Next Article in Journal
Throttling Effect and Erosion Research of Ultra-High-Pressure Grease Nozzles
Previous Article in Journal
Drying Kinetics and Physicochemical Characteristics of Dehydrated Jerusalem Artichoke (Helianthus tuberosus L.)
Previous Article in Special Issue
From Probiotic to Postbiotic: Conversion of Acerola Juice by Thermosonication
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Processes
Volume 13
Issue 8
10.3390/pr13082554
processes-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Development and Evaluation of Potential Probiotic Coconut Water Beverages: Fermentation, Storage, and Consumer Perception

by
Brenda Novais Santos
1,
Ana Lúcia Fernandes Pereira
2,
Thatyane Vidal Fonteles
3,* and
Sueli Rodrigues
3,*
1
Chemical Engineering Department, Federal University of Ceara, Campus do Pici, Bloco 709, Fortaleza 60440-900, CE, Brazil
2
Food Engineering Department, Federal University of Maranhão, Centro de Ciências de Imperatriz; Imperatriz 65915060, MA, Brazil
3
Food Engineering Department, Federal University of Ceara, Campus do Pici, Bloco 858, CEP, Fortaleza 60440-900, CE, Brazil
*
Authors to whom correspondence should be addressed.
Processes 2025, 13(8), 2554; https://doi.org/10.3390/pr13082554
Submission received: 9 July 2025 / Revised: 29 July 2025 / Accepted: 11 August 2025 / Published: 13 August 2025
(This article belongs to the Special Issue Green Technologies for Food Processing)
Browse Figures
Versions Notes

Abstract

Coconut water was explored as a plant-based substrate for the development of probiotic beverages fermented with four Bifidobacterium strains (B. animalis B-41406, B. bifidum B-41410, B. breve B-41408, and B. infantis B-41661). Each strain was tested separately in a monoculture, with the coconut water adjusted to pH 6.7 and fermented under anaerobic conditions at 37 °C for 24 h. All formulations achieved a high cell viability (>12 log CFU/mL post-fermentation) and maintained counts above 6 log CFU/mL after 42 days at 4 °C. The fermentation resulted in significant lactic acid production (up to 6.1 g/L), with moderate acetic acid accumulation, and the pH remained below 4.5, ensuring microbiological stability. The sugar consumption varied across the strains, with B. bifidum and B. breve utilizing glucose and fructose more effectively. A sensory analysis, conducted with 100 untrained panelists using a 9-point hedonic scale and the Check-All-That-Apply (CATA) method, revealed that the B. bifidum-fermented beverage had the highest acceptance, attributed to favorable descriptors such as an “ideal sweetness”, “coconut flavor”, and “ideal texture”. These findings support the application of B. bifidum in the formulation of stable, microbiologically viable, and organoleptically acceptable non-dairy probiotic beverages, highlighting coconut water as a promising functional matrix.

Graphical Abstract

1. Introduction

The development of probiotic formulations has progressed beyond traditional health-focused goals to address broader concerns, including sustainability, allergen avoidance, and the growing consumer demand for appealing, clean-label, and plant-based alternatives [1]. Within this context, non-dairy probiotic beverages offer a promising vehicle for innovation. Unlike dairy matrices, which naturally provide a buffering capacity and essential growth factors, plant-based substrates often present suboptimal conditions for probiotic survival and metabolic activity. This challenge is particularly pronounced for Bifidobacterium spp., a genus of anaerobes with a limited proteolytic capacity, narrow carbohydrate utilization profiles, and heightened sensitivity to oxygen and acid stress [2]. Although Bifidobacterium strains are among the most clinically validated probiotics, their application in plant-based fermented beverages remains restricted, often limited to co-culture strategies or encapsulated forms, thereby representing a significant bottleneck in diversifying probiotic delivery systems [3].
Nevertheless, the use of plant-based substrates naturally rich in simple carbohydrates and micronutrients, such as coconut water, may present a promising strategy to overcome these limitations. Coconut water is a sterile, isotonic liquid naturally rich in glucose, fructose, and potassium. Its low protein content and minimal buffering capacity are traditionally considered barriers for fermentation by strict anaerobes; however, when combined with optimized fermentation conditions (anaerobiosis, pH adjustment, and appropriate inoculum densities), it may serve as a viable matrix for Bifidobacterium proliferation and metabolite production [4]. Furthermore, coconut water’s refreshing sensory profile and association with wellness trends enhances its appeal for innovative functional drink formulations [5,6].
Studies exploring the fermentation of coconut water by Bifidobacterium spp. as sole starters remain scarce. Moreover, few have integrated microbiological and biochemical parameters with a sensory assessment to evaluate the overall product feasibility. Therefore, identifying Bifidobacterium strains capable of maintaining viability and contributing to desirable sensory outcomes in minimally processed plant matrices remains a significant research challenge.
The present study addresses this research gap by investigating the fermentation of coconut water with four Bifidobacterium strains (B. animalis, B. bifidum, B. breve, and B. infantis), each with distinct metabolic profiles and probiotic attributes. The research integrates the microbiological performance (viability, acid production, sugar utilization), physicochemical changes (pH, organic acids), and sensory evaluation using hedonic and Check-All-That-Apply (CATA) methodologies to comprehensively assess the feasibility and consumer perception. By identifying a strain that combines technological viability with favorable sensory attributes, this study contributes to the advancement of Bifidobacterium spp. applications in plant-based functional beverages, in line with current demands for sustainable and health-promoting food products.

2. Materials and Methods

2.1. Coconut Water

Coconut water (CW) was obtained from green coconuts (Cocos nucifera L.) purchased at the local market in Fortaleza, Brazil. CW was extracted only on the day of its use. After sanitization, the green coconuts were perforated with the help of a stainless-steel coconut opener. The CW collected was then homogenized and filtered through a Whatman n° 01 filter paper to remove fibers [5]. The pH was adjusted to 6.7 ± 0.2 to promote optimum Bifidobacterium growth. All handling steps were conducted under aseptic conditions to prevent contamination by environmental microorganisms.

2.2. Probiotic Culture Stocks and Inoculum Preparation

Four probiotic strains of Bifidobacterium spp. (B. animalis B-41406, B. bifidum B-41410, B. breve B-41408, and B. infantis B-41661)—obtained from the United States Department of Agricultural, Peoria, IL, USA, NRRL Culture Collection—were activated separately in bifidobacteria broth composed of casein (20 g/L), yeast extract (10 g/L), tomato extract (8.33 g/L), glucose (20 g/L), peptone (10 g/L), and tween 80 (2 g/L). First, the lyophilized strain was activated in screw-capped Erlenmeyer flasks (250 mL) containing 100 mL of bifidobacteria broth at 37 °C and 100 rpm for 24 h, under anaerobic conditions provided by inertization with injection of sterile N2. Sterile glycerol was added to the activated cultures (50% v/v), and then the obtained stock cultures were stored in sterile screw-capped sterile cryogenic tubes (2 mL) and frozen at −20 °C [7].
The inoculum preparation was achieved by activating 1.5 mL of the glycerol stock culture inoculated in screw-cap Erlenmeyer flasks (250 mL) containing 100 mL of bifidobacteria broth with 10 mL of 200 mM bibasic potassium phosphate buffer at pH 6.7. Activation was carried out at 37 °C and 100 rpm for 24 h, under anaerobic conditions (N2 inertization). Before inoculation, the cell concentration of each activated culture was standardized to approximately 9 log CFU/mL using spectrophotometric calibration at 600 nm (OD600), with values confirmed by serial dilution and plate counting. This ensured that each fermentation vessel received the same microbial load (2% v/v).

2.3. CW Fermentation

Coconut water was fermented in sterile 500 mL glass bottles with screw caps to minimize oxygen exposure. Each bottle contained 300 mL of coconut water, adjusted to pH 6.7 using 3 M NaOH, and inoculated with 2% (v/v) of a previously prepared inoculum. Nitrogen gas was sparged through the medium to establish anaerobic conditions. Fermentations were conducted statically at 37 °C for 24 h in a BOD incubator, with each bacterial strain inoculated in separate bottles. Samples (10 mL) were collected at 0, 12, and 24 h to analyze pH, viable cell counts, organic acids, and sugar concentrations.

2.4. Storage Stability

After fermentation, CW beverages were distributed into multiple sterile 100 mL glass bottles, each filled with 40 mL of fermented product, and stored under refrigeration at 4 °C for 42 days. To ensure anaerobic conditions, the bottles were inertized by sparging with sterile nitrogen gas. Every 7 days, one bottle was opened, and a 10 mL sample was collected to analyze pH, viable cell counts, and concentrations of sugars and organic acids, as described by [8]. After sampling, the remaining beverage was re-sparged with sterile nitrogen and the bottle was resealed with a screw cap.
A non-fermented CW sample was used as a control to assess potential microbiological contamination and enzymatic degradation. For this, 300 mL of CW was placed in a 500 mL glass bottle, and its pH was adjusted from 4.3 to 6.7 using NaOH. The control sample underwent the same fermentation and storage conditions, except without inoculation. At 7-day intervals, 10 mL aliquots were collected to monitor pH and microbial counts.

2.5. pH Analysis

The pH sample measurements were performed by direct reading, using a potentiometer (MS TECNOPON, Piracicaba, Brazil), model mPA210, calibrated with pH 4.0 and 7.0 buffer solutions at 25 °C.

2.6. Viable Cell Counts

Viable cells were counted after serial dilution in peptone water (10−1 to 10−12). After dilution, 10 µL of each dilution was placed on plates with Bifidobacterium agar (triplicate of each dilution) using the microdrop inoculation method [9]. The plates were incubated in a BOD incubator at 37 °C for 24 h, and viability was expressed as log CFU/mL.

2.7. Sugars and Organic Acids Determination

Sugars and organic acids were quantified by High-Performance Liquid Chromatography (HPLC) in an Infinity 1260 chromatograph system (Agilent, Santa Clara, CA, USA). The separation of sugars (fructose and glucose) was achieved using an Aminex HPX-87C (300 × 7.8 mm) Bio-Rad Laboratories, Hercules, CA, USA) column at 80 °C, using ultrapure water as a mobile phase with a flow rate of 0.5 mL/min. The sugars were detected by a Prostar 345 refractive index (IR) detector at 35 °C. The organic acids components (acetic acid and lactic acid) were separated in a Aminex HPX-87H column (Bio-Rad) (300 mm ~ 7.8 mm) at 65 °C, with sulfuric acid 5 mM as a mobile phase at a 0.6 mL/min flow rate. The organic acid was detected by a UV-DAD detector (Agilent) at 210 nm [10]. Identification and quantification of the compounds were performed using calibration curves as a reference.

2.8. Sensory Analysis

Sensory analysis was conducted at the Federal University of Maranhão on the 19th day, halfway through the storage period of fermented beverages. This study was approved by the Ethics Committee of the Federal University of Maranhão, Brazil (CAAE 70904717.0.0000.5087), and before the tests, the participants signed a consent form [11].
For sensory evaluation of fermented coconut water samples, 100 untrained judges of both sexes participated (69% female and 31% male). Initially, according to the methodology described by Vidal et al. (2013) [11], a free word association test was applied, and the participants were asked to write four words, descriptions, associations, thoughts, or feelings that came to mind when imagining the product titled “probiotic coconut water”. The words mentioned by at least 5% of the participants were considered for analysis.
Then, a tray containing the 4 CW beverage samples (40 mL each) fermented with B. animalis B-41406, B. bifidum B-41410, B. breve B-41408, or B. infantis B-41661—at 7 °C, coded with three-digit numbers—was given to each judge. The evaluation was carried out in individual cabins. A nine-point mixed structured hedonic scale anchored at the extremes by “(1 = extremely disliked)”; “(5 = neither liked nor disliked)”; and “(9 = extremely liked)” was used to evaluate the attributes of color appearance, aroma, flavor, and overall impression [12].
For the sensory characterization of the samples, the Check-All-That-Apply (CATA) form was created based on terms raised in other studies with coconut water and probiotic beverages [13,14]. The form contained 23 descriptive terms [15]. The judges were asked to fill out the CATA form by marking the descriptive terms they believed related to beverages. The terms considered were the following: turbid, translucent, coconut watercolor, presence of particles, strange color, dark color, fermented aroma, coconut water aroma, coconut water flavor, stale coconut water flavor, ideal sweet taste, not very sweet, very sweet, bitter taste, aftertaste, salty taste, acidic taste, ideal texture, concentrated, tasty, exotic, very acidic, and very salty.

2.9. Statistical Analysis

All fermentation experiments were performed in duplicate, and all analytical measurements (pH, cell viability, organic acids, and sugars) were performed in duplicate for each replicate. Sensory data analysis was performed using the XLSTAT program (Addinsoft, Paris, France), considering a 5% probability level for significance. For sensory data evaluated using a hedonic scale, treatments were considered as a fixed source of variation and the consumer as a random effect. The attributes were analyzed using Friedman’s non-parametric test at a 95% confidence level. To analyze the CATA data, the frequency of mention of each term was determined by counting the number of consumers who used it to describe each tested sample, and Cochran’s Q test was used to compare all the treatments. In addition, Correspondence Analysis was performed using the Chi-square method, and principal coordinate analysis was applied to the sensory terms used to describe the samples.

3. Results and Discussion

3.1. Fermentation Process

The fermentation of the coconut water by four distinct Bifidobacterium strains (B. animalis B-41406, B. bifidum B-41410, B. breve B-41408, and B. infantis B-4166) resulted in significant reductions in pH and significant increases (p < 0.05) in viable cell counts over 24 h of incubation (Table 1).
Initial pH values of the coconut water formulations ranged from 6.13 to 6.50, with slight variations. After 24 h of fermentation under anaerobic conditions at 37 °C, all formulations exhibited acidification, with final pH values between 4.37 and 4.49. This pH reduction is indicative of organic acid production, primarily lactic acid, and confirms the active microbial metabolism of all tested strains.
The magnitude of the pH reduction was relatively similar among strains, suggesting comparable acidogenic capacities under the tested conditions. However, differences in growth kinetics were observed. B. animalis showed the highest increase in viable cell counts, from 5.83 ± 0.13 to 14.95 ± 0.16 log CFU/mL, followed closely by B. breve (14.88 ± 0.17 log CFU/mL). These results suggest a robust capacity of these strains to proliferate in the coconut water matrix, despite its limited buffering capacity and low protein content. Conversely, B. infantis exhibited the lowest final viable count (12.58 ± 0.17 log CFU/mL). The ability of all strains to exceed 12 log CFU/mL after fermentation reinforces the suitability of coconut water as a non-dairy vehicle for delivering high probiotic cell loads.
The consumption of glucose and fructose, as well as the production of lactic and acetic acids, is reported in Table 2 for the coconut water fermented with Bifidobacterium spp.
While most strains metabolize the available sugars to varying extents, resulting in the accumulation of organic acids, B. animalis exhibited an unexpected increase in both glucose (from 31.33 ± 1.29 to 34.15 ± 1.17 g/L) and fructose concentrations (from 31.07 ± 1.48 to 34.53 ± 1.17 g/L). This phenomenon may reflect the delayed hydrolysis of residual polysaccharides or matrix-bound sugars not initially detectable at 0 h.
In contrast, B. bifidum and B. breve demonstrated a significant reduction in sugar contents, indicating active monosaccharide consumption (p < 0.05). B. bifidum reduced the glucose to 28.86 ± 1.34 g/L and the fructose to 29.50 ± 1.34 g/L after 24 h. Similarly, B. breve showed a comparable decrease. The abundance of residual glucose and fructose can assist in the ongoing metabolic process of the strains, leading to post-acidification in the fermented product. This also allows the preservation of sweetness, directly impacting the sensorial properties of the product [16].
Lactic acid was the predominant fermentation metabolite across all strains, with final concentrations ranging from 3.44 ± 0.10 to 3.70 ± 0.07 g/L. This profile is consistent with the activity of the Bifidus pathway (fructose-6-phosphate shunt), a distinctive metabolic route utilized by Bifidobacterium spp. that combines features of both the homo- and heterofermentative metabolism. Rather than relying solely on the Embden–Meyerhof–Parnas (EMP) or phosphoketolase pathways, the Bifidus pathway enables bifidobacteria to convert hexoses into a mixture of acetate and lactate in a theoretical molar ratio of 3:2, while yielding a higher ATP efficiency [17,18]. In this study, all strains produced low levels of acetic acid (0.27–1.19 g/L), which is favorable from a sensory perspective, as excessive acetic acid may impart undesirable sourness and astringency. B. breve exhibited the highest acetic acid concentration (1.19 ± 0.07 g/L), suggesting the strain-specific modulation of the Bifidus pathway, possibly through enhanced phosphoketolase activity or the increased flux through acetyl-CoA pathways. These results reflect the flexible metabolic strategies of Bifidobacterium spp. and their ability to balance energy production with the acid output in response to substrate availability and environmental conditions.
The distinct metabolic responses observed among the Bifidobacterium strains reveal their differential capacities for sugar utilization and acid production, which directly influence the probiotic performance and the sensory characteristics of the final product. These biochemical variations are crucial for interpreting the strain behavior during fermentation and provide a foundation for linking microbial activity to consumer acceptance.
In coconut water, the high availability of fermentable carbohydrates such as glucose and fructose supports a robust bifidobacterial metabolism. However, its low concentration of nitrogenous compounds and peptides may alter the metabolic flux through the Bifidus pathway, particularly the balance between acetate and lactate production. Such nutrient limitations can shift metabolic outputs in a strain-dependent manner, as observed in the varying acid profiles among the tested beverages. These compositional factors likely contributed to the differences in both the microbial performance and sensory perception across formulations.
The presence of nitrogen and minerals promoted a metabolic shift favoring lactic acid over acetic acid, as observed by Abascal et al. (2022) [19] in the fermentation of carrot juice. A similar effect may be inferred in this study, as B. bifidum-fermented coconut water exhibited a progressive increase in lactic acid during storage, while the acetic acid accumulation remained moderate (Table 2). This shift may reflect the strain’s adaptation to the nutrient limitations of coconut water and supports the hypothesis that lactic acid production can be modulated by the availability of micronutrients and nitrogenous compounds in the plant matrix. Thus, the acid profiles observed during FCW fermentation are not only strain-specific but also tightly linked to the biochemical characteristics of the coconut water substrate.

3.2. Storage Period

Maintaining probiotic viability during storage remains a major challenge in the functional beverage industry, especially for Bifidobacterium spp., which are highly sensitive to environmental stressors such as a low pH, dissolved oxygen, and nutrient depletion [20,21,22]. Figure 1 presents the evolution of the pH (Figure 1a) and viable cell counts (Figure 1b) over 42 days of refrigerated storage at 4 °C for coconut water (CW) beverages fermented with four Bifidobacterium strains.
As expected, a gradual decline in cell viability was observed for all strains throughout storage, as expected. Nevertheless, all beverages retained viable counts above the minimum recommended threshold of 7 log CFU/mL for probiotic efficacy by the end of the 42-day period. Among the tested strains, B. bifidum exhibited the highest residual viability (7.37 log CFU/mL), suggesting a superior capacity to withstand the acidic and low-nutrient conditions of the stored fermented coconut water (FCW). This finding aligns with a previous study indicating a strain-dependent tolerance to post-acidification stress [23].
Despite the typical association of a low pH with significant cell death, the acidity of the FCW did not critically impair the Bifidobacterium survival over the 42-day storage period. The pH values remained relatively stable within a narrow range, between 4.3 and 4.5 throughout storage, showing minimal post-acidification and thus contributing to microbial stability. This moderate acidification may reflect the attenuation or cessation of late-stage lactic acid production, likely due to the substrate depletion or inhibition by accumulated acids, or the microbial uncoupling of the growth and acid metabolism, as previously observed for B. bifidum in dairy matrices [24,25].
Acetic acid, known for its mild antimicrobial effects, was produced in relatively low concentrations and may have contributed to microbial modulation without triggering sharp declines in the pH. This balance may partially explain the absence of sharp drops in viability, especially in comparison to lactobacilli-dominated fermentations, which often experience more pronounced post-acidification and viability loss [26,27].
The control (non-fermented) coconut water sample maintained a stable pH (6.7 ± 0.2) throughout the same period, with no microbial growth detected under anaerobic incubation on Bifidobacterium-specific media, confirming the absence of contamination. This also highlights the effectiveness of the fermentation and storage conditions in preserving product microbiological safety.
Overall, the results demonstrate that coconut water can support the stability of Bifidobacterium spp. during extended refrigerated storage, with both the pH and viability parameters remaining within acceptable and functional ranges. These findings reinforce the potential of FCW as a non-dairy probiotic delivery system.
The metabolic evolution of fermented coconut water (FCW) during refrigerated storage is depicted in Figure 2, which illustrates the variation in glucose (Figure 2a), fructose (Figure 2b), lactic acid (Figure 2c), and acetic acid (Figure 2d) concentrations over a 42-day period.
As expected, the most intense production of organic acids occurred during the active fermentation phase (first 24 h), with only modest changes observed during storage, reflecting a reduced microbial metabolic activity at low temperatures.
Among the four tested strains, B. bifidum B-41410 exhibited a distinct behavior. A continuous and pronounced increase in the lactic acid content was observed throughout storage, reaching 6.12 g/L by day 42, which is higher than the concentrations maintained by the other strains, which stabilized between 4.0 and 5.0 g/L. This trend suggests the residual metabolic activity or delayed acid production by B. bifidum, which may be associated with uncoupled metabolism or stress-induced responses during refrigerated conditions [24,25]. Such behavior may also contribute to maintaining pH stability, as lactic acid continues to accumulate without sharp post-acidification effects (see Figure 1a).
Regarding acetic acid, B. bifidum and B. breve exhibited the most prominent increases during storage, both reaching final concentrations around 2.4 g/L (Figure 2d). These values are higher than those typically reported for bifidobacteria in dairy substrates and may be attributed to the strain’s heterofermentative activity or the prolonged utilization of pentose sugars and residual carbohydrates. While acetic acid contributes positively to microbial inhibition and preservation, excessive accumulation can influence sensory acceptance due to its sharp, vinegar-like notes. Therefore, monitoring acetic acid dynamics is crucial for balancing functionality and palatability.
In contrast, both glucose and fructose (Figure 2a,b) showed a slight increase during the initial days of storage, followed by a gradual decline, particularly after day 28. This pattern may indicate an early phase of sugar release, possibly from matrix-associated carbohydrates or cellular debris, followed by continued microbial activity during refrigerated storage. The continued reduction in the sugar concentration suggests that bifidobacterial cells remained metabolically active, at a slower rate, even under cold conditions. The most pronounced sugar depletion was observed in the coconut water fermented with B. breve, corresponding to its elevated acid production and supporting the hypothesis of a more resilient phenotype under the tested fermentation and storage parameters.
These observations reinforce the strain-dependent nature of the post-fermentation behavior in plant-based probiotic systems. The balance between sugar availability, acid production, and microbial survival is critical for ensuring the long-term quality and efficacy of functional beverages during the refrigerated shelf life.

3.3. Sensory Acceptance

As for word association data, a total of 147 different words were mentioned by participants when asked which first four words came to mind concerning the product named “probiotic coconut water”. The number of mentions of all words by the 100 participants was 400. All words mentioned by consumers were grouped into categories, with at least 5% of consumers being considered for analysis.
The “healthiness” category received the highest number of mentions (31.25%), with this healthiness being associated with a feeling of well-being and benefits for the intestine, which are characteristics related to consuming foods containing probiotics. Furthermore, hydration and nutritional values were related, such as the presence of mineral salts, which are nutrients present in coconut water. In this way, it can be stated that the participants in the present study have a clear idea that the product presented has a healthy characteristic. The judges also mentioned that the product, as it produces these health benefits, may have a medicinal effect, with the category “related to medicines” having 1.75%.
The second most mentioned category was “sensory characteristics” (12.25%), with flavor and appearance being the most mentioned attributes. This result reinforces the importance of sensory characteristics for the acceptance of the beverage. The judges believe the beverage presented a sweet flavor, typical of coconut water, and an acidic flavor, resulting from the fermentation. Furthermore, they also expected a turbid appearance, characteristic of coconut water.
The beverage was closely associated with the coconut fruit, with the category “characteristics related to coconut” representing 9.75% of mentions. Among the words mentioned, it can be seen that consumers imagine that flavored coconut water should be served cold. Furthermore, they related the beverage to places and situations where it can be consumed, such as on the beach and while playing sports. After this category, the highest percentage of mentions was for the “related to probiotics” category, with 7.25%. This category demonstrates the participants’ knowledge of probiotic foods by mentioning words such as microorganisms and good bacteria.
Regarding the “Product Characteristics” category, it accounted for 7.00% of the responses (Table 1), indicating that participants prefer this beverage to be refreshing and sold in small-sized packages.
The “distrust/doubt” category had 4.75% of mentions, with the main words being different, strange, exotic, unusual, and doubt. As it is a new product, there is justification for doubt on the part of consumers. The fact that it is a new product was mentioned by participants in the innovation category (4.25%). Despite the distrust, some consumers showed interest in learning about the product with mentions in the “interest/curiosity to try” category.
Another category mentioned was “relating to industrialization,” the main terms being industrialized, conservation, and industrialized product. This category is related to the consumer’s understanding that to produce these products, there must be processing that requires industrialization. In addition, consumers associated probiotic coconut water as an alternative to probiotic foods of dairy origins, according to the category “alternative to dairy products”.
Another category mentioned was the price; some participants believe the beverage would be more expensive, and others believe it is a cheap alternative to probiotics.
Furthermore, many participants mentioned that it is a natural product and may be associated with diet, which was mentioned in 1.25% of the responses. Furthermore, participants attributed hedonic attitudes to the product, which mainly had positive characteristics (tasty, delicious, good). These positive characteristics can be confirmed by the drinks’ feelings (lightness, pleasantness, smoothness, and peace).
For the sensory attributes evaluated by the hedonic scale, the averages for beverages made with B. animalis and B. bifidum were between 6.01 and 7.00 (Table 3), i.e., between the terms “slightly liked” and “moderately liked” on the hedonic scale, showing a good acceptance of beverages fermented by these microorganisms. For the coconut water fermented with B. infantis, the averages ranged between 5.34 and 6.60, between the terms “neither liked nor disliked” and “liked moderately,” showing a lower level of acceptance. For the coconut water fermented with B. breve, the averages were between 3.46 and 6.72, which refer to the terms “moderately disliked” and “moderately liked”. Therefore, for this beverage, the attributes of the aroma, flavor, and overall impression were rejected by the judges (Table 3).
Regarding the test of means, no changes were observed (p > 0.05) in the acceptance of the color and appearance of the beverages. For the attributes of the aroma, flavor, and global impression, the highest values were for coconut waters fermented with B. bifidum, followed by those fermented with B. animalis, B. infantis, and, finally, B. breve (Table 3).
The CATA methodology was used to collect information about consumers’ perceptions regarding the sensory characteristics of coconut water. According to Cochran’s Q test, there was a significant difference in the frequencies of 12 of the 23 terms presented in the CATA form, which suggests that consumers noticed differences in the sensory characteristics between the beverages. Consumers can consider such terms as the most appropriate when describing samples.
For the appearance, only the term turbid showed a difference between the beverages, with the drink fermented with B. bifidum showing a higher frequency (p < 0.05) when compared to the one fermented by B. breve.
Regarding the aroma, the coconut water fermented by B. breve and B. infantis had the highest frequencies (p < 0.05) for the term “fermented aroma” and the lowest for the term “coconut water aroma”. This greater aroma from fermentation reduced the coconut water aroma characteristics of these beverages, resulting in the lower acceptance of them according to the data from the hedonic scale (Table 3).
For the terms “coconut water flavor” and “ideal sweet taste,” the highest frequencies (p < 0.05) were for the coconut water fermented with B. animalis and B. bifidum. The coconut water with B. bifidum also had the highest frequency for the term delicious, followed by the beverage with B. animalis. These terms are related to the greater acceptance of beverages fermented by these microorganisms, as they reflect the results for the flavor obtained by the hedonic scale (Table 3).
The terms “little sweet” and “bitter taste” had higher frequencies (p < 0.05) in coconut waters fermented by B. breve and B. infantis. Furthermore, these beverages had higher frequencies for the term “acidic taste”, with the coconut water fermented by B. breve showing the highest frequency for the term very acidic. These terms indicate that the low acceptability of these beverages is related to the low sugar content of these drinks and the higher acidity. As a suggestion to improve these characteristics, some consumers indicated increasing the sweetness with the use of sweeteners.
For the term “ideal texture”, the highest frequencies (p < 0.05) were for the beverage fermented with B. animalis when compared to that fermented with B. breve. No significant differences (p > 0.05) were detected between the beverages for the following terms: translucent, coconut water color, presence of particles, strange color, dark color, the taste of old coconut water, very sweet, aftertaste, salty, concentrated, and exotic taste. Therefore, consumers associate these terms equally among all samples.
Figure 3 shows the results of the Correspondence Analysis applied to the CATA data, in which dimensions 1 and 2 explained 91.58% of the variance. Therefore, it was observed that the beverages remained close to the terms that characterize them.
The probiotic coconut water beverages were positively evaluated by the untrained sensory panel, indicating a good overall acceptance. The high residual concentrations of glucose and fructose likely contributed to the perceived sweetness of the products, which may have moderated the sensory impact of the acid production. As each Bifidobacterium strain exhibits distinct metabolic activity, even within the same genus, differences in sugar consumption and organic acid production likely underlie the perceptible variation among the beverages [28]. Organic acids play a key role in shaping flavor profiles; lactic acid, in particular, imparts a mild sourness that is generally well-received, whereas elevated acetic acid concentrations are often associated with off-flavors and vinegar-like aromas that can diminish consumer acceptance [16].
These findings are consistent with previous studies. Segura-Badilla et al. (2020) [6] reported that coconut water fermented with Lactobacillus rhamnosus exhibited desirable sensory qualities only up to a certain level of acidification, with product acceptability decreasing sharply once the pH dropped below 3.0, limiting the refrigerated shelf life to approximately 15 days. Similarly, Luckow et al. (2005) [29] observed that fruit juices fermented with Lactobacillus spp. developed atypical and medicinal flavor notes unfamiliar to consumers accustomed to dairy-based probiotic products. However, they suggested that consumer education and an increased exposure to non-dairy probiotic beverages may enhance acceptance over time.
In this context, the fermented CW beverages presented in this study demonstrate a favorable balance between microbial viability and sensory quality, likely due to the moderated acidification and retained sweetness. These characteristics may position bifidobacteria-fermented coconut water as a viable and appealing plant-based alternative in the functional beverage market.

4. Conclusions

This study demonstrates the feasibility of producing probiotic coconut water beverages using B. animalis, B. bifidum, B. breve, and B. infantis. All strains maintained viable cell counts above 7 log CFU/mL and ensured microbiological safety through sustained acidification, with pH values remaining below 4.5 during the fermentation and refrigerated storage. The fermentation process led to the accumulation of lactic and acetic acids, with only minor compositional changes observed throughout the 42-day storage period. While all strains exhibited comparable fermentation dynamics and stability, the beverage fermented with B. bifidum achieved the highest sensory acceptance, likely due to its favorable balance of residual sugars and organic acids. These findings support the use of Bifidobacterium spp. in the development of stable, non-dairy probiotic beverages and highlight coconut water as a promising matrix for delivering functional and sensorially acceptable probiotic products.

Author Contributions

Conceptualization S.R.; methodology, B.N.S. and A.L.F.P.; software, B.N.S. and A.L.F.P.; validation, S.R., A.L.F.P., and T.V.F.; formal analysis, B.N.S.; investigation, B.N.S.; resources, S.R. and A.L.F.P.; data curation, T.V.F., A.L.F.P., and S.R.; writing—original draft preparation, B.N.S.; writing—review and editing, T.V.F. and S.R.; visualization, S.R. and T.V.F.; supervision, S.R.; project administration, S.R.; funding acquisition, S.R. All authors have read and agreed to the published version of the manuscript.

Funding

The authors thank the Brazilian funding agency CNPq through the National Institute of Science and Technology of Tropical Fruits for the financial support and FUNCAP for the grants and scholarships. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—finance code 001 Brasil (CAPES).

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors also thank the ARS Culture Collection (Peoria, Il, USA) for the microbial strain donation.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Mojikon, F.D.; Kasimin, M.E.; Molujin, A.M.; Gansau, J.A.; Jawan, R. Probiotication of Nutritious Fruit and Vegetable Juices: An Alternative to Dairy-Based Probiotic Functional Products. Nutrients 2022, 14, 3457. [Google Scholar] [CrossRef]
  2. Jena, R.; Choudhury, P.K. Bifidobacteria in Fermented Dairy Foods: A Health Beneficial Outlook. Probiotics Antimicrob. Proteins 2023, 17, 1–22. [Google Scholar] [CrossRef] [PubMed]
  3. Maftei, N.M.; Bogdan, R.E.G.; Boev, M.; Marin, D.B.; Ramos-Villarroel, A.Y.; Iancu, A.V. Innovative Fermented Soy Drink with the Sea Buckthorn Syrup and the Probiotics Co-Culture of Lactobacillus paracasei ssp. Paracasei (L. Casei® 431) and Bifidobacterium animalis ssp. Lactis (Bb-12®). Fermentation 2023, 9, 806. [Google Scholar] [CrossRef]
  4. Guo, W.; Chen, M.; Cui, S.; Tang, X.; Zhang, Q.; Zhao, J.; Mao, B.; Zhang, H. Dynamics Changes in Physicochemical Properties, Volatile Metabolites, Non-Volatile Metabolites, and Physiological Functions of Barley Juice during Bifidobacterium Infantis Fermentation. Food Chem. 2023, 407, 135201. [Google Scholar] [CrossRef]
  5. Porto, E.; Alves Filho, E.G.; Silva, L.M.A.; Fonteles, T.V.; do Nascimento, R.B.R.; Fernandes, F.A.N.; de Brito, E.S.; Rodrigues, S. Ozone and Plasma Processing Effect on Green Coconut Water. Food Res. Int. 2020, 131, 109000. [Google Scholar] [CrossRef]
  6. Segura-Badilla, O.; Lazcano-Hernández, M.; Kammar-García, A.; Vera-López, O.; Aguilar-Alonso, P.; Ramírez-Calixto, J.; Navarro-Cruz, A.R. Use of Coconut Water (Cocus nucifera L) for the Development of a Symbiotic Functional Drink. Heliyon 2020, 6, e03653. [Google Scholar] [CrossRef]
  7. Pereira, A.L.F.; Maciel, T.C.; Rodrigues, S. Probiotic Beverage from Cashew Apple Juice Fermented with Lactobacillus casei. Food Res. Int. 2011, 44, 1276–1283. [Google Scholar] [CrossRef]
  8. Leite, A.K.F.; Fonteles, T.V.; Godoy Alves Filho, E.; Andrea da Silva Oliveira, F.; Rodrigues, S. Impact of Orange Juice Containing Potentially Prebiotic Ingredients on Human Gut Microbiota Composition and Its Metabolites. Food Chem. 2023, 405, 134706. [Google Scholar] [CrossRef]
  9. Herigstad, B.; Hamilton, M.; Heersink, J. How to Optimize the Drop Plate Method for Enumerating Bacteria. J. Microbiol. Methods 2001, 44, 121–129. [Google Scholar] [CrossRef] [PubMed]
  10. do Nascimento, C.S.; Santos, B.N.; Rodrigues, S. High-Intensity Ultrasound Processed Acerola Juice Containing Oligosaccharides and Dextran Promotes Lacticaseibacillus casei NRRL B-442 Growth. Int. J. Food Sci. Technol. 2022, 57, 5186–5194. [Google Scholar] [CrossRef]
  11. Vidal, L.; Ares, G.; Giménez, A. Projective Techniques to Uncover Consumer Perception: Application of Three Methodologies to Ready-to-Eat Salads. Food Qual. Prefer. 2013, 28, 1–7. [Google Scholar] [CrossRef]
  12. Lawless, H.T.; Heymann, H. Sensory Evaluation of Food; Springer: New York, NY, USA, 2010; ISBN 978-1-4419-6487-8. [Google Scholar]
  13. Freitas, H.V.; Dos Santos Filho, A.L.; Rodrigues, S.; Abreu, V.K.G.; Narain, N.; Lemos, T.D.O.; Gomes, W.F.; Pereira, A.L.F. Synbiotic Açaí Juice (Euterpe oleracea) Containing Sucralose as Noncaloric Sweetener: Processing Optimization, Bioactive Compounds, and Acceptance during Storage. J. Food Sci. 2021, 86, 730–739. [Google Scholar] [CrossRef] [PubMed]
  14. Yannam, S.K.; Patras, A.; Pendyala, B.; Vergne, M.; Ravi, R.; Gopisetty, V.V.S.; Sasges, M. Effect of UV-C Irradiation on the Inactivation Kinetics of Oxidative Enzymes, Essential Amino Acids and Sensory Properties of Coconut Water. J. Food Sci. Technol. 2020, 57, 3564–3572. [Google Scholar] [CrossRef] [PubMed]
  15. Lado, J.; Vicente, E.; Manzzioni, A.; Ares, G. Application of a Check-all-that-apply Question for the Evaluation of Strawberry Cultivars from a Breeding Program. J. Sci. Food Agric. 2010, 90, 2268–2275. [Google Scholar] [CrossRef]
  16. Yeager, S.E.; Batali, M.E.; Guinard, J.-X.; Ristenpart, W.D. Acids in Coffee: A Review of Sensory Measurements and Meta-Analysis of Chemical Composition. Crit. Rev. Food Sci. Nutr. 2023, 63, 1010–1036. [Google Scholar] [CrossRef] [PubMed]
  17. De Vuyst, L.; Moens, F.; Selak, M.; Rivière, A.; Leroy, F. Summer Meeting 2013: Growth and Physiology of Bifidobacteria. J. Appl. Microbiol. 2014, 116, 477–491. [Google Scholar] [CrossRef]
  18. Palframan, R.J.; Gibson, G.R.; Rastall, R.A. Carbohydrate Preferences of Bifidobacterium Species 71 Carbohydrate Preferences of Bifidobacterium Species Isolated from the Human Gut. Curr. Issues Intest. Microbiol. 2003, 4, 71–75. [Google Scholar]
  19. Abascal, E.; Gómez-Coma, L.; Ortiz, I.; Ortiz, A. Global Diagnosis of Nitrate Pollution in Groundwater and Review of Removal Technologies. Sci. Total Environ. 2022, 810, 152233. [Google Scholar] [CrossRef]
  20. Achi, S.C.; Halami, P.M. Bifidobacterial Probiotics through Fermented Foods. In Mining of Microbial Wealth and MetaGenomics; Springer: Singapore, 2017; pp. 267–285. ISBN 9789811057083. [Google Scholar]
  21. Shori, A.B.; Baba, A.S. Survival of Bifidobacterium bifidum in Cow- and Camel-Milk Yogurts Enriched with Cinnamomum verum and Allium sativum. J. Assoc. Arab Univ. Basic Appl. Sci. 2015, 18, 7–11. [Google Scholar] [CrossRef]
  22. Sibanda, T.; Marole, T.A.; Thomashoff, U.L.; Thantsha, M.S.; Buys, E.M. Bifidobacterium Species Viability in Dairy-Based Probiotic Foods: Challenges and Innovative Approaches for Accurate Viability Determination and Monitoring of Probiotic Functionality. Front. Microbiol. 2024, 15, 1327010. [Google Scholar] [CrossRef]
  23. Boylston, T.D.; Vinderola, C.G.; Ghoddusi, H.B.; Reinheimer, J.A. Incorporation of Bifidobacteria into Cheeses: Challenges and Rewards. Int. Dairy J. 2004, 14, 375–387. [Google Scholar] [CrossRef]
  24. Garro, M.S.; Aguirre, L.; Savoy De Giori, G. Biological Activity of Bifidobacterium longum in Response to Environmental PH. Appl. Microbiol. Biotechnol. 2006, 70, 612–617. [Google Scholar] [CrossRef]
  25. Samona, A.; Robinson, R.K.; Marakis, S. Acid Production by Bifidobacteria and Yoghurt bacteria during Fermentation and Storage of Milk. Food Microbiol. 1996, 13, 275–280. [Google Scholar] [CrossRef]
  26. Ongol, M.P.; Sawatari, Y.; Ebina, Y.; Sone, T.; Tanaka, M.; Tomita, F.; Yokota, A.; Asano, K. Yoghurt Fermented by Lactobacillus delbrueckii Subsp. Bulgaricus H+-ATPase-Defective Mutants Exhibits Enhanced Viability of Bifidobacterium breve during Storage. Int. J. Food Microbiol. 2007, 116, 358–366. [Google Scholar] [CrossRef] [PubMed]
  27. Chen, S.; Chen, L.; Chen, L.; Ren, X.; Ge, H.; Li, B.; Ma, G.; Ke, X.; Zhu, J.; Li, L.; et al. Potential Probiotic Characterization of Lactobacillus reuteri from Traditional Chinese Highland Barley Wine and Application for Room-Temperature-Storage Drinkable Yogurt. J. Dairy Sci. 2018, 101, 5780–5788. [Google Scholar] [CrossRef] [PubMed]
  28. Giri, S.S.; Sukumaran, V.; Sen, S.S.; Park, S.C. Use of a Potential Probiotic, Lactobacillus casei L4, in the Preparation of Fermented Coconut Water Beverage. Front. Microbiol. 2018, 9, 1976. [Google Scholar] [CrossRef] [PubMed]
  29. Luckow, T.; Sheehan, V.; Delahunty, C.; Fitzgerald, G. Determining the Odor and Flavor Characteristics of Probiotic, Health-Promoting Ingredients and the Effects of Repeated Exposure on Consumer Acceptance. J. Food Sci. 2005, 70, S53–S59. [Google Scholar] [CrossRef]
Processes 13 02554 g001
Figure 1. The variation in the pH (a) and viable cell counts (b) during the 42-day refrigerated storage period of potential probiotic beverages fermented with Bifidobacterium spp.
Figure 1. The variation in the pH (a) and viable cell counts (b) during the 42-day refrigerated storage period of potential probiotic beverages fermented with Bifidobacterium spp.
Processes 13 02554 g001
Processes 13 02554 g002
Figure 2. Concentrations of glucose (a), fructose (b), lactic acid (c), and acetic acid (d) during the 42-day refrigerated storage period of potential probiotic beverages fermented with Bifidobacterium spp.
Figure 2. Concentrations of glucose (a), fructose (b), lactic acid (c), and acetic acid (d) during the 42-day refrigerated storage period of potential probiotic beverages fermented with Bifidobacterium spp.
Processes 13 02554 g002
Processes 13 02554 g003
Figure 3. An illustration of potential probiotic coconut water beverages and the terms used in the first and second dimensions of the Correspondence Analysis in the CATA data.
Figure 3. An illustration of potential probiotic coconut water beverages and the terms used in the first and second dimensions of the Correspondence Analysis in the CATA data.
Processes 13 02554 g003
Table 1. pH values and viable cell counts at the fermentation process’s beginning (0 h) and end (24 h).
Table 1. pH values and viable cell counts at the fermentation process’s beginning (0 h) and end (24 h).
MicroorganismpHViable Cell Counts (log CFU/mL)
0 h24 h0 h24 h
B. animalis6.42 ± 0.02 a4.37 ± 0.06 a5.83 ± 0.13 a14.95 ± 0.16 a
B. bifidum6.13 ± 0.03 b4.49 ± 0.08 a6.36 ± 0.09 a14.27 ± 0.20 b
B. breve6.50 ± 0.02 c4.47 ± 0.09 a5.66 ± 0.22 b14.88 ± 0.17 a
B. infantis6.15 ± 0.02 b4.43 ± 0.07 a6.19 ± 0.08 b12.58 ± 0.17 c
a–c Means followed by different letters in the columns indicate a significant difference between samples using Tukey’s test (p < 0.05).
Table 2. Concentrations of glucose, fructose, lactic, and acetic acids in the fermented coconut water by B. animalis, B. bifidum, B. breve, and B. infantis.
Table 2. Concentrations of glucose, fructose, lactic, and acetic acids in the fermented coconut water by B. animalis, B. bifidum, B. breve, and B. infantis.
StrainGlucose (g/L)Fructose (g/L)Acetic Acid (g/L)Lactic Acid (g/L)
0 h24 h0 h24 h0 h24 h0 h24 h
B. animalis31.33 ± 1.29 a34.15 ± 0.66 a31.07 ± 1.17 a34.53 ± 1.48 a0.25 ± 0.04 a0.29 ± 0.03 a0.71 ± 0.04 a3.70 ± 0.12 a
B. bifidum31.33 ± 1.29 a28.86 ± 1.34 b31.07 ± 1.17 a29.50 ± 1.34 b0.25 ± 0.04 a0.48 ± 0.07 b0.71 ± 0.04 a3.70 ± 0.08 a
B. breve31.33 ± 1.29 a29.18 ± 0.88 b31.07 ± 1.17 a29.37 ± 0.75 b0.25 ± 0.04 a1.19 ± 0.04 c0.71 ± 0.04 a3.64 ± 0.07 a
B. infantis31.33 ± 1.29 a31.95 ± 0.9 b31.07 ± 1.17 a32.27 ± 1.09 a0.25 ± 0.04 a0.27 ± 0.02 a0.71 ± 0.04 a3.44 ± 0.10 b
a–c Means followed by different letters in the columns indicate a significant difference between samples using Tukey’s test (p < 0.05).
Table 3. The sensory acceptance of the color, appearance, aroma, flavor, and overall impression was measured using the hedonic scale of potential probiotic coconut water beverages.
Table 3. The sensory acceptance of the color, appearance, aroma, flavor, and overall impression was measured using the hedonic scale of potential probiotic coconut water beverages.
AttributesB. breveB. animalisB. infantisB. bifidum
Color6.53 ± 1.92 a6.92 ± 1.82 a6.60 ± 1.99 a6.81 ± 1.73 a
Appearance6.72 ± 1.73 a7.00 ± 1.71 a6.81 ± 1.82 a6.89 ± 1.73 a
Aroma4.30 ± 1.91 d6.10 ± 1.01 b5.36 ± 1.37 c6.72 ± 0.98 a
Flavor3.46 ± 1.88 d6.12 ± 1.19 b5.34 ± 1.44 c6.73 ± 1.14 a
Global impression4.17 ± 1.91 d6.01 ± 1.28 b5.44 ± 1.48 c6.62 ± 1.17 a
a–d Means followed by different letters in the lines indicate a significant difference between treatments using the Friedman test (p < 0.05).
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Santos, B.N.; Pereira, A.L.F.; Fonteles, T.V.; Rodrigues, S. Development and Evaluation of Potential Probiotic Coconut Water Beverages: Fermentation, Storage, and Consumer Perception. Processes 2025, 13, 2554. https://doi.org/10.3390/pr13082554

AMA Style

Santos BN, Pereira ALF, Fonteles TV, Rodrigues S. Development and Evaluation of Potential Probiotic Coconut Water Beverages: Fermentation, Storage, and Consumer Perception. Processes. 2025; 13(8):2554. https://doi.org/10.3390/pr13082554

Chicago/Turabian Style

Santos, Brenda Novais, Ana Lúcia Fernandes Pereira, Thatyane Vidal Fonteles, and Sueli Rodrigues. 2025. "Development and Evaluation of Potential Probiotic Coconut Water Beverages: Fermentation, Storage, and Consumer Perception" Processes 13, no. 8: 2554. https://doi.org/10.3390/pr13082554

APA Style

Santos, B. N., Pereira, A. L. F., Fonteles, T. V., & Rodrigues, S. (2025). Development and Evaluation of Potential Probiotic Coconut Water Beverages: Fermentation, Storage, and Consumer Perception. Processes, 13(8), 2554. https://doi.org/10.3390/pr13082554

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop