In vitro bioactivities of coffee brews fermented with the probiotics Lacticaseibacillus rhamnosus GG and Saccharomyces boulardii CNCM-I745
Graphical abstract
Introduction
Functional coffees, defined as coffees that have been modified to provide additional functional benefits beyond those that are inherent in coffee (e.g., caffeine), are gaining popularity amidst health and wellness trends (Euromonitor International, 2019). As a result, retail coffees fortified with protein, medium-chain triglycerides, and L-theanine have gained prominence in recent years (Euromonitor International, 2019). Within the category of functional coffees, probiotic coffees are lucrative additions, with the emergence of retail coffees fortified with probiotic Bacillus spp. spores (Dairy Reporter, 2021). Existing retail probiotic coffees are highly reliant on probiotic Bacillus spp. in the dormant spore form due to their remarkable resistance to environmental conditions, maintaining excellent viabilities at high temperatures (≥80 °C) encountered during coffee brewing (Majeed et al., 2019).
In contrast to Bacillus spp. coffee formulations which are non-fermented, we previously assessed the feasibility of fermenting coffee brews with vegetative forms of probiotics as starter cultures. Accordingly, coffee brews were fermented with the probiotics Lacticaseibacillus rhamnosus GG (formerly Lactobacillus rhamnosus GG) and Saccharomyces cerevisiae var. boulardii CNCM-I745 (Chan et al., 2020, Chan et al., 2021). In nutrient-scarce coffee brews, addition of glucose and inactivated yeast extracts was crucial to enable probiotic growth. Co-culturing with S. boulardii CNCM-I745 was also vital in maintaining L. rhamnosus GG populations above 7 Log CFU/mL for at least 14 weeks at 4 and 25 °C. Moreover, by using an untargeted metabolomic approach involving liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS/MS), we revealed that bioactive metabolites (e.g., 2-isopropylmalate, indole-3-lactate, 4-hydroxyphenyllactate, 3-phenyllactate, hydroxydodecanoic acid) were secreted by L. rhamnosus GG and S. boulardii CNCM-I745 during coffee brew fermentation. These bioactive metabolites have been reported by others to elicit wide ranging bioactivities such as antioxidant, anti-inflammatory, and anti-microbial properties (Chan, Lau, Lim, Li, & Liu, 2021).
In non-fermented probiotic Bacillus spp. coffees, in-situ metabolite production is unlikely due to the spore’s metabolically quiescent state (Bernardeau, Lehtinen, Forssten, & Nurminen, 2017). In contrast, in-situ bioactive metabolite secretion during coffee brew fermentation by L. rhamnosus GG and S. boulardii CNCM-I745 may further enhance coffee’s therapeutic benefits compared to non-fermented probiotic formulations. Indeed, extracellular secretions of proteins, motogenic factors, and polysaccharides (e.g., glucans and mannoproteins) by L. rhamnosus GG and S. boulardii CNCM-I745 have frequently been reported to exhibit in vitro and in vivo bioactivities. For example, cell-free extracellular supernatants of L. rhamnosus GG inhibited human colon cancer cell invasion (Escamilla, Lane, & Maitin, 2012), and maintained intestinal barrier integrity by preventing pathogenic Escherichia coli infection and attenuating inflammation (Wan et al., 2018, Wang et al., 2013). In parallel, cell-free extracellular supernatants of S. boulardii CNCM-I745 induced anti-proliferative activity and apoptosis in human gastric and colonic cancer cells (Chen et al., 2009, Pakbin et al., 2021), enhanced intestinal epithelial cell restitution (Canonici et al., 2012), and protected against Clostridium difficile toxin A-associated enteritis (Chen et al., 2006).
However, the bioactivities demonstrated in the above-mentioned studies are restricted to cell-free extracellular supernatants that were obtained after probiotic growth in laboratory-based media. Whether the same bioactivities can be obtained when L. rhamnosus GG and S. boulardii CNCM-I745 are grown in food matrices, particularly coffee brews, remains poorly studied. Furthermore, despite previously elucidating in situ production of bioactive metabolites by L. rhamnosus GG and S. boulardii CNCM-I745 during coffee brew fermentation (Chan, Lau, Lim, Li, & Liu, 2021), it remains unknown if production is translatable to additional health benefits compared to regular non-fermented coffee brews. For instance, coffee brews consistently display strong in vitro and in vivo activities (e.g., anti-diabetic, anti-cancer, anti-oxidant, and anti-inflammatory activities), attributed to endogenous bioactive components (e.g., caffeine, chlorogenic acids) (Hu, Wang, Zhang, & Qiu, 2019). Under these circumstances, beneficial effects arising from in situ production of bioactive metabolites may be masked by the inherently strong bioactivities of coffee brews.
Therefore, this study aimed to determine if probiotic fermentation of coffee brews would result in improved therapeutic benefits arising from in situ production of bioactive metabolites. This was achieved by comparing the in vitro health promoting properties of non-fermented coffee supernatants, against cell-free coffee supernatants fermented with the probiotics L. rhamnosus GG and/or S. boulardii CNCM-I745. In vitro properties studied were α-amylase and α-glucosidase inhibitions, anti-glycative properties, anti-proliferative activities, cellular antioxidant activities (CAA), and anti-inflammatory activities.
Section snippets
Preparation of freeze-dried coffee supernatants
Chemicals and reagents unique to this study are listed in Supplementary information A: Chemicals and reagents. Microbial cultivation and enumeration procedures have been described previously by Chan et al., 2020, Chan et al., 2021.
This study consisted of five treatment conditions: Non-supplemented and non-fermented coffee brews (N), supplemented and non-fermented coffee brews (S), supplemented coffee brews fermented with L. rhamnosus GG (S-GG), supplemented coffee brews fermented with L.
In vitro starch hydrolase inhibitory activities
To determine if in situ production of probiotic metabolites enhances the anti-diabetic effect of coffees, we evaluated the inhibitory activities of non-fermented and fermented freeze-dried coffee supernatants against α-amylase and α-glucosidase in Fig. 1.
All coffee supernatants exhibited dose-dependent inhibitions up to 28 mg/mL, beyond which no further improvements were observed. For α-amylase, inhibitions ranged from 21.7 to 59.5% at 28 mg/mL, with the strongest inhibitory effect observed for
Conclusion
This work demonstrates that although bioactive metabolites (e.g., indole-3-lactate, 4-hydroxyphenyllactate, 3-phenyllactate, 2-isopropylmalate) were produced by L. rhamnosus GG and S. boulardii CNC-I745 during coffee brew fermentation, the production did not translate to improved coffee in vitro bioactivities. By comparing our results to others who demonstrated strong bioactivities of cell-free supernatants of L. rhamnosus GG and S. boulardii CNC-I745 based on laboratory media, we have
CRediT authorship contribution statement
Mei Zhi Alcine Chan: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Visualization, Writing–original draft. Yuyun Lu: Methodology, Visualization, Writing–review & editing. Shao-Quan Liu: Conceptualization, Writing–review & editing, Supervision, Funding acquisition.
Declaration of Competing Interest
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: A patent application (PCT/SG2021/050028) has been filed for this research.
Acknowledgements
The authors would like to thank Professor Lih-Wen Deng for supplying cancer cells (MCF-7, HepG2, and HCT116) and RAW 264.7 cells. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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