The vacuum freeze-drying technology we have mastered brings great convenience to our microcapsule preparation. We only need to mix the bacterial powder with the appropriate capsule material into a solution and then mix it evenly with the core after sterilization. Finally, the mixture of the core material and the capsule material is extruded into fine drops or sprayed into the curing agent solution, slowly stirred and assimilated, and finally collect the microcapsules. What we need to ensure is that our microcapsules are enteric-soluble, as shown in figure 7.
Fig.7 Enteric-soluble microcapsules.
To ensure that the powder does not get broken down or digested elsewhere in the body, such as the stomach, we use hydroxypropyl methylcellulose capsules to bury our powder. Using hydroxypropyl methylcellulose ensures that our capsules can withstand stomach acid and disintegrate in the gut. Therefore, we designed a series of experiments according to The Chinese Pharmacopoeia to verify its enteric solubility and reliability.
According to our clinical investigation, we learned that there was an accumulation of acetic acid and lactic acid in the colon of IBD patients, and the environment was acidic. It was known that the pH of UC patients was lower than 5.7, while the pH of CD patients was also around 5.2, while the pH of the terminal colon of normal people was 7.1-7.5. Some findings also indicate that the colonic pH of UC patients may be related to the severity of the disease . Therefore, we adjusted the pH (pH=6.8, pH=6.6, pH=5.8) of artificial intestinal fluid based on the time limit test of the intestinal disintegrating capsule in The Chinese Pharmacopoeia. The disintegration of enteric-soluble hydroxypropyl methylcellulose capsules was experimentally verified under different pH conditions, as shown in figure 8.
Fig.8 Experiment for disintegration of enteric-soluble capsules.
In order to conduct targeted drug stability verification for IBD patients with different colon pH at different stages of the disease, we formulated artificial intestinal fluid with three pH gradients according to Chinese Pharmacopoeia. At the same time, we cooperated with Xbiome to conduct standardized and standardized disintegrating time limit examination of enteric-soluble capsules in their Grade C sterile laboratory by using a disintegrating instrument. According to the requirements of The Chinese Pharmacopoeia, the disintegration verification results of enteric-soluble capsules were obtained by naked eye observation, so we filmed the experimental process and results to prove our results.
First, we carried out enteric-soluble capsule embedding of bacterial powder. Due to problems in the experimental environment, we could not use our bacterial powder for the embedding experiment, so we used agar powder to simulate the powder in capsules. We used enteric-soluble hydroxypropyl methylcellulose capsules of size 2, and filled the capsules with a special spoon. The filling volume of each capsule was 100mg, as shown in figure 9.
Fig.9 Enteric-soluble capsule.
After the capsules were filled, we began step 1 of the experiment, which was checked in the hydrochloric acid solution (9:1000) without baffle for 2 hours. At this time, there should not have any crack or disintegration on each capsule shell. As shown in figure 10, the capsules in the three beakers did not disintegrate.
Fig.10 The condition of the capsules after step 1. A All beakers. B Beaker1. C Beaker2. D Beaker3.
We then took out the hanging basket, washed it with a small amount of water, and checked it in phosphate buffer solution 1, 2 and 3 in accordance with the above method, as shown in Figure 24, all capsules should disintegrate within 1 hour. If one cannot completely disintegrate, another 6 should be taken for a retest, which should meet the requirements. In the actual operation, the disintegration time only lasted for 30min. However, all capsules were found to have disintegrated, indicating that enteric-soluble capsules could disintegrate in the intestinal environment of different pH, and the experiment was successful. This experiment showed that the enteric dissolved capsules we selected could disintegrate and take effect in the intestines of IBD patients with different pH, thus confirming the reliability and stability of
Fig.11 Step 2 in progress.
Fig.12 The condition of the capsules after step 2. A All beakers. B Beaker1. C Beaker2. D Beaker3.
The above operation process is in accordance with the guidance of The Chinese Pharmacopoeia.
Fig.13 Enteric-soluble lyophilized engineered bacterial powder capsule.
So far, we have completed the design and fabrication of the drug form part of our hardware. To put it simply, in our design, we start from the practical application value of the product, starting from two different entry points of patients and pharmaceutical companies. With the concept of quality and quantity and service to patients, our products are designed and manufactured, and optimized. Our products can be directly used in the production of various preparations and added to food and beverage through different sizes of the design, which can significantly expand the application range of our products. We believe that we have not only completed a vacuum freeze-drying program of E.coli Nissle 1917 engineering bacteria with high maturity, but also made an outstanding contribution to the subsequent application of microcapsule technology in E.coli Nissle 1917 engineering bacteria.
But we hope we can do better in the future. More precise and targeted experiments and product validation are waiting for us. We believe that our work is only a tiny step, and we will have the courage to challenge the next mountain in order to create better products with the spirit of artisans.
 Ren Yan, Chen Ming et al. Influence of protectants on the survival of Lactic acid bacteria during the vacuum freeze-drying process[J].China Dairy Industry, 2013,41(09):41-45.
 Can W. Optimization of fermentation process of Anti-tumor engineering NISSLE 1917 and preparation of bacterial powder[D]. Hunan Norman University, 2017.
 Oliver, J. D. (2005). The viable but nonculturable state in bacteria. Journal of microbiology, 43(spc1), 93-100.
 Jie Z, Weiming Z, Ning L. Probiotics, Prebiotics, Synbiotics and inflammatory bowel disease[J]. Parenteral & Enteral Nutrition, 2014, 21(04):251-153+256.
 Nugent SG, Kumar D, Rampton DS, Evans DF. Intestinal luminal pH in inflammatory bowel disease: possible determinants and implications for therapy with aminosalicylates and other drugs[J]. Gut. 2001;48(4):571-577. doi:10.1136/gut.48.4.571
 陈宇洲,王东凯.炎症性肠病的结肠生理特征和结肠定位给药[J].Chinese Pharmaceutical Journal,2006(02):86-90.
 National Health Service (NHS). Inflammatory bowel disease[EB/OL]. 2020[2021.9.30].
 Group I B D, Gastroenterology C S O, Association C M. Chinese consensus on diagnosis and treatment of inflammatory bowel disease(Beijing, 2018)[J]. Chinese Journal of Practical Internal Medicine, 2018.9, 38(9):796-813.