First-Time Identification and Characteristic of Key Bacterial Strains in Kombucha Tea, Including the Newly Discovered Bacillus glycinifermentans

doi:10.21203/rs.3.rs-4997923/v1

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First-Time Identification and Characteristic of Key Bacterial Strains in Kombucha Tea, Including the Newly Discovered Bacillus glycinifermentans

https://doi.org/10.21203/rs.3.rs-4997923/v1

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Kombucha, a fermented tea drink, gained popularity for its probiotic benefits. Understanding its microbial composition, particularly the Symbiotic Culture of Bacteria and Yeast (SCOBY), is crucial for grasping the fermentation process and potential health advantages. We are reporting very first-time identification of Bacillus glycinifermentans new strain in Kombucha tea. The current research study aims to characterize three main bacterial strains part of Kombucha: Bacillus plantarum, Bacillus glycinifermentans, and Gluconacetobacter xylinus. Bacterial strains were isolated by mixing Kombucha tea with black tea. Study identified multiple bacterial strains in Kombucha, with diverse colony characteristics. Biochemical tests were performed and three isolates confirmed as fermentative bacteria, capable of producing acetic acid. ~80% conserved homology was identified among three strains Bacillus plantarum, Bacillus glycinifermentans, and Gluconacetobacter xylinus. Identifying Bacillus plantarum, Bacillus glycinifermentans, and Gluconacetobacter xylinus participates significantly in Kombucha SCOBY's microbial community. Further exploration of these microorganisms' interactions and their fermentation property could improve Kombucha's production and application as a functional food.

Kombucha is fermented tea beverage made from a Symbiotic Culture of Bacteria and Yeast (SCOBY). For decades, this particular combination of microbes has been appreciated for its health advantages, owing mostly to the tea and fermentation metabolites generated by distinct microbial populations (Kaashyap, 2021). The interaction of acetic acid bacteria (AAB), yeast, and lactic acid bacteria (LAB) not only gives kombucha its characteristic flavor and aroma, but it also enhances its potential health benefits (Wang, 2022). Acetic acid bacteria, particularly those of the Acetobacter species, plays a pivotal role in kombucha fermentation. These obligate aerobic Gram-negative bacteria belong to Alphaproteobacterial class, Rhodospirillales order, and Acetobacteraceae family (Qiu and Zhang Y, 2021). They oxidize ethanol to create acetic acid, which is crucial for the tartness of kombucha (Gomes, 2018). Gluconacetobacter xylinus, formerly known as Acetobacter xylinum, renowned for its capacity to manufacture large amounts of bacterial cellulose (BC) from a variety of carbon and nitrogen sources in liquid culture (Liu, 2018). The species under study are known for producing high-purity crystalline cellulose, making it an important member of the kombucha microbial community (Emenike, 2023). Bacillus species also known for their phenotypic characteristics and genetic architecture, making them effective biocontrol agents and plant growth boosters. These bacteria create secondary metabolites that have antibacterial and antifungal characteristics, making them useful in agricultural and biotechnological applications (Karim, 2019). Bacillus glycinifermentans, bacterium isolated from the rhizosphere of Senna occidentalis, remarkable phenotypic, antifungal, and biocontrol properties. This strain closely related to B. sonorensis and B. licheniformis, sharing probiotic and antifungal properties (Afordoanyi, 2023). Lactic acid bacteria depict essential role for the fermentation of numerous foods and drinks. They play an important role in the sensory and safety aspects of these goods (Thakur, 2017). LAB help to preserve fermented foods, such as kombucha, and give them distinct flavor. Lactobacillus plantarum, a common LAB species found in fermented foods and kombucha, is known to produce bacteriocins with antibacterial activity. (Pei and JinJ, 2020). LAB and AAB occur naturally in a range of settings, including fermented foods such as wine, beer, kefir, sauerkraut, kimchi, bread, and cacao beans (Aumiller, 2023). Their symbiotic connection during kombucha fermentation demonstrates the variety and diversity of microbial populations in fermented goods. In this study, three kombucha strains were identified and characterized: Lactobacillus plantarum 6993 (MT464040.1), Bacillus glycinifermentans (KT005408.1), and Gluconacetobacter xylinus JCM7644 (AB645737.1). According to research data available till date, no research has been published on the existence of Bacillus glycinifermentans (KT005408.1), specific strain in kombucha, underscoring the need for more intensive investigation into kombucha's microbial diversity and health benefits.

Preparation of media for bacterial culture:

SCOBY (also called mother kombucha) was purchased from Natures Store Company located in Lahore. Black tea leaves and sucrose were also purchased from local market. The inoculum for bacterial strains was prepared by boiling 100g/L sucrose in distilled water after boiling the sucrose solution was added then transferred to 1000ml glass jar, 10g/L of black tea leaves were added and steeped for 15 minutes. Tea leaves were removed by filtration and temperature of water cooled down to 30°C (Rangaswamy, 2015). Later, SCOBY culture was added and covered the glass jar with paper towel with elastic band around it and placed in dark location with temperature range of 28°C to 30°C for 10 to 15 days. Checked culture media after 15 days the layer of kombucha bacterial cellulose was formed along with kombucha Soares MG, 2021). Biochemical, and morphological characteristics of the isolated strain were determined according to Bergey’s Manual of Determinative Bacteriology (Valla).

Biochemical identifications:

The isolates were stained with gram stain according to Hucker and Conn's (1923) instructions. Bacterial strains were statically cultured in Carr medium to observe the overoxidation of ethanol for three to four days at 30 degrees Celsius. The medium's color changed from violet to yellow, and then back to violet, indicating that the ethanol had overoxidized. (Tesfaw, 2021). The isolated colonies were inoculated on agar plates containing 30 mL of ethanol, 30 g yeast extract, 20 g CaCO3, and 20 g agar per liter in order to monitor the formation of acetic acid from those colonies. The colonies' surrounding calcium carbonate suspension was considered an indication of the formation of acetic acid. (Mathew, 2019). By cultivating the isolated strains in a medium containing 30 g of glycerol, 5 g of yeast extract, and 10 g of polypeptone per liter, the production of dihydroxyacetone (DHA) from glycerol was detected. Each test tube was filled with 200 µL of Fehling's solution following a 3-day incubation period at 30°C under static culture. The emergence of orange hue in the media verified the production of DHA (Yamada et al., 1999). The medium used to investigate the formation of acid from glucose, lactose, fructose, sucrose, galactose, maltose and mannitol, contained 5 g of yeast extract, 10 g of each carbon source, and 1 liter of distilled water with the addition of 0.003% bromocresol purple (pH 6.8). The inoculated agar plates were then incubated at 30°C for a period of seven days to monitor the formation of acid from various sugar sources.

Molecular Identification of Bacterial Strains:

The bacterial isolates were cultured on HS medium at 30°C for two days, and chromosomal DNA was isolated following the technique published by Chandran Masi in 2021 (Masi C, 2021). Bacterial strains' DNA was isolated using the GeneJet Genomic DNA Purification Kit. PCR amplification was used to identify the bacterial strains obtained from Kombucha tea. Only two of the five isolates were selected for 16s rRNA identification. The universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′ GGTTACCTTGTTACGACTT-3′) were used to amplify 16s rRNA. The PCR cycle for the Expin™ Combo GP (GeneAll) was as follows: denaturation at 95°C for 30 seconds, annealing at 52°C for 30 seconds, and elongation at 72°C for 1:30 minutes. Later, the PCR product was purified and run on a 1% agarose gel. The amplified PCR products were delivered to Macrogen Company in Korea for gene sequencing. Later, NCBI Nucleotide Blast was used to conduct phylogenetic analyses.

Biochemical Profiling

Kombucha tea was mixed with black tea and sugar mixture in equal amounts and a variety of isolates were suspected as acetic acid species (yellow and pale-yellow colonies), with only 3 isolates showing clear zones on GYC media. The Carr medium mixed with bromocresol blue (100mg/ml) was used to validate the bacterial production of acid. The same medium was utilized to distinguish acetobacter from other bacteria as the acetobacter changed the medium color to yellow and eventually to green.

Table 1

Showing biochemical chracterization of bacterial strains *Bacillus Plantarum Bacillus glycinifermentans* and *Gluconacetobacter xylinus*
Biochemical Tests	Bacillus Plantarum	Bacillus glycinifermentans	Gluconacetobacter xylinus
Gram-staining	Positive	Positive	Negative
Shape	Rods	Rods	Rods
Motility	Negative	Negative	Negative
H₂Sgas production	Negative	Negative	Negative
Indole test	Negative	Negative	Negative
Methyl red test	Negative	Negative	Negative
Voges-Proskauer	Negative	Negative	Negative
Catalase test	Positive	Positive	Positive

A variety of strains were isolated from the Kombucha tea culture. The colonies vary in size, ranging from small to medium and large. The color of the colonies can be specified as white, off-white, or pale. Different colony shapes, including spherical, raised, convex, spheroid, star-shaped, rough, crinkled, and flat were observed. The isolated colonies can be found in different forms such as rods, single cells, pairs, chains, and clusters. Through biochemical testing, two isolates were identified as fermentative bacteria, which subsequently produced bacterial cellulose film. These isolates were found to be gram-positive, rod-shaped and catalse negative, the results were further confirmed by the ability of isolated bacteria to produce acetic acid through ethanol oxidation, a key characteristic of acetic acid-producing bacteria

Ethanol overoxidation of isolates was performed in CARR-media where the color change from purple to yellow, and then back to purple, indicated that acetic acid was further oxidized to CO₂ and H₂O. Suspension of calcium carbonate around the colonies in GYC media confirmed the acetic acid production by specific ethanol oxidation. Acid production from carbon sources was tested using Fructose, Glucose, Mannitol, and Sucrose, with the change in media color indicating successful fermentation. The strains gave negative results for ketogenesis from glycerol and media color didn’t change to orange after adding Fehling’s solution.

Bacterial RNA Sequencing or Bacterial Strain RNA Profiling

The Macrogen company (South Korea) sequence analysis revealed a divergent strain of Lactobacillus Plantarum 6993 (GenBank accession number MT464040.1) Bacillus glycinifermentans (GenBank accession number KT005408.1) and Gluconacetobacter xylinus JCM7644 (GenBank accession number AB645737.1). Sequence analysis shows more than 80% conserved homology based on 16sRNA analysis (GenBank accession number MT464040.1), (GenBank accession number KT005408.1) and (GenBank accession number AB645737.1). On the edges, there were two restrictions cited suggested by software analysis.

In current research study, isolated microorganisms from kombucha including AAB and (LAB) related to another study on Kombucha a fermented effervescent tea beverage that has a somewhat sweet and acidic taste. The microbial population in Kombucha is complex and fluctuates depending on the fermentation, although it’s mostly made up of AAB and yeast, however, minor numbers of lactic acid bacteria (LAB) have also been documented (Wang, 2022). Kombucha tea a fermented beverage that originated in Asia, most likely in northeastern China. The isolated AAB and LAB from kombucha in our study relates to study in which Kombucha tea is distinguished by a microbial community in which several microbial species, mostly yeasts and bacteria, coexist in a symbiotic relationship. Acetic acid bacteria are the most functional bacteria; however, lactic acid bacteria can also be detected (La China, 2021Our study matched with previously identified gram-negative bacteria Gluconacetobacter xylinus, also known as Acetobacter xylinum, a rod-shaped aerobic bacterium (Lahiri, 2021). Current research study, isolated Gluconacetobacter xylinus from kombucha, bacteria that dominate the kombucha culture are acetic and gluconic acid makers from the Acetobacter and Gluconobacter genera. Acetobacter xylinum was reclassified as Gluconacetobacter xylinus and found to be the most common bacteria in the environment, generating both acetic and gluconic acids (Coelho, R. M. D, 2020). Gluconacetobacter xylinus ZHCJ618, a bacterial cellulose synthesising strain isolated from kombucha, was chosen for commercial applications due to its high phenotypic stability and sustainable production capacity of 7.56 ± 0.57 g/L under static culturing and 8.31 ± 0.79 g/L under shaking conditions (Zhang, 2018). In the research study investigated production of Lactiplantibacillus plantarum from kombucha culture relates to the study which explained the effects of novel food processing on food-derived bioactive components by incorporating lactic acid bacteria (LAB) into kombucha fermentation. LAB integration dramatically reduced total and acetic acid levels, improving kombucha flavour, particularly with the effective strain Lactiplantibacillus plantarum (Wang, 2023).

In our study Lactobacillus Plantarum 6993 was isolated from Kombucha tea as it relates to the study of Lactiplantibacillus plantarum, formerly known as Lactobacillus plantarum, is a well-known and widely used species of lactic acid bacteria (LAB). In our study, bacteria cellulose film was produced from Lactobacillus Plantarum (MT464040.1) and in another study we found the isolation of a novel bacterial cellulose producing strain from rotten fruit, identified as Lactobacillus plantarum through 16S rRNA sequencing, is an interesting discovery. L. plantarum is commercially employed as a starting culture for various food fermentations as well as a probiotic culture. L. plantarum strains have been found to exhibit a wide range of functional qualities in the food business. We also identified the Lactobacillus Plantarum (MT464040.1) strain from kombucha, a probiotic culture. In this work, researchers looked at the features of KBC generated during green tea kombucha fermentation on days 7, 14, and 30, as well as its potential as a protective carrier of Lactobacillus plantarum, a beneficial bacterium. In our study, Lactobacillus plantarum was produced from fermentation of kombucha (Charoenrak, 2023). The unique strain of Bacillus glycinifermentans MGMM1, which was isolated from the rhizosphere of a weed plant, was examined in this study for its phenotypic traits, antifungal properties, and biocontrol potential (Afordoany DM, 2023). Bacillus licheniformis is a gram positive, rod shaped and anaerobic facultative bacteria first isolated from fermentation of Soyabean paste. Since its identification through 16S rRNA sequencing, Bacillus glycinifermentans has demonstrated a close kinship to B. sonorensis and B. licheniformis, sharing probiotic and antifungal characteristics among other traits (Zeigler, DR, 2016). Bacillus spp. have been employed as biocontrol agents against soilborne diseases due to the production of secondary metabolites with antibacterial or antifungal activities. The genome of a new strain of Bacillus glycinifermentans sp. (JRCGR-1) was sequenced and annotated in this work. The genome was searched for possible antibacterial activity genes (Karim A, 2019). In our study we isolated facultative bacterium Bacillus glycinifermentans (KT005408.1) from kombucha. No previous data is available for production of bacterial cellulose film from Bacillus glycinifermentans (KT005408.1) and strain isolation from Kombucha tea.

Kombucha's distinct microbial population, includes a combination of AAB, LAB, and other helpful bacteria, highlights its status as a remarkable fermented beverage. The interactions of these microbes not only helpful to produce kombucha characteristic flavor and aroma, but also contribute for significant health benefits. The discovery of novel strains in kombucha gives us new insights for study and application in food science and biotechnology to make the helpful modifications in human life my extensive study of Kombucha family responsible to effectively evolve multiple factors.

Author Contribution

A. Maryam Iqbal: Made significant contributions to the data analysis, work design, idea and wrote the article. B. Fatima Ali: Approved the published version and accepted responsibility for all parts of the work.C. Chou Yi Hsu: co-conceived the study.D. Ayesha Shaukat: Carried out significant intellectual substance revisions.E. Aqsa Shamim: The interpretation of reference data.

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No competing interests reported.

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You are reading this latest preprint version

First-Time Identification and Characteristic of Key Bacterial Strains in Kombucha Tea, Including the Newly Discovered Bacillus glycinifermentans

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Version 1

Abstract

Figures

Introduction

Material and Methods

Preparation of media for bacterial culture:

Biochemical identifications:

Molecular Identification of Bacterial Strains:

Results

Biochemical Profiling

Bacterial RNA Sequencing or Bacterial Strain RNA Profiling

Discussion

Conclusion

Declarations

Author Contribution

References

Additional Declarations

Status:

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