Phytic acid (PA), is an antinutritional factor, present in many legumes, cereals, and grains, which inhibits the absorption of vital dietary minerals, calcium, iron, and zinc. PA's ability to chelate these minerals can result in very insoluble salts that are poorly absorbed from the gastrointestinal tract, which results in poor bioavailability (BV) resulting in various micronutrient deficiencies like anaemia, which manifests with symptoms like fatigue, weakness, shortage of breath and dizziness or even diabetes type 1 in extreme cases. More than half of the world populations are affected by micronutrient malnutrition and one third of the world’s population suffers from anemia and zinc deficiency, particularly in developing countries.
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Our team here at ICT Mumbai is inserting BioBricked plasmids containing the phytase gene into yeast (Saccharomyces cerevisiae) cells. The yeast (Saccharomyces cerevisiae) cells can be added to any type of bread which can help improve the quality and the nutritious value of the bread.
Phytic acid (PA), also called inositol hexakisphosphate (IP6), is an antinutritional factor which inhibits the absorption of vital dietary minerals, calcium, iron, and zinc due to its strong binding ability with them. It is the primary storage form of phosphorus present in many legumes, cereals, and grains, and enters our system through these seeds. PA has the strong ability to chelate multivalent metal ions, especially zinc, calcium, and iron. The binding can result in very insoluble salts that are poorly absorbed from the gastrointestinal tract, which results in poor bioavailability (BV) of minerals. This results in various micronutrient deficiencies like anaemia which manifests with symptoms like fatigue, weakness, shortage of breath and dizziness or even diabetes type 1 in extreme cases. More than half of the world populations are affected by micronutrient malnutrition and one third of world’s population suffers from anemia and zinc deficiency, particularly in developing countries. Iron and zinc deficiencies are the major health problems worldwide. To tackle this, the iGEM team at ICT mumbai aims at Biofortification of staple diets using modern biotechnological techniques, potentially helping in alleviating malnutrition and micronutrient deficiencies in developing countries. This is to be achieved by genetically altering Saccharomyces cerevisiae using rDNA technology and synthetic biology to make it naturally produce phytase enzyme. Phytase enzymes are capable of removing the phosphates from phytate thereby deactivating it. This yeast when added to the diet can be extremely beneficial to prevent and hopefully eradicate these deficiencies in the world, especially in developing countries where biofortification is more economical than supplements.
Phytate acts as ANF for following minerals in human body
Magnesium
Calcium
Iron
Zinc
Copper
Manganese
Cobalt
Role of these minerals in the human body.
According to estimates, there will be 536.6 million people with diabetes worldwide in 2021, and that number will rise to 783.2 million by 2045, representing a prevalence of 12.2%. Diabetes was more common in people aged 75 to 79, with incidence rates being identical in men and women. According to estimates, prevalence was higher in high-income nations (11.1%) compared to low-income countries (5.5%) and in urban regions (12.1%) than in rural ones (8.3%) in 2021. Compared to high- (12.2%) and low-income (11.9%) nations, middle-income nations are predicted to see the largest relative increase in the prevalence of diabetes between 2021 and 2045 (21.1%). In 2021, it was forecast that 966 billion USD would be spent globally on diabetes-related healthcare. By 2045, that amount is expected to rise to 1,054 billion USD.
consequently, that indicates More over 10.5% of the adult population globally today has diabetes, which affects little over half a billion people. The life expectancy (LLY) of average individual with T2DM (age 65.4 years) is 18.6 years from now, compared to 20.3 years for the similar non-diabetic population, which equals LLY of 1.7 years/average person. We project that for T1DM and T2DM.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7673790/table/T1/?report=objectonly
1) Hong Sun 1,Pouya Saeedi 1,Suvi Karuranga,Moritz Pinkepank,Katherine Ogurtsova,Bruce B. Duncan,Caroline Stein,Abdul Basit,Juliana C.N. Chan,Jean Claude Mbanya,Meda E. Pavkov,Ambady Ramachandaran,Sarah H. Wild,Steven James,William H. Herman,Ping Zhang,Christian Bommer,Shihchen Kuo ,Edward J. Boyko 1Dianna J. Magliano 1:https://doi.org/10.1016/j.diabres.2021.109119, https://www.diabetesresearchclinicalpractice.com/article/S0168-8227(21)00478-2/fulltext#%20
2) )Heald AH, Stedman M, Davies M, Livingston M, Alshames R, Lunt M, et al. Estimating life years lost to diabetes: outcomes from analysis of National Diabetes Audit and Office of National Statistics data. Cardiovasc Endocrinol Metab. 2020;9:183-5.
3)Magliano D.J.,Chen L.,Islam R.M.,Carstensen B,Gregg E.W,,Pavkov M.E.,et al.Trends in the incidence of diagnosed diabetes: a multicountry analysis of aggregate data from 22 million diagnoses in high-income and middle-income settings ,Lancet Diabetes Endocrinol. 2021; 9: 203-211
A study conducted in India in 2022 found that Anaemia was higher in girls (39.6%, 48.7 million) than boys (17.6%, 23.7 million). In terms of severity, 17.6% of adolescents had mild anaemia, 10.0% had moderate anaemia and 0.9% had severe anaemia. (S. Scott et. al., 2022) https://doi.org/10.1111/mcn.13391 It is estimated that up to 17% of the global population is at risk for inadequate zinc intake, while in South Asia, up to 30% of the population may be deficient. Other areas at risk include sub-Saharan Africa and Central America. Endemic deficiency is common in up to one-third of the population in various parts of the world, primarily in Southeast Asia and sub-Saharan Africa. Zinc deficiency is also prevalent in Iran, Egypt, and Turkey, secondary to high phytate intake (L. Maxfield et. al. 2022). According to FAO(2010), India is the world’s largest producer as well as largest exporter of millets and 2nd largest producer of wheat, rice, pulses. Rice occupies 64.4% of India’s cereal exports (2013–2014). Coarse cereals (defined as cereal grains other than wheat & rice) are directly consumed in many developing countries. The nutrient content-kcal, calcium(mg), phytate(mg) per 100 g of edible portion(12% moisture) for various cereals are rice(milled,oryza sativa) 356,33,266; wheat(atta,tricium aestivum) 320,30,632; sorghum(jowar;sorghum vulgare) 334,25,549; pearl millet(bajra;penniselum glaucum) 348,42,485; maize(zeus maize) 334,26,646; finger millet(ragi;eleusine coracana) 320,350,306 respectively. From the nutritional data available for the past half a century, the dietary calcium intake of infants and children, adults, pregnant and lactating mothers are far below the RDA.
Reports show an increased intake of whole grains, pulse grains and legumes in the world from the year 2008 to 2018 in the word and in India -
This increase is owing to better awareness of the populations around the world and even in India in regards to its contribution in lowering the incidences of type 2 diabetes and aiding with celiac disease and gluten intolerances (F. Ghanbari‐Gohari et. al. 2022, M. Pierce et. al. 2021) In a 2015 international report from HealthFocus International, called “Fiber, Grains, and Gluten — A Global Perspective,” 71% of respondents (spanning 16 countries, across the Americas, Europe, and Asia) reported that they want whole grains as a source of nutrition in products. The report also found that the international awareness of ancient grains was up from 26% in 2012 to 28% in 2014, with 35% of the respondents expressing an interest in ancient grains. Fiber, a nutrient prevalent in many whole grains, was also a big area of interest for many people in the survey. (whole grain statistics - whole grains council, 2018) According to estimates, there will be 536.6 million people with diabetes worldwide in 2021, and that number will rise to 783.2 million by 2045, representing a prevalence of 12.2%. Diabetes was more common in people aged 75 to 79, with incidence rates being identical in men and women. India is the diabetes capital of the world with about 90 million cases of type 2 diabetes in India itself in the year 2022, of which, undiagnosed cases are estimated to be 51.2%. The worst part - these numbers are estimated to increase by the year 2045 to about 152 million (IDF Atlas reports, 2022). Majority of the Indian population use food grains as the main component of their diet, although there are variations in diet found across different parts of the country. The principal food grain in North India is wheat followed by rice. In the Western India, wheat comprises 50% of total grain consumption followed equally by coarse cereals & rice together. In South India, rice is consumed as the main food grain. Wheat is the staple food across India consumed using whole wheat flour(atta) in the form of unleavened flat bread(roti,chapattis) and as bakery products. Approximately, 47% urban and 57% of rural subjects’ energy intake comes from cereals for low income populations who obtain high proportion of nutrients and calories from cereals. Cereals contribute to 1 kg/per person/per month in rural India, and 0.8 kg/per person/per month(other than rice and wheat) in urban India (2011–2012). The major problem with that is the high amount of Phytic acid present in these foods which leads to the aforementioned deficiencies.
Globally, the WHO is frequently quoted as estimating that almost 2 billion people are at risk of micronutrient deficiencies (vitamins and minerals/trace elements).The latest estimate by 5 UN agencies is that 821 million people globally are undernourished, which puts them at risk of vitamin and other micro- and macro-nutrient deficiencies.So,It is high time we arrive at solutions to tackle this rising problem. Discussing, about the current solutions we have, we understand Treatment begins with oral replacement. In adults, 2 to 3 mg/kg per day or 20-40 mg daily dose often cures all clinical manifestations within 1 to 2 weeks. Even in patients with acrodermatitis enteropathica, a disease of malabsorption, oral replacement with 1 to 2 mg/kg per day is still the standard of therapy with life-long supplementation. In India there are a plethora of policies and nutrition programs to address several decades of malnutrition, but there is paucity of impact. Mid Day Meal, a nationwide initiative which serves meals to school children while they are at school, bridges the food gap, but not the nutrition gap. The focus of nutrient supplementation was aimed at calories, protein, iron and vitamin A with little emphasis on calcium and vitamin D which are essential for bone health. Apart from these, there are micronutrient supplementation programs and Food Fortification programs.
Taking supplements for the deficiencies discussed above have been observed to lead to side effects such as gastritis, constipation, nausea, hair fall and stomach cramps to name a few. Whereas it is difficult to overdo it through food sources alone. There are several methods to reduce the ANF effects of phytic acid. Traditional food preparation techniques such as soaking, fermenting, and sprouting grains, legumes, and seeds can partially degrade phytic acid and enhance mineral bioavailability. Industrial food processing techniques like milling and refining can also reduce phytic acid content. Additionally, consuming a varied diet that includes a mix of phytic acid-rich foods and foods with higher mineral bioavailability can help mitigate the effects of phytic acid as an ANF.
It's important to note that while phytic acid's antinutritional properties are well recognized, the overall impact on an individual's health depends on various factors, including dietary composition, nutrient requirements, and food preparation methods.
Implementing methods to reduce the antinutrient effect of phytic acid can present certain challenges and limitations. Some of the problems that may arise include:
Nutrient loss: The methods used to reduce phytic acid, such as soaking and refining can lead to some loss of other nutrients as well. For example, minerals and vitamins may leach out during soaking or be degraded during fermentation or refining processes. This can result in decreased overall nutrient content in the food.
Cost implications: Implementing methods to reduce phytic acid may involve additional costs. For instance, specialized equipment or facilities may be required for large-scale processing or fermentation. These added expenses can affect the affordability and accessibility of phytic acid-reduced foods, particularly in low-income populations.
Lack of consumer awareness: Despite the potential benefits of reducing phytic acid, there may be limited awareness among consumers about its antinutrient effects and the methods to mitigate them. This can result in low adoption of techniques to reduce phytic acid content in home cooking and food manufacturing. Milling and soaking
Milling is the most commonly used method to remove phytic acid from grains. This technique removes the phytic acid but also has major disadvantages as it also removes major parts of minerals and dietary fibers
Our team is inserting recombinant plasmids containing the phytase gene into yeast (Saccharomyces cerevisiae) cells. The genes were going to be for phytase extracellular and anchored expression and the plan was to decide which gene to employ next based on the yields and efficiencies of those two variations. This yeast would then be added to the flour to allow simultaneous fermentation to be used in the existing industries, transforming a process that requires two unit operations - one for fermentation and one for phytate hydrolysis - into a process that requires a single unit operation, thus reducing the time and energy consumed.
[1] F. Ghanbari‐Gohari, S.M. Mousavi and A. Esmaillzadeh, Consumption of whole grains and risk of type 2 diabetes: A comprehensive systematic review and dose–response meta‐analysis of prospective cohort studies, Food Sci Nutr. 2022 Jun; 10(6): 1950–1960 (doi: 10.1002/fsn3.2811)
[2] https://wholegrainscouncil.org/newsroom/whole-grain-statistics
[3] https://diabetesatlas.org/idfawp/resource-files/2021/07/IDF_Atlas_10th_Edition_2021.pdf
[4] Akibode, C. Sitou; Maredia, Mywish K., AgEcon search, Global and Regional Trends in Production, Trade and Consumption of Food Legume Crops, 2012 (DOI: 10.22004/ag.econ.136293)
[5] M. Pierce et. al., Associations of Total Legume, Pulse, and Soy Consumption with Incident Type 2 Diabetes: Federated Meta-Analysis of 27 Studies from Diverse World Regions, J Nutr. 2021 May 11;151(5):1231-1240 ( DOI: 10.1093/jn/nxaa447)
[6] N. P. Akah et. al., Pulse Production, Consumption and Utilization in Nigeria within Regional and Global Context, Sustainable Agriculture Research; Vol. 10, No. 2; 2021 (doi:10.5539/sar.v10n2p48)
[7] L. Maxfield; S. Shukla; J. S. Crane, Zinc deficiency StatPearls 2021 (https://www.ncbi.nlm.nih.gov/books/NBK493231/)
[8] C.V. Harinarayan, H. Akhila, and E.S.Sree, Modern India and Dietary Calcium Deficiency—Half a Century Nutrition Data—Retrospect–Introspect and the Road Ahead, Front. Endocrinol., 06 April 2021, Volume 12 - 2021 (https://doi.org/10.3389/fendo.2021.583654)
[9] Bailey RL, West KP Jr, Black RE. The epidemiology of global micronutrient deficiencies. Ann Nutr Metab. 2015;66(Suppl 2):22–33.
[10] FAO, IFAD, UNICEF, WFP, WHO The state of food security and nutrition in the world [Internet]. Rome, Italy: Food and Agricultural Organization of the UN; 2018. Available from: www.fao.org/3/i9553en/i9553en.pdf.
[11] Parrott J, Frank L, Rabena R, Craggs-Dino L, Isom KA, Greiman L. American Society for Metabolic and Bariatric Surgery Integrated Health Nutritional Guidelines for the Surgical Weight Loss Patient 2016 Update: Micronutrients. Surg Obes Relat Dis. 2017 May;13(5):727-741.
[12] Freitas BA, Lima LM, Moreira ME, Priore SE, Henriques BD, Carlos CF, Sabino JS, Franceschini Sdo C. Micronutrient supplementation adherence and influence on the prevalences of anemia and iron, zinc and vitamin A deficiencies in preemies with a corrected age of six months. Clinics (Sao Paulo). 2016 Aug;71(8):440-8.
[13] Heald AH, Stedman M, Davies M, Livingston M, Alshames R, Lunt M, et al. Estimating life years lost to diabetes: outcomes from analysis of National Diabetes Audit and Office of National Statistics data. Cardiovasc Endocrinol Metab. 2020;9:183-5.
[14] Scott S., Lahiri A., Sethi V., Arjan de Wagt, Menon P., Yadav K., Varghese M., Joe W., Vir S.C., Phuong Hong Nguyen; Anaemia in Indians aged 10–19 years: Prevalence, burden and associated factors at national and regional levels, Mother and child nutrition, October 2022, Volume18, Issue 4 ( https://doi.org/10.1111/mcn.13391)
[15] Gupta, R.K., Gangoliya, S.S. & Singh, N.K. Reduction of phytic acid and enhancement of bioavailable micronutrients in food grains. J Food Sci Technol 52, 676–684 (2015). https://doi.org/10.1007/s13197-013-0978-y
Our aim was to use the BioBrick format for the plasmids pRS426 and YIplac204 as well as the inserts, so as to produce phytase enzyme in the host organism, which we chose to be Saccharomyces cerevisiae.
Design: Amplifying the genes using a high-fidelity polymerase in a normal PCR reaction so that we could further perform restriction-digestion and then ligate them to our plasmid (pRS426GPD)
Build: Set up the PCR reaction
Test: Gel electrophoresis showed bands of much smaller length than that of the gene of interest.
Learn: PCR reaction was not successful.
Design: Since the high-fidelity enzyme (phusion) did not work, we decided to use Q5 and a combination of Taq and Pfu polymerase in varying proportions for the amplification.
Build: Set up the PCR reaction
Test: Gel electrophoresis did not show bands indicating desired length.
Learn: None of the afore mentioned polymerases worked. PCR was unsuccessful.
Design: We then decided on using a long-range polymerase for amplification of the genes.
Build: Set up the PCR
Test: Gel electrophoresis of the purified PCR product using a long-range polymerase showed bands of desirable lengths.
Learn: A long-range polymerase showed positive results for our PCR
Design: Since we had to design primers such that we could avoid the PstI site in our plasmid, we decided to amplify the individual fragments and then use splice over extension (SOE) PCR to join the fragments together. We will also be requiring an equimolar mixture of the fragments for performing SOE PCR.
Build: Set up the PCR reaction for amplification; quantify the fragment mixtures; set up SOE PCR.
Test: Bands at desirable lengths obtained for the amplification; No bands visible after the setting up of SOE PCR.
Learn: SOE PCR was not successful and needed to be troubleshooted.
Design:Producing sticky ends using REases to facilitate ligation of the fragments.
Build: Set up RD followed by gel purification
Test: Gel electrophoresis showed bands at desirable length
Learn: RD was successful
Image Credits:Team ICT-Mumbai 2022
Design:Isolate the recombinant plasmid from the transformed E. coli and insert into Saccharomyces cerevisiae
Build: Yeast transformation protocol was carried out and the culture was plated on medium – agar + YNB + complete synthetic medium w/o URA + glucose
Test: Colonies were observed on the second day after plating. Observing under the microscope confirmed it was yeast.
Learn: Yeast transformation was successful
Design:We decided to estimate the decrease in phytate content in a given sample by measuring one of the products formed after the breakdown of phytic acid upon the addition of our enzyme after intervals of half hour and 1h. We decided to use the Fiske-Subarrow method for the estimation of free inorganic phosphate in the reaction mixture after those stipulated time intervals.
Build: Carried out the protocol for the Fiske-Subarrow method, a colorimetric method for the estimation of inorganic phosphate.
Test: While preparation of the samples for the estimation, we observed our blank was also showing colour upon the addition of the reagent.
Learn: Phytic acid also gives positive results for the Fiske-Subbarrow method. An alternate method for the estimation of phytate or modifications to the existing method required.
Our intended end-product can be a wet yeast for direct use in smaller bakeries or dried yeast to be added for use for large scale bakeries; it can also be added into premixes as suggested by dietician Charolette Jhakaria, for traditional dishes like dosa or idli.
Nutrient Availability: Phytase can increase the bioavailability of essential minerals like phosphorus, iron, and zinc in baked goods, potentially addressing micronutrient deficiencies.
Healthier Diet: The enriched bread may contribute to a more nutritious diet, potentially benefiting the general public's health in the long run.
Overall, the yeast cell modification for phytase production has the potential to enhance bakery products and improve the nutritional content of daily diets, potentially benefiting both businesses and public health in India.
Enhancing the nutritional quality of livestock feed is a promising initiative with the potential to benefit consumers worldwide. By fortifying animal feed, we can ensure that the livestock raised for human consumption receive essential nutrients, ultimately improving the nutritional value of the meat and dairy products derived from them.
To adhere to regulatory guidelines, such as those outlined by the OECD, it is imperative to conduct rigorous assessments. This includes evaluating genotoxicity and health hazards associated with feed preparation. Additionally, a critical aspect is investigating the digestibility of supplemented feed. If incomplete digestion occurs, it is essential to demonstrate that any resulting metabolites are non-toxic and non-allergenic.
The outcome of these assessments will provide crucial insights into the key nutrients and potential toxicants present after supplementation, ensuring that the fortified livestock feed remains safe, nutritious, and beneficial for both animals and consumers alike.
In India, the FSSAI is the food regulations authority which states the following prerequisites for the prior approval for manufacture, storage, distribution, sale and import etc.— (1) No person shall manufacture, pack, store, sell, market or otherwise distribute or import any food or food ingredient produced from GMOs, except with the prior approval of the Food Authority. (2) The provisions of these regulations are in addition to, and not in derogation of, any other rules or regulations made under the Act.
Procedure for grant of prior approval— (1) The food business operator (manufacturer/importer) of a GM-food shall submit an application as per the format prescribed by the Food Authority along with necessary documents and fee to the Food Authority. (2) If the application is for a GMO referred to as in clause (a) of sub-regulation (2) of regulation 1, clearance from Genetic Engineering Appraisal Committee (GEAC), the national competent authority for the environmental risk assessment shall be submitted to the Food Authority.
If our intended final product receives a ‘Unique Identifier Nine-digit Code’ of the Organisation for Economic Cooperation and Development (OECD) that is approved for food use by the FSSAI, it will require no further approvals.
Our conversation with a policymaker, Dr Kapila Mehta
To overcome challenges related to the effects of fermentation on phytase and achieve desired outcomes, industries may need to implement various process optimizations. These optimizations can vary depending on the specific goals and challenges faced by each industry.
Here are some potential process optimizations:
Fermentation Conditions: Adjust fermentation conditions such as temperature, pH, and oxygen levels to create an environment conducive to phytase activity. This may involve optimizing these parameters for the specific microorganisms used.
Fermentation Duration: Optimize the duration of fermentation to balance the desired increase in phytase activity with the risk of enzyme inactivation over time. Monitoring enzyme activity throughout the process is crucial.
Enzyme Production: Develop strategies to enhance the production of phytase enzymes during fermentation. This might involve using inducers that stimulate enzyme production.
Co-Fermentation: Explore co-fermentation with microorganisms that produce organic acids or other compounds that can enhance phytase activity. This can create a more favourable environment for enzyme function.
Monitoring and Control: Implement robust monitoring and control systems to continuously assess phytase activity and adjust fermentation conditions in real-time. This can help maintain optimal enzyme activity throughout the process.
- Improved Dough Quality: Phytase can enhance dough quality, leading to better texture and volume in baked goods like bread.
- Cost Savings: It can reduce the need for costly additives, resulting in more cost-effective bakery operations along with the prospects of employing simultaneous fermentation.
- Enhanced Product Portfolio: Bakeries may diversify their product offerings with improved quality, potentially attracting more customers.
Future iGEM teams can play a pivotal role in fortifying essential nutrients in both human and animal food products by building upon our work as a foundational starting point. Our recombinant plasmid can be used to introduce genes for the synthesis of other nutrients like vitamins, to be transformed into microorganisms efficiently. iGEM teams can use this platform to engineer microorganisms for nutrient production and enhance the nutritional value of everyday foods through collaboration and innovative solutions. Our shared goal is to address global health challenges related to nutrition and improve public health by fortifying commonly consumed foods.
Our project involves genetically modifying the yeast Saccharomyces cerevisiae to express the enzyme phytase. S. ce revisiae is classified as GRAS (generally regarded as safe), and the enzyme that is being overexpressed is not toxic.
We are using an auxotrophic marker (URA3) for transforming S. cerevisiae with an episomal plasmid containing the phytase gene. For genome integration too, an auxotrophic marker (TRP1) will be used. No antibiotic markers will be used.
However, as the plasmids we are using are E. coli-S. cerevisiae shuttle vectors, the bla gene (ampicillin antibiotic marker) is present in the vector. Integration of a linear DNA fragment containing just the expression cassette and an auxotrophic marker in the yeast genome is an approach which we plan to take once we have established proof of concept.
As the constructed yeast strain will be used for leavening breads, which are baked before consumption, we do not expect release of the modified organism in the environment.
References: [1] 50374/2022/REGULATION-FSSAI [2] OECD’s Report of the task force for the safety of novel foods and feeds [3] C. Frontela, G. Ros, C. Martínez, Phytic acid content and “in vitro” iron, calcium and zinc bioavailability in bakery products: The effect of processing, Journal of Cereal Science, Volume 54, Issue 1, 2011, Pages 173-179, ISSN 0733-5210, https://doi.org/10.1016/j.jcs.2011.02.015. [4] Nuobariene, L., Arneborg, N., & Hansen, Å. S. (2014). Phytase active yeasts isolated from bakery sourdoughs. In 9th Baltic Conference on Food Science and Technology "Food for Consumer Well-Being": FOODBALT 2014 - conference proceedings (pp. 223-227). Latvia University of Agriculture. Baltic Conference on Food Science and Technology
Our proof of concept experiment consisted of experimentally determining extent of phytate breakdown in dough prepared using a millets, which have a high phytate content.
We used the method described by Heinonen and Lahti (Anal Biochem 1981 May 15;113(2):313-7. doi: 10.1016/0003-2697(81)90082-8) to determine phytate breakdown. This method is used to estimate free inorganic phosphate, which is released when phytate is broken down. Unlike the well-known Fiske-Subbarow method of inorganic phosphate determination, phytate does not cause interference in the method of Heinonen and Lahti.
We constructed a standard curve using KH2PO4 to establish the relationship between Pi and absorbance at 390 nm.
Using the equation determined from the above experiment, we determined the free Pi content of sorghum (locally known as jowar, a staple diet in rural areas of Maharashtra and Telangana states) and refined wheat flour.
We then proceeded to ferment dough using our constructed yeast strains to determine the extent of Pi liberated.
Dough was prepared using one part each of sorghum and refined wheat flour. The yeast strains were used to ferment the flour for four hours. Samples of the rising dough were taken and concentration of free Pi in the dough was determined.
As can be seen from the above table, dough fermented by strains expressing Aj and Ec phytases had 10 and 23 percent more Pi as compared to dough fermented by the control strain (P=0.005). This demonstrates that phytase breakdown was significantly higher when phytase expressing strains are used to ferment the dough.
We baked the dough to make bread, and voila!
Our experiments in the lab started the first week of June and have lasted till October. This notebook contains the account of all our highs and lows, the frustrations and the wins for the other iGEM teams and judges to go through. Due to an incredibly late start, we have fallen short of achieving everything we envisioned but nonetheless we are proud of what we have managed to do. We hope the reader finds the lab notebook insightful.
Team iGEMxICT organized a 3-day Synthetic Biology Workshop at SIRAC KJ Somaiya students. Dr. Shamlan Reshamwala's lectures covered genetic engineering basics and advancements. Hands-on lab sessions let participants apply knowledge practically, while team projects encouraged creative problem-solving. Ethical discussions highlighted responsible research. The SynBio Workshop, sponsored by GeNei, empowered participants with foundational synthetic biology understanding and practical lab skills. It promoted interdisciplinary thinking, ethical awareness, and networking for potential collaborations. The event enriched students' education and nurtured their scientific curiosity. Team iGEMxICT's 3-day SynBio Workshop, supported by GeNei and conducted at SIRAC KJ Somaiya students valuable exposure to synthetic biology principles, hands-on labs, ethical considerations, and interdisciplinary collaboration. This experience ignited participants' curiosity, preparing them for potential contributions in the scientific world.
In a dynamic hour-long session, approximately 120 students embarked on an exploration of the Basics of DNA Cloning. The workshop covered an array of pivotal topics, captivating participants with its hands-on approach and real-world applications. The workshop began by unraveling the intricacies of identifying Genes of Interest (GOIs), setting the stage for the subsequent processes. Plasmid isolation offered a tangible glimpse into the extraction of genetic material, while Restriction Digestion showcased the art of DNA cleavage at specific sites, guided by enzymes. Ligation, a genetic stitching process, led participants through the creation of recombinant molecules, demonstrating the practicality of genetic manipulation. The transformative process of Comp Cell Preparation paved the way for DNA transfer, with Transformation revealing how foreign DNA can be introduced into cells, enabling novel traits. The workshop seamlessly connected theory and practice by showcasing media broth and live cultures, bridging the gap between concept and reality. The concept of Screening underscored precision in selecting cells with successfully integrated DNA. In essence, the workshop's immersive experience kindled a passion for biotechnology and genetic engineering. As participants left with enriched knowledge and a newfound appreciation for molecular biology's potential, the workshop marked the beginning of a journey into the fascinating world of DNA cloning.
Being Food Technology students, we learnt about various micronutrients, and the essential role they play in our metabolism. While an increased importance is given to the balance of macronutrients, we saw that micronutrients are often neglected. This has led to a large chunk of our Indian population including many of our team members to suffer from deficiencies, so we wanted to target that problem.
The United Nations declared 2023 as the International year of Millets. India being one the biggest consumers and producers of millets and legumes; we decided to pursue that and enhance its nutritional content, as it is not only a staple diet for the local and rural population throughout India, but the urban population is also adapting to these grains being reduced gluten or gluten-free, among other dietary advantages.
Upon having a discussion with our PI, Dr Shamlan Reshamwala, who is working on the yeast Saccharomyces cerevisiae, he came up with the idea to work on this project. After further research into it and having conversations with multiple experts in their fields, we realised the project has potential to contribute to the deficiency and malnutrition crises in the country and the world.
To first understand the impact of our project, we had an interaction with nutritionist Charolette Jhakaria, working in Kasturba Hospital for Infectious Diseases, Mahalaxmi, which is a municipal hospital.
She emphasized that micronutrient deficiency, especially in women and children, is widespread in India. Legumes and millets, being economically accessible and part of government rations, offer a potential source of vital nutrients. However, concerns exist about the willingness to introduce genetically modified (GM) foods into diets, primarily due to perceived reduction in nutritional content with processing, and due to scepticism about GM foods within the public. Risks associated with GM foods include hormonal imbalances in adolescents and gut microbiota effects.
Certain populations, such as pregnant women, children, and geriatric patients, may be more
vulnerable to GM foods, although legumes and millets themselves are associated with poor protein digestibility.
Proper labelling specifications are crucial, including- flour standardization, ingredient combinations, allergens, sodium content, proximate analysis, and basic GM food regulations.
The project’s impact could be enhanced by creating versatile premixes with multiple cereal pulses, beneficial for addressing malnutrition, particularly in mid-day meal programs. It could also simplify nutritious food preparation, extending beyond industry applications to reach local communities. Urban areas stand to benefit the most, while extreme rural markets may require different strategies, like incorporating these ingredients into traditional recipes like Bhakri. Overall, she considered the concept healthy, with potential to further fortify and diversify its applications beyond bread.
The acceptance of GM foods varies among individuals due to genetic and health factors. Lactose intolerance and certain hormonal reactions are factors to consider, along with pricing.
Nutrition Labelling for GM Products: Proper nutrition labelling, as regulated by organizations like FSSAI, is essential when introducing GM products to the market. It should include information on sugars, cholesterol, sodium, potassium, carbohydrates, and energy content.
Impact of the Project: The project aims to address micronutrient deficiencies and reduce the risk of diseases like diabetes, pneumonia, kidney disorders, cardiovascular diseases, and neurological conditions. Collaborating with government initiatives and NGOs could enhance its impact.
Target Audience: The project can have a significant impact in both urban and rural areas as bread consumption is seeing an increase in rural areas, provided it collaborates with government programs and organizations working in the nutrition sector.
Fortification and Genetic Modification: A combination of fortification and genetic modification may be necessary to address the diverse dietary needs of different individuals.
Considerations for Implementation: To succeed, the project should gather strong scientific evidence, collaborate with relevant authorities, and address health risks associated with excessive phytate consumption.
We consulted Dr Anuradha Borkar, the Chief Medical Officer in Kasturba Hospital. She reiterated the prevalence of micronutrient deficiency in our country among various populations. She mentioned that the current treatments for deficiencies are mainly supplements. The major drawbacks for this are the exuberant costs of these supplements. For the section of the population which suffers from deficiencies due to poverty, this is not the best solution. Another way of avoiding deficiencies is to implement national fortification programs as seen in India with iodized salt and beta carotene in golden rice, etc. According to her, ultimately the cause of deficiencies is lack of awareness surrounding dietary choices, which is a major problem. For people that are poor, it is difficult to get the nutrients they require due to Anti Nutritional factors from their diets which majorly consist of millets and legumes as they are cheaper. For that, she believes our project could possibly prove to be impactful. Apart from the cost, micronutrients from a natural, dietary source have also proven to be better for us than supplements, as literature suggests that certain supplements such as Calcium, perform to a lesser degree and are even linked to heart disease. Her opinion suggested no major risks currently but a need for more research for long term use of GM foods.
Dr Rekha Singhal, HOD of Food Engineering and Technology and previously a member on the FSSAI panel on Food additives, Flavourings, Processing aids and Materials in contact with food, gave us an insight into the Food Safety guidelines, and the regulations for the approval of any food products. We gained knowledge about the various tests and standards practised at FSSAI. She gave ideas on how to incorporate our modified organism into the doughs and batters of various Indian food products, and the process modifications needed, best suitable for our legume and millet based product. She also aided us in realising the potential problems we might face while seeking approvals from governing bodies and helped provide a sense of direction for future Human Practices needed.
Dr Kapila Mehta, a renowned expert in the field, shed light on the challenges and considerations associated with regulatory approvals for genetically modified organisms (GMOs) in the food sector.
On the Regulatory Framework by FSSAI: One of the foremost challenges in the Indian food industry is the stringent regulatory framework established by the Food Safety and Standards Authority of India (FSSAI). FSSAI regulations must be adhered to rigorously; making the approval process for GMOs complex and time-consuming. Currently, there is no streamlined pipeline for obtaining approvals for commercializing such products.
On the Lack of Approved GMO-Based Products: As of now, India has not approved any commercial food products that are GMO-based. This presents a significant challenge for innovative companies and researchers seeking to introduce novel synthetic biology-based products into the market.
Multi-Step Approval Process: The approval process for synthetic biology-based products involves several stages. Dr Mehta outlines the following steps: a) Patents and Copyrights: Companies and researchers must secure patents and copyrights for their technologies, ensuring legal protection for their innovations. b) Guidelines and Lab Procedures: Strict adherence to the guidelines and laboratory procedures prescribed by FSSAI is essential to meet safety and quality standards. c) Ethical and Biosafety Approval: Products must undergo evaluation and approval by both the biosafety committee and ethical committee. These committees assess the methods and ensure that the products meet ethical standards.
Challenges in Market Launch: Launching synthetic biology-based products in the Indian market poses various challenges: a) Public Awareness and Social Implications: The Indian public is diverse, and acceptance of products containing genetically modified organisms, especially microbes in food, remains a challenge. Despite safety guidelines issued by FSSAI, consumers may be hesitant to embrace such products, as food preferences in India are often closely tied to taste and tradition. b) Economic Hurdles: Companies and researchers must evaluate whether their production processes are cost-effective. Economic feasibility is a crucial factor in gaining regulatory approval and market acceptance.
Industry Collaboration: Dr Kapila Mehta emphasized that industry collaboration is vital in the field of synthetic biology-based food products. Companies often partner with academia to harness cutting-edge technologies and then commercialize them. This collaboration allows for expertise in marketing, finance, and scale-up, which academia may lack. ClearMeat as an Example: ClearMeat, an Indian lab-based meat company, is cited as an example of a forward-thinking organization in the modified food space. They have developed cruelty-free, cost-effective media for lab-based meat production, bringing them closer to regulatory approval. Dr Kapila Mehta concluded by acknowledging that synthetic biology is a double-edged sword. While it holds tremendous potential, it is also a challenging field. She emphasized the importance of thorough validation before widespread adoption.
To get more inputs from the industry about their perspective on this, the members of our team paid a visit to Bristol bakery in Mahim. During the interview with Mr. Iliyaz, the plant manager at a Bimbo bakery product facility, several important points were discussed regarding production processes and potential collaborations. The facility mainly uses fresh yeast, along with refined wheat flour and grains like ragi jowari, aiming for healthier options with 70% grain content while also offering custom-made products. A concise project description involving the idea of inserting BioBricked plasmids into yeast cells, potentially improving efficiency and creating products with increased calcium, copper, and zinc intrigued Mr. Iliyaz and the facility is open to collaborating and exploring new yeast strains. While they haven't used genetically modified yeast yet, considering millets aligns with the Millet Year initiative, though market demand remains uncertain. In terms of regulations, lab-produced yeast faces minimal safety concerns with government approval. Marketing baked goods with a shorter shelf life due to novel yeast could be challenging, but educating consumers about its benefits is a proactive approach. If lab-produced yeast can match conventional yeast in price while offering more advantages, it holds promise, though fermentation time considerations need careful evaluation along with other ambient conditions. In essence, the plant manager's focus on innovation and collaboration, along with attention to customer preferences and market dynamics, is a driving force to help in implementation of the novel yeast into the market.
We express our deepest gratitude to Aarti Industries Ltd for their overwhelming and constant support for us as well as ICT Mumbai’s previous teams, to SD Fine Chemicals Pvt Ltd for their showing faith in us and supporting us, Mr Virendra Tulzapurkar for his immensely kind heart and helpful donation. We would also like to express our heartfelt gratitude towards all our patrons who have so kindly donated on the ketto link.
Your generosity and commitment have been instrumental in helping us achieve our goals. Your belief in our mission is a driving force behind our success, and we are truly thankful for your partnership. We look forward to continuing our journey together and making a positive impact with your invaluable support.