Ellagitannin Analysis Essay

1. Introduction

Despite the enormous efforts of the scientific and medical community, cancer still represents the second leading cause of death and is nearly becoming the leading one in the elderly [1]. It is estimated that, due to demographic changes alone, in the next 15 years the number of new cancer cases will increase by 70% worldwide [2].

The lack of effective diagnostic tools for early detection of several tumors, the limited treatment options for patients with advanced stages of cancer, and the onset of multiple drug resistance favor poor prognosis and high mortality rates. The significant, but still unsatisfactory, improvement of survival, the severe toxicity profile, and the high costs characterizing many current anticancer therapies clearly show that a threshold in terms of clinical benefit and patients’ tolerance has been reached. Thus, the identification and development of innovative, preventive as well as therapeutic strategies to contrast cancer-associated morbidity and mortality are urgently needed.

Epidemiological, preclinical, and clinical studies have generally concluded that a diet rich in phytochemicals can reduce the risk of cancer [2,3]. Due to their pleiotropism which includes antioxidant, anti-inflammatory, and antiproliferative activities as well as modulatory effects on subcellular signaling pathways, phytochemicals from edible fruits and vegetables are recognized as an effective option to counteract cancer incidence and mortality [3,4,5]. Plants constitute a primary and large source of various chemical compounds including alkaloids, flavonoids, phenolics, tannins, tocopherols, triterpenes, and isothiocyanates. Ellagitannins (ET) are an important class of phytochemicals contained in a number of edible plants and fruits recommended by the traditional medicine of a variety of cultures, both in the developing and developed countries, to treat common health problems. ET biological and nutraceutical potential has received increasing attention over the last several decades. ET exert multiple and clinically-valuable activities [4], and among them the chemopreventive, anticarcinogenic, and antiproliferative activities are being receiving growing interest and attention (Figure 1).

2. Dietary Sources, Types, and Occurrence

ET and their derivatives are noticeably contained in edible seeds, nuts, and various fruits of nutritional interests. The structures of relevant ETs and of ellagic acid are shown in Figure 2. A wide variety of fresh fruits including berries, like raspberries, black raspberries, strawberries, pomegranate, longan, and dried nuts, are renowned for their ample polyphenols concentration in the form of ET [5]. Five species of berries including raspberry, strawberry, cloudberry, rose hip, and sea buckthorn were identified by Koponen et al., [6] as significant carrier of ET in a range of 1–330 mg per 100 g of fruit. Sanguiin H-6 and lambertianin C were reported from Glen Ample raspberries and Scottish-grown red raspberries, along with some trace levels of ellagic acid [7,8]. Blackberries (fruit and seeds) have been reported for a range of ET including pedunculagin, casuarictin, sanguiin H-6 (lambertianin A), and lambertianin (C and D) [9,10,11]. Pomegranate and various fractions of the fruit are known for their cancer chemopreventive properties owing to their unique phenolics composition in the form of ET, which include punicalagin, punicalin, granatin A, granatin B, tellimagrandin I, pedunculagin, corilagin, gallagic acid, ellagic acid, and casuarinin [12].

ET, predominately those isolated from pomegranate (e.g., punicalagin), have gained a wide popularity as preventive and therapeutic ethnopharmacological approaches for cancer treatment. However, a lot more has been added to this class of compounds from fruits other than pomegranate, including raspberries, blueberries, strawberries, muscadine grapes, and longan [7,13,14,15,16,17,18]. Major phenolic fractions recovered from longan include gallic acid, ellagic acid, and corilagin, much more concentrated in the seed segment as compared to the fruit pulp and peel [17]. Good essential fatty acid composition of nuts and fairly high concentrations of ET and their derived fractions, such as ellagic acid and its glycosidic derivatives have been associated with the potential cardioprotective properties of nuts. Ellagic acid (free and total) has been reported in a range of 0.37–823 mg per 100 g of dried nuts [19]. High concentrations of a variety of ET (ellagic acid, sanguiin H2 and 6, lambertianin C, castalagin/vescalagin, galloyl-bis-HHDP glucose, pedunculagin) can be found in blackberries (Rubus sp.) [20]. Shi et al., [21] identified agrimoniin as the second highest phenolic compound of strawberries.

Irrespective of the edible fractions of fruiting plants, some inedible fractions like fruit peels, bark and foliage have also been reported as good source of hydrolysable tannins including bioactive ET [4,22]. Leaves extracts of Shepherdia argentea—a deciduous shrub commonly known as silver buffaloberry—were reported as a good reserve of gluconic acid core carrying the potential anti-HIV novel ET, such as hippophaenin A, shephagenin A and shephagenin B [23].

3. Ellagitannins—Classification and Chemistry

Tannins are unique secondary metabolites of plant phenolics with relatively higher molecular weight (300–30,000 Da) and bear the ability to generate complexes with some macromolecules, like proteins and carbohydrates [24]. Chemistry and nomenclature of the tannins is complicated by virtue of the frequent changes which parallel the advancement in this very specific field [25]. Taking into account different definitions of tannins [26,27], these compounds may be referred as either galloyl esters and their derivatives (ET, gallotannins, and complex tannins), or the oligomeric and polymeric proanthocyanidins (condensed tannins). In a broader perspective, tannins may be classified most satisfactorily and unambiguously on the basis of structural configuration and/or solubility [28]. C–C coupling of galloyl units in absence of glycosidically-linked catechin make ETs structurally different from the condensed tannins that are characterized by monomeric catechin linkages (C4–C8 or C4–C6) to generate oligomeric likewise polymeric proanthocyanidins [27]. Gallotannins and ETs constitute a major group of tannins i.e., hydrolysable tannins that are well known for their properties to hydrolyze into hexahydroxydiphenol (HHDP) or gallic acid moieties. Gallotannins are the gallic acid derivatives carrying ≥ six gallyol groups and might further be characterized on account of one or more than one digalloyl group [29].

ETs (hydrolysable tannins) on their hydrolysis yield gallic acid and ellagic acid from the compounds carrying gallyol groups and HHDP groups, respectively [28]. In vitro digestion models declare ETs to remain stable under the normal physiological condition of the stomach [30]. However, ETs hydrolysis to free ellagic acid or their degradation may proceed in the small intestine at neutral to alkaline pH [31]. Biologically, condensed tannins and gallotannins are thought to deliver relatively higher protein precipitation properties as compare to the ETs and hence are considered potential antinutritional compounds from the class of plants polyphenolics [32]. Gallotannins and condensed tannins have also been reported as oxidatively least active tannins as compared to the ETs and on the same time gallotannins and condensed tannins have also been found to reduce pro-oxidant properties of ETs [33,34].

3.1. Simple Ellagitannins

ET (M.W. 300–20,000 Da) are non-nitrogenous compounds with at least two C–C coupled galloyl units with no glycosidically-bonded catechin unit [3,35]. ET are derivatives of 1,2,3,4,6-penta-O-galloyl-β-d-glucopyranose (PGG). Structurally, ET are esters of carbohydrates and or cyclitols and also include metabolic compounds derived from oxidative cleavage of either condensed or hydrolysable tannins [27,35,36]. The presence of hexahydroxydiphenol (HHDP) in a glucopyranose ring in addition to acyl units and certain HHDP metabolites such as dehydrohexahydroxydiphenol (DHHDP), valoneoyl and chebuloyl groups constitute simple ET. Tellimagrandin I and II, pedunulagin, casuarictin, and chebulagic acid originate from the specific orientation and number of acyl groups on glucose units. Variation in HHDP group originates by linking (C–C or C–O) one or more galloyl groups to HHDP unit.

Structural diversity of ET has been reported to correlate with their carrier-plants’ taxonomy and evolutionary hierarchy [37]. More often, monomeric ET or oligomeric ET constitute the major tannic component of plant species. The monomeric compounds of the group include tellimagrandins I and II, pedunculagin, casuarictin, and potentillin. Type I hydrolysable tannins (i.e., gallotannins) carrying HHDP in stable conformation at either the 2,3 or 4,6 position on a d-glucopyranose may be referred to as a simple ET [38,39,40]. Geraniin, a type III ET, is another example of monomeric simple ET carrying a DHHDP unit linked to d-gluopyranose of HHDP unit via 1C4 conformation. Dimers of ET are generated by intermolecular oxidative coupling/condensation of simple ET.

3.2. Glycosidic Ellagitannins

Chemically, the C-glycosidic linkage of ET is established via intermolecular bonds between two monomeric units, one carrying anomeric carbon while the second one galloyl or HHDP group [3,35,41]. Most recently C-glycosidic ET including granadinin, vescalagin, methylvescalagin, castalagin, stachyurin, and casuarinin have been reported from the peel and seed fraction of camu-camu, a fruiting tree of Amazon rainforest [42]. Woody fractions of various fruits, particularly the nuts and berries, have also been observed to hold novel C-glycosidic ET (e.g., castalagin and vescalagin). Castacrenins D and F are two other forms of C-glycosidic ET isolated from the woody fraction of Japanese chestnut and carry gallic acid/ellagic acid moieties [43]. Treating vescalagin with Lentinula edodes generates quercusnins A and B that may be referred as fungal metabolites of C-glycosidic ET [44]. Castacrenins D and F isolated from chestnut wood may generate oxidative metabolites, namely castacrenins E and G, by replacing pyrogallol rings of C-glycosidic ET with cyclopentone rings [43]. Rhoipteleanins H, I, and J were reported as novel C-glycosidic ET isolated from the fruit and bark fractions of Rhoiptelea chiliantha. Structural configuration of rhoipteleanins H revealed the presence of cyclopentenone carboxy moieties that are generated by oxidation and rearrangement of C-glycosidic ET aromatic ring [45].

Condensate of C-glycosidic ET is another subclass of hydrolysable tannins, which includes rhoipteleanin J produced by the intermolecular condensation (C–C or C–O) of monomeric C-glycosidic ET followed by oxidation of aromatic rings of ET [45]. Wine aged in oak wood barrels is often reported to carry oak ET, particularly the condensation products of monomeric C-glycosidic ET. The studies infer C-glycosidic ET to play a significant role in modulation of organoleptic features of wine aged in oak wood barrels [46].

4. Ellagitannins Pharmacokinetics

A precise knowledge of phytochemicals’ pharmacokinetics is very important to exploit their health benefits, as well as the effects of their metabolites [47]. In vivo, ET, instead of being absorbed directly into the blood stream, are physiologically hydrolyzed to ellagic acid, which is further metabolized to biologically-active and bioavailable derivatives, i.e., urolithins, by the activity of microbiota in gastrointestinal (GI) tract [5,48]. The biological properties of ET, such as free radical scavenging, further depend on their metabolic transformation inside gut. ET recovered from pomegranate juice may be metabolically converted by gut microbiota to urolithin A, B, C, D, 8-O-methylurolithin A, 8,9-di-O-methylurolithin C, and 8,9-di-O-methylurolithin D, and some of these metabolites display higher antioxidant activity than the parental tannins themselves. For instance, urolithin C and D show an antioxidant capacity—as determined in a cell-based assay—which is 10- to 50-fold higher as compared to punicalagin, punicalin, ellagic acid, and gallic acid [49]. This finding suggests that intestinal transformation products of ET are likely to play a central role for the antioxidant properties at least inside the GI tract. Significant differences in urolithins’ profiles in individual human subjects feed on raspberries—a renowned source of ET—have been attributed to gut microflora, whose variations on an inter-individual basis affect their capacity of hydrolyzing ET and subsequent metabolite synthesis [48,50]. The interaction of gut microbiota composition and the host endogenous excretory system is also likely to play a further role in the observed inter-individual variability [51]. ET are highly stable under the acidic environment of stomach, and retain their composition without being hydrolyzed to simpler compounds when exposed to various gastric enzymes. Consequently the complex structure of ET impedes their gastric absorption: however, the stomach might serve as the first site of absorption of free ellagic acid and pre-hydrolyzed forms of ET.

Contrary to stomach, the neutral or alkaline environments of duodenum and small intestines, characterized by pH values ranging from 7.1 to 8.4, allow ET hydrolyzation [31,41]. In humans, ET are rapidly absorbed and metabolized, as documented by [18,52]: following ingestion of pomegranate juice (at a dose containing 25 mg of ellagic acid and 318 mg of ET), ellagic acid can be found in plasma for up to 4 h while, at later times, it is no more detectable. In contrast, another study reported that no ellagic acid could be detected in plasma during the 4 h following the juice intake [53], a discrepancy which has been attributed to inter-individual variability [54]. Ellagic acid is converted by catechol-O-methyl transferase to dimethylellagic acid, which is then glucuronidated and excreted [52].

Finally, the microbiologically metabolized fraction of ET,

1. Introduction

Ellagitannins constitute a complex class of polyphenols characterized by one or more hexahydroxydiphenoyl (HHDP) moieties esterified with a sugar, usually glucose. The HHDP group is biosynthetically formed through intramolecular, oxidative (C-C) coupling of suitably oriented neighboring galloyl residues in galloylglucoses. Ellagitannins exhibit very high structural variability due to the different ways in which HHDP residues can be linked with the glucose moiety, and especially due to their strong tendency to form dimeric and oligomeric forms [1,2]. High ellagitannin content has been reported for strawberries, cranberries, cloudberries, and red raspberries. The main raspberry ellagitannins are sanguiin H-6 (1871 g/mol) and lambertianin C (2805 g/mol). Both compounds are dimers and trimers of casuarictin (936 g/mol) respectively, which are formed by intermolecular C-O bonds between an HHDP group and a galloyl group on a neighboring monomer [1]. The content of these compounds in fresh raspberry fruit, depending on the cultivar, ranges from 360 to 750 mg/kg for sanguiin H-6 and from 280 to 630 mg/kg for lambertianin C [3]. Polyphenols, and especially ellagitannins and flavonoids, have a considerable antimicrobial potential. Many studies have shown that extracts from pomegranate and other plants rich in ellagitannins exhibit a significant inhibitory effect on Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Yersinia enterocolitica, Listeria monocytogenes, and Candida albicans, as well as on molds such as Alternaria alternata, Fusarium oxysporum, and Rhizoctonia solani [4,5,6,7].

Geotrichum candidum has been isolated from a variety of environments, including river water, air, food, and animal feed [8]. Being used in the food industry, this fungus has been subjected to numerous biotechnological studies in that area. While in the past G. candidum was usually found safe, with a low pathogenic potential, recently it has been reported to cause an increasing number of fungal infections. G. candidum is an opportunistic microorganism [9]. If present in food, this species may infect cancer patients treated with chemotherapy, HIV-positive individuals, those subjected to long-term antibiotic therapy or using corticosteroids, as well as diabetics [9,10]. Furthermore, the problem may be expected to further intensify with the increasing population of patients with immune deficiencies. Therefore, it seems necessary to seek safe antifungal compounds or preparations that could be used as natural preservatives in the food industry. The study presents the chemical characteristics of ellagitannins isolated from raspberry (Rubus idaeus L.) fruit and their in vitro and in situ antifungal activity against Geotrichum candidum.

2. Results and Discussion

2.1. Ellagitannin Identification in REP by LC-MS

Six ellagitannins were identified in the obtained raspberry extract (Table 1) in the process of gradient elution with polyphenols being purified on XAD 1600 resin. Due to the protocol applied, the extract was free of, amongst others, low molecular weight ellagitannins, ellagic acid and its conjugates, and other polyphenols, such as some anthocyanins and flavan-3-ols. Compounds with retention time of 14.57 min and 17.72 min gave a major [M − H] ion at m/z 1567.15 and a doubly charged ion at m/z 783.07. Fragmentation of the doubly charged ion gave a sequence of singly charged ions characteristic of sanguiin H-10 isomers. However, it should be noted that the first isomer fragmented to m/z 1235, which was associated with the loss of an HHDP (hexahydroxydiphenic acid) residue as described by Kähkönen et al. [11], while the second isomer fragmented to m/z 1265, which was linked to the loss of a galloylated glucose as described by Gasperotti et al. [3]. Despite the same mass, the two compounds have different structures, as reported by Gasperotti et al. [3].

The compound with a retention time of 15.13 was characterized by the presence of a doubly charged ion at m/z 1250, which fragmented to an ion at m/z 2200 losing an HHDP residue as well as to an ion at m/z 1867 losing a galloylated glucose. Based on the obtained fragmentation and literature data [3], this compound was identified as lambertianin C without one ellagic acid residue. Compounds with retention times of 19.25 and 19.75 exhibited the presence of a doubly charged ion at m/z 1401, which fragmented to m/z 1869, 1567, and 1235, indicating the presence of lambertianin C.

The occurrence of two forms suggests the presence of isomers of this compound, which were also observed by Hager et al. [12] and McDougall et al. [13]. In our case, the first compound was identified as an isomer of lambertianin C and the other one as the main (also in quantitative terms) lambertianin C found in raspberry fruit. The compound with a retention time of 20.57 min gave an ion at m/z 1869, which characteristically fragmented to m/z 1567, 1235, 935, 633, and 301, indicating the presence of sanguiin H-6. The identification of lambertianin C and sanguiin H-6 was also confirmed by comparing retention times and MS spectra of the isolated standards. The same fragmentation patterns have been reported by Gasperotti et al. [3], Hager et al. [12], and Mertz et al. [14].

2.2. Quantitative Composition of Phenolic Compounds in REP

Table 2 shows the quantitative ellagitannin composition of REP. In general, ellagitannins account for 92% of total phenolics. The other compounds, that is, flavan-3-ols and anthocyanins constitute 7.4% and 0.9% of total phenolics, respectively. The most abundant ellagitannins in REP are lambertianin C and sanguiin H-6, which collectively account for 93% of total ellagitannins (Figure 1), with the remaining ones being sanguiin H-10 isomers and lambertianin C derivatives. Ellagic acid, a product of ellagitannin hydrolysis, was not detected. The results show that the applied method of extraction (with 60% acetone) and purification (with XAD 1600 resin) leads to a product characterized by a high phenolic content, with more than 86% being hydrolysable tannins, including ellagitannins.

2.3. Inhibition of Geotrichum Candidum Growth by Ellagitannins

The strain Geotrichum candidum ŁOCK 0511 used in the presented study, had been isolated as a contaminant of non-pasteurized milk. In the ellagitannin antagonism test, this strain exhibited an inhibition zone of 10.3 ± 0.47 mm for REP and of 9.3 ± 0.47 mm and 8.3 ± 1.69 mm for LC and SH6, respectively. The MIC and MFC for REP were 10.0 mg/mL and 30.0 mg/mL, respectively, and were identical as those for the LC and SH6 fractions. REP, which is a complex preparation containing both lambertianin C (44.156 g/100 g) and sanguiin H-6 (35.917 g/100 g) (Table 3) exhibits similar antagonistic activity to the pure compounds isolated in this study (Table 3). In respect of chemical structure, sanguin H-6 is a dimer, while lamberitanin C is a trimer of casuarictin. Hence, taking into consideration their molar masses, in the MIC analysis we introduce the same mass but different amount of moles for both compounds. At the same time, the number of functional groups involved in interactions remains the same. Our research has not identified any link between the degree of polymerization or synergism of both compounds and the MIC parameter. This resulted in the same MIC parameter for the REP preparation, which included 80% of these compounds in total. It should also be noted that REP preparation also contained 7% of the flavanols, which in terms of the MIC parameter may compensate for lower ellagitannin content. Foss et al. [15] observed a similar effect, they concluded that crude extract from pomegranate and isolated compound punicalagin showed about the same value of MIC. In this case, punicalagin was the main substance of the crude extract. Moreover, application of the REP preparation in food or pharmaceutical industry for production of an extract rich in ellagitannins would be technologically easier and significantly less expensive than isolation of pure compounds. Therefore, in further study we used REP rather than individual ellagitannin fractions.

The antifungal activity of extracts from Rubia tinctorum, Carthamus tinctorius and Juglans regia against Geotrichum candidum was studied by Mehrabian et al. [16], who reported effective inhibition of fungal growth by aqueous and methanol extracts from Carthamus tinctorius (40 mm and 20 mm, respectively). However, Mehrabian et al. [16] did not specify the chemical characteristics of the plant extracts. In turn, Talibi et al. [17] studied the antagonistic activity of 43 preparations obtained from 36 plant species against Geotrichum candidum. One of the tested substances was a preparation from the leaves and shoots of Rubus ulmifolius Schott, with the MIC and MFC values of 0.3125 mg/mL and >5 mg/mL, respectively. Foss et al. [15] studied antifungal activity of crude extract and punicalagin from pomegranate against Trichophyton sp. and Microsporium sp. which are human dermatophytes. The authors determine the MIC values of crude extract of pomegranate for these dermatophytes are respectively 125 μg/mL and 250 μg/mL. Research conducted by Webster et al. [18] specified the antagonistic activity of water-based plant extracts (Heracleum maximum, Aralia nudicaulis, Glycyrrhiza lepidota, Fragaria virginiana, Populus tremuloides, Artemisia frigida, Artemisia campestris, Solidago gigantea, Alnus viridis, Potentilla simplex, Juniperus communis, Epilobium angustifolium, Betula alleghaniensis, Acorus calamus) against 23 human fungal pathogens belonging to the genera: Candida, Saccharomyces, Cryptococcus, Trichophyton, Microsporum, Epidermophyton, Aspergillus, Fusarium, Rhizopus. Researchers proved that the antifungal activity of plant extracts is individuated and it depends on the extract and the test strains. For instance, for the Fragaria virginiana extract, the MIC parameters were from 400mg/L in case of Trichophyton mentagrophytes, Microsporum canis, Epidermophyton floccosum, Aspergillus fumigatus to 50000 mg/L for Aspergillus flavus, Fusarium saloni strains. In the analysis provided by Thirach et al. [19], the antifungal activity of an ethanol extract of Acorus calamus was assessed against 28 clinical isolates of Candida sp. using a broth microdilution technique. The MIC was identified as 28,800 ± 16,320 mg/L, and Acorus calamus was interpreted in this setting as being a potential fungistatic agent.

2.4. The Fungistatic Activity (FA) of Ellagitannins and the Linear Growth Rate (T) of Filamentous Fungi in Their Presence

The fungistatic activity (FA) of ellagitannins (REP) and the linear growth rate (T) of the strain Geotrichum candidum ŁOCK 0511 in their presence was determined (Table 4). In the experiments, REP concentrations were selected based on prior studies concerning MIC and MFC.

The FA of the raspberry ellagitannin preparation against G. candidum increased with ellagitannin concentration in the growth medium. A REP concentration of 10 mg/mL led to FA of 66.4% after 24 h of incubation, which was maintained on day 6. An ellagitannin concentration lower than MIC (1.0 mg/mL) resulted in FA of 24.7% after 24 h of incubation and 13.8% on day 6. In the study of Tabei et al. [17], the FA of a preparation from Rubus ulmifolius Schott at a concentration of 5 mg/mL was 15%. The studied preparation (from Rubus idaeus L.) at a concentration of 5 mg/mL exhibited FA ranging from 49.4% to 54.5%, depending on incubation time (Table 4). The linear growth rate (T) for Geotrichum candidum ŁOCK 0511 ranged from 51.9 mm/day for an ellagitannin concentration of 1.0 mg/mL to 16.2 mm/day for 10.0 mg/mL. In contrast, in the control samples T was from 56.0 mm/day to 61.5 mm/day (Table 4). The T values obtained for G. candidum indicate effective fungal inhibition and are statistically significant with respect to the control sample.

2.5. In Situ Effect of Ellagitannin Preparation

Hydrolysable tannins are known to effectively inhibit the growth of Gram-negative bacteria, such as Salmonella sp., E. coli, Helicobacter pylori, Campylobacter sp. and Gram-positive bacteria: Staphylococcus sp., Clostridium sp., and Bacillus sp. [20]. In contrast, they do not inhibit Gram-positive bacteria, such as Lactobacillus sp., or do so only to a limited extent [21,22,23]. This gives the opportunity to use ellagitannin preparations in functional foods, especially those containing probiotics. This fact was at the core of the design of in situ tests for the inhibition of a food-contaminating fungus. The selected matrix was milk fermented by the probiotic strain Lactobacillus acidophilus ŁOCK 0842. The contaminant, Geotrichum candidum, was a pleomorphic filamentous fungus, often termed yeast-like [24]. Experiments were conducted in two variants: the ellagitannin preparation (REP) was added either prior to lactic fermentation (FMREP) or after it (FM + REP). During storage, the control exhibited the presence of Geotrichum candidum ŁOCK 0511 at a level of 5 log units (LU) throughout the storage period (Figure 2). In the FMREP sample, G. candidum counts decreased by 2 LU (to approx. 3 LU) on day 5 of storage and no live cells of this fungus were detected on day 15. In the FM + REP sample, G. candidum counts declined to 1.5 LU on day 5 of storage and no live fungal cells were detected on day 15, similarly to the FMREP sample.

Both samples supplemented with ellagitannins (FMREP and FM + REP) exhibited the same effect, that is, the absence of live G. candidum cells in 1 mL of fermented milk after 15 days of refrigerated storage. Nevertheless, in the FM + REP sample, to which the ellagitannin preparation was added following fermentation, G. candidum counts were lower by 1.5 LU than in the FMREP sample as early as on day 5 of storage. In the case of FMREP, the ellagitannin preparation was added before the process of milk fermentation. Lactobacills acidophilus

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