Background
Probiotic
What is probiotic product?
Probiotic product is generally considered as product with microbiome as main ingredient, including probiotic drugs, probiotic foods and so forth. [1]
Hill, C. et al. (2014) The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic.
The relationship with our projects?
In 2022, LZU-CHINA works on to design engineered microbe which can treat colorectal cancer. After designing and characterization of our treating microbe, the next step is to industrialize our projects. Probiotic drug, which is one of the probiotic products, is a significant part of the implementation of the future of our projects.
How to make sure the probiotic drug can work?
The consumption of live probiotic drug will be ideal for treating cancer while the physical and chemical assaults during the delivering process into the gut pose great challenge to the survival of probiotic cells. To ensure the number and activity of microbiome is the key to improve the function of probiotic drug [2].
Microencapsulation
Why to choose it?
Encapsulating the probiotic cells can alleviate the problem of the survival of probiotic cells mentioned before. Microencapsulation is broadly used in the probiotic industry in recent years. Materials such as alginate, chitosan, k-Carrageenan can be employed for the encapsulation of probiotics [3]. After searching materials and discussion, we decided to use microencapsulation as our plan for the industrialization of our probiotic drugs.
ACA (Alginate/Chitosan/Alginate)
Sodium alginate
Sodium has long been used for microencapsulation for its ability to form network structure. Alginate/Poly-L-Lysine/Alginate (APA) has been widely employed as microencapsulation for its excellent biocompatibility and biodegradability. [4]
Why choosing ACA:
However, APA is too expensive (US\$300/g) to industrialize. In recent years, scientists have found that by using chitosan to replace Poly-L-Lysine can largely decrease the price. Plus, ACA is also easy to prepare, which further simplifies the production process.
How ACA works? (Interaction between Alginate and Chitosan)
Through the attraction between positive and negative charges, Alginate and Chitosan can interact with each other. [5]
Feasibility to apply ACA to our projects:
About our projects:
Our projects are using engineered E.coli as probiotics to treat cancer. A research has demonstrated that both E.coli can survive well in ACA [6] :
Experiment
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°C.
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
First, we successfully made microcapsules, also known as ACA, Morphology of it are shown in the figure below.
For the first verified experiment, the OD value of E. coli with microcapsule protection was significantly higher than that of E. coli without microcapsule protection in both 4% bile salt solution and simulated gastric fluid (SGF), which shows that microcapsule has a good protective effect 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 .
Reference
[1] Hill, C., Guarner, F., Reid, G., Gibson, G. R., Merenstein, D. J., Pot, B., . . . Sanders, M. E. (2014). The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nature Reviews Gastroenterology & Hepatology, 11(8), 506-514. doi:10.1038/nrgastro.2014.66
[2] Centurion, F., Basit, A. W., Liu, J., Gaisford, S., Rahim, M. A., & Kalantar-Zadeh, K. (2021). Nanoencapsulation for Probiotic Delivery. ACS Nano, 15(12), 18653-18660. doi:10.1021/acsnano.1c09951
[3] Sarao, L. K., & Arora, M. (2017). Probiotics, prebiotics, and microencapsulation: A review. Crit Rev Food Sci Nutr, 57(2), 344-371. doi:10.1080/10408398.2014.887055
[4] Liu, Z., Nan, H., Jiang, Y., Xu, T., Gong, X., & Hu, C. (2022). Programmable Electrodeposition of Janus Alginate/Poly-L-Lysine/Alginate (APA) Microcapsules for High-Resolution Cell Patterning and Compartmentalization. Small, 18(10), e2106363. doi:10.1002/smll.202106363
[5] 王勇,解玉冰,马小军.(1999).壳聚糖/海藻酸钠生物微胶囊的研究进展. 生物工程进展(02). doi:10.13523/j.cb.19990203.
[6] 张宏亮.(2008).微生物细胞在壳聚糖/海藻酸盐微胶囊中的生长代谢特性研究(硕士学位论文,西北大学).https://kns.cnki.net/KCMS/detail/detail.aspx?dbname=CMFD2008&filename=2008076866.nh