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Executive summary

ProKao,we make your microbiome work for you.

We are a group of students who became inspired by the theme of their iGEM project and decided to adapt it into a real world solution. Our products allow users to harness the potential of their microbial flora to help them achieve their goals and increase their quality of life. The first product we will put on the market is a probiotic chocolate bar which contains bacterial strains that have been shown to reduce sadness and increase the ability to cope with stress. This is the first application of what will later become a platform technology that targets a variety of the user’s needs and goals using different combinations of living organisms and active substances. We have already filed the first of what will be several patent applications detailing the composition of our product and the methods required to produce it.

The market for both probiotics and mental wellness related products is expanding worldwide taking up 38% of the world's functional foods market. Despite the comforts that come with the abundance of modern life in the developed world people are far from care-free. We still have the same instincts and brain of pre-historic humans that had to constantly worry about food, shelter and predators. These instincts manifest in fight-or-flight reactions in situations where our physical well-being is no longer in question. This chronic stress has been linked with a myriad of other health problems, from high blood pressure, depression, a weakened immune system and auto-immune disorders. There are probiotics as well as numerous supplements in the market aimed at improving mental wellbeing, which is not surprising given the scale of such a concern. By targeting specific groups (stratification) or tailoring each batch according to a customer’s needs (personalisation) we’re able to leverage a growing body of research to a greater extent than ever before. Our products will help patients achieve their goals by tapping into the potential of the most neglected organ in the human body: the microbial flora, and do so with greater accuracy and control than any that have come before us.

Our first product is inspired by our iGEM project: a probiotic that helps people decrease their stress and anxiety and promote their mental well-being. The bacterial strains we will used have all been shown to produce beneficial effects in clinical studies and will be combined with other ingredients in such a way as to be tailored to their personal situation, needs and goals.

Our stratified range product will be sold at retailers that cater to the health-conscious whereas the personalised product will be specified by the customer online and made to order based on the information provided. Prokao is a product meant for daily consumption, especially the personalised version that can be changed progressively if the customer opts to sample his microbiome on a regular basis.

Over the past three months we have been advised by academics, consultants, attorneys and several people from the venture capital fund SOSVentures. Their insights have been invaluable and have motivated us to take this project beyond iGEM. Our initial funding will come from participating in a business incubator where we will develop our proof of concept. Within one year we intend to have first product on the market and start working on the next application. In three years we expect to have developed and patented optimised strains that will be used in our products as well as have begun to look into developing strains using genetic engineering techniques. In five years we expect to have our first medical treatment entering clinical trials.

We intend to start a revolution in microbial based treatment. This technology is in its infancy but has the potential to overturn the medical establishment in its current form. It will allow for continuous monitoring of chronic disease to the point of virtual cure. Our ultimate goal is to empower our customers, not only by reducing their suffering but also to help them achieve their desires.

Product

Overview

ProKao is a delicious chocolate bar that combines the goodness of chocolate and the benefits of probiotics. Our chocolate is sourced from organic and fair trade producers, and has been enhanced with billions of friendly bacteria, which have been shown to interact with your gut-brain axis, to make you happier and healthier, and help you cope with the stress of everyday life.

Once a day you will be able to indulge in our delicious chocolate bar, knowing that not only it will deliver these healthy bacteria to your gut, but also contains dietary fibers (prebiotics), which stimulate the proliferation of your natural microflora, without any added calories.

How our product came to be

The idea to create a probiotic to help with stress, anxiety and other mental health-related issues came from the core theme of our project: the gut-brain axis. Our original idea was to create genetically engineered probiotic strains but soon we realised that the current legislation doesn’t allow for the commercialisation of such products aimed at the general public, and only recently have tests with animals began taking place for pharmaceutical applications.

A three tier vision

The set of constraints we were initially faced with lead us to pivot away from focusing genetically engineered probiotics from the start. Instead we opted for the following timeline:

1. A product based on characterised strains and other GRAS (generally recognised as safe) ingredients that have a track record of efficacy and clinical data to back them up. This will be our minimum viable product, categorised as a food.
2. Once the mechanisms behind the effect of different microbiome profiles have been fully identified it will be possible to create enhanced strains. This can be done without necessarily being subjected to the tight regulations that restrict genetically engineered products, of course this would limit the scope of our action to certain techniques (deregulation, generating mutations, directed evolution, CRISPR-cas9). Another option would be to run our own experiments on particular strains we create or discover and patent them. These products would also be characterised as foods.
3. Using synthetic biology methods to enhance the production of particular compounds, introduce the genetic circuits that we have created to respond to environmental triggers and create ways of controlling the production of particular substances. This phase offers the greatest potential and the widest variety of applications; bacteria can be engineered into microbial factories that produce pharmacologically active substances of our choosing and secrete them in the desired amounts. A product such as this one is beyond the scope of what current legislation allows, and would only be considered for approval as a therapeutic and therefore be subjected to clinical trials.

Why Chocolate?

Independent studies have shown chocolate to be 3 to 4 times more effective in delivering probiotics to your gut than dairy products, as it protects them from being degraded in the passage through the stomach.

It is a delicious treat that will not only benefit your gut flora, but also has all these added benefits:

• Reduces the risk of cardiovascular disease

There are hundreds of studies that provide supporting evidence that chocolate improves the functioning of the cardiovascular system, due to its high content in antioxidants.

The flavanols in dark chocolate have been shown to increase the production of Nitric Oxide (NO), which makes the arteries relax, lowering blood pressure, and facilitating blood flow (Fraga, 2011). This also improves athletic performance, and recovery due to its anti-inflammatory properties.

In addition to this, chocolate promotes the creation of good cholesterol, which in the long term reduces the cholesterol accumulation in the arteries. (Buijsse, 2006)

• Improve Brain function and enhance mood

Dark chocolate contains compounds such as anandamide, PEA, theobromine and tryptophan, that increase serotonin and endorphin levels, which, in combination with our added probiotics, boost overall mood, and make you feel happier by encouraging the release of certain neurotransmitters such as melatonin. The consequences are increased libido, and an antidepressant effect.

The high flavonols content also improves memory and cognitive function, allowing you to be more aware, keep a sharp mind and a positive attitude (Scholey, 2013)

• It contains a high concentration of minerals

  100g of Quality Dark Chocolate (70-85%) contains:

  - 67% of the RDA for Iron.
  - 58% of the RDA for Magnesium.
  - 89% of the RDA for Copper.
  - 98% of the RDA for Manganese.
  - plenty of potassium, phosphorus, zinc and selenium.

  These are essential in building muscles, optimizing energy production, and are necessary for your immune function.

References

• Buijsse, Brian, Edith JM Feskens, Frans J Kok, and Daan Kromhout. 2006. Cocoa intake, blood pressure, and cardiovascular mortality: the Zutphen Elderly Study. Archives of internal medicine 166, no. 4: 411-417.
• Fraga, César G, María C Litterio, Paula D Prince, et al. 2011. Cocoa flavanols: effects on vascular nitric oxide and blood pressure. Journal of clinical biochemistry and nutrition 48, no. 1: 63
• Scholey, Andrew, and Lauren Owen. 2013. Effects of chocolate on cognitive function and mood: a systematic review.Nutrition reviews 71, no. 10: 665-681.

Intellectual property

From talking with our Business advisors, we realised that one of the main concerns that investors will have about us is the protection of our intellectual property, especially since the technology of our third phase product is going to be disclosed at our presentation.

Patent

We are producing the first product of its type. While there are other probiotic companies that use a chocolate matrix as their delivery mechanism, they have only patented their own method, or their encapsulation process. We only found another similar patent, by P&G that patented the probiotics + chocolate combination, but it did as a method of promoting gastrointestinal health. (Number: AU20100264448 20100624) As a result, our invention is novel and non-obvious and we believe fully-patentable.

This gave us the opportunity to patent our method, in a similar way P&G did, but with the slight difference that its aim is mood and mental wellbeing enhancement.

The relevant documents can be found below

• Filled_Form.pdf
• Receipt.pdf

Once we have developed the product further, to stages 2 and 3, we will be in the position to patent the specific engineered strains that we will add to upgrade our probiotic and make it serve the goal for which it was designed.

Trademark

During one of our policy and practice events, we were lucky enough to talk to some people with previous experience in the sector, and shared our prototype. They advised us to register our trademark as soon as possible, since we were distributing prototypes with our brand.

• Summary_of_trademark.pdf

We expect the amount of IP to grow as we diversify and develop new products, and have appointed a specific department in our functional structure to take charge of it. This department will be specifically relevant in the third phase of development

Manufacturing Operations

Prototype

With our product implementation plan in place, and a solid business model, we have developed a prototype with our engineered bacteria to test how effective the chocolate matrix is in encapsulating the bacteria and ensuring their survival for an effective delivery.

Protocol

1. Innoculate in two different Falcon tubes Nissle- TPH1 20ml of LB + Cam and shake overnight at 250rpm and 37ºC
2. Centrifuge at 40000rpm for 7min and discard the media.
3. Resuspend the pellet of one of the tubes in a solution composed of 1g of sugar and 1ml of miliQ water (A), and the one of the other in 500ul of miliQ water (B)
4. Melt 50g of dark chocolate (52%). The product also contained: Cocoa butter, Whey powder, Soya Lecithin, Polyglycerol polyricinoleate, and vanilla extract.
5. Mix 10g of chocolate with 500ul of the solution A (labeled 1.50 in the photo), and solution B (labeled 2.50).
6. Mix 10g of chocolate with 250ul of the solution A (labeled 1.25), and solution B (labeled 2.25)
7. Mix 10g of chocolate with 50ul of mili Q water for the Control

Note: the chocolate must be at under 40ºC before mixing with bacterial solution

8. Leave samples in the fridge overnight



9. Cut 5g of sample and wrap in aluminum foil for storage, to test shelf life



10. Cut 2g of sample and add to 5ml of LB in a falcon tube
11. Water bathe falcon tubes at 39ºC until the chocolate melts and mix by vortexing until a homogeneous texture is achieved
12. plate 100ul of solution in Cam LB agar plates and incubate overnight at 37ºC

The bacteria grew back on the agar plates after having been placed on the chocolate matrix overnight, which showed chocolate is an adequate media to place them. In addition, we observed a higher number of CFU in the plate of solution B, suggesting the more effective method for resuspension of the pellet.

However, from literature we know that the delivery and shelf life is best if the bacteria are microencapsulated beforehand with methods such as spray drying. There is also literature showing that when tested in a simulator of the human digestive system, probiotics in a chocolate matrix proved to reach the gut more effectively than those in Dairy (Possemiers, 2010)

This is the first step towards a more sophisticated prototype, in which we will test freeze dried bacteria against other types of microencapsulation, to optimize the delivery, as we want to be able to guarantee that at least 2 billion CFUs reach the gut and colonize it.

Our finalized chocolate bar will be a 50g single dose rectangular bar.

Scale-up Production

Manufacturing will be facilitated by outsourcing the production of our ingredients to key partners with experience in the sector. These partners will provide the already processed chocolate, and encapsulated probiotics. They will be selected according to the quality criteria with which the product identifies. This is mainly organic fair trade cocoa pods, and other more specific certifications to cater to the needs of specific sectors of the general public, such as a gluten free certification or dairy free.

This will be beneficial to us, as these companies can off-set early production, which will allow our products to reach the market sooner. This allows us to focus on the further research of the biological components needed for the development of the company to the second and third stages.

For the first stages of the business development, we would use strains whose effect is documented in literature

Production will take place following two different models, for two slightly different products

Stratified

In the stratified model, consumers identify themselves with a specific category, and select the chocolate bar that is more adequate to suit their needs.

Whereas most probiotics companies we found offered a range of different products based on flavours we would target market segments instead. We will produce a fixed number of bars with specific probiotic contents, specifically designed for each market segment. These will be differentiated in terms of age, sex, and lifestyle; which will lead to strata like: higher protein content for more athletic people, or a limited amount of lactobacillus strains for vegans.

This model has a simpler production, given that it’s a repetitive process in which machines can be programmed for a certain amount of hours to produce one specific type of bar, and then switch to a different one by just changing the proportions to be added, and producing a great amount.

Personalised

The personalized model, adds a layer of complexity to the structure. It will consist of made to order individual batches of probiotic chocolate tailored to the users goals and needs to in a way that has never been done before.

Using information supplied to us by the user we could design a probiotic fit specifically for their profile and monitor its effect by monitoring the state of the microbiome periodically. Over time more metrics and applications would be added, optimizing our ability to cater for each specific profile, and ultimately fulfilling the promise of power over all the potential that microscopic factories living within us can offer.

References

• Bravo JA, Forsythe P, Chew MV et al. (2011) Ingestion of Lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve. Proc Natl Acad Sci USA 108, 16050–16055.
• Dapoigny M, Piche T, Ducrotte, Lunaud B, Cardot JM, Bernalier-Donadille A (2012): Efficacy and safety profile of LCR35 complete freeze –dried culture in irritable bowel syndrome: A randomized , double blind study. World J Gastroenterol 18:2067-2075.
• Desbonnet L, Garrett L, Clarke G et al. (2008) The probiotic Bifidobacteria infantis: an assessment of potential antidepressant properties in the rat. J Psychiatr Res 43, 164–174.
• Hsiao EY, McBride SW, Hsien S et al. (2013) Microbiota behavioral and physiological abnormalities associated with neurodevelopmental disorders. Cell 155, 1451–1463.
• Ko CY, Lin HTV & Tsai GJ (2013) Gamma-aminobutyric acid production in black soybean milk by Lactobacillus brevis FPA 3709 and the antidepressant effect of the fermented product on a forced swimming rat model. Process Biochem 48, 559–568.
• Messaoudi M, Lalonde R, Violle N, Javelot H, Desor D, Nejidi A, et al. (2011): Assessment of psychotropic-like properties of a probiotic formulation (Lactobacillus helveticus R0052 and Bifidobacterium longum R0175) in rats and human subjects Br J Nutr 105:755-764
• Possemiers, Sam et al. (2010). Bacteria and chocolate: a successful combination for probiotic delivery."International journal of food microbiology 141.1: 97-103.
• Rao AV, Bested AC, Beaulne TM, Katzman MA, Iorio C, Berardi JM, Logan AC (2009): A randomized, double-blind, placebo-controlled pilot study of a probiotic in emotional symptoms of chronic fatigue syndrome. Gut Pathog 1:6.

Business Environment

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1. Digest at least 500 ng of each part according to the restriction digestion
2. Run the digested DNA on the gel according to gel electrophoresis protocol
3. Identify the parts that you want to ligate on a gel and cut the bands out using razor blade
4. Purify the excised bands using the commercial kit according to the manufacturer's instructions
5. Quantify the DNA yield using DNA nanodrop
6. Proceed to ligation

Marketing

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1. Prepare the PCR mix:
   - 12.5 ul of 2 x Q5 PCR master mix
   - 1.25 ul of 10 uM forward primer
   - 1.25 ul of 10 uM reverse primer
   - 2 ng of DNA to be PCRed
   - add milliQ H2O up to 25
2. Set up the PCR cycles according to the following rules:
   Initial denaturation
   - 98C for 30 seconds
   35 cycles
   - 98C for 10 seconds
   - 30 seconds at primer melting temperature
   - 72C for 30sec/kb of PCRed fragment
   Final extension
   - 72C for 2 minutes
   - Hold at 4C
3. Confirm the PCR by running 2 ul of the product on the gel according to the gel electrophoresis protocol

Management

When it comes to small companies and startups, the organizational structure has to be designed to be the most efficient way for the company to achieve its goals and objectives.

Since our company has a long term plan of several parallel projects, we decided for a matrix structure where all can be worked on in a parallel way, without getting the streams of communication to interfere with each other.

Functional Management

We decided to give our management a functional structure, centered in job functions and departments, in blue. These will be overseen by the C’s suite. They will all report to the board of directors thus centralizing the decision making, which is essential in startups.

Functional structures are specifically designed to work in heavily project- focused companies, like ours. Directors will assign each of the projects to a manager, who will coordinate the whole development, getting support from each department in each aspect of the project development. This allows each project manager to be specialized in their stream and effectively meet deadlines, thus ensuring a more rapid business development, essential in the ever-changing conditions of the current market.

Matrix Organisation

We specifically selected the matrix model because we consider that it’s the one that best allows the horizontal flow of skills and information, which, is essential in such a multidisciplinary endeavor.

It is common in the development phases, as it allows for large projects to move forward in a more organized way, since it allows employees to specialize in a project and apply their expertise in a particular field, without being removed from their department in question.

The project manager’s authority flows horizontally across departmental boundaries, but is always subjected to the board of directors.

Leadership

In terms of leadership we have advocated for a Collaborative Leadership model, which encourages equal participation across all levels, therefore giving the project managers the most responsibility on the decision making and problem solving, almost at a CEO level.

This would imply open information sharing across the company, as every department as to be on the same page about each project, thus ensuring optimum efficiency in interdepartmental collaborations. This cross departmental participation allows more creative approaches to problem solving, and idea generation, speeding up once more business development.

Resource allocation will be managed by the board of directors. Team leaders will request these proactively and provide them to their teams, for which a fluent communication is key. This ensures a fast project advance as the employees have all resources available to do their jobs efficiently.

This type of structure places more responsibility on each team member, which makes them more involved in the process, and means issues are dealt with swiftly. The fact that leaders and team members are valued equally creates a better working environment and a better flow of feedback, which allows the team to be in a constant state of improvement; and employees to constantly acquire new knowledge and experience. This is essential to respond to the challenging and highly competitive environment that current market poses.

This structure responds to employees’ seek for autonomy and engagement, and promotes professional satisfaction, and a better environment for innovation.

Financial Data

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