1. Introduction
In 2002, the United Nations’ Food and Agriculture Organization and the World Health Organization [
1] defined probiotics as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”. From that moment until the present, important efforts have been made to develop probiotic commercial products, as well as products with prebiotics and synbiotics. It is generally accepted that these are likely to provide general health benefits for humans and animals, such as restoring the disturbed gut microbiota, regulating intestinal transit, competitively excluding pathogens from adhesion sites, and producing short chain fatty acids [
2]. Consequently, the probiotic market has grown rapidly both for foods and supplements intended to enhance wellness in healthy individuals [
3]. Different microorganisms of different origins have already been used to develop commercial probiotic products. The most common commercially available strains belong to the
Lactobacillus species (
casei, acidophilus, fermentum, gasseri, johnsonii, reuteri, plantarum, paracasei, rhamnosus or
salivarius) and
Bifidobacterium species (
bifidum, breve, adolescentis, animalis, or
longum) [
4]. The probiotic market trend indicates that there is still room for new products made of alternative probiotic sources that bode extended shelf lives, chemical stability, and are reasonably priced [
5], all while reducing the risk of cholesterol problems in lactose intolerant people [
6]. Different alternative sources have been exploited, i.e., mainly non-dairy fermented food products, such as traditional fermented foods, traditional fermented drinks, vegetables, and fruit juice. It has been demonstrated that the differences in raw materials and ingredients used to prepare such products are the main factors that lead to the different available species or strains of probiotics in food sources [
7].
The main lactic acid bacteria (LAB) genera isolated from fermented plants and fermented meats is
Lactobacillus. The most common LAB genera isolated from fermented seafoods is
Enterococcus [
7]. For example, the main LAB isolated from black olives were
L. pentosus and
Leuconostoc mesenteroides and from green olives were
L. pentosus,
L. plantarum, and
L. paracasei [
8,
9].
Pediococcus pentosaceous strains were isolated from some traditional Thai fermented foods containing fish and pork [
10] or from traditional Ethiopian fermented beef sausage [
11].
Non-dairy fermented beverages, such as
Pozol,
Bushera,
Boza,
Mahewu, and
Togwa, made of cereals, millets, legumes, fruits, and vegetables, are frequently reported as good sources of probiotics [
12]. In the past decade, special attention has been paid to Kombucha, a fermented low-alcohol beverage made of tea leaves. Kombucha is reported to have several health benefits, such as antioxidant potential, antibacterial activity, and antiproliferative activity against cancer cell lines [
13]. Different sources of Kombucha were characterized for their biochemical composition and complex microbial biodiversity (characterized by the presence of acetic acid bacteria, yeasts, and lactic acid bacteria). Generally, the consortium is dominated by acetic bacteria such as
Komagataeibacter sp. and
Gluconobacter sp., as well as yeast such as
Brettanomyces, Hanseniaspora,
Saccharomyces, Torulaspora, or
Schizosaccharomyces [
14,
15]. Few studies reported on the interactions inside such complex microbial biodiversity and the focus was mainly on the acetic bacteria-yeast interaction [
16]. The LAB are often, but not always, reported in Kombucha, and the main identified genera are
Lactobacillus spp.,
Lactococcus spp., or
Leuconostoc spp. [
17,
18]. Occasionally,
Pediococcus spp. has been reported to belong to such consortium in industrial Kombucha sources [
19]. It has been demonstrated that the viability of probiotic microorganisms is more difficult to maintain in non-dairy matrices than in dairy matrices and the physicochemical parameters must be carefully controlled to guarantee the probiotic viability [
20]. Different strains of
Pediococcus spp. isolated from other traditional fermented sources were reported as potential probiotics:
P. pentosaceus from
Idly batter, a traditional fermented food in South India [
21];
P. pentosaceus and
P. acidiliactici from
Omegisool, a traditionally fermented millet alcoholic beverage in Korea [
22];
P. pentosaceous strains isolated from
Wakalim, the traditional Ethiopian fermented beef sausage [
11]; or from
Kunu-zaki, a Nigerian traditional fermented drink [
23].
In the developmental process of new probiotic food products, the selection of probiotic microorganisms is the main challenge for food industries. The selection of probiotics from different sources requests screening for non-pathogenic microorganisms, which are further evaluated for some basic properties, such as the tolerance to gastro-intestinal environments, the ability to inhibit pathogens in the gastro-intestinal tract, resistance to antibiotics, adhesion potential, etc. Another important feature for industrial use is the viability during processing treatments and storage [
24]. Our demarche targeted the screening of
Pediococcus strains, formerly isolated from industrial Kombucha, for their probiotic potential and technological aspects. On the probiotic side, the isolates were tested for their resistance to the gastro-intestinal environment (i.e., low pH, pepsin, and bile salt presence), antagonism against different groups of pathogens, safety aspects (e.g., hemolytic activity, antioxidant activity), and antibiotic resistance. The technological aspects covered tolerance to NaCl presence, as a food additive, and the influence of the freeze-drying procedure on the cell viability, as a conditioning method for probiotics’ industrial use.
4. Discussion
Nowadays, alternative isolation sources of probiotics, such as non-dairy fermented food products, are increasingly exploited. Kombucha, a fermented beverage made of tea leaves (
Camellia sinensis) is one such source because of its complex SCOBY (symbiotic consortia of bacteria and yeast). The main reported LAB in this SCOBY are
Lactobacillus spp.,
Lactococcus spp., or
Lecunosctoc spp., while
Pediococcus spp. was only occasionally isolated [
17,
18,
19]. Our goal was to test the biotechnological potential of seven
Pediococcus spp. strains isolated from a local industrial Kombucha, and identified formerly as
Pediococcus pentosaceus [
19] and
Pediococcus acidiliactici. In an initial stage, all isolates were screened for a series of probiotics properties, such as their resistance in the gastro-intestinal tract, antagonistic activity against human/animal pathogens, foodborne molds, antioxidant potential, and hemolytic activity. Two isolates, L3 (
Pediococcus pentosaceus) and L5 (
Pediococcus acidilactici), were selected as potential probiotic candidates and further investigated for their antibiotic resistance and for their behavior against some technological parameters. In terms of technological aspects, the strains were tested for their tolerance to a food preservative (sodium chloride) and for their viability after applying extreme freeze-dried conditions, which is a frequent method used industrially to process probiotics. Former data [
31] showed that L5 isolate have a good capacity to adhere in vitro to the surface of the Caco-2 cellular monolayer; it was proven that after four incubation hours, the bacterial cells start to form aggregates, suggesting a diffuse-aggregative adherence pattern.
The resistance in the gastro-intestinal tract in the presence of bile salts, pepsin, and acidic pH is one of the most important characteristics of the probiotic microorganisms.
P. pentosaceus strains isolated from
Wakalim, a traditional Ethiopian fermented beef sausage, are reported as tolerant to a pH of 3 and a 0.3% bile salt concentration [
9], while strains isolated from
Kunu-zaki, a Nigerian traditional fermented drink made from non-germinated sorghum and millet cereal grains proved to resist at the same pH but to a higher bile salt concentration of 3% [
19]. In our case, the selected strains (L3 and L5) were tolerant to a pH of 2.5, 0.3% pepsin and 0.5% bile salt concentrations.
Numerous strains isolated from fermented beverages were reported as having antagonistic effect on pathogenic microorganism, mainly due to the production of organic acids and hydrogen peroxide. LAB strains of
Lactobacillus plantarum isolated from a Turkish traditional fermented drink (
Boza) showed antagonistic activity against pathogenic bacteria such as
Listeria monocytogenes, Bacillus subtilis, Bacillus cereus, Yersinia enterocolitica, Pseudomonas aeruginosa, E. coli, Salmonella enterica Typhimurium, and
Klebsiella pneumonia, even after the neutralization of the cell free supernatant. The authors imply the presence of bacteriocin’s production [
32].
Kunu-zaki, the Nigerian traditional fermented beverage, had isolated probiotic strains of
Lactobacillus, Pediococcus, and
Lactococcus, all of which were reported with inhibitory activity on
Pseudomonas aeruginosa, Staphylococcus aureus,
E. coli, and
Enterococcus faecalis when used for active cell suspension [
23]. The authors suggested that the results were caused by bacteriocins and organic acids production.
P. pentosaceus isolated from other natural fermented sources, such as Kimchi (traditional Korean fermented vegetable), has been reported to inhibit
Listeria monocytogenes due to the production of class II bacteriocins [
33]. The species of
Pediococcus genera are recognized for their ability to produce bacteriocin. Oher strains of
P. pentosaceus isolated from paocai (a Chinese fermented vegetable) have been proven to have inhibitory activity against
E. coli and
Salmonella enterica Typhimurium [
34]. It has also proven that
P. pentosaceus strains isolated from traditional Thai meat fermented foods inhibited the growth of some pathogenic bacteria, such as
Salmonella enterica Typhimurium,
Pseudomonas aeruginosa,
Bacillus cereus, E. coli,
Staphylococcus epidermidis, or
Vibrio cholera [
10]. Our team has formerly reported bacteriocin production in the case of L5 Kombucha isolate [
19], tested against
Streptococcus thermophilus. Such strains and their bacteriocin can be used in food and feed industries as natural biopreservatives and for probiotic application to humans or livestock, including functional foods. All Kombucha isolates proved to have high inhibitory activity against the major foodborne pathogens, like
Salmonella enterica Typhimurium,
Listeria monocytogenes, Listeria ivanovii, Bacillus cereus, Proteus hauseri, as well as on resistant or methicillin sensible
Staphylococcus aureus. As a novelty, we have proven the inhibitory activity of the
P. pentosaceus isolated from Kombucha on the human emerging pathogen,
Listeria ivanovii, known as ruminants’ pathogen, as it can cause epidemic abortion, stillbirths, and encephalitis [
35].
The LAB inhibitory activity on foodborne molds has often been reported on
Lactobacillus spp. [
36] and only occasionally in the case of
P. pentosacesus or
P. acidilactici isolated from natural sources. For instance, bacteriocin produced by
P. acidilactici isolated from vacuum packed fermented meat products inhibited
A. fumigatus, A. parasiticus, F. oxyporum, and
Penicillium spp. [
37], while
P. pentosaceus isolated from malted cereals inhibited
Pencillium expansum [
38]. Strains from stored wheat samples showed antagonistic activity against different species such as
Alternaria alternata, Penicillium chrysogenum, and
Aspergillus carbonarius [
39]. Although the same authors did not report any inhibition of
P. pentosacesus on
A. niger, our experiments found that the Kombucha LAB isolates exhibited low inhibitory activity, probably due to the low pH level of the suspension (pH of 4–4.5). There might be a correlation between our results and data reported on how Kombucha itself, made of green tea, and with a final pH of 3.5, have moderate inhibitory potential on molds like
Botrytis cinerea, Aspergillus carbonarius, or
Penicillium expansum, which proved on-plate and by in vivo artificial infections on grapes [
40]. Both selected
Pediococcus isolates (L3 and L5) have moderate to high inhibitory activity against
Penicillium expansum and
Penicillium digitatum, which suggest that our strains can be used in agricultural practices to control post-harvest mold development
The radical scavenging activity of LAB isolates is due to the colonization of viable cells and their propagation in the gut. Our results are comparable with studies reported before and Kombucha
Pediococcus strains, such as L5, F2, and L3 LAB, which have an antioxidant activity of 56–58%. LAB isolated from
Neera (fermented coconut palm nectar) can reach hydroxyl-scavenging activity of 32–77% [
41], while for
Pediococcus strains from
Omegisool the DPPH radical-scavenging activity ranged between 30% and 39% [
22].
Due to safety considerations, the obtained isolates were also tested for antibiotic resistance. The transmission of antibiotic resistance genes to potentially pathogenic bacteria in the gut is a major health concern related to the probiotic application [
42]. The European Food Safety Authority (EFSA) recommends that bacterial strains harboring transferable antibiotic resistance genes should not be used in animal feeds or fermented and probiotic foods for human use. For an appropriate selection of functional strains, two main antibiotics’ groups are recommended in EFSA guidelines to be tested, such as inhibitors of protein synthesis (chloramphenicol, gentamycin, clindamycin, erythromycin, streptomycin, kanamycin, and tetracycline) and inhibitors of cell wall synthesis (ampicillin and vancomycin). In the case of LAB isolated from different natural sources, such as fermented coconut palm nectar, and belonging to other species (
Lactobacillus brevis, Enterococcus durans, Leuconostoc lactis, Enterococcus lactis, and Enterococcus faecium), chloramphenicol, vancomycin, and streptomycin were effective inhibitors [
41].
Pediococcus isolated from
Omegisool are shown to be resistant to chloramphenicol [
22]. Both Kombucha
Pediococcus isolates (L3 and L5) are sensitive to ampicillin, penicillin, erythromycin, and lincomycin, while a broad range of other antibiotics are not effective inhibitors (fluoroquinolones, amoxicillin/clavulanic acid, cephalexin, cefuroxime, amikacin, streptomycin, kanamycin, sulphamethoxazole, vancomycin, and tetracycline). In this regard, before using these isolates in food or feed formulations per EFSA guidelines, virulence and antimicrobial resistance genes should be verified to prevent the horizontal gene transfer for antibiotic resistance.
The incorporation of probiotic bacteria to food products represents a major technological challenge because of the known sensitivity of these microorganisms to salt, spices, and other substances used in its formulation. In relation to the NaCl tolerance, the most halophilic LAB (
Enterococcus ssp.,
Lactobacillus ssp.) were isolated from seafoods and fermented meats, being able to grew under a NaCl concentration of more than 22% [
7]. Meanwhile
L. fermentum and
L. plantarum isolated from fermented plants grew under NaCl concentrations of less than 6% [
43]. Strains of
P. pentosaceus isolated from various traditional Thai fermented foods containing fish and pork were reported as tolerant up to 14% NaCl in an acid medium (pH 2) with 0.3–0.5% bile salt [
10]. Our Kombucha isolates (L3 and L5) tolerated 5% NaCl. Furthermore, L5, after a period of adaptation, is tolerant to 7.5% NaCl. Thus, we concluded that L3 and L5 were resistant to salt concentrations used in industrial levels and maintained a concentration suitable for carrying probiotic effect.
According to Champagne et al. (2011) [
44], a product containing probiotic organisms is efficient if it contains a number of viable cells higher than 10
6–10
8 CFU/g. However, viability and optimum concentration of probiotic microorganism is still under debate, but the trend is to have a minimum of one billion viable cells per 100 g of product to declare it as a probiotic functional product [
45]. Lyophilization is one of the procedures used to deliver probiotics for commercial products. Regarding the lyophilization procedure, the survival rate may be improved by the use of other cryoprotectants, such as poly-glutamic acid, which was successfully used for the protection of probiotic
Lactobacillii [
45]. In the case of our
Pediococcus isolates, by the use of glucose or sucrose as cryopretectant, we obtained a good viability rate of 86–92%. In addition, lyophilization did not affect the inhibitory activity on the tested pathogenic microorganisms. The initial biomass of 10
10 CFU/mL conditioned by freeze-drying procedure, recovered levels of 10
9 CFU/g after rehydration. Rehydration is a normal industrial step when using such dried strains, and has a major influence on the CFU readings obtained. We concluded that our strains were suitable to be conditioned by lyophilization and employed industrially after rehydration.
From a larger biotechnological point of view, probiotic bacteria can also be used as ingredients in cosmetic products.
P. acidilactici strain isolated from Korean Perilla Leaf Kimchi was proven to a have direct melanin-degrading and tyrosinase-inhibiting effects, given that it has high value as a raw material for melanin degradation drugs and cosmetics [
46]. However, there are still technical barriers to incorporate live probiotics into conventional skincare products with a reasonable shelf life. Some results are reported when using
Lactobacilli, and the solution was to add probiotic ingredients which were not alive or viable to form colonies to the formulation at the end of the manufacturing process. Another strain of
P. pentosaceus isolated from kaki fruit increased the antioxidative and aging activities of the
Lavandula angustifolia extract through fermentation, so it was proposed to be used as an anti-aging agent [
47]. Our
Pediococcus isolates are expected to be further tested for such properties for further industrial application.