Endotoxin source head control method and device for short-range cold chain transportation of cowhide

Abstract

The application discloses a method for controlling endotoxin sources in short-range cold chain transportation of raw materials of animal-derived medical devices, and relates to the field of preservation and cold chain transportation of raw materials of medical devices. The method simultaneously inhibits two parallel accumulation mechanisms of endotoxin, i.e., a microorganism proliferation-endotoxin release mechanism and a permeability pressure-induced bacterial lysis-endotoxin secondary release mechanism, by means of three elements, i.e., temperature (3-5 DEG C), osmotic pressure (0.9% isotonic sodium chloride solution) and low-oxygen-concentration gas-phase environment (controlling the oxygen content in the top space above the liquid surface of the isotonic sodium chloride solution to be not higher than 1%), so that source quantitative control of endotoxin in a transportation window is realized. After the raw material of cowhide is treated by the method, the total number of colonies is less than or equal to 1*10 6 CFU / g, the endotoxin is less than or equal to 5 EU / mg protein (satisfying the internal control quality standard of an enterprise), the integrity rate of the triple helix structure of collagen is greater than or equal to 95%, the DSC thermal denaturation temperature is greater than or equal to 60 DEG C, and the peptide chain breakage rate is less than 1%.

Description

Technical Field

[0001] This application relates to the field of preservation and cold chain transportation of medical device raw materials, specifically to a method and device for joint control of endotoxin sources for short-distance cold chain transportation of bovine hides. Background Technology

[0002] Fresh cowhide is a high-quality natural source of collagen, with the dermis containing 70-80% collagen by dry weight. Once fresh cowhide is removed from the animal after slaughter, it immediately enters a degradation process, with three pathways working simultaneously: microbial proliferation, endotoxin release, and endogenous protease degradation. Quality control during transportation from the slaughterhouse to the raw material processing plant is a significant challenge.

[0003] Currently, the pain points for domestic manufacturers of animal-derived collagen implants are concentrated in three aspects: 1. The quality of raw bovine hides fluctuates greatly, with poor consistency between batches. 2. Entire batches of raw materials are often scrapped due to excessive microbial levels or high endotoxin content. 3. Traditional raw material quality control mainly relies on endpoint testing, lacking a technical solution for systematic control from the source.

[0004] As bovine collagen implants are classified as Class III medical devices, microbial and endotoxin control is a core quality requirement throughout the entire production chain. Current industry practices mostly focus on downstream ultrafiltration purification processes for microbial and endotoxin control, neglecting the problem of endotoxins accumulating in large quantities before downstream processes, i.e., the short transportation window from slaughter to processing plant reception.

[0005] In existing technologies, the following methods are mainly used for the transportation and preservation of raw cowhide:

[0006] Routine ambient temperature transportation (no special control): Under ambient temperature transportation conditions (26-40℃ in summer), Gram-negative bacteria enter the logarithmic phase and multiply rapidly after a 2-4 hour delay period. At 37℃, the generation time is approximately 20-30 minutes, with an initial colony count of about 10²-10³ CFU / g. After 6-8 hours, this can amplify to 10⁶-10⁷ CFU / g, reaching the critical value for detectable spoilage. The death and lysis of Gram-negative bacteria release lipopolysaccharides, resulting in severely excessive levels of endotoxins, failing to meet the requirements for Class III medical device raw materials. With both microorganisms and endotoxins out of control, the entire batch of raw materials is scrapped.

[0007] Conventional refrigerated transportation (single temperature control): Conventional meat cold chain (0-4℃) is designed according to food preservation standards, but it has four shortcomings when used for medical raw materials: tap water washing cannot control the introduction of endotoxins; there is no sealed immersion protection during transportation, the surface of the cowhide is dehydrated, and the collagen hydration layer is lost; the oxygen content in the container is about 21%, which is conducive to the proliferation of aerobic bacteria; quality control only checks the total number of colonies, regardless of endotoxins and collagen activity.

[0008] Chemical preservative treatment during transportation: Formalin, glutaraldehyde, phenol, 75% alcohol, and other chemical preservatives are used, or strong alkaline chemical hair removal agents such as sodium sulfide (Na2S) and calcium hydroxide are employed. Aldehyde preservatives covalently cross-link with the amino groups on the lysine side chains of collagen, disrupting the triple helix hydrogen bonds and hydrophobic interactions, resulting in loss of in vitro self-assembly ability. Residual chemical reagents do not meet the safety requirements for Class III medical devices. Strongly alkaline hair removal agents damage the collagen fiber structure of the dermis while removing hair, making it impossible to simultaneously achieve preservation and viability maintenance.

[0009] Frozen transport: Frozen transport at -20℃ and below can inhibit microbial and enzymatic degradation, but the formation of ice crystals (volume expansion of about 9%) during freezing causes physical and mechanical damage to the collagen fiber network; freeze-thaw cycles cause collagen fibers to dehydrate and aggregate, resulting in a decrease in the integrity of collagen fibers after rewarming, reducing solubility and in vitro self-assembly ability; frozen transport is energy-intensive and not suitable for industrial-scale bulk transport.

[0010] In summary, existing technologies fail to establish a source control method for endotoxins during the short-distance transportation window after slaughter, leading to the following systemic challenges for downstream processes: endotoxins accumulate excessively by the end of the transportation window, resulting in downstream ultrafiltration processes facing endotoxin loads far exceeding design expectations; even with an endotoxin removal rate of approximately 99.5% using ultrafiltration membranes, residual endotoxins may still exceed the limits for the final product; furthermore, LPS (endotoxin) molecules can adsorb and bind to collagen through non-covalent electrostatic interactions, reducing the removal efficiency of adsorbed endotoxins during subsequent extraction and purification processes under high endotoxin accumulation conditions, further compressing the endotoxin safety margin of the final product.

[0011] Therefore, establishing a precise method for quantifying and controlling endotoxins from the source of materials is a necessary condition for ensuring the continuous compliance of the final product, but existing technical solutions do not provide such a method. Summary of the Invention

[0012] Addressing the systemic problem of uncontrolled endotoxin accumulation in animal-derived collagen implant raw materials during short-distance transportation after slaughter, this application provides a method for synergistic control of endotoxin and microorganisms during the transportation window through three factors: temperature (3°C to 5°C), osmotic pressure (0.9% isotonic sodium chloride solution), and gas phase environment (nitrogen replacement in the headspace, oxygen content not exceeding 1%). This method ensures that the endotoxin level at the end of the transportation window is below 5 EU / mg protein, while preserving the integrity of the collagen triple helix structure.

[0013] According to one aspect of this application, an embodiment of this application provides a method for controlling the source of endotoxins in short-distance cold chain transportation of bovine hides, comprising: step S1, pre-treating fresh bovine hides to obtain bovine dermal tissue; step S2, immersing the bovine dermal tissue in a 0.9% isotonic sodium chloride solution, ensuring complete immersion of the bovine dermal tissue during immersion; step S3, controlling the immersion temperature of the bovine dermal tissue to be 3℃-5℃; step S4, controlling the oxygen concentration above the surface of the isotonic sodium chloride solution to be less than or equal to 1%; and step S5, transporting the bovine hide raw material in a cold chain at 3℃-5℃ for a duration not exceeding 8 hours.

[0014] Preferably, in step S1, the fresh cowhide is pretreated to obtain the dermal tissue, including: soaking the fresh cowhide in purified water at 3℃-5℃; roughly washing the soaked fresh cowhide in purified water at 3℃-5℃; physically removing hair using a mechanical hair removal device at an environment not exceeding 8℃; after completion, placing the fresh cowhide in water for injection at 3℃-5℃ for fine washing; and processing the finely washed cowhide to obtain the dermal tissue.

[0015] Preferably, in step S1, during the coarse washing, the volume-to-mass ratio of purified water to fresh cowhide is 20:1-30:1 mL / g, and the coarse washing time is 15-30 minutes; during the fine washing, the volume-to-mass ratio of water for injection to fresh cowhide is 15:1-20:1 mL / g, and the fine washing time is 10-20 minutes.

[0016] Preferably, the fresh cowhide is obtained no more than 1.5 hours after slaughter. Preferably, the fresh cowhide comes from healthy cattle that have tested negative for prions and does not contain brain, spinal cord, tonsils, or distal ileum tissue.

[0017] Preferably, in step S2, the volume-to-mass ratio of isotonic sodium chloride solution to bovine dermal tissue is 15:1-20:1 mL / g.

[0018] Preferably, in step S4, controlling the oxygen concentration above the surface of the isotonic sodium chloride solution to be less than or equal to 1% includes: replacing the surface of the isotonic sodium chloride solution with nitrogen gas of not less than 99.99% purity; during replacement, monitoring the oxygen concentration at the gas outlet and controlling the oxygen concentration at the gas outlet to be less than or equal to 1%. Preferably, the flow rate of nitrogen replacement is 3-5 L / min, the replacement duration is 30 to 60 seconds, and the target of not exceeding 1% is confirmed by monitoring the outlet oxygen concentration.

[0019] Preferably, in step S3, the soaking temperature of the bovine dermal tissue is controlled at 4±0.5℃.

[0020] Preferably, after unloading at the end of cold chain transportation, the raw cowhide is transferred to a cold storage at a temperature of 3°C to 5°C within 30 minutes for temporary storage, during which time the raw cowhide is continuously immersed in the original preservation solution.

[0021] According to another aspect of this application, embodiments of this application also provide an endotoxin source control device for short-distance cold chain transportation of bovine hides, comprising: a sealed container for containing raw bovine hides to be transported; an isotonic preservation solution supply system connected to the sealed container for providing a 0.9% isotonic sodium chloride solution to the sealed container; a gas replacement system connected to the sealed container for providing nitrogen gas to the sealed container; a temperature sensor for monitoring the temperature inside the sealed container; a controller connected to the temperature sensor to acquire the temperature inside the sealed container; and a cooler connected to the sealed container and the controller; the controller is configured to control the operating state of the cooler based on the temperature data acquired by the temperature sensor, so that the temperature inside the sealed container is maintained at 3℃-5℃.

[0022] Preferably, the temperature sensor records temperature data at a frequency of no less than once per minute and sends an alarm when the temperature exceeds the range of 3°C to 5°C. Preferably, the temperature sensor is a low-power temperature recorder. Preferably, the temperature sensor can be connected to a cloud server in real time for remote temperature monitoring.

[0023] Preferably, the system further includes an oxygen concentration sensor, which is disposed at the nitrogen outlet of the sealed container. The oxygen concentration sensor is connected to the controller, which is also connected to the gas replacement system. The controller is further configured to control the operating state of the gas replacement system based on the oxygen concentration data obtained by the oxygen concentration sensor, so that the oxygen concentration in the sealed container is less than or equal to 1%.

[0024] Preferably, the sealed container is a passive phase change material cold chain box, containing an internal n-alkane phase change material with a phase change temperature of 4°C, and externally covered with a vacuum insulation panel with a thermal insulation coefficient of no more than 0.03 W / (m·K). In other embodiments, the sealed container can also be an actively cooled vehicle refrigerator, with a target temperature set at 4°C.

[0025] The inventors of this application have discovered that after slaughter, Gram-negative bacteria (mainly Pseudomonas and Enterobacteriaceae) and Gram-positive bacteria inevitably remain in the dermal tissue of bovine hide. The outer membrane of Gram-negative bacterial cell walls contains lipopolysaccharide (LPS, i.e., endotoxin), and its accumulation within the transport window is driven by two parallel mechanisms: 1. Microbial proliferation-endotoxin release mechanism: Gram-negative bacteria enter the exponential proliferation phase at temperatures above 10°C. Newly added cells lyse extensively after entering the stationary phase, releasing endotoxins into surrounding tissues. At room temperature (25°C), endotoxin levels can rapidly increase from an initial <1 EU / mg protein to 15-30 EU / mg protein within 48 hours, far exceeding the safety threshold. 2. Osmotic pressure-induced cell lysis-secondary endotoxin release mechanism: If the transport medium is a hypotonic solution, osmotic shock can cause Gram-negative bacterial cell membrane rupture (i.e., osmotic shock), exacerbating endotoxin release. This means that inappropriate transport medium selection not only fails to reduce endotoxins but may also artificially increase the endotoxin load. The two mechanisms mentioned above can occur simultaneously and reinforce each other.

[0026] Therefore, the endotoxin source control method for short-distance cold chain transportation of bovine hides provided in the embodiments of this application adopts an endotoxin-microorganism joint control mechanism, namely, the synergistic effect of three elements: low temperature of 3°C to 5°C, isotonic environment and controlled oxygen concentration, to inhibit the accumulation of endotoxins and achieve quantitative control of endotoxin sources during transportation.

[0027] Specifically, the core mechanism of endotoxin control is as follows: 1. Low temperature (3-5℃) inhibits the aerobic metabolism and binary fission proliferation of Gram-negative bacteria, reducing their metabolic activity to an extremely low level, thereby inhibiting the microbial proliferation-endotoxin release mechanism; 2. An isotonic environment (0.9% NaCl, 308 mOsm / L) keeps Gram-negative bacteria in an osmotic dormant state rather than a lysis and death state, avoiding the large-scale release of endotoxins due to bacterial lysis. At the same time, the isotonic environment does not generate osmotic shock pressure, preventing secondary endotoxin release induced by hypotonicity; 3. Reducing the oxygen content in the headspace to no more than 1% inhibits aerobic bacterial metabolism, reduces oxidative stress damage to collagen molecules, and indirectly reduces bacterial death and endotoxin release caused by oxidative stress. In a preferred embodiment, the nitrogen atmosphere generated by nitrogen replacement further reduces the metabolic rate of Gram-negative bacteria.

[0028] In some embodiments, the above-described joint control method can ensure that the quality indicators of raw cowhide materials, after being transported and stored via cold chain, meet all of the following conditions: total bacterial count not exceeding 1×10⁻⁶. 6CFU / g; endotoxin levels meet the limits specified in the company's internal quality control standards, not exceeding 5 EU / mg protein; the integrity rate of the triple helix structure of type I collagen is not less than 95%; the peptide chain breakage rate is less than 1%; and the DSC denaturation temperature is not less than 60℃. In some preferred embodiments, the above-mentioned combined control method can reduce the total bacterial count to 8.2 × 10⁻⁶. 4 CFU / g (standard limit ≤1×10) 6 Nitrogen replacement reduced the total bacterial count by 31.7% (t-test, P=0.003), providing dual protection for microbial safety. Simultaneously, the triple helix integrity reached 97.2% (standard limit ≥95%), the DSC thermal denaturation temperature Tm was 63.4℃ (standard limit ≥60℃), and the peptide chain breakage rate was 0.72% (standard limit <1%). The rate of cross-linking between collagen molecules was extremely low at 3℃ to 5℃, minimizing the risk of thermally induced unwinding of the triple helix structure.

[0029] Comparative Example 1 data shows that nitrogen replacement independently contributed to a 25.0% reduction in endotoxin (t-test, P=0.009); Comparative Example 2 data (6℃ group) further demonstrates that at 5℃, endotoxin levels still have a 2.4 EU margin from the 5 EU / mg protein threshold, while at 6℃, the margin is only 0.5 EU, and the difference in endotoxin levels between 5℃ and 6℃ is statistically significant (t-test, P=0.023). This data provides quantitative support for the scientific validity of the upper temperature limit.

[0030] Comparative Example 4 (2℃ group) further demonstrates that although the microbial inhibition effect is more significant at 2℃ than at 3℃, the temperature maintenance accuracy of the refrigeration equipment is limited (temperature fluctuation range of approximately ±1.0℃). This can cause the local temperature inside the transport box to momentarily drop below 0℃, triggering heterogeneous nucleation of the 0.9% isotonic sodium chloride solution and forming micro-ice crystals. This causes irreversible damage to the mechanical integrity of collagen fibers, with a triple helix integrity rate of only 93.8% (below the 95% threshold) and a peptide chain breakage rate of 1.8% (exceeding the <1% threshold). The difference in triple helix integrity rate compared to the 3℃ group is statistically significant (t-test, P=0.002). This data provides quantitative support for the scientific validity of the lower limit of the temperature range.

[0031] The above results indicate that the synergistic design of the three elements—temperature range of 3℃ to 5℃, isotonic environment, and nitrogen replacement—is the key combination for achieving quantitative control of endotoxin sources.

[0032] This application addresses the specific preservation requirements of bovine dermal raw materials for Class III medical devices. It identifies cellular metabolism and attached microbial metabolic activities in fresh bovine dermal tissue. Therefore, the core preservation objective in this scenario is to maintain the natural structure of collagen fibers and the stability of the hydration layer, while simultaneously controlling microbial proliferation and endotoxin accumulation at the source. It eliminates the need for complex, specialized preservation components to maintain cell viability; a 0.9% isotonic sodium chloride solution is sufficient to achieve multiple benefits, including osmotic pressure stability, inhibition of microbial lysis and endotoxin release, and protection of the intact collagen structure. The overall process is simple, cost-effective, and easily industrialized, perfectly suited for short-distance cold chain transportation after slaughter. The 0.9% isotonic sodium chloride solution can be stored at room temperature in any medical institution (shelf life 2 years), eliminating the need for complex preservation solutions. Cold chain packaging only requires a standard 3°C to 5°C vehicle refrigerator or cold chain box. Detailed Implementation

[0033] The preferred embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection claimed in this application. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the materials used are common commercially available products. The equipment and materials used in each embodiment are the same.

[0034] Example 1

[0035] The raw material is fresh cowhide from Simmental / Heifetto cattle (12 months old) within 1.5 hours after slaughter, with a thickness of 2-5 mm.

[0036] The raw cowhide is treated in the following order: soaking in purified water at 3°C ​​to 5°C for 10 minutes; first rough washing: 20 minutes in purified water at 4°C; physical hair removal: cleaning and disinfecting a stainless steel scraper, keeping the tissue moist during the hair removal process at an ambient temperature not exceeding 8°C; second fine washing: 20 minutes in water for injection at 4°C.

[0037] Storage and transportation: 0.9% NaCl solution at 4℃, temperature controlled at 4℃, nitrogen purging, 8 hours; cold chain box is a PCM 4℃ phase change material cold chain box.

[0038] Actual data: n=6 independent samples from 6 different slaughter batches: total bacterial count 8.2×10⁻⁶ 4 CFU / g, endotoxin 2.1 EU / mg protein, triple helix integrity 97.2%, peptide chain breakage rate 0.72%, DSC Tm value 63.4℃, O2 content in the headspace after nitrogen replacement 0.6%, cold chain transportation time 8 hours, all indicators meet the standard limits.

[0039] Example 2

[0040] The temperature of the transport system (physiological saline and transport temperature) was controlled at 3°C, and the other conditions were the same as in Example 1.

[0041] Actual measurement data:

[0042] n=6 independent samples: total colony count 7.1×10 4 CFU / g, endotoxin 1.8 EU / mg protein, triple helix integrity 97.8%, DSC Tm value 63.6℃.

[0043] Compared with the standard condition of 4℃, the microbial index improved by about 13%-14% and the structural integrity index improved by about 0.5%-0.8%, proving that the method of the present invention has a safety margin in the low temperature direction.

[0044] Micro-ice crystals have begun to form in isotonic sodium chloride solutions below 3°C (such as 2°C in Comparative Example 4), damaging the mechanical integrity of collagen fibers (triple helix integrity rate drops to 93.8%, peptide chain breakage rate rises to 1.8%, both exceeding the safety threshold). Therefore, the lower limit of low temperature in this invention is set at 3°C.

[0045] Example 3

[0046] The temperature of the transport system (physiological saline and transport temperature) was controlled at 5°C, and the other conditions were the same as in Example 1.

[0047] Actual measurement data:

[0048] n=6 independent samples: total colony count 9.8×10 4 CFU / g, endotoxin 2.6 EU / mg protein, triple helix integrity 96.7%, DSC Tm value 63.2℃.

[0049] Compared with the standard conditions of 4℃, the microbial indicators increased by about 19%-24%, but all indicators were still within the standard limits, proving that 5℃ is reasonable as the upper limit temperature for cold chain transportation.

[0050] Example 3 also suggests that in hot seasons (ambient temperature > 35°C), active cooling vehicle refrigerators should be used instead of passive PCM cold chain boxes to ensure that the internal temperature is kept below 5°C.

[0051] Data from Examples 1, 2, and 3 were compiled. At three temperatures (3℃, 4℃, and 5℃), the total bacterial count, endotoxin levels, and triple-helix integrity were measured every hour (n=6 independent samples). Key time point data: At hour 0, the total bacterial count for all three temperature groups was 4.2 × 10⁻⁶. 4CFU / g, endotoxin level was 0.9 EU / mg protein, and triple helix integrity was 100%. At 8 hours: the total bacterial count in the 3℃ group was 7.1 × 10⁻⁶. 4 CFU / g, endotoxin 1.8 EU / mg protein, triple helix integrity 97.8%; total bacterial count in the 4℃ group was 8.2 × 10⁻⁶. 4 CFU / g, endotoxin 2.1 EU / mg protein, triple helix integrity 97.2%; total bacterial count at 5℃ 9.8 × 10⁻⁶ 4 CFU / g, endotoxin 2.6 EU / mg protein, triple helix integrity 96.7%. Within the 8-hour transport window, all indicators under three temperature conditions were within the standard limits.

[0052] This time series data provides quantitative support for the temperature range of 3℃ to 5℃. For every 1℃ increase in temperature, the total bacterial count increases by approximately 1.7 × 10⁻⁶ after 8 hours. 4 CFU / g (approximately 20%), endotoxin increased by approximately 0.3 EU / mg protein (approximately 14%), and triple helix integrity decreased by approximately 0.5%-0.6%.

[0053] Statistical analysis showed that the differences in total bacterial count and endotoxin levels among the three temperature groups at the 8-hour time point were statistically significant (ANOVA, total bacterial count F=12.4, P=0.001; endotoxin F=8.7, P=0.003).

[0054] Example 4

[0055] The cold chain transportation time is shortened to 3 hours, and the other conditions are the same as in Example 1.

[0056] Actual measurement data:

[0057] n=6 independent samples: total colony count 6.3×10 4 CFU / g, endotoxin 1.6 EU / mg protein, triple helix integrity 97.9%, DSC Tm value 63.5℃. All indicators in the 3-hour group were better than those in the 8-hour standard group.

[0058] Comparative Example 1

[0059] Compare the conditions under oxygen-rich air (O2≈21%) and nitrogen-replaced conditions (O2≤1% in the top space above the surface of isotonic sodium chloride solution).

[0060] Microbiological and oxidation indicators of raw bovine hides, n=6 independent samples, each sample from a different slaughter batch: total bacterial count in the aerobic group was 1.2 × 10⁻⁶. 5CFU / g, endotoxin 2.8 EU / mg, protein, MDA (oxidative marker) content 0.42 μmol / g; total bacterial count in nitrogen group 8.2 × 10⁻⁶. 4 CFU / g, endotoxin 2.1 EU / mg, protein, MDA content 0.15 μmol / g.

[0061] The differences in total bacterial count between the two groups were statistically significant (t-test, P=0.003); the differences in endotoxin levels between the two groups were statistically significant (t-test, P=0.009); and the differences in MDA content between the two groups were statistically significant (t-test, P=0.001). Nitrogen replacement reduced the total bacterial count by 31.7%, endotoxin levels by 25.0%, and MDA content by 64.3%.

[0062] The results indicate that nitrogen replacement makes a significant positive contribution to microbial control and collagen activity retention by inhibiting aerobic bacterial metabolism and reducing oxidative stress.

[0063] Comparative Example 2

[0064] To fully demonstrate the rationality of the temperature range of 3℃ to 5℃, data from a comparative experiment conducted at 6℃ were added.

[0065] The soaking temperature was controlled at 6°C, and the other conditions were the same as in Example 1.

[0066] Measured data: n=6 independent samples: total colony count 1.4×10⁻⁶ 5 CFU / g, below the standard limit of 1×10⁻⁶ 6 CFU / g, endotoxin 3.4 EU / mg protein, close to the 5 EU / mg protein threshold, retention margin only 0.5 EU / mg protein, triple helix integrity 95.9%.

[0067] A t-test was performed on the endotoxin data of the 6℃ group and the 5℃ group. The difference between the two groups was statistically significant (P=0.023), indicating that the increase in endotoxin at 6℃ was not a random fluctuation, but a systemic increase caused by the increase in temperature.

[0068] This data shows that when 6℃ is used as the transportation temperature, the endotoxin level is only 0.5 EU away from the threshold. In actual cold chain fluctuations, the temperature may rise by 0.5-1℃ instantaneously, which can easily exceed the safety threshold. Combined with the fact that the difference in endotoxin levels between 5℃ and 6℃ is statistically significant, this further proves that setting the upper limit temperature at 5℃ has a technical basis.

[0069] Comparative Example 3

[0070] The transport temperature is 25°C, the transport time is 3 hours, and the rest is the same as in Example 1.

[0071] Actual measurement data:

[0072] n=6 independent samples: total colony count increased to 3.8×10⁶ 5 CFU / g, endotoxin increased to 3.9 EU / mg protein, and triple helix integrity dropped to 91.3%, below the 95% threshold.

[0073] A comparison with Example 4 shows that even in a short-distance transportation scenario of 3 hours, low-temperature cold chain conditions remain an indispensable core technical feature.

[0074] Comparative Example 4

[0075] To fully demonstrate the rationality of the temperature range of 3℃ to 5℃, data from a comparative experiment conducted at 2℃ were further added.

[0076] An active cooling vehicle refrigerator was used with a target temperature of 2℃ (temperature control accuracy ±0.5℃, temperature fluctuation range ±1.0℃), and the other conditions were the same as in Example 1.

[0077] Actual measurement data:

[0078] n=6 independent samples: total colony count 5.4×10 4 CFU / g, below 3℃ group; endotoxin 1.5 EU / mg protein, below 3℃ group; triple helix integrity 93.8%, below 95% threshold; peptide chain breakage rate 1.8%, exceeding <1% threshold; DSC Tm value 62.1℃.

[0079] The t-test was performed on the triple helix integrity data of the 2℃ group and the 3℃ group (Example 2). The difference was statistically significant (t-test, P=0.002), indicating that the degradation of the triple helix structure under 2℃ conditions was not an accidental fluctuation, but a systematic structural damage caused by excessively low temperature.

[0080] This data illustrates that while 2℃ significantly inhibits microbial proliferation, in actual short-distance cold chain transportation, the temperature maintenance precision of refrigeration equipment is limited (typical fluctuation range ±1.0℃). The local temperature inside the transport container may momentarily drop below 0℃, triggering heterogeneous nucleation of the 0.9% isotonic sodium chloride solution (freezing point approximately -0.5℃) and the formation of micro-ice crystals. This ice crystal growth process causes irreversible mechanical damage to the collagen triple-helix fiber network, which is only 1.5 nm in diameter, resulting in a triple-helix integrity rate of 93.8% (below the 95% threshold) and a peptide chain breakage rate of 1.8% (exceeding the 1% threshold). Temperature fluctuations within the 3℃ to 5℃ operating temperature range do not drop below the freezing point, thus avoiding the aforementioned ice crystal damage mechanism. Combined with the statistically significant difference in triple-helix integrity between the 2℃ group and the 3℃ group, this further demonstrates the technical basis for setting the lower limit temperature at 3℃.

[0081] Virus and prion risk control measures:

[0082] Raw cowhide must come from healthy cattle that have tested negative for prions. The farms from which the hides originate must hold animal disease prevention certificates and have no reports of foot-and-mouth disease, mad cow disease, or other Class A animal infectious diseases within 30 days prior to slaughter. Only the dermal layer of the cowhide is used, explicitly excluding tissues with high prion concentrations, such as the brain, spinal cord, tonsils, and distal ileum. Tissues with high prion concentrations, such as the brain, spinal cord, tonsils, and distal ileum, will be sent to the customs quarantine department for prion and other high-risk testing to obtain a clear quarantine report.

[0083] After the raw cowhide arrives at the subsequent collagen extraction plant, it must undergo the following multiple processing steps, each with a specific effect on removing / inactivating viruses and prions:

[0084] Step 1: Soak in a 10% glacial acetic acid solution for 10 days.

[0085] Step 2: Pepsin digestion. In the extraction process, the enzymatic hydrolysis reaction is carried out for 7 days in a pepsin digestion solution at pH 2.5±0.1 and at 20±1℃. Pepsin is most active in a strongly acidic environment at pH 2.5 and can selectively hydrolyze denatured collagen and non-triple-helix proteins within 72 to 96 hours.

[0086] Actual test data show that after the combined use of the first and second steps of the process, the inactivation rate of the indicated virus was greater than 4 logs.

[0087] Step 3: Ultrafiltration dialysis. The collagen solution is subjected to ultrafiltration dialysis using an ultrafiltration membrane with a molecular weight cutoff of 100 kDa to remove small molecule impurities, proteins, and residual endotoxins.

[0088] The combined use of the above three steps results in a total virus removal titer of no less than 6 logs, meeting the NMPA's safety requirements for Class III medical device raw materials. This process method has been validated by the National Institutes for Food and Drug Control (NIFDC), and a virus inactivation process validation report has been obtained.

[0089] In this application, the water for injection conforms to the standards for water for injection in the 2025 edition of the Pharmacopoeia of the People's Republic of China, Part II. The 0.9% isotonic sodium chloride solution conforms to the standards for sodium chloride injection in the 2025 edition of the Pharmacopoeia of the People's Republic of China, Part II. The 0.9% isotonic sodium chloride solution can be prepared using water for injection, and the endotoxin level of the prepared solution, as determined by the Limulus Amebocyte Lysate (LAL) assay, is less than 0.1 EU / mL. The 0.9% isotonic sodium chloride solution can be stored at room temperature in any medical institution (shelf life 2 years), without the need for a complex preservation solution; cold chain packaging only requires a standard 3°C to 5°C vehicle refrigerator or cold chain box.

[0090] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0091] While specific embodiments of this application have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of this application. Those skilled in the art should understand that modifications can be made to the above embodiments or equivalent substitutions can be made to some technical features without departing from the scope and spirit of this application. The scope of this application is defined by the appended claims.

Claims

1. A method for joint control of endotoxin sources in short-distance cold chain transportation of bovine hides, characterized in that, include: Step S1: Pre-treat fresh cowhide to obtain the dermal tissue of the cowhide; Step S2: Immerse the dermal layer of the cowhide in a 0.9% isotonic sodium chloride solution, ensuring that the dermal layer of the cowhide is completely submerged during immersion. Step S3: Control the soaking temperature of the dermal layer of the cowhide to 3℃-5℃; Step S4: Control the oxygen concentration above the surface of the isotonic sodium chloride solution to be less than or equal to 1%; Step S5: Transport the raw cowhide material under cold chain conditions of 3℃-5℃, and the cold chain transportation time shall not exceed 8 hours.

2. The method according to claim 1, characterized in that, In step S1, the fresh cowhide is pretreated to obtain the dermal tissue of the cowhide, including: Soak fresh cowhide in purified water at 3℃-5℃; After soaking, fresh cowhide is roughly washed with purified water at 3℃-5℃. The hair is physically removed using a mechanical dehairing device at an temperature not exceeding 8°C. After the process is complete, the fresh cowhide is placed in water for injection at 3°C-5°C for a thorough washing. The cowhide, after being thoroughly washed, is processed to obtain the dermal layer tissue.

3. The method according to claim 2, characterized in that, In step S1, During the rough washing, the volume-to-mass ratio of purified water to fresh cowhide is 20:1-30:1 mL / g, and the rough washing time is 15-30 minutes. During the fine washing process, the volume-to-mass ratio of water for injection to fresh cowhide is 15:1-20:1 mL / g, and the fine washing time is 10-20 minutes.

4. The method according to claim 1, characterized in that, The fresh cowhide refers to cowhide obtained no more than 1.5 hours after slaughter.

5. The method according to claim 1, characterized in that, In step S2, the volume-to-mass ratio of isotonic sodium chloride solution to bovine dermal tissue is 15:1-20:1 mL / g.

6. The method according to claim 1, characterized in that, In step S4, controlling the oxygen concentration above the surface of the isotonic sodium chloride solution to be less than or equal to 1% includes: The surface of the isotonic sodium chloride solution is replaced with nitrogen gas with a purity of not less than 99.99%. During the replacement process, monitor the oxygen concentration at the gas outlet and control the oxygen concentration at the gas outlet to be less than or equal to 1%.

7. The method according to claim 1, characterized in that, Step S3: Control the soaking temperature of the cowhide dermis to 4±0.5℃.

8. A device for controlling the source of endotoxins in short-distance cold chain transportation of bovine hides, characterized in that, include: A sealed container for containing raw cowhide materials to be transported; An isotonic preservation solution supply system is connected to the sealed container, the isotonic preservation solution supply system being used to supply the sealed container with a 0.9% isotonic sodium chloride solution; A gas replacement system, connected to the sealed container, is used to supply nitrogen gas to the sealed container; A temperature sensor, used to monitor the temperature inside the sealed container; A controller, connected to the temperature sensor, is used to obtain the temperature inside the sealed container; A refrigerator, which is connected to the sealed container and to the controller; The controller is configured to control the operating state of the cooler based on the temperature data obtained by the temperature sensor, so that the temperature inside the sealed container is maintained at 3℃-5℃.

9. The apparatus according to claim 8, characterized in that, It also includes an oxygen concentration sensor, which is disposed at the nitrogen outlet of the sealed container. The oxygen concentration sensor is connected to the controller, which is also connected to the gas replacement system. The controller is also configured to control the operating state of the gas replacement system based on the oxygen concentration data acquired by the oxygen concentration sensor, so that the oxygen concentration in the sealed container is less than or equal to 1%.

10. The apparatus according to claim 8, characterized in that, The sealed container is a passive phase change material cold chain box, with an internal n-alkane phase change material having a phase change temperature of 4°C, and an external vacuum insulation panel with a thermal insulation coefficient of no more than 0.03 W / (m·K).