Nashi Pear (Pyrus pyrifolia) Pomace as a Source of Sucrose and Functional Ingredients for Kombucha Fermentation

Abstract

Kombucha, a fermented tea beverage celebrated for its unique flavor and health-promoting properties, is traditionally produced from sugared tea and a symbiotic culture of bacteria and yeast (SCOBY). In this study, Nashi pear (Pyrus pyrifolia) pomace, a nutrient-rich by-product of juice processing, was explored as a novel substrate for kombucha production, combining sustainability with functional innovation. Beverages were prepared using black tea or pear pomace with varying sugar concentrations (3%, 5%, 7% w/v) and fermented for six days at 22 °C. Physicochemical parameters, bioactive compounds, antioxidant activity, color, and microbial populations were systematically analyzed. Pomace-based kombucha exhibited higher initial pH (4.3–4.7) and higher initial titratable acidity compared to tea-based variants (pH 3.4–3.6). These values stabilized at 3.6–3.8 by the end of fermentation, ensuring safety while preserving bioactive stability. While tea kombucha had higher polyphenol content (943.81–967.74 mg GAE/100 mL) and antioxidant activity (52.22–99.87% DPPH scavenging), pear pomace kombucha offered moderate bioactivity (up to 435.13 mg GAE/100 mL and 33.52% DPPH scavenging) and distinctive color (significantly higher b* value reaching 42.7), along with robust microbial growth. The results demonstrate that Nashi pear pomace can serve as a functional, eco-friendly alternative substrate, transforming fruit processing waste into a value-added beverage with enhanced health-promoting properties. This approach highlights a sustainable pathway for circular economy practices in food production and introduces a promising direction for innovative kombucha formulations.

1. Introduction

Kombucha is a fermented beverage distinguished by its sweet-and-sour flavor, often accompanied by noticeable astringent and vinegar-like notes. It is produced through the fermentation of sugared tea extract by a symbiotic culture of bacteria and yeast (SCOBY), which metabolizes sugars into organic acids, ethanol, carbon dioxide, and various bioactive compounds. This fermentation process not only defines the sensory characteristics of kombucha but also contributes to its reported health-promoting properties [1].

Historically, kombucha is believed to have originated in China, where it was revered for its health benefits and earned the epithet “Tea of Immortality.” In vitro studies have since demonstrated a range of biological activities, including antioxidant, immunomodulatory, antihypertensive, hypolipidemic, hypoglycemic, antiproliferative, and antimicrobial effects [2,3,4]. Many of these benefits are thought to arise from the influence of kombucha on the gut microbiota, mediated by the presence of probiotic strains [5]. Over the last decade, studies and analyses of kombucha have increasingly focused on its potential beyond conventional health benefits, highlighting emerging applications in medicinal and pharmaceutical contexts [5,6,7,8,9].

The preparation of kombucha involves combining tea extract with 5–8% sugar and the SCOBY, which consists of yeasts, lactic acid bacteria, and acetic acid bacteria. The microbial composition can vary depending on fermentation conditions. Acetic acid bacteria such as Acetobacter aceti and Gluconobacter oxydans primarily produce the acidic taste, while Komagataeibacter rhaeticus forms the characteristic cellulose biofilm [8,10]. Lactic acid bacteria modulate the flavor by reducing overall acidity, with studies showing that the addition of Lactiplantibacillus plantarum decreases both acetic acid and total acid concentrations [11]. The symbiotic interaction of microorganisms involves the hydrolysis of sucrose present in the beverage by yeasts into glucose and fructose, with ethanol produced as a by-product. Subsequently, acetic acid bacteria convert these metabolites into organic acids, such as acetic acid and gluconic acid. The concentrations of these acids determine the characteristic flavor profile of the beverage [12].

Traditionally, kombucha is prepared using Camellia sinensis leaves, most commonly as black or green tea. However, over the years, a wide range of alternative plant substrates has been explored to diversify flavor, nutritional profile, and bioactive content. These include herbs such as peppermint, linden blossom, rosemary, and ginger, as well as vegetables and fruits like spinach, grape juice, cherry juice, banana peel, and pomegranate juice [1]. Modifying the sugar source can further influence the chemical and functional properties of the beverage. For example, replacing sucrose with molasses increased polyphenol content, while coconut palm sugar enhanced radical scavenging activity [13]. Such innovations not only diversify the sensory characteristics of the beverage but also create opportunities to enhance its functional properties.

Apples (Malus domestica) are widely consumed fresh and processed into juice, purées, concentrates, and alcoholic beverages. Apples are primarily composed of water (approx. 85%), carbohydrates with dietary fiber (12–14%), and, among fruits, are a rich source of polyphenols, pectin, and organic acids. Key bioactive compounds include quercetin, catechin, chlorogenic acid, and phloridzinare [14]. Similarly, pears are composed mainly of water (80–85%), sugars, particularly fructose (approx. 15%), and fiber (2%). Composition can vary between cultivars: Korean (Asian or Nashi) pears have higher water, sugar, and potassium content, while Western pears (e.g., Bartlett, P. communis) have higher fiber and calcium levels. Bioactive compounds in pears include polyphenols (phenolic acids, flavonoids) in the range from 2.0 to 5.1% in fresh fruit [15], triterpenes, and glucosides, with the highest concentrations found in leaves, seeds, and peels, followed by pulp [16].

In the production of apple-based products, substantial post-production waste is generated in the form of apple pomace, which retains high concentrations of bioactive compounds, including phenolic acids, flavonoids, and anthocyanins [17,18,19]. A similar composition can be expected in the processing residues of pears. Specifically, arbutin—a key bioactive glucoside in the Pyrus genus—is selectively concentrated in the peel, with concentrations reported to be 10 to 45 times higher than those in the fruit pulp. This makes pear pomace a potent reservoir of functional ingredients that can be valorized through fermentation [20]. Across the European Union, more than 11 million tons of apples were produced in the 2024–2025 campaign, resulting in approximately 2.2–2.75 million tons of waste [17]. This large volume of by-products provides a significant opportunity for their valorization in more beneficial ways.

When used as substrates for kombucha, Nashi pear pomaces and other alternative raw materials can directly influence the bioactive profile and functional properties of the final beverage, enhancing antioxidant, anti-inflammatory, and other health-promoting effects. Apple and pear pomace, in particular, provide a rich mixture of polyphenols, flavonoids, and other bioactive compounds that contribute to these benefits. Asian pear cultivars, particularly Pyrus pyrifolia, are recognized for their high capacity to accumulate sucrose during fruit maturation, a process driven by the increased activity of sucrose synthase (SS) and sucrose-phosphate synthase (SPS). Unlike some other pear species, the maturation of Nashi pears involves a significant accumulation of sucrose, partially due to the physiological decrease in acid invertase activity in the mature pericarp. When used as a substrate for kombucha, the pomace from these fruits provides a naturally concentrated source of sucrose along with endogenous enzymes, which may offer a more complex and nutrient-rich carbon source for the SCOBY compared to refined sugar media [21]. The replacement of traditional ingredients with such alternative substrates represents a strategic approach to the valorization of post-production food waste. From an environmental perspective, this practice directly aligns with circular economy principles by diverting organic residues from landfills, thereby reducing greenhouse gas emissions associated with waste decomposition. Economically, utilizing pear pomace as a nutrient source can significantly lower production costs by reducing the reliance on refined sugars while simultaneously eliminating the expenses related to waste disposal. Furthermore, this integration of by-products into the production cycle addresses the growing consumer demand for sustainable and eco-friendly food manufacturing processes.

Both apples (Malus domestica) and pears (Pyrus sp.) generate substantial post-production waste; in the European Union alone, apple processing resulted in approximately 2.2–2.75 million tons of pomace in the 2024–2025 campaign [17]. While apple residues are a well-documented substrate in food science, a significant research gap exists regarding the systematic valorization of Nashi pear (Pyrus pyrifolia) pomace. Due to their botanical kinship within the Rosaceae family, Nashi residues share a high concentration of bioactive compounds with apples, such as phenolic acids and flavonoids, but offer a distinct advantage: a naturally high sucrose content and endogenous enzymes driven by specific maturation physiology [21]. Unlike existing studies that typically treat fruit pomace as a mere flavoring additive, this work explores its potential as a primary, standardized carbon source for kombucha fermentation. This approach directly aligns with circular economy principles by transforming industrial residues into a high-value functional resource, thereby reducing both the environmental impact of disposal and the economic costs of exogenous sugar supplementation.

The aim of this study was to evaluate the potential of Nashi pear (Pyrus pyrifolia) pomace as a novel raw material for kombucha production and to assess how its use as a sugar source influences the physicochemical, bioactive, and functional properties of the beverage.

2. Materials and Methods

2.1. Materials

For kombucha preparation, the following ingredients were used: drinking water supplied by a local water treatment plant (source: Czyżkówko, Bydgoszcz, Poland), Nashi pear (Pyrus pyrifolia), black Ceylon tea (100% leaves; origin: Sri Lanka; William’s Manufacturing Poland Sp. z o.o., Radzymin, Poland), SCOBY, and sugar. Nashi pears and beet sugar (sucrose) were purchased from a local market in Bydgoszcz, Poland. SCOBY was obtained from a local kombucha producer (Białe Błota, near Bydgoszcz, Poland). The SCOBY was propagated in sour broth and stored at room temperature until use. The sour broth used in this study was obtained from a long-term kombucha culture maintained under laboratory conditions for approximately three years. The sour broth represents the liquid fraction of the mature fermentation, separated from the cellulose biofilm.

2.2. Methods

2.2.1. Preparation of Raw Materials and Kombucha Fermentation

To obtain Nashi pear pomace, the fruits were washed with tap water and soaked for 2 min in a chlorine dioxide solution (5 ppm). After cleaning, the pears were cut into four pieces, and the seed cores were removed and discarded. Juice was extracted from the prepared fruit using a slow juicer (Kuvings D9900, NUC Electronics Co., Ltd., Daegu, Republic of Korea). The obtained pomace was dried at 40 °C for 24 h in a laboratory dryer with forced air circulation until a dry mass of 15.90 ± 0.16 was obtained (initial dry mass 76.02 ± 0.99) (SLW 115, POL-EKO, Wodzisław Śląski, Poland). Prior to kombucha preparation, the dried pomace was stored at 3 °C. Before further processing, the total reducing sugar content in the pomace was analyzed using Bertrand’s method (as described in Section 2.2.3).

For the pomace beverage preparation, due to the volume and swelling during hydration of pomace the proportion of the amount of water added was 400 mL (T = 95 °C). The specific mass of pomace added was calculated individually for each batch based on its reducing sugar content to reach the desired initial sugar concentrations.

To prepare the black tea beverage, 1.8 g of black Ceylon tea was weighed and brewed with 200 mL of hot drinking water (95 °C for 5 min). After brewing, the leaves were separated and discarded. The tea was then cooled to room temperature (approximately 20 °C) and used for kombucha preparation.

For pomace kombucha preparation, the mass of pomace in samples was calculated based on the reducing sugar content, to obtain 3%, 5% and 7% (w/v) of sugar concentration. For tea kombucha preparation, sucrose was added to obtain final concentrations of 3%, 5%, and 7% (w/v). Subsequently, SCOBY (5% m/v) and sour broth (7.5% v/v) were added. The sour broth was used to lower the pH and accelerate the fermentation process [1,13].

Fermentation was carried out using the traditional method, in which the beverages were fermented in beakers. The fermentation temperature was maintained at 22 °C for 6 days. These fermentation parameters were selected based on established protocols to ensure optimal balance between microbial activity and the accumulation of bioactive metabolites [22,23,24]. All experiments were performed in triplicate. Sample names and abbreviations are listed in

Table 1. Explanation of sample abbreviations used in this manuscript.

Sample Abbreviation	Sample Abbreviation Explanation
T3%	Tea kombucha with 3% sugar
T5%	Tea kombucha with 5% sugar
T7%	Tea kombucha with 7% sugar
P3%	Nashi pear pomace with 3% sugar
P5%	Nashi pear pomace with 5% sugar
P7%	Nashi pear pomace with 7% sugar

.

2.2.2. Physicochemical Analysis (pH, TSS, and Titratable Acidity)

The pH of the beverages during fermentation was measured daily using a pH meter (SevenCompact S210, Mettler Toledo, Greifensee, Switzerland).

Total soluble solids (TSS) were measured daily during fermentation using an automatic refractometer (J257, Rudolph Research Analytical, NJ, USA). Prior to analysis, samples were centrifuged at 3000 rpm for 2 min (Rotina 380, Hettich, Kirchlengern, Germany) to remove suspended solids. TSS content was determined based on the refractive index and expressed as °Brix, according to the relationship between the refractive index at 20 °C and the percentage by mass of total soluble solids in a pure aqueous sucrose solution.

Titratable acidity of the beverages was determined by titration with sodium hydroxide (Chempur, Krupski Młyn, Poland) as the titrant. A 10 mL sample of fermented beverage was diluted with 10 mL of distilled water in a conical flask. A few drops of phenolphthalein indicator (Chempur, Krupski Młyn, Poland) were added, and titration was performed using 0.1 M NaOH. Results were expressed as g of malic acid per liter of beverage.

2.2.3. Determination of Bioactive Compounds and Reducing Sugars

The total phenolic content of beverages after fermentation was determined using the Folin–Ciocalteu method as described by Singleton and Rossi [25], modified by Szulc et al. [26], and described by Błaszak et al. [27]. The total phenolic content (TPC) in the analyzed sprout extracts was quantified using the Folin–Ciocalteu assay. Initially, 2 g of entire sprouts (consisting of the first leaves, stem, and root) were pulverized in a mortar. The resulting material was then combined with 40 mL of a methanol solution containing 1% acetic acid in a test tube. To facilitate extraction, the mixture was sonicated for 8 min in a chilled water bath (PS-10A Ultrasonic Waterbath Adverti, Łódź, Poland), ensuring the samples remained shielded from light. Subsequently, the extracts were centrifuged at 3500 rpm for 5 min (Rotina 380 Hettich, Kirchlengern, Germany). A 1 mL aliquot of the supernatant was then diluted with 6 mL of distilled water and reacted with 0.5 mL of Folin–Ciocalteu reagent (Chempur, Piekary Śląskie, Poland). Following a 2 min incubation period, the mixture was supplemented with 1.5 mL of saturated disodium carbonate (Chempur, Poland) and an additional 1.9 mL of distilled water. After thorough mixing using a vortex (LLG-uniTEXER 1, Meckenheim, Germany), the samples were incubated at 37 °C for 30 min (WNB 22 water bath, Memmert, Schwabach, Germany). The final absorbance was recorded at 765 nm using an HP/Agilent 8453 UV/Vis Spectrophotometer (Hewlett-Packard, Santa Clara, CA, USA). TPC results were calculated via a standard curve and reported as mg of gallic acid equivalent (mg GAE) per g of fresh weight.

The antioxidant activity of beverages after fermentation was determined using the synthetic DPPH radical (2,2-diphenyl-1-picrylhydrazyl) as described by Brand-Williams et al. [28]. To eliminate suspended solids, the extracts were centrifuged at 3000 rpm for 2 min using a Rotina 380 Hettich centrifuge (Kirchlengern, Germany). A 1 mL aliquot of the clarified supernatant was subsequently reacted with the reagents according to the protocol by Hejna et al. [29]. The antioxidant capacity was determined based on the radical-scavenging activity against DPPH radicals. The results were expressed as a percentage of radical scavenging activity (%), calculated using the appropriate calibration curve.

Reducing sugar content was determined in Nashi pear pomace before fermentation and in both fermented beverages (Nashi pear pomace kombucha and tea kombucha). Reducing sugars were analyzed using Bertrand’s method as described by Krełowska-Kułas [30]. Prior to analysis, samples were deproteinized and clarified using Carrez reagents [30]. Reducing sugar content was expressed as g of glucose per 100 mL of sample.

2.2.4. Colorimetric Measurement

Color of the beverages was determined before and after fermentation. Automated measurements were performed in the wavelength range of 380–780 nm with a 5 nm interval using a spectrophotometer (HP/Agilent 8453 UV/Vis Spectrophotometer, Hewlett-Packard, Santa Clara, CA, USA). Calculations were carried out according to the model developed by Delgado-González et al. [31]. Additionally, the determined color parameters in the LAB color system were transferred to the RGB color system and presented as a sample colors and the total color difference (ΔE) was calculated according to the formula [32]:

$$\Delta \mathrm{E}={\left[{\left({\Delta \mathrm{L}}^{\mathrm{*}}\right)}^2+{\left({\Delta \mathrm{a}}^{\mathrm{*}}\right)}^2+{\left({\Delta \mathrm{b}}^{\mathrm{*}}\right)}^2\right]}^{{\displaystyle \frac{1}{2}}}$$

where

ΔL*—the difference of L* between analyzed samples,

Δa*—the difference of a* between analyzed samples,

Δb*—the difference of b* between analyzed samples.

2.2.5. Microbial Analysis

For microbial analysis, the total viable count (TVC) and lactic acid bacteria (LAB) were determined separately to distinguish the overall microbial activity from the specific functional group of lactic acid bacteria. Ten milliliters of each sample was transferred into 90 mL of sterile physiological saline and serially diluted. Surface plating was performed using 100 µL of selected dilutions on appropriate general and selective microbiological media.

Total viable counts (TVC) were determined on Standard Agar (Merck KGaA, Darmstadt, Germany) after incubation at 30 °C for 24 h [33]. Lactic acid bacteria (LAB) were determined on MRS Agar (Merck KGaA, Germany) after incubation at 37 °C for 24 h [34].

Results were expressed as colony-forming units per milliliter (cfu/mL) of fresh matter.

2.2.6. Statistical Analysis

Statistical analysis was performed using one-way analysis of variance (ANOVA) to evaluate significant differences among the studied parameters. Tukey’s test was used for comparison of means. Statistical calculations were performed using Statistica 13.3 software. Statistical significance was set at p < 0.05. All results were presented as mean ± standard deviation of three independent replicates.

3. Results and Discussion

3.1. Acidification Dynamics and pH Stability During Fermentation

The pH values of the beverages differed between the tea infusion and the Nashi pear pomace extract (

Table 2. Change in the pH values during the fermentation.

Sample	Day of the Fermentation
	1	2	3	4	5	6
T3%	3.6 aB	3.5 aB	3.6 aB	3.2 bB	3.1 bB	3.2 bB
T5%	3.4 aB	3.1 aB	3.3 aB	3.0 aB	2.7 aB	3.0 aB
T7%	3.5 aB	3.2 aB	3.3 aB	3.0 bB	2.9 bB	3.2 abB
P3%	4.7 aA	4.1 bA	3.9 bA	3.8 bA	3.8 bA	3.8 bA
P5%	4.5 aA	4.0 bA	3.9 bA	3.8 bA	3.6 bA	3.8 bA
P7%	4.3 aA	3.7 bA	3.8 bA	3.6 bA	3.5 bA	3.6 bA

a,b—samples with the same letter in the line do not differ significantly in the same beverage during fermentation; A,B—samples with the same letter in the column do not differ significantly between different beverage variants, p < 0.05.

). On each day of analysis, tea kombucha exhibited statistically significantly lower pH values than Nashi-based variants of kombucha. On day 1, the pH of tea kombucha samples ranged from 3.4 to 3.6, whereas Nashi pear pomace samples showed higher values, ranging from 4.3 to 4.7. At the same time, no statistically significant differences were observed between samples differing in sugar concentration within the same substrate type. During fermentation, a gradual decrease in pH was observed in all analyzed samples. This is a naturally occurring process resulting from SCOBY metabolism and the production of organic acids, such as acetic and lactic acids [22,35]. In tea kombucha, statistically significant decreases in pH were observed on day 4 of fermentation for samples T3% and T7%. In contrast, for sample T5%, the decrease in pH was not statistically significant throughout the analyzed period. In all Nashi pomace kombucha samples, a significant decrease in pH was already observed on day 2 of fermentation. This indicates that the Nashi pear substrate promotes a faster initial acidification rate compared to the tea control. A similar fermentation pattern was reported by Chong et al. [23], who observed a rapid decrease in pH at the initial stage of fermentation. This phenomenon may be attributed to the rapid utilization of sucrose by yeasts during the early fermentation phase, whereas the subsequent slower decline in pH may be associated with a buffering effect resulting from interactions between synthesized organic acids and minerals present in the Nashi pear substrate [23]. At the end of fermentation, lower pH values were recorded for tea kombucha samples (3.0–3.2), while pomace kombucha samples reached pH values in the range of 3.6–3.8. The higher final pH observed in Nashi pomace-based beverages may be attributed to their significantly higher initial pH at the beginning of fermentation. Importantly, the acidic pH values obtained (<4.5) are crucial for inhibiting the growth of pathogenic microorganisms and thus ensuring microbial safety. Moreover, an acidic environment may enhance the stability of Nashi pear polyphenols, which are pH-sensitive and more susceptible to chemical degradation at higher pH values [23,36,37].

3.2. Reduction in Total Soluble Solids (TSSs) and Metabolic Activity

Statistically significant differences in total soluble solids (TSSs) were observed among the analyzed samples (

Table 3. Change in the TSS [°Brix] during the fermentation.

Sample	Day of the Fermentation
	1	2	3	4	5	6
T3%	4.0 aC	3.8 aC	3.4 bC	3.1 bC	3.2 bC	3.0 bC
T5%	6.1 aB	5.9 aB	5.7 abB	5.5 bB	5.4 bB	5.2 bB
T7%	7.8 aA	7.6 aA	7.5 abA	7.3 bA	7.2 bA	7.1 bA
P3%	2.4 aE	1.7 abE	2.2 aD	1.4 bE	1.4 bE	1.4 bE
P5%	3.1 aD	2.7 aD	1.8 bD	1.9 bD	1.7 bDE	1.7 bE
P7%	3.7 aC	3.3 aC	1.9 bD	2.1 bD	2.1 bD	2.2 bD

a,b—samples with the same letter in the line do not differ significantly in the same beverage during fermentation; A–E—samples with the same letter in the column do not differ significantly between different beverage variants, p < 0.05.

). These differences were evident both between the type of infusion (tea vs. Nashi pear pomace-based) and between sucrose concentrations. In most cases, tea kombucha samples exhibited significantly higher TSS values (day 1: 4.0–7.8 °Brix) compared to Nashi pomace-based kombuchas (day 1: 2.4–3.7 °Brix). An increase in sucrose concentration resulted in a corresponding increase in TSS values. Consequently, within the same beverage type, the lowest TSS values were recorded in samples with the lowest sucrose concentration. During fermentation, TSS values gradually decreased. In all samples, statistically significant reductions were observed after 3 days of fermentation (T3%, P5%, and P7%) or after 4 days (T5%, T7%, and P3%). The decrease in TSS can be attributed to the utilization of sucrose by the SCOBY consortium for metabolite production, primarily ethanol and organic acids. In the case of Nashi pear pomace variants, this reduction also reflects the assimilation of soluble nutrients extracted from the pear tissue, confirming that the pomace matrix effectively supports the metabolic needs of the SCOBY. This trend is consistent with the simultaneous decrease in pH observed during fermentation [35,38].

3.3. Changes in Titratable Acidity and Organic Acid Accumulation

Titratable acidity differed primarily depending on the origin of the kombucha base (

Table 4. Titratable acid concentration in fermented beverages.

Sample	Titratable Acid [mg Malic Acid/L]
T3%	1.14 d ± 0.08
T5%	1.41 c ± 0.08
T7%	1.27 c ± 0.08
P3%	2.32 b ± 0.04
P5%	4.02 a ± 0.07
P7%	3.98 a ± 0.04

a–d—samples with the same letter in the column do not differ significantly between different beverage variants, p < 0.05.

). The lowest value was observed for sample T3%, reaching 1.14 g of malic acid/L. Increasing the sucrose concentration resulted in a significant increase in titratable acidity to 1.41 and 1.27 g of malic acid/L for T5% and T7%, respectively. Significantly higher titratable acidity was determined in Nashi pear pomace-based kombucha samples compared to tea kombucha. This may be attributed to the presence of organic acids naturally occurring in the Nashi pear pomace (such as malic, citric, and quinic acids), which were extracted into the beverage matrix. As a result, the initial acid content in Nashi pear pomace-based substrates was higher than in tea infusions, contributing to greater total titratable acidity after fermentation. This increased acidity in Nashi-based variants is a significant finding, as it suggests that the pear pomace provides a more complex acid profile and enhanced biological stability without the need for additional acidification. The total concentration of organic acids in Nashi-based beverages is strongly influenced by the physiological stage of fruit ripeness [39]. During fruit development and ripening, numerous flavor-active compounds are synthesized, including organic acids such as quinic, malic, shikimic, and citric acids. These compounds contribute not only to the characteristic sensory profile of the fruit but also to its overall acid balance [40]. Importantly, the concentration and profile of organic acids change dynamically during ripening. Depending on the maturity stage, Nashi pear fruits may contain varying proportions of individual acids, as well as different sugar-to-acid ratios [39,41]. Consequently, the composition of extracts and pomace derived from Nashi pear can vary substantially, which in turn may influence the chemical characteristics of the resulting kombucha. Therefore, differences in fruit maturity may partially explain variations in titratable acidity observed in Nashi pear pomace-based kombucha samples.

3.4. Evolution of Phytochemical Profiles and Bioactive Potential

The antioxidant properties and total phenolic content (TPC) of the fermented beverages varied significantly depending on substrate type and sucrose concentration (

Table 5. Antioxidant properties of fermented beverages.

Sample	Total Phenolic Content [mg GAE/100 mL]	DPPH Scavenging Capacity [%]
T3%	962.10 a ± 41.06	52.22 c ± 4.77
T5%	943.81 a ± 38.26	65.07 b ± 2.82
T7%	967.74 a ± 27.77	99.87 a ± 0.12
P3%	199.68 d ± 7.43	18.82 e ± 0.28
P5%	284.02 c ± 9.96	18.79 e ± 1.94
P7%	435.13 b ± 45.28	33.52 d ± 0.50

a–e—samples with the same letter in the column do not differ significantly between different beverage variants.

). Tea-based kombucha samples (T3–T7%) exhibited the highest TPC values, ranging from 943.81 to 967.74 mg GAE/100 mL, with no significant differences observed despite increasing sugar levels. In contrast, Nashi pear pomace-based kombucha showed lower TPC values, ranging from 199.68 mg GAE/100 mL (P3%) to 435.13 mg GAE/100 mL (P7%), with a clear dose-dependent increase as the amount of Nashi pear pomace increased.

Antioxidant activity measured by DPPH scavenging did not follow the same trend as TPC. Tea kombucha showed a strong increase in activity with higher sugar concentrations (52.22%, 65.07%, and 99.87% for T3%, T5%, and T7%, respectively), whereas Nashi pear pomace-based kombucha exhibited lower activity overall (18.79–33.52%), with only the P7% sample showing a notable increase. Despite the marked increase in TPC from P3% to P7%, the corresponding rise in DPPH activity was relatively modest. This suggests that the antioxidant potential of Nashi pear-based beverages is driven by a different set of bioactive compounds (e.g., arbutin or specific phenolic acids characteristic of the Pyrus genus) which exhibit different scavenging kinetics compared to tea catechins. This indicates that total phenolic content alone does not fully determine the antioxidant potential of Nashi-based kombucha.

Comparable trends have been reported in studies examining the influence of substrate on kombucha bioactivity. Kombucha prepared from different tea types (green, black, Pu’er) showed an initial increase in polyphenol content and antioxidant activity during fermentation, with subsequent changes depending on substrate characteristics [42]. Similarly, kombucha made with alternative plant substrates, such as vine tea and sweet tea, demonstrated significant enhancements in both total phenolics and antioxidant capacities relative to beverages without residues [43]. Moreover, studies on kombucha supplemented with brown algae and lichen extracts revealed that alternative substrates may initially reduce measurable TPC due to interactions with the microbial matrix, stabilizing later during fermentation [44]. In our study, the lower TPC observed in the P3% Nashi pear pomace sample compared to P7% may reflect similar substrate-dependent limitations on extractable phenolics linked to the pear-derived matrix structure.

Overall, these results indicate that antioxidant activity in both tea- and Nashi pear pomace-based kombucha depends not only on the quantity of phenolic compounds but also on their composition and structure, as well as the presence of other bioactive metabolites generated during fermentation, such as organic acids and microbial-derived compounds. The lack of direct correlation between TPC and DPPH activity in the Nashi pear pomace-based beverages highlights the importance of considering qualitative differences in phenolic profiles specific to Pyrus pyrifolia residues when evaluating functional properties.

3.5. Dynamics of Reducing Sugar Utilization by the SCOBY Consortium

The level of reducing sugars after the fermentation process exhibited distinct patterns depending on the substrate type and initial sugar concentration (

Table 6. Reducing sugar content [g of glucose/100 mL] before and after the fermentation.

Sample	Before Fermentation	After Fermentation
T3%	3.00 *	1.27 e
T5%	5.00 *	3.62 c
T7%	7.00 *	6.09 a
P3%	3.00 *	2.28 d
P5%	5.00 *	3.23 cd
P7%	7.00 *	5.07 b

a–e—samples with the same letter in the column do not differ significantly between different beverage variants; *—sucrose content [%].

). While all variants experienced a decrease from the starting levels of 3.00, 5.00, and 7.00 g/100 mL, the efficiency of sugar utilization by the microbial consortium differed markedly between tea-based and Nashi pear pomace-based samples. In the variants with the lowest initial sugar content (3%), tea-based kombucha (T3%) displayed the fastest initial sugar uptake, reaching a final concentration of 1.27 g/100 mL, compared to 2.28 g/100 mL in the Nashi pomace sample (P3%). Despite the higher starting pH of the Nashi-based substrate (4.7 vs. 3.6 in tea), the drop in pH occurred earlier in Nashi pomace kombucha (day 2) than in tea-based variants (days 3–4), reflecting rapid early microbial activity supported by the nutrients and organic acids present in the pear pomace [43,44].

The accelerated sugar metabolism in all variants is associated with invertase (β-fructofuranosidase) activity, secreted by yeasts in the SCOBY, which hydrolyzes sucrose into glucose and fructose—the preferred substrates for yeast and acetic acid bacteria [42]. The interplay between Nashi pear substrate composition, invertase activity, and microbial growth explains the observed earlier acidification in pear pomace, despite its initially higher pH.

At intermediate and high sugar concentrations (5–7%), Nashi fruit pomace-based kombucha (P5%, P7%) exhibited greater overall sugar consumption than the corresponding tea-based samples (final 3.23 and 5.07 g/100 mL vs. 3.62 and 6.09 g/100 mL in T5% and T7%), This indicates that the Nashi pear matrix provides a more supportive environment for microbial activity under higher sugar loads compared to traditional tea. This may result from additional nutrients, soluble fibers, and minerals in Nashi pear, which enhance invertase activity and substrate processing efficiency [45].

Overall, these results demonstrate that both substrate type and initial sugar concentration strongly influence sugar metabolism and acidification kinetics. Tea favors rapid sugar utilization at low concentrations, while Nashi fruit pomace supports more efficient fermentation at higher sugar levels, linking invertase-mediated sucrose hydrolysis with residual sugar content, pH dynamics, and total soluble solid changes.

3.6. Impact of Substrate and Fermentation on Chromatic Characteristics

The colorimetric analysis demonstrated that substrate type was the primary factor determining the visual appearance of the fermented beverages (

Table 7. Color coordinates in the fermented beverages.

Sample	Color Coordinate			Sample Color
	L*	a*	b*
T3%	71.5	3.4	16.3
T5%	71.2	3.4	16.2
T7%	72.3	4.7	16.5
P3%	81.5	1.8	37.1
P5%	82.5	2.0	37.5
P7%	75.8	4.4	42.7

). Tea-based kombucha samples (T3–T7%) exhibited a consistent amber coloration (L*: 71–72, a*: 3–4.7, b*: 16–16.5), which is similar to the color of black tea infusions and reflects the presence of oxidized tea polyphenols, such as theaflavins and thearubigins, responsible for the characteristic brown–amber hue [45]. In contrast, pear pomace-based kombucha samples showed higher lightness (L*: 75.8–82.5) and greater yellowness (b*: 37.1–42.7), indicating a lighter, more yellow appearance due to different pigment profiles in Nashi pear pomace shown in Table 7. This distinct chromatic profile is a direct consequence of the flavonoid and carotenoid pigments characteristic of the Nashi pear skin and pulp, which differ significantly from tea-derived pigments.

Total color difference (ΔE) values emphasized perceptible differences between samples (

Table 8. The total color differences (ΔE) of the fermented beverages.

	T3%	T5%	T7%	P3%	P5%	P7%
T3%	0.0 +
T5%	0.3 +	0.0 +
T7%	1.5 +	1.7 +	0.0 +
P3%	23.2 ++	23.4 ++	22.8 ++	0.0 +
P5%	23.5 ++	23.7 ++	23.1 ++	0.4 +	0.0 +
P7%	26.8 ++	26.9 ++	26.5 ++	8.4 ++	8.1 ++	0.0 +

⁺ the observer does not notice the difference; ⁺⁺ the observer gets the impression of two different colors [46].

). Within tea kombucha, ΔE values were low (0.3–1.7), indicating barely noticeable color changes across sugar concentrations. In the Nashi pear pomace group, the ΔE between P3% and P5% was minimal (0.4), but increasing pomace to P7% caused a clearly perceptible darkening (ΔE = 8.4), suggesting enhanced pigment extraction or interactions during fermentation as reflected by the mean color values presented in the last column of Table 7. The ΔE between tea- and Nashi pear pomace-based kombuchas was exceptionally high (>22), reflecting strongly perceptible differences.

These observations are consistent with previously discussed parameters. The lighter color and higher b* values of Nashi pomace kombucha correspond with its higher pH and lower total phenolic content compared to tea kombucha, suggesting that both substrate composition and phenolic profile influence color intensity and hue [42,43]. Moreover, phenolic compound dynamics and pH shifts during fermentation have been associated with changes in pigment behavior and beverage clarity, supporting the link between fermentation parameters (pH, TSS, phenolics) and visual properties [47]. Collectively, these findings highlight that pear pomace choice and fermentation dynamics drive final color, which may influence consumer perception and acceptability [45].

3.7. Microbiological Succession: Proliferation of LAB and Total Microflora

The total viable count (TVC) of microorganisms in all fermented beverages ranged from 1.2 × 10⁶ to 3.0 × 10⁷ CFU/mL (

Table 9. The total number of microorganisms and lactic acid bacteria in fermented beverages.

Sample	TVC 1 [CFU/mL]	LAB 2 [CFU/mL]
T3%	2.1 × 106	5.3 × 102
T5%	4.5 × 106	7.6 × 102
T7%	1.2 × 106	3.6 × 102
P3%	3.0 × 106	1.0 × 103
P5%	2.5 × 106	8.1 × 102
P7%	3.0 × 107	5.4 × 103

¹—Total Viable Count; ²—Lactic Acid Bacteria.

). Presumptive lactic acid bacteria (LAB) were detected at much lower levels, ranging from 3.6 × 10² to 5.4 × 10³ CFU/mL, representing less than 1% of the total microbiota. Among the samples, P7% (Nashi pomace kombucha with 7% sugar) exhibited the highest TVC and LAB counts, suggesting that both the Nashi pear pomace substrate and elevated sugar concentration promote microbial growth during fermentation.

The observed microbial counts are characteristic of traditionally fermented kombucha beverages and fall within the ranges commonly reported in the literature [10,24,48,49,50]. Kombucha fermentation is typically dominated by yeasts and acetic acid bacteria, which constitute the majority of the microbial community, while LAB are present at lower but functionally relevant levels. This microbial balance is essential for the proper progression of fermentation and the development of the beverage’s sensory and chemical profile.

The relatively low proportion of LAB is consistent with the typical composition of a SCOBY, in which yeasts and acetic acid bacteria dominate and drive the fermentation process, producing ethanol, organic acids, and other metabolites that define the beverage’s flavor and acidity [24]. Moreover, the acidic pH values achieved during fermentation (<4.5) create conditions unfavorable for pathogenic microorganisms, further confirming the microbiological safety of the produced beverages.

The observed microbial dynamics aligned with changes in pH and total soluble solids: rapid sugar utilization during the early stages of fermentation supported microbial proliferation, while the gradual acidification of the medium created conditions that limited pathogenic growth. These results confirm that Nashi pear pomace is a highly suitable substrate for kombucha fermentation, as its specific nutrient composition supports robust microbial activity while maintaining the characteristic biological balance of the SCOBY consortium.

3.8. Study Limitations and Future Perspectives

Despite the promising results, certain limitations of the current study should be acknowledged. The primary challenge lies in the natural heterogeneity of Nashi pear pomace, as its chemical composition (sugar profile and phenolic content) may vary depending on the fruit variety, ripeness, and the specific industrial extraction method used. Furthermore, this research serves as a preliminary pilot study, and as such, the data presented in Table 7, Table 8 and Table 9 reflect the initial feasibility of the fermentation process. The authors acknowledge that the current lack of extensive biological replicates and multi-batch statistical analysis is a limitation, particularly given the inherent variability of the SCOBY consortium. While the consortium demonstrated robust growth in the pomace substrate, the stability of this microbial community over multiple fermentation cycles (back-slopping) requires further long-term investigation to ensure consistent product quality. Regarding large-scale implementation, the transition from laboratory beakers to industrial bioreactors would necessitate precise control over oxygen mass transfer and sediment management. However, the use of pomace as a primary substrate significantly reduces the need for exogenous sucrose, offering a clear economic and environmental advantage for the development of standardized, large-scale circular economy processes in the beverage industry.

4. Conclusions

The present study demonstrates that Nashi pear (Pyrus pyrifolia) pomace can serve as an effective and functional substrate for kombucha fermentation, providing a natural source of sucrose alongside bioactive compounds. Pomace-based kombucha showed higher initial pH and titratable acidity compared to tea-based beverages but stabilized at safe levels (pH 3.6–3.8), which is well below the critical threshold of 4.5, ensuring microbiological safety and inhibiting pathogenic growth. Despite lower total phenolic content and antioxidant activity than tea kombucha, pomace kombucha exhibited moderate bioactivity, efficient sugar metabolism, and a distinct light-yellow color that may enhance sensory appeal. Microbial analysis confirmed that the pomace substrate supports healthy microbial proliferation, particularly at higher sugar concentrations, reflecting the nutritional richness of the fruit by-product. Importantly, utilizing Nashi pear pomace valorizes fruit processing waste, contributing to sustainable production and circular economy practices while creating a value-added functional beverage. Overall, Nashi pear pomace is a promising eco-friendly alternative to traditional sugar sources in kombucha, combining sustainability, functional benefits, unique sensory properties, and ensured microbiological safety due to stable acidic pH levels.