Microbial biofortification is a sustainable approach to enhancing the nutritional value of foods using beneficial microbes that are responsible for the production of vitamins and nutrients during fermentation of the food. Probiotic and fermentative strains are able to produce some micronutrients such as folate (vitamin B₉), riboflavin (vitamin B₂), cobalamin (vitamin B₁₂) and menaquinones (vitamin K). This sets a transformation of rather common foods into products rich in nutrients (1, 2). The technique is of particular value in plant-based diets, in which these vitamins are often low and deficiency is widespread in many parts of the world. By calling upon microbe natural metabolism in the fermentation process, foods are enriched with increased vitamin content, more easily absorbed minerals, a higher level of proteins for digestion and bioactive molecules useful to human health (3).

Fermented foods are a major feature of world nutrition as they are staple foods in many areas of the world. Fermentation helps extend the shelf life by improving product stability, safety, and taste of the food and provides nutritional value. Across the world, large amounts of micronutrients are supplied by fermented dairy products (yogurt, kefir, cheese), cereals (sourdough bread, dosa), legumes (tempeh, miso), and vegetables (kimchi, sauerkraut) to millions of people. Starter cultures, or natural microbes, break down raw ingredients using enzymes, which increase vitamin production, eliminate anti-nutrients (such as phytates), increase mineral solubility (Figure 1) (2, 4). For example, fermented cereals can have riboflavin contents of 2.5–5 mg/kg and have had contents of 150–340 μg/100 g of folate, much higher than the content of unfermented cereals. Soy products that are fermented with Propionibacterium freudenreichii can contain 0.95–3.2 μg/100 grams of vitamin B12. Considered one of the best varieties is Bacillus subtilis natto, which produces up to 50–100 μg/100 grams of soybean consumers Vitamin K₂, vitally important for the health of bones and the heart. These benefits demonstrate the ability of fermented foods to fill micronutrient gaps, particularly in populations with limited access to nutrients by eating mostly plant foods (3, 5, 6).

Probiotics do more than just reside in fermented foods, they produce active vitamins this way and alter the nutritional profile of the food with the help of complex biochemistry. Strains of Lactobacillus, Bifidobacterium, Propionibacterium and Streptococcus thermophilus are highly renowned sources of B-group vitamins (i.e., riboflavin, folate and occasionally cobalamin) (4, 7). The synthesis of vitamin, probiotics have the effect to break down phytates which frees up the iron, zinc and calcium that are otherwise trapped into the plant cells. Their proteolytic activities enhance protein digestibility and provide needed lysine and other important amino acids to consume for those populations that may depend on mostly plant protein. Probiotics also help to support the gut health by balancing the microbiota and strengthening the intestinal barrier which increases the absorption and effectiveness of nutrients. Using probiotics for food production and as supplements are a natural and consumer friendly approach for food fortification (8, 9).

Despite promising advances in the use of probiotics to add nutrients to fermented foods, a number of research gaps make it difficult for probiotic use to become widespread. Strains like Propionibacterium freudenreichii and various lactic acid bacteria have been able to synthesize vitamins (vitamins B₂, B₉, B₁₂, and K₂) in food products, such as cheese, natto, and plant-based beverages (10). However, evidence that these vitamins are absorbed and have an impact is inconsistent, because most studies are small or short term and rarely are large and long term randomized controlled trials. Production also depends on the specific strain, fermentation conditions, food matrix and available precursors and there are hardly studies on the influence of individual differences such as the gut microbiota, genetic or health background, rendering personalized applications difficult. Additionally, the ongoing viability of probiotics is difficult during the large-scale production process, during storage and digestion (pH, oxygen, and bile) and competition from other microbes. Safety data on risks such as biogenic amines and infections, especially in vulnerable people, is also poor (11, 12).

New research is looking toward multi-omics techniques (e.g., genomics and metabolomics) for new vitamin-producing strains and for optimized mixtures of microbes and prebiotics such as inulin which can enhance stability and bioactive content. Precision fermentation with next generation probiotics, immobilization methods and sustainable ingredients (such as food by-products) are scalable solutions to adding nutrients. Longitudinal clinical trials are required to ensure benefit on immune function, metabolic health and nutritional security, however. Clear rules on approving the strains and health claims along with consumer education on the importance of the food matrix will help to get these products to market. These advances make enhancement of microorganisms with nutrients a sustainable alternative to a synthetic becoming (13, 14).

The review intends to provide an in-depth synthesis of the knowledge on microbial biofortification. It emphasizes metabolic pathways and genes involved in the production of vitamins by probiotic strains, the health benefits and effectiveness in practical use of these vitamins, and the real dictums of “bringing findings from living laboratories to a commercial product and public health intervention.” The functions of microbes in vitamin production the article covers strain specific capacity for production of folate, riboflavin, vitamin B₁₂, and vitamin K, considers how the production pathways in microbe are regulated and how they interact in microbial communities, and reviews the evidence from lab, animal and human studies, on the impact on nutrient status, gut health and immunity, of microbially produced vitamins. It also covers technological barriers, sensory barriers, and regulatory barriers, and also challenges in the development of new products and acceptance by consumers. Future directions presented in the review include genetic engineering of probiotic strains for enhanced vitamin production, personalization of nutrition through individual microbiome specific investigation, and implementation of these approaches in household structures of nutrition programs in the developing world. The interdisciplinary approach combines microbiology, nutrition and food technology to develop innovative and sustainable solutions to help reduce micronutrient deficiency around the world (1, 15).

Probiotic microorganisms enhance the nutritional value of fermented foods through a number of biochemical and physiological mechanisms (Figure 2). One of the important mechanisms is the endogenously production of vitamins in the fermentation. Some strains of probiotics, particularly lactic acid bacteria, such as Lactobacillus plantarum, Lactobacillus reuteri and bifidobacteria strain, contain the enzymatic pathway that is required to synthesize water-soluble B-vitamin complex including riboflavin (vitamin B₂), folate (vitamin B₉) and cobalamin (vitamin B₁₂). For example, lactic acid bacteria fermentation increases riboflavin contents of cereals and legumes by 2.5–5 mg/kg. Propionibacterium freudenreichii synthesize vitamin B₁₂ in soy or cereal at 0.95–3.2 μg/100 grams. Bacillus subtilis natto enhances vitamin k₂ in fermented soybean products, with reaching higher concentration (< 130–500 μg/100 g) (2, 16, 17). Some of these microbial vitamin productions contribute to the reduction of micronutrient deficiencies, especially in plant-based diets where vitamins are generally very low. Another important pathway in which probiotics increase nourishment is by increasing mineral bioavailability. During the process of fermentation, they form organic acids such as lactic and acetic acid, which lower the pH value of the food. This acidification results in the solubilisation of minerals like calcium, iron and zinc (18). Many strains also release an enzyme called phytase that degrades phytates, the substances in plants that bind minerals thereby reducing their absorption. The result is a significant reduction in phytate during the fermentation. Mineral availability is increased 10%−20% for calcium and 1.5–2.2 times higher when compared with the unfermented foods for iron and zinc. Fermented legumes and vegetables always demonstrate these enhancements, which means that probiotic–fermented foods are effective dietary sources that can be used to mitigate deficiencies in different populations (19, 20).

Probiotics also enhance the quality of the proteins by proteolysis. The lysosomes release proteases which cleave complex proteins into smaller peptides and free amino acids. This process increases essential amino acids such as lysine and leucine which are normally the deficient nutrient in cereals and legumes. In fermented chickpea flour lysine content increases from approximately 28.7 to 110.9 mg/kg; the nutritional value and digestibility of the protein is markedly increased. In the case of lentils the protein digestibility is also increased during the fermentation which affects from about 21.33 to 25.02%, which supports better nitrogen retention and utilization of nitrogen in the consumer (21, 22).

Functional metabolites are also produced during fermentation [gamma-aminobutyric acid (GABA), exopolysaccharides and short-chain fatty acids (SCFAs)]. GABA has neuroprotective benefits and blood pressure lowering benefits. SCFAs like acetate, propionate, and the butyrate, which are produced by the fermentation of the fibers that are prebiotic, are beneficial to the gut health and to the metabolism in general. The production of these compounds is dependent on the fermentation variables of pH, salt level and substrate. SCFA is at its peak when the dietary fiber content lies in the range of 2%−3% and pH lies in the range of 4.2 and 2% NaCl for maximum of GABA (23). In addition to synthesis of specific nutrients, probiotics improve nutrient absorption through the effects of probiotics on gut physiology and immunity. They exclude harmful pathogens, stimulate mucin production to strengthen the intestinal barrier and stimulate anti-inflammation cytokines. These effects help improve the environment of the gut for maximum absorption as a whole. Probiotic colonisationalso supports a balanced colonization of the microbiota, allows the metabolism of complex dietary compounds into absorbable forms and also boosts overall nutritional status. Quality and standardization of probiotic enhanced fermented foods is dependent on careful control of microbial strains, fermentation parameters and substrate composition in order to ensure the consistency of the nutrient enhancement. Standard values documented for nutrient enhancement include riboflavin levels ranging up to 2.5–5 mg/kg vitamin ruthenite irradiation, vitamin B₁₂ concentrations ranging from 0.95 to 3.2 μg/100 g, vitamin K₂ concentrations up to 100 μg/100 g, folate concentrations increased between 150 and 340 μg/100 g, calcium bioavailability increased by 10%−20%, and iron and zinc absorption increased from 1.5 to 2.2 times. Amino acid enrichment is also significant taking levels of lysine more than threefold higher in some fermented legumes. These values are benchmarks for researchers and industry stakeholders looking to develop functional foods with improved nutrient values (24, 25). Figure 3 depicts overall role of probiotics.

Probiotic strains, like bifidobacteria and other members of Lactobacillus, Bifidobacterium, Propionibacterium, and Streptococcus thermophilus are involved and indispensable for biofortification of fermented foods. They enhance these foods by creating vitamins, producing enzymes, and enhancing nutrient uptake. The outcome is increased vitamin content, better digestion, and increased absorption of minerals and proteins, which creates functional foods addressing micronutrient deficiencies. Lactobacillus types are the most versatile and best-used probiotics in this regard. The amounts they produce depend on the strain as well as the raw material (2, 17). In addition to vitamins many strains of Lactobacillus produce phytase, an enzyme that digests phytic acid that is a common antinutrient and in turn increases the availability of calcium, iron, and zinc. Their intensive proteolytic activity also contributes to the further improvement of protein quality, and essential amino acids such as lysine, leucine are liberated during fermentation. Because these functions are strain specific, manufacturer can develop a customized biofortification strategies for specific nutrients and food matrices (63, 64) (Figure 4).

Bifidobacterium species have a complementary relationship with Lactobacillus, particularly in the production of folate and the gut health. Bifidobacterium longum subspecies longum strain and Bifidobacterium breve have high folate enhancement rates for dairy and plant based fermented foods, giving 150–320 μg/100 gm; similar to Lactobacilli. They also produce organic acids, which cause a decreased pH in the food matrix, which lessens the solubility of minerals and degrades phytate levels. Although Bifidobacteria have less pronounced proteolytic activity than Lactobacillus, they favorably influence the bioavailability of nutrients indirectly, as they influence gut microbiota, leading to the increase in the absorption of nutrients produced in the gut. These synergistic roles highlight the key role of bifidobacterium in functional food biofortification (65, 66).

Propionibacterium species specifically Propionibacterium freudenrichii have become well-known as a source of vitamin B₁₂, one of the vitamins seldom created by other probiotics. This ability is important in the case of plant-based foods that lack natural sources of B₁₂. The strain can enrich soy beverages and cereals with 0.95–3.2 μg/100 gm which provides amounts of vitamin B₁₂ equivalent to recommended dietary allowances. Propionibacterium also produces propionic acid which further acidifies the food matrix and stimulates mineral availability. While their proteolytic and phytase activities are limited and lower than that of Lakobacillus, the importance of Propionibacterium in providing B₁₂ as well as enhancing mineral availability is of strategic significance in vegetarian and vegan populations (10, 67, 68). Streptococcus thermophilus is used extensively in the fermentation of yogurt and cheese. Although the level of vitamin production is very low in comparison to Lactobacillus and Bifidobacterium, it has a part to play in biofortification if used in mixed cultures with Lactobacillus delbrueckii subsp. Bulgaricus (68). This coaction results in greater production of folate and riboflavin. The acid produced by S. thermophilus decreases the pH, increasing solubility of minerals for absorption. S. thermophilus also has a moderate proteolytic activity and assets in the release of amino acids and bioactive peptides as a complement in the enhancement of nutrients. Its fast fermentation kinetics and adaptability to the substrate make it a useful starter culture for the improvement of the nutritional quality of dairy products (69, 70). Safety assessment frameworks used for engineered strains for microbial biofortification of fermented foods concentrate on risk-based assessments for the protection of human, animal and environmental health. These frameworks are based on QPS of EFSA and FAO/WHO guidelines. QPS applies to well-characterize taxa like Lactobacillus and bifidobacterium used for enrichment of foods with riboflavin or vitamin B₁₂. To qualify strains must have clear taxonomy, a safe historical record, no virulence of antimicrobial-resistance genes and prove acceptable for food use. Strains that do not conform with QPS criteria are whole genome sequenced for pathogenicity, toxin production, and assessment of the potential for horizontal gene transfer (71, 72).

Comparative studies have shown that those genera have unique and complimentary nutrient-enhancing capacities. Lactobacillus strains such as L. plantarum and L. reuteri are very versatile in their performances, secreting in large quantities B vitamins, phytase for mineral release and proteases that enhance the digestibility of proteins, with an increase of 10%−15%. Bifidobacterium is efficient in folate production and uses organic acids to mobilize minerals with its effects on gut microbial carrying over to improved absorption. Propionibacterium freudenreichii is specialized in vitamin B₁₂ synthesis assisted by propionic acid (68). Platelet-Rich Fibrin is an important plant-based biofortification microbe with a low proteolytic activity. Streptococcus thermophilus moderately synthesizes vitamins but is communal in the fermentation process so the production of nutrients is increased by synergistic activities. These genetic and metabolic characteristics differ for different strains. The genetic bases of these biofortification traits are well-characterized (73). Lactobacillus types have gene clusters for the synthesis of B group vitamins—e.g., folP and folA for folate and the rib operon for riboflavin. Many also have some enzymes such as phytase and protease to break down the antinutrients and proteins. Bifidobacterium strains possess a full folate pathway with the production of organic acids which modify the fermentation environment. Propionibacterium encode extensive clusters for cobalamin biosynthesis; more than 30 enzymatic steps are needed for active vitamin and for propionic acid production. Streptococcus thermophilus contains a smaller set of folate and riboflavin synthetic genes, acidification, and proteolysis. These genes can be associated with the measurable nutritional improvements associated with Lactobacillus; the increases have been measured as 2.5–5 mg/kg in riboflavin and up to 150–340 μg/100 grams in folate (74, 75). Excessive bifidobacterium imitation is creating similar increases in folate. Propionibacterium freudenreichii has the capacity to add 0.95–3.2 μg/100 g of vitamin B₁₂. All strains enhance calcium absorption by 10%−20% and enhance iron and zinc absorption by means of organic acid. By utilizing or co-culturing certain probiotic strains, manufacturers can use these complementary characteristics in creating functional fermented foods for micronutrient deficiency. This sustainable strategy addresses the need for more consumer-oriented natural foods with health promoting benefits and has potential to implement scalable, culturally acceptable nutritional interventions globally (68, 76). Genetically modified biofortificants e.g., propionibacterium genetically modified to produce propionate are assessed on a case-by-case basis using the frameworks established by the Office of Economic Cooperation and Development (OECD) and the Food and Safety Authority (EFSA). Key assessment elements include phenotypic stability, antibiotic resistance profiling as well as acute and chronic rodent toxicology. Rodent studies require NOAEs of >10⁹ CFU/kg. Allergenicity is anticipated by ensuring sequence homology with known allergen is less than 35% (71).

Fermented food matrices strongly influence the efficacy of probiotics to enhance the effects on nutrient and their availability and health benefits. The physical, chemical and compositional characteristics of each of the substrates determine how probiotics grow, metabolize, and synthesize nutrients during the fermentation process. Matrices include dairy, cereals, legumes, vegetables, and fruits each having different macronutrients, antinutrients, pH, water activity, and native microbes. Knowing the role of these characteristics in probiotic biofortification is essential for the adaptation of the nutrient enhancement strategy to various foods and diets (Table 4; Figure 5) (15). Dairy products like milk, yogurt and cheese offer protein-rich, buffered environments providing caseins, whey proteins, carbohydrates, lipids or minerals like calcium. These conditions are conducive to good probiotic growth and metabolic activity, and it is easy for the microbes to produce vitamins (e.g., riboflavin, folate), break down proteins into bioactive peptides and solubilise minerals. For example, the fermentation of milk by Lactobacillus helveticus increases bioactive peptide contents by as high as 300 mg/100 g, and increases folate to 200–350 μg/100 g. The high buffering capacity keeps the pH stable and favor microbial enzyme and nutrient synthesis. Calcium bioavailability is increased by 10%−20% in fermented dairy as a result of the action of casein phosphopeptides on calcium binding and intestinal absorption (77, 78).

Cereal and legume matrices, which include wheat, maize, millet, soy, chick pea and lentils, are primarily comprised carbohydrates with varying composition of proteins and fiber. Their complex structures can inhibit mineral absorption because their phytate, tannin and polyphenol content. Probiotic fermentation overcomes this by producing organic acids (reduced pH) and enzymes such as phytase, which breaks down 60%−80% of phytates to release minerals such as iron, zinc and calcium (79). Chickpea flour fermentation could increase lysine content from 28.7 to 110.9 mg/kg and increase protein digestibility by 10%−15%. There is also an increase in vitamin levels with riboflavin reaching 2.5–5 mg/kg and folate up to 340 μg/100 g in ferments cereals. The carbohydrate and fiber base works by stimulating the production of short-chain fatty acids which are responsible for supporting gut health as well as nutrient uptake (80, 81). Vegetable and fruit matrices differ as they are more acid (pH around 3–4), have high level of water contained, they are high in phytochemicals like antioxidants and phenolics. These characteristics provide selective environments that affect the survival and activities of probiotics. Fermenting cabbages or cucumbers with Lactobacillus plantarum and increases vitamin K₂ to 50–80 μg/100 g of cabbage and improves phytate breakdown which can double iron and zinc absorption organic acids produced during the fermentation process also contribute to solubilizing minerals and secretion of antioxidant metabolites provide additional benefit to the health. The low protein content that occurs naturally limits enrichment of amino acids compared to dairy and legumes (23, 81).

The microflora native to each matrix further determines biofortification results interacting in competitive or cooperative manners with supplemental probiotics. For example, spontaneous vegetable fermentations contain complex types of bacterial consortia which are able to complement the probiotic enzymes so that the degradation from phytate and the synthesis from vitamins will improve. In contrast, the pasteurized dairy permits control of probiotic dominance for practical nutritional enhancement. Substrate specific prebiotic compounds such as oligosaccharides in legumes favor the growth of bifidobacteria and synthesis of folate, and lactose in dairy products favor Lactobacillus growth and synthesis of riboflavin and subsequently metabolisms (23, 80). Fermentation conditions like the temperature, time, oxygen concentration, and the size of the inoculum interact with traits of the matrix and determine the final product that is biofortified. Optimal temperatures differ based upon matrix and strain but generally lie between 30 and 42 °C for the most efficient production of vitamins and proteolytic activities. Controlled anaerobic or microaerophilic conditions are optimal for phytase expression and organic acid formation which is important for mineral availability. Fermenting for longer periods of time in an alcoholic beer will increase the number of metabolites but may negatively affect sensory properties. Standardization of inoculum size promotes homogeneity in probiotic activity toward every pattern of release of nutrients by a particular matrix (78). Quantitative results are given to demonstrate the effects of matrices, riboflavin reaches 2.5–5 mg/kg in fermented cereals and dairy; folate, 150–340 μg/100 g in a series of legumes and sourdough bread; and at least 3.2 μg/100 g of vitamin B₁₂ in fermented soy and 50–100 μg/100 g of vitamin K₂ in vegetable and legume ferments. Mineral absorption enhancements related to matrix the bioavailability of calcium is increased by 10%−20%, while the bioavailability of iron and zinc are increased by 1.5–2.2 times in fermented cereals and vegetables. Amino acid enrichment is greatest in dairy and legume fermentations resulting in levels of essential amino acids doubling to quadrupling compared with the unfermented base (78, 81).

Microbes produce vitamins that have several health impacts on us as shown in Figure 3. These vitamins affect the micronutrient content, gut health and immunity. Studies in the lab, in animals and in humans demonstrate the role that microbe-made vitamins play in aiding body processes and preventing disease. They also demonstrate how these vitamins interact with our own flora to alter health outcomes. Cell culture models indicate how human cells absorb the vitamins produced by microbes, such as folate, riboflavin, vitamin B₁₂ and vitamin K (2). For example, some lactic acid bacteria have the ability to produce folate which assists in the synthesis and repair of DNA in cultured mammalian cells. Riboflavin by microbes enhances mitochondrial energy production by being FAD and FMN, essential redox cofactors. Microbial vitamin B₁₂, which is synthesized only by certain gut bacteria, is necessary for methylation reactions and blood cell production, which was confirmed when supplementation of cell cultures with B₁₂ improved hematopoietic cell lines. Vitamin K in particular is the bacterial form of menaquinone, which activates the enzyme gamma-glutamyl carboxylase that is required in blood clotting and bone metabolism. From in vitro experiments, greater carboxylation activity was evident when cells were exposed to microbial vitamin K (96, 97).

Animal studies provide more understanding due to simulations of complex host-microbe interactions. Germfree mice colonized with the vitaminproducers had higher levels of vitamin as compared to germfree controls demonstrating that the microbes directly contribute to the body's storage of vitamins. Mice colonized with the folateproducing Bifidobacterium had more folate in the blood and tissue, and this increased the speed of growth in the mice and enhanced brain function. Mice that were colonized with the vitamin B₁₂ producing bacteria Propionibacterium had greater haematocrit and fewer signs of anemia. Lactococcus strains producing riboflavin helped to increase antioxidant enzymes and reduce liver oxidative stress. Mice with bacteria which produced menaquinone-7 had a better calcium metabolism and bone density. These animals also displayed an improvement in immune balance and increased gut barriers, presumably due to immune cell cofactors such as microbial vitamins. However, the effects are not the same for all bacterial strains and microbial community, so not all microbes function in the same manner (98, 99).

These findings have been supported by human studies. Eating fermented foods rich in Vitamin LAB is associated with an improved status of micronutrients. One study found that consumption of probiotic drinks containing lactic acid bacillus capable of producing folate increased blood folate moderately and improved two markers of DNA methylation and cell division. Probiotics conferring a riboflavin production have been used to decrease the frequency of migraine, presumably by increasing mitochondrial energy generation (2, 100). Probiotic strains that produce vitamin B₁₂ are being tested in order to correct B₁₂ deficiency in the vegetarian population; preliminary trials indicate increased of B₁₂ in blood and improved nerve function. Foods high in vitamin K₂ or probiotics that produce vitamin K₂ have been linked to increased bone density and reduced calcification of arteries comparable to the function of vitamin K in carboxylation of proteins. Metagenomic analyses reveal that different gut enterotypes have different vitamin synthesis capabilities, which are related to vitamin concentrations found in body fluids. Yet age, genetics, where one starts and the diet affect how people will respond, and therefore individuals respond differently (101, 102). Microbial vitamins are an important contribution to the body's pool of vitamins, and in some cases 10%−50% of the daily requirement. Folate produced by the gut helps produce blood cells, brain function and the fetal. Microbial riboflavin helps keep the cells in balance with oxidation and an aid in the use of energy. Endogenous vitamin B₁₂ prevents pernicious anemia and relies on brain health. Vitamin K₂ is present in the intestine, which regulates clotting as well as bone turnover. When these vitamins are in deficit because of poor absorption and/or low diet and a brokendown vitaminproducing microbiome, people get anemia, nerve issues, osteoporosis and immune problems (103).

The gut microbiota helps to keep the gut lining strong, they provide the cofactors required for the DNA building blocks and cell growth (104). Folate impacts proteins which seal the gut cells making them less leaky and inflammatory. Riboflavin reduces oxidative stress in the gut and that protects cells against the action of bad microbes (105, 106). Vitamin B₁₂ promotes differentiation of gut cells and provides colon cell energy. Vitamin K₂ maintains the immune balance and barrier functionality in the gut, a microbiome that manufactures lots of vitamins is likely to be diverse and resilient, less favorable to harmful bacteria and inflammatory bowel disease risk is decreased. Thus, vitamin production by microbes directly supports the health of the gut as well as the rest of the metabolism (107, 108). Microbemade vitamins then also work in concert within the microbiome. Some types of bacteria produce vitamins which others lack, so there is a network of shareable nutrients. This cross feeding makes the community stronger and diversity high. Vitamins affect the communication of the bacteria such as quorum sensing and biofilm formation and also how bacteria communicate with their host immune system. In the body, microbe-vitamins are responsible for modulating the immune responses, for example vitamin B complex influences the growth of T-cells in the body among others and vitamin K stimulates enzyme activation for blood clotting and bone remodeling. The total supply of dietary and bacterial vitamins influences the immune tone i.e., making the barriers stronger and fighting infections. When vitamin production is cut and dysbiosis takes place these partnerships break down, leading to increasing the risk of inflammatory and metabolic disease. Therefore, vitamins, microbiota and immune system are a connected network necessary for health and disease prevention (109–111).

The use of vitamin producing microbial strains in food and pharmaceutical products has a number of critical challenges and limitations. These involve the viability and stability of strain during processing and storage, prevention of sensory changes that would deter consumers, and complex regulatory frameworks and standardization issues. One major issue is sustaining the viability and stability of the strains under processing, storage and distribution conditions. Microbial producers of vitamins such as folate, riboflavin, B₁₂ and K are commonly used in fermented products or probiotic products. Industrial conditions like heat, pH adjustments, oxygen, moisture and levels of nutrients can minimize strain survival and metabolic activity. Strains with a known biosynthetic ability for vitamin formation, for example Lactobacillus plantarum or species of Bifidobacterium, can be at risk of loss in the drying processes used e.g., in spray drying or freeze drying, if protective agents and process parameters are not optimized. Even the process of refrigeration can reduce the number of viable cells over time, so that after a food is consumed there are less able to produce vitamins. This loss of viability diminishes the effectiveness of both fortification in terms of effectiveness and the health benefits usually provided by probiotics in terms of viability, since active and metabolically competent cells must be present in order for vitamin synthesis to take place (112–114). In addition to these traits, the stability of the vitamin synthesis ability of the strain recognizes that there is variation, with some strains being better than others determined by genetic robustness and cellular stress responses to stay genes expressed and enzymes functioning better when under stress. Therefore, formulations may include cryoprotectants and microencapsulation and carefully controlled storage situations which increase complexity and cost. Batch to batch variability of microbial populations also makes it more difficult to achieve consistent biofortification levels for consumers. Sensory changes as a result of microbial fermentation are also one reason for a limited acceptance of the biofortified products by consumers. At the same time that metabolic activities promote vitamin elevation, they also form organic acids, volatile compounds, and peptides that change taste, aroma, texture, and appearance. For example, build-up of lactic acid will render food more action of proteases liberates peptides that can be bitter, in taste. Vitamin-producing strains such as Propionibacterium freudenreichii can create increases in B₁₂ content however generates propionic acid which produce cheesy or sharp off-flavors. Similarly, Bacillus subtilis natto is known to cause vitamin K₂ increase in fermented soy, but is nasty in terms of ammonia likeodors which may finish off palatability. These sensory characteristics, however, may be off-putting, especially when frozen foods are introduced into the market where they are less familiar. Balancing high synthesis of vitamins with low sensory influence requires a balanced choice of strains, adequate fermentation control can one working certain formulation strategies such as flavor masking or blending with other food ingredients. Acceptance studies on consumer taste indicate it rises according to the cultural background, familiarity with fermented foods and individual taste preferences. Thus, sensory optimisation remains a very important, yet difficult, component of successful vitamin biofortification (112, 115).

Stability losses during storage defeat the nutrient claims of microbial biofortification in fermented foods. These losses bring about reduced amounts of bioavailable vitamins, probiotics, and bioactive metabolites such as short chain fatty acids (SCFAs), and invalidates the original enrichment data. For instance, viable numbers of Lactobacillus and bifidobacterium are reduced one to two log CFU/gram over 4 weeks at room temperature. The decrease includes developments resulting from acidification, oxygen and a phage activity, and the count falls below the probiotic threshold of 10⁷ CFU/g. Vitamins are also degraded during storage. Riboflavin (B₂) and folate undergo a loss of 20%−40% of their content as a result of photo-oxidation and thermal lability, respectively. Provitamin A carotenoids in pearl millet ferments reduced by 30% after 28 days at 25 °C as compared with less than 15% loss when kept at refrigerated temperature. The efficacy reduction in in vivo rodent forms is proven (1, 116). Stored biofortificants elicit 25%−35% less caecal butyrate and have less shifts in clostridial, which are anti-inflammatory benefits reduced compared to fresh products. Regulatory claims must therefore incorporate a shelf life validated for stability, using methods such as using a highway to inspect the stability using, for example, the use of the techniques of the Human serum samples both techniques include monitoring of the stability of the product to verify that it provides the nutrients it claims to. Degradation destroys bioavailability and health results. Microencapsulation or lyophilization are means of decreasing such losses to as much as over 85% of activity, and support credible labeling (117).

Regulatory frameworks and standardization are also major challenges. Microbial vitamin biosynthesis and use of probiotics are covered by differing regulations in different jurisdictions, which impact on claims, labeling, safety evaluation, and manufacturing standards. Agencies like the US FDA, EFSA in Europe and others in the national authorities require the demonstration of clear evidence of safety, efficacy and quality control for using probiotic and fortified products. Vitamin production by microbes raises some unique concerns for the following reasons genetic and metabolic diversity of strains require a comprehensive characterization of biosynthetic gene clusters, demonstration of the absence of virulence factors or transferable antibiotic resistance and stability of functional characters (59, 112). Standardizing the vitamin content is challenging due to variability in strains, the dynamics of the fermentation process and the effect of the matrix, which in turn makes quality control challenging, as well as maintaining consistent dosing. Lack of harmonized standards for the labeling of microbial vitamin producers and their health claims are additional sources of regulatory complexity and can be confusing for the consumer. For example, the concentration range limits and methods for validating vitamin concentrations on a case-by-case basis vary depending on the country where products are sold, and legislations related to novel foods or supplements may put restriction on genetically modified or commonly novel microbiotabased products. Achieving regulatory approval requires comprehensive documentation of stakeholders such as strain identity, stability, bioavailability of vitamins produced and clinical proof of health benefits an extensive research and product development burden. Intellectual property issues, patents on the strains, etc., and proprietary fermentation processes also affect the access to the market and innovation. This fragmented regulatory landscape makes it hard for technologies for biofortification to be widely adopted commercially and makes scaling biofortification technologies more difficult around the world (113, 114).

In microbial vitamin biosynthesis, the future of microbial vitamin biosynthesis is to make use of advances in genetic engineering, personalized nutrition and publichealth strategies. These advances will be supported by interdisciplinary collaboration, to overcome current limits; and to maximize actual impact. Engineered strains of probiotics are a promising solution to make more vitamin production. By adding or refining pathways to synthesize folate, riboflavin, vitamin B₁₂ and vitamin K, scientists have been able to create “super – probiotic” strains which produce stable, strain-specific vitamins even when being industrially fermented and even within the gut. Technologies such as CRISPR-Cas-mediated genome editing, synthetic biology, pathway modularisation to insert delete control biosynthetic gene cluster This would mean a balance between the synthesis of the vitamins and the health and fitness of the strain (118). Engineered microbes can also be programmed to detect signals from the environment or the host and begin producing vitamins only when needed, thereby improving on bioavailability and reducing waste anyhow. By taking this genetic approach, problems of varying vitamin output and stability through processing are addressed, and is able to provide high yield probiotic products, both tailored for particular dietary or for therapeutic implementation. Integration of microbial vitamin biosynthesis with personalized nutrition and microbiome profiling is another way forward. Highresolution sequencing and metagenomic analysis to map their gut bacteria and its functional capacity for each person reveals the gaps in vitamin producing microbes that result in micronutrient shortfalls. Personalized diets or probiotic treatments can then be formulated to either restore or enhance those lost functions to improve their micronutrient status in accordance with a person's genetics, lifestyle and health profile. Advanced bioinformatics and machine learning predict the optimal microbial communities as well as vitamin dosages of the single individual. In addition, the probiotics can be combined with prebiotics and dietary fibers that selectively feed these microorganisms, making tailor-madesynbiotic formulations, which then can be used in addition. This precisionnutrition model promises to provide precision prevention and treatment of vitamin deficiencies and vitamin disorders, allowing for better efficacy without having to give vitamin supplementation unnecessarily (119, 120).

Benefits and challenges of microbial vitamin biosynthesis in low resource and public health settings growing microbial biofortification of vitamins in low resource and public health settings is a global priority. Micronutrient deficiencies impact millions of people, particularly those in underserved populations who are based on plant-based staples poor in vitamins, such as B₁₂ and K. Development of low-cost and culturally acceptable fermented foods and probiotic products enriched by microbial vitamin manufacture may sustainably address these nutrition gaps. The use of locally grown fermented systems containing vitamin producing strains adapted to local conditions, is a cost-effective, accessible alternative to the synthetic supplements. Education and capacity building programmes empower the communities to use microbial biofortification for better nutrition (2). Microbial synthesis of vitamin can also be incorporated into emergency nutrition and maternal and child health interventions, lowering illness related to vitamin deficiency. Overcoming challenges in the areas of production, storage and taste in these contexts requires constant innovations and inclusive policies, which support technology transfer and regulatory harmonization. Realization of these future directions requires research across the boundaries of microbiology, genomics, food science, nutrition, clinical medicine and regulatory science. Collaboration is important for understanding microbe-hostnutrient interactions, building robust engineered strains, optimizing fermentation and conducting rigorous clinical potentiation and clinical trials for efficacy proof of concept. Systems biology and metabolic modeling will make the optimisation of strains more efficient. New delivery methods (e.g., microencapsulation) enhance the survival of probiotics and vitamin stability. Standards, safety checks, and consumer engagement have to keep pace with the level of product quality and acceptance. Public-privates and global consortia can be used to help accelerate the pathway from research to market and to public health impact. Holistic interventions that integrate the microbial production of vitamins with more general nutritional interventions may holistically address hidden hunger (114).

Microbial biofortification through the biosynthesis of vitamins by probiotics is a sustainable and natural approach for enhancing micronutrient nutritional status on a global scale. The microbial production of essential vitamins such as folate, riboflavin, vitamin B₁₂ and vitamin K is an important part of the blend of enhanced nutrition that can be obtained from the widespread consumption of fermented foods which can provide fortified nutrient profiles without disrupting cultural food traditions. Advances in the understanding of metabolic pathways and strain-specific biosynthetic abilities is the reason for being able to acquire targeted vitamin increases in diverse foods. Research from in vitro to human findings has confirmed that the bio-efficacy of dietary microbe sources vitamins is important in improving micronutrient status, gut health and immune function. Nonetheless, challenges remain in ensuring strain stability and viability during processing and storage, managing sensory attributes to maximize consumer acceptability and navigating complex regulatory frameworks that influence standardization and product claims. Infuture highlight the possibility of advancing biosynthesis in probiotic strains by genetic engineering, personalized nutrition strategies by micropore profiling and implementation for low resources by socially acceptable fermented foods. These efforts require an interdisciplinary, collaborative approach that requires the interaction of microbiology, food technology and nutrition science as well as public health policy.