Evaluation of the Antimicrobial Efficacy of some Fermented Traditional Turkish Beverages with Probiotic Potentials

Johnson Matthey Technol. Rev., 2022, 66, (4), xxx-yyy 1/25 https://doi.org/10.1595/205651322X16388083409013 < https://doi.org/10.1595/205651322X16388083409013> <First page number: TBC> Evaluation of the Antimicrobial Efficacy of some Fermented Traditional Turkish Beverages with Probiotic Potentials Oluwaseun Temitope Aladeboyeje* Institute of Graduate Studies in Science, Istanbul University, 34116, Istanbul, Turkey Nazmiye Ozlem Sanli Section of Biotechnology, Department of Biology, İstanbul University, İstanbul, 34126, Turkey Umut Buyuk Hibrigen Biyoteknoloji, Tübitak MAM Teknoloji Serbest Bölge Şubesi Barış Mahallesi 5002 Sk. Yeni Tek. Binası A Blok 4 A/101 Gebze/Kocaeli, Turkey; Department of Molecular Biology and Genetics, Faculty of Science, Istanbul University, Istanbul 34134, Turkey *Email: <oluwaseuntemitope.aladeboyeje@ogr.iu.edu.tr, oluwaseunaladeboyeje@gmail.com> <Article History> PEER REVIEWED Received 10th September 2021; Revised 14th November 2021; Accepted 6th December 2021; Online 6th December 2021 <End of article history>


Introduction
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, texture etc.), improved nutritional values and functional properties (2,3,4). 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/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 fermentations (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, e.g. 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", e.g. kefir, 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, e.g. 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, Lactococus, and Enterococcus genera (9). Furthermore, some strains of Gram-positive bacteria in the genus Bacillus as Aladeboyeje_06a_SC ACCEPTED MANUSCRIPT 06/12/2021 Page 4 of 37 well as some yeast strains in the genus Saccharomyces are also commonly used in probiotic products (10). The FAO/WHO (11), the European Food Safety Authority (EFSA) (12) and the FDA (Food and Drug Administration) 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 health and could be used in the probiotics industry (9,10,14). 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: (i) competition for limited nutrients, (ii) competition for adhesion sites, (iii) synthesis of various antimicrobial metabolites such as organic acids, H2O2, bacteriocins, etc. and (iv) inhibition of toxin production in pathogenic microorganisms (15,16 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, these 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).

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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/Good feeling" to express the feelings experienced after drinking it (21,23,24,25,26). 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 pasteurized milk. Kefir grains is a symbiotic consortium of lactic and acetic acids-producing bacteria as well as lactosefermenting yeasts (e.g. Kluyveromyces marxianus) and non-lactose fermenting yeasts (e.g. Saccharomyces cerevisiae, S. 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/maldigestion as it contains β-galactosidase enzyme expressing organism (e.g. 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 standardized 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 viz. 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, Page 8 of 37 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 colored, 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 "1st 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 "2nd 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 3 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 nondairy 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.

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 analyzed.
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 L aliquot was taken from each dilution and spread-plated on four selective culture media namely de Man Rogosa and Sharpe (MRS) agar (Sigma-Aldrich®),

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

Spot-on-the-Lawn Technique
The antimicrobial activity of the isolates was evaluated by the spot-on-thelawn assay described by (41)

Agar Well Diffusion Assay
In order to obtain the cell-free supernatants (CFS) needed for the agar well

Results
A total of twenty-two bacterial strains comprising lactic and acetic acid bacteria were isolated from four (boza, kefir, shalgam and ayran) of the five fermented traditional Turkish beverages analyzed. 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 1.
In the Spot-on-lawn assay, the bacterial strains isolated from boza, kefir and shalgam exhibited impressive antimicrobial activity against nearly all tested indicator pathogens at varying degrees. Moreover, the results presented in Table 2  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 Lb fermentum (K13) and Gluconobacter frateurii On the other hand, in the agar well diffusion assay, the cell-free supernatants (CFS) of ten out of twenty-two isolates exhibited medium antimicrobial effects (≥ 10 ≤ 20 mm zones) against bacterial and Candida albicans pathogens (Table 3) and Lb. plantarum (S8) both with an 8.30 mm inhibition zone against P. aeruginosa and K. pneumoniae indicator strains respectively.

Discussion
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 Aladeboyeje_06a_SC ACCEPTED MANUSCRIPT 06/12/2021 Page 16 of 37 the present study. Other probiotic properties of Turkish boza and kefir were reported in a previous study by (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/or carbon dioxide 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/or 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/or 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, H2O2 etc., acquire this enviable property for probiotic potentiality and thus are a maintainable substitute to the synthetic antibiotics (46 has confirmed that the antibacterial activity and 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 Page 18 of 37 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. 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 Bacillus cereus was previously reported (55, 56). Many Lb. plantarum strains have been reported to produce bacteriocins known as plantaricins (57)  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 carbon dioxide in addition to lactic acid as by-products as against homofermentative LAB which produce only lactic acid, they possess the ability to antagonize highly pathogenic bacteria. It is, therefore, noteworthy that this is the first study to have reported the presence of L. parabuchneri in a Turkish boza drink. LAB strains that produced larger inhibition zones against E. coli, S. aureus and S. Enteritidis in a previous study were reported to be heterofermentative (61).
Comparing the results of the two methods adopted in this study, L. lactis 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)

Conclusions
Probiotics are increasingly gaining overwhelming attention from both the food industry and the 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 (Candida albicans). In total, 16 lactic acid bacteria and 2 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 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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