A cyanobacteria active extract, and a preparation method and application thereof

Abstract

This application relates to a cyanobacterial active extract, its preparation method, and its application, belonging to the field of cosmetic raw material technology. The method includes: pulverizing cyanobacterial raw material and mixing it with water to form a cyanobacterial suspension system; performing primary enzymatic hydrolysis on the cyanobacterial suspension system: adjusting the system pH to 5.0-5.5, adding a complex enzyme system composed of cellulase and pectinase, and performing enzymatic hydrolysis at 45-50℃; secondary enzymatic hydrolysis: adjusting the system pH to 6.8-7.2, adding neutral protease to continue enzymatic hydrolysis; performing low-temperature pulsed ultrasonic extraction, controlling the system temperature at 8-15℃, the ultrasonic frequency at 20-35kHz, the power density at 250-450W / L, and the duty cycle at 40-70% during ultrasonic extraction; and performing separation and purification to obtain the cyanobacterial active extract. The preparation process of this application can improve the release efficiency of cyanobacterial active ingredients while reducing heat damage and improving the extract's activity retention rate and stability.

Description

Technical Field

[0001] This application relates to an active extract of cyanobacteria, its preparation method and application, belonging to the field of cosmetic raw material technology. Background Technology

[0002] In recent years, cyanobacteria have received widespread attention in functional foods, cosmetics, and biomedicine due to their rich content of phycocyanin, polysaccharides, bioactive peptides, and natural antioxidants. Among these, phycocyanin and other bioactive substances have high nutritional and functional value, but they also suffer from poor thermal stability and are prone to structural inactivation. Therefore, high requirements are placed on the extraction process and subsequent stabilization treatment.

[0003] Existing extraction processes for active ingredients from cyanobacteria mainly include hot water extraction, solvent extraction, mechanical cell disruption extraction, and conventional ultrasound-assisted extraction. While hot water extraction is simple to operate, the high temperatures can easily denature and inactivate heat-sensitive active ingredients such as phycocyanin. Mechanical stirring or conventional cell disruption methods have limited effect on the cyanobacterial cell walls, resulting in insufficient release of intracellular active substances and low extraction yields. Although conventional continuous ultrasound extraction can improve extraction efficiency to some extent, the continuous energy input during the ultrasound process can easily cause localized heat accumulation and free radical oxidation, thereby reducing the retention rate of active ingredients.

[0004] In addition, cyanobacterial extracts obtained by existing technologies usually have problems such as large particle size, insufficient dispersibility, and poor system stability. They are prone to aggregation, sedimentation, or activity decay during storage, making it difficult to meet the requirements of functional products for long-term stability and batch consistency.

[0005] Therefore, in existing cyanobacteria extraction technologies, improving extraction yield is often accompanied by increased loss of heat-sensitive active ingredients and decreased system stability, making it difficult to simultaneously obtain cyanobacteria extraction systems with high activity retention, small particle size dispersibility, and long-term storage stability. How to improve the release efficiency of cyanobacteria active ingredients while reducing heat damage, and simultaneously ensuring extraction yield, activity retention, particle size uniformity, and long-term stability, has become a pressing technical problem to be solved in this field. Summary of the Invention

[0006] To address the aforementioned issues, this application provides a cyanobacterial active extract, its preparation method, and its application. The method provided here utilizes gradient enzymatic hydrolysis combined with low-temperature pulsed ultrasound to enhance the release efficiency of cyanobacterial active ingredients while reducing thermal damage and improving the extract's activity retention rate and stability.

[0007] This application provides a method for preparing an active extract of cyanobacteria, comprising the following steps: 1) After pulverizing the cyanobacteria raw material, it is mixed with water to form a cyanobacteria suspension system; 2) Perform gradient enzymatic hydrolysis on the cyanobacteria suspension system, including: Primary enzymatic hydrolysis: Adjust the pH of the system to 5.0~5.5, add a complex enzyme system consisting of cellulase and pectinase, and carry out enzymatic hydrolysis at 45~50℃; Secondary enzymatic hydrolysis: After the primary enzymatic hydrolysis is completed, adjust the pH of the system to 6.8-7.2, and add neutral protease to continue enzymatic hydrolysis; 3) The enzymatically hydrolyzed system was subjected to low-temperature pulsed ultrasonic extraction. During the ultrasonic extraction process, the system temperature was controlled at 8~15℃, the ultrasonic frequency at 20~35kHz, the power density at 250~450W / L, and the duty cycle at 40~70%. 4) The system after ultrasonic extraction is separated and purified to obtain the active extract of cyanobacteria.

[0008] When the ultrasonic power density is below 250 W / L, cavitation is insufficient and the cell wall breaking efficiency of cyanobacteria is low. When the ultrasonic power density is above 450 W / L, local heat accumulation is enhanced, which can easily lead to the inactivation of heat-sensitive active ingredients such as phycocyanin. Therefore, this application preferably controls the ultrasonic power density at 250~450 W / L.

[0009] Optionally, in step 2), the mass ratio of cellulase to pectinase is (1.5~2.5):(1~2), and the total amount of compound enzyme added is 1.5~3.5% of the mass of cyanobacteria raw material.

[0010] Optionally, in step 2), the first-stage enzymatic hydrolysis time is 30-60 min, and the second-stage enzymatic hydrolysis time is 20-40 min.

[0011] Optionally, in step 3), the low-temperature pulse ultrasound adopts a pulse mode of working for 5 seconds and pausing for 3 seconds, and the low-temperature pulse ultrasound processing time is 15~35 minutes.

[0012] Optionally, the microfiltration uses a microfiltration membrane with a pore size of 0.22 μm, and the ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 10 kDa. Optionally, in step 1), the cyanobacteria raw material is pulverized and then passed through an 80-120 mesh sieve.

[0013] Optionally, in step 1), the ratio of cyanobacteria raw material to water is 1:18 to 1:25 (g:mL).

[0014] Optionally, in step 1), the cyanobacterial raw material is one or more of Spirulina, Anabaena, or Nostoc. Optionally, the cyanobacterial raw material is spirulina.

[0015] Optionally, in step 2), the stirring speed during the first-stage enzymatic hydrolysis is 200~350 rpm.

[0016] Optionally, in step 2), after the secondary enzymatic hydrolysis is completed, the system is heated to 80-90°C and maintained for 5-15 minutes to inactivate the enzyme preparation.

[0017] Optionally, the ultrasonic probe is inserted 2-5 cm below the liquid surface.

[0018] Optionally, in step 4), the centrifugation temperature is 2~8℃.

[0019] Optionally, in step 4), the ultrafiltration pressure is 0.1~0.4MPa.

[0020] Optionally, in step 4), the concentrate is subjected to... 40~ Pre-freeze at 60℃ and then freeze-dry.

[0021] Optionally, the highest temperature of the system during the low-temperature pulsed ultrasound process does not exceed 15°C.

[0022] This application provides the active extract of cyanobacteria obtained by the above preparation method.

[0023] Optionally, the cyanobacterial active extract meets the following performance requirements: 1) Average particle size D50 ≤ 180 nm; 2) DPPH free radical scavenging rate ≥92%; 3) The absolute value of the Zeta potential is ≥28mV; 4) The activity retention rate is ≥90% after storage at 40℃ for 30 days.

[0024] This application provides the use of the above-mentioned active blue algae extract in cosmetic compositions.

[0025] The beneficial effects of this application include, but are not limited to: 1. According to the active extract of cyanobacteria, its preparation method and application, by using gradient enzymatic hydrolysis and low-temperature pulsed ultrasound in synergistic treatment, it is possible to improve the cell wall disruption efficiency and intracellular active ingredient release efficiency of cyanobacteria while reducing heat accumulation and heat-sensitive active ingredient inactivation during ultrasound, thereby achieving a synergistic improvement in extraction yield and activity retention rate.

[0026] 2. Based on the cyanobacterial active extract and its preparation method and application of this application, a staged enzymatic hydrolysis mechanism is adopted, which utilizes cellulase, pectinase and neutral protease to act on the cyanobacterial cell wall structure and intracellular bound components respectively, to achieve the stepwise dissociation of the cyanobacterial cell structure, avoid the degradation of active ingredients caused by a single strong cell wall disruption treatment, and improve the retention effect of phycocyanin and active peptides in the cyanobacterial active extract.

[0027] 3. Based on the active extract of cyanobacteria, its preparation method and application in this application, a low-temperature pulsed ultrasonic extraction method is adopted. The uniformity of cavitation is improved by intermittent energy input, and the temperature rise of the system is controlled by low-temperature circulation. This effectively reduces the local high temperature and oxidative damage caused by continuous ultrasound, and improves the stability of the extraction process and the stability of heat-sensitive active substances.

[0028] 4. According to the active extract of cyanobacteria and its preparation method and application in this application, a mild separation and purification process combining microfiltration, ultrafiltration and freeze drying is used to avoid further deactivation of active ingredients during the traditional high-temperature concentration process. The resulting active extract of cyanobacteria has good dispersibility, small particle size and high storage stability, which is beneficial to the application of subsequent functional products. Detailed Implementation

[0029] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments. Unless otherwise specified, the raw materials and reagents in the embodiments of the present application are all purchased through commercial channels.

[0030] The present application solution will be described below through specific embodiments.

[0031] Example 1 1) Pretreatment of cyanobacteria raw materials Select food-grade spirulina ( Spirulina platensis The raw material is a dry powder containing 58.6% protein, 13.2% phycocyanin, and ≤5% moisture. The spirulina powder is pulverized using an air jet mill and then sieved through a 100-mesh standard sieve to obtain spirulina powder with an average particle size of 125 μm. 1000 g of spirulina powder is weighed and added to 20 L of deionized water at a material-to-liquid ratio of 1:20 (g:mL). The mixture is soaked at 25℃ for 30 min, during which time it is continuously stirred at 200 rpm using a mechanical stirrer.

[0032] 2) Gradient enzymatic hydrolysis treatment 2.1) Primary enzymatic hydrolysis A composite enzyme system consisting of food-grade cellulase and pectinase was used for primary enzymatic hydrolysis. The cellulase activity was 10000 U / g, derived from Trichoderma reesei ( Trichoderma reesei The pectinase activity was 8000 U / g, derived from Aspergillus niger (…). As pergillus niger).

[0033] First, the pH of the system was adjusted to 5.2 using a 1 mol / L citrate-sodium citrate buffer solution. Then, 20 g of cellulase and 10 g of pectinase were added, with a cellulase to pectinase mass ratio of 2:1, and the total enzyme addition was 3% of the cyanobacteria mass. Primary enzymatic hydrolysis was carried out at 48℃ for 45 min, with continuous stirring at 280 rpm using a paddle stirrer during the hydrolysis process.

[0034] 2.2) Secondary enzymatic hydrolysis After the first-stage enzymatic hydrolysis, the pH of the system was adjusted to 6.9 using a 1 mol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution. Then, 30 g of food-grade neutral protease with an enzyme activity of 50,000 U / g, derived from Bacillus subtilis, was added. Bacillus subtilis Continue enzymatic hydrolysis at 50℃ for 30 min, maintaining a stirring speed of 280 rpm. After enzymatic hydrolysis, raise the temperature to 85℃ and hold for 10 min to inactivate the enzyme preparation.

[0035] 3) Low-temperature pulsed ultrasonic extraction The enzymatically hydrolyzed system was introduced into a jacketed low-temperature circulating ultrasonic reactor for low-temperature pulsed ultrasonic extraction. The ultrasonic equipment used an immersion-type titanium alloy ultrasonic probe with a diameter of 25 mm, inserted 3 cm below the liquid surface. The ultrasonic parameters were set as follows: ultrasonic frequency: 28 kHz; actual ultrasonic output power: 700 W; power density: 350 W / L; duty cycle: 60%; pulse mode: 5 s on, 3 s pause; ultrasonic treatment time: 25 min. During the ultrasonic process, an ethylene glycol low-temperature circulating system was used to control the reactor jacket temperature, maintaining the system temperature at 10 ± 2 °C, and ensuring the highest system temperature did not exceed 14 °C during treatment.

[0036] 4) Separation and purification After ultrasonic extraction, the extract was centrifuged at 8000 rpm for 15 min at 4°C (centrifuge rotor radius: 10 cm, relative centrifugal force: approximately 7150 × g). The supernatant was collected and filtered through a 0.22 μm pore size polyvinylidene fluoride (PVDF) microfiltration membrane to remove large, incompletely broken particles. The filtered extract was then concentrated using a 10 kDa molecular weight cutoff polyethersulfone (PES) ultrafiltration membrane at a pressure of 0.25 MPa and a concentration factor of 4. The concentrated extract was then placed in a [further processing facility]. Pre-freezing at 50℃ for 12 hours and then freeze-drying at a vacuum degree not exceeding 20 Pa for 48 hours yielded a blue-green active extract powder. The resulting extract was a blue-green powder with good water dispersibility.

[0037] Comparative Example 1 This comparative example is basically the same as Example 1, except that a traditional hot water extraction method is used instead of the gradient enzymatic hydrolysis and low-temperature pulsed ultrasonic extraction of this application: 1000g of Spirulina powder is weighed and added to 20L of deionized water, with a material-to-liquid ratio of 1:20 (g:mL). Extraction is carried out at 80℃ with mechanical stirring for 2 hours at a stirring speed of 300rpm. After extraction, the mixture is centrifuged at 8000rpm for 15min at room temperature, and the supernatant is collected. After filtration through a 0.22μm microfiltration membrane, the supernatant is directly freeze-dried to obtain the cyanobacterial extract.

[0038] Comparative Example 2 This comparative example is essentially the same as Example 1, except that a conventional continuous ultrasonic extraction method is used instead of the low-temperature pulsed ultrasonic extraction of this application: After completing the gradient enzymatic hydrolysis step in Example 1, the enzymatic hydrolysis system is placed in a conventional ultrasonic reactor for continuous ultrasonic treatment. The ultrasonic frequency is 28 kHz, the output power is 700 W, the power density is 350 W / L, and it operates continuously for 25 min without pulse mode or low-temperature cycle control. The highest temperature of the system during the ultrasonic process reaches 32°C.

[0039] Comparative Example 3 This comparative example is basically the same as Example 1, except that no enzymatic hydrolysis is performed, and only mechanical stirring is used for extraction: 1000g of Spirulina powder is weighed, 20L of deionized water is added, and after soaking at 25℃ for 30min, it is extracted at 50℃ for 2h using mechanical stirring at a stirring speed of 300rpm.

[0040] Comparative Example 4 This comparative example is basically the same as Example 1, except that only the first-stage enzymatic hydrolysis is performed, and the second-stage enzymatic hydrolysis is not performed: after completing the first-stage enzymatic hydrolysis step in Example 1, no neutral protease is added, and low-temperature pulsed ultrasonic extraction is performed directly.

[0041] Comparative Example 5 This comparative example is basically the same as Example 1, except that only secondary enzymatic hydrolysis is performed, and primary enzymatic hydrolysis is not performed: 1000g of Spirulina powder is weighed, the pH of the system is directly adjusted to 6.9, and 30g of neutral protease is added for enzymatic hydrolysis. The enzymatic hydrolysis conditions are: temperature 50℃; time 30min; stirring speed 280rpm.

[0042] Comparative Example 6 This comparative example is basically the same as Example 1, except that a single-step mixed enzymatic hydrolysis method is used instead of gradient enzymatic hydrolysis: 20g of cellulase, 10g of pectinase and 30g of neutral protease are added to the cyanobacteria suspension system at the same time, the pH of the system is uniformly adjusted to 6.0, and enzymatic hydrolysis is carried out at 50°C for 60min.

[0043] Comparative Example 7 This comparative example is basically the same as Example 1, except that the enzymatic hydrolysis order is different: first, the pH of the system is adjusted to 6.9, 30g of neutral protease is added, and enzymatic hydrolysis is carried out at 50°C for 30min; then, the pH of the system is adjusted to 5.2, 20g of cellulase and 10g of pectinase are added, and enzymatic hydrolysis is carried out at 48°C for 45min.

[0044] Comparative Example 8 This comparative example is basically the same as Example 1, except that in the first-stage enzymatic hydrolysis process, the mass ratio of cellulase to pectinase is adjusted to 5:1, the amount of cellulase added is 25g, and the amount of pectinase added is 5g.

[0045] Comparative Example 9 This comparative example is basically the same as Example 1, except that after completing the gradient enzymatic hydrolysis step in Example 1, the pulse mode was canceled during the ultrasonic treatment process, and continuous working mode was used for ultrasonic extraction with an ultrasonic frequency of 28kHz, a power density of 350W / L, and an ultrasonic time of 25min.

[0046] Comparative Example 10 This comparative example is basically the same as Example 1, except that after completing the gradient enzymatic hydrolysis step in Example 1, pulsed ultrasonic extraction was performed at room temperature, without using a low-temperature circulation system, and the highest temperature of the system during the ultrasonic process reached 35°C.

[0047] Comparative Example 11 This comparative example is basically the same as Example 1, except that after completing the gradient enzymatic hydrolysis step in Example 1, the ultrasonic frequency was adjusted to 45 kHz.

[0048] Comparative Example 12 This comparative example is basically the same as Example 1, except that after completing the gradient enzymatic hydrolysis step in Example 1, the ultrasonic power density was adjusted to 150W / L.

[0049] Comparative Example 13 This comparative example is basically the same as Example 1, except that after completing the gradient enzymatic hydrolysis step in Example 1, the ultrasonic power density was adjusted to 600W / L.

[0050] Comparative Example 14 This comparative example is basically the same as Example 1, except that after completing the centrifugation, microfiltration and ultrafiltration steps in Example 1, the blue-green algae extract was obtained by spray drying. The spray drying conditions are as follows: inlet air temperature 160°C; outlet air temperature 85°C.

[0051] Comparative Example 15 This comparative example is basically the same as Example 1, except that after completing the centrifugation and microfiltration steps in Example 1, a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 8kDa was used for concentration.

[0052] Comparative Example 16 This comparative example is basically the same as Example 1, except that after completing the centrifugation and microfiltration steps in Example 1, a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 12kDa was used for concentration.

[0053] Test Example 1 The performance of the cyanobacterial active extracts obtained in Example 1 and each comparative example was tested to evaluate the effects of different process conditions on the release efficiency of cyanobacterial active ingredients, retention of heat-sensitive components, antioxidant activity, dispersion stability and long-term storage stability.

[0054] The extraction yield is used to characterize the release efficiency of active ingredients from cyanobacteria. During testing, the final freeze-dried extract is weighed, and the extraction yield is calculated using the following formula: Extraction yield (%) = Mass of extract / Mass of cyanobacteria raw material × 100%.

[0055] Phycocyanin retention rate was used to characterize the retention effect of thermosensitive active ingredients during extraction. It was determined using ultraviolet spectrophotometry at wavelengths of 620 nm and 652 nm, and the phycocyanin content was calculated according to the formula. The phycocyanin retention rate was further calculated as the ratio of the extracted phycocyanin content to the phycocyanin content in the raw material, expressed as a percentage (%).

[0056] DPPH radical scavenging rate was used to characterize the antioxidant activity of the extract. The DPPH ethanol system method was used for determination, with absorbance measured at a wavelength of 517 nm. The DPPH radical scavenging rate (%) was calculated using the following formula: DPPH radical scavenging rate (%) = [1-(A1-A2) / A0]×100%, where A0 is the absorbance of the blank group, A1 is the absorbance of the sample group, and A2 is the background absorbance of the sample.

[0057] Average particle size D 50 This was used to characterize the uniformity of extract particle dispersion. Dynamic light scattering particle size analyzer was used for detection at 25℃, and the results are expressed in nanometers (nm). Wherein, D... 50 This indicates that 50% of the particles in the system have a diameter smaller than this value.

[0058] Zeta potential is used to characterize the dispersion stability of the extract system. It is detected using electrophoretic light scattering, and the results are expressed in mV. A larger absolute value of the zeta potential indicates higher dispersion stability of the system.

[0059] Long-term storage stability was evaluated using an accelerated stability test at 40℃. Samples were stored at 40℃ in the dark for 30 days, and the phycocyanin content before and after storage was measured. The activity retention rate was calculated using the following formula: Activity retention rate (%) = Phycocyanin content after storage / Phycocyanin content before storage × 100%.

[0060] All of the above tests were performed in triplicate, and the results are expressed as mean ± standard deviation, as shown in Table 1 below.

[0061] Table 1. Detection results of the extracts obtained in Example 1 and the comparative example.

[0062] Table 1 (Continued)

[0063] As shown in Table 1, the cyanobacterial active extract obtained in Example 1 exhibits superior overall performance in terms of extraction yield, phycocyanin retention rate, antioxidant activity, particle size uniformity, and long-term storage stability. Compared with the comparative examples, Example 1 not only improves the release efficiency of cyanobacterial active ingredients but also effectively reduces damage to heat-sensitive active ingredients, achieving a synergistic improvement in extraction yield and activity retention rate.

[0064] Comparative Example 1 used a traditional hot water extraction method, which achieved a certain extraction yield, but its phycocyanin retention rate was only 48.2%, and its activity retention rate was only 52.6%, significantly lower than that of Example 1. This indicates that high-temperature extraction easily leads to denaturation and inactivation of heat-sensitive active ingredients such as phycocyanin. Meanwhile, the D of Comparative Example 1... 50 The particle size reached 486 nm, and the absolute value of the Zeta potential was only 12.5 mV, indicating that the obtained system had a large particle size and poor dispersion stability, further illustrating that traditional thermal extraction methods cannot simultaneously achieve activity retention and system stability.

[0065] Comparative Example 3, without enzymatic hydrolysis, had an extraction yield of only 11.8%, significantly lower than the 21.8% of Example 1. This indicates that simple mechanical stirring is insufficient to effectively disrupt the cyanobacterial cell structure, resulting in insufficient release of intracellular active ingredients. Comparative Examples 4 and 5, which underwent only primary or secondary enzymatic hydrolysis respectively, showed lower extraction yields, DPPH free radical scavenging rates, and activity retention rates than Example 1. This demonstrates that a single enzymatic hydrolysis method is insufficient to simultaneously achieve graded cell wall dissociation and sufficient release of intracellular bound active ingredients. In contrast, Example 1 employed a gradient enzymatic hydrolysis method, utilizing cellulase, pectinase, and neutral protease to act on the cell wall structure and intracellular bound components, respectively. This effectively improved the efficiency of cyanobacterial cell wall disruption and the release efficiency of active ingredients, while reducing the activity damage caused by a single, intense cell wall disruption.

[0066] Comparative Example 6 used a single-step mixed enzymatic hydrolysis method, while Comparative Example 7 changed the enzymatic hydrolysis sequence. Although both methods improved the extraction yield, the phycocyanin retention rate, activity retention rate, and particle size stability were all lower than in Example 1, indicating that the gradient enzymatic hydrolysis sequence used in this application has significant technical value. Through staged enzymatic hydrolysis, the cyanobacterial cell structure can be dissociated step by step, avoiding mutual interference between different enzyme preparations, which is more conducive to the stable release of active ingredients and subsequent stable dispersion.

[0067] After canceling the pulse mode in Comparative Examples 2 and 9, although the extraction yield was close to that of Example 1, the phycocyanin retention rate and activity retention rate decreased significantly, indicating that continuous energy input during continuous ultrasound easily leads to local heat accumulation and free radical oxidative damage. In Comparative Example 10, without low-temperature control, the phycocyanin retention rate further decreased to 66.8%, indicating that low-temperature cycling control can effectively suppress thermal damage during ultrasound and improve the stability of heat-sensitive active substances. Therefore, Example 1, by employing low-temperature pulsed ultrasound, utilizes intermittent energy input to improve the uniformity of cavitation, and combines low-temperature cycling to reduce the system temperature rise, thereby achieving synergistic optimization of extraction efficiency and activity retention rate.

[0068] Comparative Example 12 showed a high phycocyanin retention rate, but the extraction yield was only 16.5%, indicating that the ultrasonic power was too low, resulting in insufficient cavitation and making it difficult to fully promote the release of active ingredients. While Comparative Example 13 achieved an extraction yield of 21.1%, close to Example 1, the phycocyanin retention rate decreased to 63.5%, and the activity retention rate was only 65.4%, indicating that excessive ultrasonic power would significantly damage heat-sensitive components. Therefore, the ultrasonic power density range defined in this application can achieve a better balance between extraction yield and activity retention rate.

[0069] Comparative Example 14, which used spray drying instead of freeze drying, had an activity retention rate of only 61.5%, significantly lower than that of Example 1, indicating that the high-temperature post-processing further exacerbates the deactivation of active ingredients. Example 1, by employing a mild separation and purification process combining microfiltration, ultrafiltration, and freeze drying, effectively avoids activity damage caused by high-temperature concentration and improves the long-term stability of the extract.

[0070] Comparative Example 15, after using an 8kDa ultrafiltration membrane, although D 50 The extraction yield decreased to 19.2% even with a lower molecular weight cutoff of 170 nm and a higher absolute value of the Zeta potential, indicating that a lower molecular weight cutoff easily leads to the loss of some active ingredients. In contrast, Comparative Example 16, using a 12 kDa ultrafiltration membrane, achieved an extraction yield close to that of Example 1, but the D... 50Increasing the molecular weight cutoff to 218 nm resulted in a decrease in the activity retention rate to 84.6%, indicating that a higher molecular weight cutoff can easily lead to the residue of large molecular impurities and reduce the stability of the system. However, when using a 10 kDa ultrafiltration membrane in Example 1, a higher extraction yield, smaller particle size, and higher long-term stability were simultaneously achieved, indicating that this parameter can achieve a comprehensive balance between the retention of active ingredients and the stability of the system.

[0071] In summary, this application, through the synergistic control of gradient enzymatic hydrolysis, low-temperature pulsed ultrasound, and gentle separation and purification processes, not only improves the release efficiency of cyanobacterial active ingredients but also effectively reduces thermal damage, while taking into account small particle size dispersibility and long-term storage stability, thereby obtaining a cyanobacterial active extract with high activity retention rate and stability.

[0072] Example 2 The researchers also prepared a cosmetic composition containing the blue-green algae extract of Example 1, and the preparation method is as follows.

[0073] 1) Oil phase preparation Weigh the following oil phase components by weight percentage: cetearyl alcohol 4.0%; glyceryl stearate 3.0%; caprylic / capric triglyceride 6.0%; jojoba oil 2.0%; dimethyl silicone oil 1.5%; tocopheryl acetate 0.2%. Add the above components to an oil phase container, heat at 75°C, and stir with a paddle stirrer at 300 rpm until completely melted and a homogeneous, transparent oil phase is formed.

[0074] 2) Aqueous phase preparation Weigh the following aqueous phase components by mass percentage: glycerol 5.0%; 1,3-butanediol 4.0%; xanthan gum 0.2%; disodium EDTA 0.05%; deionized water balance. First, slowly add xanthan gum to a portion of the deionized water and disperse at 500 rpm for 30 min at room temperature. Then add glycerol, 1,3-butanediol, and disodium EDTA. Heat the resulting aqueous phase to 75°C and continue stirring until the system is homogeneous and transparent.

[0075] 3) Emulsification treatment At 75℃, the oil phase was slowly added to the aqueous phase, and emulsification was performed using a high-speed emulsifier. The emulsification conditions were as follows: emulsification speed: 8000 rpm; emulsification time: 8 min. After emulsification, the mixture was stirred continuously at 200 rpm using a paddle agitator and then cooled.

[0076] 4) Adding blue algae active extract When the emulsion system cooled to below 40°C, 3.0% of the cyanobacterial active extract powder prepared in Example 1 was added. Subsequently, 1.0% panthenol, 0.8% phenoxyethanol, and 0.1% fragrance were added, and the mixture was stirred at 300 rpm for 20 minutes to ensure the cyanobacterial active extract was fully dispersed in the system. Before adding the cyanobacterial active extract, a small amount of 1,3-butanediol was used for pre-dispersion to reduce local agglomeration and improve system homogeneity.

[0077] 5) Homogenization and degassing The obtained system was homogenized using a high-pressure homogenizer. The homogenization conditions were as follows: homogenization pressure 25 MPa, homogenization twice, and after homogenization, vacuum degassing at -0.08 MPa for 15 min to obtain the blue-green active cosmetic composition. The obtained composition was a uniform blue-green cream with no obvious particle sedimentation or stratification, and had good extensibility and stability.

[0078] Test Example 2 To evaluate the skin moisturizing effect of the cosmetic composition prepared in Example 2, the skin stratum corneum moisture content was tested for cosmetic compositions from different sources of cyanobacterial extract. Specifically: Example 2 was a cosmetic composition containing the active cyanobacterial extract prepared in Example 1; Comparative Example A was a cosmetic composition prepared using the cyanobacterial extract obtained in Comparative Example 1; Comparative Example B was a cosmetic composition prepared using the cyanobacterial extract obtained in Comparative Example 6; and Comparative Example C was a cosmetic composition prepared using the cyanobacterial extract obtained in Comparative Example 10. Except for the source of the cyanobacterial extract, the formulations and preparation processes of the other cosmetic compositions were the same as in Example 2.

[0079] Twenty healthy subjects aged 22-35 years were selected. After resting in an environment with a temperature of 25±1℃ and a relative humidity of 50±5% for 30 minutes, different groups of samples were applied to the inner forearm at a concentration of 2 mg / cm². 2 The skin's stratum corneum moisture content was measured using a Corneometer before use, 2 hours after use, and 8 hours after use. The moisture content increase rate was calculated using the following formula: Moisture content increase rate (%) = (Moisture content after use - Moisture content before use) / Moisture content before use × 100%.

[0080] The test results are shown in Table 2.

[0081] Table 2. Results of Skin Moisturizing Performance Tests for Cosmetic Compositions

[0082] As shown in Table 2, the cosmetic composition prepared in Example 2 has a superior skin moisturizing effect. Compared with Comparative Examples A, B, and C, Example 2 showed a higher skin moisture content increase rate under both 2h and 8h conditions, indicating that the blue-green algae active extract prepared in Example 1 can improve the moisturizing performance and sustained moisturizing ability of the composition.

[0083] Among them, Comparative Example A, due to the use of high-temperature hot water extraction to obtain the cyanobacterial extract, experienced significant loss of heat-sensitive active ingredients, resulting in the lowest moisturizing effect. Comparative Example B, employing a single-step mixed enzymatic hydrolysis method, suffered from insufficient release efficiency of active ingredients, leading to lower moisturizing performance than Example 2. Comparative Example C, lacking low-temperature control, experienced heat accumulation during ultrasound treatment, resulting in the inactivation of some active ingredients and a decrease in sustained moisturizing ability. This demonstrates that the present application, through the synergistic control of gradient enzymatic hydrolysis and low-temperature pulsed ultrasound, can effectively improve the retention rate of cyanobacterial active ingredients and the stability of the system, thereby further enhancing the skin moisturizing effect of the cosmetic composition.

[0084] Test Example 3 To evaluate the skin barrier function improvement effect of the cosmetic composition prepared in Example 2, transepidermal water loss (TEWL) tests were conducted on cosmetic compositions from different sources of cyanobacteria extract. The sample setup for each group was the same as in Example 2.

[0085] Twenty healthy subjects were selected, and different groups of samples were applied to the inner forearm at a concentration of 2 mg / cm², twice daily for 14 consecutive days. Transepidermal water loss (TEWL) values ​​were measured before and after 14 days of continuous use using a Tewameter. The TEWL reduction rate was calculated using the following formula: TEWL reduction rate (%) = (TEWL value before use - TEWL value after use) / TEWL value before use × 100%.

[0086] The test results are shown in Table 3.

[0087] Table 3. Results of Skin Barrier Repair Performance Tests for Cosmetic Compositions

[0088] As shown in Table 3, the cosmetic composition prepared in Example 2 has a superior skin barrier repair effect. Compared with Comparative Examples A, B, and C, Example 2 can more effectively reduce transepidermal water loss, indicating that the cyanobacterial active extract prepared in Example 1 is beneficial in improving the composition's ability to improve skin barrier function.

[0089] Among them, Comparative Example A suffered from severe loss of heat-sensitive active ingredients during the thermal extraction process, resulting in poor barrier repair effect; Comparative Example B, due to the lack of gradient enzymatic hydrolysis, resulted in insufficient release of active ingredients, thus limiting its ability to improve the skin barrier; Comparative Example C, due to the lack of low-temperature control during the ultrasonic process, experienced a decrease in the stability of some active ingredients, resulting in a lower TEWL reduction effect compared to Example 2. This demonstrates that the present application, through a synergistic process of gradient enzymatic hydrolysis and low-temperature pulsed ultrasound, can not only improve the release efficiency of cyanobacterial active ingredients but also effectively reduce thermal damage and improve system stability, thereby further enhancing the skin moisturizing and skin barrier repair effects of the cosmetic composition.

[0090] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.

Claims

1. A method for preparing an active extract of cyanobacteria, characterized in that, Includes the following steps: 1) After pulverizing the cyanobacteria raw material, it is mixed with water to form a cyanobacteria suspension system; 2) Perform gradient enzymatic hydrolysis on the cyanobacteria suspension system, including: Primary enzymatic hydrolysis: Adjust the pH of the system to 5.0~5.5, add a complex enzyme system consisting of cellulase and pectinase, and carry out enzymatic hydrolysis at 45~50℃; Secondary enzymatic hydrolysis: After the primary enzymatic hydrolysis is completed, adjust the pH of the system to 6.8-7.2, and add neutral protease to continue enzymatic hydrolysis; 3) The enzymatically hydrolyzed system was subjected to low-temperature pulsed ultrasonic extraction. During the ultrasonic extraction process, the system temperature was controlled at 8~15℃, the ultrasonic frequency at 20~35kHz, the power density at 250~450W / L, and the duty cycle at 40~70%. 4) The system after ultrasonic extraction is separated and purified to obtain the active extract of cyanobacteria.

2. The method for preparing the cyanobacterial active extract according to claim 1, characterized in that, In step 2), the mass ratio of cellulase to pectinase is (1.5~2.5):(1~2), and the total amount of compound enzyme added is 1.5~3.5% of the mass of cyanobacteria raw material.

3. The method for preparing the cyanobacterial active extract according to claim 1, characterized in that, In step 2), the first-stage enzymatic hydrolysis time is 30-60 min, and the second-stage enzymatic hydrolysis time is 20-40 min.

4. The method for preparing the cyanobacterial active extract according to claim 1, characterized in that, In step 3), the low-temperature pulse ultrasound adopts a pulse mode of working for 5 seconds and pausing for 3 seconds, and the low-temperature pulse ultrasound processing time is 15~35 minutes.

5. The method for preparing the cyanobacterial active extract according to claim 1, characterized in that, The microfiltration uses a microfiltration membrane with a pore size of 0.22 μm, and the ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 10 kDa; and / or, In step 1), the cyanobacteria raw material is crushed and then passed through an 80-120 mesh sieve.

6. The method for preparing the cyanobacterial active extract according to claim 1, characterized in that, In step 1), the ratio of cyanobacteria raw material to water is 1:18 to 1:25 (g:mL).

7. The method for preparing the cyanobacterial active extract according to claim 1, characterized in that, In step 1), the cyanobacterial raw material is one or more of Spirulina, Anabaena or Nostoc. Optionally, the cyanobacterial raw material is spirulina.

8. The active extract of cyanobacteria obtained by the preparation method according to any one of claims 1 to 7.

9. The cyanobacterial active extract according to claim 8, characterized in that, The cyanobacterial active extract meets the following performance requirements: 1) Average particle size D50 ≤ 180 nm; 2) DPPH free radical scavenging rate ≥92%; 3) The absolute value of the Zeta potential is ≥28mV; 4) The activity retention rate is ≥90% after storage at 40℃ for 30 days.

10. The use of the cyanobacterial active extract as described in claim 9 in a cosmetic composition.

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