In vitro fermentation of raffinose to unravel its potential as prebiotic ingredient
Graphical abstract
Introduction
The loss of food functional properties due to the current industrial processing and the growing changes on people modern lifestyles associated with unhealthy eating behaviors are among the negative consequences of the globalization impact (Adamberg et al., 2014). As a result, the global epidemic of diet-related diseases had become a major public health issue (WHO - World Health Organization, 2018). However, the consumer awareness has increased towards the importance of using food as preventive medication, being willing to pay more for healthier eating options (Forbes, 2015). Thus, bioactive compounds produced from a natural source are currently preferred to modern therapeutic agents (Samanta et al., 2015). In particular, the use of bioactive compounds, such as prebiotics, has been shown to be very attractive given their role on modulating the gut microbiota and the subsequent beneficial effects on human and animal health over different physiological functions. The link between gut microbiota health and several different disorders, e.g. disbyosis caused by antibiotics intake or food poisoning, has been the recent focus of increased research (Kang et al., 2019; Yeung, Mocan, & Atanasov, 2018).
Recently updated by the International Scientific Association for Probiotics and Prebiotics (ISAPP), the current definition establishes a prebiotic as “a substrate that is selectively utilized by host microorganisms conferring a health benefit”, and the host can be either human or animal (Gibson et al., 2017). Prebiotic beneficial effects have been reported on the gastrointestinal tract, cardiometabolism, skeletal system, nervous system, among others (Gibson et al., 2017; Saad, Delattre, Urdaci, Schmitter, & Bressollier, 2013).
The global market demand of prebiotic is in expansion, registering 623.5 kton in 2015 and growing at a compound annual growth rate of 8.0% from 2016 to 2024 (Grand View Research, 2018). Prebiotic carbohydrates, particularly oligosaccharides, are the most widely studied and the best market established type of prebiotics. These compounds are also marketed beyond their traditional functional properties, being industrially used for instance as sugar and fat replacement, as well as taste and texture enhancement (Das, Choudhary, & Thompson-Witrick, 2019; Saremnezhad, Zargarchi, & Kalantari, 2020). Prebiotic oligosaccharides present favorable organoleptic properties, temperature and acidic stability which make them potential food ingredients (Zhao et al., 2017).
However, new potential prebiotic and less explored oligosaccharides have emerged as possible competitors, namely raffinose. Raffinose is an oligosaccharide composed by galactose, glucose, and fructose residues that can be hydrolyzed to d-galactose and sucrose by the enzyme α-galactosidase (α-GAL) (Tester & Karkalas, 2003). This trisaccharide is non-digestible to humans but can be metabolized by colon bacteria (Fernando et al., 2010), therefore being considered a prebiotic candidate. Furthermore, raffinose can be produced from cheap and abundant agro-residues such as soybean waste, holding a promising environmental and economic advantage.
Prebiotic oligosaccharides are used by the gut microbiota to growth and to produce short-chain fatty acids (SCFAs), lactate (a known precursor of SCFAs) (Flint, Duncan, Scott, & Louis, 2015), gases (mostly carbon dioxide and hydrogen), among other products (Topping & Clifton, 2001). The most common SCFAs being produced are acetate, propionate and butyrate, widely reported as key metabolites for intestinal health, benefiting also other body sites than the gut (Gibson et al., 2017).
Different approaches for testing the prebiotic potential of new compounds have been described. However, an in vitro model is considered a more suitable first-stage strategy to screen prebiotics candidates (Date et al., 2014), contrarily to in vivo studies which are expensive and time-consuming.
In agreement with the recent International Scientific Association for Probiotics and Prebiotics (ISAPP) guidelines, the in vitro models should include the use of fecal inocula and accurate high-throughput sequencing techniques to evaluate the microbial modulation. The prebiotic potential of the raffinose family of oligosaccharides has been reported using single or co-cultured microorganisms' models (Martínez- Villaluenga, Frías, Vidal-Valverde & Gómez, 2005; Ose et al., 2018). Nonetheless, in the light of ISAPP guidelines, these approaches cannot mimic the complex diversity of the intestinal microbiota (Gibson et al., 2017). Up to now, studies regarding the prebiotic effect of pure trisaccharide raffinose meeting the ISAPP requirements are scarcely reported. In this study, an in vitro batch culture model was used to evaluate the prebiotic potential of raffinose and compare it with commercial lactulose, using human fecal inocula of two adult healthy donors. Among the most widely studied and commercialized prebiotics, lactulose presents a chemical structure that better resembles raffinose, therefore comprising an interesting comparison model. Next generation sequencing techniques, namely Illumina MiSeq analysis was used to evaluate microbiota diversity. To the authors’ knowledge, this is the first study exploring the prebiotic potential of trisaccharide raffinose, used as a pure substrate and evaluated under an experimental approach compatible with the ISAPP guidelines.
Section snippets
Materials
All chemicals and media components used were of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO), including commercial raffinose, unless specified otherwise.
Fecal inoculum
Fecal samples were collected from two healthy human volunteers (Donor 1, coded D1 and donor 2, coded D2), both aged 26, who were on a non-specific Mediterranean diet, had no metabolic and gastrointestinal diseases, were non-smokers and had not received any antibiotics, pre- or probiotic supplements for at least 3 months
Substrate intake and production of lactate and short-chain fatty acids (SCFAs)
The total production of lactate and SCFAs (acetate, propionate and butyrate), and the residual substrate present in the fermentation medium are shown in Fig. 1. The fermentation of lactulose and raffinose at 10 g/L increased significantly the total amount of lactate and SCFAs (t-test student, α = 0.05) for both donors, as consequence of the selective growth-stimulation of SCFAs-producing bacteria (detailed in section 3.4). The results presented in Fig. 1 are in accordance with previous
Conclusions
The addition of trisaccharide raffinose increased the production of SCFAs and CO2, reducing the final pH of the medium and the ammonia concentration. Moreover, the presence of raffinose as substrate increased the relative abundance of Bifidobacterium and Lactobacillus species, while reducing Proteobacteria, including widely known pathogens, such as E. coli.
This study strongly suggests that raffinose holds potential prebiotic properties for human health and, subsequently, potential to be
CRediT authorship contribution statement
Cláudia Amorim: Investigation, Methodology, Writing - original draft. Sara C. Silvério: Investigation, Methodology, Writing - review & editing. Beatriz B. Cardoso: Investigation, Methodology. Joana I. Alves: Investigation, Methodology, Writing - review & editing. Maria Alcina Pereira: Funding acquisition, Resources, Validation, Writing - review & editing. Lígia R. Rodrigues: Funding acquisition, Resources, Validation, Supervision, Writing - review & editing, Project administration.
Declaration of competing interest
None.
Acknowledgments
CA an BBC acknowledge her grants (UMINHO/BPD/4/2019 and SFRH/BD/132324/2017) from Portuguese Foundation for Science and Technology (FCT). The study received financial support from FCT under the scope of the strategic funding of UID/BIO/04469/2020 unit; COMPETE 2020 (POCI-01-0145-FEDER-006684), through national funds and where applicable co-financed by the FEDER, within the PT2020 Partnership Agreement; the Projects FoSynBio (POCI-01-0145-FEDER-029549) and NewFood – Food Technologies Valorization
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2023, Food ChemistryCitation Excerpt :The flatulence-causing potential engendered the perception of α-galactooligosaccharides as undesirable components and urged numerous studies seeking approaches to remove them from soybean and other legume products (Liener, 1994). Nonetheless, α-galactooligosaccharides have been shown to exhibit potential prebiotic activity, such as reshaping bacterial composition by increasing the relative abundance of Bifidobacterium and Lactobacillus in in vitro and rodent models (Amorim et al., 2020; Xi et al., 2021). Consumption of α-galactooligosaccharides could lead to additional beneficial health effects, including decreasing total cholesterol and low-density lipoprotein cholesterol, as shown in mice fed with a high-fat diet (Chappuis et al., 2017; Dai et al., 2019) and reducing appetite and inflammation in overweight adults (Morel et al., 2015).