This study investigated Lacticaseibacillus paracasei GV17, a potentially probiotic strain, in association with the commercial culture Streptococcus thermophilus STI-12, in lactose-free fermented milk. Predictive modeling was used to estimate growth parameters and microbial viability and the technological characteristics of the fermented milk during storage. The initial concentrations of the strains were 9.80 log CFU/mL for Lc. paracasei GV17 and 9.50 log CFU/mL for S. thermophilus STI-12. After eight hours, the pH reached 4.6, and the concentrations of GV17 and STI-12 were 10.90 log CFU/mL and 11.20 log CFU/mL, respectively. The Baranyi model was fitted to the growth data, with correlation coefficients of 0.760 for Lc. paracasei GV17 and 0.852 for St. thermophilus STI-12. The maximum specific growth rates were 0.912 log CFU/h for GV17 and 0.882 log CFU/h for STI-12. Regarding technological characteristics, syneresis decreased by 8.90% after 28 days, indicating greater structural stability, while water retention capacity remained constant. The viability of LAB remained above 10.00 log CFU/mL. Lc. paracasei GV17 showed great potential for use in functional products, prompting further research. 2025 https://creativecommons.org/licenses/by/4.0/ Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( https://creativecommons.org/licenses/by/4.0/ ). This study investigated Lacticaseibacillus paracasei GV17, a potentially probiotic strain, in association with the commercial culture Streptococcus thermophilus STI-12, in lactose-free fermented milk. Predictive modeling was used to estimate growth parameters and microbial viability and the technological characteristics of the fermented milk during storage. The initial concentrations of the paracasei GV17 showed great potential for use in functional products, prompting further research. lactic acid bacteria growth kinetics mathematical modeling probiotics Brazilian Federal Agency for Support and Evaluation of Graduate Education 001 Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES financial code 001). There is no doubt that LAB are a valuable economic asset for the food industry, especially in the dairy sector [ 1 ]. Understanding strategies to prevent food spoilage and contamination by foodborne pathogens is a widely debated issue in the food industry [ 2 ]. Given their metabolic capabilities, LAB species are regarded as promising candidates to suppress the growth of foodborne pathogens and prolong product shelf life in food processing [ 3 ]. LAB are known for adapting to the dairy food matrix and contributing to the fermentation process of dairy products. The combination of commercial LAB cultures with probiotics in milk fermentation gives the product functional properties [ 4 ]. Lactic Acid Bacteria The potentially probiotic strain Lacticaseibacillus paracasei GV17 [ 10 ], from the Culture Collection of the Department of Technology and Inspection of Products of Animal Origin at the Federal University of Minas Gerais, was used (DTIPOA/UFMG, Belo Horizonte, MG, Brazil). The LAB was previously isolated from the processing of Minas artisanal cheese from the Campo das Vertentes region in Minas Gerais and identified using the MALDI-TOF spectrometry technique. For MALDI-TOF mass spectrometry, the Microflex™ system (Bruker Daltonics, Brémen, Germany) and its database were used. paracasei GV17 cocultured with the commercial strain S. thermophilus STI-12. The milk, after adding the cultures, was dispensed into sterile 50 mL bottles and fermented at 42 °C until a pH of 4.6 was reached. The selected fermentation temperature was found to be adequate for the strains, based on preliminary tests conducted on the autochthonous (GV17) and commercial (STI-12) cultures. After fermentation, the milk was cooled to 15 °C, and the gels were broken. The product was then stored at 4 °C for 28 days to evaluate LAB viability, syneresis, and water retention capacity. 2.3.2. During the 28 days of shelf life for the fermented milk, there was no statistically significant increase ( p > 0.05) in WHC. Throughout the entire storage period of the fermented milk, the viability of GV17 and STI-12 remained above 10.00 log CFU/mL ( Table 3 ). The potentially probiotic LAB strain GV17 showed an increase of approximately 1.00 log CFU/mL ( p < 0.05) in count after 14 days of refrigerated storage but exhibited no significant difference in count after 28 days. The commercial strain STI-12 did not exhibit a significant increase in viability during the storage of fermented milk at 4 °C. 4. In contrast, lower syneresis values imply a more organized structure, particularly in the fats and proteins within fermented milk [ 40 ]. The lower degree of syneresis in the fermented milk over the 28 days may be related to a possible increase in the product’s acidity. Even though the fermented milk was stored at low temperatures, the fermentation process could have still continued and further restructured the protein network [ 41 ]. Pereira et al. [ 5 ] assessed the syneresis of lactose-free milk made with a commercial culture containing Lactobacillus acidophilus LA-5, Bifidobacterium lactis BB-12, and Streptococcus thermophilus (Bio Rich ® ) and supplemented with inulin. In a study by Lacerda et al. [ 46 ], a decrease of approximately 0.3 in the pH value of fermented milk stored at 4 °C was observed, while a greater reduction occurred when the product was kept at 12 °C. In fermented milk stored at 4 °C, no significant differences in LAB count were detected throughout the shelf life. 5. Conclusions The tested strains of Lacticaseibacillus paracasei GV17, in association with the commercial culture Streptococcus thermophilus STI-12, exhibited sufficient growth to reach high cell density, enter the stationary phase, and adapt well to the Baranyi model.

Consumer demand for plant-based functional foods, especially probiotic beverages, has increased due to their health benefits and suitability as dairy-free alternatives. This study assessed, through a factorial combination, the stability of plant-based extracts (avocado, ginger, and tropical) individually inoculated with three commercial Lactobacillus strains (L. casei, L. plantarum, L. reuteri) and stored under refrigerated conditions during both primary (PSL) and secondary shelf life (SSL). Product shelf life was defined by probiotic viability, considering the functional threshold (≥6 log CFU/mL), which was maintained across all formulations throughout the storage period. Physicochemical parameters, including pH, titratable acidity, and colour, as well as volatile profile, remained stable, with only minor variations depending on the matrix and bacterial strain. Sensory evaluations (triangle and acceptability tests) confirmed that the probiotic juices were acceptable to consumers. Overall, the results demonstrate the feasibility of producing non-fermented, plant-based probiotic beverages that retain their functional properties and meet consumer sensory expectations, offering a promising alternative for vegan and lactose-intolerant individuals. Traditionally, probiotic foods on the market have been dairy based, but growing awareness of milk allergenicity, lactose intolerance, and the rise in vegan diets have driven the demand for non-dairy alternatives [ 2 , 3 , 4 ]. In response, fruits and vegetables are increasingly being explored as novel matrices for probiotic delivery, offering an attractive tool that aligns with “clean label” and vegan product tendencies [ 2 ]. Fruit and vegetable beverages represent ideal carriers for probiotics, as they are naturally rich in vitamins, antioxidants, and other phytochemical compounds [ 5 ]. Probiotic products have been linked to improved gut health, immune function, and overall well-being, which is attractive from a public health perspective [ 1 , 3 , 5 , 6 ]. Fruit juices provide an excellent base for developing probiotic beverages due to their pleasing taste, which appeals to all age groups, and their perception as Nonetheless, formulating stable, non-fermented probiotic beverages presents several challenges. Probiotic viability is a crucial concern. To be effective, probiotic foods typically require a minimum concentration of 10⁶ CFU/mL or a daily intake of 10⁸–10 11 [ 8 ], and live cell count must be maintained throughout the shelf life of the product [ 5 ]. However, the acidic pH and high organic acid content of many fruit juices can impose acid stress on probiotic bacteria, leading to gradual viability loss during refrigerated storage [ 2 , 5 ]. Additionally, other factors, like high sugar levels, salt concentration, metabolic products, dissolved oxygen, and redox potential, can negatively impact probiotic cells in juice environments [ 5 ]. Furthermore, another key challenge is sensory and physical stability. Probiotic microorganisms may impact flavour, aroma, or clarity over time [ 1 , 5 ]. This objective is rooted in the growing interest in using natural ingredients to enhance the functionality and stability of probiotics, particularly in the context of clean-label, health-conscious food and supplement products. Furthermore, the research aimed to evaluate the physiochemical stability and the maintenance of juice sensory quality during both primary shelf life (PSL) and secondary shelf life (SSL). 2. Materials and Methods 2.1. The extracts were kindly supplied by an Italian fruit and vegetable processing company. These products were selected as representative of real-market substrate for functional beverage development. 2.2. Bacterial Strain and Growth Conditions The probiotic strains used to fortify the selected extracts included the following probiotic Lactobacillus strains: L. reuteri DSM 17938 and L. plantarum LP09 obtained from a commercial suspension; and L. casei isolated from a commercial drinking yoghurt. Strains were individually propagated in De Man, Rogosa, and Sharpe (MRS, Oxoid, Milan, Italy) medium at 37 ± 2 °C for 24 h. 2.3. The cells were then harvested by centrifugation (8000× g , 4 °C, 10 min), washed twice in 50 mM phosphate buffer (PBS, 4 °C, pH 7.0), and resuspended in sterile tap water to achieve a final cell density of about 9 log colony-forming units (CFUs) per mL of solution. Each strain was then individually inoculated into the fruit extracts to obtain the fortified products, with an initial concentration adjusted to approximately 10 6 –10 7 CFU/mL. This range reflects the commonly accepted probiotic levels used in functional food formulations to ensure potential health benefits upon consumption. Freeze-drying maintains high probiotic survival rates, but protective agents are necessary to prevent cell damage [ 24 ]. This study focused on incorporating probiotics in their free form, a strategy previously reported to be effective in maintaining microbial viability in similar applications [ 2 ]. The concentration of probiotics added to the extracts was about 10 7 CFU/mL, aligning with the FAO/WHO guidelines, which recommend that probiotic products maintain a minimum of 10 6 to 10 7 CFU/mL throughout their shelf life [ 2 ]. Interestingly, the stability observed in the primary shelf life continued into the secondary shelf life. Numerous commercial and experimental products have been developed using pineapple, mango, carrot, beetroot, orange, and other tropical fruits as substrates for probiotics [ 25 ]. Probiotic viability in these matrices is highly strain-dependent and influenced by factors such as juice pH, organic acid profile, oxygen exposure, antimicrobial compounds, and processing methods [ 25 ]. These fruits are also rich in vitamins, minerals, dietary fibre, carbohydrates, bioactive compounds, and antioxidants, making them excellent and advantageous alternatives to dairy-based probiotic carriers.