Abstract
Synbiotics have been intensively studied recently to improve gut health of humans and animals. The success of synergistic synbiotics depends on the compatibility of the prebiotic and probiotic components. Certain plant extracts possess both antimicrobial and prebiotic properties representing a potential use in combination with probiotics to improve the gut health. Here, we coined the term “prophybiotics” to describe this combined bioactivity. The current study aimed to select prebiotics that are preferred as an energy source and antimicrobial plant extracts which do not inhibit the growth, of six strains of lactic acid bacteria (LAB namely; Lactiplantibacillus plantarum, Lacticaseibacillus casei, Limosilactobacillus reuteri, Lacticaseibacillus rhamnosus, Leuconostoc mesenteroides, and Pediococcus pentosaceus) in-vitro to identify compatible combinations for potential synbiotic/prophybiotic use, respectively. Their growth kinetics were profiled in the presence of prebiotics: Inulin, Raffinose, and Saccharicterpenin with glucose, as the control, using carbohydrate free MRS broth media. Similarly, their growth kinetics in MRS broth supplemented with turmeric, green tea, and garlic extracts at varying concentrations were profiled. The results revealed the most compatible pairs of prebiotics and LAB. Turmeric and garlic had very little inhibitory effect on the growth of the LAB while green tea inhibited the growth of all LAB in a dose-dependent manner. Therefore, we conclude that turmeric and garlic have broad potential for use in prophybiotics, while the prebiotics studied here have limited use in synbiotics, with these LAB.
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Introduction
A healthy gut microbiome is largely responsible for maintaining innate immunity, gut barrier functioning as well as direct and indirect exclusion of pathogens (Diaz Carrasco et al. 2019). The use of probiotics, prebiotics, and synbiotics (prebiotics + probiotics) to improve gut health of humans and animal species has been studied and reported intensively in literature (as reviewed by Yadav et al. 2022). Lactic acid bacteria (LAB) have been intensively studied and are widely used as probiotics with a wide range of beneficial properties (Ljungh and Wadström 2006). Indeed, many species of LAB are listed in the updated list of qualified presumption of safety (QPS) recommended microorganisms by European Food Safety Authority (EFSA Biohaz Panel (EFSA Panel on Biological Hazards) et al. 2023) indicating the potential use of LAB in humans and animals safely.
According to the latest consensus statement by international scientific association for probiotics and prebiotics (ISAPP), based on the components and their functional role, synbiotics are divided into two main categories namely: complementary and synergistic (Swanson et al. 2020). A complementary synbiotic is a mixture of a probiotic and prebiotic chosen to act individually to improve gut health of the host while a synergistic synbiotic is a combination of live microorganisms which have beneficial effects on a host and a substrate which can selectively stimulate the growth and activity of the chosen microorganism. The selection of components in a complementary synbiotic is relatively easier given the fact that they are expected to affect the host individually. However, the selection of a components for a synergistic synbiotic requires more carefully planned studies to select the most compatible prebiotic that effectively improves the growth and functioning of the probiotic of choice (Quintero et al. 2022). Therefore, careful screening of components in a synbiotic development is crucial for its successful application. Thus, the first objective of the current study was to determine the effect of commercial oligosaccharide-based prebiotics (Inulin, Raffinose, and Sachcharicter penin) on the growth of six strains of LAB to identify the best combinations for potential synbiotic use in terms of in-vitro growth.
As an innovative approach to synergistic synbiotics with oligosaccharide based prebiotics, plant-based second-generation synbiotics have been reviewed by Sharma and Padwad (2020). Here, the authors address the problems of using conventional oligosaccharides including, supporting growth of non-beneficial bacteria, inconsistent observations in clinical endpoints, and lack of inherent bioactivity for improving the gut health (Bindels et al. 2015; Krumbeck et al. 2016) and propose plant-based polyphenolic substrates as a better companion for probiotics in synergistic synbiotics. Among these plant-based bioactives, turmeric (Curcuma longa) (Scazzocchio et al. 2020), green tea (Camellia sinensis) (Jung et al. 2017), and garlic (Allium sativum) (Chen et al. 2019) and did not inhibit the growth of L. acidophilus (Ilham et al. 2018), L. acidophilus A001F8, L. rhamnosus A001G8, L. paracasei A002C5, L. plantarum A003A7, and L. casei A003D4 (Kim et al. 2020). In addition to that, previous literature has also shown that turmeric in combination with Lactobacillus probiotics resulted in enhanced antimicrobial activity (Kim et al. 2020) and anti-allergic inflammatory activity (Yazdi et al. 2020) while improving poultry production parameters (Kinati et al. 2022). These studies along with our current results indicate that turmeric may be a potential candidate to use in combination with Lactobacillus species without affecting bacterial growth for potential prophybiotic application.
On the other hand, existing literature has shown that green tea modulates the composition of intestinal microbiota to improve overall gut health (Chen et al. 2019), while green tea in combination with probiotics reduced the high-fat-diet-induced inflammation in mice (Axling et al. 2012) and hepatorenal syndrome in rat model (Al-Okbi et al. 2019), indicating that green tea is an excellent candidate for prophybiotic application. However, our results demonstrate that supplementation of green tea extracts at higher doses can inhibit the growth of the LAB strains used. In contrast, Story et al. (2009) found that the growth of L. acidophilus and L. gasseri was increased even at higher concentrations of green tea supplementation. Moreover, several studies have reported that the count of Lactobacillus starter cultures in yoghurt is increased with green tea supplementation (Lim 2017; Marhamatizadeh et al. 2013; Najgebauer-Lejko 2014). Interestingly, Janiak et al. (2018) claim that the variation of effects of green tea on probiotic growth could be due to the composition of polyphenolic compounds. The authors reported that the catechins (monomeric flavan-3-ols) help to modulate the growth of microorganisms more selectively than the polymeric fraction in green tea. Proanthocyanidins will inhibit microbial growth more generally and efficiently. Therefore, these findings highlight the importance of performing the individual growth curves for selected probiotic strains with a particular green tea extract when selecting the combinations for potential prophybiotic application as the LAB strains used in the current study showed sensitivity to green tea at higher concentrations.
Our results indicated that garlic extract did not inhibit the growth of most LAB strains while it displayed prebiotic effects on some strains. Interestingly, garlic has been reported to have prebiotic effects particularly on Lactobacillus species (Lu et al. 2021; Sunu et al. 2019; Sutherland et al. 2009) and Bifidobacterium species (Zhang et al. 2013). However, some contrasting results have also been found in the literature. Altuntas and Korukluoglu (2019) and Booyens and Thantsha (2013) observed that garlic extracts display antimicrobial effects on L. acidophilus and Bifidobacterium species. However, in the latter study, the inhibition of probiotic growth by fresh garlic extracts (crushing garlic cloves) was significantly higher than that of garlic powder extract. The authors suggest that it is possibly due to the presence of more active allinase enzymes in fresh cloves when compared with the powdered garlic which will produce more allicin (the active antimicrobial compound) during the extraction process. Since powdered garlic has been used in the current study, it is possible that the allicin content in our garlic extract was less than that of the study of Booyens and Thantsha which resulted in inhibition of probiotics. However, in the same study, it was shown that the sensitivity of different probiotics to garlic extract varied. Therefore, it is also possible that the strains that we have tested in the current study are more resistant to antimicrobial effects of garlic. Therefore, it is imperative to focus on the content of the antimicrobial compounds in the phytobiotics when screening for potential prophybiotic combinations. Therefore, growth curve analysis of probiotics in each case is required to develop successful potential prophybiotics.
It is also important to highlight that the effect of the supplementation of these prebiotics and plant extracts may be different in different strains of the same LAB species owing to wide metabolic differences within the strains of LAB species. Nonetheless, considering the results of the strains used in the current study, prophybiotic formulation seemed promising as plant extracts used in the current study did not inhibit the LAB studied in two out of three species. Therefore, it shows the potential to use a mixture of these LAB along with plant extracts (turmeric or garlic) to optimize the beneficial effects on the gut health of the host. However, as the LAB were very selective in their ability to exploit the commercial prebiotics as their energy source, use of a mixture of LAB with commercial prebiotics, as a synergistic symbiotic, might not be possible due to this selectivity. Nevertheless, the main constraint of prophybiotic formulations is the differences among different cultivars or different extraction systems in terms of bioactive composition. Therefore, we suggest that more future research is necessary to elucidate the potential of prophybiotic formulation, minimizing these constraints.
Conclusion
Garlic and turmeric extracts displayed non-inhibitory effects for all LAB strains studied indicating their potential to use in prophybiotic formulations in the future. Nevertheless, the commercial prebiotics displayed the potential as an energy substrate limited only to particular LAB indicating a limited use of these prebiotics in synergistic symbiotic formulation with the LAB studied.
Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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Acknowledgements
We acknowledge IC Lab, Krakow, Poland, for providing the Hidex Sense microplate reader and JHJ sp z o.o., Poland, for providing lactic acid bacteria for our experiment. We also thank Ovobime project funded by National Science Centre, Poland, and Kaesler Nutrition GmbH, Cuxhaven, Germany for supplying us the prebiotics and plant extracts (turmeric and green tea) for this study.
Funding
This research was carried out under the funding from the European Union’s Horizon 2020 research and innovation program under grant agreement no. 955374.
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All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Ramesha N. Wishna Kadawarage. The first draft of the manuscript was written by Ramesha N. Wishna Kadawarage. Funding acquisition, supervision, review, and editing was by Rita M. Hickey and Maria Siwek. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
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Wishna-Kadawarage, R.N., Jensen, M., Powałowski, S. et al. In-vitro screening of compatible synbiotics and (introducing) “prophybiotics” as a tool to improve gut health. Int Microbiol 27, 645–657 (2024). https://doi.org/10.1007/s10123-023-00417-2
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DOI: https://doi.org/10.1007/s10123-023-00417-2