Background
How to make sure the probiotic drug can work?
Ensuring the effectiveness of probiotic drugs is paramount when considering their application in Nutrient regulation, despite the formidable challenges posed by the harsh physical and chemical conditions they encounter during their journey through the gastrointestinal tract. Overcoming these challenges is crucial for preserving the viability and activity of probiotic cells. Microencapsulation has emerged as a promising solution, and in recent years, it has gained widespread recognition within the probiotic industry. Various materials, including alginate, chitosan, and κ-Carrageenan, have been explored for encapsulating probiotics [3]. After careful material evaluation and extensive deliberation, we have chosen microencapsulation as the strategy for the large-scale production of our probiotic drugs
Microencapsulation: Why Opt for It?
Microencapsulation is a technique that offers a potential solution to the survival issues encountered by probiotic cells. It provides a protective barrier for these cells during their journey through the gut. In recent times, microencapsulation has garnered considerable attention within the probiotic industry. Various materials, such as alginate, chitosan, and κ-Carrageenan, have been investigated for the encapsulation of probiotics [3]. After thorough material assessment and careful consideration, we have determined that microencapsulation is the most suitable approach for industrializing our probiotic drugs.
ACA (Alginate/Chitosan/Alginate)
Sodium Alginate:
Sodium alginate has been a longstanding choice for microencapsulation due to its ability to form a robust network structure. Alginate/Poly-L-Lysine/Alginate (APA) has been widely utilized for microencapsulation, owing to its excellent biocompatibility and biodegradability.
Why Choose ACA?
Nevertheless, the use of APA comes at a significant cost, approximately US$300 per gram, making it impractical for large-scale industrialization. In recent years, researchers have made a cost-effective discovery by replacing Poly-L-Lysine with chitosan. This substitution not only significantly reduces costs but also simplifies the production process of ACA.
How ACA Work? (Interaction between Alginate and Chitosan)
The interaction between alginate and chitosan is facilitated by attractive forces between positive and negative charges, enabling the successful encapsulation of probiotic cells.
Feasibility to apply ACA to our projects:
About our projects:
Our projects involve the use of engineered E. coli as probiotics to combat hypercholesterolemia. Some studies have demonstrated that E. coli can thrive within ACA, confirming its potential for our endeavors.
Experiment
In the case of microencapsulation, we continue to use the design of previous years
Experimental materials
Physiological saline (0.9%), 1.5% sodium alginate, 4% calcium chloride solution, 0.3% chitosan solution, E. coli.
Preparation method
All solutions for the experiments were prepared in saline.
1. Overnight activation of EcN, EcN-lac (2 copies of bacteria). After incubation to logarithmic growth period, determine the OD value of the strain and record.
2. Centrifuge the two 5ml bacterial solution at 6000rpm for 10min at 4℃.
3. Mix the centrifuged EcN with 50ml of sodium alginate solution (two) at a concentration of 1.5%, and wait until the air bubbles are eliminated.
4. Prepare 200ml of 0.3% chitosan solution with 1% acetic acid solution, dissolve it fully at 60℃ and then mix it with 20ml of 4% calcium chloride solution.
5. Aspirate the solution with a syringe (inner diameter 0.5 mm) at 20 cm from the liquid surface and stir while stirring under a magnetic stirrer in the calcium chloride-chitosan mixture (drop acceleration: 40 drops/min).
6. Wash the gumballs twice with physiological saline. After that, the gumballs were filtered out to produce sodium alginate-chitosan microcapsules, also called ACA.
7. The microcapsules are immersed in 4% sterilized calcium chloride solution at 4°C for one week. When used, preheat at 37℃ for 5min.
In vitro verifying
Validation experiment 1
1. Prepare 10ml 4% bile salt solution, 10ml PBS, 10ml SGF solution.
2. After measuring the free EcN OD value, take 500μl and suspend it with 5ml EcN(C/A)2 in 5ml PBS (control), 4% bile salt solution and SGF respectively, and incubate for 2 h at 37℃ with shaking.
3. After 2 h, the bacterial solution was aspirated, centrifuged at 6000 rpm for 10 min at 4°C, washed with phosphate buffer, and measured OD value.
4. For EcN and EcN-lac pellets, after mechanical crushing, liquefy with 1.42% sodium citrate. Centrifuge at 1500rpm/min for 10min, remove microcapsule fragments, collect the bacteriophage and measure OD.
Validation experiment 2
Take 100ul EcN-lac, 1ml EcN-lac spheres in test tube, add 5ml LB medium respectively, incubate at 37℃ for 24h and then observe the fluorescence phenomenon
Result
In our initial achievement, we have successfully crafted microcapsules, also referred to as ACA, as depicted in the illustrative image below.
Figure 2. Morphology of ACA
In our inaugural experimental verification, it was notably observed that the optical density (OD) values of E. coli, shielded by the microcapsules, exhibited a substantial increase compared to those of E. coli lacking such protective microcapsules. This effect was consistently observed in both a 4% bile salt solution and simulated gastric fluid (SGF). These results affirm the substantial protective efficacy of microcapsules on bacteria.
For the second verified experiment, fluorescence was produced in both cases, and it is evident that microcapsules do not affect the product output of E. coli .
