Team:Paris Bettencourt/Design

Should I talk first about what the problem is (malnutrition in India), or will we talk about it somewhere else?

Introduction


We decided to re-think the universal tradition of food fermentation, and extend it with new possibilities. With community acceptance in mind, we invented a product that can work sustainably in the hands of people with minimal equipment, and fits equally well into the existing infrastructure set up by the Indian government to fight malnutrition, by significantly reducing the production costs. This way we ensure that our product has the highest chances of reaching its target population. At the same time, we showed that it will be trusted by people, because it does not stricly depend on any third party and it is deeply rooted in their traditions.

Manufacturing


The aim of the manufacturing project is to find a way to grow and distribute our strains, easily and cheaply to reach as many people as possible. The product has to be simple and minimize the disruption to Indian habits.



Growth of our strains

Our project is designed for poor regions: the cheaper our product, the more people it can reach. Starting from this point, we didn't want to build labs in India, buy media and grow our strains in expensive structures. Therefore, we had to think of a cheap solution to grow the strains in a homemade media, made of common ingredients, cheap and readily available.
To avoid expensive and time-consuming treatment, an edible medium is sought. It could be directly used to cook either our distribution product or the Idli directly. The process would hence be quite simple and accessible to the population.
After some research, we selected 4 different homemade media to test with our 3 strains:
  • Water from the cooking of potato (potatoes cooked in 2L of water for 20min)
  • Water from the cooking of potato+sugar(10%)
  • Water from the cooking of rice (200ml of rice cooked in 2L of water for 10min)
  • Water from the cooking of rice+milk(10%)

We also wanted to see if the concentration in potato had an influence so for each media made out of potato water, we tried two different concentration of potatoes: 190g/L (named ???????) and 250g/L (named ???????).
For each strain, we compared the growth of the strain in the homemade media with the growth in its "normal" media (respectively YPD, M17+Glucose 1% and YEL).


Packaging and distribution


One stake of our project is to give the strains to the population. Either community labs or users grow the strains, strains needs to travel from us to the community lab and then to the final user.
To answer this problem, we tried several methods, premilinarily working on a yeast: Saccharomyces cerevisiae mcherry.
We could have just lyophilized the strains but our goal is to design something cheap and easy to do for the locals, using only ingredients they have access to and not time consuming.
We first tried to make different kind of powders, but none of it was satisfying. Powders appeared not to be very convenient.
We realised a powder wasn't the best way to distribute our strains. We found better to make portions, easy to pack, with the possibility to pack several portions together. Portions must be easy to stock.
The most efficient and ergonomic shape appeared to be a cube. Moreover, the cube will mainly be added to Idli, made of rice, so rice flour seemed to be a logical ingredient, consonant with the dish, common in India and cheap.
Little by little, we succeeded to design an easy recipe, to cook small cubes made of rice flour and water: the VitaCubes. The idea was also to be flexible to every VitaCube maker means, therefore the recipe is not very strict and can be adapted to what the people have available.



Now that we have found a convenient distribution mean, we need to be sure that it keeps our strains alive and to know how long it can be stored.
We conducted several survival test on the VitaCubes, using S.cerevisiae mCherry and L. lactis. To make these test, we were plating the liquid solution containing the strains before adding it to the flour, in order to know how many cells we put in the VitaCube. After a define period of drying, we immerse the VitaCube in 10ml of osmosed water and plated the solution obtained to count the numbers of cells that survived in the cube.
At first, we obtained very different results, even if the cubes were all plated after one day of drying. We realised the time of ??? of the cube in the water had an influence over the survival rate: the longer it was, the bigger was the survival rate. This is due to the revival of cells once back in water.
Therefore, we designed a one week experiment: we made plenty of VitaCubes on the same day, and then each day, we plated one cube. Each day, we respected the very same conditions, every step was timed.
Graphs!!!!!!!!!!
We can see that the yeasts are very resistant and have a very high survival rate, decreasing very slowly over time. The survival rate is even higher than 1 because yeasts grow very fast, so in 20 minutes in the water, they divide. In conclusion, the yeasts can be stored more than one week in a VitaCube.
For L. lactis, the survival rate is low since the first day and is decreasing very quickly every day. After 4 days of drying, the survival rate started to be very very low. Nevertheless, there are still around 105 cells in a VitaCube after one week, each is far enough to make a culture from it.(and to make Idli?)
Looking at the graphs, we can see that the mix of both strains doesn't influence their survival.



Distribution & (or?) Availability

Cost and time to create the strain (how would you call that?? Also, I need help to assess the cost and/or DALY)

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Access

An important problem we wanted to tackle with our product is the access Indian people have to sources of vitamins. Indeed, we researched what structures and programs already exist in India, and found that it was a major issue.
One of the most important governmental programs for vitamin supplementation is the Integrated Child Development Services (ICDS) program, which has existed in India since 1975. This program monitors the growth and development of children, and provides supplementary nutrition as well as education and primary health care for children under six and pregnant and lactating mothers. The program is implemented through a network of community-level anganwadi centers (AWC), which provides daily supplements both in the center as well as through take-home rations. Those supplements include doses of vitamin A and tablets of iron and folic acid, in order to prevent xerophtalmia - which can lead to blindness - and anaemia.
However, the National Family Health Survey (NFHS-3) held in 2005-2006 in India reports that only 28% of children received those supplements and health care even though 4/5 of children under six lived in a region covered by an anganwadi center. In the 6 months preceding the survey, only 1/4 of the children between 12-35 months were given vitamin A supplements - and in 9 Indian states, this number falls to less than 20%. The access to ICDS has been improved in recent years, however, although access is still far from being universal (2). According to a national survey held in 2013-14, 46% of children aged 6-59 months had received doses vitamin A.
As for folic acid, the 2013-14 survey reports only 14% of children aged 6-59 months having received iron and folic acid supplements.

Though the program holds great promise and has implemented more than a million anganwadi centers, it is clear that people’s access to them is still very limited. People have to walk to the nearest center everyday if they want to receive the supplements, which is not convenient especially in rural areas.
This observation held a major role in our design of a product that can be grown at home, in every village or household. A culture of microorganisms doesn’t require an expensive infrastructure; in fact, most Indian families have already been growing microbial cultures that are several generations old by using a part of their daily yogurt as a starter for the fermentation of the next day’s yogurt. As we have shown (Célia’s part), a microbial culture can be grown on or in a very simple medium like potato juice.
We believe that a culture of yeast and bacteria that can be grown at home and supplements fermented foods with vitamins could reach far more people than the anganwadicenters: those cultures would only need to be distributed once, which would not be very costly as the packages can be made to be very small and light and one culture would act as a starter culture for many more. In this way, the culture would be maintained in the home, without the need for people to travel long distances to have access to nutritional supplements.

Our product is also more accessible in that it doesn't have the same geographic and climate constraints as cultures of rice or other crops. Contrary to Golden Rice, the rice developed by the Swiss Federal Institute of Technology and the University of Freiburg that biosynthesizes beta-carotene and can only be grown in environments with very high water availability, a microbial culture can be grown anywhere. Even though this summer we mostly focused on microbial cultures of fermented dishes composed of rice, microbes are used to ferment any kind of cereals as well as other foods. So our product can be adapted to a very wide range of fermented dishes, and could also be used by people who don’t eat rice.



Acceptance

People's opinion

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Why idli?

Idli (or idly) is a small steamed cake made with fermented rice and black lentils (called urad dal or dal). It is a very popular breakfast food across India, mostly in the south, and is often eaten with chutney.
We wanted our product to truly fit the traditions and tastes of the people we were targeting, and idli appeared to be the ideal dish. It is a very popular staple of the Indian diet, along with the crepe-like dosa, which is made of a similar but less coarse batter. Its two ingredients (rice and dal) are very cheap and widely available resources, and idli is commonly eaten by people who lack other types of food and who suffer from vitamin deficiencies. There is even a program that distributes free rice to the poorest populations in the country, the Public Distribution System (PDS); and in the city of Chennai and its suburbs, canteens called Amma Unavagam sell idli and other foods for very low prices (Rs1 for 1 idli).

Besides its popularity and easy accessibility, the most interesting property of idli is that it is fermented. To cook idli, people soak rice and dal separately, then grind and mix them together, then let the batter ferment overnight. In the hot Indian climate, the batter ferments quickly and its volume can triple overnight.
Many studies have been made on the microbiome of idli batter. Though the strains found in idli can be highly variable from one study to another, probably because the microbiome varies from different regions, we found that Lactococcus lactis and Lactobacillus plantarum are among the most common fermentative bacteria found in idli batter, while in the yeast population, Saccharomyces cerevisiae was always present. ADD SOURCES Since we wanted our product to disrupt as little as possible the idli's taste, we chose these organisms for production of the different vitamins and to improve iron availability, instead of adding new micro-organisms that weren’t already present in the idli batter that may have influenced the microbiome in an unpredictable manner. Many people who reviewed our project said that taste was of high importance, and would prefer that we modified microbes already present in the idli rather than add new ones.
+ should we talk about the advantages of fermented foods?
Idli recipe


Taste & color

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An orthogonal GMO (is that even english??)

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Safety & Regulations

Choice of Strains

Since our product is meant to be used freely in households without containment, and to be eaten by people, safety is a major element we had to address. In this optic we chose to implement our pathways in Generally Considered As Safe (GRAS) organisms: Saccharomyces cerevisiae, Lactobacillus plantarum, Lactococcus lactis and Propionibacterium freudenreichii are all GRAS, commonly found in food.
We used Escherichia Coli to assemble our plasmids because this bacteria is easy to work with, but our goal is to have the vitamins produced by GRAS only.
A further step to our project would be to implement the pathways in wild type strains directly taken from fermented foods, instead of lab strains. or would it change anything if we did that, in regards to safety and regulations?? I don’t know


Toxicity

During the summer we worked on the synthesis of 3 vitamins: vitamin A, vitamin B2, and vitamin B12.
No toxic or adverse effects have been associated with B12, even in very large intakes, so there is no tolerable upper intake level (UL) over which the B12 becomes toxic. And this is true for all the forms of B12 present in food and supplements.
The same is not true of vitamin A, though: most vitamin pills contain retinol, which is the form of vitamin A that is used by the human body. JB je te laisse écrire c'est quoi qui est toxique et pourquoi la ß-carotene l'est pas !! :)
The vitamin B2 that our micro-organism is producing is riboflavin, which can be toxic at very high dose. However it is very unlikely that we will produce more that the daily requirement (about 1.2mg/day), contrarily to the vitamin pills that we can buy on the market and that often contain a lot more, from 10 to 100 mg. Barth do you want to add/change anything? Please do! :)

European Regulations

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Indian Regulations

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Final Product or Continuity

Someone else (Antoine ? <3) needs to fill that...

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