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Health effects and probiotic and prebiotic potential of Kombucha: A bibliometric and
systematic review

Bruna Krieger Vargas, Mariana Fensterseifer Fabricio, Marco Antônio Záchia Ayub

PII: S2212-4292(21)00457-0
DOI: https://doi.org/10.1016/j.fbio.2021.101332
Reference: FBIO 101332

To appear in: Food Bioscience

Received Date: 21 May 2021

Revised Date: 21 July 2021
Accepted Date: 23 August 2021

Please cite this article as: Vargas B.K., Fabricio M.F. & Záchia Ayub Marco.Antô., Health effects and
probiotic and prebiotic potential of Kombucha: A bibliometric and systematic review, Food Bioscience
(2021), doi: https://doi.org/10.1016/j.fbio.2021.101332.

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© 2021 Published by Elsevier Ltd.

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 1 Health effects and probiotic and prebiotic potential of Kombucha: a bibliometric

2 and systematic review

3 Bruna Krieger Vargas1,#, Mariana Fensterseifer Fabricio1,#, Marco Antônio Záchia

4 Ayub1*

6 1
Laboratory of Biotechnology (BiotechLab), Food Science and Technology Institute,

7 Federal University of Rio Grande do Sul (UFRGS), Porto Alegre, Rio Grande do Sul,

8 Brazil, Av. Bento Gonçalves, 9500, ZC 91501-970, Porto Alegre, Rio Grande do Sul,

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9 Brazil.

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1*
Corresponding author: M A Z Ayub, e-mail: mazayub@ufrgs.br
#
These authors contributed equally to this work.

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13 Abstract

14 Kombucha is a fermented beverage composed of a range of natural compounds such as

15 sugars, ethanol, organic acids, and complex microbial communities of bacteria and
16 yeasts. Based on this several biological properties are attributed to this drink. However,
17 the production of kombucha is not standardized and the final composition of the
18 beverage is highly dependent on the raw materials used and the physicochemical
19 parameters adopted in the process. As a consequence, kombuchas not only vary from
20 one producer to another but also from different batches of the same producer, making
21 the assumptions of quality and properties questionable. In this review, we explore the
22 largely unchecked relations between kombucha and its claimed health benefits. A
23 systematic review was also performed to specifically discuss the potential probiotic and
24 prebiotic effects of kombucha. Although several studies report that kombucha present
25 antimicrobial, antioxidant, detoxifying, and hepatoprotective activities, among others,
26 whereas others classify kombucha as a probiotic drink, there is a lack of scientific

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27 evidence about the content of probiotics in this drink and its possible role in the

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28 intestinal microbiota. These facts highlight the opportunities in researching and
29 modifying the microbiome composition of kombucha, possibly improving the general

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30 qualities of this so-called functional drink.
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32 Keywords: Bibliometrix; functional beverages; fermented tea; symbiotic product; gut
33 microbiota modulation; post-biotics compounds.
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35 1 Introduction

36 Kombucha is an ancient beverage obtained by the infusion or extract of Camellia

37 sinensis and sugars fermented by a symbiotic culture of microbiologically active

38 bacteria and yeasts (SCOBY) (Greenwalt et al., 2000). Kombucha is believed to have

39 originated in China, in the Manchuria region, being consumed for more than 2,000

40 years (Dorothy et al., 2020; Jayabalan et al., 2014). Traditionally, kombucha was

41 homemade, based on a very simple preparation. Initially, the tea leaves and sugar are

42 added to boiling water, leaving the mixture to rest for a few minutes. After the cooling

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43 of this solution, a starter culture, consisting of a cellulosic pellicle (known as SCOBY)

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44 and a percentage of the previously fermented batch of kombucha is added to perform

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the fermentation of the sweetened tea. In this way, kombucha tea fermentation
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46 comprises two distinct portions, as demonstrated in Figure 1: a liquid tea phase and a
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47 floating microbial cellulose pellicle layer (Chen and Liu, 2000). This fermentation stage
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48 usually takes from 7 to 14 days; at the end of it, the SCOBY is removed from the

49 kombucha along with a small volume of the ready drink to be used as a starter culture in
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50 the next fermentations (Dufresne and Farnworth, 2000). Currently, kombucha is

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51 commercially available, its consumption has been growing in several countries and it is

52 considered one of the most popular low-alcohol beverages worldwide, mainly because

53 of claims on its functional properties and health appeals (Watawana et al., 2015).

54 The production process of kombucha has not been standardized and the final

55 composition of the beverage will heavily depend on the parameters adopted, such as

56 type and concentration of tea and sugar (Shahbazi et al., 2018; Reva et al., 2015b;

57 Watawana et al., 2016), fermentation time, and temperature (De Filippis et al., 2018;

58 Tran et al., 2020), and in particular on the composition of the starter culture (Nguyen et

59 al., 2015; Villarreal-Soto et al., 2018). The latter is a complex multi-species microbial

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60 ecosystem, mostly composed of acetic acid bacteria and yeasts. Several studies show

61 that this community of microorganisms varies in great stance based on the geographical

62 origin (Chakravorty et al., 2016; Coton et al., 2017; De Filippis et al., 2018) and also

63 over time between batch fermentations of the same producer (Marsh et al., 2014).

64 However, some genera prevalently appear in most starter cultures, such as

65 Komagataeibacter and Acetobacter, among bacteria, and Saccharomyces,

66 Zygosaccharomyces, Brettanomyces/Dekkera, and Candida for yeasts (Jayabalan et al.,

67 2014; Arıkan et al., 2020). It is important to note that in some kombuchas, lactic acid

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68 bacteria (LAB) may also be present, such as Lactobacillus and Lactococcus. However,

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69 despite the importance of LAB for the probiotic properties of kombucha, this group of

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bacteria is not always present in the microbial consortium of this drink (Laureys et al.,
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71 2020; Morales, 2020; Murphy et al., 2018).
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72 The set of microbial genera included in the kombucha are responsible for reducing the
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73 initial pH of the beverage, inhibiting the proliferation of possible contaminating

74 bacteria, and also for carrying out the fermentation process (Neffe-Skocińska et al.,
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75 2017). Fermentation is initiated by yeasts that hydrolyze sucrose, converting it into

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76 glucose and fructose, ultimately producing ethanol, glycerol, and carbon dioxide. In the

77 sequence, the bacteria oxidize ethanol, producing acetic acid and transforming glucose

78 into glucuronic acid (Gaggìa et al., 2018). After the fermentation process, a refreshing,

79 slightly acidic, and carbonated drink is produced, containing a diversified content of

80 components, such as sugars (sucrose, glucose, and fructose remaining from

81 fermentation), ethanol, amino acids, organic acids (mainly acetic, gluconic, and

82 glucuronic acids), polyphenols, hydrolytic enzymes, and micronutrients, such as B-

83 complex and C vitamins, and minerals (zinc, copper, iron, manganese, and cobalt)

84 (Jayabalan et al., 2014; Kaczmarczyk and Lochyński, 2014). This nonetheless

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85 interesting composition is perhaps the basis for claims of several biological properties

86 attributed to kombucha (Kapp and Sumner, 2019).

87 This article reviews the information available in the literature on the relationship

88 between kombucha composition, consumption, and the health effects and probiotic

89 potentials attributed to it. The contribution of this bibliometric and systematic review is

90 to gather data about the biological properties of kombucha, improving knowledge about

91 this so-called functional beverage.

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 93 2 Methodology

94 This study was performed to present a review of health effects associated with

95 kombucha consumption and employed methods such as a bibliometric and systematic

96 review, according to Figure 2. The review was organized through a search for studies

97 published to date in the Scopus (scopus.com) database using the following terms: (a)

98 “kombucha” AND “health”; (b) “kombucha” AND (health” AND “effect” OR

99 “benefit*” OR “claim”); (c) “kombucha” AND “pr*biotic”, according to the

100 supplementary materials. Boolean Operators were used to find the publications

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101 containing the terms of interest on the title, abstract, or keywords of the articles

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102 available on Scopus in the years.

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The bibliometric review was conducted to present an overview of scientific production
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104 on the topic kombucha and health over the years and to identify the 50 most incident
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105 words between title, keywords, and summary among all the articles found. All
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106 publications found with the terms “a” and “b” in the English language until March of

107 2021 were exported in BibTex format and imported into Rstudio software (RStudio
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108 Team 2010) through the Bibliometrix package (bibliometrix.org). Figure 3 and Figure 4
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109 were generated by bibliometric analysis. Figure 3 was elaborated through 122 articles

110 found using the term “a'' analyzed according to the year of publication and Figure 4 was

111 developed using thematic maps (Aria and Cuccurullo, 2017), generated through the 90

112 articles found in the term “b”.

113 The systematic review was performed to explore, specifically, the general aspects in the

114 context of the probiotics and prebiotics effects of kombucha and to respond to these

115 questions:

116 ● Is kombucha a probiotic beverage in the technical definition of the term?

117 ● What is the evidence of this possible probiotic and/or prebiotic effect?

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118 A content analysis of all articles found in the database from terms “c” was performed to

119 discuss and deeply explore this topic.

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121 3 Results and discussion

122 3.1 Kombucha and its health effects

123 To guide the discussion about kombucha and its health effects, Figure 4 was generated

124 through bibliometric analysis and presents the thematic map from the analysis and

125 grouping of the 50 most recurrent words found in the literature with the term “c” (Cobo

126 et al., 2011). It is possible to observe in Figure 4 the occurrence of four main clusters,

127 among which we can find the words “tea”, “kombucha tea” and “fermented tea” in two

128 of these clusters. This results from the important association of tea in the effects of

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129 kombucha.

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130 The biological activities performed by kombucha were primarily attributed to the

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composition of the tea itself used in the preparation of this beverage. Camellia sinensis
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132 is a species in the Theaceae family, which includes pu-erh, jasmine, white, oolong,
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133 yellow, green, and black tea (Chakravorty et al., 2019). Tea is historically recognized as
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134 a healthy beverage due to the presence of antioxidants (Wang et al., 2020). The

135 distinction between these types of tea of the species Camellia sinensis is related to the
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136 degree of oxidation in the processing of tea leaves, which can significantly impact on its
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137 content and health benefits (de Almeida Souza et al., 2020).

138 Traditionally, black tea is the most used for preparing kombucha. Along with the

139 refined white sugar (sucrose), are considered the best ingredients to the proper content

140 of the kombucha (Jakubczyk et al., 2020; Kaczmarczyk and Lochyński, 2014). Black

141 tea is the most processed and susceptible to oxidation. The major compounds found in

142 this type of tea are alkaloids (caffeine, theobromine, and theophylline), and

143 polyphenols, such as catechins, theaflavins, and thearubigins (Anal, 2019). On the other

144 hand, green tea undergoes little to no processing and does not suffer oxidation reactions.

145 The term green tea can be found in the thematic map (Figure 4), configuring it as one of

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146 the most used in the articles. Gaggìa et al. (2018) in their study comparing black, green,

147 and rooibos teas found that green tea presented the most significant antioxidant

148 properties, whereas black tea showed the lowest values. In addition, green tea

149 kombucha showed less degradation of epicatechin isomers compared to black tea

150 kombucha (Jayabalan, et al., 2007). A great number of studies evaluated the choices of

151 other types of tea for fermentation, instead of the traditional black tea, in order to

152 enhance the functional properties of kombucha (Jayabalan et al., 2007; Gaggìa et al.,

153 2018; Jakubczyk et al., 2020).

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154 Over the years, investigations began to be carried out to understand the impact of tea

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155 fermentation by the microorganisms of the kombucha and the influence of this process

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on its beneficial health effects (Baschali et al., 2017; Dufresne and Farnworth, 2000).
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157 This can be seen in Figure 4 where, among the most cited words in the title, keywords,
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158 and summary of all articles analyzed, are the terms “fermentation”, “fermented tea”,
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159 “fermented drink” and “tea fungus”, also known as SCOBY, appearing in 3 of the 4

160 clusters on the thematic map.

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161 The production of fermented foods is an ancient practice among many cultures and
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162 remains as the earliest biological method of food processing and preservation (Anal,

163 2019; Marco et al., 2017). Fermentation is considered one of the first known

164 applications of biotechnology (de Almeida Souza et al., 2020). The fermentative process

165 in kombucha drink has several advantages. We can cite initially the reduced risk of

166 contamination, which are prevented by compounds formed during fermentation, such as

167 organic acids, ethanol, and bacteriocins (Marco et al., 2017). Notwithstanding, the

168 fermentation process is also responsible for many biochemical modifications of the

169 drink (Chakravorty et al., 2016). Some of these improve the sensorial profile of the

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170 beverage, developing new and complex tastes and desirable flavors, characterizing

171 kombucha, and differentiating it from the initial vegetable matrix (Marco et al., 2017).

172 Besides, fermented foods and beverages are an important nutritional source. This is

173 because these products carry potential beneficial microorganisms and their metabolites.

174 Some of these can be genetically similar to probiotics strains, providing microbial

175 stability and changes in the digestibility of nutrients, enhancing the nutritional content

176 (de Almeida Souza et al., 2020; Marco et al., 2017; Villarreal-Soto et al., 2018).

177 Fermented products have been attracting the attention of consumers mainly because of

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178 the health benefits associated with their consumption (Marco et al., 2017). Today,

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179 Kombucha is a ready-to-drink beverage easily found in several markets and has been

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related to various functional effects such as helping to establish the balance of the
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181 organism and avoiding the appearance of chronic non-communicable diseases like
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182 obesity and diabetes. The claims of “functional beverages'' and “functional food” are
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183 related to kombucha in two different clusters in the thematic map (Figure 4). In the

184 functional beverages cluster, the association of terms “fermentation” and “fermented
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185 beverages” with the word “chemical composition” was observed, demonstrating that the
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186 components generated in this drink during fermentation influence its functional

187 potential. The term “fermented beverage” also appears in the functional food cluster,

188 associated with the antioxidant compounds “polyphenols” and “flavonoids”, both

189 responsible for reducing oxidative stress and suggesting the functional appeal of

190 kombucha.

191 In view of the growing consumer demand for functional beverages, food industries have

192 been exploiting the fermentation process for the development of new functional

193 products (Baschali et al., 2017).

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194 Along with the growing interest of the population and the food industry in fermented

195 foods, research in this area has also been expanding, especially concerning kombucha.

196 The potential therapeutic properties of kombucha have become a field of interest

197 (Murugesan et al., 2009), as shown in Figure 3, which presents the increase in articles

198 published with the terms “kombucha” and “health” over years. It can be noticed that, in

199 the Scopus database, the publications in 2019 exceeded the number of 20 articles,

200 increasing in 2020 to almost 30 published works. Until March 2021, 4 articles were

201 published on the same base. One of the first articles found using these terms, by

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202 Hartmann et al. (2000), investigated the effects of chronic kombucha ingestion during

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203 an in vivo pilot study using mice. They found positive differences in appetitive

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behaviors, gross body weight, and greater longevity in animals receiving kombucha
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205 compared to the control group. Moreover, the consumption-group had longer spleens
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206 and enlarged livers. These adverse effects need more biochemical analysis for a full
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207 understanding. The hepatomegaly effect could be associated with some hepatotoxicity

208 cases in humans, but the authors stated that comparable effects and mechanisms in
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209 humans were uncertain at the time. This shows kombucha may bring health or some
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210 undesirable outcomes by its consumption.

211 Regarding positive outcomes, several health benefits make Kombucha popular

212 as a functional beverage or food (Mousavi et al., 2020; Villarreal-Soto et al., 2018).

213 Table 1 presents some in vitro and in vivo studies showing the possible biological

214 properties associated with the consumption or administration of kombucha. Vīna et al.,

215 (2013) reviewed the main physiological properties related to kombucha consumption,

216 listing, among others, antioxidant reaction, detoxifying properties, promoting immunity,

217 and energizing capacity. The latter may be related to the release of iron from the

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218 kombucha tea, increasing the hemoglobin level. As a result, tissue oxygenation is

219 favored, stimulating ATP synthesis in the body.

220 Regarding the antioxidant capacity, several studies show the association of kombucha

221 consumption with the improvement of antioxidant protection to the cells, helping in the

222 reduction of the damage caused by oxidative stress to the body (de Almeida Souza et

223 al., 2020). The terms “antioxidant activity”, “antioxidant” and “oxidative stress” appear

224 in two clusters of the thematic map (Figure 4) associated with the words “polyphenols”

225 and “flavonoids”. Antioxidant capacity is one of the best-known actions performed by

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226 phenolic compounds, and kombucha is a beverage rich in these components. For

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227 instance, Cardoso et al. (2020) screened the phenolic profile of green and black tea and

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their resulting kombuchas and observed that black tea presented a higher abundance of
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229 phenolic compounds. Also, this kombucha showed a greater diversity of these
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230 components after the fermentative process at 25 °C for 10 days. Differences in the
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231 profile of phenolic compounds were observed by ultra-performance liquid

232 chromatography coupled with an electrospray ionization quadrupole time-of-flight mass

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233 spectrometry operating in MSE mode (UPLC-QTOF-MSE) analysis wherein 127

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234 phenolics were detected in the green and black tea kombuchas (70.2 % flavonoids, 18.3

235 % phenolic acids, 8.4 % other polyphenols, 2.3 % lignans, and 0.8 % stilbenes). At least

236 103 of these phenolic components were detected for the first time in kombuchas.

237 Among them are pelargonidin 3-O-glucoside, gardenin B, lithospermic acid, and

238 oleuropein, found exclusively in black tea kombucha. These bioactive substances are

239 known for their activities, such as antitumoral and hypotensive effects and positive gut

240 microbiota modulation (Cardoso et al., 2020). Ivanišová et al., (2019) also compared the

241 phenolic profile of black tea and its kombucha after fermentation at 22 °C for 7 days.

242 They found higher content of total polyphenols in kombucha (0.42 mg gallic acid

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243 equivalent (GAE)/ml), flavonoids (0.13 mg quercetin equivalent (QE)/ml), and phenolic

244 acids (0.19 mg caffeic acid equivalent (CAE)/ml) than in black tea (0.18 mg GAE/ml;

245 0.02 mg QE/ml; 0.05 mg CAE/ml, respectively). The same author further demonstrated

246 that black tea kombucha has a significantly higher value in total polyphenol content

247 (412.25 mg (GAE)/l) compared to black tea (180.17 mg GAE/l) (Ivanišová et al.

248 2020). Similarly, Wang et al. (2020) reported an increase in the concentrations of total

249 phenol and flavonoids of kombucha during fermentation, boosting its health benefits.

250 Sai Ram et al. (2000) carried out an in vivo study, showing that phenolic compounds in

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251 kombucha, in addition to reducing oxidative stress, could decrease the

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252 immunosuppression caused by this stress condition. Based on these studies, kombucha

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would contribute to immunity and reduce inflammatory foci and cell damage,
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254 preventing the development of chronic non-communicable diseases (Vīna et al.,
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255 2013). Despite that, the phenolic content of kombucha is variable and changes
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256 according to the chemical composition of the tea before fermentation. It will also

257 depend on the concentration of carbon source, pH, temperature, inoculum size, and
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258 microbial community of kombucha (Jafari et al., 2020). The bacteria and yeasts present
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259 in kombuchas are responsible for releasing enzymes that determine the metabolites

260 generated during its fermentation, as well as degrading the polyphenols in smaller

261 molecules (de Almeida Souza et al., 2020; Jakubczyk et al., 2020). Ivanišová et al.,

262 (2019) revealed kombucha has a high concentration of phenolic metabolites, such as

263 gallic acid, chlorogenic acid, protocatechuic acid, p-coumaric acid, ellagic acid, rutin,

264 vitexin, and resveratrol. Other compounds, such as catechins, epicatechin, and

265 flavonoids are also produced by the microorganisms through biotransformation

266 (Villarreal-Soto et al., 2018; Jayabalan, Malini, et al., 2010; Ivanišová et al., 2020;

267 Mousavi et al., 2020).

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268 When analyzing the thematic map (Figure 4), it can be identified as a cluster that

269 reunites the words “bacteria”, “acid acetic bacteria”, “yeast”, “SCOBY”, “microbial

270 ecology”. In the same cluster, the terms “biotransformation” and “health effects'' appear.

271 Thereby, it can be noticed the importance of the microbial community of kombucha in

272 the generation of products with better biological action than in unfermented tea

273 (Bhattacharya et al., 2013).

274 As demonstrated by many authors, the fermentative process is responsible for the

275 increase of the bioactivity of these phenolic components. Chakravorty et al., (2016)

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276 demonstrated that fermentation of kombucha gradually enhanced its ability to eliminate

radicals, peaking on the seventh day of fermentation. Ivanišová et al., (2020) measured

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the antioxidant activity of kombucha and observed significantly higher values (1318.56
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279 mg Trolox equivalent antioxidant capacity (TEAC)/l) when compared with unfermented
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280 black tea (345.59 mg TEAC/l). Similarly, Bhattacharya et al. (2013) found that despite
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281 black tea shows potent antioxidant power itself, the scavenging activities of fermented

282 black tea on parameters such as 2,2-diphenyl-1-picryl hydrazyl (DPPH), hydroxyl, and
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283 superoxide radicals increased 18.9 %, 17.2 % and 14.97 % respectively. Ivanišová et al.
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284 (2019) also observed that kombucha after 7 days of fermentation at 22 °C showed

285 higher antioxidant capacity by ABTS and phosphomolybdenum method (1.16 mg

286 TEAC/ml and 2.04 mg TEAC/ml, respectively) when compared to black tea (0.67 and

287 0.81 mg TEAC/ml, respectively).

288 Abuduaibifu and Tamer (2019) verified that along with the enhancement of the

289 protective effect against oxidative stress of three kombuchas samples (black tea, black

290 goji berry, and red goji berry kombucha), the bioaccessibility properties also increased.

291 The total phenolic content of all samples was higher in post-digestion (gastric,

292 intestinal) than in pre-digestion. In the same way, Aloulou et al., (2012) demonstrated

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293 by histological analysis that kombucha tea had better protective actions on the pancreas

294 and liver-kidney functions on rats, compared to black tea. These in vivo improvements

295 and the greater protective capacity of the kombucha is due to the large number of

296 phenolic compounds and its products, which are responsible for inhibiting the formation

297 of reactive species and strengthening endogenous enzyme defenses (Murugesan et al.,

298 2009; Sai Ram et al., 2000; Vázquez-Cabral et al., 2017; Mulyani et al., 2019).

299 Furthermore, this characteristic may even promote curative effects, for example, in

300 conditions of hyperglycemia and hypercholesterolemia (Aloulou et al., 2012;

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301 Bellassoued et al., 2015; de Almeida Souza et al., 2020).

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302 In relation to the fermentation process, the products synthesized by microorganisms in

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kombucha may play an important function in human health caused by their detoxifying
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304 action (Anal, 2019). As for the products that are formed during the fermentative
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305 process, the most relevant and often reported is glucuronic acid, the main organic acid
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306 resulting from the oxidation of glucose in kombucha (Neffe-Skocińska et al., 2017).

307 This acid is naturally synthesized in the human liver and has detoxifying properties
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308 from its potential of binding toxin molecules and by carrying out the excretion of
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309 xenobiotics and phenols through the kidneys or intestines. Thus, kombucha assists in

310 the elimination of excess metabolites and may bring relief in situations related to the

311 accumulation of toxins in the body, such as kidney stones, rheumatism, or arthritis

312 (Dufresne and Farnworth, 2000; Kaczmarczyk and Lochyński, 2014). The relevance of

313 this organic acid can be noted by its presence on the thematic map (Figure 4).

314 The production of this important organic acid is directly related to predominant bacteria

315 in the starter culture, which can vary largely depending on the variables of the process,

316 such as pH and temperature. De Filippis et al. (2018) characterized bacterial populations

317 in kombucha fermented at 20 °C and 30 °C and evaluated glucuronic acid production,

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318 finding substantial differences between the final products. Fermentation at 20 °C

319 favored Komagataiebacter xylinus (formerly named Gluconacetobacter xylinus) growth

320 and resulted in approximately 45 mg.L-1 of glucuronic acid, whereas fermentation at 30

321 °C favored Komagataeibacter saccharivorans (formerly named Gluconacetobacter

322 saccharivorans), resulting in a production of 28 times higher glucuronic acid

323 concentrations (approx. 1300 mg.L-1). These results empathize the importance of not

324 generalizing information about kombucha, since one product may be completely

325 different from another, even when using the same starter culture.

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326 Other compounds with health-modulating potential may result from the fermentation,

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327 such as B vitamins, including folate, riboflavin, and B12 (Chamlagain et al., 2015;

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Martínez Leal et al., 2018). Nevertheless, these microbial-derived products are strain
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329 dependent and are synthesized by certain bacteria in plant and dairy foods (Russo et al.,
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330 2014). Therefore, as the microbial community of the kombucha is variable, not all
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331 kombuchas will necessarily produce these vitamins. In this sense, Wang et al. (2014)

332 identified the functional strains and quantified the functional components with
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333 hepatoprotective effects in kombucha tea (KT). The authors concluded that
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334 Gluconacetobacter sp. A4 was the microorganism able to produce significantly high

335 values of the D-saccharic acid-1,4-lactone (DSL) in the modified KT (fermented by

336 single G. sp. A4) when compared to original kombuchas, fermented by tea fungus. DSL

337 is believed to be the compound responsible for hepatoprotection conferred by kombucha

338 by assisting the glucuronic acid to exert some properties like anti-tumor, detoxifying,

339 and antioxidant (Baschali et al., 2017). These results encourage the standardization of

340 inocula in the production of kombucha, with microorganisms selected in the starter

341 culture, in order to synthesize compounds of interest.

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342 Finally, antibacterial activity also stands out on the thematic map (Figure 4), being one

343 of the most reported effects in the literature in relation to kombucha. The antimicrobial

344 action of this drink has already been reported for several microorganisms, including

345 Gram-negative and Gram-positive bacteria (Dufresne and Farnworth, 2000), the most

346 frequently reported being Escherichia coli, Staphylococcus aureus, and Helicobacter

347 pylori. The latter being the cause of gastric ulcers and other complications of the

348 digestive system (Kaczmarczyk and Lochyński, 2014). The antibacterial activity of

349 kombucha is related to the microbial community of its starter culture, acting in a

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350 symbiotic way to prevent potential contaminations, and also to the metabolites formed

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351 during the fermentation, such as catechins, ethanol, and organic acids, especially acetic

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acid (Kaczmarczyk and Lochyński, 2014; Watawana et al., 2015). This acid is reported
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353 to control the growth of pathogenic bacteria, avoiding contamination in the production
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354 of kombucha and having antifungal and antiviral properties as well (Watawana et al.,
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355 2015; Mousavi et al., 2020; Fu et al., 2015). It should also be recalled that low pH

356 values contribute to this antimicrobial action.

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357 Although several benefits are regarded to the consumption of kombucha, it is important
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358 to highlight that it may be harmful when not safely brewed and also may lead to

359 metabolic acidosis in some individuals when consumed in excess (Martini, 2018).

360 Adverse effects from kombucha consumption have been reported, such as allergies,

361 jaundice, nausea, head and neck pain (Srinivasan et al., 1997). Also, kombucha should

362 be avoided by lactating and pregnant women (Jayabalan et al., 2014).

363 Nevertheless, kombucha fulfills the consumer’s demand for healthy beverages,

364 reflecting in a market growth that is partially based on widespread information of

365 health-promoting effects (Kapp and Sumner, 2019; Kim and Adhikari, 2020). Recently,

366 some scientific studies are using kombucha as a vehicle to reduce the pathogens

17
367 associated with human illnesses (Mousavi et al., 2020), while many researchers have

368 reported the different activities of kombucha and demonstrated its potential health

369 benefits. This wide range of advantages has led to investigating the role of the

370 microbiome on health, where it is proposed that kombucha may have the ability to act

371 as a probiotic agent (Kapp and Sumner, 2019; Kim and Adhikari, 2020; Martínez Leal

372 et al., 2018; Mann et al., 2017).

373

374 3.2 Probiotic and prebiotic potential

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375 Recent studies associate the consumption of kombucha as influencing and modifying

r
376 the gastrointestinal microbiota. The human gut microbiota is complex and colonized by

377
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approximately 1•1013 to 1•1014 microorganisms (Li et al., 2016). Studies report that the
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378 phyla most identified in the colon are: Actinobacteria, Firmicutes, Bacteroidetes,
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379 Fusobacteria, and Proteobacteria (Zmora et al., 2019). When this microbial community
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380 is in dysbiosis, that is, pathogenic species are in greater concentration, an imbalance

381 situation arises, and different pathologies can originate in individuals. Because of the
ur

382 association between the deregulated intestinal microbiome and the genesis of illnesses,
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383 there is a growing interest in the modulation of microbial genera (Morales, 2020).

384 In this context, with a currently strong link between intestinal health and its direct role

385 in human health, new treatment approaches and pharmaceutical interventions for

386 disease prevention are being investigated. Thus, one of the main therapies that has been

387 implemented is the use of beneficial microbes, such as probiotics in functional foods (de

388 Almeida Souza et al., 2020; Heinen et al., 2020). According to the World Health

389 Organization (WHO), probiotics are “live microorganisms which when administered in

390 adequate amounts confer a health benefit on the host” (FAO/WHO, 2006; Gibson et al.,

391 2017; Hill et al., 2014). Probiotics are said to maintain the intestinal barrier integrity,

18
392 improve digestion and modify and select the host-microbiota (Watawana et al., 2015;

393 Kozyrovska et al., 2012). There are different mechanisms in that these microorganisms

394 perform these actions. First, they stimulate the growth of beneficial resident gut

395 microorganisms by supplying some substances generated by their metabolism. Second,

396 directly inhibit the growth of pathogenic genera through niche competition,

397 bacteriocins, or a decrease in pH. Finally, some microorganisms can indirectly interact

398 with the host epithelium and the epithelial immune system. As a result, they impact the

399 host-microbiota by controlling pro-inflammatory cytokines (Derrien and van Hylckama

f
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400 Vlieg, 2015; Marco et al., 2017). The agents most associated with the probiotic function

r
401 are lactic acid bacteria, especially Lactobacillus and Bifidobacterium, non-lactic acid

402
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species such as Bacillus cereus and Propionibacterium freudenreichii, and yeasts of the
re
403 genus Saccharomyces (species boulardii and cerevisiae) (Kozyrovska et al., 2012;
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404 Holzapfel et al., 2001). These microorganisms can assist in the generation of a healthy
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405 microbiome, restoring homeostasis in the body (Kozyrovska et al., 2012).

406 Human diets are capable of changing the composition of gut microbiota. Modifications
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407 in the food intake pattern, even for a short period of time, may increase the amount of
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408 some bacteria, while reducing the number of others, altering the whole microbial

409 community (Heinen et al., 2020). In this sense, it is observed that a typical western diet,

410 with high fat and sugar intakes, is associated with increased Firmicutes and reduced

411 Bacteroides. On the other hand, plant-based diets, with high consumption of fibers such

412 as fruits and vegetables, leads to a higher abundance of Bacteroidetes. Thereby, the

413 consumption of a diversified diet is the key to microbial homeostasis (Million et al.,

414 2018; Zmora et al., 2019).

415 The delivery of beneficial microorganisms to the gut microbiota is dependent on the

416 food matrix. Many fermented foods and beverages are related to the modification in the

19
417 indigenous microbiota. The regular ingestion of these fermented products possibly

418 enhances up to 1•104 times the microorganisms resident in the colon (Lang et al., 2014).

419 Although fermented products demonstrate being a potential vehicle to provide probiotic

420 strains, some of these are processed in a way that those beneficial microorganisms are

421 absent when consumed (Marco et al., 2017).

422 Kombucha is known worldwide as a probiotic drink, as it contains live microorganisms

423 in its composition. This beverage represents the fastest growing product in the

424 probiotics market. It is possible to find kombucha labels in several countries with the

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425 appeals of “natural”, “organic”, “raw”, “living culture”, “non-dairy probiotics”, “healthy

for your gut”, among other claims (Kim and Adhikari, 2020). However, some of these

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426

427
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statements are mostly unfounded or at least unproved. So much so, that in most parts of
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428 Europe the use of claims such as "probiotics" or “contain probiotics” is not allowed on
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429 labels (Marco et al., 2017). The presence of probiotic strains is inconsistent in
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430 kombuchas. Normally, they are not present or remain in low concentrations, mainly

431 after storage (Matei et al., 2018; Coton et al., 2017; Fu et al., 2014).
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432 Preparations or products that contain probiotic microorganisms must have an adequate
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433 quantity, making them capable of altering the intestinal microbiota through implantation

434 or colonization (Schrezenmeir and de Vrese, 2001; Kapp and Sumner, 2019). So far,

435 there is no evidence that these probiotic microorganisms are in sufficient quantity in

436 kombucha and bring some specific health benefit (Martini, 2018). Furthermore,

437 products that use probiotic microorganisms in order to confer health benefits to the

438 consumer must indicate the dosage and the time needed to obtain the desired effect,

439 based on scientific evidence obtained from studies carried out in vitro and in vivo (in

440 animals and humans) (FAO/WHO, 2006). Moreover, legislation from the country of

441 sale must be observed and may vary from place to place (FAO/WHO, 2006).

20
442 In this research, many articles mentioned kombucha as a “probiotic-containing food”,

443 without presenting scientific evidence that the microbial community indeed delivers the

444 benefits to the host (Dikeocha et al., 2020; Fu et al., 2014; Onur et al., 2019; Salafzoon

445 et al., 2018; Ranjan et al., 2020; Vilela, 2019; Chandrakala et al., 2019).This possibly

446 inadequate use of the term probiotic frequently appears and may be related to the fact

447 that several articles have reported the benefits of kombucha to health and also owing to

448 the presence of live microorganisms in the drink (Kapp and Sumner, 2019; Martínez

449 Leal et al., 2018). However, the studies suggest that the benefits come from the tea and

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450 metabolites from fermentation, such as organic acids, polyphenols, and vitamins (Kapp

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451 and Sumner, 2019). To the best of our knowledge, no studies have reported health

452
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benefits from the whole microbial community of kombucha. Notwithstanding, the
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453 microbial culture of kombucha has been used in other raw materials in order to develop
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454 new probiotic drinks (Soares et al., 2021). Mulyani et al. (2019), in their study, aimed to
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455 produce an allegedly probiotic beverage made from beat (Beta vulgaris L.) fermented

456 with kombucha culture to prevent digestive infections. The results showed that the drink
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457 had antibacterial activity against E. coli. The authors, however, did not identify or
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458 quantify the probiotic strains in the drink, and regarding antibacterial activity, it is not

459 known to what extent this effect would be observed in vivo gastrointestinal tract

460 (Lavefve et al., 2019).

461 Fermented probiotic products are produced from a complex and unstable set of

462 microorganisms, which can be modified because they remain in open environments and

463 are susceptible to variations according to the growing conditions and substrates

464 available for their development (Reva et al., 2015a). The studies reporting yeasts and

465 bacteria communities in kombucha show that there exists a huge variability between

466 products, although there are some genera that appear more frequently. In the case of

21
467 bacteria, the most predominant genera are Komagataeibacter (formerly assigned to

468 Gluconacetobacter) (Arıkan et al., 2020; Chakravorty et al., 2016; Gaggìa et al., 2018;

469 Reva et al., 2015a; Marsh et al., 2014; Villarreal-Soto et al., 2020), and Acetobacter

470 (Coton et al., 2017). Komagataeibacter species are described as bionanocellulose

471 producers, showing a large phenotypic strain diversity, strongly dependent on carbon

472 source affinities, bionanocellulose syntheses rates and composition, and strain stability

473 (Ryngajłło et al., 2019). In relation to its potential probiotic ability, it is assumed that

474 the set of central microorganisms of the kombucha could recruit some environmental

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475 bacteria, particularly of the genus Lactobacillus. Possibly, this whole microbial

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476 community favors the fermentation of kombucha for the development of a probiotic

477
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characteristic (Reva et al., 2015a). However, only a few genera of LAB may be found in
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478 this beverage, such as Lactobacillus (Chakravorty et al., 2016; Coton et al., 2017;
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479 Marsh et al., 2014; Reva et al., 2015a), Bifidobacterium (Chakravorty et al., 2016;
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480 Marsh et al., 2014), and Lactococcus (Marsh et al., 2014), all appearing to a lesser

481 amount than acetic acid bacteria. Yeast genera are more variable than bacteria, with
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482 Brettanomyces/Dekkera being the prevalent species (Coton et al., 2017; Gaggìa et al.,
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483 2018; Reva et al., 2015a; Villarreal-Soto et al., 2020), followed by Zygosaccharomyces

484 (Arıkan et al., 2020; Coton et al., 2017; Marsh et al., 2014; Villarreal-Soto et al., 2020),

485 Candida (Chakravorty et al., 2016; Villarreal-Soto et al., 2020), and Hanseniaspora

486 (Coton et al., 2017).

487 Among the most predominant bacteria and yeasts found in kombucha, some have been

488 studied regarding their potential probiotic properties. Matei et al., (2018) isolated nine

489 yeast species from pollen fermented kombucha and identified them by PCR-ITS RFLP

490 technique, aiming isolation of prospective probiotic yeasts. It was possible to identify

491 the species Dekkera bruxellensis, but no studies on probiotic potential were performed.

22
492 The same research group has studied the possible probiotic effect of five LAB isolated

493 from kombucha (Matei et al., 2018). The isolates of Pediococcus pentosaceus species

494 were subjected to on-plate screening for bacteriocin production, wherein three of five

495 strains were positive. Furthermore, isolates were tested for bile-salt resistance, with

496 three strains showing tolerance to 3 % and 6 % of bile salts. Among the five isolates, the

497 “L5” strain showed higher potential as a probiotic because it was the only bacterium

498 that showed both bacteriocin production and bile salt resistance (Matei et al., 2018).

499 This strain was further studied regarding the potential of adhesivity to Caco-2 intestinal

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500 cells and authors suggested a probiotic effect, since L5 adhered to intestinal cells in a

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501 time-dependent manner (Utoiu et al., 2018). Pediococcus is not a common genus found

502
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in kombucha and it has not been identified in studies that used next-sequencing
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503 generation (Arıkan et al., 2020; Bueno et al., 2021; Chakravorty et al., 2016; Coton et
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504 al., 2017; Gaggìa et al., 2018; Marsh et al., 2014; Villarreal-Soto et al., 2020).
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505 The variability in the kombucha microbial community demonstrates how complex is the

506 process of this fermented beverage. Although some other fermentations contain only a
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507 few dominant taxa, the fluctuations in the microorganisms of kombucha may affect the
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508 quality of this drink (Reva et al., 2015a; Marco et al., 2017). With the increase in

509 kombucha consumption, consequently, its homemade production evolved to an

510 industrial scale (Soares et al., 2021). While SCOBY can be useful as a starter culture for

511 homemade kombucha, in the industrial sector this type of inoculum is not suitable

512 because of the unrepeatable and uncontrollable qualities of the process, leading to

513 variations in the organoleptic properties, affecting the final product (Marco et al., 2017;

514 Wang et al., 2020). The beverage industries are investing in food safety and

515 improvements in the biotechnological properties of kombucha production. Standard

516 process conditions and a stable set of microorganisms throughout fermentations result in

23
517 kombuchas showing stable characteristics. The consistency of end products is a

518 primordial factor to produce high-quality food (Marco et al., 2017; Mousavi et al.,

519 2020). Thus, efforts are being made to characterize the essential microbiome of

520 kombucha in an attempt to standardize its production and also to identify the best

521 mixture of microorganisms to improve kombuchas yield (Mousavi et al., 2020; Wang et

522 al., 2020). Wang et al., (2020) developed a symbiotic microbial community for

523 kombucha with three main microorganisms, Acetobacter pasteurianus,

524 Komagataeibacter xylinus (previously named Gluconacetobacter xylinus), and

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525 Zygosaccharomyces bailii. The authors verified that this microbial set could accelerate

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526 the fermentation process of kombucha and generated a beverage with desirable

527
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qualities. Furthermore, the ability to select species of bacteria and yeasts is also an
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528 important approach to determine the metabolites generated through the fermentation
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529 process. Thereby, the modification of the kombucha inoculum can also be fundamental
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530 in improving the functional properties of this drink (Kozyrovska et al., 2012). Wang et

531 al., (2020) also concluded in their study that the designed starter culture was capable of
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532 enhancing the antioxidant content of kombucha. Similarly, Nguyen et al. (2015) carried
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533 out a screening of ideal strains for high production of glucuronic acid, in order to

534 improve the antioxidant, antimicrobial and detoxifying activities of the kombucha. The

535 authors found that the best ratio would be an initial combination of Dekkera

536 bruxellensis KN89 and Komagataeibacter intermedius KN89 (formerly named

537 Gluconacetobacter intermedius) in a 4:6 parts, respectively.

538 Another alternative strategy to obtain a probiotic kombucha could be the addition of

539 well-known and studied probiotic strains into the fermentation process, as was shown in

540 a few studies reported in this systematic review. Al-Dulaimi et al. (2018) developed two

541 kombuchas using black tea and skim milk, adding in each formulation a probiotic

24
542 culture starter, composed of two species of Lactobacillus (delbruekii and fermentum).

543 Investigating the beneficial effects of both kombuchas on some physiological and

544 biochemical parameters in rats, the authors verified that animals that were treated with

545 probiotic kombuchas had decreased concentrations of total lipid profile, ALT, AST,

546 ALP hepatic enzymes, and serum glucose, when compared to the control group. The

547 reduced serum glucose levels can be addressed to the activity of probiotic bacteria since

548 they use glucan compounds to carry out fermentation. Thus, this beverage could present

549 some beneficial actions on the liver and human body health in general. In another study

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550 using the incorporation of probiotics Bueno et al. (2021) developed a coffee kombucha

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551 added of two probiotic strains: Lactobacillus rhamnosus (LR) and Lactobacillus casei

552
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(LC). After fermentation and removal of the biofilm, the kombucha fermented from
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553 coffee was added with LR or LC, or no probiotics (control) and stored for 15 days at 4
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554 °C. The authors evaluated the survival of probiotics in the kombuchas and the microbial
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555 viabilities under simulated gastric and intestinal conditions. The authors also identified

556 the microbial diversity through next-sequencing generation on the fifteenth day of
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557 storage. Results were very interesting and showed LC viability in coffee kombucha
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558 remained without significant changes at the end of storage, whereas LG presented a

559 slight reduction of viable counts, allowing to suggest that LC and LG survive in the

560 gastric and intestinal environment. Microbial community analysis revealed that the

561 addition of Lactobacillus shifted the predominance of Acetobacteraceae family to

562 Lactobacillaceae and, although the species added was L. rhamnosus and L. casei, the

563 predominant genus in each kombucha was Lactobacillus zeae for LR and Lactobacillus

564 paracasei for LG, both strains documented as probiotics (Bueno et al., 2021).

565 On the other hand, Fu et al. (2014) evaluating the survival of specific strains in

566 kombucha after 14 days of refrigerated storage did not obtain results as positive as in

25
567 the previous upper study. The authors developed a low-cost green tea kombucha

568 (LGTK) inoculated with 5 % of starter culture composed of Saccharomyces cerevisiae

569 Meyen ex Hansen (108 CFU/mL), Komagataeibacter sp. (108 CFU.mL-1), and

570 Lactobacillus plantarum (108 CFU.mL-1), in a ratio of 1:1:1. At the end of the study

571 they verified that, under refrigeration at 4 °C, acetic acid bacteria moderately decreased

572 up to ten days of storage, whereas the survival rate of lactic acid bacteria was only 0.98

573 % on the eighth day of storage. These results reinforce the importance of the choice of

574 the microorganisms contained in the starter inoculum and their influence on the final

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575 probiotic characteristics of the beverage.

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576 Some beverage companies do not pasteurize the industrial scale kombuchas in order to

577
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maintain the drink's natural microbiome, in such a way that keeping the product raw
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578 would allow the development of a prebiotic or probiotic drink. In another approach,
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579 other companies proceed with the pasteurization process in their production and
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580 subsequently add recognized probiotic strains, such as Bacillus coagulans,

581 Saccharomyces boulardii and Lactobacillus rhamnosus, to the final kombucha

ur

582 formulation (Kim and Adhikari, 2020). It is important to note that this strategy of
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583 adding probiotics is useful depending on the country of sale regulation. In the case of

584 Brazil, for instance, the Normative Instruction for Quality and Identity Standards for

585 Kombucha does not allow the addition of strains after fermentation, as it must be added

586 at the beginning of the process (BRAZIL, 2019).

587 The criteria for probiotic strain selection involve functional, safety, technological and

588 physiological aspects (Terpou et al., 2019). Among these aspects, the kombucha

589 microbial community presents high potential as probiotic because it is non-pathogenic

590 (Arıkan et al., 2020; Chakravorty et al., 2016; Gaggìa et al., 2018), presents competitive

591 inhibition of undesirable microbiota (Ayed et al., 2017; Kaewkod et al., 2019;

26
592 Sreeramulu et al., 2000), and tolerance to acidic environments (Villarreal-Soto et al.,

593 2018). It is important to note that most probiotic products are of dairy origin. Plant-

594 based probiotic foods and beverages are less available in the market (Muhialdin et al.,

595 2021). However, with the growing knowledge about their health properties, including

596 their supposed role in boosting immunity, taking into account the recent Covid-19

597 pandemic, the demand for plant-based probiotics has increased (Antunes et al., 2020).

598 Kombucha has this advantage because it is a non-dairy drink. Its vegetable matrix used

599 in its preparation guarantees that it can be consumed by vegans, people with lactose

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600 intolerance, or by people who are reducing the consumption of products of animal

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601 origin (Panghal et al., 2018; Reva et al., 2015a).

602
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Furthermore, some authors have stated that kombucha not only can be a probiotic
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603 beverage but also acts as a prebiotic (Dufresne and Farnworth, 2000; Kozyrovska et al.,
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604 2012). Prebiotics are food-containing components that serve as a substrate for the
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605 beneficial microorganisms (Gibson et al., 2017; Hill et al., 2014). These prebiotic agents

606 are responsible for nourishing the probiotic strains, thus selectively stimulating the
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607 growth and/or activity of the microbiota resident in the human gut (Marco et al., 2017;
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608 Heinen et al., 2020).

609 Lavefve et al., (2021) studying the microbiota modulation demonstrated that kombucha

610 and especially the kombucha supernatant were able to significantly enhance the in vitro

611 growth of Bifidobacterium and Collinsella. These preliminary results showed that

612 kombucha stimulates these beneficial microorganisms, acting in a prebiotic manner.

613 Among the possible prebiotic components present in kombucha are phenolic

614 compounds. Some studies describe these phytochemicals as substances that exert

615 prebiotic-like effects, being a class of bioactive most associated with the promotion of a

616 healthy microbiome (Million et al., 2018; Dueñas et al., 2015; Tomás-Barberánet al.,

27
617 2016). This association occurs because most of the ingested polyphenols pass intact

618 through the small intestine, reaching the large intestine where they will be used as a

619 substrate by beneficial genera of the microbiota. In a reciprocal interaction, these

620 microorganisms increase the bioavailability of the polyphenols metabolizing them into

621 smaller molecules (Ozdal et al., 2016; Hollister et al., 2014). Therefore, there is an

622 enhancement of the detoxifying enzymes and improvement in the antioxidant response

623 of fermented products, protecting the organism from oxidative stress and other possible

624 chemical damages (Marco et al., 2017; Senger et al., 2016).

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625 Other classes of food components showing prebiotic properties are some

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626 polysaccharides. The cellulosic pellicle of kombucha, present in the SCOBY, could help

627
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in the growth and maintenance of the beneficial microbes in the beverage by the
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628 presence of insoluble fibers in its composition (Kozyrovska et al., 2012; Marco et al.,
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629 2017). These substances could be fermented by the microbial community of kombucha
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630 and then release some secondary metabolites, mainly short-chain fatty acids (SCFAs),

631 such as acetate, propionate, butyrate, lactate, and succinate (Salazar et al., 2016;
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632 Sonnenburg and Fischbach, 2011). These compounds are known to provide some health
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633 benefits to the host. Particularly, butyrate is known to strengthen the intestinal epithelial

634 barrier and to interact with inflammatory pathways, promoting the differentiation and

635 maintenance of the defense cells, boosting immunity (Kozyrovska et al., 2012; Kim and

636 de La Serre, 2018). Lactate, in turn, is said to reduce the reactive oxygen species in

637 intestinal enterocytes (Kahlert et al., 2016). Therefore, if a portion of the lactic acid

638 present in fermented kombucha reaches human metabolism, that organic acid may

639 provide this health protection. Additionally, some ingested microorganisms produced in

640 fermented foods might synthesize some vitamins, amino acids, and growth factors, such

641 as exopolysaccharides, which could also compete as antioxidants, stimulating the

28
642 immune system and preventing the adhesion of pathogens to the intestinal mucosa

643 (Marco et al., 2017; Gerritsen et al., 2011; Thomas et al., 2017; Makino et al., 2016).

644 All these metabolites produced by the intestinal microbiota, such as SCFA, polyphenols,

645 vitamins, and other functional compounds derived from the diet have recently been

646 called post-biotics (Lavefve et al., 2021). Post-biotic is a new term used in the area of

647 food and therefore there is no consensus on this concept yet. However, post-biotics can

648 be defined in a wide spectrum as products or by-products generated or released by

649 microorganisms during growth and fermentation in complex microbiological culture,

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650 food, or intestine (Moradi et al., 2021). These metabolites could resolve the intestinal

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651 imbalance, acting in the reestablishment of the microbiota symbiosis. Thus, post-biotics

652
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can offer positive physiological effects to the host, helping to promote their health
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653 (Klemashevich et al., 2014; Cuevas-González et al., 2020).
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654 All things considered, it is known that the claim of any artisanal kombucha as a
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655 probiotic is inaccurate since the composition and concentration of the microorganisms

656 present in the drink are unknown and change among batches depending on the
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657 fermentation variables (De Filippis et al., 2018). However, kombucha has been proven
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658 to be a potential source for isolation of probiotic bacteria in industrial or pharmaceutical

659 applications (Matei et al., 2018; Sinir et al., 2019; Utoiu et al., 2018). Also, prebiotic

660 potential and post-biotics compounds in kombucha have recently become research

661 topics. In order to have a stated functional kombucha beverage, further studies are

662 needed through the development of a defined starter culture for the fermentation of

663 kombucha, as it happens for other fermented products such as yogurt, wine, or beer.

664 Still, more studies are needed concerning the possible prebiotic and probiotic effects of

665 kombucha.

666

29
667 4 Final considerations

668 Kombucha is an important source of bioactive compounds arising from raw material

669 and increased by the fermentation process. These beneficial compounds change between

670 kombuchas and depend on several variables, such as type of raw material, time of

671 infusion, starter culture microbial community, and fermentation parameters. Therefore,

672 kombucha health-promoting properties should not be generalized, since there are so

673 many variables affecting final product composition. So far, artisanal kombucha is

674 produced through an uncontrolled fermentation process and the microbial community is

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675 complex, diverse, and variable. Also, interactions between yeasts, bacteria and their

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676 impact on human microbiota have not been understood or properly and thoroughly

677
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investigated. For this reason, kombucha cannot be considered a probiotic or symbiotic
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678 drink, unless documented probiotic strains have been added in the process. As the
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679 demand for this product is increasing, scientific studies are necessary to standardize the
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680 starter culture with well-known microorganisms, in order to control the metabolites

681 produced, standardize the final product, and develop further studies on biological
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682 activities of kombucha, establishing it as a functional symbiotic beverage.

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683

684 Conflict of Interest

685 Declarations of conflicts: none

686

687 Acknowledgments

688 This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível

689 Superior - Brasil - (CAPES) Finance Code 001.

690

691

30
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1345

f
r oo
-p
re
lP
na
ur
Jo

44
1346 Table 1. In vivo and in vitro biological properties associated with kombucha.

Type of
Biological property Method/Experimental model References
Assay
In vitro Antibacterial S.epidermidis, S. aureus, Mi. (Battikh, Bakhrouf, and
luteus, E. coli, P. aeruginosa, S. Ammar 2012)
typhimurium, L. monocytogenes

E. coli, H. influenzae (Ivanišová et al. 2020)

E. coli (Mulyani et al. 2019)

E. coli, S. sonnei, S. typhimurium, (Sreeramulu, Zhu, and

S. enteritidis, C. jejuni Knol 2000)

f
E. coli, S. aureus. B. cereus, S. (Valiyan, Koohsari, and

oo
dysenteriae Fadavi 2021)

E. coli, Salmonella (Ma et al. 2019)

r
-p
S. aureus, K. pneumoniae, E. coli, (Vitas et al. 2018)
re
P.vulgaris, P. mirabilis, B. subtilis
E. coli, S. typhimurium, M. luteus, (Deghrigue et al. 2013)
lP

S. epidermidis

E. coli, Salmonella, S. aureus, L. (Cardoso et al. 2020)

na

monocytogenes
Antifungal C. glabrata, C. tropicalis, C. sake, (Battikh, Bakhrouf, and
C. dubliniensis, C. albicans Ammar 2012)
ur

C. krusei C. glabrata, C. albicans, (Ivanišová et al. 2020)

C. tropicalis
Jo

C. albicans, A. niger (Vitas et al. 2018)

Anti-carcinogenic Human cell lines A549 (lung (Jayabalan et al. 2011)

carcinoma), U2OS (osteosarcoma),
786-O (renal carcinoma)

Human cell lines A549 (lung (Deghrigue et al. 2013)

carcinoma), Hep-2 (epidermoid
carcinoma)

Human cell lines RD (Vitas et al. 2018)

(rhabdomyosarcoma), Hep2c
(cervix carcinoma-HeLa
derivative) and murine cell line
L2OB (fibroblast)

45
 Human cell lines HCT-116 (colon (Villarreal-Soto et al.
cancer) and MCF7 (breast cancer) 2019; Villarreal-Soto et
al. 2020)

Human cell line PC-3 (prostate (Srihari et al. 2013)

cancer)

Cell lines A549 (lung (Cardoso et al. 2020)

adenocarcinoma epithelial), HCT8
(ileocecal colorectal
adenocarcinoma), CACO-2
(colorectal adenocarcinoma
epithelial)
Anti-inflammatory Human monocytic cells (THP-1) (Vázquez-Cabral et al.
differentiated to macrophages 2017)

f
5- lipoxygenase enzyme (Villarreal-Soto et al.

oo
2019)
15-lipoxygenase enzyme (Villarreal-Soto et al.

r
2020)
Antioxidant
-p
ABTS radical scavenging (Ivanišová et al. 2019;
Cardoso et al. 2020;
re
Jafari et al. 2020; Aung
and Eun 2021; Gamboa-
lP

Gómez et al. 2017)

DPPH (1,1-diphenyl 2- (Ivanišová et al. 2020;
picrylhydrazyl) radical scavenging Mulyani et al. 2019;
na

Villarreal-Soto et al.
2019; Villarreal-Soto et
al. 2020; Gamboa-
ur

Gómez et al. 2017;

Gaggìa et al. 2018; Hoon
Jo

et al. 2014; Yang et al.

2009; Aung and Eun
2021; Jakubczyk et al.
2020; Bhattacharya,
Gachhui, and Sil 2013)
Oxygen Radical Absorbance (Vitas et al. 2018;
Capacity (ORAC) Gamboa-Gómez et al.
2017)
Ferric reducing antioxidant power (Aung and Eun 2021;
(FRAP) Gaggìa et al. 2018;
Jakubczyk et al. 2020)
Reactive oxygen species induced (Yang et al. 2009;
by H2O2 Vázquez-Cabral et al.
2017)
Superoxide radical (O2-) (Hoon et al. 2014; Yang
scavenging ability et al. 2009;
Bhattacharya, Gachhui,
and Sil 2013)
Cupric ion reducing antioxidant (Jafari et al. 2020)
capacity (CUPRAC)

46
 Nitric oxide scavenging (NO) (Gamboa-Gómez et al.
2017)
Anti-hyperglicemic Glucose diffusion (Gamboa-Gómez et al.
2017)
Radioprotective Human peripheral lymphocytes (Cavusoglu and Guler
(ionizing radiation) 2010)
Cytotoxicity Human peripheral blood (Mrdanović et al. 2007)
lymphocytes
Human cell IMR90 (normal lung (Cardoso et al. 2020)
cell)
In vivo Anti- White rabbits (Alaei, Doudi, and
hypercholesterolemic Setorki 2020)
Alloxan diabetic rats (Aloulou et al. 2012)

f
oo
Wistar rats (Bellassoued et al. 2015)

r
Mice (Yang et al. 2009)

Anti-hyperglycemic
-p
Alloxan diabetic rats (Aloulou et al. 2012)
re
Female C57BL/6 mice (Gamboa-Gómez et al.
lP

2017)
Swiss albino male rats (Bhattacharya, Gachhui,
na

and Sil 2013)

Streptozotocin-induced diabetic (Zubaidah et al. 2019)
rats
ur

Normal and alloxan-induced (Shenoy 2000)

diabetic mice
Jo

Radioprotective Male albino rats (Gharib 2014)

(electromagnetic
radiation)
Brain damage Ischemic rats (Kabiri and Setorki
prevention 2016)
Anti-hyperuricemic Rats (Sukrama 2015)

Hepatoprotective Mice (Wang et al. 2014)

Male albino rats (Jayabalan, Baskaran, et

al. 2010)
Mice (Lee et al. 2019)

Microbiome Male C57BLKS db/db mice (Jung et al. 2018)

modulation
Longevity Mice (Hartmann et al. 2000)

47
 Antioxidant Male albino rats (Sai Ram et al. 2000)

Mice (Yang et al. 2009)

Prevention of Male albino Wistar rats (Lobo, Chandrasekhar

myocardial injury Sagar, and Shenoy 2017)
caused by
isoproterenol
Antiviral Swine (Fu et al. 2015)

1347

1348

1349

f
oo
1350

r
1351

1352
-p
re
1353
lP

1354
na

1355

1356
ur

1357
Jo

1358

1359

1360

1361

1362

1363

1364

1365

1366

1367

48
1368 Figure captions

1369

1370 Figure 1. Two portions of kombucha fermentation process. (A) floating microbial

1371 cellulose pellicle layer and (B) liquid tea phase.

1372 Figure 2. Methodological flowchart performed in this work.

1373 Figure 3. Scientific production over the years from articles found on Scopus with the

1374 terms “kombucha” AND “health”.

1375 Figure 4. Thematic map generated on Bibliometrix from articles found on Scopus with

f
oo
1376 the terms “kombucha” AND (“health” AND “effect” OR “benefit*” OR “claim”).

r
-p
re
lP
na
ur
Jo

49
Jo
ur
na
lP
re
-p
ro
of
Jo
ur
na
lP
re
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ro
of
Jo
ur
na
lP
re
-p
ro
of
Jo
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na
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-p
ro
of
Highlights:

 Many health benefits attributed to Kombucha make it popular as a functional

beverage.

 LAB are important for probiotic claims, but they are not always present in

kombucha.

 Kombucha microbial community has high potential for isolation of probiotic

strains.

f
oo
 Kombucha prebiotic-like compounds may stimulate probiotic strains in gut

r
microbiota.


-p
Kombucha fermentation and its microorganisms may release post-biotics
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substances.
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Declaration of conflicts of interests

Manuscript: Health effects and probiotic and prebiotic potential of Kombucha: a

bibliometric and systematic review

Bruna Krieger Vargas, Mariana Fensterseifer Fabricio, Marco Antônio Záchia Ayub

☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered

of
as potential competing interests:

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