Kombucha is a fermented beverage produced by the symbiotic activity of acetic acid bacteria and yeasts, known as the symbiotic culture of bacteria and yeast (SCOBY). It contains diverse metabolites such as organic acids, polyphenols, flavonoids and vitamins that contribute to its antioxidant and antimicrobial properties (Leal et al., 2018; Antolak et al., 2021). Despite its well-documented health benefits, the liquid form of kombucha poses limitations in stability due to continued microbial activity and degradation of bioactive compounds during storage (Kaashyap et al., 2021).

To overcome these limitations, converting kombucha into powder form via freeze drying is an effective alternative. The freeze-drying process removes water under low temperature and pressure, maintaining structural integrity, preserving bioactive stability and supporting microbial viability in shelf-stable functional products (Kumar and Mishra, 2023; Mahalakshmi et al., 2025). The use of filler or carrier materials during freeze drying is crucial for improving powder quality and preventing degradation of sensitive components. Maltodextrin is widely used for this purpose due to its low cost and excellent drying behavior; however, when used alone, it may lead to brittle particles and low solubility (Santivarangkna et al., 2008).

Natural fillers such as psyllium husk and pectin offer additional benefits beyond structural protection. Psyllium husk is a hydrophilic polysaccharide capable of forming a gel matrix that retains moisture and protects bioactive molecules (Bhat et al., 2022). Its potential as a functional ingredient has also been reported in probiotic frozen yogurt, where psyllium husk contributed to hydration capacity, water uptake and physicochemical properties of the fermented product (Katke and Deshpande, 2022). Pectin, on the other hand, serves as a stabilizer and film-forming agent that enhances encapsulation and improves the physicochemical stability of powders (Wang et al., 2020). In food and beverage systems, pectin is widely recognized as a gelling agent, thickener, emulsifier and stabilizer, with functional properties such as solubility, swelling capacity, water absorption and bulk density influencing its application in product formulation (Naveena et al., 2024).

Moreover, extending kombucha production to include rhizome-based substrates-such as turmeric-tamarind (Curcuma longa and Tamarindus indica), aromatic ginger (Kaempferia galanga) and white turmeric (Curcuma zedoaria)-has attracted attention for their rich content of curcuminoids, essential oils and phenolics, which exhibit strong antioxidant and anti-inflammatory potential (Amalraj et al., 2016; Urbanova et al., 2024). Fermentation of these substrates may enhance the release and bioavailability of these compounds, producing beverages with higher functional potential (Kim et al., 2023).

Developing instant kombucha from tea and rhizome substrates through freeze drying can thus improve both the stability and functional retention of bioactives. The incorporation of psyllium husk and pectin not only protects phenolic compounds but also introduces dietary fiber with prebiotic potential, potentially yielding a synbiotic beverage. Therefore, this study aimed to evaluate the effects of filler variations on the physicochemical and functional characteristics of freeze-dried instant kombucha and determine the most effective formulation for producing a stable, bioactive-rich product.

The study was conducted from May to September 2024 at the Laboratory of Food Science and Biotechnology, Brawijaya University, Malang, Indonesia. Jasmine tea (Tong Tji) was purchased from a local supermarket in Malang and used as the substrate for tea-based kombucha. Psyllium husk and pectin were obtained from Brataco Chemical, Indonesia, while maltodextrin (DE 10-12) was obtained from PT. Zhaveria, Indonesia. The kombucha starter (SCOBY) was obtained from the Healthy Secret Store, Malang. Rhizome ingredients-turmeric (Curcuma longa), tamarind (Tamarindus indica), galangal (Kaempferia galanga) and white turmeric (Curcuma zedoaria)-were obtained from a local herbal supplier in Malang. All reagents used in the analyses were of analytical grade.
 
Stage I- Instant tea kombucha
 
Tea kombucha was prepared by fermenting jasmine tea (Camellia sinensis) in a sucrose solution (10% w/v) inoculated with 10% (v/v) kombucha starter (SCOBY). The fermentation process was carried out for seven days at room temperature (28±2°C) under aerobic conditions. After fermentation, the liquid kombucha was filtered and subjected to freeze drying using a laboratory freeze dryer (-40°C, 0.02 mbar). Filler variations consisted of psyllium husk at concentrations of 2%, 2.5% and 3% (w/v) and pectin at 0.4%, 0.6% and 0.8% (w/v), each combined with 10% maltodextrin as the carrier agent. The mixture was frozen prior to freeze drying to produce instant kombucha powder.

The obtained powders were analyzed for physicochemical and functional parameters, including total acidity (titration method; Winandari et al., 2022), total phenolic content (Folin-Ciocalteu method; Mihai et al., 2024), antioxidant activity (IC₅₀) using the DPPH method (Molyneux, 2004), solubility time and viscosity measured using a brookfield viscometer. Lactic acid bacteria (LAB) were enumerated by the total plate count method on MRS agar (Süle et al.,  2014).

Data were analyzed using one-way analysis of variance (ANOVA) and mean comparisons were conducted using fisher’s least significant difference (LSD) test at a 95% confidence level (p<0.05). Statistical analysis was performed using Microsoft Excel 2013 and Minitab version 19.
 
Stage II- Instant rhizome kombucha
 
The optimal formulation obtained from Stage I was applied to rhizome-based kombucha made from turmeric-tamarind, galangal and white turmeric. Each rhizome was washed, sliced (1-3 mm thickness), dried in a cabinet dryer at 60°C for 6 hours and ground into fine powder. The dried rhizomes were placed in tea bags at a concentration of 0.8% (b/v), combined with jasmine tea and infused in 500 mL mineral water. During boiling for 3 minutes, sucrose (10% w/v) was added to the mixture to prepare the sweetened infusion. After cooling, the infusion was inoculated with 10% (v/v) kombucha starter, followed by fermentation for seven days at room temperature. The fermented liquid was then freeze dried using the same conditions as in Stage I.

The resulting powders were analyzed for total acidity (Winandari et al., 2022), total phenolic content (Mihai et al., 2024), total flavonoid content (Yeti and Yuniarti, 2021), antioxidant activity (IC₅₀) (Molyneux, 2004), solubility, viscosity and total LAB count (Süle et al.,  2014). Data were processed using one-way ANOVA and significant differences among treatments were determined using fisher’s LSD test at p<0.05 with Excel 2013 and Minitab 19.

Effect of filler variation on instant tea kombucha
 
The freeze-drying process effectively converted liquid kombucha into a stable powder with low moisture content and water activity, demonstrating efficient dehydration and high product stability. Among the tested filler variations, both type and concentration significantly influenced microbial viability, acidity and bioactive compound retention of the resulting instant tea kombucha powder (Table 1). Samples containing psyllium husk showed higher counts of lactic acid bacteria (LAB) than those with pectin, suggesting better microbial protection during drying. The hydrogel-like polysaccharide matrix of psyllium likely reduced dehydration stress by maintaining a micro-moisture barrier around bacterial cells, a mechanism also observed in freeze-dried lactic fermentates by Ossowski et al., (2025), who reported enhanced microbial survival and antioxidant retention in fermented peach matrices. Similarly, Barajas-Álvarez and González-Ávila (2023) demonstrated that polysaccharide-based encapsulation matrices improve probiotic viability under storage and gastrointestinal stress.



Phenolic retention and antioxidant activity were also significantly affected by filler composition. The 2% psyllium husk treatment yielded the highest total phenolic content (182.46 mg GAE/g) and the lowest IC₅₀ value (52.14 ppm), indicating stronger antioxidant capacity compared to other formulations. These findings align with Kochubei et al., (2025), who showed that microencapsulation using polysaccharide carriers effectively preserves polyphenols and green tea catechins during dehydration. The improved retention of phenolic compounds in the psyllium-treated samples can be attributed to the gel-forming ability of psyllium mucilage, which restricts oxidative degradation of sensitive phytochemicals. In contrast, pectin-based powders-4although structurally stable-exhibited higher viscosity and lower solubility, which could limit microbial activity and bioavailability. This is consistent with the results of Panou and Karabagias (2025), who reported that excessive cross-linking in pectin matrices reduces solute diffusion and phenolic release.

The physicochemical characteristics also supported this functional performance. Powders with 2% psyllium exhibited optimal viscosity (187.67 cP) and rapid dissolution (1.86 min), while maintaining balanced acidity (0.21%) and viable LAB counts (1.45 x 10³ CFU/mL). These properties are crucial for consumer acceptability and functional efficacy of instant beverages. Similar rheological advantages were highlighted in Vajdovich et al., (2025), where controlled hydration of pseudocereal-based beverages improved solubility without compromising stability. Taken together, the combination of freeze drying and psyllium incorporation effectively enhanced powder quality, maintaining both microbial viability and antioxidant integrity. Therefore, psyllium husk at 2% concentration was identified as the optimal filler formulation for further application in rhizome-based kombucha products.
 
Characteristics of rhizome-based instant kombucha
 
Application of the optimized 2% psyllium husk formulation to rhizome-based kombucha (turmeric-tamarind, aromatic ginger and white turmeric) revealed significant effects of substrate type on physicochemical, microbiological and functional characteristics depending on the substrate type (Table 2). The turmeric–tamarind kombucha exhibited the highest total phenolic content (185.73 mg GAE/g) and flavonoid concentration (89.41 mg QE/g), consistent with its rich curcuminoid composition. This sample also demonstrated the strongest antioxidant activity (IC50 = 48.32 ppm), indicating high retention of bioactive compounds. These observations are in agreement with Rojas (2024), who found that coffee pulp kombucha retained stable phenolic antioxidants after fermentation and freeze drying. Similarly, Ossowski et al., (2025) reported that the freeze-drying of fermented matrices preserves enzymatic antioxidants and phenolics more effectively than spray drying.



Among all rhizomes, the aromatic ginger kombucha achieved the highest sensory scores due to its distinctive aroma and moderate acidity (0.25-0.28%), whereas white turmeric exhibited greater viscosity (227.33 cP) and slightly higher LAB counts (3.06 x 10³ CFU/mL). The psyllium husk filler contributed to these outcomes by forming a viscoelastic network that limited oxidative degradation and moisture migration during drying. According to Panou and Karabagias (2025), interactions between pectin or soluble fibers and polyphenols can strengthen antioxidant systems through hydrogen bonding and microencapsulation effects. Psyllium, in particular, can act as a prebiotic carrier matrix, sustaining LAB viability and promoting the development of a synbiotic powder beverage-a functional synergy also emphasized in Vajdovich et al., (2025).

The overall findings suggest that the combination of fermentation, natural fillers and freeze-drying technology effectively produces an instant kombucha powder with retained bioactive compounds, viable microorganisms and favorable reconstitution properties. The integration of psyllium husk not only enhances the physicochemical and microbial stability of the product but also introduces dietary fiber with prebiotic functionality, contributing to the formulation of a stable, antioxidant-rich and synbiotic beverage. This aligns with the broader trend in functional beverage innovation toward the use of dietary fiber-based encapsulants for dual functionality-structural protection and gut health enhancement (Barajas-Álvarez and González-Ávila, 2023).

Future studies should explore the long-term storage stability of bioactive compounds and probiotic viability in instant kombucha powders. Expanding research toward in vivo models will be crucial for verifying antioxidant and metabolic health effects. Further optimization of freeze-drying parameters could enhance energy efficiency and scalability for industrial production. Additionally, exploring other prebiotic fibers and natural fillers could broaden applications to diverse fermented beverages, supporting innovation in functional food and synbiotic product development.

Freeze-dried instant kombucha formulated with psyllium husk as a natural filler demonstrated superior physicochemical stability, microbial viability and bioactive retention compared with pectin-based formulations. Psyllium husk at 2% concentration produced the best-quality powder with high phenolic content, strong antioxidant activity and good solubility, while maintaining viable lactic acid bacteria. When applied to rhizome-based kombucha, the turmeric-tamarind formulation exhibited the highest phenolic and antioxidant potential, whereas aromatic ginger provided the most favorable sensory profile. The integration of psyllium husk not only stabilizes bioactive compounds but also contributes dietary fiber, enhancing the product’s functionality as a potential synbiotic beverage.

This research was funded by the PNBP program of the Faculty of Agricultural Technology, Brawijaya University.

The authors declare no potential conflicts of interest.