Enhancing protein digestibility in red clover and lucerne seeds through lactic acid fermentation

Red clover (Trifolium pratense) and lucerne (Medicago sativa) seeds are promising alternative protein sources due to their high crude protein content and rich nutrient profile (1) . However, their potential is limited by the presence of anti-nutritional factors (ANFs) such as phytic acid, trypsin inhibitors, and chymotrypsin inhibitors (2) . These compounds interfere with protein digestion, thereby reducing protein bioavailability (3) . Lactobacillus fermentation has been proposed as a strategy to improve the nutritional quality of plant-based ingredients by enhancing protein hydrolysis, increasing the availability of bioactive compounds, and reducing ANFs. This study aimed to evaluate the effect of Lactobacillus fermentation on ANFs and protein digestion. Red clover and lucerne seed flours were fermented using three Lactobacillus strains, including L. plantarum, L. acidophilus and L. casei. ANFs, including phytic acid, trypsin inhibitory activity (TIA), and chymotrypsin inhibitory activity (CIA), were quantified using standard colorimetric assays. The INFOGEST protocol was used to evaluate in vitro protein hydrolysis (digestion), which was quantified by the o-phthaldialdehyde (OPA) method at three stages: before digestion, after the gastric phase, and after the intestinal phase. All the experiments were replicated three times (n=3) and the results were expressed as mean ± standard deviation. Statistical analysis was performed by SAS 9.4 using a two-way analysis of variance (ANOVA) with 4 legume seed samples x 4 treatments (three Lactobacillus cultures and one unfermented) on antinutritional factors and protein digestion. Fermentation significantly reduced (p < 0.05) all measured ANFs in both seed types. For example, TIA decreased by 33–82% depending on the Lactobacillus strain and seed type. Before in vitro digestion, fermented samples exhibited an average four-fold increase (p < 0.05) in protein hydrolysis compared to unfermented controls. Furthermore, the gastric and intestinal protein hydrolysis values were significantly higher (p < 0.05) in the fermented samples, indicating improved digestibility compared to the unfermented controls. Lactobacillus fermentation significantly enhanced the nutritional profile of lucerne and red clover seeds, reducing ANFs and improving protein bioavailability. The findings of this study suggest the potential application of Lactobacillus fermentation in developing nutritionally enhanced plant-based protein products using lucerne and red clover seeds.