Sweet whey can be valorized through fermentation and subsequent production of Requeson, a fresh whey cheese obtained by heat-induced protein precipitation. This study evaluated the physicochemical (moisture, water activity, protein, ash, chloride, and EPS content), textural (hardness, cohesiveness, adhesiveness, springiness, chewiness, and fracturability), sensory (overall liking and JAR saltiness), and in vitro bioactive characteristics of Requeson produced from sweet whey fermented with exopolysaccharide (EPS)-producing Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 and Streptococcus thermophilus SY-102, individually or in co-culture. Unfermented, citric-acid-acidified whey served as the control. The co-culture (FC) showed the highest EPS content (3.5 mg/g), higher moisture, lower hardness and fracturability, and greater springiness than the control. Consumer evaluation (n = 130) showed favorable acceptance of FC. Among FC formulations containing 0.5–1.5% NaCl, 0.5% NaCl yielded the highest overall liking, although it did not differ significantly from 1.0%. FC showed the highest DPPH radical-scavenging (22.76%) and ACE-I inhibitory (31.00%) activities. Overall, co-culture fermentation represents a potential strategy for producing value-added Requeson with modified technological and sensory properties and measurable in vitro bioactivities.

Requeson, a traditional Mexican whey cheese, represents a promising avenue for the valorization of dairy by‐products. However, its functional potential remains underexplored. This study evaluated the enhancement of bioactive properties in Requeson cheese through lactic acid bacteria (LAB) fermentation, comparing two monocultures ( Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 and Streptococcus thermophilus SY‐102) and a co‐culture of both strains, cultured in sweet whey. Fermented whey was used to produce Requeson cheese, and peptide‐rich extracts were analyzed for antioxidant activity (DPPH and FRAP), mineral‐chelating capacity (Fe ²⁺ , Ca ²⁺ , Mg ²⁺ ), enzymatic inhibition (ACE‐I, DPP‐IV), and antimicrobial activity against E. coli , M. luteus , and L. innocua . Liquid chromatography–tandem mass spectrometry (LC–MS/MS) was employed to identify peptides, and their potential bioactivities were predicted using the UniDL4BioPep platform. The co‐culture fermentation yielded significantly higher values across all assays, including antioxidant capacity (41.3 mg Trolox), ACE‐I inhibition (31.8%), DPP‐IV inhibition (14.2%), and antimicrobial activity. Several peptides with predicted antioxidant, antihypertensive, antidiabetic, and antimicrobial properties were exclusive to the co‐culture formulation. These findings suggest that LAB co‐culture fermentation of sweet whey is an effective strategy to develop peptide‐enriched functional Requeson cheese with health‐promoting properties. This approach supports the sustainable use of dairy by‐products and contributes to the advancement of functional dairy products in the bioeconomy.