A collagen peptide anti-inflammatory and antioxidant function evaluation method and system based on an in-vitro digestion system and immune cell linkage

Abstract

The application discloses a collagen peptide anti-inflammatory and antioxidant function evaluation method and system based on an in-vitro digestion system and immune cells. The method comprises the following steps: constructing a continuous simulation digestion system comprising a stomach section, a small intestine section and a large intestine section, and performing simulation digestion and fermentation treatment on animal-derived collagen peptides; setting a selective membrane permeation component on the side wall of the simulation small intestine module to separate the digestion side fluid and the absorption side fluid, and setting a fixed proportion shunt unit on the absorption side to distribute the absorption side fluid obtained by membrane permeation according to a preset proportion and continuously transport the absorption side fluid to an immune cell perfusion reaction cabin at a constant flow rate, so that the small molecule peptides at the absorption end can continuously stimulate the immune cells online. By detecting the immune response indexes in real time and collecting short-chain fatty acids generated in the fermentation stage of the large intestine, the comprehensive evaluation of the anti-inflammatory and antioxidant activities of the collagen peptides can be realized. The application can simulate the continuous digestion process of the collagen peptides in the body, and improve the stability and physiological relevance of the function evaluation.

Description

Technical Field

[0001] This invention relates to a method and system for evaluating the anti-inflammatory and antioxidant functions of collagen peptides in vitro, involving the interaction between the digestive system and immune cells, and belongs to the field of biotechnology. Background Technology

[0002] Collagen peptides, as important products of animal-derived collagen degradation, have attracted widespread attention for their immunomodulatory, anti-inflammatory, and antioxidant properties due to their low molecular weight, easy absorption, and rich amino acid composition rich in glycine, proline, and hydroxyproline. Existing research indicates that the bioactivity of collagen peptides is highly correlated with their molecular weight structure. Small peptides in the 1–3 kDa range typically exhibit stronger immunomodulatory and antioxidant activities; while dipeptides and tripeptides <1 kDa are rapidly absorbed, their structural information is limited, and their functional activity is unstable; although peptides in the 3–5 kDa range still have some effect, their absorption efficiency and stimulation intensity are generally lower than those of functional peptides <3 kDa. Therefore, accurately identifying the <3 kDa functional peptides actually present in the small intestine is crucial for evaluating the actual bioactivity of collagen peptides.

[0003] However, the bioactivity of orally administered collagen peptides is influenced by the continuous enzymatic hydrolysis process in the stomach, small intestine, and large intestine, as well as the rhythm of absorption-promoting small peptide formation at different stages. Constructing an in vitro evaluation system that reflects this continuous physiological process is crucial for elucidating the functional mechanisms of collagen peptides. However, current research still has significant shortcomings in in vitro absorption simulation and evaluation of immune regulatory mechanisms, making it difficult to accurately predict their true bioavailability in vivo.

[0004] Current functional studies of collagen peptides are mostly based on cell models, animal experiments, or static in vitro digestion systems. While static digestion methods, such as INFOKES, can simulate the basic chemical environment of gastric and intestinal juices, they cannot reflect the dynamic changes in pH, enzyme concentration gradients, and peristaltic mixing in the real digestive tract. Furthermore, digestion products obtained in static systems are generally present as mixed endpoint samples, making it impossible to distinguish absorbable small peptides generated at different time points during continuous enzymatic digestion, and thus failing to identify key functional peptides formed during the window of peak small peptide activity.

[0005] While commercial dynamic digestion systems such as TIM and SHIME can simulate gastrointestinal motility, they primarily focus on the digestive dynamics and lack online selective separation structures for functional peptides <3 kDa. Acquiring small peptides at the absorption end still relies on offline ultrafiltration or centrifugation, which can easily lead to issues such as peptide ratio shifts, decreased activity, and sample oxidation. Furthermore, these systems are isolated from the immune function evaluation module, failing to achieve sterile, real-time linkage between digestion products and immune cells, making it difficult to capture the immediate immune effects of peptides of different molecular weights during digestion.

[0006] Furthermore, even with a simple series connection of the dynamic digestion system and the cell evaluation system, a mismatch between the flow rate of the absorption-side products and the volume of the in vitro cell reaction system remains. The absorption-side products produced by the digestion system typically have a large flow rate, while the volume of the in vitro immune cell culture system is limited. Directly introducing all of them can easily lead to an imbalance in the stimulus dosage or instability in fluid disturbance; if samples are manually diluted, it is difficult to ensure consistency in time sequence and proportion. Therefore, how to construct a stable proportional coupling structure between the absorption-side products and the cell reaction system in the in vitro system has become a key technical problem that has not yet been solved by existing technologies.

[0007] In terms of immune evaluation, existing methods mostly employ batch loading and endpoint detection, which only reflect the average results after several hours and cannot capture the immediate and dynamic responses of immune cells to different small peptide components. For example, the peak occurrence time of immune indicators such as NO, ROS, IL-6, and TNF-α varies significantly with peptide molecular weight, and endpoint detection methods ignore a large amount of dynamic information with mechanistic significance.

[0008] Furthermore, after collagen peptides enter the large intestine, they can be further broken down by gut microbiota into short-chain fatty acids such as acetic acid, propionic acid, and butyric acid. These metabolites are associated with immune regulation to some extent. However, existing systems generally cannot simultaneously obtain information on digestive products, the composition of small peptide absorption terminals, dynamic immune responses, and large intestine fermentation metabolites, making it difficult for researchers to conduct a holistic evaluation of the complete physiological chain of "digestion-absorption-immunity-fermentation".

[0009] In summary, current technologies still lack a comprehensive system capable of simultaneously simulating the continuous digestive process of the stomach, small intestine, and large intestine, accurately capturing functional peptides <3 kDa from the absorption end of the small intestine, achieving aseptic, real-time, and dynamic evaluation of these peptides on immune cells, and combining information from large intestine fermentation products. There is an urgent need for a comprehensive evaluation system that establishes a stable coupling relationship between continuous digestive pathways, absorption interface pathways, and cellular reaction pathways in vitro. Summary of the Invention

[0010] To address the aforementioned issues, this invention provides a method and system for evaluating the anti-inflammatory and antioxidant functions of collagen peptides through in vitro digestive system and immune cell linkage, which can accurately reproduce the in vivo digestion and absorption rhythm of collagen peptides and accurately assess their immune regulatory mechanisms.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a method for evaluating the anti-inflammatory and antioxidant functions of collagen peptides in an in vitro digestive system linked with immune cells. The method utilizes an evaluation system comprising: a biomimetic stomach module, a biomimetic small intestine module, a biomimetic large intestine fermentation module, a selective permeation membrane assembly, an absorption chamber, a diversion unit, a microfluidic delivery module, a sterilization filter, an immune cell perfusion reaction chamber, and a waste collection unit, all controlled by a controller. The evaluation method includes the following steps: S1: The pH range of the gastric segment is maintained at 1.2 to 2.5 in the bionic stomach module to perform acidic digestion of collagen peptides, and the peristaltic compression device simulates gastric contraction; S2: The gastric emptying products are transported to the bionic small intestine module, and the bionic small intestine module is maintained at a pH of 5.5 to 7.2. Pancreatic juice and bile salts are added to achieve continuous enzymatic hydrolysis. S3: During the enzymatic hydrolysis of the small intestine, the digestive fluid is allowed to enter the absorption cavity through the selective permeation membrane assembly to obtain extramembrane fluid; S4: After being distributed by the diversion unit, the extracellular fluid is continuously transported by the microfluidic delivery module at a constant flow rate to the sterilization filter for filtration, and then transported to the immune cell perfusion reaction chamber for reaction. Excess fluid is discharged into the waste liquid collection unit. S5: Real-time detection of changes in immune response indicators of immune cells during perfusion is achieved through the detection module to obtain time-dependent immune response curves; S6: The small intestinal emptying products are further fed into the biomimetic colon anaerobic module for fermentation under anaerobic conditions, and the short-chain fatty acids produced by fermentation are collected. S7: The anti-inflammatory and antioxidant functions of collagen peptides were evaluated by combining small peptide dynamics, immune response, and short-chain fatty acid data.

[0012] In one embodiment of the present invention, the selective permeation membrane component has a molecular weight cutoff range of 3 to 5 kDa and adopts a tangential flow circulation method.

[0013] In one embodiment of the present invention, the microfluidic delivery module includes a constant flow micropump with a delivery rate of 1.0–1.5 mL / h; the biomimetic small intestine module has a trypsin activity of 50–150 U / mL and a bile salt concentration of 5–15 mM; and the sterilization filter has a pore size of 0.22 μm.

[0014] In one embodiment of the present invention, the collagen peptides are derived from animal connective tissue, which includes skin, bone, tendon, cartilage or a combination thereof; the extracellular fluid in the absorption cavity is output once every 5 to 10 minutes.

[0015] In one embodiment of the present invention, the short-chain fatty acids include acetic acid, propionic acid and butyric acid, the immune response indicators include NO, ROS, IL-6 and TNF-α, and the detection methods include colorimetry, fluorescence method or immunoassay.

[0016] Secondly, the present invention also provides an evaluation system for the anti-inflammatory and antioxidant function of collagen peptides linked with an in vitro digestive system and immune cells, which is applied to the aforementioned evaluation method for the anti-inflammatory and antioxidant function of collagen peptides linked with an in vitro digestive system and immune cells. The evaluation system includes a biomimetic stomach module, a biomimetic small intestine module, and a biomimetic large intestine fermentation module connected in sequence to form an in vitro digestive system. A selective permeation membrane assembly and an absorption cavity, wherein the selective permeation membrane assembly is connected to the bionic small intestine module, and the side of the assembly facing away from the bionic small intestine module is connected to the absorption cavity; A flow splitting unit is connected to the absorption cavity; A microfluidic delivery module and a sterilization filter, wherein the microfluidic delivery module is connected to the diversion unit, and the sterilization filter is connected to the microfluidic delivery module; An immune cell perfusion reaction chamber is connected to the sterilization filter; A waste liquid collection unit is connected to the diversion unit; The controller is used to adjust and control the parameters of the bionic stomach module, bionic small intestine module, bionic large intestine fermentation module, diversion unit and microfluidic delivery module.

[0017] In one embodiment of the present invention, the diversion unit includes a first branch and a second branch, the first branch being connected to the microfluidic delivery module, the second branch being connected to the waste liquid collection unit, and the diversion unit having a distribution ratio of 1:4.

[0018] In one embodiment of the present invention, the bionic stomach module has an acidic regulation unit with pH 1.2 to 2.5 and a peristaltic mixing unit. The bionic stomach module simulates gastric contraction through rhythmic pressure of 3 to 5 times / minute. The bionic small intestine module has an enzymatic hydrolysis environment with pH 5.5 to 7.2 and an enzyme solution addition unit.

[0019] In one embodiment of the present invention, the immune cell perfusion reaction chamber is used to culture immune cells and has a cell carrying area and a real-time detection window.

[0020] In one embodiment of the present invention, the biomimetic coli fermentation module forms an anaerobic environment through inert gas replacement and operates at 37 ℃, with a coli fermentation time t≥24h.

[0021] The beneficial effects of this invention are: (1) The present invention provides a method and system for evaluating the anti-inflammatory and antioxidant functions of collagen peptides by linking the in vitro digestive system and immune cells. By constructing a continuous biomimetic digestive system including the stomach, small intestine and large intestine, it realistically simulates the physical and biochemical degradation process of collagen peptides in the human body, and solves the problem that traditional static evaluation methods cannot reflect the dynamic characteristics of continuous digestion.

[0022] (2) The evaluation method and system for the anti-inflammatory and antioxidant functions of collagen peptides in the in vitro digestive system and immune cells introduces an online transmembrane capture and immune cell linkage mechanism. By setting a fixed ratio diversion structure on the absorption side, the absorption products entering the immune cell reaction system maintain a stable ratio and flow rate, which improves the stability and repeatability of immune response data.

[0023] (3) The evaluation method and system for the anti-inflammatory and antioxidant functions of collagen peptides in the in vitro digestive system and immune cells significantly improves the objectivity and physiological relevance of collagen peptide function evaluation through comprehensive evaluation of multi-dimensional indicators (small peptide distribution, immune regulation intensity, fermentation metabolites), providing reliable technical support for the development of functional animal-derived collagen peptide products. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying 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.

[0025] Figure 1 This is a schematic diagram of the structure of an evaluation system for the anti-inflammatory and antioxidant functions of collagen peptides that links the in vitro digestive system with immune cells, according to the present invention.

[0026] Figure 2 This is a dynamic curve of the NO and IL-6 immune response in Example 1 of the present invention.

[0027] In the diagram: 1. Bionic stomach module; 2. Bionic small intestine module; 3. Bionic large intestine fermentation module; 4. Selective permeation membrane component; 5. Absorption chamber; 6. Diversion unit; 7. Microfluidic delivery module; 8. Sterilization filter; 9. Immune cell perfusion reaction chamber; 10. Waste liquid collection unit; 11. Controller. Detailed Implementation

[0028] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. 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.

[0029] like Figure 1 As shown, this invention provides an evaluation system for the anti-inflammatory and antioxidant functions of collagen peptides in an in vitro digestive system linked with immune cells. The evaluation system includes a biomimetic stomach module 1, a biomimetic small intestine module 2, and a biomimetic large intestine fermentation module 3 connected in sequence. The biomimetic small intestine module 2 has a selective permeation membrane component 4 on its sidewall, which divides the module into a digestive cavity and an absorptive cavity. The absorptive cavity forms an absorptive chamber 5. A diversion unit 6 is connected to one outlet of the absorptive chamber 5. The diversion unit 6 is divided into two branches: a first branch and a second branch. The first branch is connected to a microfluidic delivery module 7. The side of the microfluidic delivery module 7 facing away from the diversion unit 6 is connected in sequence to a sterilization filter 8 and an immune cell perfusion reaction chamber 9. The second branch is connected to a waste liquid collection unit 10, where excess liquid is directly discharged for collection. A detection module is installed on one side of the immune cell perfusion reaction chamber 9, which can monitor NO, ROS, and immune-related indicators such as IL-6 and TNF-α in real time. All modules are uniformly regulated by the controller 11.

[0030] In some embodiments, the microfluidic delivery module 7 includes a constant-flow micropump with a delivery rate of 1.0–1.5 mL / h and a flow rate accuracy of ±0.1 mL / h. The sterilization unit of the sterilization filter 8 is 0.22 μm. The biomimetic stomach module 1 has an acidity regulation unit for pH 1.2–2.5 and a peristaltic mixing unit, and the biomimetic stomach module 1 simulates gastric contractions through rhythmic pressure of 3–5 times / minute. The biomimetic small intestine module 2 has an enzymatic hydrolysis environment maintaining pH 5.5–7.2 and an enzyme solution addition unit. The immune cell perfusion reaction chamber 9 is used to culture RAW264.7 or THP-1 cells and has a cell carrying area and a real-time detection module. On one side of the biomimetic small intestine module 2, fluid enters the absorption chamber 5 through the selective permeation membrane component 4, is delivered by the microfluidic delivery module 7 after being split in a fixed ratio, and flows into the immune cell perfusion reaction chamber 9 after being sterilized and filtered by the sterilization filter 8, where it reacts with RAW264.7 or THP-1 cells.

[0031] In some embodiments, the absorbent fluid is proportionally distributed via the diversion unit 6, with the first branch entering the microfluidic delivery module 7 and being delivered to the immune cell perfusion reaction chamber, and the second branch entering the waste collection unit. The preferred flow distribution ratio between the first and second branches is 1:4. This ratio is primarily determined based on the volume matching relationship between the absorbent fluid volume generated by the in vitro digestion system and the volume of the immune cell perfusion reaction system. Preliminary experiments have shown that when the proportion of fluid entering the immune cell perfusion reaction chamber is controlled at approximately 20% of the total permeabilized fluid, it ensures both a continuous and stable stimulation of immune cells by small molecule peptides and avoids disturbances in the cell culture system or fluctuations in the stimulation dose due to excessive fluid volume. When the proportion entering the cell reaction system is too high, fluid scouring and concentration instability are likely to occur; when the proportion is too low, the stimulation intensity of small peptides is insufficient, resulting in a weak immune response signal.

[0032] In some embodiments, samples from the gastric and large intestine stages can be collected offline in stages as needed for research. After aseptic processing, these samples are added to the immune cell system for comparative analysis between different digestive stages. Small molecule peptides obtained from the small intestine absorptive fluid via the selective permeation membrane component 4 can directly enter the immune cell perfusion reaction chamber 9 under aseptic conditions, making them suitable for online immune response detection. However, the gastric and large intestine systems have different technical limitations during operation, making it difficult to use the same online detection method as the small intestine absorptive side. Specifically, the gastric digestive environment is under strongly acidic conditions, accompanied by strong mechanical mixing and preliminary protein hydrolysis. The system contains a high concentration of undegraded proteins and large molecule peptides, which can easily contaminate or clog the online detection equipment, thus affecting the detection stability. For the large intestine fermentation stage, this stage requires strictly anaerobic conditions, and the system contains a large number of active intestinal flora and fermentation metabolites. Directly introducing these into the online detection equipment can easily disrupt the anaerobic environment or cause microbial contamination, thereby affecting fermentation stability. Therefore, samples from the stomach and large intestine need to be analyzed using a phased offline sampling method followed by aseptic processing and immunoassay, in order to obtain reliable immunoresponse data while ensuring the stable operation of the system.

[0033] Furthermore, this invention also provides a method for evaluating the anti-inflammatory and antioxidant functions of collagen peptides in vitro, involving the interaction between the digestive system and immune cells. This method utilizes the aforementioned evaluation system for the anti-inflammatory and antioxidant functions of collagen peptides in vitro, involving the interaction between the digestive system and immune cells. The evaluation method includes the following steps: S1: Collagen peptides were used in lyophilized powder form and dissolved in simulated gastric juice at a concentration of 10 mg / mL before the experiment. After thorough dissolution, the dissolved peptides were directly added to the biomimetic stomach module 1. The acidity adjustment unit in the biomimetic stomach module 1 controlled the pH of the system within the range of 1.2–2.5 and applied rhythmic pressure of approximately 3–5 times / minute through a peristaltic compression structure installed on the outer wall of the reaction chamber, enabling the entire system to simulate basic physiological actions such as gastric contraction, mixing, and rhythmic propulsion. Under this environment, the collagen peptides underwent preliminary hydrolysis, with some peptide chains being sheared and degraded into medium and short peptide segments of different molecular weights. The gastric segment treatment time could be adjusted according to the characteristics of the peptide raw materials, solubility, and research objectives, and was typically maintained at 1.5–2 hours.

[0034] S2: After gastric emptying, the sample enters the biomimetic small intestine module 2 at a stable flow rate. The module's internal pH adjustment unit maintains the system within the physiological range of 5.5–7.2, and the enzyme addition unit continuously replenishes pancreatic juice and bile salts. The trypsin activity is 100 U / mL, and the bile salt concentration is 10 mM, allowing the peptides to continue enzymatic hydrolysis in this region. Compared to the static system, the biomimetic small intestine module 2 allows peptides to gradually transform into smaller molecules in a flowing environment under relatively stable temperature, pH, and enzyme concentration conditions.

[0035] S3: The selective permeation membrane assembly 4, installed on the sidewall of the small intestine module, has a molecular weight cutoff range of 3–5 kDa and can operate in a tangential flow mode to reduce membrane fouling and improve the stability of small peptide capture. During enzymatic digestion, the digestive fluid from the small intestine enters the absorption chamber 5 through the selective permeation membrane assembly 4 to form the absorption-side fluid. The absorption-side fluid can be output at intervals of several minutes, with an interval of 5–10 minutes, thereby obtaining the compositional characteristics of absorbable small peptides at different time points and providing a dynamic sample source for subsequent immune cell evaluation.

[0036] S4: The absorbent fluid first enters a fixed-ratio distribution unit 6, where it is distributed to the first and second branches according to a preset ratio. The fluid in the first branch enters the microfluidic delivery module 7, which then delivers it to the sterilization filter 8. After sterilization filtration through a 0.22 μm filter membrane, it enters the immune cell perfusion reaction chamber 9. The fluid in the second branch enters the waste collection unit 10. The microfluidic delivery module 7 uses a constant-flow micropump to maintain a stable delivery rate of approximately 1.0–1.5 mL / h, ensuring that the small peptides are accurately delivered to the immune cell perfusion reaction chamber 9 in chronological order. This delivery method reduces the degradation, oxidation, or adsorption problems of small peptides caused by traditional offline processing, allowing immune evaluation to better reflect the real-time generated small peptide components.

[0037] S5: The immune cell perfusion reaction chamber 9 can culture RAW264.7 macrophages or differentiated THP-1 cells. The perfusion environment operates under constant temperature conditions, reducing the impact of flow rate fluctuations and bubble interference on cell state, allowing cells to continuously receive small peptide stimulation from different digestion stages under stable substrate conditions. The online detection module can monitor NO, ROS, and immune indicators such as IL-6 and TNF-α in real time, presenting the immune response as data curves and achieving high-time-resolution immunodynamic evaluation.

[0038] S6: After enzymatic digestion in the small intestine, the remaining substrate that is not captured by the membrane enters the biomimetic colon fermentation module 3. The biomimetic colon fermentation module 3 creates an anaerobic environment through inert gas replacement and controls the pH within the range of 6.0–7.2 to simulate the further decomposition of residual peptides by intestinal flora during fermentation. This stage produces short-chain fatty acids such as acetic acid, propionic acid, and butyric acid. Their changes can serve as auxiliary indicators for the evaluation system of this invention, used to observe the trend correlation between colonic metabolism and immune response.

[0039] S7: Evaluate the immunomodulatory effects of collagen peptides by combining small peptide dynamics, immune response, and short-chain fatty acid data.

[0040] Alternatively, immune indicators can be detected using colorimetric, fluorescence, or immunoassay methods.

[0041] Optionally, the collagen peptides are exemplified by sheepskin-derived collagen peptides from animal skin sources. However, those skilled in the art will understand that collagen peptides from various animal skin sources, such as cowhide, pigskin, horsehide, and fish skin, can be evaluated according to the method of this invention, and the process and conditions can be appropriately adjusted according to the characteristics of the raw materials.

[0042] Optionally, the parameters of this evaluation method can be adjusted according to different animal skin-derived collagen peptides, different enzymatic hydrolysis processes, or research directions. As long as the technical route of continuous digestion, online collection, fixed-ratio splitting of the absorption-side fluid, aseptic microfluidic transport, and dynamic immunomodulatory detection is maintained, a comprehensive evaluation of the immunomodulatory potential of collagen peptides can be achieved. Those skilled in the art can optimize and extend the conditions without departing from the premise of this invention.

[0043] Optionally, the specific shape, number, and relative layout of each module in the evaluation system can be adjusted according to actual application needs.

[0044] Example 1: Evaluation of in vitro continuous digestion and immunomodulation of animal skin-derived collagen peptides (using sheepskin as an example) In this embodiment, lyophilized sheepskin-derived collagen peptide powder was used as an example sample. It was dissolved in simulated gastric juice at a concentration of 10 mg / mL and then sequentially digested in a biomimetic stomach module 1, a biomimetic small intestine module 2, and a biomimetic large intestine fermentation module 3. The pH of the digestive system in the biomimetic stomach module was controlled at 2.0, and gastric contractions were simulated by a rhythmic pressure of 4 times / minute using an external wall peristaltic compression device.

[0045] After approximately 2 hours of digestion in the gastric segment, the molecular weight composition of the peptides changed significantly, with <3 kDa peptides accounting for approximately 38%, 3–5 kDa peptides accounting for approximately 34%, and >5 kDa peptides decreasing to approximately 28%. When samples from the end of the gastric segment were introduced into the immune response system, NO levels in RAW264.7 cells decreased by approximately 20% compared to baseline, and IL-6 levels decreased by approximately 10%, indicating that a certain degree of immunomodulatory effect could be generated under gastric segment conditions, but the overall inhibitory strength remained limited.

[0046] After the sample entered the biomimetic small intestine module 2, continuous enzymatic hydrolysis of the small intestine was performed at 37 ℃. The pH of the small intestine system was controlled at 6.5, bile salts were added to achieve a final concentration of 10 mM, and trypsin was added to achieve a final activity of 100 U / mL. After continuous enzymatic hydrolysis for 3 h under the above conditions, the proportion of absorbable small peptides was significantly increased, with <3 kDa small peptides accounting for approximately 73%, 3–5 kDa small peptides accounting for approximately 20%, and >5 kDa peptides further decreasing to approximately 7%, indicating a significant enrichment of small molecule peptides.

[0047] During enzymatic hydrolysis, the small intestinal digestive fluid enters the absorption chamber 5 via a selective permeation membrane component 4 with a molecular weight cutoff of 3 kDa, forming the absorption-side fluid. This absorption-side fluid is distributed by a splitting unit 6, with the first branch and the second branch having a flow ratio of 1:4. The fluid from the first branch is delivered via a microfluidic delivery module 7 at a constant flow rate of 1.2 mL / h to a 0.22 μm sterile filter 8 for sterilization and filtration before flowing into the immune cell perfusion reaction chamber 9. The fluid from the second branch enters the waste collection unit 10. The fluid from the first branch entering the immune cell perfusion reaction chamber 9 continuously stimulates RAW264.7 cells, resulting in a significant decrease in NO levels (inhibition rate of approximately 48%) and a decrease in IL-6 of approximately 31%, exhibiting a clear anti-inflammatory and immunomodulatory trend. At this time point, the small peptide composition is highly enriched in the absorbable low molecular weight range, showing a good temporal correlation with the enhanced immune response.

[0048] like Figure 2As shown, residual peptides and undigested matrix that were not captured by the membrane entered the biomimetic colon fermentation module 3 and continued fermentation for 24 h under anaerobic conditions at 37 ℃ and pH 6.8. During this stage, the peptides were further degraded by the intestinal flora, and short-chain fatty acids gradually accumulated, with the final concentrations of acetic acid, propionic acid, and butyric acid being approximately 18.5 mM, 6.4 mM, and 3.1 mM, respectively. Samples from the terminal colon were collected offline as a stage and, after aseptic treatment, were added to an immune cell system for evaluation. A decrease of approximately 54% in NO and approximately 36% in IL-6 was observed. Although the content of small peptides in the colon stage did not increase further compared to the terminal colon, the inhibition of NO and IL-6 remained at a high level in samples with higher levels of short-chain fatty acids. The trend of these changes was consistent with the accumulation of short-chain fatty acids such as butyric acid, and can serve as an auxiliary evaluation basis for the direct effects of small peptides.

[0049] In summary, animal skin-derived collagen peptides exhibited rapid small peptide generation and a prominent immunomodulatory response in the continuous biomimetic digestion-absorption side online capture-fixed ratio diversion-immunoperfusion evaluation system constructed in this invention. Particularly in the small intestine stage, the abundant accumulation of <3 kDa functional peptides closely correlated with the significant decrease in NO and IL-6, and the temporal changes in the gastric, small intestinal, and large intestine segments showed a good correspondence with the immune indicators. This embodiment verifies the accuracy and stability of the system in presenting the continuous process of collagen peptide "digestion-absorption-immunomodulation-colonic fermentation," providing a benchmark for subsequent comparisons with other embodiments or comparative examples.

[0050] Example 2: Evaluation of in vitro continuous digestion and immunomodulation of animal skin-derived collagen peptides (using bovine hide as an example) In this embodiment, bovine skin-derived collagen peptide lyophilized powder was used as an example sample. It was dissolved in simulated gastric juice at a concentration of 10 mg / mL and then sequentially digested in a biomimetic stomach module 1, a biomimetic small intestine module 2, and a biomimetic large intestine fermentation module 3. Except for the source of the raw materials, all other digestion conditions, permeabilization parameters, split ratios, microfluidic delivery conditions, and large intestine fermentation conditions were the same as in Example 1.

[0051] After approximately 2 hours of digestion in the stomach, the molecular weight composition of the peptides changed, with <3 kDa peptides accounting for approximately 34%, 3–5 kDa peptides accounting for approximately 36%, and >5 kDa peptides accounting for approximately 30%. The degree of small peptide formation was slightly lower than in Example 1. The end-stomach sample was used as a staged offline sample and, after aseptic processing, was added to an immunomodulatory system for control analysis. NO levels decreased by approximately 17%, and IL-6 decreased by approximately 8%, showing a similar but slightly weaker early immunomodulatory capacity to Example 1.

[0052] After the sample entered the biomimetic small intestine module 2, continuous enzymatic hydrolysis of the small intestine stage was performed at 37 °C. Bile salts were added to the system to achieve a final concentration of 10 mM, and trypsin was added to achieve a final activity of 100 U / mL. After continuous enzymatic hydrolysis for 3 h under the above conditions, the proportion of small peptides was further increased, with <3 kDa small peptides accounting for approximately 68%, 3–5 kDa peptides accounting for approximately 24%, and >5 kDa peptides decreasing to approximately 8%. Compared with Example 1, the enrichment of small molecule peptides was slightly lower.

[0053] During enzymatic hydrolysis, the small intestinal digestive fluid enters the absorption chamber 5 via the selective permeation membrane assembly 4, forming the absorption-side fluid. The absorption-side fluid is distributed in a 1:4 ratio via the diversion unit 6. The first branch of fluid is delivered under constant flow conditions via the microfluidic delivery module 7 to the sterilization filter 8 for sterilization and filtration before flowing into the immune cell perfusion reaction chamber 9. The second branch of fluid enters the waste collection unit 10. After the first branch of fluid entering the immune cell perfusion reaction chamber 9 continuously acts on RAW264.7 cells, NO levels decrease by approximately 43%, and IL-6 decreases by approximately 27%. The overall immunosuppressive trend is consistent with Example 1, but the inhibitory intensity is slightly weaker, consistent with the differences in the degradation characteristics of bovine skin-derived collagen peptides and their small peptide generation rates.

[0054] Residual peptides not captured by the transmembrane in the small intestine were introduced into the biomimetic colon fermentation module 3 and fermented for another 24 hours under anaerobic conditions. The final accumulation levels of short-chain fatty acids were approximately 16.2 mM acetic acid, 5.5 mM propionic acid, and 2.6 mM butyric acid, slightly lower than in Example 1. When samples from the terminal colon were taken offline as a stage and aseptically processed before being added to an immune cell system for evaluation, NO decreased by approximately 48% and IL-6 decreased by approximately 31%, with the overall trend consistent with the accumulation of short-chain fatty acids.

[0055] In summary, animal-derived collagen peptides (bovine hide as an example) also exhibited good digestive kinetics and immunomodulatory capabilities in the continuous biomimetic digestion-absorption-side online capture-fixed-ratio diversion-immunoperfusion evaluation system constructed in this invention. While their small peptide generation rate, low-molecular-weight peptide ratio, and immunomodulatory intensity were slightly weaker than those of sheepskin-derived collagen peptides, their temporal evolution trends remained consistent, further validating the applicability and stability of the evaluation system of this invention for collagen peptides from different sources.

[0056] Comparative Example 1: Stomach pH was high (pH 3.0) To compare the impact of gastric acidity deviating from the range defined in this invention on the evaluation results of collagen peptide digestion and immune regulation, the pH of the gastric digestion environment was adjusted to approximately 3.0, while the operating parameters of the remaining modules remained consistent with those in Example 1.

[0057] After 2 hours of digestion in the stomach, small peptides <3 kDa accounted for approximately 26%, small peptides of 3–5 kDa accounted for approximately 32%, and peptides >5 kDa increased to approximately 42%, significantly higher than the approximately 28% proportion of large molecular weight peptides in Example 1. This indicates that the early hydrolysis of collagen peptides was insufficient under conditions of high acidity in the stomach. Samples from the end of the stomach segment were collected offline as a stage, aseptically processed, and then added to an immune cell system for control analysis.

[0058] During the enzymatic hydrolysis process, the fluid from the small intestine digestive side enters the absorption chamber 5 through the selective permeation membrane assembly 4, and is distributed in a 1:4 ratio by the diversion unit 6. The fluid from the first branch enters the immune cell perfusion reaction chamber 9 under constant flow conditions for evaluation. NO decreased by about 29%, and IL-6 decreased by about 17%, both significantly lower than the inhibition levels in Example 1.

[0059] After 24 h of anaerobic fermentation in the large intestine, the cumulative levels of short-chain fatty acids were 13.8 mM acetic acid, 5.1 mM propionic acid, and 2.1 mM butyric acid, which were generally lower than in Example 1. Samples from the terminal large intestine were collected offline as a stage, aseptically processed, and then added to an immune cell system for evaluation. The decreases in NO and IL-6 were approximately 33% and 20%, respectively. These results indicate that when gastric acidity is high, the initial degradation of collagen peptides is insufficient, leading to limited subsequent small peptide production and consequently a decrease in the overall immunomodulatory effect.

[0060] Comparative Example 2: Gastric segment pH was low (pH 1.0) In this comparative example, the pH of the gastric digestive environment was adjusted to approximately 1.0, while the other conditions remained the same as in Example 1.

[0061] After 2 hours of digestion in the stomach segment, the proportion of <3 kDa peptides was approximately 44%, 3–5 kDa peptides approximately 30%, and >5 kDa peptides decreased to approximately 26%, indicating that the early peptide generation rate was slightly faster than in Example 1. Samples from the end of the stomach segment were collected offline as a stage and, after aseptic processing, were added to an immune cell system for control analysis. However, due to the low acidity of the stomach segment, some peptides underwent excessive hydrolysis. After the samples were further digested in the biomimetic small intestine module for 3 hours, the proportion of <3 kDa peptides increased to approximately 64%, significantly higher than in Comparative Example 1, but still lower than the approximately 73% in Example 1; 3–5 kDa peptides accounted for approximately 24%, and >5 kDa peptides approximately 12%.

[0062] During enzymatic hydrolysis, the fluid from the small intestine digestive side enters the absorption chamber 5 via the selective permeation membrane assembly 4, and is then distributed in a 1:4 ratio by the diversion unit 6. The fluid from the first branch enters the immune cell perfusion reaction chamber 9 under constant flow conditions for evaluation. Immunological evaluation results showed that the decreases in NO and IL-6 were approximately 34% and 22%, respectively, still lower than the immunosuppressive levels observed in Example 1.

[0063] After 24 h of fermentation in the large intestine, the cumulative concentrations of acetic acid, propionic acid, and butyric acid were approximately 15.1 mM, 5.6 mM, and 2.4 mM, respectively, slightly lower than in Example 1. Samples from the terminal large intestine were collected offline as a stage, aseptically processed, and then added to an immune cell system for evaluation. The decreases in NO and IL-6 were approximately 38% and 25%, respectively. The results indicate that while excessively low gastric acidity is beneficial for early degradation, it is not conducive to the formation of structurally stable small peptide combinations with superior immune activity. Overall, the evaluation results are still inferior to those under the conditions specified in this invention.

[0064] Comparative Example 3: Insufficient enzymatic digestion in the small intestine (decreased concentrations of pancreatic juice and bile salts) To investigate the effect of insufficient enzymatic digestion in the small intestine on collagen peptide absorption and immunomodulation, the amount of pancreatic juice and bile salts added in the small intestine was reduced to approximately 50% of that in Example 1, i.e., the final trypsin activity was 50 U / mL and the final bile salt concentration was 5 mM, while other conditions remained the same. After 2 hours of digestion in the stomach, the composition of small peptides was basically the same as in Example 1, with <3kDa, 3–5 kDa, and >5 kDa peptides accounting for approximately 38%, 34%, and 28%, respectively.

[0065] After 3 hours of continued enzymatic hydrolysis in the small intestine, the proportion of <3 kDa peptides only increased to about 52%, while 3–5 kDa and >5 kDa peptides still accounted for about 28% and 20%, respectively, which was significantly lower than the enrichment of small peptides in Example 1.

[0066] During the enzymatic hydrolysis process, the fluid from the small intestine digestive side enters the absorption chamber 5 through the selective permeation membrane assembly 4, and is distributed in a 1:4 ratio by the diversion unit 6. The fluid from the first branch enters the immune cell perfusion reaction chamber 9 under constant flow conditions for evaluation. The immune evaluation results show that the decrease in NO and IL-6 is approximately 26% and 16%, respectively, which is significantly weaker than in Example 1.

[0067] After 24 h of anaerobic fermentation in the large intestine, the cumulative concentrations of acetic acid, propionic acid, and butyric acid were approximately 13.1 mM, 4.7 mM, and 1.9 mM, respectively, all lower than in Example 1. The corresponding decreases in NO and IL-6 were approximately 29% and 19%, respectively. These results indicate that insufficient enzymatic digestion in the small intestine restricts the formation of absorbable small peptides, leading to a decline in immunomodulatory capacity. Furthermore, insufficient substrate for subsequent fermentation further limits metabolism and immune response in the large intestine.

[0068] Comparative Example 4: Excessive enzymatic digestion in the small intestine (increased concentration of pancreatic juice and bile salts) This comparative example increased the amount of pancreatic juice and bile salts added in the small intestine stage to approximately 150% of that in Example 1, i.e., the final trypsin activity was 150 U / mL and the final bile salt concentration was 15 mM, while other conditions remained the same. The composition of the gastric digestion products was basically the same as in Example 1, but after 3 hours of enzymatic digestion in the small intestine, the peptide composition showed a significant shift. The proportion of <3 kDa peptides increased to approximately 76%, while the proportion of 3–5 kDa peptides decreased to approximately 17%, and the proportion of >5 kDa peptides further decreased, indicating that some medium molecular weight peptides may have been further degraded due to excessive enzymatic digestion.

[0069] During enzymatic hydrolysis, the fluid from the small intestine digestive side enters the absorption chamber 5 via the selective permeation membrane assembly 4 and is then distributed in a 1:4 ratio by the diversion unit 6. The fluid from the first branch enters the immune cell perfusion reaction chamber 9 under constant flow conditions for evaluation. Immunological evaluation of the small intestine extracellular fluid showed a decrease in NO and IL-6 of approximately 39% and 25%, respectively. While this was better than under insufficient enzymatic hydrolysis conditions, it was still lower than the immunomodulatory effect of Example 1. After 24 hours of fermentation in the large intestine stage, the cumulative concentrations of acetic acid, propionic acid, and butyric acid were approximately 17.6 mM, 6.2 mM, and 2.9 mM, respectively, slightly lower than in Example 1, with corresponding decreases in NO and IL-6 of approximately 43% and 29%, respectively.

[0070] The comparative results show that although excessive enzymatic hydrolysis in the small intestine increases the proportion of small peptides <3 kDa, it disrupts the structural balance between functional peptides of different molecular weights, resulting in the immunomodulatory effect and fermentation metabolism level not reaching the optimal state under the conditions specified in this invention.

[0071] Comparative Example 5: 3–5 kDa selective permeabilization was disabled (no online small peptide capture was performed). To compare the impact of not using the 3–5 kDa selective transmembrane online collection structure on the evaluation results, this comparative example removed the selective transmembrane component and its corresponding absorption chamber and fixed-ratio shunt structure from the biomimetic small intestine module, directly collecting the mixed digested sample within the small intestinal reaction chamber. All other digestion and operating conditions remained consistent with Example 1. After 2 hours of gastric digestion, the small peptide distribution was the same as in Example 1, with <3 kDa, 3–5 kDa, and >5 kDa peptides accounting for approximately 38%, 34%, and 28%, respectively.

[0072] In the mixed sample obtained after enzymatic hydrolysis of the small intestine for 3 hours, small peptides <3 kDa accounted for approximately 49%, small peptides of 3–5 kDa accounted for approximately 26%, while peptides >5 kDa still accounted for approximately 25%, significantly higher than the proportion of residual macromolecular peptides under the permeabilization trapping conditions in Example 1. This result indicates that in the absence of a selective permeabilization structure, the sample still contains a high proportion of macromolecular peptides, making it difficult to reflect the enrichment characteristics of small peptides at the small intestinal absorption end.

[0073] When the mixed sample was used directly for immunoassay after sterilization, the decreases in NO and IL-6 were approximately 32% and 19%, respectively, both significantly lower than the inhibition levels under the online transmembrane trapping structure conditions in Example 1.

[0074] After 24 hours of fermentation in the large intestine stage, the cumulative concentrations of acetic acid, propionic acid, and butyric acid were approximately 14.2 mM, 5.0 mM, and 2.0 mM, respectively, with corresponding decreases in NO and IL-6 of approximately 36% and 23%, respectively, which were also lower than those in Example 1.

[0075] The above results indicate that removing the 3–5 kDa selective permeabilization allows unscreened macromolecular peptides to enter the immunoassay system, causing the sample composition to deviate from the small peptide enrichment characteristics, thereby weakening the immunomodulatory effect and the representativeness of subsequent fermentation evaluation, further demonstrating the necessity of the online permeabilization trapping structure of this invention.

[0076] Comparative Example 6: Using an unsuitable membrane pore size (1 kDa membrane) To illustrate the impact of membrane pore size selection on the accuracy of small peptide capture, this comparative example replaced the selective permeabilization component in the biomimetic small intestine module with a membrane having a molecular weight cutoff of approximately 1 kDa, while keeping all other conditions consistent with Example 1. The distribution of small peptides after 2 hours of gastric digestion was the same as in Example 1. After 3 hours of enzymatic hydrolysis in the small intestine, the digestive fluid from the small intestine entered the absorption chamber through a 1 kDa permeabilization membrane and was distributed in a 1:4 ratio by a diversion unit. The first branch of fluid entered the immune cell perfusion reaction chamber under constant flow conditions for evaluation.

[0077] In the resulting absorber-side fluid, peptides of <3 kDa, 3–5 kDa, and >5 kDa comprised approximately 88%, 10%, and 2%, respectively. Although the absorber-side fluid exhibited a high proportion of <3 kDa peptides, the 1 kDa membrane excessively blocked some short to medium-sized peptides with immunomodulatory effects, causing the peptide composition of the absorber-side fluid to be overly biased towards ultra-low molecular weight components, resulting in insufficient information on functional peptide structures. Immunological evaluation results showed that under these conditions, the reductions in NO and IL-6 were approximately 35% and 22%, respectively, lower than the inhibition levels observed under the 3–5 kDa permeabilization conditions in Example 1.

[0078] After 24 h of anaerobic fermentation in the large intestine stage, the cumulative concentrations of acetic acid, propionic acid and butyric acid were approximately 16.5 mM, 5.9 mM and 2.7 mM, respectively, with corresponding decreases of approximately 41% and 27% in NO and IL-6, respectively, which were still lower than those in Example 1.

[0079] The comparative results show that although a small membrane pore size can effectively block large peptides, it will limit the types of small peptides that can pass through, making the peptide spectrum of the absorbent side too simple, thereby weakening the immunomodulatory effect and the subsequent fermentation and metabolic synergistic effect. This further shows that the 3–5 kDa membrane parameters selected in this invention have better overall balance.

[0080] Comparative Example 7: Using an unsuitable membrane pore size (10 kDa membrane) To illustrate the impact of membrane pore size selection on the accuracy of small peptide capture, this comparative example replaced the selective permeabilization component in the biomimetic small intestine module with a membrane having a molecular weight cutoff of approximately 10 kDa, while keeping all other conditions consistent with Example 1. The distribution of small peptides after 2 hours of gastric digestion was the same as in Example 1. After 3 hours of enzymatic hydrolysis in the small intestine, the digestive fluid from the small intestine entered the absorption chamber through the 10 kDa permeabilization membrane and was distributed in a 1:4 ratio by the diversion unit 6. The first branch of fluid entered the immune cell perfusion reaction chamber under constant flow conditions for evaluation.

[0081] In the resulting absorber-side fluid, peptides of <3 kDa, 3–5 kDa, and >5 kDa comprised approximately 57%, 23%, and 20%, respectively. The proportion of large molecular weight peptides in the absorber-side fluid was significantly higher than in Example 1, indicating that the 10 kDa membrane could not effectively block high molecular weight peptides, causing the resulting sample to deviate from the characteristics of small peptide enrichment. Immunological evaluation results showed that the decreases in NO and IL-6 under these conditions were approximately 36% and 22%, respectively, significantly lower than the inhibition levels observed under the 3–5 kDa permeabilization conditions in Example 1.

[0082] After 24 h of anaerobic fermentation in the large intestine stage, the cumulative concentrations of acetic acid, propionic acid and butyric acid were approximately 15.8 mM, 5.7 mM and 2.5 mM, respectively, with corresponding decreases of approximately 40% and 25% in NO and IL-6, respectively, which were still lower than those in Example 1.

[0083] The comparative results show that improper selection of the permeate membrane pore size can lead to the entry of macromolecular peptide residues into the absorbent fluid, which can compromise the accuracy of the peptide composition at the absorbent end. As a result, the immunomodulatory effect and the level of short-chain fatty acid production are significantly weaker than those of Example 1, which uses a 3–5 kDa permeate membrane. This further verifies the rationality of the permeate membrane parameter settings of the present invention.

[0084] Comparative Example 8: Eliminating the fixed-ratio flow distribution structure To illustrate the impact of the fixed-ratio shunt structure on the stability of immunoassay, this comparative example, while retaining the 3–5 kDa selective permeabilization module, eliminated the fixed-ratio shunt unit on the absorption side. The fluid from the absorption side after permeabilization directly entered the microfluidic delivery module and was transported to the immune cell perfusion reaction chamber. All other conditions remained consistent with Example 1. After 2 hours of gastric digestion, the small peptide distribution was the same as in Example 1.

[0085] After enzymatic hydrolysis for 3 hours in the small intestine, the digestive fluid from the small intestine enters the absorption cavity through a 3–5 kDa permeabilized membrane. The molecular weight distribution of the resulting absorption fluid is basically the same as in Example 1, with <3 kDa small peptides accounting for about 73%, 3–5 kDa small peptides accounting for about 20%, and >5 kDa peptides accounting for about 7%.

[0086] However, due to the lack of a fixed-ratio diversion structure, all the fluid on the absorption side entered the microfluidic delivery module, and no waste discharge branch was provided. During continuous operation, the volume and concentration of the fluid entering the immune cell perfusion reaction chamber fluctuated with the rate of enzymatic digestion in the small intestine. Immunological evaluation results showed that the average decreases in NO and IL-6 were approximately 44% and 29%, respectively. Although these were close to the inhibition levels of Example 1, the fluctuation range was significantly increased in repeated experiments, and the standard deviation of the immune indicators was higher than that of Example 1.

[0087] After 24 hours of anaerobic fermentation in the large intestine, the cumulative concentration of short-chain fatty acids was basically the same as in Example 1, but the stability of the time curves of the corresponding immune indicators decreased.

[0088] The above results indicate that, without a fixed-ratio shunt structure, although enriched small peptide components can be obtained through membrane permeation, the proportion and flow rate of the absorbent fluid entering the immune cell system are difficult to maintain stably, leading to decreased repeatability of immune evaluation data. Therefore, a fixed-ratio shunt structure helps improve the stability and repeatability of the immune evaluation process.

[0089] Comparative Example 9: Microfluidic continuous perfusion was cancelled (offline batch sampling was adopted). To compare the impact of the immunoassay method on the results, this comparative study retained the 3–5 kDa selective permeabilization module and the fixed-ratio shunt structure, but removed the microfluidic continuous perfusion structure.

[0090] After enzymatic digestion for 3 hours in the small intestine, the digestive fluid from the small intestine enters the absorption chamber via a selective permeation membrane assembly and is then distributed at a 1:4 ratio via a shunt unit. The resulting first branch fluid is no longer introduced into the immune cell perfusion reaction chamber via microfluidic continuous perfusion; instead, it is collected and added to the RAW264.7 cell culture system in a static batch manner, while the remaining digestion conditions remain consistent with those in Example 1.

[0091] The distribution of small peptides after 2 hours of gastric digestion was the same as in Example 1, and the proportions of <3 kDa, 3–5 kDa, and >5 kDa peptides in the absorptive fluid of the small intestine were basically the same as in Example 1.

[0092] Because the sample was added in a single, high-concentration form, rather than acting on immune cells via a low-flow-rate, continuous perfusion method, the dynamic stimulation of cells by the small peptide was weakened. Immunological evaluation results showed that the decreases in NO and IL-6 were approximately 34% and 21%, respectively, significantly lower than the inhibition levels of approximately 48% and 31% under continuous perfusion conditions in Example 1.

[0093] When the product from the fermentation of the large intestine stage for 24 hours was treated with immune cells in a static manner, the decreases in NO and IL-6 were approximately 38% and 24%, respectively, failing to exhibit the gradually increasing immune response characteristics over time as shown in Example 1.

[0094] The above results indicate that offline batch sampling is difficult to simulate the continuous stimulation of immune cells by small peptides at the absorption end, resulting in a decrease in the amplitude and temporal resolution of the immune response. This further verifies the role of the microfluidic continuous perfusion method in constructing an immunodynamic evaluation.

[0095] Comparative Example 10: Microfluidic flow rate too low (0.3 mL / h) To investigate the impact of excessively low flow rate on the evaluation results during immune cell perfusion, this comparative example, while retaining the 3–5 kDa selective permeabilization module and the fixed ratio shunt structure, reduced the microfluidic delivery rate of the first branch fluid to approximately 0.3 mL / h, while keeping the other conditions consistent with Example 1.

[0096] After enzymatic hydrolysis in the small intestine for 3 hours, the digestive fluid from the small intestine enters the absorption chamber through a membrane and is then split at a 1:4 ratio before entering the microfluidic system. The molecular weight distribution of the resulting absorption fluid is essentially the same as in Example 1.

[0097] Due to the low perfusion flow rate, the residence time of small peptides in the delivery path is prolonged, making them more susceptible to adsorption or activity attenuation in the microfluidic tubing and interface areas. Immunological evaluation results showed that the decreases in NO and IL-6 in RAW264.7 cells were approximately 33% and 20%, respectively, lower than the 48% and 31% in Example 1.

[0098] After 24 hours of fermentation in the large intestine stage, the accumulation level of short-chain fatty acids was similar to that in Example 1, but the corresponding decreases in NO and IL-6 were approximately 36% and 23%, respectively, which were still lower than those in Example 1.

[0099] The above results indicate that when the microfluidic perfusion flow rate is too low, although the composition of small peptides does not change significantly, the effective activity is insufficient, leading to a decrease in the intensity of immune stimulation. This suggests that an appropriate perfusion flow rate plays an important role in maintaining the biological activity of small peptides.

[0100] Comparative Example 11: Microfluidic flow rate too high (2.5 mL / h) To compare the effect of excessively high perfusion flow rate on the immunoassay results, this comparative example increased the microfluidic delivery rate of the first branch fluid to approximately 2.5 mL / h while maintaining the 3–5 kDa permeabilized membrane and fixed shunt structure. All other conditions remained the same as in Example 1.

[0101] After enzymatic hydrolysis in the small intestine for 3 hours, the molecular weight distribution of the absorptive fluid was basically consistent with that in Example 1. Due to the excessively rapid perfusion flow rate, the residence time of the small peptides in the immunoreaction chamber was shortened, resulting in insufficient interaction time between immune cells and the small peptides. Immunological evaluation results showed that the decreases in NO and IL-6 were approximately 30% and 18%, respectively, which were lower than those in Example 1.

[0102] After 24 hours of fermentation in the large intestine stage, the levels of short-chain fatty acids were similar to those in Example 1, but the corresponding NO and IL-6 decreased by approximately 33% and 21%, respectively.

[0103] The comparative example shows that when the microfluidic perfusion flow rate is too high, although the small peptides can quickly enter the immune response chamber, they are difficult to form a sustained stimulating effect, thereby weakening the immunomodulatory effect, which further verifies the rationality of the perfusion flow rate range set in this invention.

[0104] Comparative Example 12: Insufficient fermentation time in the large intestine (only 6 hours) To compare the effects of insufficient colonic fermentation time on short-chain fatty acid production and immune regulation trends, this comparative example shortened the anaerobic fermentation time of the biomimetic colon module to 6 hours while retaining the 3–5 kDa selective permeable membrane component, absorption chamber, and fixed-ratio shunt structure. The conditions for the remaining gastric and small intestinal stages remained consistent with those in Example 1.

[0105] After 6 hours of fermentation, the accumulation of short-chain fatty acids was significantly lower, with acetic acid, propionic acid and butyric acid at approximately 7.8 mM, 2.9 mM and 1.1 mM, respectively, which were significantly lower than the accumulation levels in Example 1 under 24-hour conditions.

[0106] Large intestine samples were collected offline as a phase, aseptically processed, and then added to an immune cell system for evaluation. The decreases in NO and IL-6 in RAW264.7 cells were approximately 50% and 33%, respectively. Although these results were slightly higher than those in the small intestine, they were significantly lower than the 54% and 36% observed in Example 1 under 24-hour fermentation conditions, failing to demonstrate a trend of further enhancement in the later stages of fermentation.

[0107] The above results indicate that insufficient fermentation time in the large intestine limits the sufficient accumulation of short-chain fatty acids, resulting in an incomplete time-dependent immunomodulatory effect.

[0108] Comparative Example 13: Insufficient fermentation time in the large intestine (only 12 hours) To further investigate the effects of coli fermentation time on short-chain fatty acid production and immune regulation enhancement, the anaerobic fermentation time of the biomimetic coli module in this comparative example was set to 12 hours, while the other conditions remained the same as in Example 1.

[0109] After 12 hours of fermentation, the cumulative concentrations of acetic acid, propionic acid and butyric acid were approximately 12.4 mM, 4.8 mM and 2.0 mM, respectively, which were significantly higher than those after 6 hours of fermentation, but still lower than the cumulative levels in Example 1 under 24-hour conditions.

[0110] The large intestine stage samples were collected offline as a phase. After aseptic processing, they were added to the immune cell system for evaluation. The immune evaluation results showed that the decrease in NO and IL-6 in RAW264.7 cells was approximately 52% and 34%, respectively, which was enhanced in the small intestine stage, but still significantly lower than the 54% and 36% in Example 1 under the whole large intestine fermentation conditions, showing a trend of "medium fermentation time - moderate immune enhancement".

[0111] The comparative results show that the coli fermentation time has a significant time-dependent relationship with the accumulation of short-chain fatty acids and the immunomodulatory effect. Although 12-hour fermentation can produce a certain auxiliary anti-inflammatory effect, it is still difficult to achieve the optimal effect under the 24-hour condition, further proving the scientific rationality of the coli fermentation time set in this invention.

[0112] Comparative Example 14: The fermentation time of the large intestine was too long (36 hours). To further investigate the effects of prolonged coliform fermentation time on short-chain fatty acid production and enhanced immune regulation, this comparative example extended the anaerobic fermentation time of the biomimetic coliform module to 36 hours, while the conditions for the remaining gastric and small intestinal stages remained consistent with those in Example 1.

[0113] After 36 hours of fermentation, the cumulative concentrations of acetic acid, propionic acid and butyric acid were approximately 19.3 mM, 6.6 mM and 3.2 mM, respectively, showing only a slight increase compared to the cumulative levels in Example 1 under the 24-hour condition, indicating that the formation of short-chain fatty acids gradually tended to stabilize.

[0114] The large intestine stage samples were collected offline as a phase. After aseptic treatment, they were added to the immune cell system for evaluation. The immune evaluation results showed that the decrease in NO and IL-6 in RAW264.7 cells was approximately 53% and 35%, respectively. Compared with 54% and 36% under 24-hour fermentation conditions in Example 1, no further significant enhancement was observed, showing a trend of "prolonged fermentation time - immune effect tending to plateau".

[0115] The comparative results show that when the coli fermentation time exceeds 24 hours, the cumulative increase of short-chain fatty acids tends to level off, and the improvement of the immunomodulatory effect is limited. Further extending the fermentation time is unlikely to yield significant gains, indicating that 24 hours is the optimal coli fermentation time condition.

[0116] Table 1. Molecular weight distribution of peptides in the gastric / small intestinal segment (%)

[0117] Table 2. NO decrease rate (%) in each stage

[0118] Table 3. IL-6 decrease rate (%) at each stage

[0119] Table 4. Control table of short-chain fatty acid (SCFA) formation in the large intestine (mM)

[0120] As can be seen from the above Examples 1-2 and Comparative Examples 1-14, the present invention, by constructing a continuous biomimetic digestive system including the stomach, small intestine and large intestine, can simulate the continuous degradation process of collagen peptides in the human body. Compared with static digestion evaluation methods, it is more conducive to reflecting the influence of changes in digestive kinetics at different stages on the generation of small peptides.

[0121] Meanwhile, this invention incorporates a selective permeation membrane component in the biomimetic small intestine stage, combined with a fixed-ratio shunt and microfluidic continuous perfusion structure, enabling online capture and immune cell-linked evaluation of small molecule peptides on the small intestine's absorptive side. This allows the immune response to correspond to the dynamic changes in components at the permeation membrane absorptive end. Comparative analysis of conditions such as permeation pore size, enzymatic hydrolysis intensity, perfusion flow rate, and fermentation time reveals a relatively stable and consistent trend relationship between small peptide distribution, immunomodulatory intensity, and short-chain fatty acid production within the parameter range defined by this invention. Within the parameter range defined by this invention, the anti-inflammatory and antioxidant capacity of collagen peptides can be dynamically evaluated throughout the digestion process, more closely resembling the human digestive process. This ensures the accuracy, authenticity, repeatability, and stability of the evaluation, providing a foundation for future research on the anti-inflammatory and antioxidant properties of collagen peptides.

[0122] Based on the evaluation system and evaluation method provided by this invention, and by setting reasonable process parameter ranges and multi-dimensional evaluation indicators (including small peptide distribution ratio, NO and IL-6 inhibition amplitude, short chain fatty acid accumulation level, etc.), the stage changes of collagen peptides in the process of "digestion-absorption-immune response-fermentation metabolism" can be reflected more systematically. This improves the objectivity and physiological relevance of the evaluation of the anti-inflammatory and antioxidant activities of collagen peptides, and provides a technical basis for the functional evaluation and process optimization of animal-derived collagen peptide products.

[0123] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included within the protection scope of the present invention.

Claims

1. A method for evaluating the anti-inflammatory and antioxidant functions of collagen peptides in vitro, involving the digestive system and immune cells, characterized in that... An evaluation system was used, comprising: a bionic stomach module (1), a bionic small intestine module (2), a bionic large intestine fermentation module (3), a selective permeation membrane assembly (4), an absorption chamber (5), a diversion unit (6), a microfluidic delivery module (7), a sterilization filter (8), an immune cell perfusion reaction chamber (9), and a waste collection unit (10), and controlled by a controller (11); the evaluation method includes the following steps: S1: The pH range of the gastric segment is maintained at 1.2 to 2.5 in the bionic stomach module (1) to perform acidic digestion of collagen peptides and simulate gastric contraction through a peristaltic compression device; S2: The gastric emptying products are transported to the bionic small intestine module (2) to maintain the bionic small intestine module (2) at a pH of 5.5 to 7.

2. Pancreatic juice and bile salts are added to achieve continuous enzymatic hydrolysis. S3: During the enzymatic digestion of the small intestine, the digestive fluid is introduced into the absorption chamber (5) through the selective permeation membrane assembly (4) to obtain extramembrane fluid; S4: After the transmembrane extravasated fluid is distributed by the diversion unit (6), it is continuously transported by the microfluidic delivery module (7) at a constant flow rate to the sterilization filter (8) for filtration, and then transported to the immune cell perfusion reaction chamber (9) for reaction. Excess fluid is discharged into the waste liquid collection unit (10). S5: Real-time detection of changes in immune response indicators of immune cells during perfusion is achieved through the detection module to obtain time-dependent immune response curves; S6: The small intestine emptying products are further input into the biomimetic large intestine anaerobic module (3) for fermentation under anaerobic conditions, and the short-chain fatty acids produced by fermentation are collected. S7: The anti-inflammatory and antioxidant functions of collagen peptides were evaluated by combining small peptide dynamics, immune response, and short-chain fatty acid data.

2. The method for evaluating the anti-inflammatory and antioxidant functions of collagen peptides in the in vitro digestive system in conjunction with immune cells, as described in claim 1, is characterized in that... The selective permeation membrane component (4) has a molecular weight cutoff range of 3 to 5 kDa and adopts a tangential flow circulation method.

3. The method for evaluating the anti-inflammatory and antioxidant functions of collagen peptides in the in vitro digestive system in conjunction with immune cells, as described in claim 1, is characterized in that... The microfluidic delivery module (7) includes a constant flow micropump with a delivery speed of 1.0 to 1.5 mL / h; the biomimetic small intestine module (2) has a trypsin activity of 50 to 150 U / mL and a bile salt concentration of 5 to 15 mM; and the sterilization filter (8) has a pore size of 0.22 μm.

4. The method for evaluating the anti-inflammatory and antioxidant functions of collagen peptides in the in vitro digestive system in conjunction with immune cells according to claim 1, characterized in that, The collagen peptides are derived from animal connective tissue, which includes skin, bone, tendon, cartilage or a combination thereof; the extracellular fluid in the absorption chamber (5) is output once every 5 to 10 minutes.

5. The method for evaluating the anti-inflammatory and antioxidant functions of collagen peptides in the in vitro digestive system in conjunction with immune cells, as described in claim 1, is characterized in that... The short-chain fatty acids include acetic acid, propionic acid, and butyric acid; the immune response indicators include NO, ROS, IL-6, and TNF-α; and the detection methods include colorimetric, fluorescence, or immunoassay methods.

6. A system for evaluating the anti-inflammatory and antioxidant functions of collagen peptides in conjunction with the in vitro digestive system and immune cells, applied to the method for evaluating the anti-inflammatory and antioxidant functions of collagen peptides in conjunction with the in vitro digestive system and immune cells as described in any one of claims 1-5, characterized in that, It includes a bionic stomach module (1), a bionic small intestine module (2), and a bionic large intestine fermentation module (3) connected in sequence to form an in vitro digestive system; Selective permeation membrane assembly (4) and absorption chamber (5), wherein the selective permeation membrane assembly (4) is connected to the bionic small intestine module (2), and the side opposite to the bionic small intestine module (2) is connected to the absorption chamber (5); The diversion unit (6) is connected to the absorption cavity (5); Microfluidic delivery module (7) and sterilization filter (8), wherein the microfluidic delivery module (7) is connected to the diversion unit (6) and the sterilization filter (8) is connected to the microfluidic delivery module (7); An immune cell perfusion reaction chamber (9) is connected to the sterilization filter (8); Waste liquid collection unit (10) is connected to the diversion unit (6); The controller (11) is used to adjust and control the parameters of the bionic stomach module (1), the bionic small intestine module (2), the bionic large intestine fermentation module (3), the diversion unit (6) and the microfluidic delivery module (7).

7. The evaluation system for the anti-inflammatory and antioxidant functions of collagen peptides in the in vitro digestive system and immune cell linkage according to claim 6, characterized in that, The diversion unit (6) includes a first branch and a second branch. The first branch is connected to the microfluidic delivery module (7), and the second branch is connected to the waste liquid collection unit (10). The distribution ratio of the diversion unit (6) is 1:

4.

8. The evaluation system for the anti-inflammatory and antioxidant functions of collagen peptides in the in vitro digestive system and immune cell linkage according to claim 6, characterized in that, The bionic stomach module (1) has an acidic regulation unit with pH 1.2 to 2.5 and a peristalsis mixing unit. The bionic stomach module (1) simulates gastric contraction through rhythmic pressure of 3 to 5 times / minute. The bionic small intestine module (2) has an enzymatic hydrolysis environment with pH 5.5 to 7.2 and an enzyme solution addition unit.

9. The evaluation system for the anti-inflammatory and antioxidant functions of collagen peptides in the in vitro digestive system and immune cell linkage according to claim 6, characterized in that, The immune cell perfusion reaction chamber (9) is used to culture immune cells and has a cell carrying area and a real-time detection window.

10. The evaluation system for the anti-inflammatory and antioxidant functions of collagen peptides in the in vitro digestive system and immune cell linkage according to claim 6, characterized in that, The biomimetic colon fermentation module (3) forms an anaerobic environment through inert gas replacement and operates at 37 ℃, with a colon fermentation time t≥24h.

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