Description

Project Description

Overview

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.

Minerals

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.

Magnesium

It is a macronutrient which acts as an electrolyte in the human body.
Mg plays an important role in Synapse. It facilitates the electrical signaling for touch and muscle movement along the nervous system.
Human body uses Mg for structural support in cells and bones.
Mg is an enzyme cofactor for over 300 different enzymes, which activates these enzymes.These enzymes are involved in many pathways. For ex. Insulin functioning, Carbohydrate metabolism, nerve signaling, protein synthesis. It activates DNA Polymerase.
Daily recommended intake of Mg – 420 mg for men and 320 mg for women.
Deficiency of Magnesium may lead to heart problem, lung problems, neurological problems, digestive problems and diabetes
Source of Mg, green leafy vegetables, nuts, seeds, fruits, unprocessed cereals, meat.

Calcium

Ca is responsible for development and maintenance of bones, teeth formation.
Maintenance of blood circulation.
Helps in blood clotting
Muscle contraction
Maintenance of normal heart rhythm
Calcium ions initiates synaptic transmission
99% Ca stored in bones, 1%(serum calcium) circulating throughout the body.
Body has the capability to draw Ca from bones in case of deficiencies.
Daily recommendation 1000-1200 mg for female and male.
Source- kale,barley Spinach(but it acts as ANF for Ca), yogurt, salmon fish.
Deficiency leads to Osteopenia, Osteoporosis (brittle bones)
Excess Ca leads to Kidney stone, prostate cancer, constipation, weakness, fatigue, nausea, shortness of breath.

Iron

Responsible for carrying oxygen from lungs to the body.
Present in RBC (as hemoglobin)
Iron state affects activity of heart, liver, muscles, pulse action of nerve impulses
The Fe component of myoglobin supports muscle metabolism and healthy connective tissues.
People having gastrointestinal disorders such as celiac disease, Crohn’s and Colitis may be following a strict diet or experienced malabsorption of Fe or iron blood loss in gastrointestinal tract may be at risk of Fe deficiency.
Iron supplements contain Ferrous rather than the ferric form of iron. Due to its high solubility and easy bioavailability. Easy for the body to use.
Vitamin B12 +A is an iron source
Cu transports Fe through the body
Cu source Vit C
Source: green leafy vegetables, whole grains, red meat.

Zinc

Important for the immune system.
Helps heal wounds, diabetic wound healing, test smell and good vision.
Helps in age related diseases macular degeneration of eyes
Zinc deficiency symptoms - alcohol addiction, anorexia, breast feeding infants from a Zn deficient mother, chronic diarrhea, thinning hair, decreased appetite, skin rashes,mood disturbances, dry skin, impaired wound healing.
Source - meat, fish, Plants, cereals and fortified flour with zinc.

Copper

Helps formation of RBC, keeps the nervous system and immune system healthy.
Allows the formation of collagen and elastin which forms scaffolding for bones.
Acts as an antioxidant.
Deficiency may cause cardiovascular diseases, bone fracture
Source - Vitamin C, drinking water from copper vessel.

Statistics

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

data

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.

data

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 -

data

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.

Current solutions to the deficiency problem

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.

data

The issues with the current solutions to the problem

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 Solution

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.

References

[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