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Communication

‘Kombucha’-like Beverage of Broccoli By-Products: A New Dietary Source of Bioactive Sulforaphane

by
Berta María Cánovas
,
Cristina García-Viguera
,
Sonia Medina
* and
Raúl Domínguez-Perles
Laboratorio de Fitoquímica y Alimentos Saludables (LabFAS), CEBAS, CSIC, University Campus 25, Espinardo, 30100 Murcia, Spain
*
Author to whom correspondence should be addressed.
Beverages 2023, 9(4), 88; https://doi.org/10.3390/beverages9040088
Submission received: 19 September 2023 / Revised: 28 September 2023 / Accepted: 11 October 2023 / Published: 12 October 2023
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Abstract

:
The objective of this work is the development of a new fermented beverage (‘kombucha’-like), enriched with broccoli by-products as an ingredient, a source of organosulfur compounds, which could be biotransformed into more bioaccessible, bioavailable, and bioactive metabolites. The new beverages have shown variations in the physicochemical (pH, 3.6–6.3; acidity, 0.65–1.39 g/L; °Brix, 4.63–8.20). Moreover, the phytochemical characterization has demonstrated different degrees of metabolization of the glucosinolates, leached during the infusion of the plant material into isothiocyanates (sulforaphane in concentrations up to 31.39 µg/100 mL) and its metabolic derivatives (sulforaphane-N-acetylcysteine in concentrations up to 5.37 µg/100 mL). Therefore, these results demonstrate that the increase in the concentration of the bioactive compounds concentration would provide higher bioavailability and health benefits. This is especially relevant with regard to anti-inflammatory activity. Reporting additional proof of enhanced biological benefits will boost the development of new functional beverages.

Graphical Abstract

1. Introduction

Fermented beverages have been included in humans’ diets throughout history [1]. However, the advance in Food Technology knowledge in the last few decades has enabled the production of novel foods via the application of controlled microbial populations [2]. It is estimated that, at present, up to 40% of the foods consumed are fermented [3] due to its positive health effect.
These positive effects are due, to a high extent, to the presence of functional microorganisms with the metabolic capacity to transform the chemical compounds present in the food matrices, giving rise to highly bioavailable prebiotic and postbiotic compounds [4]. A wide range of these compounds have been characterized with regard to their contribution to the prevention of different pathophysiological processes [5]. Thus, currently, there are several foods and beverages based on fermentation, such as yoghurts, cheeses, breads, sauces, and fermented beverages (‘kefir’ or ‘kombucha’, obtained from the fermentation of milk and tea, respectively) [6]. In this regard, ‘kombucha’ and similar fermented beverages have broadly been studied due to their sensorial and healthy attributions [7,8].
‘Kombucha’ is a fermented beverage with thousands of years of tradition, with a significant recent increase in market demand and consumption [9]. The original development of this beverage is based on the fermentation, for 7–10 days, of green or black tea in a liquid medium and in the presence of sugar using a symbiotic culture of bacteria (acetic and, to a lesser extent, lactic) and yeasts (SCOBY- Symbiotic Culture of Bacteria and Yeast) [9]. As a result, in addition to metabolizing sugar into alcohol, the SCOBY transforms the chemical compounds in the matrix (tea) into new metabolic derivatives with improved biological capacities compared to their precursors [9]. The development of ‘kombucha’-like fermented beverages using alternative plant matrices to green or black tea would result in obtaining new “3S” (Safe, Healthy, and Sustainable) fermented beverages with alternative biological properties, providing additional health benefits to consumers [10].
Industrial activity in the agri-food sector, such as broccoli producers, is associated with the generation of large amounts of by-products, with a negative impact on the sustainability and competitiveness of sectorial industries [11]. In the search for alternative plant materials with the potential to be used as ingredients for the development of new fermented beverages, broccoli by-products have been pointed to as a very good option based on their features as sources of bioactive phytochemicals (e.g., glucosinolates (GSL), isothiocyanates (ITC), and indoles). These compounds have been demonstrated on a valuable capacity to modulate several pathophysiological situations [12], thus contributing to the diversity of biological benefits associated with broccoli consumption [13]. In this regard, sulforaphane (SFN) is of particular relevance, being the bioactive organosulfur compound most widely characterized in relation to its anti-inflammatory and anti-tumoral effects in humans [14].
Despite this potential as a source of bioactive compounds, broccoli by-products are currently affected by a gap in consolidated and sustainable methods of valorization [15]. Achieving this objective has provided stabilized material with preserved phytochemical content that could constitute the basis for the development of new, fermented “3S” beverages. The valorization of broccoli by-products in this way will increase the competitiveness of broccoli production and boost the panel of marketable fermented beverages by incorporating new nutritional and functional profiles [10].
Based on this background, the present study aims to obtain a new “3S” fermented beverage with a phytochemical profile differentiated from traditional kombucha, providing an alternative use of broccoli by-products, similar to additional ‘kombucha’-like beverages described in the literature [16], to create new added-value co-products. For this purpose, a new fermented beverage similar to ‘kombucha’, rich in organosulfur compounds, was developed and evaluated. The new beverage obtained in the frame of the present work has been characterized in relation to physicochemical parameters (°Brix, pH, and acidity), as well as the content of bioactive compounds (GLS, ITC, and indoles).

2. Materials and Methods

2.1. Reagents

Commercial standards of glucoraphanin, glucoerucin, glucoiberin, glucobrassicin, hydroxyglucobrassicin, gluconasturtin, methoxyglucobrassicin, neoglubobrassicin, and 3,4-diindolylmethane (GR, GE, GI, GB, HGB, PE, MGB, NGB, and DIM, respectively) were purchased from Phytoplan GmbH (Heidelberg, Germany). Standards of sulforaphane, sulforaphane-N-acetylcysteine, iberin, and indole-3-carbinol standards (SFN, SFN-NAC, IB, and I3C, respectively) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA), Biorbyt LTD (Cambridge, UK), and LKT Laboratories (St. Paul, MN, USA), respectively. Acetic acid and ammonium acetate were purchased from Panreac labs (Barcelona, Spain). Methanol and acetonitrile (analytical grade) were purchased from J.T. Baker (Philipsburg, NJ, USA). For the preparation of reagents and mobile phases, Milli-Q water was used (Milli-Q system (Millipore, Bedford, MA, USA).

2.2. Plant Material and Beverage Development

The broccoli by-products were obtained from broccoli plants (Brassica oleracea var. Italica, cv. ‘Parthenon’, Sakata Seeds Iberica, Valencia, Spain) grown in the winter of 2021, under agro-climatic conditions, in the semiarid area of southeastern Spain, in the experimental field of ‘La Matanza’, CEBAS-CSIC (Murcia, Spain), until developing commercial inflorescences. The stalks were transferred to the laboratory for processing (Laboratorio de Fitoquímica y Alimentos Saludables (LabFAS), CEBAS-CSIC, Murcia, Spain). The period between field sampling and laboratory processing was less than four hours so as to preserve the phytochemical burden.
In the laboratory, the stems were cut into fragments of no more than 2 cm in diameter and oven-dried to constant weight (at 40 °C for 72 h). The dried material was ground, stored under vacuum conditions, and protected from light until analysis. Quantitative profiles of organosulfur compounds (GSL, ITC, and indoles) were determined in the plant material as a reference for the phytochemical analysis of the new fermented beverage.
For the development of ‘kombucha’-like fermented beverages, two liters of broccoli stalks infusion were prepared by adding 20 g/L broccoli powder and 70 g/L sugar. The infusions were carried out for 15 min in mineral water at 90 ± 2 °C. Subsequently, the beverages obtained were allowed to cool to 25 °C for approximately 4 h and filtered through a 0.5 mm sized pore mesh to remove plant material. Once at 25 °C, a commercial SCOBY (Zygosaccharomyces lentus and Zygosaccharomyces bisporus, among other strains) was added to every two liters of infusion (broccoli ‘kombucha’-like beverage). As a control beverage, an additional two liters were also prepared by adding 20 g/L broccoli powder and 70 g/L sugar, tempered at 25 °C, without SCOBY addition. For analytical purposes, beverage samples were taken at day zero and each day for 12 days. The collected samples were filtered through 0.22 µm PVDF filters (Millipore, MA, USA) and analyzed for total soluble solids, pH, and titratable acidity (TA), as well as GSL, ITC, and indole content.

2.3. Physical-Chemical Parameters

The pH was determined as an indicator of the physicochemical quality of the broccoli-based ‘kombucha’-like beverage. Samples were analyzed in triplicate (n = 3) according to the methodology described in the literature [17,18,19]. Total soluble solids (°Brix) and titratable acidity (TA) were measured using the Pocket Brix-Acidity meter Master-Kit (ATAGO, Tokyo, Japan) and expressed as the percentage of acetic acid.

2.4. Analysis of Glucosinolates via HPLC-PDA-ESI/MSn

For the extraction of the compounds of interest, samples (100 mg) were homogenized in 1 mL of ethanol/deionized water (50:50, v/v) and extracted for 20 min at 70 °C, shaking every 5 min. The extracts were centrifuged at 4000 rpm for 5 min at 4 °C. The supernatants were collected, filtered through 0.22 µm PVDF filters (Millipore, MA, USA) and stored at −20 °C until further chromatographic analysis. Chromatographic separation and mass spectrometry analysis (negative mode) of GSL was developed according to the methodology described by Baenas et al. (2017) and Abellán et al. (2021) [17,18] (Table 1).
The identification of GSL present in the plant material and broccoli-based ‘kombucha’-like beverage was based on retention times, parent ions, and ionic products compared to previous descriptions in the literature [15]. The quantification of GSL was performed based on chromatograms recorded at 227 nm, applying standard curves freshly prepared on each day of analysis, obtaining regression coefficients (r2) ≥ 0.99 for all GSL monitored.

2.5. Analysis of Isothiocyanates and Indoles via UHPLC-ESI-QqQ-MS/

The chromatographic separation of the ITC and indoles present in the analytical extracts and the broccoli-based fermented beverage developed was performed according to the methodology described by Domínguez-Perles et al. (2014) and Baenas et al. (2017) [17,20]. In brief, we used a Zorbax Eclipse Plus C18 chromatographic column (2.1 × 50.0 mm, 1.7 µm) integrated into a UHPLC coupled with a triple-quadrupole-MS/MS detector model 6460 (Agilent Technologies, Waldbronn, Germany), operated in positive ionization mode.
The identification of the ITC and indoles was performed on the basis of their retention time, parentage, and specific fragmentation in comparison to commercial standards, as well as information gathered from metabolomic databases and the scientific literature (Figure 1). The concentration of the identified compounds was calculated on the basis of standard curves freshly prepared on each day of analysis with authentic standards, obtaining regression coefficients (r2) ≥ 0.99 for all ITC and indoles monitored.

2.6. Statistical Analysis

The results are presented as the mean ± standard deviation (n = 3). To select the statistical test, an analysis of normality and homogeneity of variance was performed using the Kolmogorov–Smirnov and Levene tests, respectively. In accordance with the normal distribution of the results obtained, paired t-tests were used to determine the significance of differences between the values recorded. The level of statistical significance was set at p < 0.05.

3. Results

3.1. Physical-Chemical Parameters

The broccoli-based fermented beverage developed showed statistically significant differences in total soluble solids (°Brix), pH, and TA compared to the control (Figure 2). While values for total soluble solids did not significantly change during the first 6 days, between days 7 and 12, in the broccoli stem-based fermented beverage, this parameter significantly decreased to reach a value of 4.80 °Brix (Figure 2A). These values are in good agreement with Guzman (2021), who described final values of 5.00 °Brix during the development of traditional ‘kombucha’ [21].
The most relevant factor for obtaining the highest concentration of total soluble solids and, consequently, the sweetness of the beverage has been attributed to the percentage of sugar, while the amount of plant material and the number of days of fermentation are not decisive for the final concentration of total soluble solids reached [21]. The total soluble solids of the broccoli-based ‘kombucha’-like beverage in comparison to the control beverage gradually decreased between days 0 and 12, indicating that the microbial activity in the newly developed fermented beverage could use the nutrients provided by the broccoli plant material and the sugar content in agreement with previous descriptions in the literature [22].
In relation to pH, the control beverage maintained constant values ranging between 6.32 and 5.03. However, the broccoli beverage rapidly and significantly reduced the values of this parameter up to 4.15, subsequently decreasing more gradually to 3.64 after twelve days, showing significantly lower values during the entire period of development of the “3S” fermented beverage (Figure 2B).
These values are in good agreement with previous studies determining the stability of ‘kombucha’ since this is favored by pH values of around 4.00 [21]. Finally, following matching evolution relative to pH, the analysis of TA showed a significant increase from day 6 after brewing (Figure 2C). The control beverage, which did not show significant changes in the TA values, maintained an average value of 0.8% acetic acid. From day 6, the broccoli ‘kombucha’-like beverage provided mean values of 1.2% acetic acid (Figure 2C). Previously, it has been suggested the modification of pH could be enclosed in the generation of organic acids using the symbiotic culture of bacteria and yeast [23].
The most relevant factor involved in the changes in TA in fermented beverages was associated with the amount of plant material/volume of water ratio used in their development. Also, the fermentation time (as can be visualized in the evolution graph for days 0–5 and 6–12) constitutes a critical factor (Figure 2C). Therefore, TA is an easily modifiable parameter through adjustments in the design of the beverage ingredients and elaboration process [21].

3.2. Glucosinolate Content of Plant Material

Prior to the development of the broccoli beverage, the GSL concentration of the stems used as raw materials, precursor organosulfur compounds, and bioactive metabolic derivatives (ITC and indoles) were analyzed (Figure 3).
This characterization revealed a high concentration of GSL in the following decreasing order of concentration: GR (2212.52 mg/kg dw) > GI (566.64 mg/kg dw) > MeGB (416.89 mg/kg dw) > GE (244.20 mg/kg dw) > GB (113.27 mg/kg dw) > NeoGB (105.94 mg/kg dw) (Figure 3). The HGB was below the limit of the quantification (LOQ) values, and the bioactive derivatives SFN, erucin, and I3C were recorded at very low levels, obtaining, in general, concentrations coincident with characterizations previously described in the literature [24].
It is important to note that SFN resulting from the enzymatic hydrolysis of GR, which is the best characterized and abundant bioactive organosulfur compound in broccoli, was almost absent in the plant material. This fact is based on the need for enzymatic hydrolysis reactions of the glucose residue present in the GSL structure. Indeed, this hydrolysis reaction is catalyzed by the β-thioglucosidase enzyme present in the plant material (myrosinase). Nonetheless, the plant enzyme is degraded to some extent during the dehydration process of the plant material, which would reduce its capacity to hydrolyze GSL towards their bioactive counterpart (ITC and indoles) [17]. Consequently, the broccoli stalks stabilized via oven-drying were to be used as an ingredient for the development of the new fermented beverage, present a high concentration of GR, as a pre-cursor of SFN, which is not hydrolyzed by plant-myrosinase.” By the version “Consequently, the broccoli stalks stabilized via oven-drying could be used as an ingredient for the development of the new fermented beverage, presenting a high concentration of GR, as a pre-cursor of SFN, which is not hydrolyzed by plant-myrosinase. Alternatively, this GSL could be hydrolyzed by the enzymes with β-thioglucosidase activity of microbial origin [25], tentatively including the various bacteria/yeast species and strains making up the SCOBY. In this regard, the actual microorganism responsible for the β-thioglucosidase activity needs to be clarified.

3.3. Bioactive Organosulfur Compounds Content of Broccoli Kombucha

Based on the analysis of the broccoli-based fermented beverage developed in relation to the organosulfur bioactive compounds, the absence of all GSL was recorded in both the control samples and the broccoli-based fermented beverages, which are of special relevance for GR, the most abundant GSL in the broccoli by-products. However, SFN, the hydrolysis product of GR, was found in high concentrations, while the precursor GSL (GR) was not found in the beverages (Figure 4).
The SFN concentration increased from day 2 in the fermented beverage developed using broccoli stalks as a plant-based ingredient, reaching its highest concentration on day 5 (31.39 µg/100 mL). This concentration rapidly decreased in the following days until it reached unquantifiable levels on day 9. This evolution allowed the detection of, in the broccoli-based beverage, concentrations significantly higher than the control (where no quantifiable levels of SFN were found) between days 1 and 7 (Figure 4A). It is important to highlight that these concentrations were slightly lower than those previously described in fermented beverages, such as beer, in which between 14.19 and 18.85 µmol/L have been reported [10,26].
In the plant material, the GSL are hydrolyzed enzymatically to give rise to ITC and indoles with the concourse of myrosinase, a plant present in specific vacuoles of the plant cells after the disruption of the plant material integrity. Concerning the major GSL described in broccoli, namely GR, several factors could modulate the efficiency of its enzymatic hydrolysis to form SFN, including pH, temperature, or the presence/absence of cofactors [27]. Beyond the plant’s β-thioglucosidase, during the fermentation of the plant (broccoli) material, the microbial populations involved in this process also accounted for the enzymatic capacity to hydrolyze GSL to form ITC and indoles [10,26]. Accordingly, the differences detected between ‘kombucha’-like beverage and the previously characterized broccoli-based beers could be attributable to the metabolic capacity of the different microorganisms used in their development, the concentration of the GR precursor in the raw material, and the processing conditions [26].
Thus, the optimum pH for maximum plant β-thioglucosidase activity varies between 6.00 and 7.00, while the newly developed beverage maintained it between 4.00 and 5.00. These values could decrease the enzymatic activity of the plant myrosinase, thus reducing the formation of SFN. Nevertheless, these pH values would constitute a valid range for the activity of the microbial β-thioglucosidases, whose optimal pH is in the range 5.20–7.60, thus allowing the hydrolysis of GR to form SFN [25,28,29].
An additional factor that could influence the transformation of GSL into ITC is the presence in the matrix of the new fermented beverage of free iron (Fe2+ and Fe3+), since this is a necessary co-factor for the optimal activity of the enzyme β-thioglucosidase [26]. In this regard, this factor should be taken into consideration in future formulations of the new fermented beverage to optimize the concentration of the bioactive compound of interest. The fact that this new broccoli-based fermented beverage presents a valuable concentration of bioactive SFN that is not found in traditional green/black tea-based ‘kombucha’ provides a new product designed with an additional and differential biological scope that could meet well with market demand.
Also, presumably, as a result of bacterial and yeast metabolism, SFN was metabolized to sulforaphane-N-acetylcysteine (SFN-NAC), whose maximum concentration was reached after 7 days of processing (5.37 µg/100 mL) and as a result of the decrease in the concentration of free SFN. After its formation, the level decreased to 2.90 µg/100 mL (Figure 4B), remaining at constant levels until the end of day 12 for finalizing the development of the broccoli stalks-based fermented beverage. Currently, the metabolization of SFN to SFN-NAC as a result of microbial metabolism has not been described, which should be explored in the near future, especially due to the interest that the bioactivity of this compound has aroused in recent years, as it is a powerful anti-inflammatory compound [30,31].

4. Conclusions

The development of a new ‘kombucha’-like fermented beverage based on broccoli by-products provides an alternative dietary source of bioactive organosulfur compounds present in this vegetable, which have been widely studied in relation to their beneficial effects on health. Thus, this study of the conversion of the individual GSL generated as a result of the microbial fermentation of a broccoli by-product infusion has provided evidence of the transformative potential of this type of processing and the improvement in the health properties of foods. Concerning the evolution of the quality and compositional parameters considered in the present characterization, the development process of traditional ‘kombucha’ is enclosed to fermentation for 7 to 14 days, which coincides with the higher concentration of bioactive compounds generated as a result of the microbial metabolism of the different GSL. These results make it possible to describe new applications of broccoli by-products for the production of value-added products as a source of dietary bioactive compounds.

Author Contributions

Conceptualization S.M., C.G.-V. and R.D.-P.; methodology, S.M. and R.D.-P.; formal analysis, B.M.C. and R.D.-P.; investigation, B.M.C., S.M. and R.D.-P.; resources, C.G.-V. and S.M.; writing—original draft preparation, B.M.C.; writing—review and editing, S.M., C.G.-V. and R.D.-P.; supervision, S.M. and R.D.-P.; project administration, C.G.-V. and S.M.; funding acquisition, C.G.-V. and S.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the AGROALNEXT Program, which has been financed by MCIN, with NextGenerationEU funds and the Seneca Foundation, with funds from the Autonomous Community of the Region of Murcia (CARM) both of them via the project PRTR-C17.I1.

Data Availability Statement

Data are unavailable due to privacy issues.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. UPLC-QqQ-MS/MS parameters for the identification and quantification of isothiocyanates and indoles present in the fermented broccoli beverage in positive ionization mode.
Figure 1. UPLC-QqQ-MS/MS parameters for the identification and quantification of isothiocyanates and indoles present in the fermented broccoli beverage in positive ionization mode.
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Figure 2. Evolution of Brix (A), Ph (B), and titratable acidity (% acetic acid) (C) of the broccoli fermented beverage vs. control beverage during the 12 days of processing. The results are presented as the mean ± standard deviation (n = 3). According to the t-test performed, differences were detected at the statistical significance level of *** p < 0.001.
Figure 2. Evolution of Brix (A), Ph (B), and titratable acidity (% acetic acid) (C) of the broccoli fermented beverage vs. control beverage during the 12 days of processing. The results are presented as the mean ± standard deviation (n = 3). According to the t-test performed, differences were detected at the statistical significance level of *** p < 0.001.
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Figure 3. Glucosinolate, isothiocyanates, and indoles content (mg/kg dw) of dehydrated broccoli stalk used as a raw material for making fermented broccoli beverage. The results are presented as the mean ± standard deviation (n = 3). LOQ, limit of quantification.
Figure 3. Glucosinolate, isothiocyanates, and indoles content (mg/kg dw) of dehydrated broccoli stalk used as a raw material for making fermented broccoli beverage. The results are presented as the mean ± standard deviation (n = 3). LOQ, limit of quantification.
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Figure 4. Evolution of the sulforaphane (A) and sulforaphane-N-acetylcysteine (B) content (µg/100 mL) of the broccoli fermented beverage vs. control beverage during the 12 days of processing. The results are presented as the mean ± standard deviation (n = 3). According to the t-test performed, differences were detected at the following statistical significance levels: * p < 0.05, ** p < 0.01, and *** p < 0.001.
Figure 4. Evolution of the sulforaphane (A) and sulforaphane-N-acetylcysteine (B) content (µg/100 mL) of the broccoli fermented beverage vs. control beverage during the 12 days of processing. The results are presented as the mean ± standard deviation (n = 3). According to the t-test performed, differences were detected at the following statistical significance levels: * p < 0.05, ** p < 0.01, and *** p < 0.001.
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Table 1. Parameters applied to the analysis of the qualitative profile of individual glucosinolates in plant material (broccoli (Brassica olreacea var. italica L. cv. ‘Parthenon’)) stems and broccoli beverages via HPLC-PDA-ESI/MSn working in negative ionization mode.
Table 1. Parameters applied to the analysis of the qualitative profile of individual glucosinolates in plant material (broccoli (Brassica olreacea var. italica L. cv. ‘Parthenon’)) stems and broccoli beverages via HPLC-PDA-ESI/MSn working in negative ionization mode.
GlucosinolateRetention Time
(min)		Parental Ion (m/z [M-H]−)Ionic Product (m/z MS2[M-H]−)
Glucoiberin (GI)5.3422259, 97
Glucoraphanin (GR)6.0436372, 259, 97
Hydroxyglucobrassicin (HGB)16.2463285, 241, 97
Glucoerucine (GE)18.9420259, 97
Glucobrassicin (GB)20.5447404, 259, 97
Gluconasturtine (PE)23.0422259, 97
Methoxyglucobrassicin (MGB)25.6477259, 97
Neoglucobrassicin (NGB)27.8477446, 259, 97
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MDPI and ACS Style

Cánovas, B.M.; García-Viguera, C.; Medina, S.; Domínguez-Perles, R. ‘Kombucha’-like Beverage of Broccoli By-Products: A New Dietary Source of Bioactive Sulforaphane. Beverages 2023, 9, 88. https://doi.org/10.3390/beverages9040088

AMA Style

Cánovas BM, García-Viguera C, Medina S, Domínguez-Perles R. ‘Kombucha’-like Beverage of Broccoli By-Products: A New Dietary Source of Bioactive Sulforaphane. Beverages. 2023; 9(4):88. https://doi.org/10.3390/beverages9040088

Chicago/Turabian Style

Cánovas, Berta María, Cristina García-Viguera, Sonia Medina, and Raúl Domínguez-Perles. 2023. "‘Kombucha’-like Beverage of Broccoli By-Products: A New Dietary Source of Bioactive Sulforaphane" Beverages 9, no. 4: 88. https://doi.org/10.3390/beverages9040088

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