Sensory Assessment, Nutritional and Antibacterial Effect of Kombucha Incorporated with Citrus grandis (L.) Extract

Kombucha is a fermented tea beverage made from a symbiotic culture of bacteria and yeast known as SCOBY and it has a high concentration of bio active chemicals generated from plant material (tea, juices, herb extracts) as well as microbial metabolic activity (Kapp and Summer 2019; Antolak et al., 2021). The tea fungus originated in East Asia and was introduced to Europe from eastern Siberia, also known by other names, such as Chinese and Japanese mushroom. During the fermentation process of kombucha, several metabolites are created, which are responsible for its beneficial effects (Jakubczyk et al., 2020).  It is a symbiotic culture of bacteria and yeast that ferments regular tea for around two weeks to generate a high-end fruity workout beverage. In addition to being a low-sugar, pro-biotic-rich drink, kombucha provides the advantages of the tea used in its preparation, including its high antioxidant content (Hsieh et al., 2021). After fermentation, kombucha contains sugars, tea polyphenols, organic acids, ethanol, amino acids, vitamins (C and B), minerals and hydrolytic enzymes (Kapp and Summer, 2019; Laavanya et al., 2021).

Citrus grandis (L.) or pomelo, is an underutilized fruit in Assam with significant potential for enhancing livelihoods and nutritional security (Sharma et al., 2004). Despite its nutritional and economic benefits, pomelo is rarely cultivated. Studies emphasize its potential to improve food security and offer economic advantages (Barua et al., 2019; Kumar et al., 2019). Pomelo’s antioxidant properties make it suitable for health supplements and functional foods. The increasing health consciousness among people has significantly altered consumption habits, leading to a surge in demand for fruit-based beverages in the market. (Pavithra and Mini, 2023). Blending of seasonal fruits and flowers in the form of food products can be a cost-effective way to fulfill everyday nutritional requirements (Singh and Sharma, 2019 and Sahrawat and Chaturvedi, 2022).

It is evident that Kombucha tea beverages will be a good alternative to carbonated beverages due to their therapeutic and health benefits. The product can easily replace many expensive and artificial antbacterial additives used in the food industry. It can also be an alternative to the use of artificial or synthetic additives in food production because in recent years this is becoming a major issue and problem for the concern for the consumers. Therefore, the present research was taken up to investigate the sensory attributes, nutritional composition, total phenol and flavonoid content, antioxidant and antibacterial activity and vitamin C content of kombucha incorporated with Citrus grandis (L.) extract.

Preparation of kombucha
 
In the present study, raw materials for the preparation of kombucha tea were collected in January 2024. Tea leaves, sugar and fresh Citrus grandis fruit were procured from the local market of Guwahati and SCOBY (symbiotic culture of bacteria and yeast), which was ordered from Peepal Farm Products online. The whole experiment was conducted within a period of 5 months. The value-added kombucha was prepared using a previously described method with slight modification (Zubaidah et al., 2018 and Soto et al., 2019). Plain kombucha was prepared in 1000 ml of water which was boiled at 100^oC for 5-6 min. Sugar (100 g) and 3 teaspoon of tea leaves (three teaspoons) were added to water. After preparation of the tea, it was maintained at room temperature. The prepared tea was poured into a glass jar and SCOBY was added to the starter tea was mixed together. It was further fermented for 2 weeks at room temperature. For the Citrus grandis juice, 700 ml of fruit juice was extracted from approximately four medium-sized grapefruits. Sugar (300 g) was added to the juice to turn it into concentrated syrup and refrigerated in the jar until further use. After fermentation of plain kombucha for 2 weeks, SCOBY was removed and plain kombucha and Citrus grandis (L) were infused together and refrigerated for 1 day. 

Formulation of kombucha treatments
 
A total of five formulations were developed in different variations, with a control formulation coded as C, containing 100% Raw or Plain Kombucha. The ratio of plain kombucha and Citrus grandis (L) extract for Treatment 1 (T1) was 80:20, for Treatment 2 (T2) was 70:30, for Treatment 3 (T3) was 60:40 and for Treatment 4 (T4) was 50:50 (Table 1). After mixing with fruit extract, the treatments were left to ferment for one more day so that both the flavors mixed well together and then served for sensory evaluation (Fig 1).
 






Sensory evaluation of the formulated Citrus grandis (L) kombucha
 
In the present study, a score card was made consisting of a table utilizing the hedonic ratings on 9 points scale from like extremely to dislike extremely. Sensory evaluation of the kombucha was performed by 35 semi- trained panelists.  The most sensory-acceptable treatment of Citrus grandis (L) kombucha was chosen for nutritional analysis and shelf-life investigations at ambient and refrigerated temperatures.
 
Determination of physical parameters
 
Physical parameters, such as total soluble solids (TSS) and pH, were determined using a hand refractometer (ERMA) and digital pH meter (SYSTRONICS). To determine the pH of the developed product, a pH meter was used and the reducing sugar content was determined by the Lane-Eynon Titration Method (AOAC method) using standard procedures (Sewwandi, 2020).
 
Determination of nutrient content
 
Proximate analysis for total fat, protein and carbohydrate content was performed using AOAC (2005) methods. Mineral content (iron, manganese, copper, etc.) was determined by Atomic Absorption Spectrophotometer (SpectrAA 220, Varian, USA).Antioxidant activity was assessed using the DPPH method, calculating % inhibition with the formula:
 

 

                  
IC50 values were derived from a sigmoid curve using nonlinear regression.

Total phenolic content (TPC) was determined via the Folin-Ciocalteu method at 765 nm, while total flavonoid content was measured colorimetrically at 510 nm.  Vitamin C was analyzed through HPLC (Agilent 1220 Infinity II) using a C18 column and a methanol-water mobile phase (30:70), with readings taken at 280 nm after sample filtration. This approach ensures accurate nutrient and bioactive compound analysis.
 
Antibacterial activity of the developed sample against Escherichia coli
 
Antibacterial activity was assessed using the broth microdilution method (Eloff, 1998) to determine the Minimum Inhibition Concentration (MIC). Overnight bacterial cultures were adjusted according to McFarland standards. Kombucha extracts were dissolved in acetone (10 mg/ml), with 100 μl added to the first well of a 96-well plate and serially diluted with water. E. coli dilutions were prepared and transferred to the plate. Antibiotics were added to the 11^th row, while the 12^th row served as a control.  Azithromycin was used as a positive control and acetone was used as a solvent control. After it was kept in an incubation at 37^oC for 24 hours, MIC was visually determined as the lowest concentration inhibiting bacterial growth. The shelf life of the product was studied and was done for 7 days by checking the microbial count in serial dilution method of the developed sample. Nutrient agar was used as a media to check the growth of the microorganisms.
 
Statistical analysis
 
All sensory evaluation data were statistically analyzed in triplicate. According to descriptive statistics, means were evaluated and are represented as mean±SD.

Sensory evaluation of the developed kombucha
 
After sensory or organoleptic evaluation of the developed product, all the data were compared and analyzed using the Mean Acceptability Scores (Table 2). Among all five formulations, the most selected sample was T4, which contained equal amounts of Plain Kombucha (50%) and Citrus grandis (L) (50%) extract. 


 
Physical parameters
 
As shown in Table 3, there was no significant change in the total soluble Solid from 1^st day of fermentation (8.18) to 10^th day of fermentation (8.21). The Reducing sugar (Sugar as Sucrose) for the developed Citrus grandis (L) Kombucha was found to be 4.20 g /100 g on 1^st day and 4.34 g / 100 g on 7^th day. The pH of the developed Citrus grandis (L) kombucha was approximately 5.59, which was quite acidic on 10^th day with a pH value of 3.35. The change in total soluble solids and reducing sugars in kombucha has been observed in similar studies (Wispen et al., 2021). The pH of kombucha typically starts from 4.9 to 5.5 for black and green teas. As fermentation progressed, the pH dropped rapidly, reaching below 4.6 early on to prevent microbial contamination. By the end of primary fermentation, the pH usually falls between 2.5 to 3.5 for all fruit juice (Kitwetcharoen et al., 2023).


 
Determination of nutrient composition
 
From Table 4 it was observed that the developed Citrus grandis (L) Kombucha had a fat content of 2.25 g /100 g, total protein content 0.04 g /100 g and the total carbohydrate 4.7 g /100 g. The iron, manganese and zinc contents of the developed product were 4.609 mg, 2.320 mg and 0.245 mg, respectively. The vitamin c content of the sample was 0.1049 µg /ml. DPPH scavenging activity was observed at 120 ug / ml in compared with that at different concentrations (74.33 %) (Fig 3). The IC 50 value of the developed sample was found to be 42.16 mg vit C/ml. The total phenol content and total flavonoid content assays recorded value of 83.40 mg/ml and 262.24 mg/ml respectively.





Present research is in consonance with a similar study in which researchers found significant levels of minerals such as calcium (1.5 to 4.0 mg), magnesium (1.0 to 2.5 mg), potassium (10 to 30 mg) and phosphorus (0.5 to 1.5 mg) per 100 Ml in different kombucha samples (Villarreal Soto et al., 2018; Suhre et al., 2021 and Içen et al.,  2023). Studies have reported that the vitamin C content in kombucha can increase significantly during fermentation, especially when enriched with ingredients such as jujube, where the vitamin C content could reach up to 0.00711 µg/100 mL (La Torre et al., 2024). The antioxidant data aligns with those of other studies where the researchers revealed that the content of antioxidant compounds ranged between 70.62% and 94.61% DPPH scavenging activity for their kombucha samples (Jakubczyk et al., 2020). The total phenol content (412.25 mg/l) was recorded for bamboo leaf kombucha fermented with leaf residues as recoded by Xiong et al., (2023). which is higher than the value recorded in the present study. This variation may be due to the effect of temperature and the processing method adopted.
 
Antibacterial activity of the developed sample against Escherichia coli
 
From the Fig 4 it was observed that the developed kombucha showed activity against the selected pathogen i.e E coli in dose dependent manner which can be compared with standard antibiotic i.e Azithromycin. The lowest concentration, that is, less than 50%, also showed the minimum inhibitory concentration. Previous researchers have also reported strong antibacterial activity of kombucha against certain pathogens, such as E. coli and Staphylococcus aureus in their previous studies (Al-Mohammadi et al., 2021).



From the microbial plate count method, it showed that after fermentation, the developed kombucha could be stored at room temperature for 7 days after which microbial growth was observed. In similar study for soursop kombucha researchers observed a decreased microbial growth after storing the kombucha at 4^oC. It is also observed that prolonged storage in appropriate temperature may have high potential to improve the quality, metabolites content and biological activity of kombucha beverage  (Tan et al., 2020).

The present study was carried out to develop an underutilized fruit, Citrus grandis (L) of Assam, to make people aware of the nutritional and health benefits it possesses and to make this fruit available in their diet as well as in the market by transforming it into various forms of appealing food products. Selected and pre-processed fruit extracts of Citrus grandis (L) were used to develop a fermented drink known as kombucha. Therefore, this study aimed to develop low-cost, easily available nutritious food products incorporating Citrus grandis (L) that can be utilized to determine their organoleptic quality, bioactive properties and different methods and processes of value addition further processed to higher commercial levels.  The popularity of kombucha is increasing because of its beneficial effects on human health, where the beverage is known to be rich in bioactive components that can be digested, metabolized and absorbed by the body. Also, it was thought that the use of different berry fruits as a raw material in the production of Kombucha tea will be more preferred by consumers in terms of flavor, taste and functional properties.

The authors acknowledge Assam down town University, Guwahati, Assam for laboratory infrastructure facilities.
 
Author’s contribution
 
All authors significantly and directly contributed to data collection, data analysis, data presentation and discussion. The authors are responsible for the content and writing of this manuscript.
 
Disclaimers

The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Ethics statement
 
Not applicable.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.