Lactic acid bacteria (LAB) are a group of microorganisms significant industrial relevance, particularly in the food and pharmaceutical sectors. They are widely used to produce fermented functional foods, enhancing the bioavailability of various bioactive compounds and contributing to the sensory and safety attributes of food products through their antimicrobial and biopreservative properties (Meruvu and Tellioglu Harsa, 2023; Evivie et al., 2017; Ayed et al., 2024). LAB naturally inhabits diverse ecological niches, including fermented foods, the oral cavity, and the gastrointestinal tract, where they promote host health by maintaining the balance of the intestinal microbiota. These attributes have led to their widespread use as probiotics (Abdel Tawab et al., 2023; Anjana and Tiwari, 2022; Bernal-Castro et al., 2024).

In recent years, the valorization of agro-industrial by-products for biotechnological applications has emerged as a sustainable alternative for waste management. These by-products, generated during food processing, are rich in essential nutrients and bioactive compounds, making them attractive substrates for the development of high-value-added products. Their use not only represents an economically viable alternative but also contributes to sustainability strategy for waste reduction and circular economy development (de la Rosa et al., 2019; Gonçalves et al., 2023). With global food waste estimated to exceed 1,052 million tons by 2022, contributing between 8 and 10% of global greenhouse gas emissions, innovative waste management strategies are urgently needed (Forbes et al., 2024).

LAB can produce lactic acid (LA) as the main product of carbohydrate fermentation. Agro-industrial residues containing lactose (from dairy) (Popova-Krumova et al., 2024; Turner et al., 2017), glycerol (from biodiesel) (Kano et al., 2022; Patel, 2012) fructans from agave (Martha-Lucero et al., 2025), and cellobiose (from cellulose hydrolysis) have shown promise as substrates for lactic acid production (Adsul et al., 2007), these options offer dual benefits: valorizing waste and supporting sustainable production processes.

Lactic acid is an organic acid recognized as GRAS (generally recognized as safe) by the FDA and has applications across multiple industries. In the food sector, it is used as a food preservative and sefety (Zapaśnik et al., 2022) and acidulant (Ameen and Caruso, 2017). In the chemical industry, it serves as a pH regulator (Zhou, 2023), solvent, cleaning agent (Kim et al., 2022), and precursor for biodegradable polymers like polylactic acid (PLA) (Banerjee et al., 2023; Swetha et al., 2023; de França et al., 2022). Additionally, it finds applications in cosmetics and pharmaceuticals, including skin treatments (Chuang et al., 2023), controlled drug delivery (Boisgard et al., 2017), and immunostimulants (Jiang et al., 2024).

Lactic acid exists as two optical isomers: L-lactic acid and D-lactic acid. In the context of biodegradable polymers, the mechanical properties and biodegradability of polylactic acid (PLA) are significantly influenced by the ratio of these enantiomers. Specifically, the D/L lactic acid ratio modulates the crystallinity, thermal stability, and degradation rate of the resulting polymer. Consequently, considerable research has been directed toward the selective production and purification of each isomer to meet the specific requirements of biopolymer applications (Augustiniene et al., 2022; Zhao et al., 2013). While chemical synthesis of lactic acid results in a racemic mixture, microbial fermentation offers a viable alternative for obtaining high-purity L-lactic acid. However, wild-type LAB strains, such as Lactobacillus plantarum, naturally produce a racemic mixture. Thus, it is critical to investigate the isomeric composition of lactic acid produced by various strains and substrates to optimize its industrial applicability.

Various species of Lactobacillus spp., including L. acidophilus, L. plantarum, L. casei, and L. rhamnosus, have been extensively applied in food biotechnology for the fermentation of diverse substrates, particularly in the development of symbiotic functional foods. Beyond their nutritional applications, these strains are gaining relevance in industrial and biomedical contexts due to their ability to synthesize bioactive metabolites, including lactic acid isomers, exopolysaccharides, and antioxidant compounds (Gao et al., 2025; Frumuzachi et al., 2024; Plessas et al., 2024; Palencia-Argel et al., 2024).

In addition to their recognized safety and probiotic status, certain LAB genera such as Lactiplantibacillus, Lacticaseibacillus, and Bifidobacterium exhibit notable antioxidant properties. These effects are mediated through the production of antioxidant enzymes, glutathione, and exopolysaccharides, which contribute to the mitigation of oxidative stress and the modulation of cellular signaling pathways such as Nrf2/NF-kB (Guerrero-Encinas et al., 2021; Ruiz-Gonzalez et al., 2024). These traits further support their suitability for health-oriented biotechnological applications.

To guide the rational selection of strains and conditions for targeted isomer production, the use of model substrates including monosaccharides, disaccharides, and oligosaccharides enables precise characterization of substrate preferences and fermentative capacities in LAB. This strategy is particularly relevant when designing processes that utilize complex carbon sources such as lignocellulosic hydrolysates or agro-industrial by-products rich in unconventional sugars.

Despite the extensive literature on glucose and lactose metabolism in LAB (Iskandar et al., 2019; Arefi et al., 2024; Wasewar et al., 2004), the fermentative behavior of probiotic strains in response to alternative carbon sources such as rhamnose, arabinose, agave-derived fructooligosaccharides (FOS), and high-degree polymerization fructans (FAMP) remains underexplored. These sugars are abundant in agave residues and other dietary fibers and offer great potential as functional substrates in sustainable fermentation processes.

Therefore, this study evaluates the growth performance and lactic acid isomer production (L- and D-lactic acid) of five probiotic strains cultured on selected model substrates derived from agro-industrial by-products. The results aim to support the development of efficient and sustainable bioprocesses aligned with circular economy principles.

Lactic acid bacteria (LAB) can produce lactic acid as a primary metabolite through the fermentation of hexoses. Homofermentative LAB uses glycolysis (Embden-Meyerhof-Parnas pathway) to convert a single hexose molecule into two molecules of lactic acid, generating two ATP molecules while maintaining a balanced NAD⁺/NADH redox state. This could explain the higher lactic acid production observed in L. casei (Cui and Qu, 2021), L. plantarum (Gänzle, 2015), and B. animalis (Cui et al., 2019).

In contrast, heterofermentative LAB such as Lactobacillus reuteri (Årsköld et al., 2008) and L. rhamnosus (Morita et al., 2009) utilise the pentose phosphate pathway due to the absence of fructose-1,6-bisphosphate aldolase. As a result, these strains produce not only lactic acid but also CO₂, ATP, and either ethanol or acetic acid per glucose molecule. In this context, the conversion of acetyl phosphate to ethanol requires the oxidation of two NADH molecules, maintaining redox balance. Conversely, the formation of acetic acid allows for the regeneration of NAD⁺ and the production of an additional ATP molecule.

Our findings suggest that lactic acid isomer production (L- or D-form) is influenced by the bacterial strain and the type of substrate fermented. Certain strain-substrate combinations favour specific metabolic pathways that result in high-purity L-lactic acid, while others promote D-lactic acid production. On the other hand, the low performance in the proliferation or growth of some heterofermentative LAB, such as L. reuteri, may be due not only to the metabolic implications of assimilating a complex carbohydrate source, but also to the acidic characteristics of the lactate produced by LAB that may act as growth inhibitor, like the case studied in the strain L. reuteri JCM1112 (Ichinose et al., 2020).

Lactic acid is an important bio-based platform compound, which can be divided into D-lactic acid and L-lactic acid according to its optical rotation (Wang et al., 2021). The high proportion of L-lactic acid obtained from L. rhamnosus fermenting agave fructans, L, casei with cellobiose and lactose, and B. animalis with glucose, may be attributed to two key factors: (1) enzyme specificity and (2) metabolic regulation, indicating dominant L-lactate dehydrogenase (L-LDH) activity under favourable substrate conditions. Under these circumstances, the activity or expression of D-lactate dehydrogenase (D-LDH) is reduced, leading to the near-exclusive production of L-lactic acid.

Metabolic regulation may also dictate the dominance of homolactic versus heterolactic fermentation depending on the substrate. In L. rhamnosus, L. casei, and B. animalis, homolactic fermentation is prevalent (Cui et al., 2019; Gänzle, 2015; Cui and Qu, 2021), meaning most pyruvate is channelled toward L-lactic acid formation rather than alternative products such as ethanol or acetic acid.

Agave fructans are fructose-based polymers that must first be hydrolyzed into fructooligosaccharides (FOS), free fructose, and glucose for microbial utilization (Morales-Landa et al., 2024; Cui et al., 2021; Biedrzycka and Bielecka, 2004). L. rhamnosus and L. casei possess fructofuranosidase or β-fructosidase enzymes capable of breaking down these polymers. The resulting monosaccharides enter glycolysis via the fructose-6-phosphate (F6P) pathway, bypassing the metabolic branching of the pentose phosphate pathway and favouring L-lactic acid production. Similarly, cellobiose, a disaccharide composed of two glucose units linked by a β-1,4 bond, is hydrolyzed by β-glucosidase in L. casei, enabling glucose entry into glycolysis via glucose-6-phosphate (G6P) and promoting rapid L-lactate formation.

In B. animalis, glucose metabolism primarily follows the bifid shunt, also known as the fructose-6-phosphate phosphoketolase pathway, which is unique to the genus Bifidobacterium. In this route, fructose-6-phosphate is cleaved by F6PPK into acetyl phosphate and erythrose-4-phosphate. The conversion of acetyl phosphate to acetate or lactate depends on NADH availability and L-LDH activity. Under conditions of high NADH regeneration such as during glucose fermentation, B. animalis favors lactate over acetate production, improving conversion efficiency (González-Rodríguez et al., 2013; Pokusaeva et al., 2011).

In contrast, L. reuteri and L. plantarum appear to exhibit greater D-LDH activity when fermenting lactose, favoring D-lactic acid production (Morita et al., 2008; Goffin et al., 2004). This behavior may result from differences in LDH gene regulation or intracellular conditions such as NAD⁺/NADH ratios and carbon flux. These factors can shift the metabolic equilibrium toward D-lactic acid synthesis.

The L and D forms of lactic acid produced by LAB fermentation play a significant role in the synthesis of biodegradable polylactic acid. There is a great interest in the biomedical area in the fields of tissue regeneration and repair, in the application of biomaterial technologies (Capuana et al., 2022; Alavi et al., 2023), in the cosmetic area for skin care and treatment of melanogenesis (Huang et al., 2020), to mention a few examples.

Overall, these findings highlight the metabolic complexity of lactic acid biosynthesis in probiotic strains and underscore the importance of strain-substrate selection in directing fermentation toward high-purity L-lactic acid.

The growth behavior and lactic acid production of probiotic bacteria are strongly influenced by the type of substrate used. Glucose, fructose, and disaccharides such as cellobiose and sucrose are the most efficient in promoting both bacterial growth and lactic acid synthesis. Furthermore, enzymatic specificity and metabolic regulation play critical roles in determining the L-/D-lactic acid ratio. Notably, L. rhamnosus, L. casei, and B. animalis show a clear preference for L-lactic acid production. These results provide valuable insight into fermentation processes through strategic selection of both LAB strains and carbon sources, with implications for food biotechnology but for the medical field. Our findings ensure a follow-up in the study with substrates originating from by-products of the food industry, aimed at promising uses and biomedical applications, thus promoting the utilisation of these sources, which are important actions in the circular economy.