Kombucha fermentation using commercial kombucha pellicle and culture broth as starter

Fermentation is one of the oldest methods to improve food value and preservation via a low-cost energy conservation system. The biochemical changes introduced during fermentation may change the composition of food, thereby imparting new biological activity or increasing digestion and absorption. Recently, fermentation has been used to extract or produce active compounds from plant materials used in the food or beverage industry. Mixed cultures of various microorganisms such as yeast, lactic acid bacteria (LAB), acetic acid bacteria (AAB), and koji fungi (Aspergillus oryzae) have been employed in traditional fermentation industries (Makwana & Hati, 2019).


Introduction
Fermentation is one of the oldest methods to improve food value and preservation via a low-cost energy conservation system. The biochemical changes introduced during fermentation may change the composition of food, thereby imparting new biological activity or increasing digestion and absorption. Recently, fermentation has been used to extract or produce active compounds from plant materials used in the food or beverage industry. Mixed cultures of various microorganisms such as yeast, lactic acid bacteria (LAB), acetic acid bacteria (AAB), and koji fungi (Aspergillus oryzae) have been employed in traditional fermentation industries (Makwana & Hati, 2019).
Mixed cultures have the advantage of being able to utilize a wide range of enzymes and complex mixtures of carbohydrates, proteins, and fats for fermented food production (Dai et al., 2017). Likewise, the appropriate strain can alter or degrade toxic or noxious compounds that may be present in the fermentation substrate. During food fermentation by mixed microorganisms, yeast produces alcohol, and bacteria produce lactic and organic acids, changing the environment from aerobic to anaerobic; thus, they complement each other and prevent the growth of unwanted microorganisms. Therefore, the inhibitory compounds, anaerobic conditions, and low pH caused by mixed cultures hinder the production of undesirable mold and bacteria (Hesseltine, 1992).
In Korea, kombucha refers to a black tea fungus and also to a fermented beverage obtained by symbiotic fermentation (bacteria and yeast) of the main raw materials of black tea and sugar (Lee & Kim, 2000). Kombucha is a healthy carbonated beverage made by fermenting sugar with SCOBY (symbiotic culture of bacteria and yeast) in extracts such as green/black tea (Amarasekara et al., 2020). SCOBY is a culture product of bacteria and yeast used in the manufacturing of various foods and beverages such as kombucha, vinegar, sourdough, and kefir (Gallegos et al., 2016;Laureys et al., 2020). Kombucha is a beverage produced by Saccharomyces and Brettanomyces, which are yeasts that use sugar to produce alcohol, and Acetobacter that use alcohol to produce various organic acids including acetic acid and gluconic acid. It is characterized by the formation of a pellicle, a gel-like membrane of cellulose produced by a symbiotic strain on the surface of the kombucha culture (Jayabalan et al., 2014).
Although despite the various physiological effects of kombucha, its application and utilization in the industry are remarkably low. This is mainly due to insufficient analysis of microbial species, distribution, and metabolites produced during the fermentation of kombucha, which occurs through symbiosis of microorganisms. In this study, in order to industrialize, standardize, and secure the safety of kombucha, the first kombucha fermentation strain and strain distribution were confirmed the changes in several biochemical markers and antioxidant properties of kombucha fermentation were measured for a period of 3 weeks. Through this study, we intend to provide data that must be secured for the mass production and industrialization of kombucha.

DNA extraction and metagenomic sequencing of 16S rRNA
The liquid samples were centrifuged at 5,000 × g for 10 min, and the supernatant was removed. Kombucha genomic DNA was extracted using the DNeasy PowerFood Microbial kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The concentration and purity of metagenomic DNA extracted from kombucha samples were measured using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
For preparation of the MiSeq library amplicons, the 16S rRNA V3-V4 variable region of DNA extracted from kombucha samples was amplified. After the first polymerase chain reaction (PCR), the PCR products were purified and a quality-controlled PCR product was used as a template for the second PCR. Nontarget products of purified products from the second PCR were eliminated using Agencourt AMPure XP Beads (Beckman Coulter, Inc.), and the concentration and purity of quantified PCR amplicons were measured using GX caliper HS DNA 1000 (PerkinElmer, Waltham, MA, USA) and the NanoQuant pro200 instrument (Tecan, Mannedorf, Switzerland). Sequencing was carried out at Chunlab, Inc. (Seoul, Republic of Korea) using the MiSeq system (Illumina, San Diego, CA, USA).

Analytical method
In order to measure the changes in components during kombucha fermentation, the total sugar and reducing sugar contents were measured using modifications of the phenol-sulfuric acid method (Dubois et al., 1956) and the method reported by Miller (1959), respectively. At this time, glucose was used as a standard when measuring total sugar and reduced sugar. In addition, the polyphenol content was measured by modifying the method proposed by Singleton et al. (1999) using gallic acid as a standard. The organic acid analysis was performed using a Phenomenex Luna C18 column (250 × 4.6 mm, 5 μm) using a mixture of potassium dihydrogen phosphate (pH 2.4) and methanol (97:3) as the mobile phase (flow rate 1.0 mL min -1 ) with a UV detector (220 nm). DSL was determined by HPLC analysis using a YMC C18 column (250 × 4.6 mm, 5 μm). Elution was performed using a mobile phase of 3% methanol in 20 mM KH 2 PO 4 (pH 2.4) at a flow rate of 1 mL min -1 , and the elution profile was monitored at 210 nm with a column temperature of 35 °C (Chakravorty et al., 2016).

Assay of radical scavenging activity
The DPPH radical scavenging ability was measured according to the method proposed by Cheng et al. (2006) with some modification. After dissolving the sample in ethanol, 800 μL was taken and mixed with 200 μL of DPPH (0.15 mM) to measure absorbance at 517 nm after 30 minutes. ABTS radical scavenging ability was measured using the method proposed by Re et al. (1999). Manufactured 7 mM ABTS in which 2.45 mM potassium persulfate was dissolved, it was left in the dark for 12-16 hours, and then diluted with distilled water so that the absorbance at 414 nm was 1.4-1.5. After reacting 10 μL of the sample with 200 μL of ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6sulfonic acid, Sigma-Aldrich) radical solution for 60 minutes, absorbance was measured at 414 nm.

Statistical analysis
Data are presented as mean ± standard deviation (SD) and the difference between the groups were analyzed using a Student's t-test, and differences within the groups were analyzed using one-way analyses of variance (ANOVA) with Tukey's post hoc test. p values < 0.05 were considered significant.

Results and discussion
Microbial communities were compared using two types of commercial kombucha (Kom-1 and Kom-2). In kombucha, the major phyla were Proteobacteria (35.60% or 78.1%) and Firmicutes (64.06% or 15.57%). Among the microorganisms belonging to the phylum Proteobacteria, Komagataeibacter hansenii and Gluconobacter oxydans were the main species ( Figure 1). However, in the phylum Firmicutes, there was a difference between major species in kombuchas. Main species of kombucha Kom-1 were Oenococcus oeni (25.60%), Lactobacillus (23.1%), and Sporolactobacillus shoreae (13.04%). Main species of kombucha Kom-2 were Lactobacillus delbrueckii (6.54%), Lactobacillus casei (5.45%), and Ralstonia pickettii (5.81%) (Figure 1). Five bacterial phyla (Actinobacteria, Bacteroidetes, Deinococcus-Thermus, Firmicutes, and Proteobacteria) were reported from five types of kombucha pellicles. Among them, it was reported that Proteobacteria was the main bacterial phylum (Marsh et al., 2014). The most dominant genus in kombucha was Gluconacetobacter, which represented 85% or more bacteria in most kombucha. However, the Acetobacter group was found to make up less than 2%. There was also a kombucha in which up to 30% of the Lactobacillus group, known to be non-dominant strains, was detected in kombucha (Marsh et al., 2014). Traditionally, in kombucha, bacteria such as Acetobacter, Gluconacetobacter, and Lactobacillus are the most common species (Trovatti et al., 2011;Yang et al., 2010). Oenococcus oeni, which is rarely observed and is resistant to acid in kombucha (Figure 1), is known to be the most well-known LAB species in wine and apple cider (Breniaux et al., 2018). Although rarely detected in other environments, it has recently been reported in kombucha (Coton et al., 2017). Sporolactobacillus shorea was found in some fermenting materials such as the tree bark in Thailand with spore-forming LAB, but it was first found in kombucha. It shows a different microbiota from the kombucharelated strains reported so far. Microbiota from different pellicles in Europe and the United States observed the highest levels of LAB in Ireland (Marsh et al., 2014). It reports the difference in microbiota of kombucha by region. It is known that these differences in kombucha community are due to differences in pH, temperature, and fermentation substrate.
The kombucha microbial community consist a cellulose biofilm (kombucha pellicle) and a basic liquid or soup (culture broth), so kombucha was prepared using kombucha pellicle and culture broth. Previous reports have shown that kombucha fermentation may differ depending on the inoculum used in the microbial community (Malbaša et al., 2011). Changes in the microbial community were measured after kombucha fermentation by inoculum with different microbiota. (Figure 2). When comparing the phyla, Kom-P (kombucha fermented by pellicle) accounted for 99.77% of Proteobacteria and 0.18% of Firmicutes, while Kom-F (kombucha fermented by fermentation broth) accounted for 99.24% of Proteobacteria and 0.53% of Firmicutes. Commercial kombucha (Kom-C) used as a starter accounted for 78.18% of Proteobacteria, 15.57% of Firmicutes, and 6.13% of Actinobacteria. The bacterial diversity of kombucha fermented by inoculating culture broth was higher than that of pellicle. Chakravorty et al. (2016) also reported higher bacterial diversity in the broth than biofilm (pellicle).
Gluconobacter oxydans, which was produced using pellicle as a starter, accounted for 95.25% of the main fermentation strain in Kom-P. However, kombucha that used fermented broth as a starter contained Komagataeibacter hansenii as the main fermentation strain, which accounted for 76.69% in Kom-F. Komagataeibacter hansenii, which accounts for 62.88% of commercial Kombucha (Kom-C), was the main fermentation strain. Komagataeibacter hansenii, identified as the main fermentation bacterium in the second half of fermentation, and also known as Gluconacetobacter hansenii, was identified as an acetic acid-producing bacterium, and is known as a producer of highly crystalline cellulose known as bacterial cellulose. In the heatmap, which plots the abundance and correlation between each OTU at the phylum and species level, the species with the highest number of sequence reads differed  between samples, as shown in Figure 2, kombucha fermented with the culture broth had similarity to commercial kombucha at the phylum and species levels. Similarity was found between Kom-F and KOM-C in the Komagataeibacter and Gluconobacter genera, which were identified as major fermentation strains. In kombucha fermentation, it seems to be more appropriate to use kombucha fermentation starter than pellicle alone.
To confirm the difference between kombucha using a pellicle corresponding to the biofilm as a starter and kombucha using a culture broth as a starter, changes in components according to the culture time were measured. Changes in total sugar and reducing sugar content were measured (Figure 3). Changes in total sugar tended to decrease with the increase in fermentation time. Changes in reducing sugar content showed a tendency to increase with fermentation time in Kom-F, while Kom-P slightly increased with fermentation time and then decreased. The total sugar content decreases as fermentation progresses because the fermented strain uses sugar, and because of the tendency for a portion of the reducing sugar to increase as the sugar, which is a non-reducing sugar, is degraded and is converted into a reducing sugar. It is thought that the total sugar content decreases as it is converted to reducing sugar or used by the fermentation strain. Changes in reducing sugar content tended to increase as sugar, a non-reducing sugar, was degraded and converted into reducing sugar. However, the tendency for the levels of reducing sugar to increase and then decrease in Kom-P was the result of reducing sugar, a degradation product of sugar, being used for fermentation in the late fermentation period. Because black tea extract is used as the raw material for kombucha, the initial polyphenol content is high and tends to decrease somewhat by fermentation. In this study, the total polyphenol content was gradually reduced with the fermentation time, which is somewhat different from the reported increase in polyphenol content during fermentation (Jayabalan et al., 2008a, b). The increase in polyphenol content in fermentation occurs because the tea polyphenols containing kombucha seem to be degraded into smaller molecules by enzymes produced by microorganisms involved in the fermentation of kombucha. In particular, in the case of kombucha fermented by Candida sp., such as Candida tropicalis, which is known to be able to degrade complex polyphenols, it is considered that their overall content increases during fermentation because complex polyphenols are enzymatically degraded into small molecules (Ettayebi et al., 2003). However, the kombucha starter used in this study does not seem to have any microorganisms involved in the production of enzymes that can hydrolyze polyphenols into smaller molecules, such as Candida sp.
During kombucha fermentation, acetic acid production tended to increase with increasing fermentation time. The amount of acetic acid produced in Kom-F was higher than that in kombucha fermented with pellicle (Kom-P) as a starter (Figure 4). In addition, the amount of lactic acid increased with the fermentation time in Kom-F, but in the case of Kom-P, lactic acid levels increased until day 7 and then gradually decreased (Figure 4). Kombucha has a sour taste in the early fermentation period, but when the fermentation time increases, the taste changes to a light vinegar-like taste. The main components responsible for this taste are acetic acid and lactic acid, as well as various organic acids (Blanc, 1996). AAB involved in kombucha fermentation use sugar as a carbon source to produce one of the major metabolites, acetic acid. The major AAB involved in kombucha synthesis are Acetobacter, Gluconobacter, and Gluconacetobacter (Komagataeibacter) (Jayabalan et al., 2014). In this experiment, there was a difference in the strains that produce acetic acid due to the difference in starters. The major AAB of Kom-P were Gluconobacter oxydans (95.25%), and the major AAB of Kom-F were Komagataeibacter hansenii (76.69%), Gluconobacter oxydans (13.4%), and Komagataeibacter europaeus (5.73%) (Figure 1). The difference in acetic acid and lactic acid production due to the starter difference seems to be due to the difference in the strains involved in fermentation.
The production of glucuronic acid and DSL is shown in Figure 5. When kombucha fermentation used pellicle as a starter, glucuronic acid production at the 7th day of fermentation showed the highest value at 15.2 mM and then decreased gradually thereafter. However, in Kom-F, glucuronic acid production tended to increase as fermentation time increased. L-Glucuronic acid is another major organic acid found in kombucha metabolites, and production differs depending on the raw material used for preparing kombucha (black tea, green tea, tea manufacture waste) (Jayabalan et al., 2007). In addition to glucuronic acid, the main active compound in kombucha is DSL. The change in DSL production during fermentation tended to increase with increasing fermentation time ( Figure 5). Glucuronic acid and DSL produced during kombucha fermentation contribute to detoxification. In the liver, glucuronic acid combines with toxins to remove them. In addition, DSL inhibits glucuronidase (an enzyme suspected to be the cause of colorectal cancer) in the intestine (Diez & Cabezas, 1979). DSL serves to inhibit the reuptake of toxins by aiding the breakdown of polysaccharides bound by the enzyme (Kim et al., 1995). Among kombucha fermentation strains, Gluconobacter species can use sugar, but Acetobacter and Gluconacetobacter species use glucose and fructose, the yeast-mediated degradation products of sucrose (Mamlouk & Gullo, 2013). Therefore, it appears that glucuronic acid increases in fermentation time in Kom-F, which is a predominant bacterium of the genus Gluconacetobacter (Figure 2). In addition, as the main strain of Kom-P, Gluconobacter (Figure 2), uses sucrose directly, it seems that the glucuronic acid content increases and then decreases in the early stage of fermentation. Gluconacetobacter (Yang et al., 2010) and LAB (Wang et al., 2010) are reported to have a strong DSL production capacity. Therefore, it seems that DSL production in Kom-F with many of these strains is high. It seems that the strains involved in kombucha fermentation are important for glucuronic acid and DSL production. . Changes in acetic acid, lactic acid glucuronic acid and DSL contents during kombucha fermentation with different starters. Kom-P: Kombucha fermented using commercial kombucha (Kom-C) pellicle as a starter; Kom-F: Kombucha fermented using commercial kombucha (Kom-C) fermentation broth as a starter. Different letters show significant differences within the group at p < 0.05. *p < 0.05 indicates a significant difference from other kombucha at the same culture time. Figure 5. Changes in glucuronic acid and DSL contents during kombucha fermentation with different starters. Kom-P: Kombucha fermented using commercial kombucha (Kom-C) pellicle as a starter; Kom-F: Kombucha fermented using commercial kombucha (Kom-C) fermentation broth as a starter. Different letters show significant differences within the group at p < 0.05. *p < 0.05 indicates a significant difference from other kombucha at the same culture time. kombucha fermentation using isolated strains. Therefore, future research should focus on fermentation using strains isolated from kombucha.
The radical scavenging activity of kombucha tea represents its potential as a beverage rich in antioxidants. As a result of measuring changes in ABTS or DPPH radical scavenging activity during kombucha fermentation, the ABTS radical scavenging activity tended to decrease with increasing fermentation time ( Figure 6). However, DPPH radical scavenging activity increased within 7 days of fermentation and then decreased (Kom-P) or remained constant (Kom-F). The antioxidant activity of kombucha has been attributed to polyphenols, ascorbic acid, and DSL. Thus, kombucha has been observed to have higher antioxidant activity than unfermented tea, which is known to be due to the modification of low molecular weight polyphenol components by enzymes produced by bacteria and yeast during fermentation. However, in this study, the amounts of polyphenols produced during fermentation tended to decrease slightly with fermentation time. This seems to be due to the fact that, as explained above, there are very few Candida species actively produce enzymes involved in the degradation of polyphenols. Jayabalan et al. (2008a, b) reported that the total phenolic compound, the scavenging activity for DPPH radicals, the inhibitory activity for superoxide radicals and hydroxyl radical-mediated linoleic acid were increased during kombucha fermentation, but the reducing power, hydroxyl radical scavenging ability (ascorbic acid-iron EDTA), and anti-lipid peroxidation capacity were reduced. The radical scavenging activity is determined by the fermentation time, the type of tea material, and the characteristics of the metabolites present in the kombucha fermentation strain. The difference in the radical scavenging capacity of kombucha is closely related to the microorganisms involved in fermentation. Microbial diversity affects the formation of metabolites and shows differences in radical scavenging capacity (Liu et al., 1996).

Conclusion
Currently, kombucha is produced on a small scale through uncontrolled fermentation. To meet the consumer demand, it is important to ensure the reliability of kombucha fermentation. In addition, it is important to establish the process used for Figure 6. Changes in DPPH and ABTS radical scavenging activity during kombucha fermentation with different starters. Kom-P: Kombucha fermented using commercial kombucha (Kom-C) pellicle as a starter; Kom-F: Kombucha fermented using commercial kombucha (Kom-C) fermentation broth as a starter. Different letters indicate significant differences within the group at p < 0.05. *p < 0.05 indicates a significant difference from other kombucha at the same culture time.