Applmicrobiol 02 00005 v3

Review
Advances in Kombucha Tea Fermentation: A Review

Laura M. Nyhan 1 , Kieran M. Lynch 1 , Aylin W. Sahin 1 and Elke K. Arendt 1,2, *
1 School of Food and Nutritional Sciences, University College Cork, T12 YN60 Cork, Ireland;
[email protected] (L.M.N.); [email protected] (K.M.L.); [email protected] (A.W.S.)
2 APC Microbiome Ireland, University College Cork, Western Road, T12 K8AF Cork, Ireland
* Correspondence: [email protected]; Tel.: +353-21-4902064; Fax: +353-21-4270213
Abstract: Kombucha is a carbonated, slightly acidic beverage traditionally produced by the fer-
mentation of sweetened tea by a symbiotic culture of bacteria and yeast (SCOBY). The microbial
community of kombucha is a complex one, whose dynamics are still not fully understood; however,
the emergence of culture-independent techniques has allowed a more comprehensive insight into
kombucha microbiota. In recent times, advancements have been made towards the optimisation
of the fermentation process, including the use of alternative substrates, defined starter cultures
and the modification of fermentation parameters, with the aim of producing an innovative bever-
age that is improved in terms of its physiochemical, sensory and bioactive properties. The global
kombucha market is rapidly increasing, with the rising popularity of the tea attributed in part to
its purported health benefits, despite the lack of research in human subjects to substantiate such
claims. Accordingly, the incidence of kombucha home-brewing has increased, meaning there is a
requirement for individuals to recognise the potential hazards associated with fermentation and the
relevant preventative measures to be undertaken to ensure the safe preparation of kombucha. The
aim of this review is to provide an update regarding the current knowledge of kombucha production,
microbiology, safety and marketing.

Keywords: kombucha; tea; beverage; fermentation; bacteria; yeast
Citation: Nyhan, L.M.; Lynch, K.M.;
Sahin, A.W.; Arendt, E.K. Advances
in Kombucha Tea Fermentation: A
Review. Appl. Microbiol. 2022, 2, 1. Introduction
73–103. https://doi.org/10.3390/
Kombucha tea is a non-alcoholic, naturally carbonated beverage prepared by ferment-
applmicrobiol2010005
ing a solution of sweetened tea with a kombucha culture containing bacteria and yeast. It is
Academic Editor: Fani Mantzouridou believed to have originated in northeast China over 2000 years ago, with the consumption
of kombucha first recorded in 220 BC in Manchuria [1]. Following this, it is reported to have
Received: 21 December 2021
travelled to Russia and Eastern Europe in the 1800s and beyond this to Western Europe and
Accepted: 14 January 2022
North Africa during World War II [2]. Kombucha is known by several names including
Published: 15 January 2022
haipao, teakwass, tea fungus, Manchurian mushroom and kambotscha [3].
Publisher’s Note: MDPI stays neutral Kombucha is produced by fermenting sweetened tea using a symbiotic culture of bac-
with regard to jurisdictional claims in teria and yeast (SCOBY), also known as a pellicle. Acetic acid bacteria (AAB) of the genera
published maps and institutional affil- Komagataeibacter, Acetobacter and Gluconobacter and yeasts such as Zygosaccharomyces spp.,
iations. Saccharomyces spp. and Brettanomyces spp. are the core drivers of kombucha fermenta-
tion [4,5], although lactic acid bacteria (LAB) such as Lactobacillus and Leuconostoc have also
been systematically isolated from the beverage [6,7]. Traditionally, culture-based methods
were relied upon for characterisation of the microbial composition of kombucha [4,8], how-
Copyright: © 2022 by the authors.
ever the emergence of culture-independent techniques such as metabarcoding has allowed
Licensee MDPI, Basel, Switzerland.
This article is an open access article
for a more in-depth analysis of kombucha microflora and their metabolic activities and
distributed under the terms and
interactions [6]. During fermentation, yeasts hydrolyse sucrose to glucose and fructose as a
conditions of the Creative Commons
by-product of ethanol fermentation, while AAB then convert ethanol and glucose into acetic
Attribution (CC BY) license (https:// acid and gluconic acid, respectively [9]. Other metabolites formed as a result of kombucha
creativecommons.org/licenses/by/ fermentation include lactic, citric, malic and glucuronic acids; vitamins; minerals; and
4.0/). phenolic compounds [10,11]. Black or green tea and sucrose are the traditional substrates
Appl. Microbiol. 2022, 2, 73–103. https://doi.org/10.3390/applmicrobiol2010005 https://www.mdpi.com/journal/applmicrobiol

Appl. Microbiol. 2022, 2 74
for kombucha fermentation; however, in recent years there has been a shift towards the use
of alternative carbon and nitrogen sources, with the aim of increasing the functionality of
the final beverage.
The global kombucha market has seen significant growth in recent years, standing at
USD 1.84 billion in 2019, with a projected growth rate of 23.2% by 2027 [12]. The rise in
popularity of kombucha is in part attributed to its purported health benefits, having been
cited as having such abilities as boosting the immune system, alleviating IBS symptoms,
aiding in weight loss and reducing blood pressure, to name but a few. However, most
of these claims are unsubstantiated and lack scientific evidence, with few clinical trials
carried out to date [13]. Moreover, there have even been reports of toxicity associated with
over-consumption of kombucha, particularly in immunocompromised individuals [14–16].
The rising popularity of kombucha as a ‘healthy’ product has resulted in an increased
incidence of home-brewing of the beverage, often using a SCOBY obtained from another
household as a starter [17]. Thus, it is imperative that individuals are aware of the potential
hazards associated with domestic kombucha production and the preventative measures
that should be taken for safe kombucha fermentation [18].
The objective of this review is to provide an update on the current knowledge and
research in the area of kombucha. The traditional production and fermentation processes
of kombucha will be discussed, along with advancements in the area, such as the use of
alternative substrates and adjustment of fermentation parameters. An overview of the
microbial composition will be given, with a focus on the emergence of culture-independent
methods for microbial characterisation. The safety of kombucha production and associated
risks will be outlined, in addition to the purported health benefits and recent progress in
the area of human clinical trials. Finally, an overview of the kombucha market will be
provided, alongside an analysis of some of the commercial kombucha products.
2. Kombucha Tea Production

The typical process of kombucha preparation and fermentation is outlined in Figure 1.
Under aerobic conditions, kombucha symbiosis can convert sweetened tea into a slightly
carbonated, mildly sour and refreshing beverage. The fermentation process essentially
exploits the symbiotic relationship between bacteria and yeast. Sucrose is hydrolysed into
glucose and fructose by the yeast, resulting in the production of carbon dioxide and ethanol;
in turn, LAB utilise glucose and fructose molecules to produce lactic acid, while AAB
utilise glucose and ethanol to produce gluconic acid and acetic acid, respectively. Typically,
kombucha is prepared by fermenting sweetened black or green with previously fermented
liquid tea broth (10–20% v/v) or a tea fungus pellicle. The substrate is incubated statically
under aerobic conditions for 7–14 days at 20 ◦ C–30 ◦ C [9].
Sucrose is by far the most common carbon source used in kombucha fermentation,
both because of its ability to provide glucose and fructose for microbial metabolic pathways
and its low cost and convenient availability. There is a general consensus that the optimal
sucrose concentration lies between 5–10% w/v [19,20], a range within which the majority
of kombucha fermentations are performed. The use of tea in kombucha fermentation is
primarily cited as providing a source of nitrogen and minerals for kombucha microorgan-
isms, with dried tea leaves containing 21–28% protein and amino acids contributing about
6% w/w of the extract solids. Additionally, the reported levels of glycine, valine, lysine,
leucine, alanine, methionine and threonine in tea protein exceed even those of soy proteins
(g/100 g protein) [21,22]. Black and green teas sourced from leaves of Camellia sinensis (L.)
are traditionally used for kombucha fermentation. For home fermentations, the process is
considered complete once the desired level of sourness is achieved, while for experimental
or industrial investigations, the fermentation endpoint may be determined by measure-
ment of the pH, total titratable acidity (TTA), alcohol content and sugar concentration. It
is generally accepted that pH of an optimally fermented kombucha (in terms of flavour)
should fall between pH 2.5 and 3.5 [9,23] while a TTA of ~4 g/L produces a traditional
kombucha beverage of high sensory value [2,24].
Appl. Microbiol. 2022, 2 fermented kombucha (in terms of flavour) should fall between pH 2.5 and 3.5 [9,23] while
75
a TTA of ~4 g/L produces a traditional kombucha beverage of high sensory value [2,24].
1. An overview of kombucha preparation

Figure 1. preparation and
and fermentation.
fermentation.
2.1. Alternative Substrates

2.1. Alternative Substrates
Although sucrose is the most commonly used carbon source in kombucha fermenta-
Although sucrose is the most commonly used carbon source in kombucha
tion, the influence of alternative carbohydrate sources and their respective concentrations
fermentation, the influence of alternative carbohydrate sources and their respective
on the fermentation process has been investigated (Figure 2). Early studies by Reiss [24]
concentrations on the fermentation process has been investigated (Figure 2). Early studies
investigated the fermentation characteristics of kombucha teas containing sucrose, glucose,
by Reiss [24] investigated the fermentation characteristics of kombucha teas containing
lactose or fructose, with the results showing that the sugar type used distinctly affected the
sucrose, glucose, lactose or fructose, with the results showing that the sugar type used
ethanol and lactic acid formation; however, the concentration of the sugars did not have a
distinctly affected the ethanol and lactic acid formation; however, the concentration of the
significant impact. Malbasa et al. [25] found that kombucha produced using molasses as a
sugars did not have a significant impact. Malbasa et al. [25] found that kombucha
substrate had higher total acidity, higher lactic acid content and a significantly larger yield
produced using molasses as a substrate had higher total acidity, higher lactic acid content
of biomass than the sucrose-sweetened kombucha tea, attributed to the greater availability
and
of a significantly
nitrogen present larger
in the yield of biomass
molasses. thanthe
However, thesensory
sucrose-sweetened
properties ofkombucha tea,
the obtained
attributed to the greater availability of nitrogen present in the molasses. However,
kombucha beverage were not satisfactory, with a sweet, caramelised taste and dark colour. the
Recently, another study investigated molasses as an alternative substrate to sucrose, alonga
sensory properties of the obtained kombucha beverage were not satisfactory, with
sweet,
with caramelised
coconut taste and
palm sugar. darktocolour.
Similar Recently,
previous another
observations study
[25], investigated
kombucha molasses
produced with
molasses had a higher total acidity level than that of the sucrose-containing kombucha,
while notably kombucha tea fermented with coconut palm sugar demonstrated the highest
antioxidant activity and phenolic content, highlighting its potential for use as a functional
beverage [26]. The roles of complex carbohydrate sources in kombucha fermentation have
also been studied, with the authors investigating the influence of sucrose substitutes such
as Jerusalem artichoke, malt extract and jaggery [27–30]. Of note is a recent study where
germinated corn was used as a substrate to produce a no-added-sugar kombucha beverage,
Appl. Microbiol. 2022, 2 the first investigation of kombucha fermentation of exclusively raw seeds or grains with no 77
additional carbon or nitrogen sources. The authors proposed that grains could provide the
nutritional requirements for a ‘healthy SCOBY’, while the soluble fibre could be a suitable
substitute
beneficial for
in tea alkaloids.
terms of theirThe final pHcompounds,
functional value of the beverage
but they was
can similar to that ofthe
also stimulate a
commercial kombucha product, while 57.6% of consumers preferred the germinated
increased formation of organic acids such as glucuronic acid under favourable corn
kombucha
fermentationover the commercial
conditions [50–52].beverage [31].
Figure 2. Examples of alternative carbon and nitrogen sources used in kombucha fermentation.
Figure 2. Examples of alternative carbon and nitrogen sources used in kombucha fermentation.
2.2. Adjustment of Fermentation

Despite black and green Conditions
tea being well documented in terms of their functional
compounds, such
Typically, as their flavonoids,
kombucha fermentationspolyphenols and saponins,
are performed in recent times7–14
for approximately attention
days,
has turnedthis
although towards the use in
varies greatly ofthe
alternative
literature,tea or nitrogen
with reports ofsources (Figurebeing
fermentations 2), with the
carried
aim
out of
forproducing a beverage
up to 60 days [27]. Thericheffects
in bioactive
of the compounds.
fermentation Alongtime on with
the black
final and green
kombucha
tea, the processing
beverage have beenof Camellia sinensis
well-studied, (L.)results
with the leaveshighlighting
also produces its white (non-oxidised),
significant impacts on
oolong (semi-oxidised) and pu-erh (post-oxidised) teas. A comparative
the product’s bioactive, sensory and microbial characteristics. Vohra et al. [27] reported analysis of the
influence of each of these tea types on kombucha fermentation revealed
an increase in antioxidant activity of the kombucha beverage during the first seven days that both oolong
tea kombucha (OTK)
of fermentation, which and white
was tea kombucha
attributed (WTK) showed the
to the biotransformation highest antioxidant
of phytochemicals into
capacities
polyphenols during the fermentation,
by enzymes secreted by while OTK, WTKculture.
the kombucha and pu-erh tea kombucha
However, (PTK)
a decreasing all
trend
had higher bioaccessability values than green or black tea kombucha.
was observed over the remaining 53 days of fermentation. A similar scenario was Differences between
teas were attributed
observed by Zofia ettoal.factors such as the
[53], whereby thetea leaf processing
antioxidant method,
effect of kombuchamaturation period
was highest at
and relative abundance of phenolic compounds [32]. The functional properties
day 14 of fermentation but was significantly lower on day 28. Additionally, the 14-day- of oolong
tea were also
fermented highlighted
kombucha by Tanticharakunsiri
proved to be less cytotoxicet al.
to[33],
skinresulting
cells thaninthe
increases in the total
28-day-fermented
beverage, with the authors speculating that prolonged fermentation may result in the
accumulation of harmful levels of organic acids and other metabolites. Based on similar
observations, Muhialdin et al. [26] recommended a fermentation period of 12 days for
optimal antioxidant activity and phenolic content. The microbial composition of
kombucha can also be influenced by fermentation, with an in-depth study by Chakravorty
phenolic content (TPC) and total flavonoid content (TFC) of the kombucha beverage by 6.6%
and 32.26%, respectively, compared to 3.4% and 12.48% with mint tea. Teas such as yerba
mate, rooibos and Zijuan have also recently been investigated as alternative kombucha
substrates, with studies highlighting their superiority compared to black tea kombucha in
terms of their increased antioxidant activity, reduced oxidative stress and improved sensory
characteristics [34–36]. Interest in the use of herbal teas and medicinal plants as kombucha
substrates has also increased, with several studies attempting to harness their associated
health benefits and functional properties. Early attempts to use these as kombucha tea
alternatives proved unsuccessful, with herbal teas such as peppermint, lime blossom, mint,
camomile, rosemary and sage deemed as unsuitable substrates due to the negative effects
of their volatile oils, including their poor acidification, interference with the growth of
kombucha microbes due to the presence of antimicrobial compounds and storage instability
of the beverage [37]. However, Velićanskí et al. [38] found that kombucha could be suitably
produced using peppermint or thyme teas, resulting in comparable or shorter fermentation
lengths [38], while Tanticharakunsiri et al. [33] and Zhang et al. [39] successfully obtained
sensorially acceptable kombucha beverages using mint tea as a substrate. The first use of
yarrow (a plant containing over 100 bioactive compounds) as a kombucha substrate was
reported by Vitas et al. [40], with the authors exploiting the medicinal properties of the
plant to produce a beverage that was characterised as having antimicrobial, anticancer,
and antioxidant potential. Other such herbs and plants tested as kombucha substrates
include ginger, lemon balm, oak and nettle leaf [38,41–46]. Fruits and vegetables have also
emerged as potential kombucha substrates, with researchers again focusing on those well-
characterised in terms of their bioactive compounds and potential health benefits. Such
studies have investigated the use of raw materials such as spinach, grape juice, cherry juice,
banana peel and pomegranate juice [45,47–52]. Not only are these substrates beneficial in
terms of their functional compounds, but they can also stimulate the increased formation
of organic acids such as glucuronic acid under favourable fermentation conditions [50–52].
2.2. Adjustment of Fermentation Conditions

Typically, kombucha fermentations are performed for approximately 7–14 days, al-
though this varies greatly in the literature, with reports of fermentations being carried
out for up to 60 days [27]. The effects of the fermentation time on the final kombucha
beverage have been well-studied, with the results highlighting its significant impacts on
the product’s bioactive, sensory and microbial characteristics. Vohra et al. [27] reported
an increase in antioxidant activity of the kombucha beverage during the first seven days
of fermentation, which was attributed to the biotransformation of phytochemicals into
polyphenols by enzymes secreted by the kombucha culture. However, a decreasing trend
was observed over the remaining 53 days of fermentation. A similar scenario was observed
by Zofia et al. [53], whereby the antioxidant effect of kombucha was highest at day 14 of
fermentation but was significantly lower on day 28. Additionally, the 14-day-fermented
kombucha proved to be less cytotoxic to skin cells than the 28-day-fermented beverage,
with the authors speculating that prolonged fermentation may result in the accumulation
of harmful levels of organic acids and other metabolites. Based on similar observations,
Muhialdin et al. [26] recommended a fermentation period of 12 days for optimal antioxi-
dant activity and phenolic content. The microbial composition of kombucha can also be
influenced by fermentation, with an in-depth study by Chakravorty et al. [7] revealing
significant changes in microbial community and diversity over 21 days. Candida initially
dominated yeast in the liquid portion of the kombucha; however, a shift in dominance to
Lachancea spp. on day 7 of the fermentation was observed. In addition, bacterial diversity
was higher in the kombucha liquid than in the pellicle, peaking on the seventh day and
subsequently decreasing. Interestingly, a similar situation was observed with the bio-
chemical properties of the kombucha, with the radical scavenging ability, reducing power
and polyphenol content reaching maximum levels at day 7, indicating a link between the
microbial composition and biochemical activity. Finally, the effect of fermentation time
on the sensory characteristics of the final beverage must also be considered. In general,
a fermentation period of 6–7 days appears to result in a sensorially acceptable beverage,
while increasing this often results in a vinegar-like sour flavour [24,34,43,47,48,54].
The fermentation temperatures vary greatly amongst the literature; however, the ma-
jority of kombucha fermentations are performed in the range of 20–30 ◦ C. As the optimum
growth temperatures of yeast and bacteria differ, it is not surprising that the temperature
profile can greatly influence the biochemical reactions taking place during fermentation,
with studies showing that the temperature is more impactful on fermentation kinetics than
the inoculum concentration [55–57]. Additionally, the metabolic activity of the yeast is
critical to provide substrates for LAB and AAB in the kombucha culture; thus, fermenta-
tions must be performed at a temperature suited to yeast growth [58]. De Filippis et al. [59]
investigated the microbial compositions of kombuchas fermented at 20 ◦ C and 30 ◦ C. The
results showed that there was a higher level of diversity in the pellicle at 30 ◦ C, while
this temperature also promoted the growth of several LAB and AAB species. Significant
differences in microbiota composition of the kombucha beverages were reported. Interest-
ingly, the prevalence of two different species of Gluconacetobacter (Komagataeibacter) was
strongly dependent on the fermentation temperature, with G. xylinus (K. xylinus) and
G. saccharivorans (K. saccharivorans) dominating at 20 ◦ C and 30 ◦ C, respectively. Addition-
ally, the increased levels of gluconic and glucuronic acids at 30 ◦ C positively correlated
with the presence of G. saccharivorans. Antioxidant activity during fermentation is another
factor that is primarily affected by temperature, with Hur et al. [60] commenting that as
temperature increases, so too does the polyphenol content, up until a point where too high
of a temperature will result in polyphenol oxidation.. The optimal fermentation tempera-
ture will also be determined by the kombucha substrate, as evidenced by Vitas et al. [57],
whereby the sensory acceptability of kombucha produced using stinging nettle or winter
savoury plant was highly dependent on the herbal tea used, with the kombucha beverages
scoring highest when produced at 43 ◦ C or 40 ◦ C, respectively.
3. Microbial Diversity
Kombucha fermentation consists of metabolically active bacteria and yeast, which
thrive in two mutually non-exclusive compartments: the fermented liquid and the pellicle
or biofilm floating on it [7]. The microbial composition of kombucha varies greatly from
one batch to another and is dependent on the origin, substrate and fermentation conditions
(Table 1). Whilst no yeast has been found to be universally associated with kombucha
fermentation, species from genera such as Zygosaccharomyces, Saccharomyces, Brettanomyces,
Pichia and Candida are amongst the most common isolates [4,8,61–63]. AAB appear to
dominate the microbial community, with the main isolated species affiliated with the
Komagataeibacter, Acetobacter and Gluconobacter genera. Komagataeibacter xylinus (formerly
Gluconacetobacter xylinus) is thought to be one of the most important species associated with
kombucha fermentation, due to its superior cellulose-synthesising ability [64–66]. LAB are
not consistently present; however, the systematic isolation of species from genera such as
Lactobacillus, Leuconostoc and Bifidobacterium has been reported [35,67,68].
Notwithstanding the widespread applicability and popularity of culture-based tech-
niques, studies reliant on such methods are limited in that only minor parts of complex
microbial communities are generally culturable, while less-abundant microbes are often
overshadowed by dominating ones [7]. Moreover, the potential for high-throughput anal-
ysis is severely reduced, with only a finite number of isolates ever being investigated [6].
The emergence of culture-independent techniques has allowed for a deeper insight into
the complex microbial population of kombucha and the metabolic interactions occurring
within [6,7,67]. Marsh et al. [6] were the first to perform a high-throughput sequence-based
analysis of the fungal and bacterial populations of five kombucha pellicles and the resultant
fermented tea. Contrary to previous studies [4,8,62], reads assigned to Gluconacetobacter
(>85%) greatly exceeded those of Acetobacter (<2%), which may partially have been due to
the reclassification of Acetobacter xylinum to Ga. xylinus (now known as K. xylinus) previ-
ously. The authors also noted the possibility that reads assigned to Gluconacetobacter may
belong to the genus Komagataeibacter, as microbial databases had not yet been updated to
reflect the reclassification at the time of publication. Interestingly, a considerable abundance
of LAB (Lactobacillus, Bifidobacterium, Lactococcus, Leuconostoc) was detected, a novel finding
considering Lactobacilli had been isolated from kombucha in just two studies previous to
this [68,69], while the presence of Leuconostoc had not previously been reported. Zygosac-
charomyces was the dominant yeast in the fermented beverage, while nine yeast species that
had not before been associated with kombucha, including L. fermentati, K. marxianus and
Wallemia sebi, were also detected. Zygosaccharomyces was also identified as the dominant
yeast in a study by Arikan et al. [70], which used a combination of whole-metagenome
shotgun (WMS) and amplicon sequencing to characterise the microbial composition of
kombucha. Through gene prediction and gene cluster screening, copper-binding peptides
were detected, which the authors speculated may be responsible for the copper biosorp-
tion characteristics of kombucha and its toxicity when brewed in metal vessels. Lachancea
and Kluveromyces were also isolated from kombucha by Chakravorty et al. [7]; however,
in contrast to findings by Marsh et al. [6], Zygosaccharomyces was absent, with the yeast
community instead being dominated by Candida, the first observation of this phenomenon.
In this study, both D1-D2 LSU and ITS2 genes were sequenced, an approach that is thought
to better elucidate the dynamics of the yeast community than sequencing of just a single
gene. Until addressed by Coton et al. [67], culture-independent techniques had not been
utilised to identify kombucha bacterial communities to species-level. Using partial 16S
rRNA gene sequencing (~1500 bp), members of the Acetobacteraceae family (Gluconobacter,
Gluconacetobacter, Acetobacter) were identified as the dominant bacterial species, followed
by Lactobacteriaceae. Interestingly, the substrate choice impacted the bacterial composition
of the fermented tea, with O. oeni and Lb. nagelii being present in both black and green tea
kombuchas and an additional species (Lb. satsumensis) being detected in just the black tea
fermentation. Dekkera, Hanseniaspora and Zygosaccharomyces were identified as the dominant
yeasts, while the tea type did not appear to influence the yeast community. Conversely,
Gaggìa et al. [35] found that the tea substrate choice (black, red or rooibos) did indeed affect
the fungal diversity but did not impact the bacterial composition, with Komagataeibacter
spp. dominating all kombuchas. Notably, the SCOBY archetype has also been shown to
influence the microbial composition of kombucha, with Harrison and Curtin [71] observing
differences in the diversity of microbiota between the upper and lower layers of the pellicle.
The top layer was more enriched in bacterial and fungal cell numbers, with increases of
3 log CFU/mL and 2 log CFU/mL, respectively, compared to the bottom SCOBY layer. As
the SCOBY usually floats to the surface of the kombucha during fermentation, parameters
such as oxygen and nutrient availability and temperature vary between the upper and
lower surfaces, likely influencing the microbial composition. In this study, the dominating
microorganisms of the upper SCOBY layer were determined to be Komagataeibacter and
Brettanomyces, taxa which both have an affinity for oxygen; in contrast, the abundance of
Lactobacillus in the lower pellicle was significantly higher, most likely due to its prefer-
ence for a high-nutrient, low-oxygen growth environment. The microbiota of the pellicle
also appears to be subject to a selection process during the fermentation, with Gaggìa
et al. [35] reporting that the abundance of yeasts in the fermented tea did not match that of
the biofilm.
Table 1. Studies investigating the microbial composition of kombucha.
Yeast Lactic Acid Acetic Acid Fermentation Fermentation Fermentation

Bacteria Bacteria Culture Inoculum Type Substrates Temperature Time Reference
Brettanomyces, Pellicle and liquid

Zygosaccharomyces, n.d. n.d. Dependent n/a n/a n/a [5]
Saccharomyces, Candida (n = 34)
Acetobacter (A.) Pellicle (3–4 layers) 67.5 g/L sucrose,

Zygosaccharomyces sp. n.d. Dependent 20 ◦ C–22 ◦ C 60 d [8]
xylinum and liquid (10% v/v) 1.5 g/L tea
Zygosaccharomyces (Z.) bailii, A. aceti, A. Pellicle and liquid
Saccharomyces (S.) cerevisiae, n.d. pasteurianus Dependent n/a n/a n/a [4]
Brettanomyces (B.) bruxellensis (n = 3)
Z. bailii, Schizosaccharomyces
(Sc.) pombe, Torulospora Pellicle and liquid 100 g/L sucrose,
delbreuckii, Rhodotorula n.d. n.d. Dependent 20 ◦ C–22 ◦ C 14 d [63]
mucilaginosa, B. bruxellensis, (10% v/v) 5.4 g/L black tea
Candida (C.) stellata
Defined starter
culture of yeasts and 70 g/L sucrose,
Isolate 5/3, isolate 7/2, n.d. Isolate 5, isolate 9 Dependent 28 ◦ C 11 d [61]
isolate 2/1 AAB (1 × 107 – 7.5 g/L winter
savory tea
4 × 108 CFU/mL)
Defined starter
culture of yeasts 7% sucrose,
Zygosaccharomyces sp., S.
n.d. Acetobacter Dependent (5.8 × 104 CFU/mL) 1.5 g/L black or 28 ◦ C 10 d [62]
cerevisiae green tea
and AAB
(105 CFU/mL)
5% sucrose,
Defined starter 7.5 g/L black tea,
S. cerevisiae Lactobacillus (Lb.) Gluconacetobacter sp. Dependent culture of yeasts, 30 ◦ C 90 h [72]
plantarum green tea or tea
LAB and AAB (1:1:1) powder
Dekkera (D.) bruxellensis, D.
anomala, Z. bisporus, Z. lentus,
Pichia (P.) kudriavzevii,
Davidiella tassiana, Wallemia sebi, Lactobacillus,
Lachancea (L.) fermentati, Lactococcus, Acetobacter, Pellicle (10% w/v) 10% sucrose,
Independent 0.49% black tea 23 ◦ C 10 d [6]
Leucosporidiella fragaria, Leuconostoc, Gluconacetobacter and liquid (10% v/v)
Kazachstania unispora, Bifidobacterium
Kluyveromyces marxianus,
Naumovozyma castelli,
Hanseniaspora (H.) vineae
Table 1. Cont.

Candida, Lachancea,
Kluyveromyces, Debaryomyces,
Pichia, Waitea, Eromothecium,
Meyerozyma, Zygowilliopsis, Lactobacillus, Komagataeibacter, Pellicle and liquid 10% sucrose, 5
Saccharomyces, Weissella, Independent g/L black tea 28 ◦ C 21 d [7]
Gluconobacter (10% v/v)
Saccharomycopsis, Hanseniaspora, Bifidobacterium
Kazachstania, Starmera,
Merimbla, Sporopachydermia,
Sugiyamaella
Komagataeibacter Pellicle (3% w/v) 10% sucrose,
Z. bailii n.d. Dependent 2% tea Room temp 28 d [73]
(K.) saccharivorans and liquid (10% v/v)
A. lovaniensis, A.
okinawensis, A.
peroxydans, A
syzgii, A.
C. boidinii, D. anomala, D. tropicalis,
bruxellensis, H. valbyensis, Gluconacetobacter
Wickerhamomyces anomalus, P. (Ga.) eurapaeus, Dependent
Oenococcus (O.) Ga. hansenii, Ga. Sweetened black
membranifaciens, S. cerevisiae, S. oeni, Lb. nagelii, L. intermedius, Ga. and n/a n/a 8d [67]
uvarum, Torulaspora satsumensis and green tea
liquefaciens, Ga. independent
microellipsoides, Z. bailii, Z. xylinus,
florentina
Gluconobacter (G.)
cerinus, G.
oxydans,
Tanticharoemia
sakaeratensis
10% sugar source
(Aspartame, bees’
Lactobacillus, honey, glucose,
Dekkera, Pichia, Leuconostoc, Gluconacetobacter, Caryota urens
Independent Pellicle (3% w/v) honey or 24 ◦ C 7d [74]
Zygosaccharomyces Lactococcus, Acetobacter
Bifidobacterium Palmyrah
jaggery), 10 g/L
black tea
Lactobacillus, Acetobacter, Ga. Dependent Pellicle (20 g/L) and 100 g/L sucrose,
n.d. Lactococcus, xylinus, Ga. and 10 g/L black or 20 ◦ C or 30 ◦ C 21 d [59]
Streptococcus saccharivorans independent liquid (3% v/v) green tea
Table 1. Cont.

80 g/L sucrose,
Lactobacillaceae, K. intermedius, K. Dependent
B. bruxellensis, Z. parabailii Leuconostocaceae, rhaeticus, Ga. Pellicle (3% w/v) 8 g/L black, 27 ◦ C 14 d [35]
and and liquid (10% v/v) green or
Streptococcaceae entanii independent
rooibos tea
Komagataeibacter
sp. DS1MA.62A, 10% molasses in
B. bruxellensis n.d. Dependent Liquid (10% v/v) acetate buffer Room temp 6d [75]
K. xylinus, K. (200 mM,
saccharivorans, Ga. pH 4.75)
saccharivorans
Zygosaccharomyces, Z. bailii n.d.

Komagataeibacter,
Independent Pellicle (100 mm2 ) 10 g/L sucrose,
28 ◦ C 15 d [70]
K. rhaeticus and liquid (10% v/v) 6 g/L tea
A. maolroum, A.
pasteurianus, A.
pomorum, A.
tropicalis, K.
C. arabinofermentans, B. rhaeticus, K. 70 g/L sucrose,
n.d. xylinus, K. Independent Pellicle (2% w/v) 25 ◦ C 14 d [76]
bruxellensis, Sc. pombe, Z. bailii europaeus, K. and liquid (2% v/v) 10 g/L black tea
intermedius, G.
oxydans,
Gluconacetobacter
sp. SXCC-1
Defined starter
culture of Z. bailii
(105 CFU/mL), A.
A. pasteurianus, pasteurianus 100 g/L sucrose,
Z. bailii n.d. Dependent 29 ◦ C 10 d [77]
Ga. xylinus 8 g/L black tea
(105 CFU/mL) and
Ga. xylinus
(106 CFU/mL)
Brettanomyces,
Zygosaccharomyces, Starmerella, Komagataeibacter,
Lachancea, Saccharomycetales, Lactobacillus, Acetobacter,
Oenococcus Gluconobacter, Independent Pellicle (n = 103) n/a n/a n/a [71]
Candida, Hanseniaspora, Pichia,
Ascomycota, Kregervanrija Gluconacetobacter
Table 1. Cont.

Acetobacteraceae, 70 g/L glucose

K. rhaeticus, K. Pellicle (4% w/v) and fructose 30 ◦ C
B. bruxellensis, Z. bisporus n.d. hansenii, K. Independent 15 d [78]
and liquid (10% v/v) mixture (1:1),
xylinus 0.5% green tea
Defined starter
A. tropicalis, A. culture of yeasts,
D. bruxellensis, H. uvarum, Z. Lb. nagelii, O. oeni okinawensis, K. 55 g/L blond 25 ◦ C
bailii hansenii, G. Independent LAB and AAB sugar, green tea 27 d [79]
oxydans (105 CFU/mL of
each)
B. bruxellensis, H. valbyensis, S. A. papayae, Ga. 60 g/L sucrose,
n.d. takamatsuzukensis Dependent Liquid (12% v/v) 26 ◦ C 3d [80]
cerevisiae 1% black tea
Note: Genus and species designations are shown as published; n.d.: not detected; n/a: information not available.
Use of Defined Starter Cultures

The use of defined strain starter cultures for kombucha fermentation is rare and has
been the focus of few research articles to date; thus, the transfer of the kombucha pellicle or
fermented liquid from one batch to the next remains the primary method of inoculation.
Although sufficient for home-brewing, such a method may be problematic for industrial-
scale production of kombucha due to uncontrolled shifts in microbial composition and
biological activities and a lack of reproducibility [77]. Malbaša et al. [62] investigated the
use of two different starter cultures for kombucha fermentation; the first a combination
of Saccharomyces cerevisiae and mixed AAB (starter culture 1; SC1) and the other a mix of
Zygosaccharomyces spp. and AAB (SC2). It was reported that SC1 resulted in a kombucha
beverage with significantly higher levels of antioxidant activity than SC2 when black tea
was used as a substrate; however, the native kombucha consortium (SCOBY) displayed
superior antioxidant activity on green tea. Fu et al. [72] discussed the use of equal amounts
(108 CFU/mL) of S. cerevisiae Meyen ex E.C. Hansen, Gluconacetobacter spp. and Lb. plan-
tarum, strains previously reported to be optimal for kombucha fermentation [81]. The
kombucha beverage produced using green tea exhibited the highest antioxidant potential,
although microbiological analysis of the kombucha stored at 4 ◦ C for 14 days showed poor
survival of Lb. plantarum (0.98%) compared to that of S. cerevisiae (73.97%) or Gluconacetobac-
ter spp. (54.09%). A tailor-made kombucha consortium consisting of two LAB species, four
AAB species and three yeast species previously isolated from kombucha and thought to be
the main drivers of the fermentation process was recently designed by Savary et al. [79]. The
authors carried out an in-depth analysis of the biochemical, physicochemical and microbial
parameters of fermentation and were the first to report on the coupling of fluorescent in
situ hybridisation (FISH) probes with confocal laser scanning microscopy (CLSM) and
scanning electron microscopy (SEM) to monitor the progressive formation of the kombucha
biofilm. The results showed that AAB dominated the biofilm, with evidence to suggest
that yeast and bacterial species may also have contributed to its formation, a phenomenon
that has previously been described in the literature [82–84]. Interestingly, a correlation
between shifts in yeast, LAB and AAB dominance and changes in the volatile profile of
the kombucha was established. The production of aldehydes during the early stages of
fermentation was linked to high abundance levels of H. uvarum, O. oeni and Z. bailii, with
ethanol, esters, and diacetyl formation during days 11–14 correlated with high AAB counts
in the biofilm. Lastly, a shift in dominance towards D. bruxellensis, A. tropicalis and Lb.
nagelii during the late stage (days 20–27) of the fermentation correlated with increased
contents of organic acids, esters, and ketones. A study by Wang et al. [77] compared the
biochemical and sensory properties of kombucha prepared using a SCOBY to one prepared
using a defined starter culture of AAB (A. pasteurianus and Ga. xylinus) and yeast (Z. bailii).
The use of the starter culture accelerated the fermentation and resulted in a beverage with
increased levels of phenolic compounds and comparable sensorial characteristics to those
of the traditionally prepared kombucha. Although research in this area is lacking, the use of
defined starter cultures is a promising alternative to the traditional kombucha inoculation
method, allowing for customisation of the microbial composition and enhancement of the
sensory and functional properties of the tea.
4. Potential Health Benefits

4.1. Clinical Trials
The rising popularity of kombucha is due in part to its purported health benefits, such
as the improvement of depression and anxiety, prevention of cancer, reductions in blood
pressure and cholesterol, prevention of hair loss, and weight loss; however, many of these
claims are based off anecdotal evidence and testimonials and very few can be substanti-
ated with actual scientific evidence. Furthermore, the validation of such claims in human
clinical trials is rare, with a systematic literature review performed in 2018 identifying just
one uncontrolled empirical human subject study investigating the effects of kombucha
consumption on health [13]. During the writing of this review, we performed a similar
search of clinicaltrials.gov and the EU clinical trials register (www.clinicaltrialsregister.eu)

(accessed on 8 December 2021) to establish whether any kombucha-associated human
subject studies had been registered since 2018. The search identified three clinical trials
investigating the effects of kombucha on human health, one of which is still ongoing
(Table 2). Bergström [85] evaluated the effect of daily consumption of either living kom-
bucha (intervention group), heat-sterilised kombucha (placebo group) or tap water (control
group) on the gut microbiota, with the results revealing that kombucha consumption had
no significant effect on the microbial communities of the participants. Prior to the trial, all
participants were required to undertake a 2-week ‘washout period’, during which they
were prohibited from consuming foods with high amounts of living microorganisms, such
as probiotic drinks, yoghurts, kombucha or fermented products. These dietary restrictions
continued throughout the 3-week trial period and for an additional 10 days post-trial;
however, no details were provided regarding the diet of the participants throughout the
study. Therefore, differences in the subjects’ dietary patterns could have affected the out-
come of the trial. The second study investigated the hyperglycaemic therapeutic effect of
kombucha consumption on 12 human participants, while the third trial is also focused on
the potential impacts of kombucha on blood sugar levels. Unfortunately, data and findings
from these trials were not available. Although the increased interest in kombucha research
with human subjects is encouraging, the lack of advancements in this area is remarkable
considering the significant rise in the commercialisation of kombucha and the widespread
belief that the beverage supports health [13]. Consequently, the purported health benefits
of kombucha are based solely on in vitro and in vivo studies and looks set to remain this
way until significant progress is made in the form of rigorous clinical trials.
4.2. In Vitro and In Vivo Studies

The antimicrobial activity of kombucha has mainly been attributed to the high organic
acid content and correspondingly low pH of the beverage [11]. Several studies have re-
ported antimicrobial effects against both Gram-positive and Gram-negative bacteria such
as Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, Escherichia coli and Klebsiella
pneumoniae [40,89–92]. Antimicrobial activity is typically determined using endpoint meth-
ods such as disc diffusion assays or minimum inhibitory concentration (MIC) assays that
have inherent advantages but provide insight as to how a compound impacts bacterial
growth over time, particularly in a liquid or food matrix. Al-Mohammadi et al. [93] demon-
strated the effect of kombucha tea on pathogen growth in BHI broth and fruit juices, with
the results revealing a significant decrease in cell numbers of E. coli and S. aureus in straw-
berry, apple, guava and tomato juices, and in all cases complete growth inhibition within
96 h when treated with 2% or 4% kombucha tea. The authors also observed that neutralised
kombucha also inhibited bacterial growth, suggesting that the antimicrobial activity of
the kombucha tea was not solely due to acetic acid content or low pH but also bioactive
compounds such as alkaloids, heterocyclic compounds and esters produced during fer-
mentation. Ayed et al. [48] came to a similar conclusion regarding their investigation of the
antimicrobial activity of kombucha tea against a range of bacteria. Kombucha has also been
shown to exert antifungal activity against yeasts such as C. albicans, C. krusei, C. galbrata and
A. fumigatus [94,95]. Moreover, the fermented tea has even been characterised as possessing
antiviral potential against foot and mouth disease virus (FMDV) [96]; however, no other
studies to date have documented the antiviral effect of kombucha.
Table 2. Details of controlled human intervention trials examining the effects of kombucha on human health (data correct as of December 2021).
Study Title Study Status Participants Study Type Intervention Treatment Location Source
Participants will be divided into three
groups, in which one is given living
The effect of the fermented tea Interventional;
kombucha (intervention), one
beverage kombucha on the Completed (2018) 42 randomised allocation; Lund University, Sweden [85,86]
heat-sterilised kombucha (placebo) and
oral and gut microflora double-blind
one tap water (control). Dosage: one
bottle (330 mL) daily for 3 weeks
Evaluating the effects of
Either ginger kombucha (intervention)
kombucha as a Interventional;
or ginger water (placebo) will be given
hyperglycaemic therapeutic Completed (2020) 12 randomised allocation; Georgetown University, USA [87]
to subjects for weeks 1–4 followed by
agent within diabetic human double-blind
the reciprocal beverage for weeks 6–10
subjects (completed)
At first visit, participants will be
divided into four groups, in which one
is given commercial kombucha
(intervention 1), one brewed kombucha
The effect of kombucha on Interventional; (intervention 2), one tea (control 1) and
blood sugar levels in humans Recruiting 20 (planned) randomised allocation; one tap water (control 2). At each University of Missouri, USA [88]
(recruiting) single-blind (participant) subsequent visit, subjects will be
randomly allocated into one of the
remaining groups until they have
completed each intervention treatment
Dosage: 8 oz (237 mL)
Kombucha has been reported to have a hepatoprotective effect on non-alcoholic fatty

liver disease (NAFLD), a liver disorder defined by abnormal lipid retention in >5% of
hepatocytes (steatosis), which can progress to cirrhosis or hepatocellular carcinoma if not
properly managed [97]. Cardoso et al. [98] reported that kombucha tea promoted the
modulation of lipid metabolism in rats fed a high-fat, high-fructose diet; subsequently, a
reduction in the severity of steatosis from grade 2 to grade 1 was observed, an outcome the
authors attributed to phenolic compounds and organic acids present in the tea. A similar
inference was made in another study, whereby it was suggested that kombucha influences
lipid metabolism and protects hepatocytes from lipid toxicity in mice with NAFLD, with
liver restoration promoted by the reductions in inflammation and fibrosis [99]. Contrary to
these findings, Urrutia et al. [100] reported that kombucha did not affect the lipid profiles
of mice fed a cafeteria diet (high fat and high sugar); however, consumption of the tea
(5 mL/kg of body weight) did result in increased satiety and subsequent weight loss
compared to the control group, which consumed a saline solution.
The anti-diabetic effect of kombucha has also been tested. Zubaidah et al. [101] re-
ported a 31–59% reduction in fasting plasma glucose levels of diabetic rats to which snake
fruit (a fruit of the Salak palm tree) kombucha was administered orally for 28 days. This de-
crease in blood glucose levels was thought to be due to the protective and repairing effects
of antioxidants in kombucha on pancreatic beta cells, and consequently improved insulin
secretion. A later study by the same group found that the anti-diabetic activity of snake
fruit kombucha was comparable to that of metformin, highlighting the beverage’s potential
as an alternative hyperglycaemic therapeutic agent [102]. The anti-inflammatory and im-
munomodulatory properties of kombucha have been associated with phenolic compounds
produced during the fermentation process. Experimental autoimmune encephalomyelitis
(EAE) is an animal model in which common pathological symptoms of the inflammatory
disease multiple sclerosis (MS) are simulated [103]. Haghmorad et al. [104] observed sup-
pression in the incidence and clinical manifestation of the disease following kombucha
treatment, attributed to the inhibition of IFN-γ and IL-17 inflammatory cytokines. Vázquez-
Cabral et al. [44] observed the suppression of pro-inflammatory cytokines TNF-alpha and
IL6 release from macrophages following treatment with oak kombucha (kombucha infused
with oak leaves), possibly due to the higher catechin and quercetin glucuronide contents in
the beverage than in traditional kombucha, highlighting how the fermentation substrate
can also influence the functional properties of the tea [44]. Sknepnek et al. [105] produced
novel kombucha beverages using Coriolus versicolor and Lentinus edodes mushrooms
as substrates in place of tea, reporting for the first time the immunomodulatory effects
of crude polysaccharide extracts. The results suggested that the consumption of such
polysaccharide fractions may suppress IL-4 and IL-5, key Th2 cytokines involved in the
development of asthma and allergic reactions; however, further in vitro and in vivo studies
are required to confirm the authors hypotheses.
5. Safety of Kombucha
5.1. Potential Toxicity
Despite being promoted as ‘healthy’ and the ‘elixir of life’, as previously mentioned
there is little evidence in the form of human clinical trials to substantiate such claims.
Kombucha consumption has been associated with toxic effects in some cases. For example,
Gedela et al. [15] described a patient presenting with nausea, jaundice and dark-coloured
urine following consumption of significant amounts of kombucha for the month prior to
her admission. Upon inspection, she was diagnosed with moderately active hepatitis, most
likely due to drug-induced liver injury. Similar cases of hepatoxicity associated with kom-
bucha consumption have also been reported in the literature [17,106]. Srinivasan et al. [14]
reported possible side effects of kombucha consumption such as nausea, headaches and
jaundice in four different patients, while other conditions that may be linked to kombucha
consumption include renal failure [16], myositis [107] and pellagra [108]. However, it
should be noted that each of these were isolated cases involving single patients, with
no substantial evidence implicating kombucha as the sole source of illness or infection.

For example, the patient who presented with pellagra had also been taking zinc and
calcium supplements and Acidophilus tablets and had a history of medical problems,
including dermatitis, mononucleosis and malnourishment [107]. In addition, some of the
other patients also had a history of medical conditions such as alcoholism [14], diabetes,
hypothyroidism [15,17] and HIV [16]; thus, it cannot be ruled out that their illness and
symptoms were not the result of pre-existing medical issues. It should also be considered
that in most cases the kombucha consumed was home-brewed, which raises questions
regarding the safety of the kombucha; it is possible that an improper kombucha preparation
process could result in contamination and adverse medical effects. Thus, it is imperative
for kombucha producers to recognise and address the potential hazards associated with
kombucha fermentation.
5.2. Risk Analysis of Kombucha Production

Although mainly used in industrial food settings, a Hazard Analysis and Critical
Control Point (HACCP)-based risk analysis can be applied to the domestic production
of kombucha (Table 3). As can be seen, most potential hazards linked with kombucha
preparation are biological in nature. Despite tea not being commonly associated with
foodborne illness, pathogens such as Salmonella spp., Bacillus spp. Escherichia coli and
Clostridium perfringens can survive in low-moisture environments and have been detected
in dried herbs and spices at the point of retail [109–111]. It has also been shown that black
and green tea can have microbial loads of up to 105 CFU/g and may contain bacterial and
fungal species such as Aspergillus, Shigella and Pseudomonas [112,113]. Boiled water should
be used for kombucha preparation to ensure any vegetative pathogens in the water, tea and
sugar are killed. The sweetened tea mixture should be inoculated immediately once cooled,
as the risk of contamination is at its greatest at this point. Home-brewing of kombucha
often results in the propagation and sharing of tea fungus between households [17,114], a
practice that increases the risk of biological contamination with the growth of moulds such
as Aspergillus and Penicillium, with the subsequent release of mycotoxins being of particular
concern [5,11,114]. To avoid this, a commercial culture should always be used for the first
brew and any kombucha that shows signs of mould contamination or fails to reach a pH of
≤4.2 during fermentation should be discarded immediately (along with the SCOBY). In
general, the presence of pathogenic bacteria in kombucha is rare due to its low pH and
the inability of microorganisms to tolerate such an environment. However, the kombucha
‘mushroom’ (SCOBY) has been implicated in cases of anthrax, with patients applying the tea
fungus to body parts as a painkiller; laboratory analysis subsequently showed that although
Bacillus anthrax could not be detected due to the high level of bacterial contamination,
samples inoculated with species were able to support its growth [115]. Recently, Brewer
et al. [116] demonstrated the ability of Shiga toxin-producing E. coli (STEC) and Salmonella
to survive in some brands of home-brew kits. Initially inoculated at 6–7 log CFU/mL,
cell counts of both pathogens were reduced to <0.3–1.85 log CFU/mL after 10–14 days of
fermentation in most brands, but alarmingly the numbers of Salmonella and STEC increased
within one day and remained stable throughout the 14-day fermentation period in three
brands of kombucha kits. Although both microorganisms were inoculated at levels much
higher than what would be expected to be present as a result of natural contamination,
the study highlights the requirement for strict hygiene and sanitary practices during
kombucha brewing.
Table 3. HACCP-based risk analysis of kombucha production (adapted from [18,117]).
Step Procedure Hazard Type Potential Hazard Preventative Measure

1 Boil water Biological Presence of pathogens in water Boil water to kill pathogens
Use clean and sanitised utensils and vessels. Ensure fermentation and
Dissolve sugar and steep tea in Introduction of contaminants prior preparation areas are clean and sanitary. Cover fermentation vessel
2 Biological
boiled water. Cool heated tea. to fermentation with a porous cloth. Cool tea to 20 ◦ C within 2 h and start
fermentation as soon as possible.
Use a commercial culture for first fermentation. Do not reuse a
Inoculate kombucha with SCOBY Use of contaminated SCOBY or liquid
3 Biological culture with signs of mould contamination. Do not reuse a culture
and liquid kombucha kombucha inoculum
which did not reach a pH of ≤4.2 in a previous fermentation.
Ensure fermentation areas are clean and sanitary. Cover fermentation
Introduction or growth of contaminants
Biological vessel with a porous cloth. Ferment aerobically to ensure acetic acid
during fermentation
production and ferment until a pH ≤ 4.2 is reached
Leaching of chemical contaminants from Use food-grade fermentation vessels such as glass, stainless steel,
Chemical
fermentation vessel due to low pH of kombucha high-density polyethylene (HDPE) or propylene (PP)
4 Ferment for 7–10 days
Excessive production of organic acids due to Stop fermentation before excess acid is produced. The pH of the final
Chemical
prolonged fermentation product should be ≥2.5
1 Stop fermentation before excess ethanol is produced OR allow

Excessive production of ethanol due to
Chemical fermentation to proceed and include an additional manufacturing
prolonged fermentation
step to reduce ethanol (e.g., distillation)
Use clean and sanitised utensils and vessels. Ensure fermentation and
Remove SCOBY. Filter kombucha Contamination of kombucha. Contamination of preparation areas are clean and sanitary. Use SCOBY in new
5 Biological
(optional) SCOBY if not stored/re-used correctly kombucha fermentation or store refrigerated in fresh sweetened tea
until use.
Continued fermentation and production of
6 Refrigerate Biological Refrigerate at 4 ◦ C to prevent continued fermentation
excess alcohol and acetic acid
Excess consumption could result in toxicity Ensure pH of final product is ≥2.5. Do not consume more than the
7A Consume at home Chemical
or acidosis daily recommended amount.
Leaching of chemical contaminants from
Chemical Use suitable food-grade packaging materials
fermentation vessel due to low pH of kombucha
7B Package for retail sale
Introduction of contaminants from
Biological Ensure packaging materials are clean and sanitised
packaging materials
1 Applicable to commercial kombucha producers where ethanol levels may be required to be below a specified limit to be classed as non-alcoholic.
In terms of chemical hazards, metabolic acidosis is a concern associated with kom-

bucha consumption. Such a condition results from an accumulation of organic acids in the
blood, a potentially fatal situation, particularly for those susceptible to acidosis [18]. In Iowa
in 1995, two patients presented with severe metabolic acidosis and elevated blood lactic
acid levels. Subsequent investigations showed that patient 1 had consumed approximately
4 oz (125 mL) of 7-day-fermented kombucha daily for the previous two months, while pa-
tient 2 had recently increased their daily consumption level and fermentation time to 12 oz
(375 mL) and 14 days, respectively. The pellicles used by each patient were derived from
the same mother pellicle; however, the microbiological analysis showed that pathogenic
or toxin-producing microorganisms were not detected, and a causal relationship between
kombucha consumption and the patients’ elevated blood acid levels could not be defini-
tively established. [114]. A prolonged fermentation period may result in overproduction of
organic acids that pose a possible hazard to the consumer; thus, it is recommended that the
fermentation time does not exceed ten days, with a desired final pH of ≥2.5 [18,117]. As
is the case with any food or beverage, excess consumption may lead to health problems;
thus, kombucha should be consumed in moderation. Alcohol produced during kombucha
fermentation can also be classed as a potential chemical hazard, particularly for at-risk
populations such as children and pregnant women, for whom even low levels of alcohol
consumption can have severe consequences [118,119]. In general, no measures are taken
to reduce ethanol production during home brewing of kombucha; however, consumers
should be aware of the potentially high alcohol levels in such brews. As commercial kom-
bucha producers often require ethanol contents to be below a specified threshold for the
beverage to be classed as non-alcoholic, the fermentation may be stopped after a specified
period or the alcohol may be removed post-fermentation. Kombucha is a raw product, and
accordingly there is a risk of continued fermentation and subsequent increases in organic
acids and ethanol levels if the beverage is not refrigerated. Commercial producers may opt
to include a pasteurisation step post-fermentation; however, most choose not to so as to
retain the status as a ‘raw’ and accordingly ‘healthier’ product. Chemical contamination
of kombucha may also occur if the fermentation is performed in unsuitable containers
or vessels. Examples of food-grade containers suitable for kombucha production include
those made from glass, stainless steel, high-density polyethylene (HDPE) or propylene (PP),
while lead-containing materials such as ceramic or crystal should be avoided due to the risk
of leaching toxic constituents from the container. Such an incident has been reported, with
a married couple requiring chelation therapy for lead poisoning after daily consumption
of kombucha for six months; the patients had brewed the kombucha tea in a ceramic pot,
leading clinicians to hypothesise that the acidic tea had leached the heavy metal from the
internal pot glazing [120]. However, such an occurrence is rare, with a similar situation not
having been reported in the literature since [18].
6. Kombucha Market
6.1. Market Analysis and Current Trends
The global kombucha market stood at USD 1.85 billion in 2019 and is forecasted to
increase to USD 10.45 billion by 2027, a projected compound annual growth rate (CAGR)
of 23.2%. North America dominated the global market in 2019, with a revenue share of
approximately 52%, while Europe is set to hold the second-largest market share in the
coming years. In addition, substantial market growth is also being witnessed in Asia–
Pacific, the Middle East and Africa [12]. Formed in 2012, the Kombucha Brewers Institution
(KBI) is a non-profit trade association of commercial kombucha brewers globally that strives
to advance the industry through research, communication and legislation. As of December
2021, 215 commercial kombucha companies across the globe were registered with KBI, with
the majority in North America (69.8%), followed by Europe (16.3%) (Figure 3). Unchanged
from 2019, the numbers of registered kombucha companies in North America, Latin
America, Asia–Pacific and Europe are dominated by Western USA, Mexico, Australia and
Spain, respectively. In recent times, there has been increased interest from leading beverage
companies seeking to expand their product portfolio and capitalise on the significant growth
of the kombucha market. In 2016, PepsiCo acquired California-based company KeVita,
a leading fermented beverage company that currently has 4 product lines (Master Brew
Kombucha, Sparkling Probiotic Drink, Apple Cider Vinegar Tonic and Prebiotic Shots)
spanning 28 flavours [121]. In June 2018, Clearly Kombucha was acquired by Molson
Coors Brewing Company, a strategic step to strengthen the company’s non-alcoholic
beverage offering [122]. Coca-Cola added its first line of kombucha products in 2018
by acquiring Organic and Raw Trading Co. (producers of MOJO Kombucha), adding to
its ever-increasing Australian beverage portfolio of 165 products and 25 brands at the
time [123]. Coca-Cola’s continued interest in the kombucha market resulted in a $20 million
equity investment in Health-Ade Kombucha in 2019 [124], a company that later sold a
Appl. Microbiol. 2022, 2 controlling stake to longstanding partner First Bev [125]. In 2018, Peet’s Coffee (a USA 93
company producing ready-to-drink (RDT) coffee) acquired a majority stake in Revive
Kombucha for an undisclosed amount [126].
Figure 3. Number of Kombucha Brewers International (KBI) registered companies and their respec-
Figure 3. Number of Kombucha Brewers International (KBI) registered companies and their
tive regions. Data correct as of December 2021.
respective regions. Data correct as of December 2021.
Several trends have been highlighted as driving factors for the significant growth
6.2.
inCommercial
the kombucha Kombucha
market.Products
Recently, consumer preference has shifted towards low or
non-alcoholic drinks due
Table 4 provides to the mortality
an overview of somerate associated
global with harmful
commercial kombuchaalcohol consump-
brands and their
tion [127]. Consumers are adopting healthier lifestyles, and the surging popularity of
product details, data which were collected from company websites or online retail sites.
fermented kombucha beverages is further encouraging this shift. The rising popularity of
As can be seen, the main product claims made are ‘raw’, ‘organic’, ‘vegan’, ‘non-GMO’
functional beverages has also contributed towards increased interest in kombucha, with
and ‘gluten-free’,
functional while
beverages most ahave
holding 76.4%similar base
share in the ingredients of filtered
overall beverage marketor in purified
the U.S inwater,
black
2017or green
[12] tea,European
and the cane sugar and kombucha
functional cultures.
beverage market According
projected to the
to increase IFIC from
by 6.5% Food and
Health Survey
2021–2025 2021
[128]. [129], 72%
Although of consumers
the COVID-19 are attempting
pandemic to limit many
negatively impacted or avoid sugar, with
industries,
20%
thedoing so based
fermented on medical
beverage advice. Accordingly,
market witnessed ‘sweetness
a slight increase as consumerreinvented’ is one of the
demand shifted
10towards immunity-boosting
Key Trends forecasted beverages
for 2022 amid
[130],rising
withrates
someof coronavirus
kombuchainfection;
producersfor ex-
already
embracing this and providing ‘zero-’ or low-sugar options or replacing traditional sugar
with artificial sweeteners such as stevia leaves or erythritol. In such cases, sugar is still
added at the beginning as a carbon source for the kombucha culture but is depleted during
fermentation, with sweeteners added to replace the sweet taste (Gutsy Kombucha, KÖE
ample, WonderBrew Kombucha (Malaysia) had surpassed its 2018 sales figures by June
of 2019 just by increasing its online presence. Additionally, kombucha beverages are now
more readily available through various distribution channels, including supermarkets and
convenience stores, health stores and online retailers. The range of innovative kombucha
flavours available is also attracting increased consumer attention, with companies offer-
ing novel flavours such as lavender, watermelon, peach and passionfruit alongside their
original kombucha beverages [12].
6.2. Commercial Kombucha Products

Table 4 provides an overview of some global commercial kombucha brands and their
product details, data which were collected from company websites or online retail sites.
As can be seen, the main product claims made are ‘raw’, ‘organic’, ‘vegan’, ‘non-GMO’
and ‘gluten-free’, while most have similar base ingredients of filtered or purified water,
black or green tea, cane sugar and kombucha cultures. According to the IFIC Food and
Health Survey 2021 [129], 72% of consumers are attempting to limit or avoid sugar, with
20% doing so based on medical advice. Accordingly, ‘sweetness reinvented’ is one of the
10 Key Trends forecasted for 2022 [130], with some kombucha producers already embracing
this and providing ‘zero-’ or low-sugar options or replacing traditional sugar with artificial
sweeteners such as stevia leaves or erythritol. In such cases, sugar is still added at the
beginning as a carbon source for the kombucha culture but is depleted during fermentation,
with sweeteners added to replace the sweet taste (Gutsy Kombucha, KÖE Kombucha,
Kevita MasterBrew, The GUTsy Captain Kombucha, Remedy Kombucha). Most producers
offer kombucha beverages in a range of flavours, with fruits (e.g., strawberry, raspberry, wa-
termelon, blueberry, lemon) and herbs (e.g., ginger, mint, sage, rosemary) and combinations
thereof the most common options. Along with different flavours, some manufacturers offer
different product ranges to provide beverages that are distinctly different from traditional
kombucha. For example, GT’s Living Energy offers three different classes of kombucha:
the ‘classic’ range, which encompasses traditional kombucha in a range of flavours; the
‘synergy’ range, which provides a smoother taste to new drinkers; ‘hard’ kombucha, which
includes more sophisticated flavours and has an ABV (% v/v) of 3%. In addition, Brew Dr.
and Aqua ViTea have capitalised on the current cannabidiol (CBD) trend in the USA, both
launching a CBD-infused kombucha beverage range.
Of the 26 kombucha products surveyed, most were marketed as raw, with just one
(Biona Organic, Surrey, UK) stating that the beverage underwent pasteurisation. Several
brands also add additional live cultures, with Bacillus spp. appearing to be a popular choice
amongst kombucha producers. Specifically, both GT’s Living Energy Kombucha and MOJO
Kombucha use Bacillus coagulans GBI-30 6086, while Kevita MasterBrew Kombucha utilises
B. coagulans MTCC 5856, commercially available strains that are well-documented in terms
of their resistance to low pH, making them excellent candidates for survival in the acidic
kombucha environment [131–133]. Interestingly, Majeed et al. [134] recently highlighted
the remarkable survival of B. coagulans MTCC 5856 in tea (99.76% viability) and addition-
ally demonstrated that inclusion of the strain after brewing did not significantly impact
the sensory profile of the beverage. Other live cultures commonly used in commercial
kombucha products include Bacillus subtilis, Lb. plantarum and S. boulardii.
Table 4. Examples of commercial kombucha products on the market.
No. of
Country of Calories Sugar Alcohol
Brand Name Products/Flavours Base Ingredients Product Claims
Production (kcal/100 mL) (g/100 mL) (% v/v)
Available
Black tea, green tea, cane sugar, kiwi
juice, live cultures (S. boulardii: 4 billion Classic: 0.5–1%; Organic; Vegan;
GT’s Living Foods 50 (12 Classic; 31
USA CFU, Lactobacillus bacterium: 4 billion ≤31 2.5–4.2 Hard: 3%; Synergy: Non-GMO;
Kombucha Synergy; 7 Hard)
CFU, Bacillus coagulans GBI-306086: <0.5% Gluten-Free; Raw
1 billion CFU)
Filtered water, green tea, black tea,
Organic; Vegan;
kombucha culture (yeast and bacteria),
Wonder Drink Non-GMO; Gluten-free;
USA 5 cane sugar, prebiotic corn fibre ≤14 2.3–2.8 <0.5%
Kombucha BPA free; Low-sugar;
(xylo-oligosaccharides), natural flavour,
Non-alcoholic; Raw
stevia leaf extract
Filtered water, green tea, black tea,
Organic; Vegan;
24 (13 Original; 6 white grape juice, cane sugar, live
Humm Non-GMO; Gluten-free;
USA Zero; 5 Whole30 kombucha cultures, natural flavours, ≤18 2.4–3.9 <0.5%
Kombucha Keto-friendly;
Approved) Bacillus subtilis (2 billion CFU),
Non-alcoholic; Raw
vitamin B12
Purified sparkling water, black tea,
sugar, active culture, erythritol, natural
Organic; Vegan;
KÖE flavour, lime juice concentrate, citric
USA 7 ≤9 2.3 <0.5% Non-GMO; Gluten-free;
Kombucha acid, ascorbic acid, B. coagulans, fruit
Non-alcoholic; Raw
and vegetable juice, stevia extract, green
tea extract, green coffee extract
Filtered water, green tea, black tea, cane Vegan; Non-GMO;
Rowdy Mermaid
USA 9 sugar, live cultures ≤14 2.5–2.8 <0.5% Gluten-free;
Kombucha
(Lactobacillus plantarum) Non-alcoholic; Raw
Filtered water, kombucha culture, black
tea, green tea, natural flavour, cane Organic; Non-GMO;
Kevita Master Brew
USA 15 sugar, B. coagulans LactoSpore MTCC ≤17 3.3–3.6 <0.5% Kosher;
Kombucha
5856, black tea essence, caffeine (green Non-alcoholic; Raw
coffee bean extract), stevia leaf extract
Table 4. Cont.
No. of
Country of Calories Sugar (g/100 Alcohol
Production (kcal/100 mL) mL) (% v/v)
Available
Organic; Non-GMO;
Brew Dr. 13 (10 Classic; 3 Filtered water, green tea, cane sugar,
USA ≤19 2.9–3.6 <0.5% Gluten-free; Kosher;
Kombucha Tranquil (CBD)) live kombucha culture (yeast, bacteria)
Alcohol-extracted; Raw
Filtered water, active culture, cane
sugar, black tea, green tea, ginger root, Organic; Non-GMO;
Clearly Kombucha USA 4 natural flavours, fruit and vegetable ≤5 0.0–1.2 <0.5% Gluten-free; Kosher;
juice, live probiotic (B. coagulans, Non-alcoholic; Raw
I billion CFU)
Filtered water, green tea, black tea, cane Organic; Vegan;
Health-Ade 22 (15 Original; 7
USA sugar, live kombucha cultures (yeast ≤17 2.7–3.6 Trace amounts non-GMO;
Kombucha Health-Ade Plus)
and bacteria) Gluten-free; Raw
Organic; Vegan;
Aqua ViTea 11 (9 Original; Filtered water, black tea, green tea, cane Non-GMO; Gluten-free;
USA ≤15 1.3–2.1 <0.5%
Kombucha 2 CBD) sugar, kombucha culture Paleo;
Alcohol-extracted; Raw
Dominga Filtered water, green tea, cane
Mexico 4 n/a n/a n/a Raw
Kombucha sugar, SCOBY
Organic; Vegan;
Gutsy 8 (6 Classic; Filtered water, black tea, cane sugar,
Canada ≤14 0.0–3.7 <0.5% Non-GMO;
Kombucha 2 No Sugar) living cultures (bacteria and yeast)
Gluten-free; Raw
Hoochy Booch Filtered water, green tea, cane sugar, Organic; Vegan; Gluten
Canada 10 8 2.1 Trace amounts
Kombucha kombucha culture free; Raw
Vitae Volcanic mineral water, green tea, cane Organic; Vegan; Gluten
Spain 9 ≤16 1.6–3.7 <0.5%
Kombucha sugar, kombucha culture free; Lactose-free; Raw
Filtered water, cane sugar, green tea Organic; Vegan; Gluten
The GUTsy Captain 15 (9 Traditional;
Portugal (0.4%), stevia leaves, kombucha ≤18 0.0–4.4 <0.5% free; Low-calorie;
Kombucha 6 Zero)
cultures, Bacillus coagulans Non-alcoholic; Raw
Table 4. Cont.
No. of
Country of Calories Sugar Alcohol
Production (kcal/100 mL) (g/100 mL) (% v/v)
Available
11 (6 Classic
Organic; Vegan; Gluten
Kombucha Bottles; Filtered water, sencha green tea, golden
SynerChi Kombucha Ireland ≤38 0.0–4.5 <0.5% free; Dairy free; Soy-free;
3 Kombucha Cans; cane sugar, kombucha cultures
Non-alcoholic; Raw
2 Kombucha Shots)
Organic; Vegan;
Holo Filtered purified water, green tea, black
Ireland 2 ≤7 3.1 <1% Non-GMO; Gluten free;
Kombucha tea, sugar, kombucha cultures
Dairy free; Raw
Leave Your Sword Filtered water, tea, beetroot sugar,
Netherlands 7 n/a 4.5 2.5% Raw
Kombucha kombucha culture
Organic; Vegan; Gluten
Pure spring water, chun-mee green tea,
Equinox Kombucha UK 12 ≤18 2.5–4.3 <0.5% free; Plant-based;
raw cane sugar, kombucha cultures
Non-alcoholic; Raw
Tea herbal extract (natural mineral
water, green tea, black tea, mate leaves,
lime blossom, lemon verbena,
cornflower blossom, lemon balm, mint, Organic; Vegan;
Biona
UK 3 nettle, woodruff, elder blossom, ≤18 4.7 <0.5% Non-alcoholic;
Kombucha
raspberry leaves, marigold blossom, Pasteurised
blackberry leaves, liquorice root), raw
cane sugar, kombucha cultures, natural
carbon dioxide
15 (6 Activated
Organic; Vegan;
MOJO Kombucha; 4 Gut Water, sugar, tea, kombucha culture,
Australia ≤22 1.7–2.4 <0.5% Low-sugar;
Kombucha Shot; 3 Kombucha B. coagulans GBI-30 6086 (1 billion CFU)
Non-alcoholic; Raw
Soda; 2 Superbooch)
Sparkling water, sugar, black tea, green Organic; Vegan; Gluten
Remedy Kombucha Australia 13 tea, wild kombucha culture, erythritol, 4 0.0 <0.5% free; Non-alcoholic;
steviol glycosides Raw; Halal
Naughty Booch Kombucha apple cider blend, lime juice,
Australia 2 ≤36 1.4–2.1 4.6% Vegan; Low-sugar
Kombucha monk fruit, natural flavours
Table 4. Cont.
No. of
Country of Calories Sugar (g/100 Alcohol
Production (kcal/100 mL) mL) (% v/v)
Available
Swig New Purified Wanaka alpine water, sugar,
4 n/a n/a n/a Organic; Raw
Kombucha Zealand green tea, black tea, kombucha culture
Filtered water, green tea, black Ceylon
BomBooch
China 6 tea, raw unrefined cane sugar, ≤19 1.7 <0.5% Raw
Kombucha
SCOBY culture
Wild Filtered water, tea leaves, cane sugar,
Malaysia 17 11 1.5 <1% Organic; Raw
Kombucha kombucha culture
n/a: information not available.
As shown in Table 4, most kombucha beverages have an ABV value of <0.5%, keeping
in line with guidelines regarding the classification of non-alcoholic beverages, although
these vary depending on geographical region. Some manufacturers have chosen to embrace
the alcoholic nature of kombucha fermentation, such as GT’s Living Energy Hard Kom-
bucha (3% ABV), Leave Your Sword Kombucha (2.6% ABV) and Naughty Booch Alcoholic
Kombucha (4.6% ABV). There have been several controversies surrounding the alcohol
contents of commercial kombucha products, with some found to contain levels exceeding
values stated on the label. As such, these products would no longer be classed as non-
alcoholic and would be subject to alcohol taxation. As already mentioned, due to the raw
nature of the kombucha product, there is a possibility of fermentation post-manufacture;
therefore, although the alcohol content may be below the threshold limit upon leaving
the producer, the ABV may exceed the permissible limit if not correctly stored during
distribution. A recent study by Jang et al. [135] measured the ethanol contents of 684 kom-
bucha samples collected from processers and purchased from retailers in British Columbia,
where beverages >1% ABV are considered alcoholic. They found that 31.5% of samples
contained ethanol levels that exceeded the regulatory limits of 1% ABV, while four teas
(0.6%) were reported to contain >3% ABV, more than five times their stated alcohol content.
This is neither an uncommon nor a recent phenomenon, with kombucha products being
recalled worldwide since 2010 due to false advertising of alcohol contents [136–138]. The
KBI organisation is actively pursuing legislation (H.R.2124—117th Congress) that would
see the level at which taxation would occur increase from 0.5% to 1.25% ABV, while still
abiding by the health and safety requirements applicable to non-alcoholic beverages [139].
7. Conclusions and Future Perspectives

The global popularity of kombucha is rising significantly, with increases in both
household and commercial production of the fermented tea. The beverage’s microbiological,
sensory, and functional compounds vary and are highly dependent on the substrate used,
the fermentation conditions and the microbial diversity of the kombucha consortium.
Sucrose and black or green tea are the primary fermentation substrates; however, the use
of alternatives has been investigated. Different substrates can positively or negatively
influence the kombucha beverage, affecting the microbial growth, metabolite production
and functionality. Yeasts and AAB are the core microbiota of the kombucha community,
while LAB have also been identified systematically. Microbial diversity at genus and species
levels can vary between consortiums in different geographical regions and even within
the same region. Yeasts play a key role in making substrates available to AAB through
a mutualistic relationship, while AAB utilises such substrates to produce organic acids
and other metabolites, which positively modify the environment for yeast. The emergence
of culture-independent techniques has allowed for a deeper insight into the diversity
of the complex microbial community and the interactions taking place therein. The use
of defined starter cultures to reproduce a stable kombucha community is a promising
alternative to traditional kombucha fermentation; however, this area of research remains
largely understudied. The safety of kombucha is typically guaranteed by its low pH;
however, producers and consumers should still be aware of the risks, such as biological
contamination and secondary fermentation. There is a widespread belief that kombucha
is a health product; however, its purported benefits have been investigated using animal
models and human cell lines, while little in the way of research on kombucha’s function in
human subjects has been performed to date. Future studies should focus on the modulation
of kombucha’s sensory and functional characteristics through a deeper understanding of
the communication and interactions between members of the microbial community. In
addition, research efforts should be directed towards the use of defined starter cultures,
particularly to facilitate a shift toward industrial-scale production of kombucha.
Author Contributions: Conceptualisation, E.K.A.; investigation, L.M.N.; writing—original draft

preparation, L.M.N. and K.M.L.; writing—review and editing, L.M.N., K.M.L., A.W.S. and E.K.A.;
supervision, E.K.A. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: This work has been sponsored by and performed in collaboration with AB-
InBev’s GITeC—The Core Global Research and Development Centre. The authors would like to thank
Stuart Wilkinson, Luk Daenan and Patrick O’ Riordan for their support of this work. The authors
would also like to thank William Organ and Audrey Byrne for their assistance throughout the writing
of this manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Review

Advances in Kombucha Tea Fermentation: A Review

Appl. Microbiol. 2022, 2, 73–103. https://doi.org/10.3390/applmicrobiol2010005 https://www.mdpi.com/journal/applmicrobiol

2. Kombucha Tea Production

1. An overview of kombucha preparation

2.1. Alternative Substrates

2.2. Adjustment of Fermentation

2.2. Adjustment of Fermentation Conditions

Table 1. Studies investigating the microbial composition of kombucha.

Yeast Lactic Acid Acetic Acid Fermentation Fermentation Fermentation

Brettanomyces, Pellicle and liquid

Acetobacter (A.) Pellicle (3–4 layers) 67.5 g/L sucrose,

Yeast Lactic Acid Acetic Acid Fermentation Fermentation Fermentation

Yeast Lactic Acid Acetic Acid Fermentation Fermentation Fermentation

Zygosaccharomyces, Z. bailii n.d.

Yeast Lactic Acid Acetic Acid Fermentation Fermentation Fermentation

Acetobacteraceae, 70 g/L glucose

Use of Defined Starter Cultures

4. Potential Health Benefits

search of clinicaltrials.gov and the EU clinical trials register (www.clinicaltrialsregister.eu)

4.2. In Vitro and In Vivo Studies

Kombucha has been reported to have a hepatoprotective effect on non-alcoholic fatty

no substantial evidence implicating kombucha as the sole source of illness or infection.

5.2. Risk Analysis of Kombucha Production

Table 3. HACCP-based risk analysis of kombucha production (adapted from [18,117]).

Step Procedure Hazard Type Potential Hazard Preventative Measure

1 Stop fermentation before excess ethanol is produced OR allow

In terms of chemical hazards, metabolic acidosis is a concern associated with kom-

6.2. Commercial Kombucha Products

Table 4. Examples of commercial kombucha products on the market.

7. Conclusions and Future Perspectives

Author Contributions: Conceptualisation, E.K.A.; investigation, L.M.N.; writing—original draft

Funding: This research received no external funding.

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