From Lab to your Gut
We believe that content is not the only important thing. How it looks from the outside is as important as what is inside. So, in this section we are dissecting the external aspects of our product from packaging to consumption. For an introduction, we are engineering a natural probiotic E. coli Nissle 1917 (ECN) to have the ability as an antibiotic. For further detail of our system see our Design
What is probiotic?
According to WHO/FAO, probiotics are living microorganisms which if given in sufficient quantities can provide health benefits to the host. In consequence, several properties are needed to be fulfilled by a probiotic: therapeutic effects, stability in gastric acid and bile salt, adherence and colonization ability. Considering these properties, the most common microorganisms used as probiotics are Lactobacillus sp. and Bifidobacterium sp. Other less common microorganisms include Bacillus coagulans, Streptococcus thermophilus, Enterococcus faecium and Saccharomyces cerevisiae, Probiotics (MUST!) survive the upper gastrointestinal tract and then work primarily in the gut through a wide range of mechanisms.1,2
The proposed mechanisms (Figure 12)
are adhesion and nutrients competition, antimicrobial compound synthesis, and immune modulation.1
Figure 12. Probiotic's mechanism of action
Probiotics’ faces (packaging)
Probiotics come in various faces (re: forms) from foods, drinks, and supplements. Simplified, the packaging is available in two primary forms, which are fermented foods and supplements. Fermented foods can come from a number of dairy products, with yogurt as the most common being. Other forms of food format probiotics are extremely diverse from chocolate, crackers, cereals, snack, chips, granola, etc. Probiotic supplements consist of dried (lyophilized) bacteria in powder, capsule, or tablet form. Principally, improvement of probiotic activity depends on its viability and survivability, The advantage and disadvantages of each delivery system, it presents in the table below.1,2
Table 3. Advantages and disadvantages of available probiotics delivery system1
- Affordability and easy availability
- Fermentation increase cell population and cost efficiency
- Ease of incorporation into daily patterns
- Additional nutritional benefits
- Enhanced bacterial survival
- through upper GI tract (100× fewer bacteria can be given per dose)
- Effective in the upper GI tract
- It contains dairy proteins and lactose
- Taste can be an issue
- Not suitable when traveling (refrigeration is needed)
- shorter shelf life
- Not suitable for vegans
- Ease of administration
- Contain no binders
- Not therapeutic in upper GI tract (unless opened or chewed)
- May contain allergenic excipients
- Higher cost
- Ease of administration
- Effective in the upper GI tract
- May contain allergenic or otherwise problematic binders and excipients (e.g., gluten)
- Higher cost
- Effective in the upper GI tract
- Dosages can be easily adjusted
- It can be incorporated into foods or drinks
- Contain no binders
- wettability and dispersibility issue
What do consumers perceive?
Drug consumption may become challenging, especially regarding patients’ compliance in long term medication. So, the mode of delivery of probiotics has to consider what consumers prefer.
A previous report in Belgium for 503 respondents from healthy adult people and Inflammatory Bowel Disease (IBD) patients found 60% of them choose the probiotic product as a food product (e.g., dairy products) over the pharmaceutical product or food supplements. The intention for participants who know about functional food groups are 2.4 times less likely to prefer the supplements and three times less likely to choose a pharmaceutical product over the functional food group. In a review by Aziz et al in 2016, they recommended the use of probiotic foods rather than probiotic supplements. Pill fatigue due to probiotic supplement is a major concern that shifts the paradigm to develop a natural consumption of probiotics through dairy products. It is believed that the mode of the delivery system affects consumer preference about probiotic products.2–4
Our mini survey
The aforementioned studies suggest that a different method of delivery through non pill/tablet/capsule is prefered. We did a mini survey to find out the preferences towards probiotics' mode of delivery in our surroundings. For further detail see the Business
So, what do we choose?
When we compare, it turns out that only 100 times fewer bacteria are needed in dairy products than probiotic supplements, like pills, to achieve the same amount of bacteria that live in the lower intestine.1 This reason, although we are not aiming to deliver the bacteria to the intestine, shows that dairy products are the ideal delivery system to carry our engineered E. coli Nissle 1917 (ECN).
Consumer is the king from the business perspective, so it is important to take into account their perspectives and behaviors towards available probiotic packages. As we see from the literature and also from our mini survey, people preferred a friendly “drug” delivery and chose dairy products (foods) over pills/tablets/capsules. So, yes we are designing our product as a yogurt as the most widely available and acceptable products.
There are some packaging options that enable a mixture of shelf stable liquid and probiotics just before consumption (using straw or bottle cap). We are aiming to pack our product in that manner, separating the ECN and our juice containing arabinose as the proteinase K inducer, and mix them before the time of administration. Ofcourse, there must be a little delay before it can be consumed.
Industrial and practical aspect
As it is important to take into account our genetic circuit especially the inducer of our system. Since the Proteinase-K production is triggered by L-arabinose, we pack the arabinose in a separated column before it is intended to be consumed. For optimum induction, 10 mM (0,15% w/v) of L-arabinose is prefered. Since the presence of ammonia induces secretion through production of autolysis protein and acts as a trigger of the PGLa-AM1 system, it is important to eliminate ammonia from our product. Furthermore, as our juice will contain milk, the absence (or very little amount) of ammonia is an indicator of little contamination of bacteria. We have ensured that our product (at the very least our transformed bacteria) will have no dangerous substances that will affect human health and pose risk to the environment. By designing the kill switch system we are preventing the possible negative effect of mutation in our engineered ECN. See our Design
Further studies are needed to determine the efficient amount of bacteria. However, according to our modelling, approximately 8.300 proteinase-K (~ 29 kDa each) and 20.000 PGLa-AM1 (~2 kDa each) per single ECN are secreted 30 minutes and 4 hours post induction by ammonium respectively. To achieve the therapeutic level of proteinase-K (25 μg/mL) it is estimated 2.8 billion ECN are needed to disperse the biofilm that is formed by 107
CFU/mL H. pylori. It is should be noted that the concentration of 25 μg/mL may not be the minimal therapeutic concentration since available study did not evaluate lower concentrations.
While for PGLa-AM1 (1 μg/mL in vitro) as much as 15 billion ECN are needed to effectively inhibit the growth of H. pylori. The same study by Zhang et al. reported the PGLa-AM1 has an effective dose of 40 mg/kg for mice in two divided doses daily for 7 days which results in 100% clearance of H. pylori. Converting the dose to human equivalent dose (HED), we get 3.25 mg/kg HED. For a 60 kg man, each day around 195 mg is needed per day. However, since the Proteinase-K may digest some of available PGLa-AM1, higher dose is needed. Based on our modelling, to reach the MIC of 1 μg/mL in the presence of Proteinase-K, as many as 500 mg PGLa-AM1 is needed. To produced this amount of PGLa-AM1, enormous amount of E. coli are deemed (more than the amount of bacteria in our body!!!). Unfortunately since the data regarding Proteinase-K activity especially against AMP are lacking, our model is an oversimplification and we can not determine the minimal dose and the minimal number of E. coli to achieve the therapeutic range before any in vitro and in vivo study are done.
Product design and consumption
Our product will be packed as shown in Figure 13
. It contains two containers: (1) probiotic powder and (2) arabinose containing juice. For the consumer convenience, the container is 75 ml in volume. The two components need to be mixed before consumption. As the arabinose needs time to induce the production of a significant amount of Proteinase-K, our product can be consumed after approximately 3.5 hours. A biodegradable material for the container will be preferred. More research is needed to determine the best biodegradable material that is suitable as our container but polylactic acid as one of the most used materials can be considered.
Figure 13. Product design of HELICOSTRIKE
To make a probiotic on an industrial scale there is a long way to go. In vitro, in vivo, and clinical trials have to be done followed by several product registration steps. Many aspects are needed to be considered to design an efficient system for large scale production of probiotics with modified and engineered ability for biofilm dispersion and infection eradication.
We are discussing some legal processes in the Integrated Human Practice section. In short, a legal permission for our product as a probiotic has to be obtained before we start to register our product as an antibiotic. For antibiotic purposes, of course, well designed clinical trials are needed. However, in some highly prevalence H. pylori infection populations and for prevention purposes, being registered as a probiotic product is an enormous step. So our product is not only made for those who are symptomatically infected by H. pylori, but to reach those who haven’t developed any symptoms.
In order to register our product and make it publicly available, our products have to be registered and permitted by “Badan Pengawas Obat dan Makanan” or shortly BPOM. BPOM is responsible for controlling the circulation of food and drugs in Indonesia. It’s analogue to the USA’s FDA. Some steps need to be done to get permission to introduce our products in the market. Registering products in BPOM can be done in two ways: offline by visiting the BPOM office, or via online through e-Registration apps. These are steps that need to be done!
- Before that, we need to have Industry Permit from the Indonesia Department of Industry and Food Trading.
- Registering via online needs company registration, so register our Company first in e-Registration application.
- Login in e-Registration apps, fill in the necessary details such as product registry data and product food composition data.
- After completing the food composition data, we can ask for an analysis of the result based on the food category.
- Several supplementary documents can also be uploaded such as nutritional facts of our products.
- We need to pay for the product registration before advancing to the next step.
- After that, submit all the data and wait until you get the Letter of Acceptance (Surat Persetujuan Pendaftaran).
These steps require us to register our company first. Below are several steps that needed to be done in order to register our company before requesting BPOM permission for food safety.
- Register our company through e-Registration apps (https://e-reg.pom.go.id/ereg/index.php/register).
- Fill in our company details and factory details based on the company registered data and company status.
- After that, submit the food categories that are made in our factory.
- Upload the needed documents such as scan of NPWP (Tax Register) and Industry Permit from the Government.
- Wait until the approval of company registration is given via email.
And so our team needs to register the company first before requesting BPOM permission for our products' safety via e-Registration apps. Having permission to distribute our products is a must in Indonesia to ensure our consumers safety and to be widely accepted by people.
- Khalighi A, Behdani R, Kouhestani S. Probiotics: A Comprehensive Review of Their Classification, Mode of Action and Role in Human Nutrition. In: Rao V, Rao LG, editors. Probiotics and Prebiotics in Human Nutrition and Health [Internet]. InTech; 2016 [cited 2021 Sep 4]. Available from: http://www.intechopen.com/books/probiotics-and-prebiotics-in-human-nutrition-and-health/probiotics-a-comprehensive-review-of-their-classification-mode-of-action-and-role-in-human-nutrition
- Fenster K, Freeburg B, Hollard C, Wong C, Rønhave Laursen R, Ouwehand AC. The Production and Delivery of Probiotics: A Review of a Practical Approach. Microorganisms. 2019 Mar 17;7(3):83.
- Jacobs S, Wim V, Sas B. Mode of delivery of probioticsConsumers’ preference and its determinants - tks | publisher, event organiser, media agency [Internet]. TKS Publisher. [cited 2021 Sep 11]. Available from: https://www.teknoscienze.com/tks_article/mode-of-delivery-of-probioticsconsumers-preference-and-its-determinants/
- Terpou A, Papadaki A, Lappa IK, Kachrimanidou V, Bosnea LA, Kopsahelis N. Probiotics in Food Systems: Significance and Emerging Strategies Towards Improved Viability and Delivery of Enhanced Beneficial Value. Nutrients. 2019 Jul 13;11(7):1591.