Untargeted LC-QTOF-MS/MS based metabolomics approach for revealing bioactive components in probiotic fermented coffee brews.

Amidst trends in non-dairy probiotic foods and functional coffees, we recently developed a fermented coffee brew containing high live counts of the probiotics Lacticaseibacillus rhamnosus GG and Saccharomyces boulardii CNCM-I745. However, probiotic fermentation did not alter levels of principal coffee bioactive components based on targeted analyses. Here, to provide therapeutic justification compared to other non-fermented coffee brews, we aimed to discover postbiotics in coffee brews fermented with L. rhamnosus GG and/or S. boulardii CNCM-I745. By using an untargeted LC-QTOF-MS/MS based metabolomics approach coupled with validated multivariate analyses, 37 differential metabolites between fermentation treatments were putatively annotated. These include the production of postbiotics such as 2-isopropylmalate by S. boulardii CNCM-I745, and aromatic amino acid catabolites (indole-3-lactate, p-hydroxyphenyllactate, 3-phenyllactate), and hydroxydodecanoic acid by L. rhamnosus GG. Overall, LC-QTOF based untargeted metabolomics can be an effective approach to uncover postbiotics, which may substantiate additional potential functionalities of probiotic fermented foods compared to their non-fermented counterparts.

In vitro bioactivities of coffee brews fermented with the probiotics Lacticaseibacillus rhamnosus GG and Saccharomyces boulardii CNCM-I745.

Previously, we demonstrated the production of bioactive metabolites (e.g., indole-3-lactate, 4-hydroxyphenyllactate, 3-phenyllactate, 2-isopropylmalate) by the probiotics Lacticaseibacillus rhamnosus GG and Saccharomyces boulardii CNCM-I745 during coffee brew fermentation. However, it remains unclear if in situ production of bioactive metabolites confers additional health benefits to coffee brews. Here, we aimed to investigate the in vitro bioactivities of freeze-dried cell-free coffee supernatants fermented with L. rhamnosus GG and/or S. boulardii CNCM-I745, compared to non-fermented coffee supernatants. In vitro bioactivity assays pertained to α-amylase and α-glucosidase inhibition, antiglycative activities, anti-proliferation against human cancer cell lines (MCF-7, HCT116, and HepG2), cellular antioxidant activities, and anti-inflammatory activities. We demonstrated that non-fermented coffee supernatants displayed weak starch hydrolase inhibition (IC50 > 36.00 mg/mL), but otherwise displayed strong anti-glycative (IC50 0.71-0.74 mg/mL), anti-proliferative (IC50 0.45, 0.36, and < 0.5 mg/mL for MCF-7, HCT116, and HepG2 respectively), cellular antioxidant (85,844.22 µmol quercetin equivalents/100 g coffee supernatant), and anti-inflammatory activities (35.7% reduction in nitrite production at 0.13 mg/mL). In all the assays tested, probiotic fermented coffee supernatants exhibited very similar bioactivities compared to non-fermented coffee supernatants, and improvements were not observed. Overall, in vitro bioactivities of coffee brews were not improved via in situ metabolite production by L. rhamnosus GG and/or S. boulardii CNCM-I745. Therefore, bioactive metabolites produced during probiotic-induced food fermentations may not necessarily confer additional health benefits compared to non-fermented counterparts.

Growth, survival, and metabolic activities of probiotics Lactobacillus rhamnosus GG and Saccharomyces cerevisiae var. boulardii CNCM-I745 in fermented coffee brews.

Amidst rising demand for non-dairy probiotic foods, and growing interest in coffees with added functionalities, it would be opportune to ferment coffee brews with probiotics. However, challenges exist in maintaining probiotic viability in high-moisture food products. Here, we aimed to enhance the viability of the probiotic bacteria, Lactobacillus rhamnosus GG, in coffee brews by co-culturing with the probiotic yeast, Saccharomyces cerevisiae var. boulardii CNCM-I745. The yeast significantly enhanced the viability of L. rhamnosus GG, as bacterial populations beyond 7 Log CFU/mL were maintained throughout 14 weeks of storage at 4 and 25 °C. In contrast, the single culture of L. rhamnosus GG suffered viability losses below 6 Log CFU/mL within 10 weeks at 4 °C, and 3 weeks at 25 °C. Growth and survival of S. boulardii CNCM-I745 remained unaffected by the presence of L. rhamnosus GG. Volatile profiles of coffee brews were altered by probiotic metabolic activities, but co-culturing led to suppressed generation of diacetyl and ethanol compared to single cultures. Probiotic fermentation did not alter principal coffee bioactive compounds and antioxidant capacities; however, declines in peroxyl radical scavenging capacities were observed after ambient storage. Overall, we illustrate that yeasts are effective in enhancing probiotic bacterial viability in coffee brews, which may be useful in developing shelf stable probiotic food products.

Growth, survival, and metabolic activities of probiotic Lactobacillus spp. in fermented coffee brews supplemented with glucose and inactivated yeast derivatives.

Amidst rising interest in non-dairy probiotic foods, and growing global coffee consumption patterns, it would be opportune to ferment coffee brews with probiotics, yet it remains unexplored. In this study, we aimed to develop a fermented coffee beverage rich in live probiotics, by supplementing nutrient-deficient coffee brews with glucose and inactivated yeast derivatives. This was followed by fermentation with single probiotic bacteria cultures (Lactobacillus rhamnosus GG, L. paracasei Lpc-37, L. plantarum 299v, and L. acidophilus NCFM), and subsequent storage at 4 and 25 °C. We demonstrated that nutrient supplementation was essential in supporting probiotic growth and survival in coffee brews, as viabilities above 7 Log CFU/mL could not be sustained longer than 2 weeks in non-supplemented coffees. In contrast, viabilities above 7 Log CFU/mL were maintained for 10 weeks by L. rhamnosus GG and L. paracasei Lpc-37 in supplemented coffees stored under refrigeration. Probiotic metabolic activities led to consumption of glucose, glutamic acid, and alanine, with simultaneous formation of lactic acid, 3-methylbutanoic acid, and diacetyl. Nevertheless, endogenous coffee volatiles, bioactive components, and antioxidant capacities were retained. Overall, we illustrate the potential functionalities of probiotic fermented coffee brews, arising from high probiotic live counts and retention of major coffee bioactive components.