A Mineral Boost Could Transform How Probiotic Bacteria Ferment Milk
A carefully chosen mineral combination has dramatically improved the growth and acid production of a probiotic bacterium in milk, while also reshaping hundreds of small molecules produced during fermentation. The finding, reported for the strain Lactiplantibacillus pentosus 9D3, suggests that a relatively simple change in a fermentation recipe could help manufacturers produce functional dairy foods more efficiently. The researchers found that adding manganese and magnesium to milk enabled the bacterium to reach nearly four times the cell increase observed in unsupplemented milk, while lowering the milk’s pH to a level typical of yogurt-style drinks. The study used genome-guided nutrient selection and two complementary high-resolution mass-spectrometry techniques to reveal how the bacterium adapts to the chemically demanding milk environment.
L. pentosus is a lactic acid bacterium found in fermented foods, plant materials and the gastrointestinal tract. It has attracted attention as a potential probiotic starter because some strains can produce lactic acid, antimicrobial compounds and gamma-aminobutyric acid, or GABA, a bioactive molecule involved in nervous-system signaling. Strain 9D3 was originally isolated from Thai fermented spider weed and has previously shown promise in plant-based fermentation. Yet transferring a plant-associated bacterium into milk is not straightforward. Milk contains abundant protein, lactose, fat and minerals, but it does not necessarily provide every vitamin or cofactor that a fast-growing microbial cell needs in the right amount or form. In ordinary milk, the researchers observed only a modest rise of about 0.5 logarithmic units in viable bacterial counts after 24 hours, far below the growth achieved in a nutrient-optimized laboratory medium.
To identify possible nutritional bottlenecks, the team first examined the complete genome of strain 9D3. The genome contains 3,356 protein-coding genes, with 1,883 assigned functions through the Kyoto Encyclopedia of Genes and Genomes, or KEGG, a database used to connect genes with biochemical pathways. The analysis indicated that the bacterium had incomplete predicted pathways for vitamins B2, B3, B6, B7, B9 and B12. These vitamins can act as cofactors, the molecular helpers required by enzymes to transfer electrons, rearrange chemical groups or build essential cellular components. The researchers also selected manganese, magnesium, iron and zinc because these ions are associated with metal-dependent enzymes and other functions in lactic acid bacteria. The strategy was not to add every possible nutrient indiscriminately, but to match missing or potentially limiting functions in the genome with the known chemistry of milk.
The vitamin experiments produced a surprisingly restrained response. Adding riboflavin, pyridoxine, biotin, folic acid or cyanocobalamin produced little meaningful improvement in either bacterial counts or acidification over the tested range. Niacinamide, the form of vitamin B3 used in the experiments, actually became mildly inhibitory at higher concentrations. At 10 milligrams per liter, the final bacterial count fell to 7.18 log colony-forming units per milliliter, compared with 7.54 in unsupplemented milk, and the final pH remained relatively high at 6.06. The researchers suggest that the strain’s natural ability to make or recycle some vitamins, combined with the vitamins already present in milk, may have been sufficient for short-term growth. Excess B3 could instead disturb redox metabolism, the network of reactions that balances electron transfer inside the cell. The result highlights a crucial point for food biotechnology: a nutrient predicted to be missing from a genome is not automatically a nutrient that must be added to the food.
The minerals produced a much stronger effect, particularly manganese. Supplementing milk with 10 to 50 milligrams per liter of manganese ions increased bacterial growth and acidification in a dose-dependent manner. At 50 milligrams per liter, the culture reached 8.63 log CFU per milliliter after 24 hours and reduced the pH to 4.78. Higher manganese levels continued to lower the pH slightly, reaching 4.68 at 200 milligrams per liter, but did not significantly increase the final number of viable cells. Magnesium showed a similar but somewhat weaker pattern. The best result came at 100 milligrams per liter, where the culture reached 8.42 log CFU per milliliter and a pH of 4.87. Iron and zinc, by contrast, had little effect under the conditions tested, and higher iron concentrations reduced growth. These differences indicate that the bacterium was not suffering from a general shortage of trace elements. Instead, its performance in milk appeared to be constrained by particular mineral requirements.
Manganese and magnesium were then combined to test whether their effects would complement one another. Manganese is involved in bacterial defenses against oxidative stress and supports the activity of several enzymes, while magnesium stabilizes ATP and nucleic acids and participates in enzyme activation and carbohydrate metabolism. In the fortified milk, the researchers used 50 milligrams per liter of manganese and 100 milligrams per liter of magnesium. They tested inoculum levels ranging from 2 to 10 percent by volume and found that a 6 percent inoculum provided an efficient balance between starter culture use and cell multiplication. After 24 hours, the culture reached 8.72 log CFU per milliliter, with a net increase of 1.87 logarithmic units and a final pH of 4.54. Increasing the inoculum to 8 or 10 percent pushed the final count only slightly higher, to 8.81 and 8.89 log CFU per milliliter, while the net population increase barely improved. The likely explanation is that dense cultures rapidly consume available nutrients and accumulate acids or other inhibitory products, limiting further proliferation.
The researchers next asked whether the mineral boost changed only the number of bacteria or also the chemistry of the fermented milk. They compared three groups: unfermented milk, fermented milk without mineral supplementation and fermented milk containing manganese and magnesium. The team combined gas chromatography coupled to high-resolution accurate-mass mass spectrometry with ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. The two platforms detect different chemical families. Gas chromatography is particularly useful for volatile or chemically derivatized small molecules, while liquid chromatography can separate a broader range of polar compounds, lipids and other metabolites before they enter the mass spectrometer. Together, the methods identified 299 metabolites after duplicate signals were removed. Only 89 were shared across all three groups, while each treatment also contained unique compounds, indicating that fermentation and mineral supplementation produced distinct biochemical fingerprints.
Statistical analysis revealed clear separation among the three milk groups in principal-component analysis, a technique that compresses complex datasets into a smaller number of dimensions while preserving the largest sources of variation. Fermentation altered amino acids, peptides, carbohydrates, organic acids, fatty-acid derivatives, glycerophospholipids and nucleotide-related compounds. The mineral-fortified milk showed its own pattern, including changes in lipid-associated metabolites such as glycerophospholipids and fatty-acid derivatives. Some compounds, including norvaline and hydroxytetradecanoyl-CoA-related molecules, were more abundant in fermented samples, whereas compounds such as galactose 1-phosphate and several sugar-associated intermediates declined. These shifts are consistent with the bacterium consuming lactose-derived sugars, breaking milk proteins into peptides and amino acids, and redirecting carbon through energy-generating and biosynthetic pathways. Pathway analysis pointed to purine metabolism, pyrimidine metabolism, galactose metabolism, propanoate and butanoate metabolism, amino-sugar pathways, fatty-acid processing and the citrate cycle.
The results do not yet prove that the altered metabolites make the milk healthier, tastier or more beneficial to people. Many detected compounds could not be assigned confidently to established chemical classes, a common limitation of untargeted metabolomics because databases do not contain every microbial transformation product. The pathway analysis also relied on a human pathway library because microbial databases provide limited coverage for complex fermented-food samples. In addition, the experiments used one bacterial strain, one milk substrate and laboratory-scale conditions. The researchers did not directly measure fermentation speed, sensory quality, shelf life, post-acidification or health effects in animals or humans. Even so, the study offers a striking demonstration of precision fermentation: genome information can identify plausible nutritional weaknesses, controlled supplementation can improve microbial performance, and high-resolution chemical profiling can reveal the metabolic consequences. Before the approach reaches commercial products, those consequences will need to be tested through targeted metabolite measurements, molecular experiments, storage studies and clinical or functional evaluations.
Subject of Research: Genome-guided mineral supplementation and metabolomic adaptation of Lactiplantibacillus pentosus 9D3 during bovine milk fermentation
Subject of Research: Chemistry
Article Title: Integrated GC-HRAM-MS and UHPLC-QTOF-MS metabolomics reveal mineral-induced metabolic adaptation of Lactiplantibacillus pentosus 9D3 during milk fermentation
Article References: Tunsagool, P., Kemsawasd, V., Kwandee, P., Popluechai, S., Whanmek, K., Intawong, S., Phucharoenrak, P., Temviriyanukul, P., & Kittibunchakul, S. (2026). Integrated GC-HRAM-MS and UHPLC-QTOF-MS metabolomics reveal mineral-induced metabolic adaptation of Lactiplantibacillus pentosus 9D3 during milk fermentation. Food Chemistry: X, 38, Article 104329. https://doi.org/10.1016/j.fochx.2026.104329
Image Credits: AI Generated
DOI: 10.1016/j.fochx.2026.104329
Keywords: Lactiplantibacillus pentosus, milk fermentation, manganese, magnesium, probiotic bacteria, metabolomics, precision fermentation, lactic acid bacteria
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SCIENMAG. (August 28, 2026). Metabolomics Reveals Mineral-Driven Adaptation in Lactiplantibacillus pentosus During Milk Fermentation. https://scienmag.com/metabolomics-reveals-mineral-driven-adaptation-in-lactiplantibacillus-pentosus-during-milk-fermentation/
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Tags: acid production and pH regulation in probiotic fermentationfunctional dairy food productionfunctional dairy foods developmentGABA biosynthesis in fermented foodsGABA production in fermented milkgenome-guided nutrient optimizationgenome-guided nutrient selection in fermentationhigh-resolution mass spectrometry in dairy researchhigh-resolution mass spectrometry in microbial studiesimpact of manganese and magnesium on probiotic growthLactiplantibacillus pentosus metabolic adaptationmetabolomics analysis of milk fermentationmetabolomics in dairy sciencemilk fermentation optimizationmineral supplementation in dairy fermentationplant-derived probiotic strains in dairy applicationsplant-derived probiotics in milk fermentationprobiotic bacteria acid productionprobiotic bacteria milk fermentationprobiotic fermentation enhancementprobiotic strain 9D3 from Thai fermented foodsrole of manganese and magnesium in probiotic growth
