Fresh Milk Exosome Extraction via Membrane and Phase Separation
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Solution Overview
Problem
Existing methods for extracting exosomes from fresh milk are limited in scale, efficiency, and purity, and are hindered by the presence of milk fat particles, which are insoluble and difficult to remove.
Innovation Solution
A method involving a hydrophilic and lipophobic nano-interfacial net membrane to filter out fat particles, followed by ultrafiltration with a tangential flow device, phase separation using an aqueous two-phase solution of polyethylene glycol and dextran, and final purification with a cross-linked agarose gel column to achieve large-scale exosome extraction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If ultracentrifugation or sucrose density gradient centrifugation is used for exosome extraction, then exosomes can be separated, but the extraction scale is limited and the process is time-consuming
Solution Approach 1:
The patent extracts and removes fat particles from fresh milk using a hydrophilic and lipophobic nano-interfacial net membrane before exosome extraction. This preliminary removal of interfering substances enables subsequent large-scale extraction methods to work effectively, resolving the contradiction by separating the fat removal function from the exosome extraction function.
Solution Approach 2:
The patent employs a multi-step extraction protocol that combines membrane filtration, ultrafiltration, and phase separation in a single integrated process. This multi-functional approach achieves both high purification quality and large-scale productivity, as each step serves multiple purposes: fat removal, concentration, and exosome recovery.
2Quantity of substance
If conventional extraction methods are used, then exosomes can be obtained, but the recovery rate is limited and purity does not meet requirements
Solution Approach 1:
The patent divides the extraction process into distinct sequential steps: (1) fat particle removal by nano-interfacial net membrane, (2) ultrafiltration concentration, and (3) aqueous two-phase separation. Each segment optimizes for specific quality metrics, and their combination achieves both high recovery rate and high purity simultaneously.
Solution Approach 2:
The patent uses an aqueous two-phase system (polyethylene glycol and dextran) as an intermediary medium to separate exosomes from other milk components. This intermediary phase selectively partitions exosomes, achieving high purity while maintaining high recovery rates that conventional single-step methods cannot achieve.
3Productivity
If high-speed centrifugation is used for exosome separation, then exosomes can be concentrated, but the process requires complex equipment and causes damage to exosomes
Solution Approach 1:
The patent replaces high-speed centrifugation (mechanical force) with aqueous two-phase separation (density-based partitioning). This substitution achieves effective exosome concentration without the mechanical stress and equipment complexity of ultracentrifugation, preserving exosome integrity while maintaining productivity.
Solution Approach 2:
The patent changes the separation parameter from mechanical centrifugal force to density-based phase partitioning. By using polyethylene glycol and dextran to create density differences, the system achieves exosome concentration through gentle phase separation rather than high-speed rotation, maintaining exosome reliability.
4Device complexity
If fat particles in fresh milk are not removed, then the extraction process is simpler, but fat particles interfere with exosome extraction and cannot be removed by ordinary filtration
Solution Approach 1:
The patent extracts and removes fat particles from fresh milk using a hydrophilic and lipophobic nano-interfacial net membrane before exosome extraction. This preliminary removal of interfering substances enables subsequent large-scale extraction methods to work effectively, resolving the contradiction by separating the fat removal function from the exosome extraction function.
Solution Approach 2:
The patent employs a hydrophilic and lipophobic nano-interfacial net membrane with specific pore characteristics to selectively remove fat particles. The porous structure with tailored surface properties allows efficient fat particle removal while preserving exosomes, achieving high extraction efficiency without overly complicating the process.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The method enables high-purity exosome extraction with minimal damage, allowing for large-scale production without high-speed centrifugation, and achieves nearly 100-fold concentration with minimal sample loss and chemical denaturation.
Implementation Method 1
filtering the fresh milk by a hydrophilic and lipophobic nano-interfacial net membrane to remove cell fragments and fat particles
Implementation Method 2
performing ultrafiltration by means of a tangential flow ultrafiltration device with an ultrafiltration membrane having a pore size of 100-300 kDa to concentrate the clarified fresh milk
Implementation Method 3
performing phase separation after mixing the concentrated liquid with an aqueous two-phase solution prepared by combining polyethylene glycol and dextran, collecting an enriched exosome solution in the lower phase
Implementation Method 4
separating the concentrated exosomes by a column using highly cross-linked agarose filler to remove other impurity proteins
Data Source
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AI summary
A method for large-scale extraction of exosomes from fresh milk including: filtering fresh milk by using a hydrophilic and lipophobic nano-interfacial net membrane to remove cell fragments and fat particles in the fresh milk, and performing ultrafiltration by means of a tangential flow ultrafiltration device using an ultrafiltration membrane having a pore size of 100 kDa to 300 kDa to concentrate the clarified fresh milk; mixing the concentrated liquid with an aqueous two-phase solution prepared by combining polyethylene glycol and dextran, then performing phase separation, collecting an enriched exosome solution in the lower phase, and repeating one or two times to concentrate exosomes; further separating the concentrated exosomes by means of a column using a highly cross-linked agarose filler to remove other impurity proteins, and separating out exosomes and external vesicles having different diameters. The method includes simple purification process, without the need for high-speed centrifugation, and tens liters or more of fresh milk can be processed at a time, the extraction purity is high, external vesicles having various particle sizes can be effectively separated, and a large-scale extraction can be achieved with minimal damage to exosomes.