New Fermented Beverage from Orange Peel By-Products Containing Bioactive Flavanones

Abstract

The increasing popularity of fermented beverages, such as kombucha, has prompted the search of alternative ingredients with distinct functional and sensory properties. Orange (Citrus sinensis L. Osbeck) peel, an abundant by-product of the citrus industry, represents a valuable natural source of flavanones associated with multiple health benefits, offering a suitable substrate for fermentation. In this context, the present study proposes the valorisation of this by-product through the development of a new fermented beverage analogous to kombucha, rich in bioactive flavanones. During the fermentation process, variations were observed in physicochemical quality parameters (pH (4.86–2.91), titratable acidity (maximum 0.45% as acetic acid), and total soluble solids (TSS) (6.90–7.05 °Brix), as well as in the fermentation metabolites and substrates: sucrose (73.99–45.75 g/L), fructose (0.98–6.87 g/L), glucose (1.60–1.35 g/L), ethanol (0.06–0.24 g/L), and acetic acid (0.45–3.00 g/L). On the other hand, the initial total flavanone content (11.85 mg/100 mL), of which 70% corresponded to hesperidin, decreased during fermentation but then remained stable, reaching a final concentration of 5.72 mg/100 mL. Overall, these results highlight the potential of orange peel by-products for the development of innovative fermented beverages with a high content of bioactive flavanones, which are distinct from conventional tea-based kombucha. Moreover, this strategy represents a potential approach for this citrus waste valorisation, contributing to improved resource efficiency and supporting the transition towards a circular economy.

1. Introduction

The consumption of fermented foods has increased substantially in recent years, primarily due to their minimal processing and the multiple health benefits associated with the intake [1]. Regarding these beverages, kombucha, a traditional tea fermented drink, has a prominent position, due to its attractive sensory profile, characterised by its acidity and slight effervescence, which is a result of the metabolic activity of the diverse and variable symbiotic culture of yeasts and bacteria (SCOBY) [2]. This consortium typically includes yeast genera such as Saccharomyces, Zygosaccharomyces, Brettanomyces, Candida, Torulaspora, Pichia, Hanseniaspora, and Schizosaccharomyces, as well as acetic acid bacteria (AAB), including Komagataeibacter, Acetobacter, and Gluconobacter [3,4,5]. During the fermentation process, the yeasts hydrolyse sucrose into the monosaccharides glucose and fructose, which are subsequently metabolised into ethanol via glycolysis. Later, this alcohol is oxidised into acetic acid by AAB [6]. The ethanol concentration in kombucha beverages is generally low, typically remaining below 0.5% (v/v) in commercially available products. However, higher levels may be observed depending on the fermentation parameters, such as time, temperature, and sugar content. In this context, the actual alcoholic strength by volume must only be indicated on the label when it exceeds 1.2% (v/v) [7]. Moreover, this fermented drink is characterised by a high content of bioactive compounds, including (poly)phenols such as catechins, derived from green or black tea, as well as other healthy pre- and postbiotics, produced during fermentation. This phytochemical profile is mainly determined by the raw materials used, the microbiological composition of the SCOBY, and the fermentation conditions [3,8,9,10]. These qualities have increased interest in the consumption and production of this beverage, with expectations of reaching a global market of $7.05 billion by 2027 [11]. In this context, the exploration of alternative plant materials for the production of kombucha ‘analogues’ is of particular interest, as these materials could confer functional and sensory attributes that differ from those of the conventional black or green tea-based ones [3,12].

Continuing our research on the development of novel 3S (Safe, Salubrious and Sustainable) foods [13,14], including the valorisation of agri-food by-products, new kombucha formulations represent a promising alternative, offering citrus industry residues a particularly abundant resource. It is estimated that 54.84% of global citrus production is attributable to Citrus sinensis (sweet orange), with an annual output exceeding 158 million tonnes. As a consequence of the industrial juice and derived products processing, of this fruit, discarded peel accounts for up to 60% of the total weight of the fruit, thereby becoming one of the most noteworthy by-products generated by this sector [15,16]. Nevertheless, although orange peel currently has some industrial value, due to its concentration in essential oils, aromatic compounds, simple sugars, pectin, cellulose, minerals, and vitamins, as well as bioactive compounds such as carotenoids and flavanones, this low-cost raw material remains underused [17,18,19,20]. Among bioactive compounds, the 7-O-glycoside flavanones, identified in orange peel, hesperetin and naringenin derivatives (hesperidin and narirutin, respectively) are present in the highest proportions, followed by isosakuranetin (didymin) and the 3,5-diglycoside, hesperetin 7-O-rutinoside-3′-glucoside [17,21,22,23]. These flavonoids have been associated with anti-inflammatory, anti-cancer, antioxidant, and cardioprotective properties, thereby enhancing their relevance as valuable alternative matrices for kombucha analogue production, promoting the consumption of 3S foods [24,25]. Even if the use of other citrus peel, such as sour orange (Citrus x aurantium L.) [12] and mandarin (Citrus nobilis) [26], has been briefly referenced in the literature for kombucha production, it has to be taken into consideration the different bioactive profile and that they do not represent such a significant sustainability problem. The use of orange peels for producing a kombucha-like beverage enabled the valorisation of this waste into a functional drink rich in vitamin C and total flavonoids, with maximum concentrations observed on day 9 of fermentation, after which total flavonoid levels gradually declined [26]. Although the use of alternative substrates such as leaves, fruits, flowers, mushrooms, truffles, and agri-food residues in kombucha production has been investigated, demonstrating its feasibility and potential to improve the functional properties of the final beverage [27], most studies have focused on general physicochemical parameters and total phenolic content. Detailed characterisation of specific bioactive compounds, such as flavanones and their evolution during fermentation of citrus residues, remains largely unexplored, something to consider since flavanones may modulate microbial dynamics during the fermentation process, contributing to the inhibition of pathogenic microorganisms and the promotion of the proliferation of bacteria with pre or probiotic potential [28].

Based on these premises, the main objective of the present study was to develop a new healthy non-standard 3S fermented beverage, with a distinctive phytochemical profile, compared to traditional kombucha, together with an extra valorisation of orange peel waste. This study is focused on the beverage’s physicochemical parameters (pH, titratable acidity, and TSS) and chemical composition (flavanones, sugars, ethanol, and acetic acid content), as well as their evolution throughout the fermentation process in response to interactions with the microbial consortium (SCOBY), thereby contributing novel insights into the valorisation of citrus by-products.

2. Materials and Methods

2.1. Chemicals and Reagents

Hesperidin (hesperetin 7-O-rutinoside) was purchased from Merck (Darmstadt, Germany) and formic acid and methanol from Panreac (Barcelona, Spain). The preparation of reagents was undertaken using a Milli-Q water system (Milli-Q system, Millipore, Bedford, MA, USA). All LC-MS-grade solvents were acquired from JT Baker (Phillipsburg, NJ, USA). Kits required for the determination of sucrose, glucose, fructose, acetic acid, and ethanol were supplied by BioSystems S.A. (Barcelona, Spain). Instrumental solutions and reagents were prepared in accordance with the manufacturer’s instructions. Sugar (sucrose (98%)) was provided by AB Azucarera Iberia S.L. (Madrid, Spain).

2.2. Plant Material and Beverage Preparation

Navel oranges (Citrus sinensis L. Osbeck) were supplied by Frutas Salmarsa (Cartagena, Spain) in November 2024. Upon arrival at the laboratory, fresh orange peels (albedo and flavedo) were separated. The orange peel was cut into uniform pieces approximately 1–2 cm in length and 0.5 cm in width, providing a sufficient surface area for the extraction of bioactive compounds and facilitated microbial access during fermentation.

For the development of the kombucha beverage, 50 g of fresh orange peel (flavanone content of 10.02 mg/g (2.57% hesperetin 7-O-rutinoside-3′-glucoside, 11.16% naringenin 7-O-rutinoside, 80.31% hespertin 7-O-rutinoside, and 5.96% isosakuranetin 7-O-rutinoside) was brewed in 1 L of mineral water (n = 3) at 90 °C for 15 min (OPK). Subsequently, the infusions were filtered through a gauze with a pore size of 0.5 mm, and sucrose was added at a concentration of 70 g/L, according to Cánovas et al. [13]. A control beverage (CK), without orange peel, and the same amount of sugar, was also prepared. Both were left to cool at room temperature (25 ± 2 °C) for ~60 min. Subsequently, a commercial SCOBY (Brava Drinks SL, Murcia, Spain), composed primarily of strains from the genera Komagataeibacter, Saccharomyces, and Zygosaccharomyces according to the manufacturer, with 5% starter liquid (parameters summarised in

Table 1. Starter liquid parameters.

Parameter	Values
pH	2.54
TA (% of acetic acid)	0.54
TSS (°Brix)	2.20
Sucrose (g/L)	4.87
Glucose (g/L)	0.99
Fructose (g/L)	3.38
Ethanol (g/L)	0.42
Acetic acid (g/L)	4.40

), was added. At this stage, samples were collected for each drink (day 0). The beverages in glass containers were then covered with breathable cloth, allowing passive gas exchange with the environment, and left to ferment for 10 days under darkness in an incubator set at room temperature (25 ± 2 °C). Triplicate samples were collected at two-day intervals and stored at −20 °C for subsequent analysis.

2.3. Physicochemical Parameters

Physicochemical parameters, pH, titratable acidity (TA), and °Brix (TSS), were determined according to Cánovas et al. [14]. The pH was measured using a pH-meter (GLP 21; Crison Ltd., Barcelona, Spain). TA (expressed as a percentage of acetic acid using conversion of equivalent weight = 0.9375) and Total Soluble Solids (TSS—expressed as °Brix) were recorded using a Pocket Brix-Acidity Meter (Citrus) (PAL-BX/ACID1, Atago Co., Ltd., Tokyo, Japan) at room temperature (25 °C ± 2 °C).

2.4. Analysis of Sugars, Ethanol, and Acetic Acid Content

The concentrations of sucrose, fructose, glucose, ethanol, and acetic acid were analysed using a Y15 Automatic Biochemistry Analyser (Code 83106, BioSystems, Barcelona, Spain) by enzymatic methods according to previous methodology by Cánovas et al. [13].

2.5. Qualitative and Quantitative Analysis of Flavanones

The identification and quantification of flavanones were performed by HPLC-DAD-ESI/MSn according to the methodology previously described by Salar et al. [19]. Briefly, samples were filtered through 0.22 µm PVDF filters (Millipore, MA, USA) and flavanones quantified as hesperidin at 280 nm with calibration curves freshly prepared each day of analysis. Results were expressed as milligrams per 100 millilitres of kombucha analogue beverage (mg/100 mL).

2.6. Statistical Analysis

The results were expressed as mean ± standard deviation (SD) (n = 3). A paired t-test was performed to identify significant differences between the two beverages (CK and OPK). In addition, the homogeneity test and analysis of variance (ANOVA) were performed to identify significant differences between the different fermentation days for each beverage. Tukey’s multiple range test was applied as a post hoc categorisation test. The statistical analysis was conducted using SPSS 29.0 (LEAD Technologies, Inc., Chicago, IL, USA), and the level of statistical significance was set at p < 0.05.

3. Results and Discussion

3.1. Physicochemical Parameters

In order to ascertain the effect of the fermentation process on the global quality parameters of the beverages, pH, TA (% acetic acid), and TSS (°Brix) values were monitored (Figure 1).

The OPK beverage demonstrated significantly lower pH values compared to CK throughout the experimental period, even at day zero. It is worth mentioning that even if equal proportions of starter liquid were added to both beverages, to ensure optimal pH conditions at the start of fermentation, this initial discrepancy can be attributed to the acidic nature of the fruit by-product, as well as the release of organic acids from the orange peel into the medium during the infusion process [3,8]. A significant decrease in pH (~40%) was observed in the OPK beverage during the fermentation process, reaching final values of 2.91 ± 0.05 (Figure 1). These values are within the accepted pH range for kombucha, considered safe for human consumption (2.50–4.20), since lower values could have negative health effects and are associated with excessive acetic acid concentrations [13,28]. This pH range would also have benefits for food safety, inhibiting the growth of bacterial pathogens such as Listeria monocytogenes [29]. On the other hand, CK samples only displayed a moderate decrease in pH (~12%) at day 2 of fermentation. As previously described, this decrease may be due to the SCOBY metabolism that produces compounds such as gallic acid from sucrose, independently of the matrix [4].

Titratable acidity is also a relevant physicochemical parameter in the production of fermented beverages, indicating flavour quality and preservation. It is noteworthy that a significant increase was observed in OPK from the fourth day until the end of this study, reaching final values of 0.4 ± 0.02% (Figure 1), which aligns with the typical trends reported for this type of beverage [26,30]. However, no acidity values (<LOD) were recorded for the CK beverage on any of the sampling days. Higher titratable acidity not only indicates the inhibition of the growth of pathogenic microorganisms but lest also the influence on enzymatic activity, promoting fermentation and contributing to the safety of the beverage [6,8,10]. The decrease in pH, along with the increase in TA in fermented beverages, is predominantly attributed to the synthesis of organic acids due to the metabolic activity of the microorganisms involved in the fermentation process [8,12,31,32]. Microbial metabolism requires the presence of certain compounds and nutrients, which are primarily provided by the plant matrix. These nutrients are lacking in the CK, explaining the absence of acidity in the control beverage as previously reported [31,32]. It is important to note that the acidity of kombucha beverages is a parameter that is susceptible to modification to align with consumer preferences, given that, as evidenced in the OPK beverage (Figure 1), fermentation time has a decisive influence on its evolution [14,28].

Concerning TSS content (°Brix), only slight differences were observed between the two beverages, which were more pronounced on day 6 (<10%), with a higher OPK content (Figure 1). However, these differences do not represent a meaningful difference in beverage quality [29,33]. These values are commonly associated with the sugar content of the beverage, which tends to decrease during the fermentation process. However, this parameter reflects the total concentration of organic and inorganic substances dissolved in the medium, including sugars and other compounds. Consequently, it may fluctuate throughout the fermentation process and even increase, depending on the substrates consumed and the metabolites produced by the different metabolic pathways [2,13,34].

3.2. Sugars, Ethanol, and Acetic Acid Content

Concerning the specific molecules directly responsible for the nutritional profile and the biological value of the beverage, sugars, ethanol, and acetic acid were measured, in accordance with Cánovas et al. [13]. In this respect, sugars are the main source of energy and carbon for microbial metabolism [8], so the sucrose, fructose, and glucose content of the beverages was determined throughout the fermentation process (Figure 2).

No significant differences in sucrose content were observed between OPK and CK until day 4 of fermentation, as both beverages were prepared by adding the same concentration of sucrose (70 g/L), according to conventional kombuchas [8]. From the fourth day onwards, a progressive decrease in sucrose content was observed in the OPK beverage, reaching an overall reduction of ~38%. This decrease can be attributed to the hydrolysis of the sucrose into the monosaccharides glucose and fructose, a process catalysed by yeast metabolism [6]. The absence of this phenomenon until the fourth day of fermentation in the OPK beverage could be attributed to the adaptation phase to the medium conditions that yeasts require before beginning the assimilation of carbohydrates [13]. Nevertheless, in the case of the CK beverage, no significant decrease in sucrose content was observed during fermentation, which suggests that microbial activity may have slowed down. This emphasises the importance of providing not only sucrose as a substrate but also a suitable plant-based matrix to support proper microbial growth and metabolism.

The hydrolysis process of sucrose that occurred in OPK resulted in a significantly higher concentration of both monosaccharides, fructose and glucose, in this beverage compared to CK throughout the study (Figure 2). The OPK beverage exhibited an increase in fructose content on the fourth day of fermentation, coinciding with the degradation of sucrose, with an upward trend during the remainder of the experimental period, reaching values of 6.87 ± 1.50 g/L. Nevertheless, the glucose content of this beverage did not demonstrate significant differences between the initial and final stages of fermentation, with the exception of certain intermediate fluctuations. The stability of the glucose content, despite its release during the fermentation process, can be attributed to the consumption of this monosaccharide in the subsequent metabolic pathways, as the bacteria and yeasts that comprise the SCOBY act synergistically [28]. Conversely, in the absence of the hydrolysis process in CK, the fructose content remains stable (below 0.12 g/L) in this beverage, while only a slight increase (0.20 to 0.36 g/L) in glucose was observed, during the fermentation process (Figure 2). The low sugar metabolism observed in CK indicates that the microbial consortium requires not only carbohydrates as an energy source to maintain optimal fermentation activity but also minerals or nitrogen sources derived from the plant material to support proper fermentation [13,31,32]. In this context, the depletion of free amino nitrogen in the fermentation medium severely restricts microbial growth and prevents de novo protein synthesis. This highlights the essential role of an adequate nitrogen source in sustaining high population densities and metabolic activity [35]. Additionally, yeasts such as S. cerevisiae secrete amino acids under nitrogen-rich conditions, enabling cross-feeding that supports the growth of lactic acid bacteria. This highlights the metabolic interdependence required for kombucha fermentation [36,37].

During the fermentation process, glucose is metabolised into ethanol, a pathway characteristic of yeast. In this process, glucose is glycosylated into pyruvate, and subsequently into ethanol and carbon dioxide [2,28]. In this study, the ethanol level was significantly higher in OPK than in CK during fermentation (Figure 3), with statistical differences becoming apparent from day 4. It is important to note that the initial ethanol content in the beverages is primarily attributable to the added starter liquid, which remained constant for both beverages until the fourth day of fermentation, since when a significant increase in the ethanol content of the OPK beverage was observed, which corresponded to the rise in glucose availability, reaching levels of 0.24 ± 0.03% g/L, at the end of fermentation period, consistent with those previously reported for tea kombucha by Chakravorty et al. [30]. During the fermentation process, ethanol levels can reach a plateau state, or even decrease, in line with other studies carried out in kombucha analogues based on fruits as alternative substrates [2,3]. However, this condition does not necessarily imply a decrease in the production of this alcohol but rather its use as a substrate for the synthesis of acetic acid by AAB, which could also explain the lower ethanol content in OPK compared to CK on the second day of fermentation [31,38]. On the other hand, a significant decrease in ethanol content was observed in the CK beverage on the fourth day of fermentation, and it subsequently remained stable until the end of this study (Figure 3). Consequently, the control of this parameter is one of the primary challenges in the kombucha production to ensure a final concentration below the threshold that would qualify for an alcoholic beverage (1.2% (v/v) ethanol) [13].

As previously stated, ethanol is oxidised by AAB under aerobic conditions to produce acetic acid, the predominant acid in this type of beverage [4,6]. Consequently, the enhanced availability of this alcohol resulted in OPK demonstrating a higher acetic acid content in comparison to CK from the sixth day of fermentation (Figure 3). From that day, a significant increase in the concentration of this acid was observed in the OPK beverage, reaching a maximum of 3.00 ± 0.43 g/L at the end of the fermentation process, similar to the observations documented by Cardoso et al. [10] in traditional green tea and black tea kombucha, while remained stable in the CK samples. The concentration of acetic acid may vary in this category of fermented beverages due to several factors, such as fermentation time, the microaerophilic conditions created by cellulose production, and the diversity of microbiological species [13,39]. Despite the absence of a regulatory framework in Spain, the Brazilian government has specific legislation that establishes acceptable concentration of volatile acidity for kombucha within the range of 1.80 to 7.81 g/L of acetic acid, making it one of the few existing regulations worldwide [40], with our beverages falling within the recommended range. It is important to note that the presence of acetic acid in adequate concentrations is key to ensuring food safety, since its antimicrobial properties would inhibit the growth of pathogenic microorganisms [6,11,28].

3.3. Qualitative and Quantitative Analysis of Flavanones by HPLC-DAD-ESI/MSn

(Poly)phenols represent a category of bioactive compounds that have been associated with numerous health benefits, mainly antioxidant, anti-inflammatory, antimicrobial, and anti-carcinogenic properties [11]. Within this group, orange peel contains a significant quantity of flavonoids, specifically flavanones, predominantly present in their glycosylated form [9]. Nevertheless, is worth noting that only 2.36% of the total flavanones in fresh peel was extracted during the infusion process, yielding an initial concentration of 11.85 mg/100 mL in OPK. This initial level decreased by approximately ~46% on the second day of fermentation and subsequently remained stable until the end of this study. This is due to the fact that, once in the fermentation medium, flavanones are exposed to degradation caused by various factors, including the concentration of the starter liquid used, the decrease in pH, exposure to oxygen (which may promote oxidation), or their absorption by microorganisms [13,34,41].

The quantitative analysis of individual flavanones identified in this study (Figure 4) was consistent with the flavanone content reported previously in the orange peel [42]. The predominant compound in the OPK beverage was identified as hesperetin 7-O-rutinoside (hesperidin), which accounted for approximately 70% of the total flavanone content. It is important to highlight that all individual compounds exhibited a behaviour similar to that observed in the total flavanone content, with a significant reduction on the second day of study, by ~45% in hesperidin (initial concentration 8.28 mg/100 mL). On the other hand, the rest remained below 3 mg/100 mL throughout the studied period.

The decrease in glycosylated flavanone content in this type of beverage can be attributed to the conversion of these compounds into molecules with a simpler structure. During the fermentation process, the microorganisms present in the microbial consortium release enzymes (such as β-glucosidase or α-rhamnosidase) capable of hydrolysing glycosidic bonds, resulting in an increase in free aglycones [1,43,44,45]. It has been demonstrated that various microbial species commonly present in SCOBY, including lactic acid bacteria, AAB, and yeasts such as Candida tropicalis or S. cerevisiae, contribute to the enzymatic degradation of flavonoids [1,30,46,47]. In this regard, the microbial composition of SCOBY and its interactions could play a decisive role not only in the physicochemical profile of the beverage but also in its polyphenol content and the chemical form in which these are found [28,48].

Due to the instability of these compounds and the presence of sugars in the glycosidic bonds [11,49], it is possible that, in the initial period of fermentation, the microorganisms may follow alternative metabolic pathways using these compounds as a source of nutrients and energy [44,47,50]. As fermentation progresses, once the yeast adaptation phase is complete, these microorganisms begin to obtain energy through the hydrolysis of sucrose, thus contributing to the stability of the flavanone content, a phenomenon that could be observed in the OPK beverage from day 4 of fermentation onwards (Figure 2 and Figure 4). Furthermore, the concurrence of the onset of sucrose degradation and the stability of the flavanone content could also be attributed to the increase in glucose available in the medium, since previous research has shown that the presence of this monosaccharide can inhibit the activity of certain deglycosylating enzymes [51].

These results are aligned with those reported by Noronha et al. [43], who also observed an increase in microbial degradation of flavonoids during the initial stages of black tea kombucha fermentation, resulting in the production of lower molecular weight phenolics. Therefore, the initial reduction in the glycosylated compounds does not necessarily imply their complete loss. Instead, it may be the result of a modification in their structure, which, in certain cases, may result in improved bioavailability and bioefficacy of certain flavonoids [48].

In that context, it is worth mentioning that flavanones have been reported to exert diverse bioactivities associated with multiple health benefits, including anti-inflammatory effects through the reduction of pro-inflammatory cytokine production, as well as antioxidant activity by neutralising reactive oxygen species and preventing free radical formation [24,52]. Specifically, hesperidin, the main compound in the drink, has been investigated for its potential effects in the prevention of diverse diseases and disorders, including cancer, type 2 diabetes, and cardiovascular disease [25,52]. It is also remarkable that flavanones have the capacity to modulate the gut microbiota by promoting the proliferation of beneficial bacteria. This effect, combined with the probiotic potential of these fermented beverages, represents a promising dietary strategy for supporting gastrointestinal health and maintaining microbial homeostasis [24,28].

4. Limitation

While the current study concentrated on flavanone detection and evolution, the absence of microbial characterisation (e.g., viable counts and population dynamics) is a limitation that must be addressed in future research.

5. Conclusions

The use of orange peel by-product as an alternative substrate for producing kombucha analogue resulted in a novel fermented beverage with a distinctive phytochemical profile compared to traditional tea-based kombucha. Notably, the beverage contained high levels of flavanones, bioactive compounds mainly occurring in citrus fruits, which remained stable at elevated concentrations throughout fermentation, suggesting its potential as a healthy beverage.

The qualitative and quantitative analysis of flavanones in this fermented beverage opens up a new line of research, allowing for further biochemical and technological investigations into the functionality of such beverages. Beyond its compositional advantages, this approach represents an innovative valorisation strategy for citrus by-products. Although the overall mass of orange peel residues is not substantially reduced, their transformation into a fermented beverage containing functional compounds contributes to the circular economy by generating added value from agri-food by-products. By combining food innovation with waste valorisation, this kombucha analogue would not only respond to the current growing demand for natural and health-promoting beverages but also contribute to sustainability and advance progress in the agri-food sector.