Germination Increases the Glucomoringin Content in Moringa Sprouts via Transforming Tyrosine.

Moringa seeds are an excellent dietary source of phytochemicals (i.e., glucosinolates, GSLs; isothiocyanates, ITCs) with health-beneficial effects. Although numerous studies have been conducted on moringa seeds, the effect of germination on the regulation of GSLs remains scarcely explored. The present study investigated the dynamic changes of GSLs in moringa seeds during germination (at 25, 30, and 35 °C for 6 days in the dark) through an untargeted metabolomics approach and compared the antioxidant capacity of ungerminated and germinated moringa seeds. Our results showed that germination significantly increased the total GSL content from 150 (day 0) to 323 µmol/g (35 °C, day 6) on a dry weight (DW) basis, especially glucomoringin (GMG), the unique glucosinolate in moringa seeds, which was significantly upregulated from 61 (day 0) to 149 µmol/g DW (35 °C, day 4). The upregulation of GMG corresponded to the metabolism of tyrosine, which might be the initial precursor for the formation of GMG. In addition, germination enhanced the total ITC content from 85 (day 0) to 239 µmol SE/g DW (35 °C, day 6), indicating that germination may have also increased the activity of myrosinase. Furthermore, germination remarkably increased the total phenolic content (109-507 mg GAE/100 g DW) and antioxidant capacity of moringa seeds. Our findings suggest that moringa sprouts could be promoted as a novel food and/or ingredient rich in GMG.

The thermal degradation of glucomoringin and changes of phenolic compounds in moringa seed kernels during different degrees of roasting.

The effect of heat treatment on the abundant bioactive compounds in moringa seed kernels (MSKs) during different degrees of roasting remains sparingly explored despite the flour of roasted MSKs has been incorporated into the human diet (e.g., cakes, cookies, and burgers) as a substitute to enrich the nutritional content. Therefore, we investigated the impacts of different roasting conditions (e.g., temperature and duration) on bioactive compounds (e.g., glucosinolates (GSLs), phenolic acids and alkaloids) and antioxidant capacity of MSKs. Our results showed that light and medium roasting increased the glucomoringin (GMG, the main GSL in MSKs) content from 43.7 (unroasted MSKs) to 69.7-127.3 µmol/g MSKs (dry weight), while excessive/dark roasting caused thermally-induced degradation of GMG (trace/undetectable level) in MSKs, resulting in the formation of various breakdown products (e.g., thiourea, nitrile, and amide). In addition, although roasting caused a significant reduction of some phenolic compounds (e.g., gallic, chlorogenic, p-coumaric acids, and trigonelline), other phenolic acids (e.g., caffeic and ferulic acids) and alkaloids (e.g., caffeine, theobromine, and theophylline) remarkably increased after roasting, which may contribute to the enhanced total phenolic content (up to 2.9-fold) and antioxidant capacity (up to 5.8-fold) of the roasted MSKs.

High-Fat Diet-Induced Decreased Circulating Bile Acids Contribute to Obesity Associated with Gut Microbiota in Mice.

The altered circulating bile acids (BAs) modulate gut microbiota, energy metabolism and various physiological functions. BA profiles in liver, serum, ileum and feces of HFD-fed mice were analyzed with normal chow diet (NCD)-fed mice after 16-week feeding. Furthermore, gut microbiota was analyzed and its correlation analysis with BA was performed. The result showed that long-term HFD feeding significantly decreased hepatic and serum BA levels, mainly attributed to the inhibition of hepatic BA synthesis and the reduced reabsorption efficiency of BAs in enterohepatic circulation. It also significantly impaired glucose and lipid homeostasis and gut microbiota in mice. We found significantly higher bile salt hydrolase activity in ileal microbes and a higher ratio of free BAs to conjugated BA content in ileal contents in HFD groups compared with NCD group mice, which might account for the activated intestinal farnesoid X receptor signaling on liver BA synthesis inhibition and reduced ileal reabsorption. The decreased circulating BAs were associated with the dysregulation of the lipid metabolism according to the decreased TGR5 signaling in the ileum and BAT. In addition, it is astonishing to find extremely high percentages of taurocholate and 12-OH BAs in liver and serum BA profiles of both groups, which was mainly attributed to the high substrate selectivity for 12-OH BAs of the intestinal BAs transporter during the ileal reabsorption of enterohepatic circulation. This study revealed a significant effect of long-term HFD feeding on the decreased circulating BA pool in mice, which impaired lipid homeostasis and gut microbiota, and collectively resulted in metabolic disorders and obesity.

Metabolomics analysis of okara probiotic beverages fermented with Lactobacillus gasseri and Limosilactobacillus fermentum by LC-QTOF-MS/MS.

In this study, okara was fermented with probiotic strains Lactobacillus gasseri LAC 343 and Limosilactobacillus fermentum PCC, respectively. Significant increases in cell count (by 2.22 log CFU/mL for LAC and 0.82 log CFU/mL for PCC) and significant decreases in pH (by 1.31 for LAC and 1.03 for PCC) were found in fermented okara slurry. In addition, strain LAC tended to produce amino acids, while strain PCC depleted most amino acids. An untargeted metabolomic-based approach using liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was used to further understand the compositional changes and potential health benefits by identifying bioactive metabolites in fermented okara slurry. We successfully identified various beneficial bioactive compounds including γ-aminobutyric acid, indolelactic acid, d-phenyllactic acid, and p-hydroxyphenyllactic acid which had differences in fold-changes in okara slurry fermented with different strains. Our study indicated the feasibility of using probiotics to ferment okara for novel functional food development.

Effect of oxygen supplement on post-mortem metabolic profile of shrimp during cold storage.

Shrimp, renowned for its exceptional nutritional value, holds a pivotal position within the realm of aquatic products. The supplementation of extra oxygen to shrimp throughout the entire supply chain has found application within the commercial seafood market. In this study, a dual-platform metabolic analysis, coupled with multivariate data analysis, was employed to discern the impact of supplementary oxygen. Furthermore, this approach facilitated the construction of the post-mortem metabolic profile of shrimp during cold storage. A noticeable decrease of alcohols, ketones and carbohydrates which are related to the energy metabolism in shrimp has been found during cold storage, compared to the fresh shrimp. The degradation of nutritional amino acids was alleviated in shrimp after 4 h of extra oxygen supplement. Furthermore, a higher concentration of identified fatty acids, integral to lipid metabolism and functioning as flavor compounds was observed in shrimp subsequent to oxygen supplementation. Therefore, the additional oxygen supplementation exerted influence on multiple metabolic pathways, including nitrogen metabolism, amino acid and peptide metabolism, nucleotide metabolism, carbohydrate metabolism, and lipid metabolism. This study has constructed a comprehensive post-mortem metabolic profile of shrimp during cold storage, thereby establishing a theoretical foundation for the utilization of oxygen supplements in the preservation of seafood.

Untargeted LC-QTOF-MS/MS-based metabolomics of spent coffee grounds alcoholic beverages fermented with Lachancea thermotolerans and Lactiplantibacillus plantarum.

Spent coffee grounds (SCG) is a solid waste generated from coffee brewing. We recently developed a novel SCG hydrolysate-derived alcoholic beverage fermented with Lachancea thermotolerans Concerto and Lactiplantibacillus plantarum ML Prime. To further understand the potential health benefits of the fermented SCG hydrolysate alcoholic beverage, an untargeted metabolomics-based approach (UPLC-QTOF-MS/MS) was applied to detect and identify bioactive metabolites especially low molecular weight compounds. Our results showed that, compared to yeast monoculture-fermented SCG alcoholic beverages, yeast-lactic acid bacterial coculture enriched the beverage with a range of bioactive compounds especially aromatic and branched-chain amino acid derivatives (e.g., 4-hydroxyphenyl lactic acid, phenyl lactic acid, indole lactic acid, (S)-(-)-2-hydroxyisocaproic acid, and 4-hydroxyphenyl ethanol). Although some endogenous phenolic compounds were metabolized during fermentation, many phenolic metabolites (e.g., vinyl phenols, dihydrocaffeic acid, 3,4-dihydroxybenzoic acid, 4-hydroxycoumarin) were produced. Our study provided a theoretical basis for further valorization of SCG hydrolysates from the health benefits point of view and the findings may be extended to other fermented products.

Brassica rapa subsp. Chinensis juice enhances Bacillus subtilis selectively in leafy green production.

Bacillus subtilis (BS) is a well-known beneficial microorganism for plants but is not competitive in the plant rhizosphere microbiome. We report the selective support of Brassica rapa subsp. Chinensis (Xiao Bai Cai) juice (XBCJ) on BS both in hydroponic nutrient solution and the plant rhizosphere of lettuce. After 2 weeks of being inoculated in the lettuce rhizosphere, the Bacillus population was enumerated at 3.30 ± 0.07 log CFU/unit in the BS group and at 5.20 ± 0.39 log CFU/unit in the BS + XBCJ group (p < 0.05). Accordingly, lettuce crops from the BS + XBCJ group were significantly higher than the control group for all of the tested biomass-related parameters (p < 0.05). The treatment did not significantly affect the texture, colour, moisture contents, total phenolic contents, or antioxidant activities of the lettuce crops (p > 0.05). Non-target ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) suggested that phenolic compounds could be the key class of phytochemicals being responsible for the selectivity. High-throughput RNA-based 16S rRNA gene sequencing and analysis were performed to depict the influence of BS and XBCJ over the global microbiome compositions of plant rhizosphere.

Transforming Spent Coffee Grounds' Hydrolysates with Yeast Lachancea thermotolerans and Lactic Acid Bacterium Lactiplantibacillus plantarum to Develop Potential Novel Alcoholic Beverages.

In the present work, the modification of spent coffee grounds (SCG) hydrolysate composition by mixed cultures of a non-Saccharomyces yeast, Lachancea thermotolerans, and a lactic acid bacterium, Lactiplantibacillus plantarum, as well as their interactions, were evaluated. It was found that L. plantarum inhibited the growth and survival of L. thermotolerans as compared with that in the yeast alone. On the other hand, the growth and survival of L. plantarum was slowed in sequential fermentation, but not in co-culture. Compared with co-culture, higher ethanol content, less residual sugars, and less acetic and succinic acids were found in sequential fermentation. In addition, lower amounts of caffeine and phenolic acids (e.g., ferulic, caffeic, and p-coumaric acids) were obtained in mixed (co- and sequential) cultures with corresponding levels of volatile phenols relative to the yeast monoculture. Moreover, co-culturing resulted in the highest contents of total alcohols (ethanol excluded) and total esters. Therefore, mixed culturing of L. plantarum and L. thermotolerans presented positive effects on the chemical constituents of fermented SCG hydrolysates, which might be a new alternative approach to valorizing the SCG into novel alcoholic drinks with different ethanol and flavor constituents.

Enzymatic acylation improves the stability and bioactivity of lutein: Protective effects of acylated lutein derivatives on L-O2 cells upon H2O2-induced oxidative stress.

The instability of lutein has limited its wide application especially in the food industry. In this study, enzymatic acylation of lutein with divinyl adipate was investigated. Three new acylated lutein derivatives, lutein-3-O-adipate (compound 1), lutein-3'-O-adipate (compound 2) and lutein-di-adipate (compound 3), were identified and their stabilities and bioactivates were evaluated. Notably, compounds 1-3 showed better thermal, light stability and stronger scavenging capacity to ABTS radical cation (ABTS+) and hydroxyl radical (OH). Most importantly, these acylated lutein derivatives exhibited excellent protective effects on L-O2 cells upon hydrogen peroxide (H2O2)-induced oxidative stress. In particular, the acylated lutein derivative termed compound 3 prevented cellular oxidative stress via restraining the overproduction of reactive oxygen species (ROS), thereby increasing related antioxidant enzymes activity and inhibiting apoptosis by mitochondria pathway. Our research provides important insights into the application of acylated lutein derivatives in food, cosmetic, and pharmaceutical products.

Integrated metabolomics analysis of Lactobacillus in fermented milk with fish gelatin hydrolysate in different degrees of hydrolysis.

Dual-platform metabolomics combined with multivariate data analysis was used to investigate the effects of adding fish gelatin (FGH) at different degrees of hydrolysis (DH) on the growth and metabolic pathways of different species of Lactobacillus in fermented milk. The results showed that the promotion effect of FGH on Lactobacillus was related to the species of probiotics. The corresponding metabolic pathways also changed, with the promotion of Lactobacillus by FGH mainly regulated through amino acid metabolism, lipid metabolism, and nucleotide metabolism pathways. The excess DH inhibited the growth of L. paracasei by adjusting its metabolic state through reducing nucleotide requirements, allocating protein resources, and adopting a stress response. In conclusion, this study revealed the effectiveness of dual-platform metabolomics in explaining the metabolic mechanisms of probiotics, providing theoretical support and a scientific basis for the development of functional fermented foods.

Elucidating the inhibitory mechanism on polyphenol oxidase from mushroom and melanosis formation by slightly acid electrolysed water.

It has been revealed that slightly acid electrolysed water (SAEW) could delay enzymatic browning and melanin formation in food. In this work, multi-spectroscopic methods and UHPLC-Q-TOF-MS were combined to study the underlying reason. The reversible mixed-type inhibition mode of HOCl (main components in SAEW) was determined. The ground state complex formation quenched the intrinsic fluorescence of polyphenol oxidase (PPO) and it was stable at lower temperature. The PPO conformational change (transformation from α-helix to ß-sheet) induced by SAEW was confirmed by 3D fluorescence and Circular dichroism (CD) spectrum. Moreover, the driving force of the interaction between HOCl and PPO was hydrogen bond, which was validated by the molecular docking result. Besides, the formation of melanin related compounds including dihydroxyphenylalanine (DOPA), dopaquinone, dopachrome, 5,6-dihydroxyindole-2-carboxylic acid (DHICA), 5,6-dihydroxyindole (DHI), and 5,6-indolequinone were significantly inhibited by SAEW treatment. These results demonstrated the potential of SAEW as a PPO inhibitor in the food industry.

Alteration of volatile compounds profile of brewers' spent grain by bath-ultrasonication and its combination with conventional water-bath and autoclave treatment.

The study aimed to investigate the capability of bath-ultrasonication and its combination with conventional water-bath and autoclave treatment in modifying the volatile composition of brewers' spent grain (BSG). It was hypothesized that the treatments modified the volatile composition of BSG due to the sonochemical modification. The results demonstrated that the treatments intensified the desirable odor and removed the undesirable one which might allow the possibility of masking and renewing the odor perception of BSG. Besides the influence on odor perception related compounds, it is worth to highlight that the treatments eliminated herbicidal compounds such as (E,E)-2,4-heptadienal and (E)-2-hexenal which might be present from herbicidal treatment. Combination of bath-ultrasonication with autoclave treatment modified the volatile aldehydes while its combination with conventional water-bath generated the same profile as it was in untreated BSG. Time elevation on bath-ultrasonication had no significant impact on the amount of ketones and alkanes, while the fluctuation occurred as an impact of thermal exposures. Moreover, the treatment reduced the amount of alcohol and increased the fatty acids. In conclusion, bath-ultrasonication and its combination with thermal exposure modified the volatile compositions of BSG.

Effects of sequential inoculation of Lachancea thermotolerans and Oenococcus oeni on chemical composition of spent coffee grounds hydrolysates.

Spent coffee grounds (SCG) disposal is an environmental problem. These residues from coffee brewing and instant coffee production have potential to produce novel alcoholic beverages. SCG valorization through sequential alcoholic and malolactic fermentation was investigated using a yeast, Lachancea thermotolerans Concerto and a lactic acid bacterium (LAB), Oenococcus oeni Lalvin 31 in this study. Our results showed that sequential inoculation prevented early death of yeast confronted when simultaneous inoculation was adopted, allowing for growth and persistence of both yeast and LAB till the end of fermentation. Adequate ethanol production (4.91 ± 0.13 %, v/v) with low residual sugar content was also attained. In addition, relatively lower levels of acetic, lactic, and succinic acids were produced by sequential inoculation than that of simultaneous inoculation. Furthermore, SCG hydrolysates fermented via sequential inoculation had the widest variety of volatiles (e.g. esters and ketones). Overall, our results indicated that sequential inoculation of L. thermotolerans and O. oeni in SCG hydrolysates might be a way to develop novel beverages with pleasant flavor profiles.

Fermented shellfish condiments: A comprehensive review.

Fermented shellfish condiments are globally consumed especially among Asian countries. Condiments, commonly used as flavor enhancers, have unique sensory characteristics and are associated with umami and meaty aroma. The main reactions that occur during fermentation of shellfish include proteolysis by endogenous enzymes and microbial activities to produce peptides and amino acids. The actions of proteolytic enzymes and microorganisms (predominantly bacteria) are found to be largely responsible for the formation of taste and aroma compounds. This review elaborates different aspects of shellfish fermentation including classification, process, substrates, microbiota, changes in both physicochemical and biochemical components, alterations in nutritional composition, flavor characteristics and sensory profiles, and biological activities and their undesirable impacts on health. The characteristics of traditional shellfish production such as long duration and high salt concentration not only limit nutritional value but also inhibit the formation of toxic biogenic amines. In addition, this review article also covers novel bioprocesses such as low salt fermentation and use of novel starter cultures and/or novel enzymes to accelerate fermentation and produce shellfish condiments that are of better quality and safer for consumption. Practical Application: The review paper summarized the comprehensive information on shellfish fermentation to provide alternative strategies to produce shellfish comdiments that are of better quality and safer for consumption.

Biovalorization of Market Surplus Bread for Development of Probiotic-Fermented Potential Functional Beverages.

Bread wastage is a growing concern in many developed countries. This research aimed to explore the biovalorization of market surplus bread for the development of probiotic-fermented beverages in a zero-waste approach. Bread slurries with different initial total solid contents were inoculated with probiotics Lacticaseibacillus rhamnosus GG (LGG) and Saccharomyces cerevisiae CNCM I-3856, alone and in combination. Our results showed that, of all percentages tested, 5% (w/w, dry weight) initial total solid content resulted in better growth of the probiotics and higher cell counts, while the texture of bread slurries with concentrations higher than 5.0% was too thick and viscous for bread beverage developments. In addition, the development of probiotic-fermented bread beverages was feasible on various types of bread. Furthermore, food additives (sweetener and stabilizer) did not affect the growth of LGG and S. cerevisiae CNCM I-3856 in both mono- and co-culture fermentation. During shelf life measurement, co-inoculation of LGG with S. cerevisiae CNCM I-3856 significantly improved the survival of LGG compared to the mono-culture at 5 and 30 °C, demonstrating the protective effects provided by the yeast. Our study suggests the potential of using market surplus bread as raw materials to deliver live probiotics with sufficient cell counts.

Dietary Organosulfur-Containing Compounds and Their Health-Promotion Mechanisms.

Dietary organosulfur-containing compounds (DOSCs) in fruits, vegetables, and edible mushrooms may hold the key to the health-promotion benefits of these foods. Yet their action mechanisms are not clear, partially due to their high reactivity, which leads to the formation of complex compounds during postharvest processing. Among postharvest processing methods, thermal treatment is the most common way to process these edible plants rich in DOSCs, which undergo complex degradation pathways with the generation of numerous derivatives over a short time. At low temperatures, DOSCs are biotransformed slowly during fermentation to different metabolites (e.g., thiols, sulfides, peptides), whose distinctive biological activity remains largely unexplored. In this review, we discuss the bioavailability of DOSCs in human digestion before illustrating their potential mechanisms for health promotion related to cardiovascular health, cancer chemoprevention, and anti-inflammatory and antimicrobial activities. In particular, it is interesting that different DOSCs react with glutathione or cysteine, leading to the slow release of hydrogen sulfide (H2S), which has broad bioactivity in chronic disease prevention. In addition, DOSCs may interact with protein thiol groups of different protein targets of importance related to inflammation and phase II enzyme upregulation, among other action pathways critical for health promotion.

Enzymatic treatment, unfermented and fermented fruit-based products: current state of knowledge.

In recent years, food manufacturers are increasingly utilizing enzymes in the production of fruit-based (unfermented and fermented) products to increase yield and maximize product quality in a cost-effective manner. Depending on the fruits and desired product characteristics, different enzymes (e.g. pectinase, cellulase, hemicellulase, amylase, and protease) are used alone or in combinations to achieve optimized processing conditions and improve nutritional and sensorial quality. In this review, the mechanisms of action and sources of different enzymes, as well as their effects on the physicochemical, nutritional, and organoleptic properties of unfermented and fermented fruit-based products are summarized and discussed, respectively. In general, the application of enzymatic hydrolysis treatment (EHT) in unfermented fruit-based product helps to achieve four main purposes: (i) viscosity reduction (easy to filter), (ii) clarification (improved appearance/clarity), (iii) better nutritional quality (increase in polyphenolics) and (iv) enhanced organoleptic characteristic (brighter color and complex aroma profile). In addition, EHT provides numerous other advantages to fermented fruit-based products such as better fermentation efficiency and enrichment in aroma. To meet the demand for new market trends, researchers and manufacturers are increasingly employing non-Saccharomyces yeast (with enzymatic activities) alone or in tandem with Saccharomyces cerevisiae to produce complex flavor profile in fermented fruit-based products. Therefore, this review also evaluates the potential of some non-Saccharomyces yeasts with enzymatic activities and how their utilization helps to tailor wines with unique aroma profile. Lastly, in view of an increase in lactose-intolerant individuals, the potential of fermented probiotic fruit juice as an alternative to dairy-based probiotic products is discussed.

Enzymatic acylation of lutein with a series of saturated fatty acid vinyl esters and the thermal stability and anti-lipid oxidation properties of the acylated derivatives.

Lutein was enzymatically acylated with saturated fatty acid vinyl esters of different lengths of carbon chain (C6 -C14 ) under the action of Candida antarctica lipase B (Novozyme 435). The acylation reaction was optimized by considering substrate molar ratio, reaction solvent, type of enzyme, and reaction time. The highest yield (88%) was obtained using the Novozyme 435 to catalyze the acylation reaction of lutein and vinyl decanoate (lutein/vinyl decanoate molar ratio of 1/10) for 16 h in methyl tert-butyl ether. Ten lutein esters were synthesized, isolated, and purified, which were characterized by Fourier-transform infrared spectroscopy, high-resolution mass spectrometry, and nuclear magnetic resonance spectroscopy. We found that the acylation of lutein improved its antioxidant capacity in lipid system and thermal stability. Our study extended the potential application of lutein in lipophilic food, cosmetic, and pharmaceutical industries. Practical Application: Enzyme acylation of lutein improved its antioxidant capacity in lipid system and thermal stability, extended its potential application in food, cosmetic, and pharmaceutical industries. In addition, our study also provided a new perspective and cognition for the further development and utilization of lutein.

Targeted and Nontargeted Metabolomics of Amino Acids and Bioactive Metabolites in Probiotic-Fermented Unhopped Beers Using Liquid Chromatography High-Resolution Mass Spectrometry.

Beer is one of the most popular beverages in the world. The increased popularity of craft beers has led to the development of unique beers that are alcohol-free, gluten-free, low calorie, or with functional properties through fermentation with probiotic microorganisms. In this study, functional unhopped beers were evaluated by utilizing probiotics (Lacticaseibacillus paracasei Lpc-37 and ibSium Saccharomyces cerevisiae CNCM I-3856) as starter cultures. The metabolites produced by probiotics were investigated using a nontargeted metabolomics approach and identified against metabolomics databases (Kyoto Encyclopedia of Genes and Genomes (KEGG), Human Metabolome Database (HMDB), Yeast Metabolome Database (YMDB), METLIN tandem mass spectrometry (MS/MS)). Derivatives of branched-chain (leucine) and aromatic amino acids (phenylalanine, tryptophan, and tyrosine) were enriched (one-way analysis of variance (ANOVA) p < 0.05) in probiotic-fermented unhopped beers, especially tryptophan metabolites. In addition, the synergistic effects of yeast-lactic acid bacteria (LAB) interactions led to further enrichment of higher acids such as (S)-(-)-2-hydroxyisocaproic acid, phenyllactic acid, hydroxyphenyllactic acid, and indolelactic acid. The potential pathways for the formation of novel bioactive tryptophan metabolites (indole and indoleacrylic acid) by LAB were elucidated. Altogether, probiotic LAB-fermented unhopped beer showed the highest antioxidant capacity and total phenolic content. This work provides the basis for the discovery of bioactive metabolites in probiotic-fermented foods.

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.