Brewer’s Spent Grain as a Substrate for Lactic Acid Fermentation: Contrasting Performance of Lacticaseibacillus rhamnosus and Limosilactobacillus fermentum

Brewer’s spent grain (BSG), the major by-product of the brewing industry, has attracted growing interest in valorisation through lactic acid bacteria fermentation. However, research has largely focused on Lactiplantibacillus plantarum, while other species have mainly been investigated using enzymatic hydrolysates rather than intact solid BSG. This study compared the solid-state fermentation performance of Lacticaseibacillus rhamnosus ATCC 7469 and Limosilactobacillus fermentum ATCC 9338 on BSG over 10 days. In vitro antioxidant, antidiabetic, anti-obesity, and antimicrobial activities were assessed every two days and integrated into a global bioactivity score. Lacticaseibacillus rhamnosus successfully colonised the substrate, reaching 9.62 log10 CFU g−1 on Day 2 and reducing the pH to 3.6. Antioxidant capacity peaked on Day 2, whereas antidiabetic and anti-obesity activities peaked on Day 10. Bactericidal activity was also confirmed in a bread-simulating matrix, and the global bioactivity score identified Day 2 as the highest-scoring endpoint. In contrast, Limosilactobacillus fermentum failed to establish a successful fermentation, with polymicrobial contamination observed from Day 4 onwards. To investigate whether substrate sterilisation could improve Limosilactobacillus fermentum fermentation performance, autoclaving and a two-step heat treatment were evaluated. Although autoclaving effectively sterilised the substrate, it reduced the number of detectable proteins by nearly 25%, whereas the two-step heat treatment preserved the protein profile but did not support successful fermentation by Limosilactobacillus fermentum. Overall, these findings identify Lacticaseibacillus rhamnosus as a promising candidate for BSG valorisation and underscore the importance of strain selection, as fermentation performance can vary substantially among lactic acid bacteria.