Introduction

Various traditional and folk medicines have used many antimicrobial substances from such sources as microorganisms, animals and plants [1].

More recent interest in natural food ingredients has been an important factor in expanding studies on less known horticultural plants. Various berries containing high amounts of bioactive phytochemicals, which have noticeable long-term physiological effects, have been the focus of a number of recent studies [2, 3].

Generally, fruit extracts have been reported to exhibit antibacterial, antifungal and insecticidal properties under laboratory conditions [4, 5]. Moreover, fruits and berries are important dietary sources of such phenolic compounds as phenolic acids, flavonoids, stilbenes and lignans [6]. While little is known about the quantity of polyphenols consumed daily throughout the world, some information is available about the most studied group, the flavonols, the consumption of which has been estimated at 20–25 mg/day in the United States, Denmark and Holland [7]. In Italy, consumption ranges from 5 to 125 mg/day, with a mean value of 35 mg/day [8]. People who eat several servings of fruits and vegetables a day likely have a total polyphenol intake of about 1 g/day. Polyphenols have received much attention since being reported to have a positive influence on human health [9]. Epidemiological studies have repeatedly shown an inverse association between the risk of chronic human diseases and the consumption of a polyphenol-rich diet [10]. Polyphenol consumption limits the incidence of coronary heart diseases, as these substances are potent inhibitors of LDL oxidation, which is considered to be a key mechanism in the development of atherosclerosis [7].

Polyphenols may be protective against cardiovascular diseases because of their antioxidant, anti-platelet, and anti-inflammatory effects, and also because they increase HDL and improve endothelial function [11]. Moreover, other epidemiological studies and associated meta-analyses strongly suggested that long-term consumption of diets rich in plant polyphenols offered some protection against development of cancers, diabetes, osteoporosis and neurodegenerative diseases [12, 13].

Such fruits as grapes, apples, pears, cherries and berries contains up to 200–300 mg polyphenols per 100 g of fresh weight. In food, polyphenols may contribute to the bitterness, astringency, colour, flavour, odour and oxidative stability. The distribution of phenolics in plants at the tissue, cellular and sub-cellular levels is not uniform [14].

Among the polyphenols, the flavonoids are the most studied, mainly because a sub-group, the anthocyanins, have shown great bioactivity [14]. The anthocyanins exhibit a wide range of antioxidant protection and therapeutic benefits, including improved visual acuity and cognitive behaviour, as well as anti-inflammatory, antimicrobial and anti-age properties [15, 16]. Berry anthocyanins offer therapeutic and pharmacologic benefits, in particular, significant chemoprotective, anti-platelet aggregation and anti-angiogenic properties. Berry anthocyanins have been identified as novel cardioprotectants, beneficial in reducing age-associated oxidative stress, improving neuronal and cognitive brain function, as well as ocular health, and protecting genomic integrity [15, 17]. The purple-black fruits of elderberries (Sambucus spp. L.) are one of the richest sources of anthocyanins and phenolic compounds among small fruits [16]. Recently, extracts of both European, black or common elderberry (Sambucus nigra L.) and American elderberry (Sambucus canadensis L.) demonstrated significant chemopreventive properties, showing the potential to control enzymes commonly associated with various forms of cancer [18]. Because red grape (Vitis vinifera L.) contains a high amount of anthocyanins, which provide interesting colour, it is used in the food industry as an ingredient in healthy juices that are visually appealing to consumers [19]. The concentration and profile of anthocyanins in red grapes varies with species, variety, maturity, seasonal conditions, production area, viticultural practice and yield. Plums are the most numerous and diverse groups of fruit tree species [20], but the amount of research data does not reflect this abundance and there is only limited knowledge about anthocyanin composition in European plums (Prunus domestica L.).

In the production of probiotic functional foods, polyphenols do not interfere with the fermentation processes that take place in the food matrix, but they can play a key role in stimulating the metabolic activity of microorganisms, as well as improving viability in vitro and in vivo of probiotic starter cultures [21].

The majority of dietary anthocyanins are not absorbed at the upper GI tract, and hence reach the intestinal microbiota where they are biotransformed into their metabolites. This conversion is often essential for absorption and modulates the biological activity of these dietary compounds, not only due to the direct bioactivity of the products of microbiota metabolism, which is different from that of the parent compounds, but also because of their prebiotic activity in modulating the microbiota composition [22]. The phenolic influence on growth and viability of lactic acid bacteria was studied by Rodriguez and co-workers [23]. Only a few studies are available regarding the effect of polyphenols on starter and probiotic bacteria growth and viability. Lactobacillus casei, L. plantarum, L. fermentum, L. rhamnosus, L. acidophilus, L. bulgaricus, Bifidobacterium lactis, B. breve, B. infantis and B. bifidum [24] are the most studied probiotic species. In a study of the effect of phenolic compounds with antioxidant properties on L. casei, as a representative of probiotic microorganisms, it was shown that pure antioxidant compounds and fruit extracts had a stimulatory effect on its growth [25].

The first of three objectives of this study was to evaluate the total content of phenolics, anthocyanins, and flavonoids in extracts from Italian red grape skins, plum skins and elderberry parts, specifically the skin alone, the fruit (pulp, skin and seeds), and the skin and seeds together. Second, the antioxidant activity and inhibitory capacity of the five fruit extracts toward some pathogenic bacteria were evaluated. Third, the ability of the five fruit extracts to stimulate growth of some probiotic bacteria was tested in order to determine their prebiotic potential.

Materials and methods

Fruit extract preparation and characterization

The plums (Prunus domestica L.) and Italian red grapes (Vitis vinifera L.) were purchased from a local market, while the elderberry fruits (Sambucus nigra L.) were collected from the local botanical garden. Each fruit type was transported to the lab and manually peeled. Each type of fruit underwent washing, drying, lyophilizing and grinding, that is grape peel, plum peel, and three categories of elderberry parts. Specifically, they were washed three times with distilled water, dried at room temperature and then lyophilized using an ALPHA 1–4 LD plus Freeze Dryer (Martin Christ, Osterode, Germany). The freeze-dried products were ground using a mini-blender (Philips, Suffolk—Glemsford, England) and stored at − 20 °C.

The smell, colour and granule dimensions of the lyophilized fruit powders were noted, the latter two visually, and the water activity and moisture content were determined, the former with the use of an AQUA LAB 4TE Decagon Device (Hopkins Ct. Pullman, USA) and the latter with the use of an Ohaus Thermo-balance moisture analyser (Nanikon, Switzerland).

Ethanolic fruit extract

All fruit extracts were prepared using 70% ethanol to reach a final concentration of 250 mg/ml, then filtered and stored at − 20 °C.

Determination of total phenolic content

Total phenolic content was determined in aliquots of each fruit extract by a modified Folin-Ciocalteu assay [26], using gallic acid as reference standard. All spectrophotometric data were acquired using a Shimadzu UV–visible spectrophotometer (Shimadzu Europa GmbH, Duisburg, Germany). Absorbance was read at 765 nm and results were expressed as gallic acid µg/ml equivalents using a gallic acid standard curve.

Determination of monomeric anthocyanin content

Total monomeric anthocyanin content was determined spectrophotometrically using the pH-differential method [27]. Two salt solutions were employed, namely potassium chloride, pH 1.0 (0.025 M) and sodium acetate buffer, pH 4.5 (0.4 M), and the absorbance was read at 510 and 700 nm. Total anthocyanins were calculated using the following equation:

$$ {\text{Total}}\,{\text{anthocyanins}}\,({\text{mg}}/{\text{l}}) = \frac{{A \times M_{\text{W}} \times {\text{DF}} \times 10^{3} }}{\varepsilon \times 1}, $$

where A = (A 520nm − A 700nm)pH 1.0 – (A 520nm − A 700nm)pH 4.5, M W (molecular weight) = 449.2 g/mol for cyanidin-3-glucoside (cyd-3-glu), DF = dilution factor, l = path length in cm, ɛ = 26,900 molar extinction coefficient, in l × mol−1 × cm−1 for cyd-3-glu and 103 = factor for conversion from g to mg. Results of monomeric anthocyanins were expressed as cyanidin-3-glucoside equivalents in mg/l.

Determination of total flavonoid content

The total flavonoid content of the fruit extracts was determined using a modified colorimetric method [28]. Briefly, 0.25 ml of diluted fruit extracts were mixed with 1.25 ml of distilled water and, subsequently, with 0.075 ml of 5% sodium nitrite solution and allowed to react for 5 min. Then 0.15 ml of 10% aluminium chloride was added and allowed to further react for 6 min before 0.5 ml of 1 M sodium hydroxide was added. Distilled water was added to bring the final volume of the mixture to 3 ml. The absorbance of the mixture was immediately measured at 510 nm against a prepared blank using a Double Beam UV/visible UV-1601 spectrophotometer (Shimadzu, Kyoto, Japan). The flavonoid content was determined by a catechin standard curve and expressed as µg/ml catechin equivalents.

Antioxidant activity assay

Antioxidant activity was determined using a DPPH (2,2-diphenyl-1-picrylhydrazyl) method [29] in which samples were allowed to react with 3.9 ml of daily prepared DPPH solution for 90 min at room temperature, in a dark place. The absorbance (A) of the resulting solution was measured at 517 nm. Blank values were subtracted from samples and standard values and a linear regression for the Trolox standards were constructed. For the antioxidant activity, the inhibition (%) was calculated with the following formula: \( I(\% ) = \frac{{A_{\text{blank}} - A_{\text{sample}} }}{{A_{\text{blank}} }} \times 100 \). The higher the inhibition, the higher the antioxidant activity.

Antimicrobial activity against pathogens and probiotic bacteria

Pathogens/probiotic strains and growth conditions

Standard pathogenic bacteria were purchased from ATCC and DSM, while Listeria strain was isolated from foods by IZS (Istituto Zooprofilattico Sperimentale, Umbria-Marche, Italy). The probiotic strains Lactobacillus rhamnosus IMC 501® and Lactobacillus paracasei IMC 502® [30], their combination, named SYNBIO®, as well as Lactobacillus plantarum IMC 509 [31, 32], were provided by Synbiotec S.r.l. (Camerino, Italy). All the strains, their origins and cultural conditions are described in Table 1.

Table 1 Target strains used in this study, their origin, cultural and growth conditions
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Agar well diffusion method

Potential mechanisms involved in the inhibition of pathogenic bacterial growth were investigated by a well diffusion assay [32]. A concentration of 0.5 McFarland (1.5 × 108 CFU/ml) of each target strain was prepared, then inoculated on the surface of the specific media (Table 1), after which 10 μl of each fruit extract was added. The 70% ethanol was used as control sample. After incubation the inhibition zone was measured. The results are expressed as difference between the inhibition zone given by the fruit extracts (IZFE) and the inhibition zone given by the ethanol (IZEt), in order to have the exclusive effect of inhibition of just the active compounds extracted from the lyophilized fruit powder.

Minimum inhibitory concentration (MIC)

The initial concentration of the fruit extracts was of 250 mg/ml and a minimum concentration of 0.24 mg/ml was reached. MICs were determined by a modified version of the broth micro-dilution method [33].

Growth kinetics of probiotic strains

Experimental design

Investigation of the growth kinetics of each culture was conducted using a modified de Man, Rogosa, Sharpe broth (mMRS) fermentation by L. rhamnosus IMC 501®, L. paracasei IMC 502®, SYNBIO® and L. plantarum IMC 509, in the presence of the fruit extracts (10 g/l). MRS broth medium (Liofilchem, Roseto degli Abruzzi, Italy) containing glucose at a concentration of 10 g/l was used as positive control (CP). In particular, the maximum specific growth rate (μ) for each culture was calculated using the following equation:

μ = (InX 2 − InX 1)/(t 2 − t 1), where X 2 and X 1 are the cell concentrations at times t 2 and t 1. Mean doubling time (Td) was calculated as Td = In2/μ [34].

Optical density and pH determination

The optical density (OD) of each culture was monitored each hour using a Double beam UV/visible spectrophotometer at λ = 560 nm, while the pH was measured with a JENWAY 3510 pH meter (Stone Staffordshire, UK) every hour, in triplicate.

Determination of probiotic bacteria viability

The viability of probiotic bacteria was determined by the standard plate method and expressed as CFU/ml. For each sample, 500 μl were taken and serially diluted in saline solution. Ten-fold serial dilutions were used in counting bacteria by the pour plate technique in MRS agar, after aerobic incubation at 37 °C for 72 h.

Antioxidant and antimicrobial activities of new functional combinations

Preparation of fruit extract

To determine antioxidant and antimicrobial activity, the extracts of plum skin, Italian red grape skin and different parts of elderberries previously prepared were used in combination with several probiotic strains: L. rhamnosus IMC 501®, L. paracasei IMC 502®, their combination 1:1 (SYNBIO®) and L. plantarum IMC 509.

Preparation of cell-free supernatants of probiotic strains

Cell free supernatant (CFS) of the probiotic strains was prepared to be tested as follows. Lactobacilli were grown in MRS broth for 24–48 h at 37 °C. Each culture was centrifuged at 10,000 rpm for 30 min and filtered using a 0.20 μm porous membrane (Sigma, St. Louis, MO). In order to determine the antioxidant and antimicrobial activity of the Lactobacillus strains in combination with the fruit extracts, each CFS probiotic strain was mixed 1:1 with each fruit extract.

Antioxidant and antimicrobial activity

The antioxidant activity of each fruit extract, CFS of probiotic lactobacilli, and their combination 1:1 (fruit extract: probiotic CFS) was investigated by the DPPH method, described previously, while their bacterial-inhibitory activity was investigated by agar well diffusion method against all pathogen strains described in Table 1.

Statistical analysis

All experiments were carried out in duplicate and each sample was analysed in duplicate. The results were expressed as mean ± standard deviation (SD). For each experiment and where applicable, a negative and a positive control were prepared. Significant differences between mean values were determined by Tukey’s test after one-way analysis of variance (1-way ANOVA) using the GraphPad PRISM® 5.1 program. A P value less than 0.05 was considered statistically significant.

Results and discussions

Lyophilized fruit powder characterization

The visual description (colour and aspect), the water activity [35] and moisture content determination are shown in Table 2. Regarding the water activity, elderberry skin and seeds extract showed the lowest value (0.195), while the highest Aw was registered for the plum skin extract (0.412).

Table 2 Physical and chemical properties of the lyophilized fruit powders
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Determination of total phenolic content

The total phenolic content, expressed as µg of gallic acid per g of extract powder, was determined using a gallic acid standard curve and is presented in Table 3. Regarding the concentration of gallic acid in the extracts, the highest amount was shown in the elderberry skin extract (1005.02 µg/g), followed by the elderberry fruits and Italian red grape skin extracts, with a concentration in gallic acid of 474.95 and 275.39 µg/g, respectively. As for the plum skin and the elderberry skin and seeds extract, the concentration of the total phenolics did not exceed 122 µg/g.

Table 3 Concentration of total polyphenols (TP), total anthocyanins (TA), total flavonoids, antioxidant activity and TA/TP ratio of the fruit extracts
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Determination of monomeric anthocyanin content

The total monomeric anthocyanin of all the tested samples ranged between 42.15 and 381.14 μg/g of extract powder. The elderberry fruit extract had the highest concentration in cyanidin-3-glucoside (381.14 μg/g), followed by elderberry skin and Italian red grape skin extracts with a value of 284.28 mg/ml of cyanidin-3-glucoside and 236.95 μg/g, respectively. The plum skin extract showed the smallest content of monomeric anthocyanins (42.15 μg/g), as reported in Table 3.

Determination of total flavonoid content

The flavonoid content was correlated with the total phenolic content and ranged from 29.39 µg of catechin/g in the plum skin extract to 534.51 µg of catechin/g in the elderberry skin extract (Table 3).

Determination of antioxidant activity

The antioxidant activity of all fruit extracts was tested determining the anti-radical activity against free-radical DPPH. All extracts displayed antioxidant activity and the highest values were demonstrated by the elderberry fruit and Italian red grape extracts, with 90.19 and 89.86%, respectively (Table 3). The elderberry skin extract also provided very good antiradical activity against DPPH, with a percentage of 83.53%, while the plum skin extract, with a value of 35.01%, showed the lowest antiradical activity.

Antimicrobial activity against pathogens and probiotic bacteria

Agar well diffusion method

Figure 1 shows the antimicrobial activity of fruit extracts against several potential pathogen strains (Gram-positives, Gram-negatives and yeast). The antimicrobial activity of an active compound has been classified on the basis of the pathogen’s resistance or sensitivity, according to measurement of inhibition zone [36]: when IZ < 5 mm it is considered resistant (R), when IZ = 5–20 mm it is considered sensitive (S) and when IZ > 20 mm it is considered super-sensitive (SS).

Fig. 1
figure 1

Effect on growth of pathogenic and probiotic bacteria (Bc B. cereus DSM 345, Lprc L. paracasei IMC 502®, Lp L. plantarum IMC 509, Lrhm L. rhamnosus IMC 502®, Lm L. monocytogenes 306, Sa S. aureus ATCC 25923, SY-SYNBIO®, Ec E. coli ATCC 13706, Ca C. albicans ATCC 10261)

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As shown in Fig. 1, all extracts showed inhibitory activity against the potential pathogen strains tested. The extracts had very good growth inhibitory activity against B. cereus DSM 345, especially the elderberry skin and seeds extract, which had an inhibition zone higher than 18 mm. Regarding S. aureus ATCC 25923 and E. coli ATCC 13706, the extracts displayed almost the same inhibitory activity, the inhibition zone ranging between 10 and 15 mm. Except for C. albicans ATCC 10261, which was resistant to all fruit extracts, the other pathogens exhibited sensitivity when in contact with the fruit extracts.

Figure 1 also indicates that the fruit extracts seem to stimulate growth of probiotic strains. All the probiotics were stimulated in the presence of fruit extracts, except L. paracasei IMC 502®, which was slightly stimulated by the presence of extracts or, in some cases, such as elderberry skin and elderberry skin and seeds extracts, was slightly inhibited. The elderberry skin extract expressed the highest stimulatory effect on the growth of L. rhamnosus IMC 501®. The other fruit extracts also stimulated the growth of L. rhamnosus IMC 501®, except for elderberry skin and seeds extract, which instead expressed slight inhibition. In the case of L. plantarum IMC 509 and SYNBIO®, which is the 1:1 combination of L. rhamnosus IMC 501® and L. paracasei IMC 502®, there was a tendency to growth stimulation in the presence of fruit extracts.

Determination of minimum inhibitory concentration (MIC)

Table 4 shows the MIC of all 5 fruit extracts and ethanol tested to evaluate the inhibitory effect level on several microorganisms. Among the Gram-positive bacterial strains, B. cereus DSM 345 and S. aureus ATCC 25923 were highly sensitive to all three elderberry extracts and the Italian red grape extract, with values ≤ 15.63 mg/ml. Regarding Gram-negative bacteria, E. coli ATCC 13706 was sensitive to elderberry skin and Italian red grape skin extracts, with a MIC value of 7.81 mg/ml, while C. albicans ATCC 10261 was inhibited only by elderberry skin extract at a concentration of 3.91 mg/ml.

Table 4 Antimicrobial activity of fruit extracts on several bacterial strains expressed as minimal inhibitory concentration (MIC)
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Growth curve of several probiotic strains

Growth kinetics

Table 5 shows fermentation starting time, according to the fermentation process of probiotics, and all the growth curves followed the tendency of the positive control (glucose), in which the pH decreased as the lactobacilli grew and multiplied. Table 5 shows that all five fruit extracts can be used by the probiotic strains tested as prebiotic substrate, with behaviour similar to that of the positive control. Table 6 reports the mean doubling time (hours) of each probiotic strain and of the SYNBIO® combination when grown in mMRS enriched with one of the fruit extracts, that is, mMRS with elderberry skin, mMRS with plum skin, mMRS with elderberry skin and seeds, mMRS with elderberry fruits and mMRS with Italian red grape skin. Mean doubling time was used as a measure of the effectiveness of the polyphenol/anthocyanin-rich fruit extracts in modulating the growth rate. In general, doubling times of the tested probiotics in the presence of the fruit extracts were similar to those of the positive control.

Table 5 Fermentation starting time (h) of L. rhamnosus IMC 501®, L. paracasei IMC 502®, SYNBIO® and L. plantarum 509 in mMRS enriched with several fruit extracts
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Table 6 Doubling time of Lactobacillus strains in mMRS enriched with fruit extracts
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Determination of probiotic concentration

Probiotic bacterial concentration, expressed as CFU/ml, was monitored during the process of fermentation of all probiotics tested, using different modified media enriched with fruit extracts, as described above. Overall, the presence of fruit extracts improved the probiotic counts. In all cases, the bacterial concentration followed the same trend as the positive control (glucose) (Table 7).

Table 7 Viable counts (expressed as Log CFU/ml) of L. rhamnosus IMC 501®, L. paracasei IMC 502®, SYNBIO® and L. plantarum IMC 509 in mMRS containing different fruit extracts
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Antioxidant and antimicrobial activities of new functional combinations

Antioxidant activity

As illustrated in Fig. 2, the combined formulation with plum skin extract and all four probiotics showed higher antioxidant activity than did the extract alone. The same situation was also observed for the elderberry skin and seeds combined with the probiotic strains. Even if each individual probiotic strain demonstrated low antioxidant activity, the ability to increase the antioxidant activity of the plum skin extract and the elderberry skin and seeds extract was significantly higher than the extract activity alone.

Fig. 2
figure 2

Antioxidant activity of fruit extracts in combination with probiotics (filled square-Fruit extract alone, filled square-Fruit extract with L. rhamnosus IMC 502®, filled square-Fruit extract with L. paracasei IMC 502®, filled square-Fruit extract with SYNBIO®, filled square-Fruit extract with L. plantarum IMC 509)

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Antimicrobial activity

Applying the previous agar well diffusion method, the antimicrobial activity of the combinations of the five fruit extracts with each probiotic strains was analysed. The combination between probiotics and fruit extracts did not demonstrate potentiated/increased antimicrobial activity against any of the pathogen strains tested (data not shown).

Conclusions

Consumers are increasingly interested in foods that contain high levels of bioactive compounds that help prevent various diseases. Flavonoid compounds of plant origin have gained considerable attention due to their functional and nutritional benefits, including anti-inflammatory, antimicrobial activity and other salutary effects [37].

The results of this study provide enough evidence that fruit extracts that are rich in polyphenols, especially anthocyanins, have beneficial effects on human health. The fruit extracts were tested for their composition in bioactive compounds, such as polyphenols, flavonoids and anthocyanins. Among all the fruit extracts, the highest concentration in polyphenols and flavonoid compounds was found in elderberry skin extract, followed by the elderberry fruits and Italian red grape skin extracts. High concentrations of anthocyanins, expressed as cyanidin-3-glucosides, were found in elderberry skin and elderberry fruits extracts, followed by Italian red grape skin extract. Regarding the anthocyanin content, a lower concentration was found in plum skin extract and elderberry skin and seeds extract. It is known that the higher concentration in phytochemicals, especially anthocyanins, the higher the antioxidant activity. All the fruit extracts expressed strong antioxidant capacity, being able to combat the free radicals that are the major contributors to aging and diseases like cancer, heart disease, decline of the brain function and decline of the immune system. As expected, the highest antioxidant activity was expressed by the elderberry fruits, elderberry skin and Italian red grape skin extracts, followed by the extracts of elderberry skin and seeds and of plum skin.

All fruit extracts also revealed antimicrobial properties with an inhibitory effect against all the pathogenic strains tested, except for C. albicans ATCC 10261, which was resistant, given an inhibition zone less than 5 mm. The highest inhibition was observed in the cases of B. cereus DSM 345, S. aureus ATCC 25923 and E. coli ATCC 13706. The elderberry skin and seeds extract had a very good inhibitory effect against the growth of B. cereus, with an inhibition zone of almost 20 mm, thus making B. cereus almost super-sensitive in the presence of this extract. The fruit extracts had a moderate inhibitory effect against L. monocytogenes 306.

While all 5 fruit extracts had an inhibitory effect against pathogens, it was interesting and surprising to note that they had a stimulatory effect on the growth of the probiotic strains tested (L. rhamnosus IMC 501®, L. paracasei IMC 502®, their 1:1 combination, named SYNBIO® and L. plantarum 509). The highest stimulatory effect on growth was observed in the case of L. rhamnosus IMC 501® in the presence of elderberry skin extract. The fruit extracts did not have a good stimulatory effect on L. paracasei IMC 502®, but there was a satisfactory stimulatory effect on the SYNBIO® blend of L. rhamnosus IMC 501® and L. paracasei IMC 502®, specifically in the presence of fruit extracts rich in anthocyanins, the highest stimulatory effect being observed in the presence of elderberry fruit extract, followed by the Italian red grape skin extract.

To verify the real stimulatory effect of fruit extracts on the growth of probiotics, the growth curves of L. rhamnosus IMC 501®, L. paracasei IMC 502®, the SYNBIO® combination and L. plantarum 509 were determined in order to evaluate the prebiotic potential of the extracts. All fruit extracts were used as carbon source by the probiotics during the in vitro fermentation, thus their growth were stimulated in the presence of these anthocyanin-rich fruit extracts. Growth curves similar to those of the positive control were observed in the media enriched with Italian red grape skin extract for all probiotics tested and a similar trend was also observed in media enhanced with elderberry fruit extract. The pH followed the normal trend-line of fermenting lactobacilli, the values decreasing with the growth of probiotic cells.

When considering antimicrobial treatments for diseases, it is important to reflect on the possible impact upon beneficial microorganisms, because many antibiotic treatments cause a detrimental reduction in the natural microflora population, and thus the overpopulation of non-beneficial bacteria. This research demonstrates that the probiotic species have the resistance and, surprisingly, were stimulated by the fruit extract treatments, compared to pathogenic species. The results suggest that anthocyanins and their metabolites may exert a positive modulation on the intestinal bacterial population.

Therefore, the present findings indicate that anthocyanins appear to be stable in food matrices. With the goal of creating a functional food product or a synbiotic food formulation, the antioxidant capacity and antimicrobial activity of several probiotic strains in combination with several anthocyanin-rich fruit extracts were tested. The results demonstrate improved antioxidant potential of the probiotic-fruit extract combinations.

When it comes to pro-health food, maximal retention of bioactive compounds during processing is strongly desired, since the beneficial biological properties of food are to some extent due to their active constituents. Quantification of the anthocyanin profile provides useful information in the effort to optimize the fruit extracts, as well in the choice of processing techniques, storage methods and raw materials that best limit losses and changes, in order to improve the pro-health potential of these red fruit extracts.

The results of this study indicate that all five fruit extracts tested, in particular the elderberry extracts and the Italian red grape extract, have high phenolic (anthocyanin) content and elevated antioxidant activity. Further studies are needed to determine how anthocyanins from different fruit sources behave after consumption and to explore the relationship between a polyphenol/anthocyanin-rich diet and gut microbiota composition.

In conclusion, elderberry, like all red foods, can be considered quite beneficial for human health because of the antioxidant and antimicrobial compounds it contains.

In fact, the elderberry investigated here showed appreciable antioxidant activities and supported the growth of L. rhamnosus IMC 501®, L. paracasei IMC 502®, SYNBIO® combination and L. plantarum 509. Therefore, this variety of plant can be very useful in reducing free radical induced damage, enhancing immune-competence and reducing inflammation mediated pathologies.

While interesting and in some cases surprising, the results of this study have limitations, dictated by the in vitro testing, single culture microbial growth and inhibition experiments, and focus on chemical antioxidant capacity. They do not mimic the complex, competitive gut environment or take fully into account biological antioxidant activity.

An in vivo study on human subjects will follow to confirm these interesting results and to provide more information on the significant bioactive qualities of elderberry.