Polyphenols for restoration of the intestinal microbiota

Abstract

The present invention relates to a potent prebiotic product based on polyphenols that exhibit stimulatory effects on S. boulardii growth, and inhibitory effects on the growth of C. albicans. Polyphenol compounds have been identified and the optimal concentration of pure polyphenol compounds that exhibit the most potent stimulatory effect on yeast growth has been determined. Caffeic, chlorogenic and cinnamic acids, as well as apigenin and luteolin, exert positive effects on the S. boulardii growth. Moreover, the synergistic effect of mixtures of polyphenolic compounds on S. boulardii has been demonstrated. The same mixtures almost completely inhibit the growth of C. albicans. The present invention discloses the selective stimulation of some and the inhibition of other types of microorganisms by certain polyphenol compounds. The present invention provides prebiotic preparations containing polyphenol compounds, synbiotic preparations containing S. boulardii and polyphenol components, which provide wide possibilities for application in the food and pharmaceutical industry.

Description

[0001] POLYPHENOLS FOR RESTORATION OF THE INTESTINAL MICROBIOTA

[0002] TECHNICAL FIELD

[0003] The present invention belongs to the field of biotechnology. Particularly, the present invention is related to chemical compounds having significant prebiotic potential. In addition, it is related to the use of chemical compounds for modification of the fungal microbiota. More particular, the present invention relates to polyphenol compounds, which, in optimal concentration, provide growth stimulation of a probiotic yeast, Saccharomyces boulardii, as well as growth inhibition of a pathogenic yeast belonging to the genus Candida, such as Candida albicans.

[0004] TECHNICAL PROBLEM

[0005] The technical problem solved by the present invention is how to improve the probiotic effect and the growth of yeast, Saccharomyces boulardii. Another technical problem solved by the present invention is how to inhibit the growth of a pathogenic yeast belonging to the genus Candida, such as Candida albicans. Further technical problem solved by the present invention is how to restore the balance of the intestinal microbiota, and at the same time provide stimulation of S. boulardii and inhibition of Candida albicans growth.

[0006] BACKGROUND OF THE INVENTION

[0007] Probiotics are live microorganisms that have positive effects on a host’s health. Saccharomyces cerevisiae van boulardii (S. boulardii) is the only probiotic yeast, which is characterized by a good safety profde. It is clinically proven that S. boulardii can be used for the prevention of gastrointestinal diseases, including travelers' and antibiotic-induced diarrhea, and that it has a positive effect on inflammatory bowel disease, and Helicobacter pylori eradication, among other conditions. The mechanisms by which S. boulardii exerts positive effects on human health include improvement of the integrity of the intestinal barrier, its immunomodulatory properties, as well as competition with pathogens, and the synthesis of antimicrobial peptides. Despite the genetic closeness to the S. cerevisiae yeast, the strains are different, and only S. cerevisiae var boulardii has probiotic activity. S. boulardii has unique phenotypic characteristics and physiological properties and is characterized by a greater resistance to the conditions in the gastrointestinal tract, a different optimal temperature range (37°C vs 30°C for S. boulardii and S. cerevisiae, respectively), as well as survival in an environment with a lower pH value (Pais P, et al., (2020). Saccharomyces boulardii: What Makes It Tick as Successful Probiotic? J. Fungi 6(2), 78.).

[0008] Studies have shown that S. boulardii exhibits an inhibitory effect on the growth of pathogenic bacteria and yeasts. It has been proven that the probiotic yeast inhibits the growth, adhesion, and biofilm formation of C. albicans by secreting capric acid, and it is assumed that the interactions between S. boulardii and Candida fungi contribute to the establishment of healthy, balanced microbiota (Mukherjee, P.K., et al., (2015). My cobiota in gastrointestinal diseases. Nature Reviews Gastroenterology & Hepatology, 12(2), 77-87.).

[0009] In addition to the use of probiotics in the regulation of the microbiota structure and the exhibition of positive effects on human physiology, dietary interventions in the form of prebiotic preparations and dietary modifications have great potential in the prevention and treatment of chronic diseases, as well as maintaining the stability of microbiota in different niches (e.g. gut, skin, vaginal, oral). Prebiotic is a substrate that is selectively utilized by host microorganisms conferring a health benefit.

[0010] Polyphenols represent the largest group of plant secondary metabolites, and they are most abundant in fruits, vegetables, cereals, coffee, tea, herbs, and medicinal plants. There is a large amount of data on the positive effects of polyphenol intake on human health, and among the most significant characteristics of these compounds are antioxidant and anti-inflammatory activity, antimicrobial, antitumor, cardioprotective, neuroprotective, and immunomodulatory activity (Kumar Singh, A., et al., (2019). Beneficial Effects of Dietary Polyphenols on Gut Microbiota and Strategies to Improve Delivery Efficiency. Nutrients, 11(9), 2216).

[0011] In order to improve the probiotic effect of yeast, Saccharomyces boulardii, and optionally, at the same time inhibit the growth of a pathogenic yeast belonging to the genus Candida, such as Candida albicans, the chemical components of polyphenols are identified, and their optimal concentrations for achieving the most potent prebiotic effects are determined. A synbiotic is a mixture comprising live microorganisms and substrate(s) selectively utilized by host microorganisms that confer a health benefit on the host. A product containing identified polyphenols may be used as a prebiotic product. Moreover, a product containing identified polyphenols in combination with S. boulardii may be used as a synbiotic product. Both products contribute to the host’s health by maintaining the balance of intestinal microbiota in addition to the direct effects on the host (antioxidant, immunomodulatury etc).

[0012] SUMMARY OF THE INVENTION

[0013] The present application discloses the identification of polyphenol compounds present in significant quantities in extracts of medicinal plants, especially yarrow, which exert a stimulatory effect on the growth of the probiotic yeast S. boulardii. Namely, the addition of phenolic acids such as, caffeic, chlorogenic, and cinnamic acid, and flavonoids such as apigenin, luteolin, and their glycosides has demonstrated positive effects on the growth of the yeast S. boulardii. Moreover, the addition of a mixture of polyphenolic compounds, particularly from the group of phenolic acids, i.e. chlorogenic and caffeic acid in the optimal concentration exhibits a synergistic effect on the growth stimulation of S. boulardii. Furthermore, a high degree of S. boulardii growth stimulation was also observed after the addition of flavonoid compounds, such as apigenin, to the mixture of phenolic acids.

[0014] The polyphenol compounds when applied individually suppressed the growth of the pathogenic fungus Candida albicans. Moreover, the application of mixtures of the polyphenol compounds exhibited a synergistic inhibitory effect on the growth of C. albicans. Some mixtures exhibited complete inhibition of the growth of the pathogenic yeast. Importantly, mixtures of the polyphenol compounds demonstrated a stimulatory effect on the growth of S. boulardii. Moreover, the present invention demonstrated both the stimulatory effect on the growth of probiotic yeast as well as the inhibitory effect on the growth of C. albicans, in the mixed culture of S. boulardii and C. albicans. The present application discloses the selected polyphenol compounds with the most potent prebiotic effect, mixtures thereof, and their optimal individual concentrations for modifying the Saccharomyces! Candida growth in pure culture and co-culture. The polyphenol compounds disclosed in the present application may be used in medicinal products for restoring the balance of the microbiota. Particularly, the present invention discloses synbiotic preparations comprising the probiotic yeast S. boulardii and the prebiotic polyphenol components, which synbiotic preparations may be further used in the food and pharmaceutical industry but also in agriculture.

[0015] In the present invention, chemical compounds from the group of polyphenols with stimulatory effects on the growth of the probiotic yeast S. boulardii are disclosed for the first time. The selected polyphenols’ inhibitory effects on the growth of C. albicans are also disclosed for the first time. The present invention discloses the synergistic effect of a mixture of polyphenolic compounds on the growth of probiotic yeast. Results from several studies have shown that the individual polyphenol compounds applied in optimal concentrations stimulated the growth of S. boulardii by a maximum of 3.97-fold compared to the control, while the growth stimulatory potential of mixtures comprising the polyphenol compounds has shown a 20-fold increase. Additionally, the application of the mixtures comprising the polyphenol compounds resulted in, in some cases, complete inhibition of C. albicans growth in individual cultures, as well as in the mixed culture with probiotic yeast S. boulardii.

[0016] Moreover, the present disclosure provides a standardized preparation, i.e., a preparation with precisely defined components and their weight ratios within the preparation. Obtaining the standardized preparations was not possible so far using naturally derived materials as starting components, such as yarrow extract.

[0017] Thus, the present invention provides the standardized preparation, for use as prebiotic and / or synbiotic. Due to the stimulative effect of these polyphenol compounds on the growth rate of S. boulardii, this procedure could be used also for the efficient production of live biomass in a liquid broth.

[0018] DETAILED DESCRIPTION OF INVENTION

[0019] The present invention describes the influence of polyphenol compounds on the growth of the yeasts S. boulardii, S. cerevisiae, and C. albicans and the identification of compounds with the most potent prebiotic potential. For the identification of polyphenol compounds with a stimulatory effect on the growth of S. boulardii, and an inhibitory effect on the growth of C. albicans, commercially available polyphenol compounds were used. Polyphenols were selected based on the fact that they are present in yarrow in significant quantities. Yarrow polyphenols that were selectively utilized by S. boulardii were previously identified by the HPLC-GC method as phenolic acids.

[0020] The study was carried out using ten polyphenolic substances predominantly present in yarrow, using a concentration range from 3.12 to 50.00 pg / mL. The polyphenol substances include phenolic acids: cinnamic, chi orogenic, caffeic, and benzoic acids, and flavonoids: cyanidin chloride, luteolin, luteolin 7-O-glycoside, apigenin 7-O-glycoside, apigenin, and kaempferol. Phenolic acids and flavonoids were added to the microbiological medium for yeast growth, individually and in combinations. Thus, the product for restoring the balance of the intestinal microbiota according to the present invention must contain at least one polyphenol selected from the group containing caffeic acid, chlorogenic acid, cinnamic acid, apigenin, apigenin -7- glucoside, luteolin, luteolin-7-glucoside, and kaempferol, or the combination thereof.

[0021] The yeast growth density on a microbiological medium with polyphenol compounds was compared to the control sample (containing only the microbiological medium). The results are shown as the ratio of the concentrations of microorganisms, expressed as CFU / mL, in the medium containing phenolic compounds, and in the control sample.

[0022] For the first time, the stimulatory effect of particular polyphenol compounds on the growth of S. boulardii was demonstrated, where the degree of stimulation depended on the concentrations applied. The highest degree of growth stimulation was achieved using caffeic and chlorogenic acid in lower concentrations (<12 pg / mL), and the results indicate a dose-dependent effect of the tested acids. The results of testing the influence of polyphenol compounds on yeast growth indicate a significant degree of S. boulardii growth stimulation after the addition of apigenin, luteolin, and their glycosides. In addition, a synergistic effect of various combinations of polyphenol compounds has been demonstrated.

[0023] The prebiotic effect of polyphenols on the growth of different yeast strains such as S. cerevisiae and S. cerevisiae var. boulardii was also examined. The results indicated the existence of significant differences in the achieved effect. Moreover, contrasting the stimulation of S. boulardii growth, the lower or no stimulatory effect and even the inhibitory effect of a mixture of polyphenol compounds on the growth of S. cerevisiae was observed. Examination of the antimicrobial activity of yarrow polyphenols directed to the growth of the pathogenic yeast C. albicans confirmed their potential in suppressing the growth of this opportunistic pathogen. The inhibitory activity of polyphenols was enhanced by the application of mixtures of polyphenols, with the complete inhibition of the growth of C. albicans frequently reported in repeated experiments.

[0024] Polyphenol compounds according to the present invention exhibit a selective growth stimulation of the probiotic yeast S. boulardii, with a parallel growth inhibition of C. albicans. In general, a greater degree of growth stimulation of S. boulardii and growth suppression of the pathogenic yeast C. albicans was observed after the application of lower concentrations such as 6.25 pg / mL of polyphenol compounds compared to higher concentrations, such as 50 pg / mL.

[0025] Further, a difference in the effect of glycosylated flavonoids compared to non-glycosylated apigenin and luteolin was also observed, thus indicating the importance of choosing an adequate form of a polyphenol compound that can be used in prebiotics. Particularly, apigenin 7-0- glycoside showed a less significant effect on the growth stimulation of S. boulardii compared to the aglycone component, and also a glycosylated form of apigenin had positive effects on the growth of C. albicans. Based on the observed data, it is evident that both prebiotic and antimycotic activities depend on glycosylation.

[0026] The present application discloses the use of prebiotic preparations based on polyphenols, and a synbiotic product containing S. boulardii and polyphenols. Prebiotic preparations based on polyphenols in combination with probiotic S. boulardii yeast may be used for restoring the balance of the microbiota and inhibiting the growth of the fungus C. albicans. Also due to the stimulative effect of the polyphenols on the growth rate of S. boulardii, this principle can be used for more efficient production of alive biomass of S. boulardii in liquid broth supplemented with chosen polyphenols.

[0027] 1. Yeast growth stimulation test

[0028] The effect of the addition of polyphenol compounds, which are present in yarrow in significant amounts on the growth of S. boulardii (Saccharomyces cerevisiae var. boulardii HANSEN CBS 5926), S. cerevisiae, and C. albicans ATCC 24433 was tested using tryptone soy broth (TSB) as a liquid growth medium. TSB was prepared by adding 6 grams of commercially available yeast extract powder (Torlak, Serbia) to commercially available TSB powder (Torlak, Serbia). Growth stimulatory activity was assessed using the dilution method and plating the microorganisms on tryptone soy agar (TSA) plates made by adding 2% agar to TSB. For the dilution series, a previously prepared physiological solution using Sodium chloride (Centrochem, Serbia) was used. Growth media was finally prepared by boiling and sterilization by autoclaving at 120°C for 30 minutes. TSA substrates were dried at 37°C for 24 hours before inoculation.

[0029] The effect on yeast growth was tested by using commercially available polyphenol compounds in concentrations of 3.12, 6.25, 12.50, 25.00, and 50.00 pg pure compound / mb TSB, and pure TSB was used as a control. The prepared liquid media and the control were inoculated with freshly grown yeasts until a final concentration of ±5 x 106CFU / mL was reached. Upon overnight incubation at 37°C for 24h for S. boulardii and C. albicans, and S. cerevisiae dilution series were prepared. Plates were seeded by adding 10 pl dilution series to 1 square cm of surface and were incubated at 37°C for 24h for S. boulardii and C. albicans, while S. cerevisiae was incubated for 48h at 30°C, and then colonies were counted. The growth in mixed yeast cultures was performed using HiCrome™ Candida differential agar, and the results were read after 48 hours of incubation at 37°C. The experiments were carried out in minimum duplicate (biological replicate), and the results are shown as the degree of yeast growth stimulation or inhibition which was calculated as the ratio between the growth on the test medium and the control sample and expressed as the mean value of the number of colonies with standard deviation.

[0030] The following examples are provided to illustrate the present invention.

[0031] Example 1 : Growth stimulation of S. boulardii yeast

[0032] Stimulation of S. boulardii growth by the addition of pure polyphenol compounds to the growth medium was tested by adding ten different polyphenol components at different concentrations ranging from 3.12 to 50.00 pg / mL and compared to the growth in the control sample (pure TSB). Of all the tested compounds, only benzoic acid did not stimulate the growth of probiotic yeast (Tables 1 and 2), while the degree of stimulation for the other polyphenols varied depending on the concentrations. The highest degree of stimulation was shown by caffeic acid (3.97 folds) when added at a concentration of 6.25 pg / mL, while chlorogenic acid showed the highest stimulation (3.28 folds) when added at a concentration of 12.50 pg / mL to the growth medium. Table 1. Growth density of S. boulardii in the TSB medium with the addition of three phenolic acids and benzoic acid Among the tested flavonoids: luteolin, apigenin, cyanidin chloride, kaempferol, luteolin-7- glucoside and apigenin-7-glucoside, apigenin and apigenin-7-glucoside showed the greatest growth stimulatory effect on S. boulardii (3.79 and 3.70-folds, respectively, Table 2). The optimal concentration of both apigenin and its glucoside was 12.50 pg / mL, although only a slightly lower level of stimulation was achieved at 6.25 pg / mL. The prebiotic effect was drastically reduced when these two compounds were added in higher concentrations. Luteolin and luteolin-7-glucoside also stimulated the growth of S. boulardii, and the addition of these polyphenols increased the density of S. boulardii by 2.28 and 3.20-folds, respectively. The addition of glucoside molecules had a strong effect on the prebiotic activity of luteolin, although the effect was only evident for the two lowest concentrations tested (3.12 and 6.25 pg / mL). The optimal concentration of luteolin was 12.5 pg / mL, while for luteolin-7-glucoside it was 6.25 pg / mL. The results of the experiment indicate that several flavonoids and phenolic acids can stimulate the growth of S. boulardii when given in adequate concentration. There was a strong effect of the concentration of flavonoids on the level of stimulation, although stimulation was observed for all tested concentrations (in the range from 3.12 to 50 pg / mL).

[0033] Kaempferol also exhibited a stimulatory effect on the growth of S. boulardii, but to a slightly lesser extent, and a maximum stimulation of 2.69-fold was achieved with a final concentration of 12.5 pg / mL (Table 2). Among the tested flavonoids, the prebiotic effect of cyanidin was the least. Although a 1.69-fold growth stimulation was evident when this compound was added to the growth medium at a concentration of 12.5 pg / mL, this was at the limit of significance (p-value 0.0595). The addition of cyanidin in other tested concentrations resulted in an even weaker effect, which suggests that this compound does not have a significant effect on the growth of S. boulardii.

[0034] Table 2. Growth density of S. boulardii in TSB medium with the addition of flavonoids

[0035] To examine the potential synergistic effects of polyphenols on the growth of S. boulardii, mixtures of polyphenol compounds were applied at concentrations in which the individual compounds exhibited the most potent stimulatory effects. The most potent stimulatory effect was achieved by a mixture containing caffeic acid 10-30 wt%, chlorogenic acid 30-50 wt% and apigenin or cinnamic acid 40-60 wt%. Moreover, the preferred ratio between caffeic acid, chlorogenic acid and apigenin or cinnamic acid is 1:2:2.

[0036] In one aspect, the product according to the present invention is a capsule containing between 100 and 1500 mg in total of one or more polyphenol that stimulates S. boulardii growth. In another aspect, the product according to the present invention is a capsule containing a mixture of polyphenols. Preferably, the capsule contains 200 mg of caffeic acid, 400 mg of chlorogenic acid, and 400 mg of apigenin. By keeping the same or similar mass ratio various product formulations are possible.

[0037] The results indicate that the combination of different compounds can exhibit both synergistic and antagonistic effects (Table 3). The highest, 5-fold, stimulation was observed after the addition of a polyphenol mixture containing caffeic (6 pg / mL) and chlorogenic acid (12 pg / mL). A slightly smaller effect was observed when apigenin or cinnamic acid was added to the mixture of caffeic and chlorogenic acid at a concentration of 12 pg / mL, while the mixture of all three phenolic acids: caffeic, chlorogenic, and cinnamic with apigenin resulted in suppression of S. boulardii growth. Moreover, the preferred ratio between caffeic acid, chlorogenic acid, or apigenin is 1 :2.

[0038] Table 3. Growth density of S. boulardii in TSB medium with the addition of combinations of different polyphenols with proven growth stimulations activity

[0039] Example 2: S. cerevisiae growth stimulation assay

[0040] The effect of the addition of pure polyphenol compounds on the growth of the industrial brewer's yeast Saccharomyces cerevisiae was investigated to determine whether there are differences in the growth stimulation of S. cerevisiae in relation to S. cerevisiae van boulardii. Although S. boulardii is a genetic variant of the S. cerevisiae species, many characteristics distinguish the probiotic from industrial yeast, such as survival in gastrointestinal conditions (S. boulardii survives low pH, gastrin, and bile acids).

[0041] In the experiment, the effect of polyphenols, such as caffeic acid, chlorogenic, and cinnamic acid, apigenin, apigenin 7-0 glucoside, cyanidin chloride, and luteolin on the growth of S. cerevisiae was tested. The results indicate a lower level of stimulation compared to that observed for S. boulardii yeast. All tested phenolic acids stimulated the growth of S. cerevisiae, but the level of stimulation was lower than that observed for S. boulardii. The highest degree of stimulation was recorded for caffeic and chlorogenic acids, but in higher concentrations compared to S. boulardii (Table 4). Table 4. Growth density of S. cerevisiae in TSB medium with the addition of three phenolic acids

[0042] The addition of four flavonoids: apigenin, apigenin 7-0 glucoside, cyanidin chloride, and luteolin showed non-consistent effects on the growth density of S. cerevisiae yeast, and the highest observed suppression was about 54%, while the highest stimulation of growth density was 90% (Table 5). These results indicate that the effects of flavonoids observed are specific to S. boulardii and cannot be applied for the entire species or the entire Saccharomyces genus. This is probably most clearly illustrated by the example of cyanidin chloride which stimulated S. cerevisiae at all tested concentrations, while it did not show a significant effect on the growth of S. boulardii at tested concentrations.

[0043] Table 5. Growth density of S. cerevisiae in TSB medium with the addition of four flavonoids

[0044] Differences in the effects of the addition of polyphenols on the growth of S. boulardii and S. cerevisiae were also observed when applying mixtures of selected polyphenols (Table 6). Surprisingly, all tested mixtures of polyphenols inhibited the growth of S. cerevisiae i.e., the growth was strongly suppressed in five of the six tested mixtures. Only in the presence of a mixture of caffeic acid (6 pg / mL) and chlorogenic acid (12 pg / mL) did S. cerevisiae grow at densities similar, albeit a bit lower, to those observed in the control microbiological medium. All other mixtures of polyphenols strongly suppressed the growth of S. cerevisiae. Regarding the response to the mixture of polyphenols, S. cerevisiae showed a behavior that was closer to that of C. albicans than that of S. boulardii. The obtained data provide evidence that polyphenols selectively stimulate the growth of S. boulardii and that their effect is strain-specific.

[0045] Table 6. Growth density of S. cerevisiae in TSB medium with the addition of combinations of different polyphenols with proven growth stimulations activity of S. boulardii

[0046] Example 3 : Growth assay of C. albicans

[0047] The effect of polyphenol compounds, which are present in yarrow extract in significant amount, on the growth of the pathogenic yeast C. albicans was investigated by the addition of seven different polyphenol components to the growth medium. In previous experiments, it was determined that benzoic acid did not stimulate the growth of probiotic yeast, and the effect of this compound was not tested on C. albicans. The results indicate the inhibition of the growth of C. albicans for most applied concentrations of phenolic acids, except for the application of cinnamic acid in concentrations of 3.12 and 12 pg / mL when a slight stimulation of yeast growth was recorded (Table 7). Caffeic and chlorogenic acid exhibited antimicrobial activity against the pathogenic fungus C. albicans for all tested concentrations, and the highest degree of growth inhibition was recorded when adding cinnamic acid at a concentration of 50 pg / mL (66% growth inhibition) and caffeic acid at a concentration of 3.12 pg / mL (62% growth inhibition).

[0048] Table 7. Growth density of Candida albicans in TSB medium with the addition of three phenolic acids

[0049] The results of testing the effect of the addition of four flavonoids to the growth medium on the cell density of the pathogenic yeast C. albicans indicate that the effect of the flavonoids was not strong (Table 8). Of all the tested compounds, only apigenin 7-0 glucoside stimulated the growth of C. albicans at all tested concentrations, while for the other tested compounds, the effect was concentration-dependent. Luteolin and apigenin showed a general trend of suppressing the growth of C. albicans, although at certain concentrations this effect was reduced. Apigenin present at a concentration of 12.5 pg / mL stimulated the growth of the pathogenic yeast but to a lesser extent compared to the stimulation of the probiotic yeast (1.44 vs. 3.79-fold stimulation). Table 8. Growth density of Candida albicans in TSB medium with the addition of four flavonoids

[0050] Examination of the effects of different combinations of yarrow polyphenols on the growth of C. albicans indicated a significant degree of suppression of the growth for all tested mixtures (Table 9). Polyphenol compounds were tested in concentrations that proved to be optimal for S. boulardii and a high degree of growth inhibition was constantly observed. Mixtures containing caffeic acid (6 pg / mL) and apigenin (12 pg / mL) exhibited complete suppression of C. albicans growth (Table 9). Table 9. Growth density of C. albicans in TSB medium with the addition of combinations of different polyphenols with proven growth stimulations activity of S. boulardii Example 4: Examination of yeast growth in mixed culture

[0051] The growth test in the differential HiCrome™ medium allowed monitoring of the effects of polyphenols in a mixed culture of C. albicans and S. boulardii. Phenolic acids and apigenin were used in concentrations with the most potent stimulatory effect on the growth of S. boulardii (Table 10). The results indicate a synergistic stimulatory effect of caffeic and chi orogenic acid, as well as caffeic and cinnamic acid on S. boulardii growth, with parallel significant suppression of C. albicans growth in a mixed yeast culture. Moreover, almost complete inhibition of growth was observed in a mixed culture, indicating the retention of the antimicrobial effect of polyphenols in more complex microbial communities.

[0052] Table 10: Growth stimulation test of C. albicans and S. boulardii yeasts mixed culture in differential HiCrome™ medium

Claims

Patent claims1. A product for restoring the balance of the intestinal microbiota containing one or more polyphenols selected from the group containing caffeic acid, chlorogenic acid, cinnamic acid, apigenin, apigenin -7-glucoside, luteolin, luteolin-7-glucoside, and kaempferol.

2. The product according to claim 1, containing 100 - 1500 mg of polyphenols.

3. The product according to claim 1 or 2, containing caffeic acid, chlorogenic acid, and apigenin or cinnamic acid.

4. The product according to claim 2 or 3, containing caffeic acid 10-30 wt%, chlorogenic acid 30-50 wt%, and apigenin or cinnamic acid 40-60 wt%.

5. The product according to claim 3, containing caffeic acid, chlorogenic acid, and apigenin or cinnamic acid in a weight ratio 1 :2:2.

6. The product according to claim 1 or 2, containing caffeic acid and chlorogenic acid or apigenin.

7. The product according to claim 6, containing caffeic acid and chlorogenic acid or apigenin in a weight ratio 1:2.

8. The product according to claims 1 to 7, further containing S. boulardii.

9. Use of the product according to claims 1 to 7 as a stimulant for S. boulardii growth in a liquid broth and the production of its biomass.

10. Use of the product according to claims 1 to 7 as a prebiotic.

11. Use of the product according to claim 8 as a synbiotic.

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