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Frontiers in Drug Discovery ·

Volume 6 - 2026 | https://doi.org/10.3389/fddsv.2026.1856085

Minireview Kefir as a source of antimicrobial compounds against bacterial pathogens

  • Silvia Tenorio-Salgado1*corresponding author
  • Alejandra Sánchez-Canché1
  • Ernesto Perez-Rueda2*corresponding author
  1. 1Tecnológico Nacional de México, Instituto Tecnológico de Mérida, Merida, Yucatan, México
  2. 2Unidad Académica del Estado de Yucatán, Mérida, Yucatán, Mexico
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Abstract

Milk kefir is a fermented drink traditionally consumed by humans for approximately 3,000 years, shaped by a bacterial and yeast consortium, with probiotic capacity. During the fermentation process, the microbial community synthesizes a broad-spectrum of compounds working synergically, as organic acids, bacteriocins, exopolysaccharides, and other bioactive metabolites that inhibit diverse bacterial or fungal pathogens like Salmonella typhimurium, Pseudomonas aeruginosa or Fusarium oxysporum. These compounds are mainly produced by the kefir’s lactic acid bacteria and modifies the environment and disrupt pathogen cell membranes or promote pores into the cell walls in pathogenic bacteria, among other mechanisms. Therefore, in this review, we explore the antimicrobial bioactive compounds produced by the kefir bacteria and associated yeast, such as organic acids, bacteriocins, bioactive peptides, and exopolysaccharides, among others, not only acting as individual molecules but also as results of a complex combination of metabolic products.

Keywords

1 Introduction

The use of milk fermented products has been known to humanity for centuries, dating back to the Bronze Age (Liu et al., 2024). In the last decade, research focused on various fermented food products has increased to identify compounds for health benefits, such as yogurt, kefir, kimchi, and sauerkraut. In this context, milk kefir is a fermented product characterized by a complex microbial consortium, in which lactic acid bacteria, acetic acid bacteria, and yeasts are the predominant groups (Pogačić et al., 2013; Rosa et al., 2017; Zhou et al., 2009). However, the dominant bacterial populations vary as a function of the fermentation substrate, culture maintenance method, geographic and climatic conditions, cultural practices, and the type of milk used (Marsh et al., 2013; Prado et al., 2015). Milk kefir has been associated with reduction of blood pressure (Klippel et al., 2016), immunoregulation (De Montijo-Prieto et al., 2015), and antiallergic, antitumoral, antimicrobial, and anti-inflammatory effects (Azizi et al., 2021; Baars et al., 2023; Santini et al., 2020). During kefir fermentation, the bacterial strains in the grains produce a wide diversity of bioactive compounds, which are proposed to confer its health benefits. One mechanism supporting these benefits is antimicrobial activity that could derive from different mechanisms, including organic acids, bioactive peptides, bacteriocins, and exopolysaccharides (Rosa et al., 2017). Therefore, in this mini review, we address the shortfall about the microbial composition of kefir grains, the main bioactive compounds produced, and health effects. We also, explore the antimicrobial bioactive compounds produced by the kefir microbiome, bacteria, and associated yeast, such as organic acids, bacteriocins, bioactive peptides and exopolysaccharides, among others, not only acting as individual molecules but also as result of a complex combination of metabolic products.

2 Microbial composition of milk kefir grains

The microbial diversity of kefir grains has identified over 50 distinct microbial species (Bourrie et al., 2016; Nejati et al., 2020; Prado et al., 2015), including multiple species of lactic acid bacteria (LAB), such as Lactobacillus helveticus, L. kefiranofaciens, Lactococcus lactis; non-LAB bacteria, as Acetobacter sp, Bifidobacterium sp, Streptococcus sp, and a broad array of yeasts, as Kluyveromyces marxianus, Candida spp., and Kazachstania spp., among others (Marsh et al., 2013). In this context, Walsh et al. (Walsh et al., 2023) analyzed 64 kefir grains sourced from 25 different countries, identified a consistent pattern of domination by representatives from the species L. helveticus or the sub-species L. kefiranofaciens subsp. Kefiranofaciens, L. lactis subsp. Lactis or L. cremoris subsp. Cremoris in each grain kefir (Walsh et al., 2023). Therefore, the presence and relative abundances of Bacteria and Eukarya in the consortia influence the production of more bioactive compounds, such as has been also reported in water kefir, where the coculture between L. kefiranofaciens and Saccharomyces cerevisiae enhances the production of kefiran, a polysaccharide with antimicrobial activity (Cheirsilp and Radchabut, 2011). In summary, this bacterial and eukaryal heterogeneity highlights the dynamic and adaptable nature of the kefir grains and underscores the necessity of detailed taxonomic analysis to fully characterize the composition and functional properties of this fermented product. Figure 1a.

Antimicrobial properties identified in kefir milk grains. **(a)** bacterial and eukaryal organisms identified in metagenomic analysis); **(b)** Non-ribosomal peptides and polyketides (plipastatin, fengycin, and bacillaene; **(c)** Exopolysacharides; **(d)** Antimicrobial peptides; and **(e)** organic acids (lactic acid, acetic acid and butyric acid).
Figure 1. Antimicrobial properties identified in kefir milk grains. (a) bacterial and eukaryal organisms identified in metagenomic analysis); (b) Non-ribosomal peptides and polyketides (plipastatin, fengycin, and bacillaene; (c) Exopolysacharides; (d) Antimicrobial peptides; and (e) organic acids (lactic acid, acetic acid and butyric acid).

In summary, kefir grains contains a large bacterial and yeast symbiotic community, that may remain relatively stable when properly maintained (Magalhães et al., 2010); however, its composition, as well as the resulting metabolic and volatile-compound profiles, are influenced by the grain’s origin and the processing parameters, among other conditions (González-Orozco et al., 2022). In this regard, kefir grains modulate microbial competition through both direct and indirect mechanisms. For example, Lactobacillus kefiranofaciens and Lentilactobacillus kefiri, inhibit bacterial pathogens such as Listeria monocytogenes, and S. typhimurium (Likotrafiti et al., 2015; Zanirati et al., 2015). Furthermore, in vitro and in vivo studies have validated and corroborated the antimicrobial potential of kefir and its components; such as kefir-derived Lactobacillus strains, including L. casei 17U, and L. kefiranofaciens 8U inhibit multiple pathogens, including Pseudomonas aeruginosa and L. monocytogenes among others (Zanirati et al., 2015); whereas kefir microbial mixtures provided protection against enterocolitis, diarrhea, and epithelial invasion by pathogens (Bolla et al., 2016). Therefore, kefir’s antimicrobial activity is the sum of the synergistic effects of low pH, organic acids, short chain fatty acids production, bioactive peptides, and exopolysaccharides (Tingirikari et al., 2024).

3 Main bioactive compounds detected in milk kefir grains

During milk kefir fermentation, the action of microbiota facilitates the hydrolysis of milk macromolecules, transforming them into metabolites with functional activity. While the final composition is subject to diverse components, such as cell envelope proteinases of microorganisms, especially the Lactobacillus, that catalyze the first step of hydrolysis of milk proteins into peptides like casein (Meltretter et al., 2008; Solieri et al., 2018; Wang et al., 2023); matrix flora, mainly associated with the presence of Lactic Acid Bacteria that consume lactose and produce lactic acid, drive the pH of the milk steadily downward, and yeasts, molds, and non-starter LAB; and acid-induced cleavage, that cause structural changes and peptide bond hydrolysis of the protein matrix, triggered by acidification, releasing bioactive peptides. Therefore, an average of 700 metabolites has been identified after fermentation (Cui et al., 2023; Sánchez-Rodríguez et al., 2024). In this regard, an exhaustive literature lookup revealed a high abundance of bioactive compounds, such as kefiran, bacteriocins, antibiotics, peptides and organic acids, associated with milk kefir grains. Table 1.

TABLE 1. Main bioactive compounds identified in kefir.

CompoundTypeSourceTargetMechanismEvidenceReferences
KefiranExopolysaccharidesLactobacillus kefiranofaciensStreptococcus pyogenes, P. aeruginosa, E. coli, S. aureus, and S. typhimuriumDisruption of the cell membrane through induced pore formationin vitro by using lipid membrane modelsBarbosa et al., 2011; Georgalaki et al., 2021; Ghasemlou et al., 2012; Zajšek et al., 2013
BacteriocinsBacterial peptides ribosomally synthesizedGram-positive bacteria: Lactobacillus sp, Lactococcus spK. pneumoniae, C. difficile, S. aureus, L. monocytogenes, P. aeruginosa, S. enteritidis, E. coli, B. cereus, Enterococcus fecalis, Cronobacter sakazakiiPores in the membrane, inhibition of cell wall synthesis (e.g., nisin), and interference with nucleic acid or protein synthesis (e.g., Lactocin 27)In vitro studies; metagenomic evidence in kefir (genes for helveticin, zoccin)Powell et al., 2007; González-Orozco et al., 2023; Upreti and Hinsdill, 1975; Martínez et al., 2008; Cole et al., 2025; Tian et al., 2026
PeptidesSmall peptides (6–50 residues)Milk kefir Turkey and BrazilE. coli, K. pneumoniae, P. aeruginosa, B. cereus, B. subtilisPhysical disruption of the bacterial membrane (multi-target)Inhibition assays of pathogenic bacteria; active peptides (MALDI-TOF); 16 bioactive protein sequencesDalabasmaz et al., 2023; De Lima et al., 2018; Ebner et al., 2015
Organic acidsShort- and long-chain carboxylic acidsLactic bacteria: Lactobacillus spp., Bifidobacterium spp., Lactisaceibacillus casei, L. coryniformis, L. plantarumS. arizonae, S. typhimurium, E. coli, Penicillium expansum, Aspergillus parasiticus, and Candida sppH decrease, passive penetration of the undissociated form, cytoplasm acidification, cation chelation (Ca2+, Mg2+), and ATP depletionLactic acid concentrations of in kefir supernatant with antagonistic effectGut et al., 2022; Kök Taş et al., 2025; Chen et al., 2021, Gamba et al., 2015

In the following, we describe the most abundant compounds identified in diverse milk kefirs, and their potential to be used as novel antimicrobial molecules.

3.1 Bacteriocins

The antimicrobial activity of kefir grains against pathogenic bacteria and some fungi is associated with the production of diverse compounds, as bacteriocins. In this regard, bacteriocin producers identified in the consortium are mainly from the LAB group, such as Lactobacillus plantarum or Lactococcus, isolated from milk kefir grains. Bacteriocins are bacterial peptides ribosomally synthesized. In general, they are small cationic molecules between with molecular weight less than 5 kDa to >30 kDa, and many of them exhibit extensively post-translationally modified structures encoded on complex gene clusters, whereas others have simple linear structures (Dillen et al., 2026; Kumariya et al., 2019; Sugrue et al., 2024) Several studies have demonstrated their activity against a range of infectious diseases caused by pathogens, as Klebsiella pneumoniae and Clostridioides difficile (Cole et al., 2025) Stenotrophomonas maltophilia (Paškevičius et al., 2022), Streptococcus sp, Staphylococcus aureus methicillin resistant, L. monocytogenes (Tian et al., 2026; Wasfi et al., 2018)), P. aeruginosa, Salmonella enteritidis, Escherichia coli, Bacillus cereus, Enterococcus fecalis and Cronobacter sakazakii (formerly Enterobacter sakazakii) (Pineda et al., 2024; Tian et al., 2026).

The bacteriocin classification considers the phylogeny of the producer bacterium as the primary criterion, distinguishing two principal classes: bacteriocins from Gram-positive bacteria, e.g., lantibiotics, and bacteriocins from Gram-negative bacteria, e.g., colicins, and microcins, among others (Alvarez-Sieiro et al., 2016; Dillen et al., 2026; Sharma et al., 2021; Solis-Balandra and Sanchez-Salas, 2024; Zimina et al., 2020). Table 2.

TABLE 2. Classification of gram-negative bacteriocins.

ClassDescriptionReferences
ColicinsBiomolecules with a molecular weight between 30 and 80 kDa, generally produced by E. coli strains that harbor a plasmid called colicinogenicNegash and Tsehai, 2020; Pérez-Ramos et al., 2021
MicrocinsBiomolecules low-molecular-weight bacteriocins of 1–10 kDa with a highly stable molecular structure that is active at a wide pH range, not very sensitive to the proteases’ activity, and thermoresistantPérez-Ramos et al. (2021)

Gram-positive bacteriocins, mainly produced by Lactobacillus and Staphylococcus are subclassified into three groups based on the presence of post-translational modifications (class I) or their absence (class II), while having a third class that presents tertiary structure (Negash and Tsehai, 2020; Sharma et al., 2021). Class I, includes small heat-stable post-translationally modified peptides (<10 kDa), as lantibiotics, identified in kefir-derived Lactiplantibacillus plantarum and L. lactis (Alvarez-Sieiro et al., 2016; Dillen et al., 2026), with a molecular weight less than 5 kDa, thermostable, and a high abundance of post-translational modifications, including unusual amino acids, as lanthionine, β-methyl lanthionine, and dehydrated amino acids. In turn, this class of bacteriocins is subdivided into two subdivisions: class Ia, with structures that are polar, with a positive net charge; and class Ib, including uncharged and negatively charged bacteriocins (Negash and Tsehai, 2020). Table 3.

TABLE 3. Classification of Gram-positive bacteriocins.

Class*DescriptionSubclassDescriptionReferences
Class ILantibiotics, with a molecular weight less than 5 kDa, thermostable, and a high abundance of post-translational modifications, including unusual amino acids, as lanthionine, β-methyl lanthionine, and dehydrated amino acidsIaStructures that are polar, with a positive net chargeNegash and Tsehai, 2020; Sharma et al., 2021
IbUncharged and negatively charged bacteriocins
Class IIIncludes small molecules with a molecular weight of <10 kDa. This class of bacteriocins is characterized by minimal or no post-translational modificationsIIaPeptides with activity against Listeria sp, a pathogenic bacterium that causes food-borne diseaseAntoshina et al., 2022; Sharma et al., 2021
IIbThese bacteriocins are characterized by their heterodimer function, where two distinct peptides act synergistically to exert antimicrobial activity
IIcIncludes peptides with a circular structure
IIdLinear peptides with not activity against Listeria
Class IIIBacteriocins with high-molecular weight, >30 kDa, and thermolabileIIIaLytic capacityJozala et al., 2015; Negash and Tsehai, 2020
IIIbNo-Iytic capacity

Class I: presence of post-translational modifications. Class II: absence of post-translational modifications. Class III: possesses tertiary structure.

Class II, includes heat-stable small molecules with a molecular weight of <10 kDa. This class of bacteriocins is characterized by minimal or no post-translational modifications, and it is subdivided into four subclasses. Subclass IIa includes peptides with activity against Listeria sp, a pathogenic bacterium that causes food-borne disease. Subclass IIb bacteriocins are characterized by their heterodimer function, where two distinct peptides act synergistically to exert antimicrobial activity. Subclass IIc includes peptides with a circular structure, and finally, subclass IId considers linear peptides with not activity against Listeria (Antoshina et al., 2022; Sharma et al., 2021).

Finally, class III includes large, heat-labile unmodified bacteriocins with high-molecular weight, >30 kDa, and thermolabile. These are subdivided into bacteriocins with lytic capacity and non-lytic but still inhibit target cells (Jozala et al., 2015; Negash and Tsehai, 2020).

The classification of Bacteriocins produced by Gram-negative is mainly associated with biocompounds produced by E. coli and other enterobacteriaceae, where only two types of bacteriocins have been isolated to date, such as Colicins and Microcins (Negash and Tsehai, 2020; Pérez-Ramos et al., 2021).

3.2 Mechanism of action

Several bacteriocins produced by lactic acid bacteria isolated from milk kefir grains, kill target cells by creating pores in the membrane, although alternative mechanisms, such as inhibition of cell wall synthesis and interference with nucleic acid or protein synthesis, have also been described (Hassan et al., 2012; Kumariya et al., 2019; Ríos Colombo et al., 2018).

Gram-negative bacteria have an inner membrane and an anionic lipopolysaccharide-rich outer membrane, whereas Gram-positive bacteria lack an outer membrane; however, they possess a thick peptidoglycan layer decorated with negatively charged teichoic acids (Mihaylova-Garnizova et al., 2024; Yeaman and Yount, 2003). Despite these structural differences, both bacterial types present a negatively charged surface. This feature allows bacteriocins with positive charge and/or with hydrophobic domains bind electrostatically to negative charged bacterial surfaces and then insert into the membrane, forming pores and causing rapid cell death (Kim et al., 2025). Figure 2. While most bacteriocins produced by lactic acid bacteria derived from kefir-milk can form pores, others employ different mechanisms of action; some bind with high affinity to lipid II, a key component of peptidoglycan biosynthesis in the cell wall; upon binding, they inhibit cell wall synthesis (Islam et al., 2012). Nisin, produced by L. lactis, is a bacteriocin with a dual mechanism: it binds to lipid II, disrupting the cell wall by pore formation and preventing peptidoglycan synthesis (Islam et al., 2012). Bacteriocins interfere with intracellular processes after entering the cell, such as protein synthesis. For example, Lactocin 27, produced by L. helveticus strain LP27, is an example; it inhibited primarily protein synthesis without affecting deoxyribonucleic acid and ribonucleic acid synthesis or adenosine 5′-triphosphate levels (Otunba et al., 2022; Upreti and Hinsdill, 1975; Wang et al., 2023). Another example is lactococcin 972, a bacteriocin that inhibits septum biosynthesis in L. lactis rather than forming pores in the cytoplasmic membrane (Martínez et al., 2008). Evidence of bacteriocin production in kefir grains has also been reported by several studies. Powell et al. (2007) characterized bacteriocin ST8KF produced by L. plantarum (formally Lactiplantibacillus plantarum) isolated from a milk kefir, whereas González-Orozco et al. (2023) and Tenorio-Salgado et al. (2021) (González-Orozco et al., 2023; Tenorio-Salgado et al., 2021) identified genes associated with bacteriocin synthesis, as helveticin and zoocin, in metagenomic analyses of kefir grain samples.

Bacteriocins that form pores across the cellular membrane, exhibit three states. In State I, bacteriocins are soluble in aqueous solution. In State II, bind to the outer membrane bilayers. State III, a stable transmembrane alpha-helix is formed, increasing the pore formation. Modified from (Deacon et al., 2015).
Figure 2. Bacteriocins that form pores across the cellular membrane, exhibit three states. In State I, bacteriocins are soluble in aqueous solution. In State II, bind to the outer membrane bilayers. State III, a stable transmembrane alpha-helix is formed, increasing the pore formation. Modified from (Deacon et al., 2015).

3.3 Nonribosomal peptides and polyketides

Nonribosomal peptides (NRP) have a characteristic fatty acid chain connected to the N-terminal end. They are cyclic or lineal compounds, containing non-protein amino acids, small heterocyclic rings, and other unusual peptide chain modifications (Wasmuth et al., 2025). There is a wide range of NRPS, and an extremely extensive range of biological activities and pharmacological properties that are not synthesized by ribosomes (Tajbakhsh et al., 2017). In addition, polyketides are a large group of natural biomolecules and are biosynthesized from acyl-CoA units by different polyketide synthases (PKSs), which display a wide diversity of functional domains and mechanisms of action between fungi and bacteria (Xu et al., 2021) Figure 1b.

Kefir grains microbiota possess the genetic potential to produce polyketides and nonribosomal peptides, and many secondary metabolite biosynthetic gene clusters have been predicted by bioinformatic tools. Tenorio-Salgado et al. (2021) predicted the presence of biosynthetic gene clusters for these secondary metabolites, including plipastatin, fengycin, and bacillaene, in metagenomic analyses of two Mexican kefir grain samples, and González-Orozco et al. (2023) detected contigs involved in exporting polyketides in milk kefir metagenomes of Texas. Recently, in a Bacillus velezensis Bac-9 strain isolated from milk kefir, was predicted that 55% of the genome encodes for non-ribosomal peptides (NRP), where 36% correspond to polyketides (PKs) and 9% to NRP + PK hybrids (Pérez-Rueda et al., 2025). From these NRPs, the expression of the genes involved in the biosynthesis of iturin, bacilysin and mycosubtilin was corroborated by RT-PCR; however experimental validation to determine their functional roles, is further necessary.

3.4 Polysaccharides

Polysaccharides are polymeric complex carbohydrates formed by long chains of more than 10 monosaccharides. Diverse studies have shown that most lactic acid bacteria isolated from kefir milk grains can produce high levels of exopolysaccharides (Kaur and Dey, 2023; Wang et al., 2023). Figure 1c.

In this regard, kefiran (KE), a water-soluble branched glucogalactan composed of equal proportions of D-glucose and D-galactose, being characterized by the repetition of a pentasaccharide unit with one or two monosaccharide branches in random positions in its structure, is mainly produced by L. kefiranofaciens in milk kefir (Ghasemlou et al., 2012; Zajšek et al., 2013). There is evidence that KE exhibits a broad range of biological activities, such as antitumoral, antifungal, and antibacterial properties. In addition, KE has an immunomodulation effect, epithelial protection, wound healing, anti-inflammatory, and antioxidant action (Correia et al., 2022; Tan et al., 2020). These molecular features are directly linked to kefiran’s functional behavior in aqueous systems, such as thickening and gel-forming capacity (Wang et al., 2008). Agar diffusion methods reveal activity of KE extract against Candida albicans and multiple bacteria, with the highest potency observed against Streptococcus pyogenes. Aditionally, bacterial viability assays confirm its bactericidal effects on P. aeruginosa, E. coli, S. aureus, and S. typhimurium (Georgalaki et al., 2021). KE exhibits a multifaceted nature and beneficial attributes, making it a promising candidate for further exploration in various scientific fields. Its antimicrobial activity is related to disruption of the cell membrane through induced pore formation (Barbosa et al., 2011), and kefiran concentrations ranged between 11.4 and 22.7 μmol/L, could induce membrane defects formed in supported bilayer lipid membrane due to the penetration of kefiran and pore formation, like a detergent (Barbosa et al., 2011). In this regard, the authors suggested a mechanism involving hydrogen bonding between the carbohydrate and the phosphate head group of the phospholipid with a carpet-like model of interaction. The overall results can be contributed to direct molecular interactions between the prebiotic oligosaccharides and the cell surfaces, which can be related to the biological activity of kefiran in several experimental models (Barbosa et al., 2011). It also protects intestinal epithelial by preventing B. cereus-induced necrosis, F-actin disorganization, and microvilli effacement in Caco-2 cells (Georgalaki et al., 2021).

Recent reports showed that the exopolysaccharides extracted from bacteria-derived from milk kefir grains have a significant reducing power activity that increases with concentration. The antioxidant activity of a polysaccharide depends mainly on its structural characteristics, including molecular weight, chain conformation, monosaccharide content, and configuration of the glycosidic linkage, among others, and is probably not due to one single factor but to the interaction of several factors (Liang et al., 2016; Radhouani et al., 2018). In this regard, docking studies highlight the robust binding affinity of kefiran for key antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), improving the fatty acid composition and decreased the de novo lipogenesis index (Hammami et al., 2025). Finally, the exopolysaccharide kefiran also has demonstrated potential antimicrobial properties in wound infections against S. aureus in a rat model and it could exert biological activity to prevent damage in human enterocytes by Bacillus cereus B10502 in Caco-2 cells line, and an inhibitory effect on the formation of biofilms by E. coli O157, among other bacterial pathogens (González-Orozco et al., 2022). Therefore, the antimicrobial mechanism of the EPS may be associated with an induction of the immune response, probably increasing the production of IgA + B cells and the production of cytokines in the gut lamina propria and blood serum (Vinderola et al., 2006).

In summary, kefir grains produce exopolysaccharides, where kefiran is the main compound (Lai et al., 2025). However, it also produces other exopolysaccharides such as levan, and heteropolysaccharides rich in glucose, galactose, and rhamnose. It can also produce homopolysaccharides (glucans/fructans) that are complex carbohydrates, or polymers, made from a single type of monosaccharide, a simple sugar unit. They can be either linear or branched and serve as either storage or structural compounds (Fels et al., 2018; Han et al., 2016; Zajšek et al., 2011).

3.5 Antimicrobial peptides

Antimicrobial peptides are small peptides, typically ranging between 6 and 50 residues, produced by various organisms that destroy a wide range of microorganisms, including bacteria, fungi, and viruses (Zhang et al., 2022). In this regard, milk kefir is a rich source of antimicrobial peptides, such as those with potential hypertensive activity due to angiotensin converting enzyme inhibition (Amorim et al., 2019), antimicrobial, immunomodulatory, opioid and anti-oxidative functions (Dallas et al., 2016); and, antimicrobial, immunomodulating and opioid activity (Ebner et al., 2015); among others. Recently, the purification of 46 peptides from a Turkish milk kefir grains cultured with bovine milk was reported using reversed-phase high-performance liquid chromatography, of which 13 showed 100% inhibition against E. coli, K. pneumoniae, and P. aeruginosa, among other pathogenic bacteria (De Lima et al., 2018). Similarly, Dalabasmaz et al. (2023) identified 1942 peptides using MALDI-TOF in a brazilian kefir grains cultured with sheep’s milk (Dalabasmaz et al., 2023). From these, 13 peptides exhibited antimicrobial activity against P. aeruginosa, Bacillus cereus, and Bacillus subtilis. They also identified 257 peptides using ultra-high-performance liquid chromatography coupled with mass spectrometry; of these, 230 sequences are unique to kefir grains, sixteen of which had been previously described as bioactive, with antihypertensive, immunomodulatory, antioxidant, antithrombotic, and antimicrobial effects (Ebner et al., 2015). Figure 1d.

Antimicrobial peptides have a significantly lower incidence of resistance compared to conventional antibiotics. This is because antimicrobial peptides employ a multi-target mechanism, primarily the physical disruption of the bacterial membrane, in contrast to the single-target mechanism of traditional antibiotics (Zheng et al., 2025).

In summary, due to its natural versatility and characteristics, antimicrobial peptides produced by milk kefir grains represent an affordable and sustainable tool against pathogenic microorganisms.

3.6 Organic acids

Fermentation is essential in food production and for the biological synthesis of diverse products such as, solvents, functional metabolites, and organic acids (Rymuszka and Gorczynska, 2026). During milk fermentation, lactic acid bacteria transform lactose and citrate into a variety of organic acids, such as lactic acid, acetic acid, propionic acid, succinic acid, and citric acid. These compounds do not only decrease the pH to inhibit the pathogenic bacterial growth, but also provide foods their unique taste, increasing their flavor and helping digestion (Punia Bangar et al., 2022). Figure 1d.

Lactic acid is the predominant end-product of lactose metabolism in lactic acid bacteria, which is fermented via the Embden-Meyerhof pathway to generate adenosine triphosphate (ATP), and lactic acid as a byproduct of NAD+ regeneration. Its function is to decrease the milk pH (∼6.7 to less than 4.6), causing casein coagulation and inhibition of the growth of pathogens. The isomers produced depend on the bacterial populations; for example, L (+)-lactic acid is produced by Lacticaseibacillus casei, whereas L. coryniformis produces D (-)-lactic acid, and L. plantarum, a racemic mixture (Khawam et al., 2026; Punia Bangar et al., 2022).

A second organic acid commonly identified in milk kefir grains is acetic acid that is produced primarily by heterofermentative bacteria, such as Lactobacillus spp and Bifidobacterium spp. It confers a sharp flavor to fermented foods, but it can produce undesirable tastes in excessive amounts. Acetic acid is produced in lower concentrations compared to lactic acid during lactic acid bacteria fermentations, and it exhibits a stronger antimicrobial effect in comparison with lactic acid against molds and yeasts (Kök Taş et al., 2025).

A third organic acid identified in kefir is citric acid, a product naturally present in milk (∼0.15%). This compound is consumed by many lactic acid bacteria during fermentation, and produces acetic acid, contributing to key aroma compounds, such as diacetyl, buttery aroma, acetone, and 2,3-butanediol (Kök Taş et al., 2025; Smit et al., 2005). Indeed, citric acid exhibits inhibitory activity against different foodborne pathogens and spoilage microorganisms. It is also used as a preservative in food and beverages by decreasing the pH (Akbas and Ölmez, 2007; In et al., 2013). Its antimicrobial mechanism includes chelation of divalent cations, such as Ca2+ and Mg2+, from the outer membrane. Additionally, at low pH, in its undissociated form, citric acid can penetrate the cell membrane and acidify the cytoplasm, disrupting metabolic functions and cellular homeostasis (Amato et al., 2020; Burel et al., 2021; Hu et al., 2019).

Short-chain organic acids are crucial metabolites in fermented products like kefir grains. Those are aliphatic carboxylic acids with a short hydrocarbon chain, generally composed of less than six carbon atoms, are the main end products of the milk kefir fermentation process. Although present in low concentrations, short-chain organic acids are crucial for the complex flavor profile (Kovanda et al., 2019). Propionic acid and butyric acids exhibit antimicrobial effects against pathogenic bacteria. The antimicrobial activities of butyric acid have been widely reported against Gram-negative and Gram-positive bacteria, such as E. coli, K. pneumoniae, Salmonella typhimurium, and Clostridium perfringens (Mallo et al., 2012; Salsali et al., 2008; Stecher and Hardt, 2011).

Minor organic acids also produced in fermented foods include pyruvic acid; a metabolic intermediate found in fresh fermented milk. Succinic acid is another organic acid generated as a product of the tricarboxylic acid cycle, amino acid and carbohydrate oxidation and lipolysis. These acids originate from lipolysis, carbohydrate metabolism, and amino acid metabolism. Hippuric acid, present in milk, decreases during fermentation, and it is converted into benzoic acid, a natural preservative (Sun et al., 2023).

3.7 Mechanism of action

The organic acids, as lactic and acetic, are produced in the environment, some of the molecules dissociate to H+ and anions. The pH of the environment, pK of the acid, concentration and other factors will determine the concentration of dissociated and undissociated molecules present. This cause and increase in transmembrane protons and pH gradients, as well as a concentration gradient of undissociated cations (H+) and anions. The lipid bilayer is permeable to lipophilic acetic, and lactic acids, facilitating their diffusion into the cytoplasm. The intracellular pH relatively alkaline, and the acids being soluble in the internal aqueous environment, the undissociated molecules will dissociate, producing protons and anions. When protons exceeding the buffering capacity are produced, they are transported out through the membrane-bound proton pumps, depleting the energy reserves. Therefore, there is a lowering of intracellular pH, which in turn denatures proteins and destabilizes structural and functional components of the cells, interfering with viability and growth (Mira et al., 2024; Ray, 2019; Ricke, 2003; Surendran Nair et al., 2017). In this context, organic acids have potent antimicrobial potential, mainly at acidic pHs. The food industry has been taking advantage of these properties for years, using them as preservatives to inhibit the growth of pathogenic or spoilage-causing fungal and bacterial species. One of those molecules is acetic acid, which has been used as an antiseptic primarily to treat infected wounds in burn patients (Mira et al., 2024). During milk fermentation there is production of organic acids, mainly lactic and acetic acids; however, many others have also been detected, such as butyric, propionic, and formic acids (Iraporda et al., 2017; Puerari et al., 2012).

Therefore, the antimicrobial properties of kefir grains are linked with the presence of the undissociated form of organic acids (Yassunaka Hata et al., 2023). For instance, Gut et al. (Gut et al., 2022) observed antagonistic effect of supernatants of two different traditional milk kefir grains on Salmonella arizonae and S. typhimurium after 24 and 48 h fermentation, the inhibition was attributed to action of lactic acid as kefir cell-free supernatant contained high concentrations of lactic acid ranging from 83.59 to 229.92 mm, and probably to the presence of acetic acid (Gut et al., 2022), reinforcing the role of these compounds to growth inhibition.

Finally, previous studies on S. Typhimurium and E. coli 0157: H7 reported the bactericidal effect of lactic acid, suggesting that the mechanism involves the disruption of the outer cell membrane making it permeable and thus enhanced the activity of other antibacterial molecules (Alakomi et al., 2000). In this regard, lactic acid should be able to potentiate the apparent antimicrobial activity of other components against Gram-negative bacteria. Therefore, in fermented low-pH products obtained by lactic acid bacteria, numerous metabolites are present that are too lipophilic or too large to effectively penetrate the Gram-negative bacterial outer membrane but that could possibly do so in the presence of lactic or another organic acid. Besides, a plethora of cryptic antimicrobials acting in synergy with lactic acid such as that high concentrations of lactic acid sensitizes Gram-negative bacteria to bacteriocins, as nisin, produced by bacteria isolated from milk-kefir (Tian et al., 2026).

4 Concluding remarks

Milk kefir is a natural source of different classes of molecules with potent antimicrobial activity against pathogenic microorganisms. This antimicrobial activity could be attributable to the synergic mechanism between the produced compounds identified in the consortium, and not solely to a single antimicrobial secreted during fermentation. Consequently, kefir grains represent an important source for obtaining antimicrobial compounds with biotechnological potential.

Despite the human health benefits associated with kefir compounds, exist several limitations to interpret these findings due to substantial heterogeneity in kefir preparation, dosage, intervention duration, study populations, outcome measures (Ebner et al., 2015), and the precise contribution of each bacterial or bioactive compounds (Amorim et al., 2019; Brasil et al., 2018). Larger, well-designed clinical trials with standardized kefir interventions, including detailed reporting of microbial composition and viable counts, consistent dosing, longer interventions, and, where feasible, double-blind designs with matched sensory controls, are needed to better define kefir’s efficacy in specific populations and health conditions.

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Footnotes

Edited by: Alan Talevi, National University of La Plata, Argentina

Reviewed by: Vishal Manjunatha, McKee Foods, United States

Pak Heri, Lampung University, Indonesia

Statements

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The author EP-R declared that they were an editorial board member of Frontiers at the time of submission. This had no impact on the peer review process and the final decision.

Funding

The author(s) declared that financial support was received for this work and/or its publication. This work was funded by Dirección General de Asuntos del Personal Académico-Universidad Nacional Autónoma de México (IN206326). There was no additional external funding received for this study. The funders did not have additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author contributions

ST-S: Conceptualization, Investigation, Writing – original draft, Writing – review and editing. AS-C: Data curation, Methodology, Writing – review and editing. EP-R: Data curation, Funding acquisition, Writing – original draft, Writing – review and editing.

Acknowledgements

We thank Israel Josué Novelo Zel, Israel Sanchez-Dominguez and Ana Cecilia Perez Arteaga for their technical support.

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Citation

Tenorio-Salgado, S., et al. (2026). Minireview Kefir as a source of antimicrobial compounds against bacterial pathogens. Frontiers in Drug Discovery, Volume 6. https://doi.org/
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6 - 2026

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Copyright © 2026 Tenorio-Salgado, Sánchez-Canché and Perez-Rueda.

License: https://creativecommons.org/licenses/by/4.0/

*Correspondence: Silvia Tenorio-Salgado; Ernesto Perez-Rueda

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