-activation time is 0.5-1.5h;

[0024] Preferably, the pre-activation temperature in step 1) is 35°C and the pre-activation time is 1 hour;

[0025] Preferably, the ratio of the added mass of Haematococcus pluvialis in step 2) to the inoculation mass of the bran koji is (2-4):2;

[0026] Preferably, the ratio of the added mass of Haematococcus pluvialis in step 2) to the inoculation mass of the bran koji is 3:2.

[0027] Preferably, the aerobic coexistence culture temperature in step 2) is 25-30℃, and the aerobic coexistence culture time is 1-3h.

[0028] Preferably, the aerobic coexistence culture temperature in step 2) is 25-30℃, and the aerobic coexistence culture time is 1.5h.

[0029] Preferably, the sealed fermentation temperature in step 3) is 25-30℃, and the sealed fermentation time is ≥5 days;

[0030] Preferably, the sealed fermentation temperature in step 3) is 28°C, and the sealed fermentation time is 5-10 days;

[0031] And / or, the sealed fermentation includes a headspace oxygen depletion stage and an anaerobic fermentation stage.

[0032] This invention provides a method for extracting carotenoids, which uses the above-described method of using biological fermentation to break the cell wall of Haematococcus pluvialis for pretreatment.

[0033] Preferably, the pretreatment of Haematococcus pluvialis further includes the steps of centrifuging Haematococcus pluvialis and then extracting it with ethanol or acetone to obtain carotenoids;

[0034] Optionally, ultrasonic extraction may be used in the ethanol or acetone extraction step.

[0035] Ultrasound can accelerate the dissolution rate of carotenoids.

[0036] Preferably, the carotenoid is astaxanthin and / or astaxanthin ester.

[0037] The present invention also provides a nutritional beverage, the raw materials of which include the cell wall broken Haematococcus pluvialis fermentation broth or fermentation supernatant obtained by the above-described method of breaking the cell wall of Haematococcus pluvialis through biological fermentation.

[0038] The technical solution of this invention has the following advantages:

[0039] (1) The method for breaking the cell wall of Haematococcus pluvialis by bio-fermentation provided by the present invention includes: adding bran koji to Haematococcus pluvialis that has been pretreated by high temperature sterilization or Haematococcus pluvialis that has not been pretreated for sealed fermentation, and obtaining Haematococcus pluvialis fermentation broth with broken cell wall after fermentation; the present invention uses bran koji fermentation to break the cell wall of Haematococcus pluvialis, which can effectively break the cell wall of Haematococcus pluvialis. In the latter case, the fermentation hardly affects carotenoid components such as astaxanthin.

[0040] (2) The method for breaking the cell wall of Haematococcus pluvialis using biological fermentation provided by this invention includes the following steps when Haematococcus pluvialis is not sterilized at high temperature: 1) Inoculating bran koji into a carbon source aqueous solution for pre-activation; 2) Adding Haematococcus pluvialis to the pre-activated bran koji solution in step 1) for aerobic coexistence culture; 3) After aerobic coexistence culture in step 2), sealing fermentation is carried out, and after fermentation, a cell wall-broth of broken Haematococcus pluvialis fermentation is obtained. This invention uses bran koji fermentation to break the cell wall of Haematococcus pluvialis, including adding carbon source to bran koji for pre-activation. The addition of carbon source provides initial nutrition for the development of microbial community, meeting nutritional needs. The bran koji community proliferates and secretes various cell wall degrading enzymes, providing a basis for continuous hydrolysis. At the same time, bran koji is a common food hydrolysis bacteria source, and the resulting fermentation liquid has a fragrant and rich aroma, which can be used to develop nutritional beverages. After pre-activation, Haematococcus pluvialis is added for co-cultivation in an aerobic environment, allowing the microbial community composition to adapt to environmental changes and aiding in the cell wall disruption of Haematococcus pluvialis. This invention utilizes microbial fermentation in bran koji to effectively break down the cell walls of Haematococcus pluvialis while minimally affecting carotenoid components such as astaxanthin. The resulting fermentation broth has a rich aromatic flavor and can be used in the development of nutritional beverages. Attached Figure Description

[0041] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0042] Figure 1 The operation method and results of Example 1 of the present invention are shown in the figure. In the figure, a is the operation method, b is the relative recovery rate of carotenoids with Comparative Example 1 as a reference, and c is the odor evaluation result. In the figure, S8 represents Example 1, C1 represents Comparative Example 1, and r1, r2, and r3 represent three reviewers.

[0043] Figure 2 The carotenoid recovery results of Example 5 of the present invention (with Comparative Example 1 as a reference); S8 in the figure represents Example 5, and C1 represents Comparative Example 1;

[0044] Figure 3 The operation method and results of Comparative Example 2 of this invention are shown in the figure. In the figure, a is the operation method, b is the relative recovery rate of carotenoids with Comparative Example 1 as a reference, and c is the odor evaluation result. In the figure, C1 represents Comparative Example 1, and r1, r2, and r3 represent three reviewers.

[0045] Figure 4 The operation method and results of Comparative Example 3 of this invention are shown in the figure. In the figure, a is the operation method, b is the relative recovery rate of carotenoids with Comparative Example 1 as a reference, and c is the odor evaluation result. In the figure, C1 represents Comparative Example 1, and r1, r2, and r3 represent three reviewers.

[0046] Figure 5 The figures show the surface morphology of Haematococcus pluvialis cells; a1 and a2 are two fields of view of the cell surface morphology of the original Haematococcus pluvialis powder; b1 and b2 are two fields of view of the cell surface morphology of the Haematococcus pluvialis powder after sterilization at 120°C for 15 min; c1 and c2 are two fields of view of the cell surface morphology of the Haematococcus pluvialis powder after sterilization at 120°C for 15 min and standing for 7 days; d1 and d2 are two fields of view of the cell surface morphology after treatment in Example 5; e1, e2, and e3 are three fields of view of the cell morphology after ethanol extraction (extraction according to Example 4) of the cells treated in Example 5.

[0047] Figure 6 The changes in Ast and Ast-E between Example 1 and Comparative Example 1 are shown in the figure, where "*" and "**" represent P<0.01 and P<0.05, respectively; C1 represents Comparative Example 1, Fer represents Example 1, and T60 and T120 are two control samples, namely, the changes in the relative abundance of astaxanthin and its esters of Haematococcus pluvialis freeze-dried samples sterilized at 60℃ and 120℃ and then ground (according to Comparative Example 1) (with Comparative Example 1 as a reference);

[0048] Figure 7 The differential volatile components and relative abundance of the pre-activated Angel baijiu koji solution in Example 1 of this invention are identified; in the figure, jiuqu_1, jiuqu_2 and jiuqu_3 represent parallel samples of the pre-activated Angel baijiu koji solution; F_L_1, F_L_2, F_L_3, F_L_4 and F_L_5 are parallel samples of the fermentation supernatant of Example 1;

[0049] Figure 8 This is the result of ethanol extraction of astaxanthin and / or astaxanthin ester in Example 4 of the present invention; 1, 2, 3, 4, 5, 6, 7, and 8 in the figure represent the extraction results of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, and 8th extractions, respectively.

[0050] Figure 9 This is the screening result of strains or curds for cell wall disruption of Haematococcus pluvialis in Experiment Example 4 of this invention. Detailed Implementation

[0051] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0052] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0053] The Angel Yeast koji used in the following examples was purchased from Angel Yeast Co., Ltd. (Yichang City, Hubei Province, China);

[0054] Haematococcus pluvialis was purchased from Zhongke Yuhong Biotechnology Co., Ltd.: Fresh Haematococcus pluvialis sludge was transported under dry ice freeze, then repackaged and freeze-dried to obtain freeze-dried powder. The freeze-dried powder was passed through 40-mesh and 100-mesh sieves. The freeze-dried powder that passed through the 40-mesh sieve but was retained by the 100-mesh sieve was stored for later use.

[0055] Example 1

[0056] This embodiment provides a method for disrupting the cell wall of Haematococcus pluvialis using bio-fermentation, such as... Figure 1 As shown in a, it includes the following steps;

[0057] 1) Inoculate 1g of Angel Yeast baijiu starter into 50mL of glucose aqueous solution (2wt%) for pre-activation at 35℃ for 1h. Figure 1 Phase I of a;

[0058] 2) Dispense 10 mL portions of the pre-activated Angel Yeast solution from step 1) into clean glass bottles, add 300 mg of Haematococcus pluvialis powder, and incubate aerobically at 25°C for 1.5 hours. Figure 1 Phase II of a;

[0059] 3) After aerobic coexistence culture in step 2), sealed fermentation is carried out at a fermentation temperature of 28℃ for 120 hours. The sealed fermentation process includes the oxygen depletion stage in the headspace (corresponding to...). Figure 1 The third stage of a) and the anaerobic fermentation stage (corresponding to Figure 1 (Stage IV of a) After fermentation, the cell wall broken Haematococcus pluvialis fermentation broth is obtained.

[0060] Example 2

[0061] This embodiment provides a method for breaking the cell wall of Haematococcus pluvialis using biological fermentation, including the following steps;

[0062] 1) Inoculate 0.5g of Angel Baijiu starter into 50mL of glucose aqueous solution (1wt%) for pre-activation at 32℃ for 1.5h;

[0063] 2) Dispense 10 mL portions of the pre-activated Angel baijiu yeast solution from step 1) into clean glass bottles, add 200 mg of Haematococcus pluvialis to each bottle, and carry out aerobic coexistence culture at a temperature of 25°C for 3 hours.

[0064] 3) After aerobic coexistence culture in step 2), sealed fermentation is carried out at a fermentation temperature of 25℃ for 10 days. The sealed fermentation process includes the oxygen depletion stage in the top space and the anaerobic fermentation stage. After the fermentation is completed, the cell wall broken Haematococcus pluvialis fermentation broth is obtained.

[0065] Example 3

[0066] This embodiment provides a method for breaking the cell wall of Haematococcus pluvialis using biological fermentation, including the following steps;

[0067] 1) Inoculate 2.5g of Angel Baijiu starter into 50mL of glucose aqueous solution (5wt%) for pre-activation at 36℃ for 0.5h.

[0068] 2) Dispense 10 mL portions of the pre-activated Angel Baijiu yeast solution from step 1) into clean glass bottles, add 500 mg of Haematococcus pluvialis to each bottle, and carry out aerobic coexistence culture at a temperature of 30°C for 1 hour.

[0069] 3) After aerobic coexistence culture in step 2), sealed fermentation is carried out at a fermentation temperature of 30℃ for 6 days. The sealed fermentation process includes the oxygen depletion stage in the top space and the anaerobic fermentation stage. After the fermentation is completed, the cell wall broken Haematococcus pluvialis fermentation broth is obtained.

[0070] Example 4

[0071] This embodiment provides a method for extracting astaxanthin and / or astaxanthin esters, comprising: taking the cell-wall-broken Haematococcus pluvialis fermentation broth from Example 1, centrifuging to obtain the Haematococcus pluvialis precipitate at the bottom, then adding ethanol (9 mL) for 10 min of extraction, with ultrasonic assistance during the extraction process, centrifuging to obtain the supernatant, adding 9 mL of ethanol to the residue again, and repeating this process 7 times to achieve complete extraction. The results are as follows. Figure 8As shown in the figure, as the number of extractions increases, the color of the residue gradually fades from red. The red color represents astaxanthin and / or astaxanthin esters. By the 8th extraction, the residue has almost completely faded, indicating that astaxanthin and / or astaxanthin esters have been completely extracted. This further illustrates that the method in Example 1 can break the cell wall of Haematococcus pluvialis.

[0072] Example 5

[0073] This embodiment provides a method for disrupting the cell wall of Haematococcus pluvialis, including the following steps:

[0074] High-temperature sterilization (120℃, 15 min) was employed. 300 mg of *Haematococcus pluvialis* was mixed with 10 mL of water and sterilized. After cooling, 30 mg of Angel Yeast koji was inoculated, and sealed fermentation began at 28℃ for 120 h. The sealed fermentation process included an oxygen depletion stage in the headspace and an anaerobic fermentation stage. After fermentation, a cell-wall-broth-broken *Haematococcus pluvialis* fermentation broth was obtained. Electron microscopy was performed, and the results are as follows: Figure 5 As shown in d1 and d2.

[0075] The obtained Haematococcus pluvialis fermentation broth with broken cell walls was extracted with ethanol according to the method in Example 4, and then observed under an electron microscope. The results are as follows. Figure 5 As shown in e1, e2 and e3.

[0076] Set up a control group at the same time, such as Figure 5 In the diagram, a1 and a2 are two fields of view of the cell surface morphology of the original Haematococcus pluvialis powder; b1 and b2 represent two fields of view of the cell surface morphology of the Haematococcus pluvialis powder after sterilization at 120°C for 15 min; c1 and c2 represent two fields of view of the cell surface morphology of the Haematococcus pluvialis powder after sterilization at 120°C for 15 min and standing for 7 days.

[0077] The above results demonstrate that the method of the present invention can disrupt the cell walls of Haematococcus pluvialis.

[0078] Comparative Example 1

[0079] This comparative example provides a method for completely disrupting the cell wall of Haematococcus pluvialis by grinding, including the following steps:

[0080] Grind 300 mg of Haematococcus pluvialis using a 20 mL homogenizer, add 2-5 mL of acetone solvent, and maintain relative motion between the grinding rod and the grinding sleeve at 100 rpm for 1 hour. The ground algal powder is then diluted to 100 mL with acetone and can be used as a control for complete extraction experiments.

[0081] Comparative Example 2

[0082] This comparative example provides a method for disrupting the cell wall of Haematococcus pluvialis, including the following steps:

[0083] Pasteurization (60℃, 30 min) was performed by mixing 300 mg of Haematococcus pluvialis with 10 mL of water and then sterilizing. After cooling, Angel Yeast baijiu starter at concentrations of 30 mg, 60 mg, 90 mg, 120 mg, and 150 mg was inoculated. Seal fermentation was then initiated at 28℃ for 120 hours, including a stage where the oxygen in the headspace was depleted (corresponding to...). Figure 3 The third stage of a) and the anaerobic fermentation stage (corresponding to Figure 3 (Stage IV of a) After fermentation, the cell wall broken Haematococcus pluvialis fermentation broth is obtained.

[0084] Comparative Example 3

[0085] This comparative example provides a method for cell wall disruption of Haematococcus pluvialis, including the following steps:

[0086] Pasteurization (60℃, 30 min) was performed by mixing 300 mg of Haematococcus pluvialis with 10 mL of water (containing 1% glucose) and then sterilizing. After cooling, Angel Yeast koji (30 mg, 60 mg, 90 mg, 12 mg, and 150 mg) was inoculated. Following inoculation, aerobic co-cultivation was carried out at 25℃ for 2 hours. Figure 4 Phase II of the process: After aerobic coexistence culture, sealed fermentation begins at 28℃ for 120 hours. The sealed fermentation process includes a stage where the oxygen in the headspace is depleted (corresponding to...). Figure 4 The third stage of a) and the anaerobic fermentation stage (corresponding to Figure 4 (Stage IV of a) After fermentation, the cell wall broken Haematococcus pluvialis fermentation broth is obtained.

[0087] Experimental Example 1

[0088] Comparative Example 1 was ground and then diluted to 100 mL with acetone solution. The supernatant was collected for later use. The fermentation broths of Examples 1, 5, and Comparative Examples 2 and 3 were freeze-dried to obtain dried powder, which was then extracted directly with 100 mL of acetone at 100 rpm under light for 2 hours. Comparative Example 1 represents a recognized method for complete cell disruption. The total integrated area of ​​the liquid chromatogram of the supernatant of Comparative Example 1 within the 14-28 min interval was used as a control. The percentage values ​​were obtained by comparing the total integrated area of ​​the liquid chromatogram of the supernatant of the fermentation broths of Examples 1, 5, and Comparative Examples 2 and 3 within the 14-28 min interval with Comparative Example 1. The average value of parallel experiments was taken, and the relative recovery content of carotenoids in Examples 1, 5, and Comparative Examples 2 and 3 was compared and analyzed. The results are as follows: Figure 1 b in Figure 2 , Figure 3 b in Figure 4As shown in b in the figure. The above results show that the relative recovery content of carotenoids in Example 1 of the present invention is significantly higher than that in Comparative Examples 2 and 3.

[0089] The method for HPLC chromatographic analysis of the supernatant obtained from the products of Examples 1, 5, and Comparative Examples 2 and 3 is as follows:

[0090] Take 1 mL of the supernatant and filter it through a 0.22 μm filter membrane for liquid chromatography analysis. HPLC chromatographic conditions: Waterse2695 with a 2998 PDA detector; YMC C30 4.6 × 250 mm column; mobile phase and elution program (A: water; B: methanol; D: methyl tert-butyl ether): 0 min, 4% A, 81% B, 15% D; 15 min, 4% A, 66% B, 30% D; 23-27 min, 4% A, 16% B, 80% D; 29-35 min, 4% A, 81% B, 15% D. Injection volume: 20 μL; detection wavelength: 480 nm; column temperature: 35 °C; flow rate: 1 mL / min.

[0091] Experimental Example 2

[0092] Non-targeted metabolomics analysis based on UHPLC-MS was conducted to analyze the chemical differences between astaxanthin and its esters extracted from Haematococcus pluvialis after treatment with Example 1 and Comparative Example 1 (i.e., raw Haematococcus pluvialis powder). Two control samples, T60 and T120, were also set up to represent the changes in Haematococcus pluvialis sample components caused by conventional heat treatments at 60℃ and 120℃, respectively.

[0093] like Figure 6 As shown, this is a global view of the chemical differences between astaxanthin and its ester phases obtained by different treatment methods. The initial algal powder extract after grinding in Comparative Example 1 is denoted as C1, and the extract after treatment in Example 1 is denoted as Fer. To compare with the state of algal powder after pasteurization and high-temperature sterilization, extracts obtained from grinding Haematococcus pluvialis after pasteurization (60℃, 30 min) and high-temperature sterilization (120℃, 15 min) were also compared and denoted as T60 and T120, respectively. Compared with Comparative Example 1 (C1), the fold change in the abundance of astaxanthin and its ester-related molecules in the Fer / C1 group was less than 2, indicating that most components did not undergo significant changes.

[0094] Experimental Example 3

[0095] The aroma of the Haematococcus pluvialis fermentation broth obtained in Example 1 was evaluated, and the volatile components and relative abundance of the components in the fermentation supernatant were identified.

[0096] Aroma evaluation test standards and test results are as follows: Figure 1 c in Figure 3 c in Figure 4 As shown in c, the aroma evaluation of Example 1 was the highest, while that of Comparative Example 2 and Comparative Example 3 was poor.

[0097] The results of Example 5 are compared with Comparative Example 1 (C1). Although adding yeast after high temperature and high pressure sterilization can break the cell wall, it cannot maintain a good aroma, and the aroma score is <10.

[0098] Identification of volatile components and relative abundance of components in fermentation supernatant (Example 1) as follows: Figure 7 As shown, the supernatant obtained by centrifugation was analyzed by headspace solid phase microextraction (GCMS), with pre-activated Angel baijiu koji solution as a control. 37 differentially volatile components were screened by the OPLS-DA differential analysis model (S-plot, VIP>1). The molecules of each substance were identified by the NIST2017 spectral library. Based on the physicochemical odor characteristics of the differentially volatile molecules, this invention suggests that the strong sweet aroma of the fermentation supernatant is caused by characteristic sweet volatile substances such as ethyl octanoate, hexyl formate, and phenylethyl butyrate.

[0099] The above results indicate that although Example 5 completely disrupted the cell wall, it had an unpleasant odor, and the high-temperature sterilization treatment affected the components. While Comparative Example 3 improved the odor, Comparative Examples 2 and 3 failed to effectively disrupt the cell wall. In contrast, the cell wall disruption method provided by this invention yields a fragrant fermentation broth that effectively disrupts the cell wall while barely affecting the functional components within Haematococcus pluvialis. This method enables cell wall disruption through bio-fermentation, and the fermentation broth is also beneficial for development and application in nutritional beverages.

[0100] Experiment Example 4

[0101] This experimental example provides a method for screening strains or cultivars for cell wall disruption of Haematococcus pluvialis, including the following steps:

[0102] 1) Weigh out 300mg of Haematococcus pluvialis powder, add 10mL of pure water, sterilize at 121℃ for 20min, then transfer to a clean bench for inoculation or koji preparation, and incubate at 28℃ for one week. The 12 hydrolytic bacterial sources used include lactic acid bacteria (S1), Bacillus subtilis (S2), Lactobacillus plantarum (S3), Pichia pastoris (S4), yeast (S5), *Aspergillus cristatus* (S6), *Aspergillus niger* (S7), Angel Yeast koji (S8), Gutian red yeast rice (S9), *Aspergillus oryzae* powder (S10), *Mucor koji* powder (S11), and acetic acid bacteria powder (S12). Single-strain inoculation uses activated high-concentration bacterial solutions, while koji inoculation is done by direct addition. All 12 hydrolytic bacterial sources are commercially available products.

[0103] 2) After the sample from step 1) is freeze-dried for 7 days, weigh 30mg of powder and add 6mL of acetone for extraction.

[0104] Control groups C2 and C3 were set up simultaneously. C2 was the sample sterilized at 120℃ for 15 minutes; C3 was the sample sterilized at 120℃ for 15 minutes and stored for 7 days.

[0105] The filtering results are as follows Figure 9 As shown, the algae powder with broken cell walls has a distinct deep red color, such as... Figure 9 It can be seen that the hydrolyzed koji of S7-S12 can break the cell wall of Haematococcus pluvialis, among which S8 Angel Baijiu koji has the darkest color and the best cell wall breaking effect on Haematococcus pluvialis.

[0106] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art can make other variations or modifications, or expand the range of parameter operations, based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for disrupting the cell wall of Haematococcus pluvialis using biological fermentation, characterized in that, The method includes the following steps: The bacterial strain or koji was added to a carbon source aqueous solution for pre-activation at 32-36 °C for 0.5-1.5 h to obtain a pre-activated solution. The pre-activated solution was then added to a pre-treated Haematococcus pluvialis solution that had undergone high-temperature sterilization. Haematococcus Pluvialis ) or untreated Haematococcus pluvialis ( Haematococcus Pluvialis The algae were subjected to aerobic coexistence and sealed fermentation treatment, and the cell wall broken Haematococcus pluvialis fermentation broth was obtained after the sealed fermentation treatment was completed. The strains or kojis are Aspergillus niger, Angel baijiu koji, Gutian red yeast rice, Aspergillus oryzae powder, Mucor koji powder and / or acetic acid bacteria powder. The steps of the aerobic coexistence treatment are as follows: the pre-activated solution is added to the Haematococcus pluvialis pretreated by high-temperature sterilization. Haematococcus Pluvialis ) or untreated Haematococcus pluvialis ( Haematococcus Pluvialis They were co-cultured for 1-3 hours at a temperature of 25-30℃ under aerobic conditions. The steps of the sealed fermentation treatment are as follows: after the aerobic coexistence treatment, the aerobic coexistence treatment product is sealed for fermentation at a temperature of 25-30 °C for 5-10 days. The sealed fermentation includes a headspace oxygen depletion stage and an anaerobic fermentation stage.

2. The method for disrupting the cell wall of Haematococcus pluvialis using bio-fermentation according to claim 1, characterized in that, The high-temperature sterilization refers to sterilizing Haematococcus pluvialis at 80℃-160℃ for >0 minutes and ≤120 minutes.

3. The method for disrupting the cell wall of Haematococcus pluvialis using bio-fermentation according to claim 2, characterized in that, The high-temperature sterilization refers to sterilizing Haematococcus pluvialis at 120°C for 15 minutes.

4. The method for disrupting the cell wall of Haematococcus pluvialis using bio-fermentation according to claim 1, characterized in that, The inoculation amount of the strain or koji is 1-5 wt% of the total mass of the carbon source aqueous solution; And / or, the mass concentration of the carbon source in the aqueous solution is 1-5 wt%.

5. The method for disrupting the cell wall of Haematococcus pluvialis using bio-fermentation according to claim 4, characterized in that, The strain or starter culture is Angel Yeast Baijiu starter culture.

6. The method for disrupting the cell wall of Haematococcus pluvialis using bio-fermentation according to claim 4, characterized in that, The inoculation amount of the strain or koji is 2 wt% of the total mass of the carbon source aqueous solution.

7. The method for disrupting the cell wall of Haematococcus pluvialis using bio-fermentation according to claim 4, characterized in that, The carbon source in the aqueous solution has a carbon source concentration of 2 wt%.

8. The method for disrupting the cell wall of Haematococcus pluvialis using bio-fermentation according to claim 1, characterized in that, The ratio of the added mass of Haematococcus pluvialis to the inoculation mass of the bacterial strain or cultivar is (2-4):

2.

9. The method for disrupting the cell wall of Haematococcus pluvialis using bio-fermentation according to claim 1, characterized in that, The ratio of the added mass of Haematococcus pluvialis to the inoculation mass of the bacterial strain or cultivar is 3:

2.

10. A method for extracting carotenoids, characterized in that, The method for pretreating Haematococcus pluvialis using bio-fermentation as described in any one of claims 1-9 is used.

11. The method for extracting carotenoids according to claim 10, characterized in that, The pretreatment of Haematococcus pluvialis also includes centrifugation to separate Haematococcus pluvialis and extraction with ethanol or acetone to extract carotenoids.

12. The method for extracting carotenoids according to claim 11, characterized in that, Ultrasonic extraction is used in the ethanol or acetone extraction steps.

13. The method for extracting carotenoids according to any one of claims 10-12, characterized in that, The carotenoids are astaxanthin and / or astaxanthin esters.

14. A nutritional beverage, characterized in that, The raw materials include the cell wall broken Haematococcus pluvialis fermentation broth obtained by the method of breaking the cell wall of Haematococcus pluvialis using biological fermentation as described in any one of claims 1-9.

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