Breast milk is the gold standard for infant nutrition. However, nearly 38% of infants worldwide cannot receive adequate breastfeeding for various reasons, placing them at higher health risks. Current infant formulas fall short in mimicking the key functions of breast milk, particularly in immune support, protein digestion, and lactose tolerance. To address this global challenge, our team “ProBabyotics” has developed an innovative solution: a bio-manufacturing platform built on the safe probiotic chassis Escherichia coli Nissle 1917 (EcN). Through three core modules, this platform precisely replicates the essential functions of breast milk—an Immune Enhancement Module (2′-FL synthesis to foster a beneficial gut microbiota and strengthen immune defense), a Protein Hydrolysis Module (trypsin expression to pre-digest casein and reduce allergy risks), and a Lactose Tolerance Module (β-galactosidase production to break down lactose and alleviate intolerance). This multifunctional system not only enables cost-effective production of critical nutrients but also holds the promise of a next-generation infant formula that is safer, more digestible, and functionally comprehensive, providing an accessible and sustainable health solution for millions of families worldwide who are unable to breastfeed.

Figure 1: The overall diagram of the functions of the three products from our program

Our project began with the deeply personal story of one of our team members.

Due to a lack of sufficient breast milk, his childhood was marked by persistent digestive problems caused by infant formula. This not only impacted his health but also left his mother with a profound sense of guilt and helplessness. Her lament, "Watching my child in discomfort, yet feeling powerless to help," became a heavy responsibility that our team now carries.

This story fostered a deep sense of empathy within our team: when breastfeeding is not an option, a family bears the dual burden of difficult nutritional choices and immense emotional stress. It is this firsthand experience of hardship that drives our determination to shield others from the same struggle. Thus, Team "ProBabyotics" was born. We are determined to harness the power of synthetic biology to systematically optimize current infant formulas, bringing them closer to breast milk in both functionality and safety. Our goal is to provide a better, more reliable alternative for families for whom breastfeeding is not a viable option.

Breast Milk: The Irreplaceable Gold Standard

Undoubtedly, breast milk is the most ideal natural food for infants. Particularly for infants aged 0-6 months, breast milk not only contains all the nutrients required for growth and development but also possesses excellent properties for digestion and absorption, making it the supreme choice for the healthy growth of infants and young children. Furthermore, breast milk is rich in natural antibodies and immunoactive substances, which can effectively prevent common illnesses such as respiratory infections, allergic reactions, and diarrhea(Rollins et al., 2016). Considering the aspects of safety, nutritional value, and cost-effectiveness, breastfeeding remains the optimal choice whenever circumstances permit.

Figure 2: The Nutritional and Functional Disparity Between Breast Milk and Infant Formula(Andreas et al., 2015)

The Reality Gap: Why Formula is a Necessity

However, the reality is often far from ideal. According to UNICEF data, approximately 38% of infants globally are not exclusively breastfed, with this figure exceeding 50% in some developing countries (UNICEF, 2023). The reasons for this are multifaceted:

1.Some mothers are unable to breastfeed due to medical conditions such as HIV infection or severe postpartum depression.

2.Workplace pressures and the lack of breastfeeding-friendly environments compel many mothers to wean their infants prematurely.

3.Mothers of premature infants often face challenges such as insufficient milk supply or the infant's weak sucking ability.

For these families, infant formula becomes an indispensable necessity.

Figure 3: Geographical Distribution of Breastfeeding Rates Worldwide(Liu et al., 2021)

The Three Core Challenges of Current Formulas

Challenge 1: The Missing Guardian - Human Milk Oligosaccharides (HMOs)

Oligosaccharides (HMOs) are critical immune guardians in breast milk that can significantly reduce the risk of infant intestinal infections(Donovan et al., 2016). However, due to prohibitive technological costs and regulatory restrictions, HMOs have long been absent from most infant formulas. In China, for instance, it was not until 2024 that core HMOs like 2'-fucosyllactose (2'-FL) were approved for use as food nutritional fortifiers, whereas they have been widely available in international brands for years. Now that policy has approved their use, the key challenge has shifted to how to economically and sustainably scale up HMO production—and this is precisely the core problem our project aims to solve using synthetic biology.

Figure 4: China's New Regulations on 2'-Fucosyllactose (2'-FL)

Challenge 2: The Digestive Hurdle - Casein Protein

The primary protein source in infant formula is cow's milk, of which approximately 80% is casein. Unlike the predominant and easily digestible whey protein in breast milk, casein exhibits unique physicochemical properties that pose a significant challenge to an infant's immature digestive system(Lönnerdal et al., 2014) . Upon entering the acidic gastric environment of an infant, casein coagulates to form large, hard, and dense curds. This process leads to delayed gastric emptying, meaning the stomach takes longer to process food and pass it to the intestines. Prolonged retention of food in the stomach can increase intragastric pressure, leading to a higher incidence of reflux and regurgitation. Furthermore, these hard-to-digest casein curds can cause discomfort and bloating. Therefore, how to make the casein in formula as gentle and easily digestible as breast milk protein is the second core challenge we are determined to overcome.

Figure 5: Digesting challenges of casein in infants' intestines

Challenge 3: The "Sweet" Burden - Lactose Intolerance

Approximately 30% of infants experience symptoms of lactose malabsorption, with a particularly high prevalence among Asian populations (Lin et al., 2022). This condition stems from a congenital deficiency of the enzyme β-galactosidase in the intestinal mucosa. Unable to metabolize lactose, an osmotic imbalance occurs, leading to bacterial fermentation(Heyman et al., 2006). The clinical manifestations include bouts of diarrhea, incessant crying, and associated gastrointestinal discomfort.

The Common Barrier: The High-Cost Wall

Although high-end formulas fortified with HMOs like 2'-FL or utilizing extensively hydrolyzed proteins are available on the market, their prohibitive prices create a significant barrier to nutritional equity. Consequently, in resource-limited regions, infants often consume only standard, lower-cost formulas, making them more susceptible to micronutrient deficiencies that can impair their growth and development.

Figure 6: The disadvantages of current formulas in the market

The Three Key Compounds in Infant Formula

2′-Fucosyllactose

Figure 7: Chemical Structural Formula of 2'-Fucosyllactose

In the pursuit of developing infant formulas that more closely mimic human breast milk, scientists have identified a pivotal component: 2'-fucosyllactose (2'-FL). As one of the most abundant Human Milk Oligosaccharides (HMOs), its concentration is surpassed only by lipids and lactose, making it a core element responsible for the functional properties of breast milk.

Figure 8: Proportion of 2′-FL among HMOs in breast milk(Bode et al., 2012)

2'-FL is a key functional oligosaccharide in human milk. It is not digested by the infant's own enzymes but travels intact to the intestine. There, it functions as a crucial prebiotic, selectively promoting the proliferation of beneficial bacteria and thereby helping to establish a healthy gut microbiota balance. Concurrently, it acts as a "decoy," binding to and preventing pathogens from adhering to intestinal cells, which directly reduces the risk of infection. These combined actions support the maturation of the infant's immune system and overall health. These unique functions are precisely the central focus of current advanced research in infant formula(Weichert et al., 2013).

Figure 9: Functions of 2′-FL

However, current methods for obtaining 2'-FL still face significant limitations.

Presently, the primary production methods for 2'-FL include extraction from human milk, chemical synthesis, natural extraction, and enzymatic synthesis(Zhu et al., 2022). However, direct extraction from human milk is costly and not scalable, while chemical synthesis involves complex procedures and the use of toxic reagents. Glycosylated products naturally extracted from cow's milk have extremely low concentrations and significant structural differences, rendering them functionally inadequate. Although enzymatic synthesis can achieve higher purity under mild conditions, it remains constrained by the high cost of glycosyl donors and the reliance on highly efficient glycosyltransferases.

Figure 10: Challenges in Producing 2′-FL

Trypsin

Approximately 80% of the protein in infant formula is bovine casein. In the acidic environment of an infant's stomach, it forms dense, indigestible curds. Existing methods to improve casein digestibility, including physical, ultrasonic, and enzymatic treatments, are limited by nutrient loss, lack of precision, or poor compatibility with human milk proteins.

Figure 11: Challenges in the digestion of Casein

Our solution is to introduce Trypsin as a "protein digester." Trypsin is a highly specific serine protease, primarily secreted by the pancreas and activated in the intestine. It plays a critical role in the initial hydrolysis of proteins by specifically cleaving peptide bonds C-terminal to basic amino acid residues such as lysine (Lys) and arginine (Arg)(Holscher et al., 2018). This highly specific protease acts like a pair of precise "biological scissors" during the milk pre-treatment stage, breaking down the complex structure of casein into smaller, more easily absorbable peptides. In this way, we can fundamentally reduce the difficulty of protein digestion, making the formula gentler on the infant's gut.

Figure 12: The 3D structure of trypsin

β-Galactosidase

Lactose intolerance is a prevalent condition affecting approximately 70% of the global population and is particularly common among infants in Asia. These infants lack sufficient levels of the enzyme β-galactosidase, which is necessary to break down lactose(Vandenplas et al., 2013). Consequently, undigested lactose ferments in the intestine, leading to symptoms such as diarrhea, abdominal pain, and crying, which severely impacts their nutrient absorption and healthy development.

Figure 13: Lactose intolerance

Our solution directly addresses this enzymatic deficiency. We utilize engineered bacteria to produce β-galactosidase, which is used to pre-treat the raw milk during the formula manufacturing process. This enzyme efficiently hydrolyzes lactose into easily absorbable glucose and galactose. As a result, even infants with lactose intolerance can safely consume the formula, receiving its full nutritional and energy benefits without experiencing digestive discomfort.

Figure 14: Structure of β-Galactosidase

To address the core nutritional and functional challenges of current infant formulas, our team, "ProBabyotics," has utilized synthetic biology tools to design and construct a multifunctional biomanufacturing platform. This platform is based on the safe probiotic chassis, E. coli Nissle 1917 (EcN), and is designed to modularly produce the core functional components for a next-generation infant formula we have named "2'F-Luxe."

Our platform operates through the synergy of three key modules:

Module 1: Immune Enhancement (2'-FL): This module aims to efficiently synthesize the key human milk prebiotic, 2'-fucosyllactose (2'-FL), in our engineered bacteria to mimic the natural immune-protective functions of breast milk.

Module 2: Protein Hydrolysis (Trypsin): This module is designed to produce the specific protease, Trypsin, which will be used to pre-digest the large, indigestible casein macromolecules in milk into smaller, more easily absorbable peptides.

Module 3: Lactose Tolerance (β-galactosidase): This module focuses on producing β-galactosidase to pre-hydrolyze lactose during the production phase, thereby addressing the issue of lactose intolerance in infants.

Through these three modules, we can independently produce 2'-FL, Trypsin, and β-galactosidase. By integrating these components into the future manufacturing process for "2'F-Luxe," we aim to create a truly innovative infant formula that emulates the core advantages of human milk, offering enhanced safety and improved digestibility.

Chassis - EcN

To ensure the safety and efficacy of our solution, we have selected E. coli Nissle 1917 (EcN) as the chassis microorganism for our entire biomanufacturing platform. EcN is a probiotic with a century-long history of safe application and has recently emerged as a prominent strain for Live Biotherapeutic Products (LBPs), demonstrating immense potential in the field of synthetic biology(Sonnenborn et al., 2016).

Our choice of EcN as the chassis is based on several key advantages. Firstly, unlike most E. coli strains that release endotoxins (which can trigger inflammatory responses), EcN's endotoxin-free characteristic ensures a high biosafety profile for both the organism itself and its metabolic products—a critical factor for a chassis microorganism. Secondly, as one of the most well-characterized model organisms, EcN possesses a comprehensive and well-established genetic engineering toolkit, including a wide array of plasmid vectors and sophisticated expression control systems. This enables us to construct and optimize our target gene circuits with high efficiency and stability. It is upon these strengths that we are able to produce 2'-FL, Trypsin, and β-galactosidase in this safe and controllable chassis, providing a solid and reliable foundation for our project.

Figure 15: Escherichia coli nissle 1917

Modular Design and Engineering Cycles

We constructed three modules in our chassis microorganism for the production of 2'-FL, β-galactosidase, and trypsin, respectively.

Module 1: Immune Enhancement — High-Efficiency Biosynthesis of 2'-FL

To emulate the critical immune-protective function of human milk in our infant formula, we designed and constructed a biological system for the efficient de novo synthesis of 2'-fucosyllactose (2'-FL). The core of this system is an expression cassette containing five key enzyme-encoding genes, designed to convert common intracellular metabolites into the high-value product, 2'-FL.

1. Design of the Core Metabolic Pathway

Our design strategy was to reconstruct a complete metabolic pathway in our engineered bacteria, redirecting flux from central carbohydrate metabolism toward 2'-FL synthesis. This pathway begins with the endogenous metabolite mannose-6-phosphate and proceeds through a series of enzymatic reactions involving five key enzymes. These enzymes are encoded by the genes manB, manC, gmd, ful, and futC, and their specific functions are illustrated in Figure 16 below.

Figure 16: The genetic circuit diagram of the enzymes we use for 2'-FL product on

Figure 17: The system of 2'-FL

Figure 18: The function of each gene(Lee et al., 2012)

2. System Optimization through DBTL Cycles

Following the initial validation of our pathway's feasibility, we executed four successive rounds of the Design-Build-Test-Learn (DBTL) cycle to systematically optimize 2'-FL production. This iterative process was divided into two distinct phases:

First, the initial two DBTL cycles were dedicated to assembling and refining the core biosynthetic pathway. Through this process, we determined that the complete pathway, comprising the five genes manB, manC, gmd, ful, and futC, was essential for achieving the highest 2'-FL yield.

With this optimal gene set established, we conducted two subsequent DBTL cycles focused on further enhancing productivity. These efforts targeted remaining bottlenecks through two key strategies: (1) optimizing the chassis organism for improved metabolic flux and (2) introducing fusion tags to the enzymes to increase their stability and efficiency.

1st Iteration: Optimizing the Chassis to Preserve a Key Substrate

Learn & Design: Our analysis revealed that in the initial chassis, E. coli BL21(DE3), the endogenous β-galactosidase (encoded by the lacZ gene) was degrading the lactose we supplied as a substrate. This diversion of the precursor pool limited the final 2'-FL yield. Based on this finding, we decided to switch the chassis.

Build & Test: We transferred the entire synthesis system into the DH5α strain, which naturally lacks a functional lacZ gene. Since this strain cannot break down lactose, the supplied substrate was more efficiently channeled into 2'-FL synthesis, resulting in a significant increase in production.

2nd Iteration: Enhancing a Key Enzyme's Performance with a Fusion Tag

Learn & Design: Further analysis identified that the expression efficiency and stability of the final enzyme in the pathway, the human-derived FutC (α-1,2-fucosyltransferase), had become the new rate-limiting step. To enhance its performance, we devised a protein engineering strategy.

Build & Test: To overcome the expression bottleneck of the FutC enzyme, we fused a thioredoxin A (TrxA) tag to its N-terminus. The TrxA tag, known for its high-solubility properties, effectively prevented the misfolding and aggregation of the fused FutC protein within the cell. This allowed the majority of the FutC protein to exist in a soluble, active state rather than forming non-functional inclusion bodies. This design proved successful, leading to a significant boost in 2'-FL production.

Figure 19: DBTL Engineering Cycle Diagram

Through this series of rigorous engineering designs and iterative cycles, we successfully constructed an efficient and stable 2'-FL biosynthesis system, laying a solid foundation for its future application in our infant formula.

Module 2: Protein Hydrolysis — Production and Functional Verification of Recombinant Trypsin

To address the challenge of digesting the large casein macromolecules present in infant formula, we designed a biological system for the production of the specific protease, trypsin. Trypsin can precisely cleave casein, breaking it down into smaller, easily absorbable peptides, thereby reducing the digestive burden on infants.

1. Design Challenge and Solution: From Inactive Zymogen to Functional Protease

Expressing active trypsin directly in E. coli presents two major challenges. First, trypsin's potent proteolytic activity can lead to 'auto-digestion' or degradation of host proteins, resulting in failed expression or cell death. Second, its complex tertiary structure, which includes multiple disulfide bonds, is difficult to fold correctly in the reducing environment of the E. coli cytoplasm.

To overcome these challenges, we adopted a two-step strategy: safe intracellular production followed by in vitro activation.

Step 1: Safe Expression of Inactive Trypsinogen.

We chose to express the inactive precursor of trypsin, known as trypsinogen. Trypsinogen contains an N-terminal propeptide that 'seals' its catalytic active site, rendering it inert during intracellular expression. This prevents any toxicity to the host cell. We constructed the expression element shown in the figure below, utilizing the strong T7 promoter to drive high-level expression of the trypsinogen gene in E. coli.

Figure 20: The genetic circuit diagram of trypsin

Step 2: In Vitro Refolding and Autocatalytic Activation.

Due to high-level expression, the trypsinogen predominantly accumulated as inactive inclusion bodies within E. coli. We designed a precise in vitro processing workflow to "awaken" it:

Denaturation and Refolding: First, we used high concentrations of urea and DTT to denature and solubilize the inclusion bodies, fully unfolding the misfolded protein chains. Subsequently, we transferred the protein into a refolding buffer containing a redox pair (such as cystine/cysteine) via dilution. This process guided the protein chains to refold correctly, restoring their native three-dimensional conformation.

Autocatalytic Activation: The refolded trypsinogen was then placed in a slightly alkaline buffer containing Ca²⁺. The Ca²⁺ ions stabilize its conformation and induce a small number of trypsinogen molecules to undergo self-cleavage, removing the N-terminal propeptide and converting them into active trypsin(Zhao et al., 2015). This newly formed trypsin then rapidly catalyzes the activation of the remaining bulk of trypsinogen, creating a highly efficient positive feedback loop.

Functional Verification

To confirm that our recombinant trypsin possessed the expected catalytic activity, we performed an activity assay. The experimental results demonstrated that our prepared recombinant trypsin exhibited potent catalytic capability, confirming its potential to efficiently hydrolyze casein. This validates its suitability as an ideal "protein digester" for application in the production of infant formula.

Through this strategic approach of "safe intracellular expression, precise extracellular activation," we successfully overcame the core challenges of heterologous trypsin expression, providing a stable and highly efficient source of the functional enzyme for subsequent applications.

Module 3: The Lactose Tolerance Module - Building the β-Galactosidase System

The lacZ gene encodes β-galactosidase, which catalyzes the breakdown of lactose into glucose and galactose, enabling bacteria to utilize lactose as a carbon source(Panesar et al., 2006). To address the problem of lactose intolerance, we successfully cloned the lacZ gene from E. coli K-12 and constructed its expression system in a pET28a vector. This work lays a solid foundation for the future production of a functional enzyme preparation capable of degrading lactose.

Figure 21: The genetic circuit diagram of β -galactolactase

Our project extends beyond theoretical validation and laboratory results. We are committed to transforming the potential of "ProBabyotics" into a tangible solution that can genuinely improve the nutritional status of infants worldwide. To this end, we have developed a comprehensive implementation blueprint that spans from the user to the product and ultimately to industrial-scale production.

1. Target Users & Product Form

Our product, "2'F-Luxe," is an advanced infant formula developed in strict adherence to the global consensus that "breast milk is the gold standard for infant nutrition." Therefore, this product is not positioned as a substitute for breast milk, but rather as a scientific and safe nutritional support solution for infants when breastfeeding is not feasible or is insufficient.

Our core target users are families who are unable to exclusively breastfeed for specific reasons and who have a high demand for scientifically advanced infant nutrition. This primarily includes:

Families Facing Objective Feeding Challenges:

• Insufficient or Interrupted Milk Supply: This includes mothers with physiological conditions leading to insufficient milk production, those with specific illnesses requiring medication incompatible with breastfeeding, or those unable to consistently breastfeed due to objective reasons like work-related separation.
• Infants with Special Health Needs: This includes premature infants, those with specific metabolic disorders, or those who are intolerant to certain components in breast milk (though rare), all of whom require specially formulated nutritional support.
• Infants with Digestive and Absorption Issues: This is targeted at infants who exhibit symptoms of poor digestion, protein allergies, or lactose intolerance even when on mixed feeding or standard formula feeding regimens.

Our Ethical Commitment & Stance

We explicitly emphasize that "2'F-Luxe" formula is not intended for, nor do we encourage its use by, families who are capable of exclusive breastfeeding but choose not to for personal preference. Our mission is to leverage synthetic biology to provide a scientific, evidence-based nutritional solution that most closely mimics the functional benefits of breast milk for those families who have no other choice, thereby bridging the nutritional gap when breastfeeding is not an option.

Product Form and User Experience:

Our final product will be presented as a complete infant formula in a can, branded as "2'F-Luxe." It will feature the following characteristics:

Ease of Use: The can will include clear, illustrated instructions for preparation, ensuring parents can mix the formula with the correct proportions and water temperature for their baby.

Information Transparency: The packaging will clearly label the content of core functional ingredients like 2'-FL and pre-hydrolyzed proteins. It will also feature a QR code linking to our iGEM project wiki, educating consumers about synthetic biology and its applications in improving lives.

Assured Safety: All ingredients will comply with the infant food safety standards of various countries.

We have designed a user manual for the "2'F-Luxe" infant formula and milk for our target customers.

Figure 22: Instruction for milk powder

2. Industrial Manufacturing Blueprint

To advance "2'F-Luxe" from the laboratory bench to the market, we have designed a four-step industrial manufacturing process:

Step 1: Modular Engineering Strain Construction2'-FL Production Strain: The optimized 2'-FL synthesis operon (containing manB, manC, gmd, fcl, and TrxA-futC) will be stably integrated into the food-grade safety chassis, E. coli Nissle 1917.

Functional Enzyme Production Strains: Dedicated engineered strains for the high-level expression of trypsinogen and β-galactosidase will be constructed separately.

Step 2: Large-Scale Fermentation and Product SeparationThe three engineered strains will be cultivated in high-density fed-batch fermentations in large-scale bioreactors, using cost-effective carbon sources such as glycerol and lactose.

Through downstream processing techniques like centrifugation and filtration, the fermentation supernatant containing 2'-FL and the cell pellets containing the two target enzymes will be collected separately.

Step 3: Purification and Preparation of Functional Components2'-FL: High-purity 2'-FL powder will be obtained from the fermentation broth using purification techniques such as chromatography and crystallization.

Enzyme Preparations: The collected cell pellets will undergo lysis to release the intracellular enzymes. The trypsinogen will be processed through inclusion body washing, denaturation, refolding, and activation. Finally, both trypsin and β-galactosidase will be prepared as highly active enzyme formulations.

Step 4: Final Product Integration and Manufacturing

High-quality raw milk will be pasteurized.

Under mild conditions, our self-produced trypsin and β-galactosidase will be added sequentially to achieve precise pre-hydrolysis of the casein and lactose in the milk.

The high-purity 2'-FL powder, along with other essential vitamins and minerals, will be blended into the treated milk base.

The final "2'F-Luxe" infant formula powder, which is nutritionally complete and easy to digest and absorb, will be produced through processes like homogenization and spray drying.

Figure 23: Industrial-Scale Production Process

3. Safety and Future Application Considerations

Safety is the cornerstone of all infant products and the highest principle guiding our project design. Our solution incorporates meticulous safety considerations from the chassis selection to the final product form.

A Safe Biological Chassis and a Cell-Free Production Model

GRAS-Certified Chassis Strain: Our chosen chassis, E. coli Nissle 1917 (EcN), is a probiotic with a century-long history of safe use and is Generally Recognized as Safe (GRAS)(Magalhães et al., 2007). Its endotoxin-free nature provides a reliable safety foundation for the entire biomanufacturing process.

Principle of No Live Bacteria Addition: Our design strictly adheres to the principle of "component addition" rather than "live bacteria addition." In future industrial production, the final ingredients added to the infant formula will be highly purified 2'-FL, trypsin, and β-galactosidase, free from any viable engineered bacteria or their genetic material (DNA). This "cell factory" model, where the production organism is separated from the final product, fundamentally eliminates the risk of introducing genetically modified organisms (GMOs) into the food chain, ensuring the biological safety of the product.

4. Differentiated Product Line Strategy: Navigating Regulatory Requirements

Following consultations with legal experts, we learned that the addition of different functional components places a product into distinct regulatory categories. To ensure our technological advancements can benefit infants with diverse needs more quickly and broadly, we have planned two distinct product pathways:

Product Line 1: Functional Infant FormulaCore Component: Addition of 2'-FL synthesized via our biological method.

Regulatory Positioning: According to Announcement No. 8 of 2024 by the National Health Commission (NHC) of China, 2'-FL has been approved for use as a food nutritional fortifier. Therefore, an infant formula supplemented with 2'-FL can be registered as a conventional or functional food. This approval pathway is relatively mature, enabling a faster route to market to meet the widespread demand for immune enhancement in infants.

Product Line 2: Food for Special Medical Purposes (FSMP)Core Components: In addition to 2'-FL, the formula will be pre-treated with trypsin and β-galactosidase.

Regulatory Positioning: Because this product includes enzyme preparations with clear therapeutic functions—addressing specific medical issues like protein maldigestion and lactose intolerance—it will be classified as a Food for Special Medical Purposes (FSMP). Its development, production, and market launch will be subject to more stringent clinical validation and approval processes. This product line will precisely serve infants with special requirements for protein digestion and lactose tolerance, such as premature infants or those with allergies.

Through this dual-product-line strategy, we have not only ensured the overall safety of our project but have also charted a clear, pragmatic, and viable path for its future commercialization and differentiated market application.

Figure 24: The Two 2'-FL Products

1. Functional Mimicry, Beyond Basic Nutrition

The Status Quo: Conventional infant formulas lack Human Milk Oligosaccharides (HMOs), offering insufficient immune protection.

Our Advantage: Our system enables the high-efficiency production of 2'-FL. This key HMO actively modulates the gut microbiota and inhibits pathogen adhesion, providing infants with active immune protection that is analogous to that conferred by breast milk.

2. Precision Biotechnology to Solve Digestive Issues

The Status Quo: Physical or chemical methods used to treat milk proteins can degrade essential nutrients or leave behind unwanted chemical residues.

Our Advantage: We utilize biosynthesized trypsin and β-galactosidase to gently yet efficiently break down casein and lactose through highly specific enzymatic action. This approach ensures nutritional integrity while avoiding safety risks, precisely solving the critical challenges of protein indigestion and lactose intolerance.

3. Cost-Effectiveness to Advance Nutritional Equity

The Status Quo: Premium formulas supplemented with a single functional ingredient, such as chemically synthesized 2'-FL, are often prohibitively expensive, placing them out of reach for many families.

Our Advantage: Our synthetic biology platform utilizes inexpensive feedstocks like glycerol and lactose to achieve the scalable production of three functional components through microbial fermentation. This strategy fundamentally breaks down cost barriers. It makes it possible to produce a comprehensively functional, high-end formula that rivals breast milk at an affordable price, allowing its benefits to reach a much wider population of infants.

Figure 25: Project Advantage

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