Evaluation of the Antimicrobial Efficacy of some Fermented Traditional Turkish Beverages with Probiotic Potentials
Evaluation of the Antimicrobial Efficacy of some Fermented Traditional Turkish Beverages with Probiotic Potentials
The first study to evaluate antimicrobial properties of isolates from five beverages against a broad spectrum of human microbial pathogens
Turkey is a home country for a good number of fermented beverages derived from milk, cereals, fruits and vegetables, and several studies have reported the probiotic potentiality of these beverages. Probiotics, otherwise known as beneficial microorganisms possess the ability to exert antimicrobial effects, which is one of the most important selection criteria for their use in commercial products. In the current study, the antimicrobial activities of potential probiotic bacteria isolated from five fermented traditional Turkish beverages (boza, kefir, ayran, shalgam juice and hardaliye) were evaluated. The bacterial isolates were morphologically characterised and genotypically identified by 16S rRNA gene sequence analysis. The antimicrobial effects of the isolates against selected human pathogens were assessed using spot-on-the-lawn and agar well diffusion assays. 18 of the 22 strains displayed varying degrees of antagonism against the tested pathogens. Amongst the isolates, the strongest antimicrobial effects were exhibited by strains from boza, kefir and shalgam which can be attributed to their greater microbiota diversity. Strain specificity in the activities of the obtained isolates and specificity with the different indicator pathogens tested was observed. The antimicrobial effects exhibited by boza, kefir and shalgam isolates offer a promising health benefit to consumers of these fermented probiotic products.
Fermented foods are defined as foods and beverages produced via controlled microbial growth, and by the conversion of food components through enzymatic actions (1). Fermentation enhances the preservation of foods as well as enables the transformation of raw materials into a new product with unique sensory properties (taste, aroma and texture), improved nutritional values and functional properties (2–4). Foods and beverages that are prepared via fermentation processes constitute an important part of human nutrition in virtually every food culture around the globe (3). Fermented or pickled fruits and vegetables, fruit juices, tea leaves, cereals, roots and tubers are very popular and widely consumed in many regions of Europe, Asia, the Americas, Africa and Middle East (5). Generally, several genera of lactic acid bacteria (LAB) including Lactobacillus, Streptococcus and Leuconostoc are predominant in fermented foods, but other bacteria as well as yeast and fungi also contribute to food fermentation (6). There are two main methods by which foods are fermented. First, foods can be fermented naturally, also known as ‘spontaneous ferments’ or ‘wild ferments’, a process whereby microorganisms are naturally present in the raw food ingredient or processing environment, for example, sauerkraut, kimchi and certain fermented soy products (7). On the other hand, foods can also be fermented by the addition of starter cultures, often referred to as ‘culture-dependent ferments’, for example kefir and kombucha (6). One method of preparing a culture-dependent ferment is ‘back-slopping’, a process in which a little amount of a previously fermented batch is inoculated into the raw food, for example sourdough bread (1).
With a surging interest in gastrointestinal health in recent years, the consumption of fermented foods containing live microorganisms has gained popularity as an important dietary strategy for improving human health (1, 7). This popularity may not be unconnected with the rising consumer awareness about the concept of probiotics. Probiotics (from Greek: meaning ‘for life’) are “Live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” (8). Human probiotic microorganisms mostly belong to the Lactobacillus, Bifidobacterium, Streptococcus, Lactococcus and Enterococcus genera (9). Furthermore, some strains of Gram-positive bacteria in the genus Bacillus as well as some yeast strains in the genus Saccharomyces are also commonly used in probiotic products (10). The Food and Agriculture Organization (FAO) and World Health Organization (WHO) (11), the European Food Safety Authority (EFSA) (12) and the US Food and Drug Administration (FDA) have stipulated different criteria such as safety, functionality, technological usefulness and the absence of the risk of acquired resistance to antibiotics for probiotic strain selection that would exert beneficial effects on human or animal health and could be used in the probiotics industry (9, 10, 13, 14). Generally, one of the most important properties among the selection criteria is that a probiotic strain must produce antimicrobial substances and be antagonistic to pathogens. Antimicrobial activity of probiotics may be manifested by one or a combination of the following mechanisms including: (a) competition for limited nutrients; (b) competition for adhesion sites; (c) synthesis of various antimicrobial metabolites such as organic acids, hydrogen peroxide, bacteriocins; and (d) inhibition of toxin production in pathogenic microorganisms (15, 16). For instance, LAB produce lactic acid as well as other organic acids; thus, lower the environmental pH and consequently inhibit the growth of bacterial pathogens (17, 18).
Although commercially-produced fermented foods usually serve as carriers for probiotic bacteria (6), that a particular food or beverage is produced by fermentation does not necessarily imply that it contains live microorganisms. For example, bread, wine, beer and distilled alcoholic beverages are produced by yeast fermentation, but the producing organisms are either inactivated by heat (in the case of bread and some beers) or are physically removed by filtration or other means (in the case of wine and beer). Moreover, many fermented foods are heat-treated after fermentation to extend shelf-life or enhance food safety. Hence, soy sauce, sauerkraut and other fermented vegetables are made shelf-stable by thermal processing. Even non-thermally processed fermented foods may notwithstanding contain low levels of live and viable microorganisms simply due to unfavourable environmental conditions which decrease microbial populations over time (6). Furthermore, it is also pertinent to note that the absence of live microorganisms or probiotics in the final fermented product does not preclude a positive functional role. Several studies have reported some mechanisms through which fermented foods may exert beneficial effects on health and disease whether they contain live microorganisms or not. For instance, food-fermenting microbes may produce vitamins or other bioactive molecules in situ or inactivate anti-nutritional factors and yet be absent at the time of consumption (6). Prebiotics and other components found in fermented foods may also exert health benefits (1, 19). In addition, fermentation-derived metabolites may exert health benefits such as in the case of LAB which generate polyamines and bioactive peptides in both dairy and non-dairy fermented foods with potential effects on immune, metabolic and cardiovascular health (20).
There are sufficient historical and scientific records that have shown Turkey to be an origin country for a decent number of fermented probiotic beverages (21). Moreover, several fermented traditional foods are produced in many regions of Turkey from products that are indigenous to particular regions. Kefir, ayran, boza, hardaliye and shalgam juice seem to be the most widely known fermented traditional Turkish non-alcoholic beverages. The first two products are produced from milk while the last three are obtained from cereals, fruits and vegetables respectively, and their microbiota is composed mainly of LAB (22).
Kefir is a smooth, slightly foamy, whitish, viscous, slightly acidic, slightly carbonated mildly alcoholic fermented beverage that is widely consumed in Turkey (23, 24). The name kefir is derived from the Turkish word ‘keyif’ meaning ‘joy, pleasure or good feeling’ to express the feelings experienced after drinking it (21, 23–26). Kefir can be produced by fermentation of different kinds of milk including cow, ewe, goat or plant-derived milk (26). It is traditionally produced by adding a starter culture known as ‘kefir grains’ to pasteurised milk. Kefir grains is a symbiotic consortium of lactic and acetic acids-producing bacteria as well as lactose-fermenting yeasts (for example, Kluyveromyces marxianus) and non-lactose fermenting yeasts (for example, Saccharomyces cerevisiae, Saccharomyces unisporus) housed within a polysaccharide and protein matrix called kefiran (25, 27). Kefir has been reported to be tolerated well by people with lactose intolerance and maldigestion as it contains β-galactosidase enzyme expressing organism (for example K. marxianus) which hydrolyses lactose, and thus reduces lactose concentrations in the beverage (7).
Ayran is an indigenous Turkish fermented milk beverage which is more or less a ‘national drink’ widely consumed across the length and breadth of Turkey (21). It is produced in either of two ways, i.e. by the addition of water to yoghurt (homemade) or by the addition of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus as starter cultures to standardised milk for fermentation (industrial production) (28). Basically, ayran is generated by blending yoghurt with 30–50% of water and 0.5–1% of salt (29).
Boza is a cereal-based fermented beverage produced by a combination of yeast and lactic acid fermentation of millet, maize, wheat, rye, rice semolina or flour and mixed with sugar or saccharine (30). It is a viscous liquid with a pale yellow colour and sweet slightly sharp to slightly sour taste which is widely consumed in Turkey due to its pleasant taste, flavour and nutritional properties (31). It is also popular and widely consumed in several Balkan countries on a daily basis (26, 32). In Turkey, boza is usually served alongside cinnamon powder and roasted chickpeas, and is generally considered to be beer’s ancestor (30). Boza is produced at both an artisanal and industrial scale (33). Its preparation typically involves six stages: milling of raw materials (cereals), boiling, straining, cooling, sugar addition and fermentation. A previously fermented boza is used as inoculum for the fermentation stage (21, 30, 31). Lactic acid fermentation by LAB produces antimicrobial metabolites such as lactic acid and increases acidity which supplies preservative effect whereas the metabolites from alcoholic fermentation by yeasts bring about the mouthfeel and odour of boza (26, 30, 31). Boza is a good source of fibre, carbohydrate, protein and vitamins including riboflavin, thiamine, niacin and pyridoxine (26, 30).
Hardaliye is a kind of fruit-based fermented non-alcoholic traditional beverage which originates from Thrace, the European part of Turkey, and is produced from red grape juice and 0.2% crushed black mustard seeds (34, 35). Hardaliye is mostly homemade by the traditional method. The ingredients are pressed and allowed to ferment at room temperature for 5–10 days in wooden or plastic barrels (22, 26, 33). Benzoic acid is sometimes added as a preservative especially at the industrial scale (26). Owing to its rich LAB flora, hardaliye has been described as a non-dairy probiotic beverage which aids digestion and also helps in the prevention of coronary heart disease (36). The nutritional value, functional properties and health benefits of hardaliye are derived from its ingredients and fermentation process. For example, grapes are rich in phenolic contents and provide strong antioxidant effects for the human body and thus, inhibit cancer cells formation, whereas the oils from mustard seeds exert medicinal effects on circulatory disorders, common cold and bronchitis as well as possess antimicrobial properties (34).
Shalgam juice (Şalgam) is a vegetable-based red coloured, cloudy and sour beverage produced by LAB and yeast fermentation of a mixture of black or purple carrots (Daucus carota), turnips (Brassica rapa), bulgur (broken wheat) flour, salt, sourdough and water (37). It is widely consumed in many cities across the Mediterranean region of Turkey but has also in recent years become popular in metropolises such as Istanbul, Ankara and Izmir (38). Production comprises two stages with the first one termed ‘first fermentation’ which involves mixing bulgur flour (3%), salt (0.2%) and sourdough (0.2%) together with water and allowing it to ferment at room temperature for 3–5 days. In the second stage, cleaned and chopped black carrots (10–20%), sliced turnips (1–2%), salt (1–2%) and water are then added to the extract obtained from stage one and left to undergo a ‘second fermentation’ for 3–10 days in a wooden barrel (37). The juice is then filtered and packaged in suitable containers, and chilli powder may also be added depending on consumer preference (26). Shalgam is typically made on a home-scale and consumed within three months from production time, although it is also produced commercially with an extended shelf life of 1–2 years using preservatives (26). Shalgam juice is a highly nutritional beverage due to its high mineral, amino acid, polyphenol and vitamin contents (37, 39).
There are quite a few studies and review papers that have reported the microbiological, chemical, nutritional and some probiotic properties of fermented traditional Turkish foods and beverages (22, 26). However, no study reporting the antimicrobial efficacies of the potential probiotic microbiota of these five major beverages (boza, kefir, hardaliye, ayran and shalgam) was encountered in the literature. The current study, therefore, aimed to consolidate past works on the probiotic properties of the selected beverages specifically by investigating the antimicrobial activities of their microbiota against selected standard pathogens, thereby helping to validate their widely acclaimed beneficial effects on human health. Ultimately, the present study aims to contribute to public knowledge of fermented functional foods and probiotics, increase awareness of the Turkish populace about the health benefits derivable from their own indigenous fermented dairy and non-dairy beverages. It is hoped that in the long run this knowledge and awareness will result in increased consumption of these functional food products and, hence, result in an improved general public health.
2. Materials and Methods
2.1 Isolation, Identification and Characterisation of Target Microorganisms from Beverage Samples
Sampling was done by purchasing commercially produced and prepackaged fermented Turkish beverages from retail locations. Three different brands from each of the five beverages (boza, kefir, hardaliye, ayran and shalgam) were analysed.
Tenfold serial dilutions of the beverage samples were prepared up to 10–6 with sterile 0.5% peptone water accordingly. To isolate the target microorganisms 100 μl aliquot was taken from each dilution and spread-plated on four selective culture media namely de Man, Rogosa and Sharpe (MRS) agar (GranuCult®, Sigma-Aldrich®, Merck, Germany), Bifidobacterium selective medium (BSM Agar, NutriSelect® Basic, Sigma-Aldrich®, Merck, Germany), M17 agar and Sabouraud dextrose agar (SDA, with the addition of 0.05 gchloramphenicol–1). MRS and BSM plates were incubated for 72 h at 37°C in anaerobic jars (OxoidTM, Thermo ScientificTM, UK) containing gaspack (AnaeroGenTM, OxoidTM, Thermo ScientificTM, UK) (oxygen level <1%, CO2 level 9–13%). M17 agar and SDA plates were incubated aerobically at 28–30°C for 72 h. After the incubation periods, colonies were randomly picked from the plates and sub-cultured on fresh plates of the same media. Morphologically characterised pure isolates were stored in the appropriate broths (MRS, BSM, M17 and yeast extract peptone glycerol (YEPG) broth media) containing 20% glycerol at –20°C and –80°C until antimicrobial assays. For all subsequent assays, isolates were propagated twice and activated in the corresponding media at the appropriate incubation conditions.
Preliminary phenotypic and morphological characterisation of the isolates was performed with catalase test and Gram-staining reaction to select the target groups of bacteria. Gram-stained smears were examined microscopically. Subsequently, molecular identification by 16S rRNA gene sequencing was performed on phenotypically characterised isolates. Bacterial genomic DNA was extracted from overnight cultures, using a bacterial genomic DNA isolation kit (Hibrigen, Turkey) according to the manufacturer’s guidelines. Polymerase chain reaction (PCR) amplifications of the genomic DNA fragments of approximately 1500 base pairs (bp) of the 16S rRNA gene were performed with a 2X PCR master mix (Hibrigen) as described by (40), using the universal primers 16S rRNA (forward: 5′-TGGAGAGTTTGATCCTGGCTCAG-3′; reverse: 5′-TACCGCGGCTGCTGGCAC-3′).
PCR amplification of the 16S rRNA genes and subsequent purification of the amplicons were performed as previously described by (40). The PCR amplification reactions contained 50 ng of DNA, 12.5 μl 2X master mix, 0.3 mM; of each primer in a final volume of 25 μl. Amplification reactions were performed as follows: an initial denaturation step at 94°C for 2 min which was followed by 35 cycles at 94°C for 1 min, then at 55°C for 1 min, 72°C for 1.5 min and one last cycle at 72°C for 7 min for the final elongation step. The PCR products were then analysed by electrophoresis in 1% agarose gels in tris-borate-EDTA (TBE) buffer with ethidium bromide and visualised by ultraviolet (UV) light. The PCR products were sequenced in an Applied BiosystemsTM 3130 Genetic Analyzer (Thermo Fisher ScientificTM, USA). The results of DNA sequencing runs were assembled using the Chromas software. In order to identify the strains, the sequences of each lactobacilli strain were compared with those available in the databases of the National Center for Biotechnology Information (NCBI) using the basic local alignment search tool (BLAST®) program.
2.2 Evaluation of the Antimicrobial Activities of the Isolates
The antimicrobial activities of the obtained isolates were evaluated against selected common human microbial pathogens using spot-on-the-lawn and agar well diffusion methods. All strains used were sourced from the American Type Culture Collection (ATCC® ), and all pathogens were activated for 24 h on trypticase soy agar (TSA, OxoidTM) prior to every assay. The standard indicator pathogenic strains used in this study are as follows: Acinetobacter baumannii (ATCC® 19606TM), Bacillus cereus (ATCC® 14579TM), Escherichia coli (ATCC® 25922TM), Klebsiella pneumoniae (ATCC® 4352TM), methicillin-resistant Staphylococcus aureus (MRSA) (ATCC® 33591TM), Pseudomonas aeruginosa (ATCC® 9027TM), Salmonella typhi (ATCC® 14028TM), Staphylococcus aureus subsp. aureus (ATCC® 6538TM), Staphylococcus epidermidis (ATCC® 12228TM), vancomycin-resistant Enterococcus (VRE) faecium (ATCC® 51299TM) and Candida albicans (ATCC® 10231TM).
2.2.1 Spot-on-the-Lawn Technique
The antimicrobial activity of the isolates was evaluated by the spot-on-the-lawn assay described by (41) with slight modification. A 2 μl inoculum taken from overnight broth culture was spotted in triplicates on the surfaces of their corresponding isolation media, i.e. MRS, BSM, M17 and SDA plates. The spots were allowed to dry for 1 h at room temperature after which the plates were incubated appropriately. Incubation of M17 and SDA plates was done at 30°C, MRS and BSM at 37°C in anaerobic jars containing gaspack for 24 h. After colony development, the spots were overlaid with 10 ml of soft (0.75%) TSA seeded with 1% (v/v) of an active overnight culture of the target pathogenic strain which had been adjusted to McFarland 0.5 turbidity standard (1–1.5 × 108 colony forming units (CFU) ml–1). The overlaid plates were then incubated under appropriate conditions for 24 h to 48 h. After 24 h to 48 h of incubation, measurements of inhibition zones around the isolate colonies were taken from the outer edge of those colonies to the outer edge of the clear zones. Inhibition zones of >20 mm, 10–20 mm and <10 mm were interpreted as strong, intermediate and weak inhibitions respectively.
2.2.2 Agar Well Diffusion Assay
In order to obtain the cell-free supernatants (CFS) needed for the agar well diffusion assay, 24 h activated isolates were grown in MRS, BSM (anaerobically at 37°C), M17 and YEPG (aerobically at 30°C) broths respectively for 24 h. The cells were harvested by centrifugation of cultures (8000 rpm at 4°C for 15 min). The obtained supernatants were sterilised by filtration (0.22 μm pore size, Millipore, USA).
The antimicrobial efficacy of the potential probiotic isolates was then further examined using the agar well diffusion technique (42). Briefly, 20 ml of Mueller Hinton agar (MHA) medium cooled to 45°C was vigorously mixed with 200 μl of an overnight culture of each indicator pathogen (adjusted to 1–1.5 × 108 CFU ml–1 by McFarland 0.5 standard), poured into petri dishes and allowed to solidify. Then 50 μl aliquots of the prepared supernatant (CFS) was placed in a 6 mm well excavated in the agar. Plates were maintained at 4°C for 1 h prior to incubation in order to allow for diffusion of the supernatant into the agar. Inhibition of the indicator pathogens was evaluated after incubation at 37°C for 24 h. The antimicrobial activity was expressed as the diameter of inhibition zones around the wells. Inhibition zones ≥10 mm were regarded as positive.
A total of 22 bacterial strains comprising lactic and acetic acid bacteria were isolated from four (boza, kefir, shalgam and ayran) of the five fermented traditional Turkish beverages analysed. No microbial strain was isolated from hardaliye. Boza and kefir contained the largest microbiota loads with eight isolates each followed by shalgam having five, which is due to their various raw materials and fermentation processes. The names of the 22 isolates as identified by molecular sequence analysis of their 16S rRNA and their distribution in the studied fermented traditional Turkish beverages are presented in Table I.
In the spot-on-the-lawn assay, the bacterial strains isolated from boza, kefir and shalgam exhibited antimicrobial activity against nearly all tested indicator pathogens at varying degrees. Moreover, the results presented in Table II clearly show that the obtained probiotic isolates displayed antagonistic activity against both Gram-positive (B. cereus, MRSA, S. aureus, S. epidermidis, VRE faecium) and Gram-negative bacteria (A. baumannii, E. coli, K. pneumoniae, P. aeruginosa, S. typhi). Lactobacillus nagelii (B24), Lactobacillus parabuchneri (B25) and Acetobacter peroxydans (B27) isolates obtained from boza displayed the strongest antagonistic effects with ≥20 mm diameter inhibition zones against all tested bacterial pathogens. However, they exhibited no antifungal activity against C. albicans. MRSA was highly sensitive to Lb. nagelii (B24) and Lb. parabuchneri (B25) isolates from boza as well as to Lactobacillus fermentum (K13) and Gluconobacter frateurii (K26) isolates from kefir with 26.00 mm, 22.10 mm, 26.40 mm and 25.90 mm inhibition zones, respectively, but minimally sensitive to Lactococcus lactis (S2) obtained from shalgam (12.00 mm zone). L. lactis strains B7 and B10 isolated from boza showed medium antimicrobial activity against the bacterial pathogens and Candida albicans with ≥ 10 mm ≤ 20 mm diameter inhibition zones. L. lactis (B7) strain exhibited the strongest inhibitory effect (28.60 mm zone) while L. lactis (B10) and Lb. fermentum (K13) strains displayed medium antagonism producing 16.80 mm and 11.90 mm inhibition zones respectively against C. albicans. G. frateurii (K26) and Lb. fermentum (K13) strains isolated from kefir also showed a very strong antibacterial activity creating 20.80–30.10 mm diameter inhibition zones against five bacterial pathogens comprising both Gram-positive (VRE, MRSA) and Gram-negative (P. aeruginosa, K. pneumoniae and E. coli). In contrast, another Lb. fermentum strain K12 also obtained from kefir displayed a weak antagonistic effect (<10 mm zones) against the same pathogens. The strain L. lactis (S2) isolated from shalgam exhibited a medium inhibitory effect (9.60–15.70 mm zones) on all indicator bacterial pathogens except A. baumannii.
On the other hand, in the agar well diffusion assay, the CFS of 10 out of 22 isolates exhibited medium antimicrobial effects (≥ 10 mm ≤ 20 mm zones) against bacterial and C. albicans pathogens (Table III). However, P. aeruginosa was strongly inhibited by Lb. fermentum (B4), Lactobacillus pentosus (S5) and Lactobacillus plantarum (S8) strains with 25.10 mm, 23.40 mm and 19.10 mm diameter zones respectively. The CFS of Lb. fermentum (B6) with a 25.40 mm zone of inhibition displayed the strongest antagonistic activity against the yeast pathogen C. albicans. Furthermore, L. lactis strains (K18 and A22), Lb. fermentum strains (B4 and K13), Lb. plantarum strains (K14 and K15) had medium inhibitory effects with 10.20–15.80 mm zones on the pathogenic yeast C. albicans. The weakest antibacterial effects were displayed by the supernatants of L. lactis (K18) and Lb. plantarum (S8) both with an 8.30 mm inhibition zone against P. aeruginosa and K. pneumoniae indicator strains respectively. Representative images of the isolates are shown in Figures 1–3.
Among the known mechanisms of action by which probiotics exert their beneficial effects on human health is antagonism against microbial pathogens via the production of antimicrobial metabolites. In the present study, the antimicrobial efficacy of some potential probiotic bacteria isolated from fermented traditional Turkish beverages against 11 common human pathogens has been evaluated. Thus, with respect to antimicrobial effects, the potentials of boza, kefir and shalgam to bestow some probiotic health benefits to the consumers have been validated through the present study. Other probiotic properties of Turkish boza and kefir were reported in a previous study (43). Kefir was also reported in a previous study to exhibit antimicrobial activity which is attributable to the lactic acid, acetic acid, bacteriocins, hydrogen peroxide, acetaldehyde, volatile acids, diacetyl and CO2 produced by the bifidobacteria and LAB strains present in the drink (44). Although ayran is the most widely consumed amongst the fermented Turkish beverages examined, its inherent LAB strains exhibited no significant antimicrobial effects. No lactic acid bacterial or other potential probiotic strain was isolated from commercially available hardaliye in this study. This reason is thought to be due to the presence of the preservative benzoic acid which is usually added at the start of the fermentation process. It has, therefore, been determined that commercial hardaliye lacks probiotic properties considering the fact that probiotics must be found alive and in sufficient quantities in final products. In order to retain the potentially probiotic natural microflora of commercial hardaliye, production without the use of benzoic acid or other chemical preservatives is recommended. In the same vein, no live LAB strain was found in some brands of commercial shalgam that were examined. Similar to hardaliye, the reason is also thought to be due to the presence of preservatives and additives such as sodium benzoate which is declared in the ingredients list on label.
Pathogenic bacterial multidrug resistance as well as biofilm formation have led to the ineffectiveness of the antibiotics available in the treatment of infections whereas the application of probiotics has been considered functional in preventing and counteracting biofilm-related infections (45). Antagonism by antimicrobial metabolites has been considered as an important property in the selection of potential probiotics for the maintenance of a healthy microbial balance in the gut. LAB, mostly the lactobacilli due to their capacity to alienate bacterial pathogens via the production of some antimicrobials such as organic acids (mainly lactic acid), bacteriocins and hydrogen peroxide, acquire probiotic potential (46). Lactic acid typically diffuses into pathogenic bacterial cell which disrupts the cell membrane integrity thereby causing damage to it as well as retarding their metabolic processes and preventing growth (47). Within the scope of this study, the antimicrobial activities of LAB and acetic acid bacteria strains isolated from fermented Turkish beverages were assessed by two methods namely spot-on-the-lawn and agar well diffusion methods.
Through this study, it has been possible to evaluate the potential beneficial effects of fermented traditional Turkish beverages on human health as regards antimicrobial efficacy. 18 strains namely: Lb. fermentum (B4), L. lactis (B7), Lb. fermentum (B6), L. lactis (B10), Lb. fermentum (K13), Lb. fermentum (K12), Lb. nagelii (B24), Lb. parabuchneri (B25), L. lactis (S2), L. lactis (K18), L. lactis (A22), Lb. plantarum (K14), Lb. plantarum (K15), Lb. fermentum (B3), Lb. pentosus (S5), Lb. plantarum (S8), G. frateurii (K26) and A. peroxydans (B27) exhibited varying degrees of antimicrobial activities against the tested pathogens. Furthermore, differences in the antibacterial effect of different strains of the same species of isolates were observed. This result has confirmed that the antibacterial activity and potential health benefits imparted by probiotic bacteria are strain-specific rather than being species- or genus-specific. It is therefore crucial to note that no single strain will provide all the proposed benefits, not even strains of the same species, and also not all strains of the same species will be effective against defined health conditions (48, 49). This specificity has also been confirmed by previous studies in terms of the microorganism strain, the metabolites it produces, and even the susceptibility pattern of the test organism to antimicrobials (50). It therefore goes without saying that in order to obtain maximum health benefits, the addition of a mixture of various strains of probiotic organisms to diets is imperative.
Many bacteriocins isolated from LAB are usually active against Gram-positive bacteria while Gram-negative bacteria generally exhibit little sensitivity to bacteriocins. The difference in resistance between Gram-positive and Gram-negative bacteria may be due to differences in the cell envelopes (51). However, production of lactic acid in high concentrations in combination with bile salts has an inhibitory effect on the growth of pathogenic Gram-negative bacteria in the intestinal tract (52). The present study has reported antagonistic activity of LAB strains against both Gram positive and negative bacteria alike. This finding is in accordance with a similar study (53) which reported that the CFS of a Lb. plantarum strain displayed broad spectrum antimicrobial activities against Gram-positive and negative bacteria as well as against yeast C. albicans. P. aeruginosa , K. pneumoniae and E. coli were reported to be sensitive at medium levels to Lb. plantarum and L. lactis strains isolated from Turkish boza and B. cereus (a Gram-positive bacterium) was also strongly inhibited by the same LAB strains (54). Also, the inhibitory effect of Lb. plantarum and Lb. fermentum strains used as starter organisms in the production of bread and bakery products against rope-forming B. cereus was previously reported (55, 56). Many Lb. plantarum strains have been reported to produce bacteriocins known as plantaricins (57) which are compounds that are highly diverse in their activities and structures and have been reported to be particularly active against gastrointestinal as well as food-borne pathogens (58). In the present study, L. lactis strains (B7 and B10), Lb. fermentum strains (K12 and K13), G. frateurii ( K26), Lb. nagelii (B24), Lb. parabuchneri (B25), A. peroxydans (B27) and L. lactis (S2) isolates showed better antibacterial activity by spot-on-the-lawn method. The excellent antibacterial activity reported of Lb. parabuchneri against all indicator pathogens in this study is in contrast to the results obtained by Meira et al. (59) who had reported that Lb. parabuchneri strain isolated from a Brazilian ovine cheese had little inhibitory effect against all tested pathogens. Lb. parabuchneri is an obligatory heterofermentative bacterium occasionally isolated from cheeses (60). Considering that heterofermentative LAB typically ferment glucose to yield ethanol, acetic acid and CO2 in addition to lactic acid as byproducts as against homofermentative LAB which produce only lactic acid, they possess the ability to antagonise highly pathogenic bacteria. It is, therefore, noteworthy that this is the first study to have reported the presence of Lb. parabuchneri in a Turkish boza drink. LAB strains that produced larger inhibition zones against E. coli, S. aureus and Salmonella enteritidis in a previous study were reported to be heterofermentative (61).
Comparing the results of the two methods adopted in this study, L. lactis strains (B7 and B10) as well as Lb. fermentum (K13) strain showed antifungal activity against C. albicans by spot-on-the-lawn method, but the same effect was not observed with agar well method. The lack of positive results by agar well diffusion method may be due to the indiffusibility of the secreted molecules since this is the basis of agar well assay. In contrast, Lb. plantarum (K14, K15), Lb. fermentum (B3, B4, B6), L. lactis (K18), Lb. pentosus (S5) and Lb. plantarum (S8) strains showed antimicrobial activity by agar well diffusion but no antagonism was recorded with spot-on-the-lawn method. Since they showed negative results by spot-on-the-lawn method, it is thought that the isolates may not secrete any primary or secondary metabolites other than bacteriocins. Therefore, spot-on-the-lawn method has been found in this study to be more effective than agar well diffusion method in the determination of antimicrobial activity. This finding was consistent with the results reported by (62) who investigated the antagonistic effects of LAB strains against Gram-negative bacteria using these two methods and found spot-on-the-lawn to be more effective. However, utilising the probiotic strain Bifidobacterium bifidum against S. enterica serovar Enteritidis , agar well diffusion method was demonstrated to be better in determining antagonism than the other two methods (disk diffusion and spot-on-the-lawn) employed (63). The inhibitory activity on tested bacteria by the spot-on-the-lawn method is seen as better, but it could be as a result of the synergy of all metabolites lactic acid, acetic acid, diacetyl and bacteriocin, as they are being produced during the assay period (64). The variation in antibacterial activities as depicted by different authors might be due to the number of the CFU of the LAB used (in spot-on-the-lawn) and the quantity of culture supernatant used (in agar well diffusion) as well as the activity and diffusibility of the bacteriocins possessed in it (46, 65).
Probiotics are increasingly gaining attention from both the food industry and academia. Probiotic foods constitute a significant part of the functional foods market worldwide. The presence of these good microbes in fermented food products all over the world, and their ability to combat pathogenic and spoilage microbes using different mechanisms of action, has been validated with several scientific studies. From the perspective of antimicrobial health benefits, the present study consolidates past studies demonstrating the probiotic potentials of fermented traditional Turkish beverages. The study has substantiated the antimicrobial efficacy of the potential probiotic strains isolated from kefir, boza and shalgam against an array of human pathogens comprising both Gram-positive and negative bacteria as well as yeast (C. albicans). In total, 16 LAB and two acetic acid bacteria isolates were found to be antagonistic at varying degrees against the tested bacterial and yeast pathogens. In the current study, boza and kefir followed by shalgam were found to be more effective in terms of antimicrobial activity against human pathogens. The outstanding antimicrobial efficacy of boza and kefir in particular is believed to be connected with the greater diversity of their microflora as well as the absence of chemical preservatives. It has, therefore, been determined that boza, kefir and shalgam compared to other fermented traditional Turkish beverages analysed, are the most promising probiotic candidates. In addition, going by the results obtained in this study as well as previous studies on other probiotic properties, boza, kefir and shalgam may also be applicable as bio-therapeutics or nutraceuticals against bacterial and yeast infections in humans, although in vivo studies would be useful in validating their efficacy. Due to the presence of beneficial nutrients and substances such as probiotics and antioxidants, boza, kefir, ayran, shalgam and hardaliye can also be generally regarded as functional foods. Other probiotic properties of the microbiota of these fermented Turkish beverages have been reported individually in previous studies. However to the best of our knowledge, this is the first study that evaluated the antimicrobial properties of the isolates obtained from all five beverages against a broad spectrum of human microbial pathogens. The study has to a large extent achieved its objective.
Future research efforts should be directed toward animal testing and clinical trials to better evaluate the antimicrobial as well as overall probiotic effects of Turkish boza, kefir and shalgam on humans. Furthermore, suitable biopreservatives for commercial hardaliye and shalgam drinks should be investigated in order to retain their beneficial microflora whilst extending their shelf lives. Alternatively, probiotic strains and starter cultures resistant to benzoic acid may be investigated in further studies for fortification of commercial hardaliye since no LAB strain was isolated from any hardaliye sample which is thought to be due to the presence of chemical preservative. Ayran which is a ‘national drink’ is also recommended for fortification with carefully selected and promising probiotic strains from the Bifidobacterium and Lactobacillus genera in order to enhance its probiotic functional qualities.
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Oluwaseun Temitope Aladeboyeje is an international award-winning microbiologist with a first class Master’s degree from Istanbul University, Turkey. His undergraduate research thesis at the University of Ilorin, Nigeria was ‘highly commended’ – rated in the top 10% worldwide in the 2014 Global Undergraduate Awards held in Dublin, Ireland. His areas of research interest include probiotics, fermented foods microbiology, food safety, microbial pathogenesis, antimicrobial resistance and new drug development. He plans to commence his PhD degree programme in 2022.
Nazmiye Ozlem Sanli currently serves as Assistant Professor in the Department of Biology, Faculty of Science, Istanbul University. She received her PhD at Istanbul University in 2009. Her main area of research is biofilms in man-made water systems and their control strategies, the efficacy of industrial biocides and treated materials in different applications such as healthcare, white goods, postharvest storage areas and agriculture. She carries out applied industrial research projects through collaboration with different sectors.
Umut Büyük is currently pursuing his doctorate in Istanbul University, Faculty of Science, Department of Molecular Biology and Genetics. His main research area is human genetics, molecular biology of genetic diseases and diagnosis of genetic diseases. He also has an enterprise that manufactures molecular biology consumables.