Freeze-Drying Encapsulated Cells Using Cryoprotectant Mixture
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Solution Overview
Problem
The freeze-drying process often results in significant mortality of bacterial cells due to loss of membrane integrity and denaturation of macromolecules, which affects the stability and viability of probiotic bacteria during storage and passage through the gastrointestinal tract.
Innovation Solution
A method involving at least two consecutive incubation steps with increasing concentrations of a cryoprotectant mixture of skim milk, glycerol, and trehalose is used to protect encapsulated bacterial or yeast cells during freeze-drying, enhancing their structural integrity and viability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If freeze-drying is performed without cryoprotectant treatment, then the process is simple and fast, but significant mortality of bacterial cells occurs due to loss of membrane integrity and denaturation of macromolecules
Solution Approach 1:
The method applies preliminary cryoprotectant treatment to the encapsulated cells before freeze-drying. The cells are incubated with cryoprotectant solution containing skim milk, glycerol, and trehalose for 30 minutes to 2 hours, allowing the cryoprotectants to penetrate and protect the cells. This preliminary action prevents membrane damage and macromolecule denaturation during subsequent freeze-drying, significantly improving cell viability without requiring complex equipment modifications
Solution Approach 2:
The invention uses cryoprotectant solution as an intermediary substance between the cells and the harsh freeze-drying environment. The solution containing skim milk (3-8% w/v), glycerol (0.5-2% w/v), and trehalose (5-13% w/v) acts as a protective medium that mediates the interaction between cells and freezing conditions, preventing direct damage from ice crystal formation and osmotic stress while maintaining cell integrity throughout the process
2Duration of action of stationary object
If encapsulated cells are subjected to freeze-drying, then storage stability is improved, but structural integrity of encapsulation material is compromised leading to cell damage
Solution Approach 1:
The method optimizes multiple parameters of the cryoprotectant solution to achieve both structural protection and storage stability. The specific concentration ranges of skim milk (3-8% w/v), glycerol (0.5-2% w/v), and trehalose (5-13% w/v) are carefully controlled to prevent encapsulation material degradation during freeze-drying while ensuring long-term storage stability. The pH value and incubation time are also optimized to maintain structural integrity throughout the process
3Reliability
If cryoprotectant concentration is increased to improve cell protection, then cell viability increases, but the cost and complexity of the process increase
Solution Approach 1:
The invention uses a composite cryoprotectant system combining three different substances with complementary protective mechanisms: skim milk provides buffering capacity and membrane stabilization, glycerol acts as a penetrating cryoprotectant preventing ice crystal formation, and trehalose forms a glassy matrix protecting macromolecules. This composite approach achieves superior cell viability protection at moderate concentrations compared to single agents, maintaining process simplicity while maximizing protective efficacy
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
This method significantly increases the viability and shelf-life of encapsulated cells by maintaining the structural integrity of the encapsulation material and protecting the cells from damage during the freeze-drying process.
Implementation Method 1
A method involving at least two consecutive incubation steps with increasing concentrations of a cryoprotectant mixture of skim milk, glycerol, and trehalose is used to protect encapsulated bacterial or yeast cells during freeze-drying
Implementation Method 2
The evaporation (sublimation) of the frozen water in the material is usually carried out by reducing the surrounding pressure to allow the frozen water in the material to sublimate directly from the solid phase to the gas phase
Implementation Method 3
freeze-drying, also known as lyophilisation, lyophilization, or cryodesiccation, can be defined as cooling of liquid sample, resulting in the conversion of freezable solution into ice
Data Source
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AI summary
The disclosure provides a method of freeze-drying encapsulated cells, the method comprising at least two consecutive incubation steps, wherein the encapsulated cells are incubated in each incubation step in an incubation solution containing cryoprotectant over a suitable period of time, wherein the concentration of cryoprotectant in the incubation solution is increased with each subsequent incubation step. The disclosure also provides freeze dried cells that are obtained by this method as well as various uses of these cells as pharmaceutical, food additive or additive in cosmetics. The disclosure also provides a composition that contains skim milk, glycerol and a carbohydrate.