Introduction

Foodborne pathogens have gained much attention because of frequent outbreaks of serious safety issues. In general, foodborne illnesses are characterized by acute conditions such as hemorrhagic colitis and hemolytic uremic syndrome (caused by Escherichia coli O157:H7), listeriotic and abnormalities (caused by Listeria monocytogenes), bacterial gastroenteritis (caused by Campylobacter, Salmonella, and Vibrio parahaemolyticus), chronic arthritis (caused by Shigella and Salmonella), and even colon cancer (caused by Salmonella) (Buchanan et al., 2017; Li et al., 2019; Luo et al., 2018; Ssemanda et al., 2018). The World Health Organization (WHO) has reported around 600 million cases of foodborne diseases and 420,000 related deaths occurring annually due to pathogens (Shan et al., 2019).

Conventional physical treatment may efficiently deactivate foodborne pathogens but influence the functional and sensory properties of foods. Chemical agents, including antibiotics, also have been used widely to reduce microbiological contamination done by foodborne pathogens. However, misuse of antibiotics has contributed to the large spread of foodborne pathogens that have become resistant to multiple antibiotics (Caniça et al., 2019; Liao et al., 2020; Yuan et al., 2020). Consumption of contaminated food products could transfer resistant genes from antibiotic-resistant microorganisms to the human body through mobile genetic elements, leading to foodborne diseases, and hence enormous pressure on medical system (Das et al., 2017). This led researchers to develop novel methods to fight foodborne pathogens.

Recently, in order to reduce the heavy burden of foodborne diseases, natural antiseptic strategies have been increasingly developed to control foodborne pathogens (Ghazy et al., 2021; Kim et al., 2021; Savas et al., 2020; Zhu et al., 2021). Kombucha is known as anacidic and sweet refreshing beverage obtained from fermented sugared tea (Cardoso et al., 2020) or other substrates such as grape juice (Ayed et al., 2017), soursop (Tan et al., 2020), yarrow (Vitas et al., 2018), coffee (Bueno et al., 2021), and milk (Kruk et al., 2021). This fermentation is carried out by a characteristic consortium of yeasts, acetic acid bacteria, and often, but not always, lactic acid bacteria (Figure 1) (Coelho et al., 2020). The popularity of kombucha has spread widely due to its high nutritive and healthy values (Jayabalan et al., 2014; Leal et al., 2018).

Figure 1. Kombucha fermentation and its antimicrobial activities.

Although some studies have summarized the biological activities of kombucha (Coelho et al., 2020; Jayabalan et al., 2014; Leal et al., 2018), knowledge of its antimicrobial activity against foodborne pathogens and potential antimicrobial mechanism is still limited. In the context of above discussion, this review aimed to provide an overview of the current knowledge related to chemical and microbiological compositions of kombucha obtained by fermenting various substrates, the potential antimicrobial agents found in kombucha, and the limitations of kombucha in the food industry. In addition, need for developing antimicrobial agents from kombucha is discussed for potential applications. A comprehensive online literature review was conducted from January 2021 to July 2021 by using the following keywords on the web of science: kombucha and/or foodborne pathogens. In addition, for each selected publication, the reference section was examined to identify additional relevant publications. The information provided in this review indicates that kombucha could serve as an alternative approach to antibiotics to control pathogens.

Knowledge of Kombucha: Microbiological and Chemical Composition

Microbiological composition of kombucha

The difficulty in understanding kombucha fermentation is due to the high diversity of microorganisms and their complex interactions (May et al., 2019). Yeasts such as Zygosaccharomyces, Dekkera, Pichia, Hanseniaspora, Zygowilliopsis, Candida, and Saccharomyces, found in kombucha appear to be complicated, with the dominant species changing during the fermentation process (Coton et al., 2017; Villarreal-Soto et al., 2018). Yeasts have high fermentative ability to hydrolyze sucrose into fructose and glucose by action of invertase, followed by the generation of ethanol via glycolytic pathway. However, high concentration of alcohol is harmful to microorganisms through modifications to the structures, functions, and integrities of cellular membranes (Teoh et al., 2004). Ethanol can be reduced by aldehyde and alcohol dehydrogenase produced by acetic acid bacteria that oxidize it and produce acetic acid via the Krebs cycle (Gomes et al., 2018). This acetic acid conversion could be stopped when the desired taste of the beverage is achieved. The acidic conditions in kombucha protect the system from the invasion of pathogenic microorganisms (Coelho et al., 2020). The acetic acid bacteria contained in kombucha is mostly represented by Komagataeibacter, Acetobacter, Gluconacetobacter, and Gluconobacter (Arıkan et al., 2020; Tran et al., 2020). Genera of Komagataeibacter and Gluconacetobacter can also synthesize cellulose chains to form a surface biofilm by oxidation reactions with glucose dehydrogenase. This biofilm protects microorganisms from extreme environmental challenges such as ultraviolet (UV) radiation (Coelho et al., 2020). In addition, lactic acid bacteria (Lactobacillus, Lactococcus, Leuconostoc, and Oenococcus) have been reported to be frequently isolated and identified in kombucha from different areas (Marsh et al., 2014). It must be mentioned that the presence of lactic acid bacteria could improve the survival and biological functions of acetic acid bacteria (Nguyen et al., 2015; Pei et al., 2020). However, the microbial composition of kombucha samples, described in separate studies, is affected by various factors, including the incubation temperature and time, raw materials, source of sugar, and selected starter (Morales, 2020; Neffe-Skocińska et al., 2017). Hence, it is not easy to find out an exact consortium of kombucha for industry applications.

In most studies, the production of kombucha was carried out on a low scale, while vital fermentation parameters, such as oxygenation and sugar content distribution, are ignored (Arıkan et al., 2020; Cardoso et al., 2020; Kaewkod et al., 2019). To unravel a core microbial composition essential for controlling the fermentation of kombucha, Coton et al. (2017) conducted a study to explore microbial composition from kombucha fermentation on industrial level. Dominated yeasts are identified as Dekkera, Hanseniaspora, and Zygosaccharomyces, while Gluconobacter oxydans, Gluconacetobacter europaeus, Acetobacter peroxydans, and Gluconacetobacter saccharivorans are dominant acetic acid bacteria found in kombucha.

Chemical components of kombucha

High nutritive values of kombucha are partly attributed to its microbial community established during the fermentation of kombucha (Coelho et al., 2020). As shown in Table 1, the chemical analysis of kombucha has confirmed the presence of various compounds, such as organic acids (tartaric acid, malic acid, citric acid, acetic acid, quinic acid, oxalic acid, citric acid, succinic acid, D-glucuronic acid, and ascorbic acid), sugars (sucrose, glucose, and fructose), minerals (manganese, zinc, copper, cobalt, nickel, and lead), water-soluble vitamins (C, B₁, B₂, B₆, and B₁₂), and ethanol (Ivanišová et al., 2020; Kaewkod et al., 2019). However, the chemical composition of kombucha varies depending on the microbiological composition of the culture used for kombucha fermentation, types of substrates, and fermentation conditions (Kaewkod et al., 2019; Neffe-Skocińska et al., 2017).

Antimicrobial activities

Scientific studies have correlated antimicrobial activity of kombucha with acids production, polyphenols as well as the presence of lactic acid bacteria (Kaewkod et al., 2019; Morandi et al., 2020; Verrillo et al., 2021). However, the exact mechanism of action of kombucha on pathogenesis is still debatable.

Acids

Recent studies have indicated that the antimicrobial activity of kombucha against foodborne pathogens is mostly attributed to its low pH value, especially in the presence of acetic acid. It is known that acetic acid can inhibit a number of microorganisms by cytoplasmic acidification and accumulation of dissociated acid anion to toxic levels (Velićanski et al., 2014). For example, Steinkraus et al. (1996) showed that the inhibition effects of kombucha against E. coli, Helicobacter pylori, and S. aureus were attributed to the high concentration of acetic acid. The significance of organic acids found in kombucha was also pointed out by Kaewkod et al. (2019), who determined that kombucha tea showed antibacterial activity against E. coli, Salmonella Typhi, Vibriocholera, and Shigella dysenteriae, while neutralized kombucha did not reveal any antimicrobial activity against these microorganisms. Moreover, the antibacterial activity of heat-denatured kombucha indicates that this thermo-stable antimicrobial agent could be used in food products to control thermophilic spore-forming bacteria (Kaewkod et al., 2019). Tan et al. (2020) proved that acetic acid in kombucha could penetrate Gram-positive bacteria cells more easily than Gram-negative bacteria because of its lipophilic characteristics, and the highest proportion of microbial growth inhibition against E. coli and S. aureus was 99.83% and 100%, respectively. However, Sreeramulu et al. (2000) confirmed the presence of antimicrobial compounds in kombucha other than acetic acid and large proteins, as the antimicrobial activities against E. coli, Shigella sonnei, Salmonella typhimurium, S. enteritidis, and C. jejuni were shown even at neutral pH. Silva et al. (2021) also indicated that bioactivity could be more related to compounds present in kombucha than the total acidity produced during fermentation. Different varieties and quantities of substrates used in the production of kombucha result in beverages with different levels of organic acids and antimicrobial activity.

Polyphenols

Besides organic acids, tea-derived phenolic compounds have been described as potential antimicrobial agents that regulate the composition of intestinal microbes to prevent foodborne diseases by destabilizing microbial cell surface and cytoplasmic membranes (Verrillo et al., 2021). A total of 127 phenolic compounds were identified in the green and black tea kombucha by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS), most of which were flavonoids (70.2%), followed by phenolic acids (18.3%), other polyphenols (8.4%), lignans (2.3%), and stilbenes (0.8%) (Cardoso et al., 2020). The high diversity of phenolic compounds in kombucha is due to their bio-transformation or degradation in tea varieties by enzymatic action or low pH during the fermentation of kombucha (Cardoso et al., 2020; Kaewkod et al., 2020).

Mizuta et al. (2020) proved the antibacterial activity of green tea kombucha fermented at different periods, even at low concentrations, against Alicycobacillus species, including Alicyclobacillus hesperidum, Alicyclobacillus herbarius, Alicyclobacillus cycloheptanicus, Alicyclobacillus acidiphilus, and Alicycobacillus acidoterrestris in orange juice. After exposure to the polyphenolic fractions of kombucha, the cell wall disruption and bacillus integrity deformation of Alicycobacillus species were observed by scanning electron microscopy (SEM). In addition, the increased antimicrobial activity of kombucha was due to the presence of metabolites produced by the kombucha consortium. In another study, the fermented sugared black tea exhibited potent bactericidal activity against enteric bacterial pathogens, including S. flexneri, E. coli, S. Typhimurium, and V. cholera. Isorhamnetin and catechin were proved as the main antimicrobial agents in the polyphenolic fraction of kombucha (Bhattacharya et al., 2016). It is hypothesized that the presence of hydroxylation at positions 5 and 7 of the A ring and position 3 of the C ring, and free hydroxyl group(s) in the B ring of flavonoids contributes to the antimicrobial activity of polyphenolic compounds. Moreover, in vivo studies have been carried out to confirm the promising antimicrobial activity. For example, Bhattacharya et al. (2020) suggested that these two polyphenolic fractions from kombucha can significantly inhibit the motility and gene expressions (motY and flaC) of V. cholera related to flagellar regulatory, and prevent bacterial colonization in intestinal epithelial cells at sub-inhibitory concentrations. On the other hand, phenolic compounds are reported to increase the abundance of probiotics, maintain intestinal homeostasis, and prevent the infections caused by foodborne pathogens (Faria et al., 2014).

Nevertheless, there are some studies that conflict the antibacterial activity of polyphenols. For example, Chou et al. (1999) reported that the tea fermentation process decreased its antibacterial activity with increasing fermentation time, which indicated that the antibacterial activity of tea-derived phenolic compounds could be partly destroyed by reduction in the concentration of catechins during enzymatic oxidation.

Lactic acid bacteria

Some studies have proved that the antimicrobial activity of kombucha is not exclusively due to organic acids and phenolic compounds but possibly because of lactic acid bacteria and their biologically active components such as bacteriocins (Alizadeh et al., 2020; Kadyan et al., 2021). The most important antimicrobial mechanism is the production of acid by lactic acid bacteria, as acidification may alter cell metabolism by damaging enzymes and the substructure and function of cell walls and membranes, interrupting nutrient absorption and inhibiting protein synthesis (Gao et al., 2019). In addition, bacteriocins are antimicrobial agents produced by diverse bacterial species that can alter the membrane by corrupting potassium ion and ATP and cause cell failure to balance intracellular pH (Simons et al., 2020). Therefore, the antimicrobial activity of bacteriocins against foodborne pathogens has gained increasing interest for their applications in the food industry.

Pediococcus pentosaceus and Pediococcus acidiliactici isolated from kombucha showed high antimicrobial activity against S. enterica Typhimurium, L. monocytogenes, Listeria ivanovii,B. cereus, Proteus hauseri, and S. aureus. Moreover, their antimicrobial activities were also proved against foodborne molds, including Penicillium expansum and Penicillium digitatum (Diguta et al., 2020). In another study, a novel bacteriocin produced by Lactobacillus plantarum isolated from the kombucha consortium was found to have antibacterial activity against Gram-negative (E. coli) and Gram-positive bacteria (Listeria innocua, Bacillus subtilis, L. monocytogenes, Clostridium butyricum, B. cereus, Bacillus megaterium, Micrococcus luteus, Brochothrix thermosphacta, and S. aureus) because of increasing cell membrane permeability and release of potassium ions (Pei et al., 2020).

However, the only concern is that the resistance genes may be transferred from pathogens to lactic acid bacteria, and the consumption of lactic acid bacteria carrying resistance genes may pass these genes to humans.

Interestingly, kombucha was reported to modulate gut microbiota in humans and its subsequent effect on health status. Jung et al. (2018) reported that genera of Allobaculum, Turicibacter, and Clostridium involved in the pathogenesis of non-alcoholic fatty liver disease were significantly decreased, whereas Lactobacillus increased after the consumption of kombucha, which could suppress accumulation of fat in the human liver. However, more clinical trials are required to evaluate the effects of kombucha on human microbiome.

It is worth mentioning that the antimicrobial activity of kombucha is not always positively affected by the fermentation process. Silva et al. (2021) proved that antimicrobial agents could be more related to the compounds present in tea infusion than to those produced during the fermentation process.

Toxicity and Limitation of Kombucha

Some pathogenic microorganisms, including Salmonella enterica, C. albicans, and Penicillium. spp, were isolated and identified from kombucha prepared without hygiene environments (Villarreal-Soto et al., 2020). Migration of ceramic containers used to store kombucha was reported to cause lead poisoning and gastrointestinal toxicity (Phan et al., 1998). Allergic reactions, nausea and dizziness, lactic acidosis, headache, and other potential toxicities have been reported in individuals (Jayabalan et al., 2014). Therefore, further studies involving more in vivo trials are required to validate the toxicity of ingredients found in kombucha.

Conclusion and Perspectives

Antibiotics are widely used to control infections caused by foodborne pathogens. However, the emergence of antibiotic-resistant bacteria has increased with the overuse of antibiotics. Therefore, towing to their health benefits and antimicrobial activities, natural products such as kombucha could be considered as effective, cheap, and easily available alternatives against foodborne pathogens without negative effects on the health of consumers. However, control of kombucha production is mostly empiric despite the increasing knowledge provided by scientific community, and its antimicrobial property is also not well understood. Besides the organic acids, phenolic compounds, and lactic acid bacteria, alcohol and aldehydes are other important antimicrobial agents found in kombucha but not considered. Utilization of omics technology is encouraged to further reveal the molecular-level antimicrobial mechanisms. Moreover, clinical trials are required to confirm the effects of kombucha consumption on gastrointestinal microbiota composition and health risks in humans.