Revision as of 10:04, 19 November 2015

Ferment It Yourself

iGEM Paris-Bettencourt 2O15

Introduction
Distribution & Availability
Acceptance
Safety & Regulations
Continuity

Introduction

Despite many efforts from the indian government and NGO to decrease the micronutrient deficiencies in India, there are two major limitations that prevent the vast majority of the population to access sufficient sources of vitamins and iron or phytic acid: cost, and distribution. Our project proposes an elegant solution to address both those issues, via a cost-effective biosynthesis of vitamins and phytic acid by micro-organisms, which can be cultivated in everyone’s home without any need of external infrastructure.

We decided to re-think the universal tradition of food fermentation, and to expand it with new possibilities. With community acceptance in mind, we developed 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 designed our product to engender people's trust, as its use is sustained without strictly depending on any third party and it is deeply rooted in their traditions.

Distribution & Availability

The Integrated Child Development Services (ICDS) programme

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.

Vitamins in everyone’s home
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. A great part of our project was dedicated to find a cheap and edible medium in which the microbes could be cultivated at home, and to manufacture a simple and practical way to distribute our engineered yeast and bacteria (See Manufacturing page). We found that a microbial culture could be grown in a very simple medium like potato juice, and that the yeast could be lyophilized, mixed with rice flour and water, and distributed in the form of small cubes.

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 anganwadi centers: 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.

Amma Unavagam
Another way to cheaply give people access to micronutrient-rich foods could be to implement our product in the popular canteens called Amma Unavagam (meaning “Mother Restaurants) which are being developed by the Government in the state of of Tamil Nadu, mostly in the city of Chennai. Those canteens offer people dishes like idli and dosa, both made of fermented rice, for the very cheap price of Rs. 1 for an idli ($0.01).
Those canteens are visited by thousands of people. If they were using our product during the fermentation of their rice, those people would benefit from the micronutrients produced by the engineered strains.

An adaptable solution
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

Since the very beginning of our project, we researched which solution would be convenient and acceptable to them, by asking them about their habits and opinions. For this we interviewed local indian citizens, and well as local authorities in India (find the details on the Acceptance page).

We found out that people in India don’t want GMO to be associated with farmers, and they don’t want them to be dependent on one company that would have monopoly on the product. This fitted well with the idea of strains that can be grown at home by everyone. By focusing on yeast and bacteria that are kept in pots and that can be added in a dish at the user’s convenience, we are not targeting the farmers specifically, and we are not affecting the crops at all; and we are giving people an independence and way to chose at any time whether or not they want to eat the GMO.

Most of the indian people we interviewed also told us that they would try our idea of adding a vitamin-producing bacteria, as long as it is proven efficient and harmless for human health. They said that a slight change in the recipe of a dish (aka adding the yeast a bacteria so that they would ferment and produce the vitamins in the dish) would not bother them as long as the taste is not changed.
In order to not affect the taste, we chose to modify micro-organisms already present in the dish instead of adding new ones, in order to not disrupt the microbiome of the fermented dish. To check if high amounts of vitamins could change the taste, we also tasted idlis (fermented rice cakes) in which vitamin A, B2 and B12 bought in a supermarket had been added in quantities equivalent to the required daily amount, and did a blind test: people found no difference in taste at all with and without vitamins.

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. 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.

Idli recipe

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.

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, and which can be toxic at very high dose. The ß-carotene produced by our yeast though is not toxic, since the body only converts what it need into retinol.
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.

European and Indian Regulations

In order to evaluate the feasibility of our product’s implementation, we researched the european and indian regulations concerning the production and distribution of genetically engineered micro-organisms.
We found that the EU directive 90/219/EC of the EC relative to the contained use of GMO would allow production of this product within the european market. This directive is enforced in each of the EU member’s national regulations.

In India, the FSSIE (Food Safety and Standard Authority in India) told us the safety needed to be established in order for our product to be authorized. Both Dr. A. K. Sharma from the FSSAI and Dr. Sunita Grover the Dairy Microbiology Division at the National Dairy Research Institute advised us to chose micro-organisms that were already present in the fermented foods we were targeting, which we did. These organisms are all in Risk Group 1 (Unlikely to cause human disease.), and have the GRAS status (Generally Considered As Safe).
We also found out that the Indian law currently doesn’t allow GM microorganisms because of the use of antibiotic markers that makes them unsafe to eat.

But according to Samir K. Brahmachari, former director of the Council of Scientific and Industrial Research in India, our product has a greater chance to be authorized if there is no more live bacteria in the final form of the dish, that is actually eaten. We checked this affirmation and found out this regulation:

‘...food stuffs...derived from Living Modified Organisms where the end product is NOT a Living Modified Organism are exempted from mandatory approval of the Genetic Engineering Approval Committee.’

Food Safety and Standards Authority of India (FSSAI)
Notification no. S. O. 1519(E) dated 23-8- 2007 in the Gazette of India

Since the idli is steamed for 10 to 15 minutes before it is eaten, we made an experiment to assess the presence of yeasts and bacteria in the steamed idli, and found out that the steaming process effectively sterilizes the idli and eliminates the microbes present in the batter.

Plates of different media, 36h after being inoculated with idli batter, before and after the batter was steamed for 10 minutes

We can see than nothing grew in the plates inoculated with steamed idli batter, which indicates that all the micro-organisms present in the batter were killed during the steaming process.

Continuity

As we wanted people to feel that our product really belong to them, we designed it in the form of an open framework that can be modified and improved easily. Based on precious input from the target population and by the Indian community of Paris, this new chassis is made for being manufactured by factories as well as community labs, so people can be sure they will always keep their independance. It allows to reduce the cost of research and development by abstracting the most technical functions and to easily make organisms that can be homegrown and used for nutrient production. This way we hope that people will feel ownership on the product and trust that it only serves their own interests.

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 <br><br>
-In India, the FSSIE (Food Safety and Standard Authority in India) told us the safety needed to be established in order for our product to be authorized. Both Dr. A. K. Sharma from the FSSAI and Dr. Sunita Grover the Dairy Microbiology Division at the National Dairy Research Institute advised us to chose micro-organisms that were already present in the fermented foods we were targeting, which we did. These organisms are all in Risk Group 1 (Unlikely to cause human disease.), and have the GRAS status (Generally Considered As Safe).
+In India, the FSSIE (Food Safety and Standard Authority in India) told us the safety needed to be established in order for our product to be authorized. Both Dr. A. K. Sharma from the FSSAI and Dr. Sunita Grover the Dairy Microbiology Division at the National Dairy Research Institute <a href="https://2015.igem.org/Team:Paris_Bettencourt/Acceptance">advised us to chose micro-organisms that were already present in the fermented foods</a> we were targeting, which we did. These organisms are all in Risk Group 1 (Unlikely to cause human disease.), and have the GRAS status (Generally Considered As Safe).
 <br>We also found out that the Indian law currently doesn’t allow GM microorganisms because of the use of antibiotic markers that makes them unsafe to eat.
-<br>But <a href="https://2015.igem.org/Team:Paris_Bettencourt/Acceptance">according to Samir K. Brahmachari</a>, former director of the Council of Scientific and Industrial Research in India, our product has a greater chance to be authorized if there is no more live bacteria in the final form of the dish, that is actually eaten. We checked this affirmation and found out this regulation:
+<br><br>But <a href="https://2015.igem.org/Team:Paris_Bettencourt/Acceptance">according to Samir K. Brahmachari</a>, former director of the Council of Scientific and Industrial Research in India, our product has a greater chance to be authorized if there is no more live bacteria in the final form of the dish, that is actually eaten. We checked this affirmation and found out this regulation:
 <br><br><div align="center"><i>‘...food stuffs...derived from Living Modified Organisms where the end product is NOT a Living Modified Organism are exempted from mandatory approval of the Genetic Engineering Approval Committee.’</i></div>
 <div align="right">Food Safety and Standards Authority of India (FSSAI)<br>

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