Room temperature storage of stem cell exosomes for use in skin care compositions

Abstract

The application relates to the field of biological medicine technology and discloses normal-temperature preservation of stem cell exosomes in skin-care composition application, which comprises the following steps: obtaining a secondary drying time according to sample temperature and shelf temperature; obtaining a moisture step degree according to the difference of residual moisture content of a pressure increasing rate; obtaining a moisture continuous removal index according to the residual moisture content, combining the moisture step degree to obtain a comprehensive moisture removal efficiency; obtaining an activity loss degree according to protein activity and the comprehensive moisture removal efficiency, and then obtaining a water content critical threshold value to construct a damage sensitivity; obtaining a damage index according to the protein activity, the comprehensive moisture removal efficiency and the damage sensitivity, and selecting a target pressure increasing rate; and performing stem cell exosome production application in skin-care composition according to the secondary drying time, the water content critical threshold value and the target pressure increasing rate. The application prolongs the normal-temperature preservation duration of stem cell exosomes in skin-care composition application by regulating the secondary drying process.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the room temperature preservation of stem cell exosomes in skin care compositions. Background Technology

[0002] Stem cell exosomes, as core effector substances in stem cell paracrine signaling, are nanoscale extracellular vesicles with a lipid bilayer structure and a diameter between 30-150 nm. They are rich in key bioactive molecules such as growth factors, cytokines, proteins, and microRNAs. In skincare formulations, exosomes, with their extremely small particle size and natural biocompatibility, can effectively overcome the limitations of traditional active ingredients' low transdermal penetration. By mediating signal communication between skin cells, they precisely drive fibroblast proliferation, promote extracellular matrix regeneration, and inhibit inflammatory aging. They exhibit biological activity surpassing conventional biochemical ingredients in areas such as damaged barrier repair, deep anti-aging, and pigment regulation, making them a key technological pathway for developing next-generation precision biological repair formulations.

[0003] Stem cell exosomes are extremely sensitive to the environment due to their nanoscale lipid bilayer structure and the active signaling molecules loaded inside. They are prone to membrane rupture, physical aggregation, or rapid degradation and inactivation due to residual enzymes in room temperature aqueous solutions. Currently, the industry generally uses freeze-drying technology to convert exosomes into dormant dry powder to reduce residual moisture and chemical reaction rates. This balances the need to maintain biological activity and the convenience of room temperature storage for cosmetics without relying on extremely low temperature cold chains.

[0004] Existing freeze-drying processes primarily convert exosomes into dormant dry powder through three stages: pre-freezing, primary sublimation, and secondary desorption. Given the low efficiency of traditional secondary drying, vacuum pressure fluctuation technology is introduced. This technology artificially creates periodic pressure fluctuations in a vacuum environment, utilizing micro-circulation convection of gas to break the static boundary layer on the material surface, thereby significantly improving heat and mass transfer efficiency and ensuring that exosomes achieve extremely low residual moisture levels. However, frequent and significant pressure changes generate substantial physical shear force losses, leading to mechanical fatigue in the fragile exosome nanomembrane structure. Especially when process control is inadequate, if the pressure jumps too rapidly, the trace amounts of air trapped inside the micropores of the freeze-drying support may undergo violent volume expansion. This instantaneous internal impact can directly cause exosome membrane rupture, resulting in the loss of biosignal activity. Summary of the Invention

[0005] This invention provides a method for room temperature preservation of stem cell exosomes in skincare compositions, addressing the problem of loss of biosignal activity caused by unreasonable vacuum pressure fluctuations and process parameters during existing secondary drying processes. The specific technical solution adopted is as follows: This invention proposes a method for preserving stem cell exosomes at room temperature in skincare compositions, comprising the following steps: The shelf temperature and sample temperature at each moment were obtained, and the residual moisture content and protein activity were obtained for several cycles at several pressurization rates during the drying process. The timing for the second drying step is determined based on the abrupt temperature rise of the sample and its proximity to the shelf temperature. During the vacuum pressure fluctuation process after reaching the second drying time, the degree of moisture step at each pressurization rate is obtained based on the difference between the residual moisture content at the pressurization rate and the previous pressurization rate; the moisture removal index at each pressurization rate is obtained based on the continuous decrease of the residual moisture content at each pressurization rate; and the comprehensive moisture removal efficiency at each pressurization rate is obtained by combining the degree of moisture step. Based on the differences in protein activity between pressurization rates and the changes in overall water removal efficiency, the degree of activity loss at each pressurization rate is obtained; a critical water content threshold is obtained based on the degree of activity loss; a damage sensitivity for each pressurization rate is constructed based on the critical water content threshold; and a damage index for each pressurization rate is obtained based on the protein activity at each pressurization rate, the overall water removal efficiency, and the damage sensitivity. The target pressurization rate is selected based on the damage index of the pressurization rate; the production control of stem cell exosomes in the skin care composition is carried out based on the secondary drying time, the critical water content threshold, and the target pressurization rate.

[0006] Furthermore, the specific method for determining the secondary drying time based on the abrupt temperature rise of the sample and its proximity to the shelf temperature includes: For any given moment, obtain the difference between the sample temperature at that moment and the previous moment, and record the maximum value between the difference in sample temperature and 0 as the degree of temperature change at that moment. The absolute value of the difference between the sample temperature and the shelf temperature at any given moment is recorded as the sublimation endpoint gap at that given moment. The linearly normalized result of the ratio of the temperature change at any given moment to the sublimation endpoint gap is denoted as the degree of intervention at any given moment. The moment when the degree of intervention exceeds the preset intervention threshold is recorded as the second drying moment.

[0007] Furthermore, during the vacuum pressure fluctuation process after reaching the secondary drying time, the degree of moisture step at each pressurization rate is obtained based on the difference in residual moisture content between the pressurization rate and the previous pressurization rate. The specific method includes: After reaching the second drying time, the second drying stage begins, involving vacuum pressure fluctuations. For any pressurization rate, the difference between the residual moisture content after the last cycle of the previous pressurization rate and the residual moisture content after the first cycle of the previous pressurization rate is obtained. The ratio of the difference to the residual moisture content after the last cycle of the previous pressurization rate is recorded as the moisture step degree of the previous pressurization rate.

[0008] Furthermore, the specific method for obtaining the moisture removal index for each pressurization rate based on the continuous decrease in residual moisture content at each pressurization rate is as follows: For any cycle at any pressurization rate, the difference between the residual moisture content of the previous cycle and the current cycle is recorded as the degree of moisture reduction in the current cycle. The minimum degree of water reduction across all cycles at the pressurization rate is denoted as the degree of sustained dehydration at the pressurization rate. The difference between the residual moisture content after the last cycle of the pressurization rate and the first cycle of the pressurization rate is recorded as the first difference. The product of the first difference and the degree of continuous dehydration of the pressurization rate is recorded as the moisture removal index of the pressurization rate.

[0009] Furthermore, the specific method for obtaining the comprehensive moisture removal efficiency is as follows: For any pressurization rate, the product of the water step degree of the pressurization rate and the water removal index is denoted as the overall water removal efficiency of the pressurization rate.

[0010] Furthermore, the specific method for determining the degree of activity loss at each pressurization rate based on the differences in protein activity between pressurization rates and the changes in overall water removal efficiency includes: For any given pressurization rate, the difference between the protein activity after the last cycle of the preceding pressurization rate and the protein activity after the last cycle of the preceding pressurization rate is denoted as the activity loss degree of the given pressurization rate. The inversely proportional normalized result of the absolute value of the difference between the preceding pressurization rate and the overall water removal efficiency of the given pressurization rate is denoted as the dehydration limit index of the given pressurization rate. The product of the activity loss degree of the given pressurization rate and the dehydration limit index is denoted as the dehydration inactivation degree of the given pressurization rate.

[0011] Furthermore, the specific method for obtaining the critical water content threshold based on the degree of activity loss includes: The residual water content after the last cycle of the previous pressurization rate corresponding to the pressurization rate with the greatest degree of dehydration and inactivation is taken as the critical water content threshold.

[0012] Furthermore, the specific method for constructing the damage sensitivity for each pressurization rate based on the water content critical threshold is as follows: For any pressurization rate, the difference between the critical water cut threshold and the mean of the residual water cut of all cycles corresponding to the pressurization rate is obtained. The maximum value between the difference and 0 is used as the damage sensitivity factor of the pressurization rate. The result of an exponential function with base e and exponent of the damage sensitivity factor of the pressure boost rate is taken as the damage sensitivity of the pressure boost rate.

[0013] Furthermore, the specific method for obtaining the damage index for each pressurization rate based on protein activity and overall water removal efficiency at the pressurization rate, combined with the damage sensitivity, includes: For any pressurization rate, obtain the inversely proportional normalized result of protein activity after the last cycle of the pressurization rate, and record the ratio of the inversely proportional normalized result to the overall water removal efficiency of the pressurization rate as the basic damage index of the pressurization rate. The product of the basic damage index of the boost rate and the damage sensitivity is denoted as the damage index of the boost rate.

[0014] Furthermore, the target boost rate is specifically obtained as follows: Among all boost rates, the boost rate with the smallest damage index is selected as the target boost rate.

[0015] The beneficial effects of this invention are as follows: Addressing the problem of loss of biosignal activity caused by unreasonable vacuum pressure fluctuations during the secondary drying process in the production of exosome-based skincare compositions, this invention first obtains the timing of the secondary drying by varying shelf and sample temperatures. This ensures that the exosomes have passed the unstable period of ice crystal sublimation and are beginning to be stably supported by a protective agent, avoiding the risk of collapse. Furthermore, it analyzes the changes in residual moisture content of the samples corresponding to different pressure change slopes. The overall moisture removal efficiency at each pressurization rate also affects protein activity. Based on the protein activity at each pressurization rate and the overall moisture removal efficiency, combined with the aforementioned damage sensitivity, this invention obtains a damage index for each pressurization rate, balancing residual moisture content and protein activity, and accurately obtaining the target pressurization rate. This invention controls the production of stem cell exosomes in skincare compositions by controlling the secondary drying time, critical moisture threshold, and target pressurization rate, thus extending the room-temperature storage time of stem cell exosomes in skincare compositions. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a schematic diagram illustrating the room temperature preservation process of stem cell exosomes in a skincare composition, as provided in an embodiment of the present invention. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Please see Figure 1 The diagram illustrates a process flow chart for room temperature preservation of stem cell exosomes in skincare compositions according to an embodiment of the present invention. The method includes the following steps: Step S001: Obtain the shelf temperature and sample temperature at each moment, and obtain the residual moisture content and protein activity for several cycles at several pressurization rates during the drying process.

[0020] It should be noted that the purpose of this embodiment is to precisely control the balance between the pressurization rate and the preservation of protein activity in the vacuum pressure fluctuation process, obtain the optimal intervention time to ensure the stability of the scaffold, and combine the comprehensive water removal efficiency with the damage sensitivity factor constructed based on the water content critical threshold to quantitatively evaluate the damage index under different pressurization rates. In this way, the optimal process parameters can be screened to achieve extremely low residual water to inhibit chemical degradation and avoid protein conformational collapse caused by excessive dehydration. This ensures that stem cell exosomes can maintain structural integrity and biological signal activity in the skin care composition, and significantly prolong their storage time at room temperature.

[0021] It should be further explained that, in order to obtain the optimal process parameters, this embodiment first needs to systematically acquire data in an experimental scenario, in order to provide an objective basis for subsequent logical analysis.

[0022] Specifically, a standard 10 ml vial is used as the container, and each vial is precisely filled with 3 ml of stem cell exosome skin care composition solution, which contains a freeze-drying protectant. The experimental samples were placed evenly in a matrix in the central area of ​​the freeze dryer shelf. Five samples were selected at the central location as temperature indicator bottles. A thin-film thermocouple sensor was placed at the bottom center of the bottle to collect the sample temperature in real time. At the same time, a shelf temperature sensor was placed in close contact with the shelf surface in that area to collect the shelf temperature at each moment. The monitoring software was set to take 30 seconds as a time interval and the average temperature of the samples in all temperature indicator bottles in the freeze dryer at each moment was recorded as the sample temperature at each moment. The sample temperatures at all moments were linearly normalized. Select a vial at a specific location as a spectral indicator vial, and make close contact between the end of the NIR probe and the side wall of the vial; In the secondary desorption stage, after entering the secondary drying stage, the system sequentially switches the pressurization rate, with a set range covering gradients of 0.5 Pa / s, 1 Pa / s, 2 Pa / s, 3 Pa / s, 4 Pa / s, 5 Pa / s, 6 Pa / s, 7 Pa / s, 8 Pa / s, 9 Pa / s, 10 Pa / s, 11 Pa / s, and 12 Pa / s. For each set pressurization rate, 30 complete pressure increase / decrease cycles are continuously executed. After each cycle, raw NIR spectra are acquired immediately. Through spectral preprocessing and quantitative analysis models, residual water content and protein activity data reflecting the integrity of the protein's secondary structure are extracted at each cycle node. The method for obtaining residual water content and protein activity from raw NIR spectra is existing technology, and the specific method will not be described here. Pa / s refers to Pascals per second.

[0023] Step S002: Based on the sudden temperature rise of the sample and its proximity to the shelf temperature, the secondary drying time is determined.

[0024] It should be noted that in exosome research, the timing of pressure fluctuation intervention and the slope of pressure change need to be confirmed sequentially. First, determining the timing of intervention is to ensure that the exosome has passed the unstable period of ice crystal sublimation and has begun to be stably supported by the protective agent to avoid the risk of collapse. Then, determining the pressure slope is to detect the fatigue threshold of the nanoscale membrane structure against physical shear force, ensuring the functional standard of no membrane rupture and no leakage, thereby achieving both the integrity of exosome biological signals and extremely low residual water.

[0025] It's important to further clarify that choosing the timing of vacuum pressure fluctuation intervention essentially involves identifying the critical point between the primary drying and sublimation stage and the secondary drying and desorption stage. At this point, most of the ice crystals inside the vial have disappeared, and the exosomes are supported by the protective agent within a porous framework, achieving peak physical stability and the ability to withstand certain pressure fluctuations. For substances like stem cell exosomes, which have extremely high requirements for microstructure, intervention too early can lead to product collapse or vial spraying, while intervention too late negates the purpose of accelerating dehydration. During the primary drying stage, the sample temperature will remain at a relatively low plateau of approximately -25°C due to sublimation endothermic effects. When the ice crystals are largely depleted, the sample temperature will rise rapidly; the optimal intervention point is when the sample temperature rises and approaches the shelf temperature.

[0026] Specifically, for any given moment, the difference between the sample temperature at that moment and the previous moment is obtained, and the maximum value between this difference and 0 is recorded as the degree of temperature change at that moment. The absolute value of the difference between the sample temperature and the shelf temperature at that moment is recorded as the sublimation endpoint gap at that moment. The linear normalized result of the ratio of the temperature change at that moment to the sublimation endpoint gap is denoted as the intervention level at that moment. It should be noted that if the sublimation endpoint gap is less than or equal to the preset gap safety threshold, the intervention level is directly assigned a value of 1, where the preset gap safety threshold is 0.1. This embodiment is described using this as an example. The moment when the degree of intervention is greater than the preset intervention threshold is recorded as the second drying moment; where the preset intervention threshold is 0.9, this embodiment is described using this as an example.

[0027] Step S003: During the vacuum pressure fluctuation process after reaching the secondary drying time, the degree of moisture step at each pressure rate is obtained based on the difference in residual moisture content between the pressure rate and the previous pressure rate; the moisture removal index for each pressure rate is obtained based on the continuous decrease in residual moisture content at each pressure rate; and the comprehensive moisture removal efficiency for each pressure rate is obtained by combining the degree of moisture step.

[0028] It should be noted that after reaching the second drying point, the second drying stage begins, which requires cycling at different pressurization rates. After 30 cycles at the previous rate, the moisture content usually reaches a plateau at that rate. If, in the first cycle after switching to a higher pressurization rate, the residual moisture content suddenly drops sharply, it indicates that the increased rate has overcome the resistance to moisture diffusion, and the moisture drying level shows a step increase.

[0029] Specifically, after reaching the second drying time, the process enters the second drying stage, where vacuum pressure fluctuations are performed. For any pressurization rate, the difference between the residual moisture content after the last cycle of the previous pressurization rate and the residual moisture content after the first cycle of the previous pressurization rate is obtained. The ratio of this difference to the residual moisture content after the last cycle of the previous pressurization rate is recorded as the moisture step degree of the pressurization rate. It should be noted that if the residual moisture content after the last cycle of the previous pressurization rate is 0, the moisture step degree of the pressurization rate is directly assigned to 0. It should be noted that the moisture step degree of the first pressurization rate is calculated as follows: the difference between the residual moisture content at the second drying time and the residual moisture content after the first cycle of the first pressurization rate is obtained. The ratio of this difference to the residual moisture content at the second drying time is recorded as the moisture step degree of the first pressurization rate.

[0030] It should be noted that if the water level jump is large, it means that there is still water inside the exosome scaffold that can be squeezed out by the pressure difference; if the water level jump is small, it means that the water is mainly constrained by the intermolecular adsorption force, and increasing the pressure rate has no effect, so the rate should be stopped.

[0031] It should be noted that when switching to a higher pressurization rate, the step drop in moisture content only proves that the instantaneous pressure jump has successfully overcome the diffusion resistance of the current material surface. However, if subsequent cycles cannot maintain an efficient dehydration trend, it indicates that the rate has not substantially improved the overall heat and mass transfer efficiency. In this case, the increased pressurization rate only generates ineffective mechanical pressure redundancy. This redundant physical shear force will cause unnecessary mechanical fatigue to the fragile nanomembrane structure of exosomes, and may even directly lead to membrane rupture and loss of biological activity without significant drying gain. Therefore, the true effectiveness of the rate must be evaluated in conjunction with the continuous removal capacity.

[0032] Specifically, for any cycle within any pressurization rate, the difference between the residual moisture content of the previous cycle and that cycle is recorded as the degree of moisture reduction in that cycle; it should be noted that the degree of moisture reduction in the first cycle within that pressurization rate is not calculated. The minimum degree of water reduction across all cycles at that pressurization rate is denoted as the sustained dehydration degree at that pressurization rate. The difference between the residual moisture content after the last cycle at the pressurization rate and the first cycle at the pressurization rate is recorded as the first difference. The product of the first difference and the degree of continuous dehydration at the pressurization rate is recorded as the moisture removal index of the pressurization rate.

[0033] It should be noted that when the moisture removal index of the pressurization rate is large, it means that the overall decrease in residual moisture content is relatively high in all cycles at that pressurization rate, and this high degree of decrease is continuous. When the moisture removal index of the pressurization rate is small, although the rate is increased, the overall dehydration speed remains unchanged, and the extra mechanical pressure is redundant and may even damage the protein.

[0034] It should be noted that when analyzing the overall moisture removal efficiency of pressurization rate, it is necessary to pay attention not only to the step-like nature of moisture removal capacity at pressurization rate, but also to the continuity of moisture removal.

[0035] Specifically, for any pressurization rate, the product of the degree of moisture step at that pressurization rate and the moisture removal index is denoted as the overall moisture removal efficiency of that pressurization rate.

[0036] Step S004: Based on the differences in protein activity between pressurization rates and the changes in overall water removal efficiency, obtain the degree of activity loss at each pressurization rate; obtain the critical water content threshold based on the degree of activity loss; construct the damage sensitivity for each pressurization rate based on the critical water content threshold; obtain the damage index for each pressurization rate based on the protein activity at each pressurization rate and the overall water removal efficiency, combined with the damage sensitivity.

[0037] It should be noted that water can protect and maintain the structural integrity of proteins, preventing conformational collapse and loss of recognition function during drying. However, it is also a medium that causes chemical degradation, oxidation, and enzymatic reactions at room temperature. Therefore, in the preparation of skin care compositions, it is necessary to obtain the critical residual water content. This involves removing free water to form a stable glassy matrix and blocking molecular motion, while avoiding the stripping of essential bound water from the protein surface due to excessive dehydration and high-intensity pressure fluctuations. This balances the physical stability for long-term preservation at room temperature with the preservation of the biological activity of stem cell signaling molecules. Therefore, the critical water content threshold must be determined first.

[0038] It should be further explained that since each pressurization rate segment involves multiple pressure increase and decrease cycles, the protein activity after the last cycle represents the final stable state of the exosomes after undergoing sufficient mechanical fatigue at that specific stress level. By calculating the difference in protein activity at the end of adjacent rate segments, the background effects accumulated in the previous process can be effectively eliminated, thereby accurately quantifying the marginal activity loss caused by the increase in the slope of pressure variation.

[0039] Specifically, for any pressurization rate, the difference between the protein activity after the last cycle of the previous pressurization rate and the protein activity after the last cycle of the current pressurization rate is denoted as the activity loss degree of that pressurization rate. It should be noted that the activity loss degree of the first pressurization rate is calculated as follows: the difference between 1 and the protein activity after the last cycle of the first pressurization rate is denoted as the activity loss degree of that pressurization rate; the inversely proportional normalized result of the absolute value of the difference between the previous pressurization rate and the overall water removal efficiency of the current pressurization rate is denoted as the dehydration limit index of that pressurization rate; the product of the activity loss degree of the pressurization rate and the dehydration limit index is denoted as the dehydration inactivation degree of that pressurization rate.

[0040] It should be noted that when the degree of activity loss reaches its maximum, it means that the mechanical shear force generated by the pressurization rate is violently coupled with the current low water state, leading to the collapse of the hydration protection that maintains the integrity of the protein structure and irreversible mechanical fatigue of the membrane structure. The dehydration limit index reflects the bottleneck of water removal. When water removal becomes extremely difficult and activity begins to decline sharply at the same time, the degree of dehydration inactivation will reach its peak. Since this damage occurs at the current rate stage, it means that the endpoint of the previous rate stage is the lowest residual water content limit that exosomes can tolerate before large-scale structural damage occurs. Therefore, using the residual water content at this safe point as the critical water content threshold can define the turning point from the stable plateau phase to the damage outbreak phase of biological activity.

[0041] Specifically, the residual water content after the last cycle of the pressurization rate corresponding to the pressurization rate with the highest degree of dehydration and inactivation is used as the critical water content threshold.

[0042] It should be noted that before reaching the critical water content threshold, there is no significant loss of biological activity, and the focus is mainly on the overall water removal efficiency and protein activity under steady-state conditions. However, once the critical water content threshold is reached, there is a high probability of protein inactivation due to the influence of the aquatic environment, so it is necessary to significantly improve the damage sensitivity.

[0043] Specifically, for any pressurization rate, the difference between the critical water cut threshold and the mean of the residual water cut of all cycles corresponding to that pressurization rate is obtained. The maximum value between this difference and 0 is used as the damage sensitivity factor for that pressurization rate. The result of an exponential function with base e and exponent of the damage sensitivity factor at that boost rate is taken as the damage sensitivity at that boost rate.

[0044] It should be noted that the damage index of each pressurization rate is mainly composed of the protein activity after the last cycle of each pressurization rate and the overall water removal efficiency of each pressurization rate. The overall water removal efficiency of each pressurization rate represents the water removal benefit brought by the pressurization rate. The greater the overall water removal efficiency, the more beneficial it is for the room temperature preservation of stem cell exosomes in the subsequent application of skin care compositions. The protein activity after the last cycle of each pressurization rate represents the activity loss directly exhibited by the protein at that pressurization rate to achieve the current water removal efficiency. Therefore, in order to comprehensively address the protein damage caused by the water environment, a damage sensitivity factor needs to be introduced to further amplify the damage index of the pressurization rate.

[0045] Specifically, for any pressurization rate, the inversely proportional normalized result of the protein activity after the last cycle at that pressurization rate is obtained. The ratio of this inversely proportional normalized result to the overall water removal efficiency of that pressurization rate is recorded as the basic damage index of that pressurization rate. It should be noted that if the overall water removal efficiency of that pressurization rate is 0, then the pressurization rate is determined to be a high-risk parameter with zero benefit, and that pressurization rate will not be included in subsequent analysis and judgment. The product of the basic damage index of the boost rate and the damage sensitivity is denoted as the damage index of the boost rate.

[0046] Step S005: Select the target pressurization rate based on the damage index of the pressurization rate; control the production of stem cell exosomes in the skin care composition based on the secondary drying time, the critical water content threshold, and the target pressurization rate.

[0047] It should be noted that the damage index of the pressurization rate reflects the balance between the dehydration benefit and the protein activity cost of that pressurization rate. A smaller damage index indicates that the pressurization rate has a high cost-effectiveness, that is, a large degree of water removal is achieved with minimal damage to protein activity. This indicates that the stress level is within the safe range of exosome mechanical tolerance and the drying efficiency is excellent. On the other hand, a larger damage index indicates that the physical shear force generated by this rate is too severe relative to its dehydration contribution, or that it causes severe protein conformational collapse or membrane fatigue in the current low water content environment. This is a high-risk, low-return process parameter.

[0048] Specifically, among all boost rates, the boost rate with the lowest damage index is selected as the target boost rate; In the specific production process, the secondary drying time is obtained in step S002. After the secondary drying time is reached, the primary drying is immediately switched to secondary drying. Vacuum pressure fluctuation technology is used in the secondary drying process, and the target pressure increase rate is used as the pressure increase rate parameter of the vacuum pressure fluctuation technology. During the production process, the NIR probe is continuously monitored. If the real-time residual moisture content is less than the critical threshold, the system will automatically fine-tune the pressure fluctuation frequency according to the damage sensitivity factor to avoid stripping the essential bound water on the protein surface.

[0049] It should be noted that the linear normalization in this embodiment is min-max normalization; this embodiment adopts... The model is used to represent the inverse proportional relationship and for normalization processing. As input to the model, To prevent overparameters with a denominator of 0, implementers can set inverse proportional functions and normalization functions according to the actual situation.

[0050] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Room temperature preservation of stem cell exosomes in skincare compositions, characterized in that, The method includes the following steps: The shelf temperature and sample temperature were obtained at each moment, and the residual moisture content and protein activity were obtained for several cycles at several pressurization rates during the drying process. The timing for the second drying step is determined based on the abrupt temperature rise of the sample and its proximity to the shelf temperature. During the vacuum pressure fluctuation process after reaching the second drying time, the degree of moisture step at each pressurization rate is obtained based on the difference between the residual moisture content at the pressurization rate and the previous pressurization rate; the moisture removal index at each pressurization rate is obtained based on the continuous decrease of the residual moisture content at each pressurization rate; and the comprehensive moisture removal efficiency at each pressurization rate is obtained by combining the degree of moisture step. Based on the differences in protein activity and the changes in overall water removal efficiency between pressurization rates, the degree of activity loss at each pressurization rate is obtained; based on the degree of activity loss, a critical water content threshold is obtained; based on the critical water content threshold, a damage sensitivity for each pressurization rate is constructed; based on the protein activity and overall water removal efficiency at each pressurization rate, combined with the damage sensitivity, a damage index for each pressurization rate is obtained. The target pressurization rate is selected based on the damage index of the pressurization rate; the production control of stem cell exosomes in the skin care composition is carried out based on the secondary drying time, the critical water content threshold, and the target pressurization rate.

2. The method for room temperature preservation of stem cell exosomes in skincare compositions according to claim 1, characterized in that, The method for determining the secondary drying time based on the abrupt temperature rise of the sample and its proximity to the shelf temperature includes the following specific steps: For any given moment, obtain the difference between the sample temperature at that moment and the previous moment, and record the maximum value between the difference in sample temperature and 0 as the degree of temperature change at that moment. The absolute value of the difference between the sample temperature and the shelf temperature at any given moment is recorded as the sublimation endpoint gap at that given moment. The linearly normalized result of the ratio of the temperature change degree at any given moment to the sublimation endpoint gap is denoted as the intervention degree at any given moment. The moment when the degree of intervention exceeds the preset intervention threshold is recorded as the second drying moment.

3. The method for room temperature preservation of stem cell exosomes in skincare compositions according to claim 1, characterized in that, During the vacuum pressure fluctuation process after reaching the secondary drying time, the degree of moisture step at each pressurization rate is obtained based on the difference in residual moisture content between the pressurization rate and the previous pressurization rate. The specific method includes: After reaching the second drying time, the second drying stage begins, involving vacuum pressure fluctuations. For any pressurization rate, the difference between the residual moisture content after the last cycle of the previous pressurization rate and the residual moisture content after the first cycle of the previous pressurization rate is obtained. The ratio of the difference to the residual moisture content after the last cycle of the previous pressurization rate is recorded as the moisture step degree of the previous pressurization rate.

4. The room temperature preservation of stem cell exosomes in skincare compositions according to claim 1, characterized in that, The method for obtaining the moisture removal index for each pressurization rate based on the continuous decrease in residual moisture content at each pressurization rate includes the following specific steps: For any cycle at any pressurization rate, the difference between the residual moisture content of the previous cycle and the current cycle is recorded as the degree of moisture reduction in the current cycle. The minimum degree of water reduction across all cycles at the pressurization rate is denoted as the degree of sustained dehydration at the pressurization rate. The difference between the residual moisture content after the last cycle of the pressurization rate and the first cycle of the pressurization rate is recorded as the first difference. The product of the first difference and the degree of continuous dehydration of the pressurization rate is recorded as the moisture removal index of the pressurization rate.

5. The room temperature preservation of stem cell exosomes in skincare compositions according to claim 1, characterized in that, The specific method for obtaining the comprehensive moisture removal efficiency is as follows: For any pressurization rate, the product of the water step degree of the pressurization rate and the water removal index is denoted as the overall water removal efficiency of the pressurization rate.

6. The room temperature preservation of stem cell exosomes in skincare compositions according to claim 1, characterized in that, The method for determining the degree of activity loss at each pressurization rate based on the differences in protein activity between pressurization rates and the changes in overall water removal efficiency includes the following specific methods: For any given pressurization rate, the difference between the protein activity after the last cycle of the preceding pressurization rate and the protein activity after the last cycle of the preceding pressurization rate is denoted as the activity loss degree of the given pressurization rate. The inversely proportional normalized result of the absolute value of the difference between the preceding pressurization rate and the overall water removal efficiency of the given pressurization rate is denoted as the dehydration limit index of the given pressurization rate. The product of the activity loss degree of the given pressurization rate and the dehydration limit index is denoted as the dehydration inactivation degree of the given pressurization rate.

7. The room temperature preservation of stem cell exosomes in skincare compositions according to claim 1, characterized in that, The specific method for obtaining the critical water content threshold based on the degree of activity loss is as follows: The residual water content after the last cycle of the pressurization rate corresponding to the pressurization rate with the highest degree of dehydration and inactivation is taken as the critical water content threshold.

8. The room temperature preservation of stem cell exosomes according to claim 1 in skin care composition applications, characterized in that, The specific method for constructing the damage sensitivity for each pressurization rate based on the water content critical threshold is as follows: For any pressurization rate, the difference between the critical water cut threshold and the mean of the residual water cut of all cycles corresponding to the pressurization rate is obtained. The maximum value between the difference and 0 is used as the damage sensitivity factor of the pressurization rate. The result of an exponential function with base e and exponentiated by the damage sensitivity factor of the pressure boost rate is taken as the damage sensitivity of the pressure boost rate.

9. The room temperature preservation of stem cell exosomes according to claim 1 in skin care composition applications, characterized in that, The method for obtaining the damage index for each pressurization rate based on protein activity and overall water removal efficiency at the pressurization rate, combined with the damage sensitivity, includes the following specific steps: For any pressurization rate, obtain the inversely proportional normalized result of protein activity after the last cycle of the pressurization rate, and record the ratio of the inversely proportional normalized result to the overall water removal efficiency of the pressurization rate as the basic damage index of the pressurization rate. The product of the basic damage index of the boost rate and the damage sensitivity is denoted as the damage index of the boost rate.

10. The room temperature preservation of stem cell exosomes according to claim 1 in skin care composition applications, characterized in that, The target boost rate is specifically obtained as follows: Among all boost rates, the boost rate with the smallest damage index is selected as the target boost rate.

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