Team:KCIS NewTaipei/Implementation

Implementation

IMPLEMENTATION IMPLEMENTATION
In this iGEM project, we hope to incorporate the use of D-licious, our specialized fermentation starter, into the daily diets of people around the world. We aim for our D-licious fermentation kit to take advantage of the importance of fermented foods in multiple cultures to alleviate and ultimately cure vitamin D deficiency around the world. Through our product, we hope to combat the issue of vitamin D deficiency in the healthiest, most convenient, and enjoyable way possible.
We want to make our product as practical as possible so that consumers will accept D-licious as a worthwhile investment for their health. To verify the importance of this project and justify our product, we started by conducting a public survey on people’s opinions about vitamin D deficiency. While the majority of our responses were from Taiwan, we received responses from 6 continents, and most responses reached the same consensus. Out of the 1355 total survey results, 25% responded that over the past two weeks, they were exposed to sunlight for less than one day while another 25% were exposed for less than 3 days. We also found that although around 53.7% of individuals that responded recognize that vitamin D deficiency is an issue, very few take any action against it besides spending a few more hours under sunlight. With the COVID lockdown, outdoor activities dropped drastically, further reducing sunlight exposures and therefore increased the risk of vitamin D deficiency. After confirming the need for a product for vitamin D deficiency, we, the KCIS_iGEM team present our solution.

*Fig 1. The survey results demonstrate that most people spent fewer than three days outdoors in the past two weeks due to the COVID-19 pandemic in 2021, which increases the risk of vitamin D deficiency. (survey results collected from 6/5~6/19)
*Fig 2. Half of those who responded were unsure about the problems associated with Vitamin D, reflecting the lack of public awareness on the issues concerning vitamin D deficiency.
Fermentation is one of the methods by which cells generate energy and maintain redox balance in anoxic environments (Catalanotti et al., 2013). Yeasts and lactic acid bacteria play crucial roles in the metabolism of alcoholic fermentation and lactic acid fermentation respectively, in which a carbohydrate is converted into an alcohol or an acid (Maicas, 2020) (National Research Council (US) Panel on the Applications of Biotechnology to Traditional Fermented Foods, 1992). Fermentation increases acid and alcohol content and decreases water, inhibiting the growth of spoilage microorganisms by creating a harsh environment difficult for their survival. Therefore, the yeasts and lactic acid bacteria are employed by people worldwide to preserve and produce food including wine and beer as well as pickles, yogurts, and kimchi.

The process of fermentation varies for different fermented foods. Fermentation processes of vegetables, including dill pickles and sauerkraut, can be conducted by dry salting or brining, whereas products such as yogurt and kombucha require the addition of starter culture to the food materials. However, our D-licious fermentation starter can be added to both kinds of fermentation food to increase the growth of certain target bacteria (Downshiftology, 2019).

Fermentation starters are bases used during the commercial production of some fermented foods to ensure the consistency in properties of the products, including the acidity and aroma. The microbial cultures consist of specific cultivation media and a specific mix of essential microorganisms that would be inoculated to perform the fermenting process (Starter Culture - an Overview | ScienceDirect Topics, 2017). The fermentation starters of our products would be in the form of powder. The manufacture of them starts with heat treatment of the medium, in which the original microorganisms and the dissolved oxygen are eliminated. The specific bacteria culture is then inoculated to the heated-treated media cooled down to a suitable temperature for bacteria growth. During incubation, the bacteria multiply and perform the process of fermentation until the starter culture is cooled down to inhibit further growth, which helps maintain the high activity of the bacteria. Finally, with methods such as freezing, the starters are preserved for storage (CULTURES AND STARTER MANUFACTURE, 2019).
*Fig 3. The manufacture of fermentation starters follows the processes of heat treatment, cooling, inoculation, incubation, and preservation with each of the steps conducting under suitable temperatures respectively.
The D-licious fermentation kit includes tools and recipes for homemade fermentation food along with a powdered fermentation starter incorporating probiotics specifically engineered to upregulate the activation of the vitamin D the users consume/intake.

Several tools are included in the D-Licious fermentation kit to assist fermentation processes at home, including fermentation jars, a pounding tool/tamper, waterless airlock tops, glass, weights, and recipes. The fermentation jars provide containers of food materials in which the probiotics in the starter perform the fermentation process. The pounding tool helps to crush large food materials such as cucumbers into smaller pieces. The airlock, which should be attached to the glass and the lid, maintains an anaerobic atmosphere for fermentation, preventing the air from entering the jars while allowing the release of CO2 generated during fermentation. The weights help hold the food in place during the fermentation process. To suit the preference of each user, we also include the details of the steps for making each specific fermented food (yogurt, pickles, and kimchi) in the recipes.

*Fig 4. Overview of basic contents included in our fermentation kit
To utilize the D-Licious fermentation starter, users can start with the preparation of food materials, of which the process would vary with the type of food. Specifically, milk would be heated and vegetables would be sliced into appropriate pieces. After setting the materials to the temperature suitable for the growth of bacteria, the fermentation starter could be added (along with salt/ saltwater in terms of vegetable food materials) and left for incubation. Finally, the fermented food could be frozen for preservation.
*Fig 5, Overview of the main processes of fermentation
Although our engineered fermentation starter can be applied all around the globe with different cultures, lifestyles, and diets, we believe that it is still important to further analyze higher risk groups of vitamin D deficiency; this will allow us to maximize our product’s potential and identify populations more in need of our product. By identifying the target user groups in need of our product, D-Licious could potentially be more than just a part of a fermented food diet but a probiotic treatment for vitamin D deficiency. While individuals fulfilling these requirements may experience more severe vitamin D deficiency symptoms, this product is available to all individuals without diseases or intestinal damage.
Taiwanese Local Target Users
Vitamin D deficiency is a serious health issue around the world; Taiwan is one of the countries most significantly impacted. According to a survey conducted by Chang Gung Memorial Hospital that examines the 25(OH)D levels of a total of 3954 individuals, despite the high level of sunlight expected in subtropical regions, vitamin D deficiency is prevalent in Taiwan. Specifically, in urban areas of northern Taiwan, behavior factors such as the tendency to stay indoors for work, the application of makeup on the skin, and the increasing air pollution limit people’s exposure to sunlight, resulting in a higher vitamin D deficiency prevalence rate of 22.4% (25(OH)-D concentration < 20 ng/mL) (Lee et al. 2019). The issue could potentially worsen due to the recent COVID-19 lockdown, which has prevented people from outdoor activities. The decrease in exposure to sunlight might cause a higher risk of vitamin D deficiency in Taiwanese. Therefore, our product is especially suitable for local citizens under these circumstances.
Global General Target Users
Infants and Toddlers (6 months~3 years old)
An urban primary care clinical study measured the prevalence rate of vitamin D deficiency among 365 healthy infants and toddlers, the results were shocking. Out of all participants, 52.1% were shown to have levels of vitamin D below the accepted optimal threshold (Gordon et al., 2008). From this, we see that newborn babies to toddlers up to 3 years of age are all high-risk groups for vitamin D deficiency. Although breast milk provides a blend of vitamins, proteins, and fat, the amount of vitamin D in breast milk is far less than the minimum required for a healthy infant (Breastmilk information, 2021). Also, the nutrition of breast milk depends on the mother’s condition, therefore vitamin D levels in the milk of deficient individuals are further reduced. The infant or toddler, therefore, requires to intake extra amounts of vitamin D from other food sources which generally have minimal levels of vitamin D compared to supplements or sunlight (CDC, 2019). Vitamin D limitations in food sources as well as infants’ lack of sunlight exposure all contribute to their development of rickets, muscle weakness, and fractures (Canadian Paediatric Society, 2021). Considering how deficient amounts of vitamin D hinder infants and toddlers’ motor development and put them at risk of vitamin D related diseases, we decided to target infants from 6 months (can start yogurt intake in small quantities) up to toddlers of 3 years old (Can Babies Have Yogurt: Answers for Parents, 2018).
Older Adults/Seniors
According to a study in Singapore, out of 134 subjects ( ≥ 65 years old), 115 patients (85.6%) presented low serum 25-hydroxyvitamin D3 (25OHD3) levels. Another clinical report has also shown that out of 217 consecutively geriatric hospitalized patients, approximately 96% of all have serum 25(OH)D below the optimal concentrations of ≥30 ng/ml (Boettger et al., 2018). Risk factors for the elderlys’ vitamin D deficiency issues include decreased VDR expression, decreased vitamin D production in the skin, decreased kidney function for 1,25(OH)2D production, and several others. In older individuals, aging results in lower vitamin D production in the human skin due to the decrease in 7-DHC which further slows down the response to UVB rays when exposed to sunlight. When a person ages, renal functions decline as well, leading to decreased activity of renal enzymes, such as 1α hydroxylase that converts 25OHD into 1,25(OH)2D. Moreover, ones with age ≥80 often have glomerular function rates (GFR) under 50 mL/min, highly lowering the production of 1,25(OH)2D levels. While these all have major contributions to relationships between aging and vitamin D deficiency, substrate deficiency also plays a significant role in vitamin D deficiency in seniors. This type of deficiency is caused by the diet or lack of sunlight exposure, decreasing 25(OH)D that limits 1,25(OH)2D production (Gallagher, 2013). Due to all factors above, seniors are especially at risk with vitamin D deficiency, and can therefore be listed as one of the main target users for our engineered fermentation starter.
Individuals with Darker Skin
Skin color has high biological significance and is associated with the regulation of Vitamin D in human bodies. Individuals with darker skin have higher risks of vitamin D deficiency as well. According to the Division of Health Equities at Beckman Research Institute of City of Hope, about 70% of African Americans suffer from vitamin D deficiency, causing them to be more vulnerable to cancer and cardiovascular diseases than people with lighter skin tone. The pigmentation causing the darker skin tone is a natural barrier against the UV light penetrating through the skin. Specifically, the large amounts of melanin, which is responsible for the tanning of the skin, in the epidermal layer undermine the skin’s ability to absorb sunlight and produce vitamin D. Furthermore, scientists also found that three regions of genes in African Americans that associate with the regulation of skin pigmentation also has a strong connection to vitamin D deficiency, therefore leading to the requirement of more intense sunlight exposure to synthesize sufficient amounts of Vitamin D. As a result, Individuals with darker skin tone are the potential target users for our products.
Obese Individuals
Obesity, a chronic health condition, has displayed strong bonds with vitamin D deficiency. Recent reports revealed prevalence rates of vitamin D deficiency in the obese population of up to 90% (UC Health). Studies have revealed that increasing BMI and body fat is associated with lower 25(OH)D concentrations, high PTH (parathyroid hormone) concentrations, and low 1,25(OH)2D levels (Vanlint, 2013). Reasons for connections between vitamin D deficiency and obesity may be due to obese individual’s lower participation in outdoor activities, leading to minimal sunlight exposure. Other scientific theories also suggest how obese subjects experienced lower increases in plasma levels of 25(OH)D after oral supplementation or exposure to UVB rays because of vitamin D distributing into excessive amounts of adipose tissues (AT) which are responsible for accumulating and retaining fat-soluble vitamins. Furthermore, several also pointed out possible correlations of impaired hepatic 25-hydroxylation with lower 25(OH)D concentrations. As one common chronic liver disease in obesity, non-alcoholic fatty liver disease (NAFLD) has been found to be responsible for causing liver cirrhosis, causing the damage, inflammation, or necrosis in the liver which may also contribute to low 25(OH)D. While the reasons for associations between obesity and vitamin D remain unclear, there is no doubt about a cause-effect relationship between the two (Vranić et al., 2019).
Among the 1027 responses, 64.5% replied that they are not willing to try our product mainly because of their concern for its safety. Coincidentally, the remaining 35%, who replied that they might try our product, have the least amount of exposure to sunlight. Taking our product is, therefore, an alternative to balance the amount of vitamin D in their body.
*Fig 6. The survey results show that more information is still needed for people to trust the safety of our product.
Generally, probiotics, butyrate, and fermented food all have potential negative health implications if over consumed. However, if consumed in safe/adequate amounts, fermented food made from D-licious starters not only trains a more healthy and diverse gut microbiome but also upregulates the downstream effects of vitamin D that would benefit health. Users can refer to our safety page for more detailed and specific information concerning the safety of our product.
To let our products be promoted globally, we research a variety of information to complete our marketing plan. We analyzed and searched for similar products in the current market, locking specific customers in and out of the marketplace. Considering changes in community and time, we try to find ways of fitting into our current societal environment. And try to find out what the position of our product is. What is the difference between our product and the others?
(For more information, please click here.)
In order to achieve commercialization globally, we compiled information from different sources, gathered public opinions, and created marketing plans. Through our product’s hardware design and recipes customized for various fermentation foods, D-licious will be effectively and unwittingly incorporated into people’s daily diets in the real world.
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