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Development of flavored kombuchas with Amazonian fruits: bioactive compounds evaluation and antioxidant capacity

Abstract

Using Amazonian fruits to flavor kombuchas is a promising proposal, as it adds nutritional value to the drink. This work sought to develop kombucha flavored with Amazonian fruits and evaluate the bioactive compounds and antioxidant capacity of the formulations. Three kombucha formulations were prepared using green tea (Camellia sinensis) and three Amazonian fruits: cupuassu (Theobroma grandiflorum), tapereba (Spondias lutea L.) and bacuri (Platonia insignis). Kombucha fermentations were evaluated before and after the insertion of nectars through the analysis of phenolic compounds, vitamin C and antioxidant capacity. Analyzes of pH, total sugars, acetic acid, ethanol, and microbiological characterization of final formulations were also carried out. For the first fermentation, were found values of phenolic compounds and antioxidant capacity of 30.60 ± 0.93 mg EAG/L and 295.02 ± 5.59 µmol ET/mL, and the formulation with tapereba showed the highest values for total phenolic compounds (34.92 ± 12.25 mg EAG/L), antioxidant capacity (320.57 ± 9.53 µmol ET/mL) and vitamin C (198.25 mg/100g). Thus, the formulations developed had a crucial nutritional appeal to stimulate consumption by the population, in addition to enabling the valorization and addition of commercial value to the Amazonian fruits used.

Key words
Cupuassu; Bacuri; Flavoring; Fermentation; Vitamin C; Symbiotic Colony

INTRODUCTION

Kombucha is a fermented food originated in Asia, based on black tea or sweetened green tea (5-10% sugar) and a biofilm of bacteria and yeast called SCOBY (Symbiotic Colony of Bacteria and Yeast) (Marco et al. 2021MARCO ML ET AL. 2021. The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on fermented foods. Nat Rev Gastroenterol Hepatol 18: 196-208.). The final product is obtained after at least 14 days of the mixture fermentation. It is important not to confuse this product with tisanes (infusion with other herbs), produced by infusing dry or fresh leaves, flowers, or roots of other plants (Freitas et al. 2022FREITAS A, SOUSA P & WURLITZER N. 2022. Alternative raw materials in kombucha production. Int J Gastron Food Sci 30:100-128.). Large-scale consumption of this beverage is recommended due to the high antioxidant capacity due to the bioactive compounds present in tea after fermentation, which makes it beneficial to health (Dutta & Paul 2019DUTTA H & PAUL SK. 2019. Kombucha Drink: Production, Quality, and Safety Aspects. Product Manag Beverag 1: 259-288.), in addition to other benefits, such as reduction and control of cholesterol and diabetes levels (Bellassoued et al. 2015BELLASSOUED K, GHRAB F, MAKNI-AYADI F, PELT JV, ELFEKI A & AMMAR E. 2015. Protective effect of kombucha on rats fed a hypercholesterolemic diet is mediated by its antioxidant activity. Pharm Biol 53: 1699-1709., Hosseini et al. 2015HOSSEINI SA, GORJIAN M, RASOULI L & SHIRALI S. 2015. A Comparison between the Effect of Green Tea and Kombucha Prepared from Green Tea on the Weight of Diabetic Rat. Biosci Biotechnol Res Asia 12: 141-146.).

Kombucha is made using various subtypes of Camellia sinensis, such as black tea and green tea. However, other inputs are part of its production, both in the first fermentation and the second fermentation (tasting stage), such as fruit pulp, plant extracts, other teas, and spices (Zubaidah et al. 2022ZUBAIDAH E, DEA EC & SUJUTI H. 2022. Physicochemical and microbiological characteristics of kombucha based on various concentration of Javanese turmeric (Curcuma xanthorrhiza). Biocatal Agric Biotechnol 924: 102467.). Thus, flavouring kombucha with different types of fruit, such as those of Amazonian origin, is a promising proposal, considering that some fruits from the region have a high amount of bioactive compounds with high antioxidant activity, which can add nutritional value to the food drink (Santos et al. 2018SANTOS RMB, CHAGAS PC, ROCHA JHMV, CHAGAS EA, PANDURO MHP, LOZANO RMB & RODRIGUEZ CA. 2018. Cadeia de produção do camu-camu (myrciaria dubia (kunth) mc vaugh): o caso das regiões produtoras de loreto e ucayali na amazônia peruana. Interciencia 43: 261-268.).

In addition, to obtain a quality fermented beverage, the proportion of its components (tea, sugar, and culture) must be well adjusted. Therefore, it is noteworthy that the amount of sugar (sucrose) used directly interferes with the acidity and pH of the product since the variable acts as a substrate for the generation of carbon dioxide, ethanol, and organic acids by microorganisms (Nascimento & Lima 2019NASCIMENTO LCN & LIMA MDE. 2019. Influência de diferentes fontes de açúcar sobre as propriedades físicas do kombucha. Em: Congresso Brasileiro de Engenharia Química em Iniciação Científica. Uberlândia. Retrieved from: https://www.semanticscholar.org/paper/influ%c3%8ancia-de-diferentes-fontesde-a%c3%87%c3%9acar-sobre-as-nascimentolima/43c338e081e018ff5221d23e96e13556169f9a17.
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). The organic compounds produced by kombucha give the drink a functional character, as many studies show the relationship between a healthy diet and the consumption of its compounds (such as flavonoids), in addition to attesting to its role in combating chronic diseases such as obesity, acting as an inflammation modulator (Dário & Weschenfelder 2020DÁRIO PA & WESCHENFELDER S. 2020. Benefícios e características da Kombucha: Uma revisão. Rev Bras Agrotec 10: 01-12.). The long history of consumption of this beverage has global appeal and has demonstrated through the scientific literature antimicrobial effects and benefits to the general health of the consumer (Mohsin et al. 2022MOHSIN AZ, NOR NAN, MUHIALDIN BJ, ROBY BH, MAHMOODABADL M, ASYILAMARZLAN A, HUSSAIN N & HUSSIN AS. 2022. The effects of encapsulation process involving arabic gum on the metabolites, antioxidant and antibacterial activity of kombucha (fermented sugared tea). Food Hydrocoll Health 2:100-130.).

Several Amazonian fruits have great commercial appeal for consumption due to their flavor, color, and intrinsic health benefits that are associated with nutritional and antioxidant properties, such as cupuaçu (Alvarez et al. 2017), bacuri (Yamaguchi et al. 2021YAMAGUCHI KKL, DIAS DS, LAMARÃO CV, CASTELO KFA, LIMA MS, ANTONIO AS, CONVERTI A, LIMA ES & VEIGA-JUNIOR VF. 2021. Amazonian Bacuri (Platonia insignis Mart.) Fruit Waste Valorisation Using Response Surface Methodology. Biomolecules 11: 1-2.) and taperebá (Aniceto et al. 2021ANICETO A, MONTENEGRO J, CADENA R DS & TEODORO AJ. 2021. Physicochemical Characterization, Antioxidant Capacity, and Sensory Properties of Murici (Byrsonima crassifolia (L.) Kunth) and Taperebá (Spondias mombin L.) Beverages. Molecules 26: 1-13.). Combining these characteristics with the already recognized properties of kombucha allows the development of commercially attractive biotechnological products with high nutritional potential.

Although there are already many commercial kombuchas, there are practically no products flavored with Amazonian fruits. For that reason, this work demonstrates innovation by developing three different kombucha formulations, with the inclusion of recognized Amazonian fruits, with the premise of obtaining new products with a differentiated taste, high antioxidant and nutritional capacity.

MATERIALS AND METHODS

Materials

The green tea used was obtained from the company CHÁ DÕ® (São Paulo, Brazil) and the SCOBY colony was donated from the city of Belém. Sugar (União, São Paulo, Brazil) and fruit pulp from Petruz® (Belém, Brazil) were purchased from a supermarket chain in the city of Belém.

Methods

Tea preparation

To prepare the tea, 2 liters of water were boiled at 100°C, leaving it in the boiling phase for 3 minutes so that all the chlorine present was removed (Silva et al 2021). 14 g of Camellia Sinensis herb (green tea) was added and left to infuse for 15 minutes, with the addition of 40 g of sugar. The preparation was filtered and the substrate (mixture of tea + sugar) was left to cool at room temperature.

First fermentation

The first fermentation was carried out by adding the SCOBY to the previously prepared and sweetened tea. The SCOBY used already contained a small amount of green tea, in which it is normally stored. The mixture was added to a 2 L beaker at 25 °C and the container was closed and stored for 7 days (Figure 1).

Figure 1
The production stages of kombucha fermentations.

Characterization of the first fermentation process

The following analyzes were performed: determination of pH, using a calibrated benchtop pHmeter (Onda Digital®), titratable total acidity (using a standardized 0.1 N NaOH solution), total soluble solids with a digital refractometer (Kasvi®), all analyzes followed AOAC (2019)AOAC - ASSOCIATION OF OFFICIAL ANALYTICAL CHEMISTS. 2019. Official Methods of Analysis, 17th ed, AOAC International, Arlington..

Nectar and syrup preparation

The nectars were standardized based on legislation (Brazil 2013BRAZIL. 2013. Leis, Decretos, Resoluções, Portarias. Instrução Normativa no 42 de 11 de setembro de 2013. Alteração do artigo 3º da Instrução Normativa no. 12, de 4 de setembro de 2003. Brasília/DF: Ministério da Agricultura, Pecuária e Abastecimento, 2 p.). 100 g of pulp were used for all the preparations, while the amounts of sugar and water were defined by preliminary tests, generating the final formulations (Table I). For the bacuri nectar, 150 mL of water and 10 g of sugar were used, and for the taperebá nectar, 100 mL of water and 15 g of sugar. The cupuaçu pulp was made into syrup, to which 100 mL of water and 50 g of sugar were added. All the mixtures were homogenized, filtered and stored at room temperature.

Table I
Composition of Formulations.

Second fermentation

For the second fermentation, 3 bottles of polyethylene terephthalate with a capacity of 500 mL were used, in which the product of the first fermentation was added: “initial kombucha” and 20% nectar or syrup, values shown below in Table I (Kombucha Brewers International, 2021). The container was closed, homogenized, and placed in an environment without contact with light for a period of 24-36 hours.

Fermented products characterization

Initial kombucha and flavored kombuchas pH, acidity, soluble solids and reducing sugars

The pH, total titratable acidity and soluble solids were determined using the same parameters explained in the previous topic. Furthermore, analyses of reducing sugars were performed by DNS in a spectrophotometer (Cirrus 80 PR, Femto®) at 540 nm. For such analysis, a 1.0 g/L glucose solution was used for the calibration curve. A 2 N NaOH solution and a DNS reagent solution were prepared, containing 1 g of 3,5-dinitro salicylic acid, 30 g of double sodium potassium tartrate, and 20 mL of NaOH 2N solution following the methodology described by Miller (1959)MILLER GL. 1959. Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Anal Chem 31: 426-428..

Total phenolic compounds and antioxidant capacity

The analysis of total phenolic compounds followed the Folin-Ciocalteu method described by Singleton & Rossi (1965)SINGLETON VL & ROSSI JA. 1965. Colorimetry of Total Phenolics with Phosphomolybdic Phosphotungstic Acid Reagent. Am J Enol Vitic 16: 144-158., and it took place in a microplate reader (NovoStar, BMG LabTech®) with a filter of 725 nm. A calibration curve was prepared with gallic acid (Sigma-Aldrich ®) at a concentration ranging from 25-200 µg/mL. The results were expressed in mg of gallic acid equivalents (GAE)/mL or mg of extract.

The DPPH method (stable free radical 2,2-diphenyl-1-picrylhydrazyl) followed the method described by Macedo et al. (2011)MACEDO JÁ, BATTESTIN V, RIBEIRO ML & MACEDO GA. 2011. Increasing the antioxidant power of tea extracts by biotransformation of polyphenols. Food Chem 126: 491-497. to assess the antioxidant capacity of kombuchas. The analyses and reactions took place in a 96-well microplate reader (NovoStar, BMG LabTech®) at an absorbance of 520 nm. The reaction mixture followed the sequence: 50 µL of the sample under study was mixed with 150 µL of DPPH reagent (Sigma-Aldrich®) prepared at 0.2 mM in methanol. After the reaction period (90 min), DPPH decolorization was measured. The results were expressed as μmol Trolox equivalent (TE)/mL of kombucha.

Vitamin C content

The test was carried out according to Strohecker (1967)STROHECKER RL & HENNING H M.1967. Análises de vitaminas: Métodos comprovados. Madrid: Editora Paz Montalvo, 428 p., who based it on Tillmans’ titrimetric method, in which the ascorbic acid content is measured by reducing the reagent 2,6-dichlorophenolindophenol (DFI). Solutions of 1% oxalic acid, 0.2% sodium 2,6-dichlorophenolindophenol and 50 mg/mL ascorbic acid were used. After standardizing the DFI solution, 4 mL of the sample extracts were collected and added to a 125 mL erlenmeyer flask with 46 mL of deionized water, which was titrated with the standardized DCFI solution until a persistent pink color appeared for 15 seconds. The results were expressed in mg of vitamin C/100g of extract.

Ethanol content by HPLC

Ethanol was quantified in the samples using the methodology described by Oliveira et al. (2020)OLIVEIRA JAR, CONCEIÇÃO AC, SILVA MARTINS LH, MOREIRA DKT, PASSOS MF & KOMESU A. 2020. Evaluation of the technological potential of four wastes from Amazon fruit industry in glucose and ethanol production. J Food Process Eng 44: 1-11.. A 300 mm × 7.8 mm column (HPX-87H,Aminex®) contained in a liquid chromatograph (1260 Infinity, Agilent®) was used. This equipment had an IR refractive index detector and UV-vis DAD. The mobile phase used was 0.05 M H2SO4 with an eluent flow rate of 0.6 ml/min at 30°C. 15 μl of the sample were injected and the total analysis time was 30 min.

Microbiological analyzes

Aerobic mesophiles, molds, yeasts and thermotolerant coliforms were analyzed. 25 ml of sample was diluted in 225 ml of distilled water for all counts. The mesophile count was carried out according to Apha (1992)APHA - AMERICAN PUBLIC HEALTH ASSOCIATION. 1992. Compendium of methods for the microbiological examination of foods 3. ed, Am Public Health Assoc, Washington, 1219 p., by taking 1 ml from three or more dilutions and placing it in a Petri dish with Plate Count Agar (PCA) culture medium (Kasvi ®), followed by incubation in an oven at 35°C for 48 hours. Counting was carried out on a Quebec counter and plates containing 30 to 300 colonies were selected.

For molds and yeasts analyses, the DRBC agar base medium (Kasvi®) was used. 0.01 µL of sample was inoculated onto plates with solidified medium and spread with a Drigalski loop. The plates were incubated in an oven at 37°C for 72 hours (Apha 1992APHA - AMERICAN PUBLIC HEALTH ASSOCIATION. 1992. Compendium of methods for the microbiological examination of foods 3. ed, Am Public Health Assoc, Washington, 1219 p.). Finally, thermotolerant coliforms were analyzed using Lauryl Sulphate broth (Kasvi®). The presumptive test was carried out by immersing the inverted Duran tube inside a test tube with the sample and incubating it in an oven at 37°C for 24 to 48 hours (Silva et al 2017).

Statistical Analysis

For statistical evaluation, all the samples were tested in triplicate and the means and standard deviations were calculated. A comparison was performed between the samples using the Tukey test (p ≤ 0.05), using the Minitab 16.1.1 statistical program.

RESULTS AND DISCUSSION

Characterization of the fermentation process of initial kombucha

Figure 2 shows the parameters evaluated over the 14 days of the first fermentation. According to the figure, it was possible to verify a standard behavior of the kombucha fermentation processes, in which there is, over time, a reduction in the values of soluble solids (°Brix), a reduction in the pH values with a consequent increase in acidity, caused mainly by the increase in acetic acid levels (Zubaidah et al. 2018ZUBAIDAH E, DEWANTARI FJ, NOVITASARI FR, SRIANTA I & BLANC PJ. 2018. Potential of snake fruit (Salacca zalacca (Gaerth.) Voss) for the development of a beverage through fermentation with the Kombucha consortium. Biocatal Agric Biotechnol 13: 198-203.).

Figure 2
Variation of values of Brix° (a), pH (b) and Acidity (c) during the first.

Figure 2. Variation of values of Brix° (a), pH (b) and Acidity (c) during the first fermentation.

The average values of soluble solids along the first ranged from 1.2±0.00-0.4±0.00 °Brix; for the pH values, the range of 3.28 ± 0.005 – 3.13 ± 0.025 was found and finally, the increase in acidity during the analyzed period (0.18 ± 0.01- 0.41 ± 0.00 g.100 mL-1), the decrease in soluble solids, and the increase in acidity occurred due to the consumption of sucrose during the fermentation process and the production of organic acids, such as acetic acid, by aceto-acid bacteria present in the symbiotic colony (Muzaifa et al. 2022MUZAIFA M, ROHAYA S, NILDA C & HARAHAP KR. 2022. Kombucha Fermentation from Cascara with Addition of Red Dragon Fruit (Hylocereus polyrhizus): Analysis of Alcohol Content and Total Soluble Solid. In: International Conference on Tropical Agrifood, Feed and Fuel (ICTAFF-2021). Samarinda, Indonesia. Retrieved from: https://www.atlantispress.com/article/125968331.
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).

Physicochemical characterization of initial kombucha and flavored kombuchas

The values quantified in the characterization of traditional and flavored kombuchas, shown in table II, are in accordance with the safety and quality standards for the type of fermented beverage produced, provided for in Normative Instruction No. 41 (Brazil 2019BRAZIL. 2019. Ministério da Agricultura, Pecuária e Abastecimento. Estabelece o Padrão de Identidade e Qualidade da Kombucha em todo território nacional (Instrução Normativa n° 41, de 17 de setembro de 2019). Diário Oficial da República Federativa do Brasil.) and international legislation, according to Kombucha Brewers International – Code of Practice (Kombucha Brewers International 2021KOMBUCHA BREWERS INTERNATIONAL. 2021. Retrieved from: https://kombuchabrewers.org/.
https://kombuchabrewers.org/...
), about pH range, titratable acidity and amount of sugars.

Table II
Physical-chemical characterization of initial kombucha and flavorings.

Table II. Physicochemical characterization of initial kombucha and flavored kombuchas.

The pH values directly linked to acidity, present in Table II, maintain an inversely proportional relationship with the fermentation time so that more extended fermentation periods promote acidification of the medium and a consequent decrease in pH. Therefore, the data obtained for kombucha in the first fermentation (KF1) (pH = 3.2) and flavored drinks with pH values of 3.18; 3.10 and 2.98 for KB, KC and KT, respectively, have a significant decrease (p ≤ 0.05), which is coherent with the longer fermentation time and the addition of sugars from the nectars.

Acetic acid is one of the most prevalent organic compounds in kombucha, a result of the fermentation process of bacteria present in the drink, and responsible for the vinegary flavor (Amarasinghe 2018AMARASINGHE H, WEERAKKODY NS & WAISUNDARA VY. 2018. Evaluation of physicochemical properties and antioxidant activities of kombucha “Tea Fungus” during extended periods of fermentation. Food Sci Nutr 6: 659-665., Khosravi et al. 2019KHOSRAVI S, SAFARI M, EMAM-DJOMEH Z & GOLMAKANI MT. 2019. Development of fermented date syrup using Kombucha starter culture. J Food Process Preserv 43: 13872.). In the present work, the acetic acid calculated for kombucha KF1 e KT were similar (0.39 ± 0.00 and 0.39 ± 0.11 g acetic acid.mL-1), and statistically different when compared to the other flavored kombucha (KC= 0.49 ± 0.03 and KB=0.41 ± 0.01), which demonstrated higher acidity. The data obtained were higher than those determined by Abuduaibifu and Tamer (2019)ABUDUAIBIFU A & TAMER CE. 2019. Evaluation of physicochemical and bioaccessibility properties of goji berry kombucha. J Food Process Preserv 43: 1-14., in their flavored kombuchas with goji berry and black tea substrate and those observed by Neffe-Skocińska et al. (2017)NEFFE-SKOCIŃSKA K, SIONEK B, ŚCIBISZ I & KOŁOŻYN-KRAJEWSKA D. 2017. Acid contents and the effect of fermentation condition of Kombucha tea beverages on physicochemical, microbiological and sensory properties. CyTA - J Food 15: 601-607. (1.42 to 1.52 g. L-1).

The soluble solids analisis (°Brix) for the initial kombucha (1.0 ± 0.00) demonstrated lower amount than what is found in the literature, in studies with green tea-based kombucha at the end of the fermentation process (Treviño-Garza et al. 2020TREVIÑO-GARZA M, GUERRERO-MEDINA AS, GONZÁLEZ-SÁNCHEZ RA, GARCÍA-GÓMEZ C, GUZMÁN-VELASCO A, BÁEZ-GONZÁLEZ JG & MÁRQUEZ-REYES JM. 2020. Production of Microbial Cellulose Films from Green Tea (Camellia Sinensis) Kombucha with Various Carbon Sources. Coatings 10: 1132.). For Akarca (2021)AKARCA G. 2021. Determination of Potential Antimicrobial Activities of some Local Berries Fruits in Kombucha Tea Production. Braz Arch Biol Technol 64: 1-15., the significant reduction in soluble solids is associated with the hydrolysis of sucrose into the monosaccharides glucose and fructose, consumed by microorganisms for the production of different metabolites. However, there was a significant increase (p ≤ 0.05) in preparations with the Amazonian fruits (KC=4.01 ± 0.00; KT=2,00 ± 0.00; KB=2.00 ± 0.00), which was expected from the flavoring process.

Sugar is an essential element in the preparation of kombucha and its quality and quantity may or may not boost the fermentation process (Martínez et al. 2018MARTÍNEZ LJ, SUÁREZ LV, JAYABALAN R, OROS JH & ESCALANTE-ABURTO A. 2018. A review on health benefits of kombucha nutritional compounds and metabolites. CYTA J Food 16: 390-399.). Therefore, the reducing sugars estimated in the flavorings were significantly increased (p ≤ 0.05) compared to the initial kombucha (Table II).

The present work demonstrated lower values of reducing sugars than those found in the literature, such as Laver’s kombucha (Porphyra dentata), which presented 0.45 g.L-1 (Aung & Eun 2021AUNG T & EUN JB. 2021. Production and characterization of a novel beverage from laver (Porphyra dentata) through fermentation with kombucha consortium. Food Chem 350: 129-274.). In this view, to Neffe-Skocinska et al. (2017), fructose and glucose development through yeasts fermentation, the chosen substrate and the amount of sugar added during the process can increase the amount of reducing sugars.

The lowest value of ethanol we observed in KF1, which refers to kombucha without flavoring. The insertion of fruit pulps allowed the addition of sugars capable of promoting the chemical transformation characteristics. Thus, it is possible to verify the ethanol concentation varied between 0.54 - 5.07 g.L-1 , with KC being the most alcoholic, probably due to the higher content of added sugars in the form of commercial sucrose, and transformed by the action of scoby yeast, in ethanol. Nhan et al. (2020)NHAN HTH, VY CTT, NHAT NT & LINH VTK. 2020. Development of fermented beverages from white mulberry juice using the kombucha consortium. J Tech Educ Sci 60: 44-57. in their fermentation with the addition of typical fruit of Vietnam, found an ethanol value within the range obtained for developed formulations (0,88 g.L-1). Generally, Kombucha’s ethanol content generally ranges from 3.6 to 10 g/L-1, including values observed for developed kombuchas (Jayabalan et al. 2014JAYABALAN R, MALBAŠA RV, LONČAR ES, VITAS JS & SATHISHKUMAR M. 2014. A Review on Kombucha Tea-Microbiology, Composition, Fermentation, Beneficial Effects, Toxicity, and Tea Fungus. Compr Rev Food Sci Food Saf 13: 538-550., Greenwalt et al. 2000GREENWALT CJ, STEINKRAUS KH & LEDFORD RA. 2000. Kombucha, the fermented tea: Microbiology, Composition, and Claimed Health Effects. J Food Prot 63: 976-981.).

Besides, the highest levels of ethanol in the amazonian fruits flavored kombuchas is significant (p ≤ 0.05), when compared to the initial kombucha. Table III shows the results found for the analysis of phenolic compounds and antioxidant capacity.

Table III
Analysis of Phenolic Compounds and Antioxidant Activity.

The amount of phenolic compounds in kombucha is related to several factors, such as the type of tea used, the amount of sugar, fermentation time, and temperature (Antolak et al. 2021ANTOLAK H, PIECHOTA D & KUCHARSKA A. 2021. Kombucha Tea-A Double Power of Bioactive Compounds from Tea and Symbiotic Culture of Bacteria and Yeasts (SCOBY). Antioxidants 10: 15-41.). The values found for the phenolic compounds in the present work did not show any statistical difference between them at 95 % statistical confidence. It’s important to notice that there was an increase in total phenolic compounds in flavored formulations compared to KF1, which is explained by the addition of fruit nectars and the phenolic compounds biotransformation. Besides, the values estimated were smaller than those observed in the literature for other fruits (Dada et al. 2021DADA AP, LAZZARI A, CESTÁRIO ACO, SILVA DS, SARAIVA BR, ROSA CILF & PINTRO PTM. 2021. Caracterização de kombucha elaborada a partir de chá verde. Res Soc Dev 10: 1-21., Gramza-Michaiowska et al. 2016GRAMZA-MICHAłOWSKA A, KULCZYŃSKI B, XINDI Y & UMIENNA M. 2016. Research on the effect of culture time on the kombucha tea beverages antiradical capacity and sensory value. Acta Sci Pol Technol Aliment 15: 447-457., Kayisoglu & Coskun 2021KAYISOGLU S & COSKUN F. 2021. Determination of physical and chemical properties of kombucha teas prepared with different herbal teas. Food Sci Technol 41: 393-397.).

The bacuri flavored kombucha (KB) showed the highest value of total phenolic compounds (38.3 ± 0.93 mg GAE.L-1) compared to the formulation with tapereba (KT) (34.92 ±12.25 mg GAE.L-1) and cupuassu (KC) (33.40 ± 1.60 mg GAE.L-1). This fact may be related to the different phenolic profiles of each fruit and how these phenols behave in the face of a biotransformation process during fermentation (Wang et al. 2020WANG S, ZHANG L, QI L, LIANG H, LIN X, LI S, YU C & JI C. 2020. Effect of synthetic microbial community on nutraceutical and sensory qualities of kombucha. Int J Food Sci Tech 55: 3327-3333.).

Regarding antioxidant activity, when comparing the result obtained by kombucha from the first fermentation (295.02 ± 5.59 µmolET.mL-1) to some literature, our result was lower than that of Zou et al. (2021)ZOU C, LI RY, CHEN JX, WANG F, GAO Y, FU YQ, XU YQ & YIN JF. 2021. Zijuan tea-based kombucha: Physicochemical, sensorial, and antioxidant profile. Food Chem 363: 130-322.. Antioxidants are sensitive to some environmental factors, such as pH; temperature and oxidation time, and are usually related to the amount of phenolic molecules in the studied sample (Degirmencioglu et al. 2021 and Tang et al. 2022TANG Z ET AL. 2022. A Review on Fruit and Vegetable Fermented Beverage-Benefits of Microbes and Beneficial Effects. Food Rev Int 31:1-38.). The KB formulation showed the highest antioxidant activity value (307.98 ± 2.51 µmolET.mL-1), followed by the KT (295.39 ± 3.89 µmolET.mL-1) and KC (295,02 ± 5,59 µmolET.mL-1), similar to what was observed for total phenolic compounds.

Figure 3 shows the vitamin C estimated at the initial and final stage of the first fermentation, the fruit nectars and the same stages of the flavored kombucha formulations.

Figure 3
Quantification of vitamin C during kombucha production.

Figure 3. Quantification of vitamin C during kombucha production.

Regarding the quantification of vitamin C (Figure 3), after adding fruit nectars, the vitamin C levels of all formulations increased significantly (p ≤ 0.05). At the end of second fermentation, it was observed increased levels of vitamin C in all developed formulations, similarly when Annona muricata was used by Candra et al. (2021)CANDRA A, PRASETYO BE & TARIGAN JB. 2021. Study of vitamin C level of soursop leaves (Annona muricata l.) and galactomannan utilization in kombucha during fermentation. Em: The International Conference On Chemical Science and Technology (ICCST-2020): Chemical Science and Technology Innovation for Better Future. Medan, Indonesia.. The flavor with the highest concentration was kombucha with tapereba (398.38 ± 2.22 mg.100 mL-1), followed by bacuri flavoring (375.25 ± 2.22 mg. 100 mL-1) and finally the fermented drink with cupuassu (360.57 ± 13.12 mg.100 mL-1).

The value obtained for flavored kombucha with cupuassu, the sensitivity to sudden changes in temperature and light may be responsible for the decreased vitamin C levels in the drink. This fact was demonstrated by Vieira et al. (2000)VIEIRA MC, TEIXEIRA AA & SILVA CLM. 2000. Mathematical modeling of the thermal degradation kinetics of vitamin C in cupuaçu (Theobroma grandiflorum) nectar. J Food Eng 43: 1-7., who evaluated the kinetics of the degradation of ascorbic acid present in cupuassu nectar, which proved to be unstable to various external factors.

After the first fermentation (KF1T0), the vitamin C decreased from 112.98 ± 4.44 to 99.25 ± 1.70 mg.100 mL-1 (KF1TF). The values calculated can be explained by the micronutrient instability in an acidic medium, such as the present in kombucha, in which the pH ranges between 3 and 6 (Bishop et al. 2022BISHOP P ET AL. 2022. Kombucha: Biochemical and microbiological impacts on the chemical and flavor profile. Food Chem Adv 1: 1-20.).

Despite that, the quantified values of vitamin C in flavored kombuchas were promising, since they supply the daily amount that should be ingested by an adult (Brazil 2005BRAZIL. 2005. Ministério da Saúde. Resolução-RDC Nº 269, de 22 de Setembro de 2005. Diário Oficial da União. Retrieved from: https://bvsms.saude.gov.br/bvs/saudelegis/anvisa/2005/rdc0269_22_09_2005.html.
https://bvsms.saude.gov.br/bvs/saudelegi...
). In addition, the total micronutrient found was higher compared to cashew apple juice (NEPA 2011NEPA - NÚCLEO DE ESTUDOS E PESQUISAS EM ALIMENTAÇÃO. 2011. Tabela de Composição dos Alimentos. Universidade Estadual de Campinas, 4ª Ed., 161 p.).

Microbiological assessment

With the premise of guaranteeing food safety and the quality of the products developed in this study, the final formulations were evaluated from a microbiological point of view. The results are shown in Table IV.

Table IV
Microbiological Analysis.

The fermented beverage kombucha still does not have a specific legislation for the microbiological standard, being used for comparison with the parameters found in other literature. Brunini et al (2019), found a similar count (1,3.102 UFC/100mL) of mesophilic bacteria, molds and yeast found in this present study. In relation to thermotolerant coliforms, there was no colony count, similar to what happened in the study by Yikmis &Tuggum (2019)YIKMIŞ S & TUĞGÜM S. 2019. Evaluation of microbiological, physicochemical and sensorial properties of purple basil kombucha beverage. Türk Tarım-Gıda Bilim ve Teknoloji dergisi 7: 9. of kombucha based on purple basil (Ocimum basilicum L.).

These findings ensure compliance with the hygiene standards during the kombucha preparation, since the presence of the investigated microorganisms is associated with failures in the cleaning, disinfection, transport, storage, and temperature control during the manufacturing process of the products (Bruini 2019BRUINI B. 2019. Aspectos físico-químicos e microbiológicos no processo de fabricação da Kombucha. Revista Engenho 11: 48-67., Silva et al. 2017SILVA N, JUNQUEIRA VCA, SILVEIRA NFA, TANIWAKI MH, GOMES RAR & OKAZAKI MM. 2017. Manual de Métodos de Análise Microbiológica de Alimentos. 5ª ed., São Paulo: Blucher, 560 p.).

CONCLUSIONS

From this work, it was observed that the flavored kombuchas had high amounts of vitamin C, an important micronutrient in the human diet and necessary for the functioning of the immune system, as well as satisfactory levels of phenolic compounds and antioxidant capacity. In addition, the physicochemical characteristics and microbiological analysis of the fermented products proved that they were developed in accordance with the quality and safety standards laid down in the legislation.

As a result, it can be understood that the flavored formulations added nutritional and functional properties to the original fermented beverage that increased its potential for commercialization. Therefore, the association between the chosen fruits and traditional kombucha proves to be a sustainable and promising alternative for combining a probiotic fermented food with the diversity of bioactives found in fruit species from the Amazonian Region.

Finally, it is necessary to carry out sensory and consumption tests to complement the analysis of these new products, but our work is a tool to initiate further studies on kombuchas flavored with Amazonian fruits.

ACKNOWLEDGMENTS

The Federal University of Pará and the Faculty of Nutrition for providing a PIBIC research grant.

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Publication Dates

  • Publication in this collection
    15 July 2024
  • Date of issue
    2024

History

  • Received
    29 Mar 2023
  • Accepted
    7 Apr 2024
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