Determination of Bioactive Compounds, Antioxidant, and Sensory Properties of Beverages from Mulberry, Yacon, and Lemon Fruits Produced by Natural Fermentation

The increased consumption of functional foods has been linked with the management and control of non-communicable diseases. There is limited information on the effects of the biochemical and sensory properties of beverages from the natural fermentation of mulberry, yacon, and lemon fruit beverages. This study aimed to determine the bioactive, antioxidant, and sensory properties of mulberry, yacon, and lemon fruit beverages taken at various stages of natural fermentation. The samples were made from a combination of mulberry fruits, yac¯ on root, and lemon fruits in the ratios T0 (90:0:10), T1 (60:30:10), and T2 (30:60:10), respectively. The samples were fermented and checked for the different parameters on different days, which include: 0, 5, 10, and 21 days. The bioactive compounds (flavonoids and phenolics) were measured using the Aluminium chloride colorimetric method and the Folin-Ciocalteau method, respectively. The antioxidant (FRAP and DPPH) was done with the ferric reducing antioxidant power assay and 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay, respectively. The sensory properties were assessed by panelists and were evaluated using the 9-point hedonic scale. The results indicated that total flavonoid concentration (TFC) ranged from 73.12 mg/LQE to 222.30 mg/LQE and the total phenolic concentration ranged from 193.94 mg/L GE to 297.27 mg/L GE. The DPPH ranged from 49.94% to 81.26% and the FRAP ranged from 35.77 AAE to 42.89 AAE. There was no significant difference between the different formulations, but a significant difference was observed between the different days of fermentation. The sensory properties showed variations between the different formulations, with treatment T0 giving significantly higher levels for most attributes than the other treatments. In conclusion, a combination of fruits with optimum fermentation enhances bioactive and antioxidant properties that make it a suitable functional food. 

Introduction

The global burden of non-communicable diseases (NCDs) is increasing daily. The World Health Organization reports that 74% of deaths globally are associated with non-communicable diseases, and 86% of these deaths occur in low-or middle-income countries [1]. NCDS risk factors include physical inactivity, unhealthy diets, and substance use. Non-communicable diseases include: cancer, diabetes, mental health problems, and cardiovascular problems. The antioxidants help to fight these NCDs by neutralizing free radicals that cause cellular damage and oxidative stress.

In Kenya, over a third of the deaths are associated with NCDs, and more than 50% of the reported cases are associated with lifestyle problems [2]. Dietary factors are lifestyle issues associated with the occurrence of NCDs. This is also associated with a lack of dietary balance and eating fast foods with fewer nutrients. Poor incorporation of fruits rich in antioxidants may lead to an increase in the incidence of these diseases [3]. Most fruits are perishable, and developing their beverages may help increase their longevity and availability in the market for consumption.

Mulberry and yacōn fruits are important for health because of their nutritional contents. Mulberry fruit contains more than 80% water and is rich in glucose, sucrose, fructose, flavonoids, anthocyanins, phenolic acids, and other bioactive compounds [4]. These bioactive compounds provide functional health-promoting properties, including antioxidant, anti-atherosclerosis, anti-diabetic, anti-microbial, anti-inflammatory, neuroprotective, antitumor, hepatoprotective, and immunomodulatory properties [5].

Anthocyanins, flavanols, flavanones, and phenolic acids are bioactive compounds that promote antioxidant properties by binding to hydrogen peroxide radicals that cause NCDs. Mulberry fruit has anti-diabetic properties [4]. This is achieved by reducing β-cell viability, inhibiting apoptosis, and restoring glucose recovery [6]. Moreover, Martins et al. [7] revealed that people who consume mulberry fruits have improved health outcomes, as the fruit contain bioactive compounds that boost body functions, prevent cardiovascular diseases, prevent chronic diseases such as cancer, reduce inflammation, and improve brain function.

Yacōn is a Peruvian apple that originated in South America. Yacōn (Smallanthus sonchifolius) is a root tuber with health-promoting properties. It is considered a functional food because of its biologically active compounds, which provide physiological benefits beyond the nutritional functions [8]. In vivo studies have shown that it lowers blood glucose levels and acts as an antioxidant, anti-inflammatory, and anticancer [9]. Yacōn is rich in fructo-oligosaccharides, triterpenoids, saponins, flavonoids, polychronic, and inulin, which have potential health-promoting properties [10], [8]. Yacōn is a nutritious tuber that can be incorporated into various products, owing to its potential benefits.

Fruits and fruit-based beverages have been proven to be the sources of most bioactive compounds. However, most fruits are highly perishable, and there is a need for their preservation for long-term storage. The most common method of preservation is thermal processing, which has some disadvantages owing to nutritional losses. There is a need to consider the methods of processing the fruits, as some methods destroy bioactive compounds [6]. Thermal processing of fruits causes significant losses of bioactive compounds, whereas non-thermal processing of fruits may not cause significant loss of bioactive compounds. Fermentation is a non-thermal processing method and has been identified as one of the processes that increases the shelf-life of a product and enhances microbiological safety [11]. According to Punthi and Jomduang [12], the natural fermentation of mulberry fruit retains high profiles of bioactive compounds compared to other non-thermal processing techniques, such as pulsed electric field, irradiation, and cold plasma. Natural fermentation prolongs the process of fermentation, allowing the bioprocessing of existing microorganisms to fully utilize the substrate [12].

Information on the effects of natural fermentation on the bioactive compounds of beverages from different fruit mixtures is limited This study aimed to determine the levels of bioactive compounds and antioxidant, and sensory properties of beverages from mulberry fruits, yacōn, and lemon mixtures produced through natural fermentation.

Materials and Methods

Raw Material Collection

Ripe mulberry fruits (Morus nigra) and fully mature yacōn roots were obtained from the Center for Food and Naturopathy (CeFoNa) Demonstration Farm (Dekut). Lemon fruit was purchased from Ox-Farm Organic Farm in Nyeri County. They were all cleaned and in running water before storage at −18°C for two days before fermentation. Both the mulberry fruit and yacōn were washed in sodium hypochlorite and rinsed with sterile water to eliminate any surface microbial load. Thereafter, they were stored at −18°C for two days before fermentation.

Chemicals and Reagents

All standards and chemicals were purchased from Multi-Gen Laboratory Supplies (Nairobi, Kenya).

Preparation of the Beverage and Fermentation

Experimental Design

The project used a Completely Randomized Design in which three treatments were prepared by mixing mulberry fruits, yacōn tubers, and lemons using the proportions (%) indicated in Table I. The samples were subjected to natural fermentation.

Table I. Preparation of Beverage Formulation

Treatments	T0 (Control)	T1	T2
Mulberry	90	60	30
Yacōn tuber	0	30	60
Lemon	10	10	10

Natural Fermentation

Fermentation of different samples was performed according to the method of [13], with slight modifications. Before fermentation, the raw materials were thawed for 8 h. Yacōn tubers and lemon fruit were cut into small pieces of approximately 1 mm. The mulberry fruit was used as a whole. The different fruits were combined according to the proportions listed in the table above. The different samples were fermented at room temperature for 21 d with no addition of starter cultures. Samples from the treatment groups were collected on 0, 5^th, 10^th, and 21^st day and used for analysis. The collected samples were decanted, and the supernatants were stored at −20°C awaiting chemical analysis.

Chemical Analysis

Determination of Bioactive Compounds (Total Phenolic Concentration and Total Flavonoid Concentration)

Total phenolic concentration was determined using the method described by [14], with slight modifications. Folin-Ciocalteau reagent was used to form a blue complex, which was quantified using a UV spectrophotometer (UV-1600). One milliliter of sample was diluted with distilled water at a ratio of 1:5. 0.5 mL of the mixture was mixed with 2.5 mL of Folin-Ciocalteau reagent. The solution was vortexed for two minutes and allowed to settle for another 5 min. Two milliliters of 7.5% sodium carbonate was added, and the mixture was incubated for 30 min. The absorbance was measured at 765 nm. The standard curve was prepared using gallic acid, and was used to quantify the results, which were expressed as milligrams of gallic acid equivalents (mg GAE) per gram of the fermented mulberry fruit-yacōn root beverage drink.

The UV spectrophotometer (UV-1600) and aluminum chloride colorimetric method, as described by Kwaw et al. [15] with slight modifications, were used to determine the total flavonoid concentration. 300 µl NaNO₂ (50 g/l) and 4 mL distilled water were added to 1mL fermented juice and vortexed for one minute. The mixture was allowed to stand for 5 min, then 1 mL of Aluminium chloride was added, vortexed, and allowed to stand for 5 min. Subsequently, 2 mL of 1 mol/l NaOH and 2.4 mL of distilled water were added. The mixture was incubated at 25°C with a 2-minute intermittent shaking. After 10 min, the absorbance was measured at 510 nm using a UV spectrophotometer (UV-1600). Total flavonoid levels were expressed as milligrams of quercetin equivalents per milliliter of fermented juice.

Determination of Antioxidant Property

DPPH

The 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity (DPPH-SA) was measured to determine the antioxidant activity of the beverage. It was determined using the method described by Kwon et al. [16] with some slight modifications. In summary, 60 µL of fermented beverage was added to 2.1 mL of a 0.1 mM solution of DPPH prepared in methanol. The mixture was incubated in the dark for 30 min at 25°C, and the absorbance was read at 517 nm. The control group was comprised fermented beverages without DPPH. The percentage of DPPH-SA in the fermented beverage was calculated as follows:

$$\begin{array}{r}\mathrm{\%}DPPH-SA=(Acontrol-Asample)/Acontrol\times 100\end{array}$$

Ferric Reducing Antioxidant Power (FRAP)

The method described by Kwon et al. [16] was used to determine the FRAP of the fermented beverage drink, with slight modifications. 2.5 mL of (0.2M Phosphate) was prepared and pH adjusted to 6.6 with 10% sodium hydroxide, and added to 1 ml of the sample. After vortexing for 2 min, potassium ferricyanide (2.5 mL) was added to the solution, and the mixture was vortexed for 2 min. The mixture was then incubated at 50°C for 20 min After 20 minutes, 2.5 mL of 10% trichloroacetic acid was added. From the mixture, 2.5 mL was obtained from the mixture; and deionized water (2.5 mL) added. The supernatant formed a blue color after adding 0.3 mL ferric chloride, and the results were read at 700 nm. Ascorbic acid was used as the standard, and the results are expressed in milligrams of ascorbic equivalent (AAE) per 100 g.

Sensory Analysis

Sensory analysis was performed by 50 panelists; and samples with unique codes were provided. The parameters considered include: color, appearance, aroma, and overall acceptance. The parameters were analyzed using a 9-point hedonic scale: 9 = like extremely, 8 = like very much, 7 = like moderately, 6 = like slightly, 5 = neither like nor dislike, 4 = dislike slightly, 3 = dislike moderately, 2 = dislike very much, 1 = dislike extremely [17].

Ethical Considerations

The research was conducted following the ethical guidelines, and the research received ethical approval from Dedan Kimathi University of Technology Scientific Ethics Review Committee (DEKUTSERC/ISREC/03422/090). The participants were aged 20–26 years and were from Dedan Kimathi University of Technology. They were informed by a notice and voluntarily participated in the research process. A total of 50 research participants joined the study. Data confidentiality and privacy were ensured. The research process was explained to the participants, and after they understood it, they were required to sign an informed consent form.

Statistical Analysis

Data were captured using Microsoft Excel version 2019. The data collected in triplicate are expressed as the means ± standard deviation. Minitab software version 18 (Minitab Inc., Pennsylvania, USA) was used to analyze the data. Two-way analysis of variance (ANOVA) was performed and the means of separation were calculated using the Fisher Pairwise Comparison and LSD method at a 95% confidence interval, with a significance difference of p ≤ 0.05. General linear models were used to determine the interactions between treatment contents and fermentation days.

Results and Discussion

Determination of Phenolic Compounds

The total phenolic concentration (TPC) of the different samples are presented in Table II. There were no significant differences between the different treatments (T0, T1, andT2) in the levels of total phenolic composition for the different days of fermentation. The TPC level increased with increasing fermentation days. For treatment T0, the TPC ranged from 193.94 to 275.94; T1 from 216.79 to 297.27; and T2 from 182. 73 to 231.45. The significant increase in TPC with fermentation time is associated with the conversion of complex phenolic compounds to simpler forms;, thus; enhancing their availability in beverages with increased fermentation time [18]. Lin et al. [19] revealed that the synthesis of phenolic compounds, such as hydroxybenzoic acids, hydroxycinnamic acids, and flavonoids, such as quercetin, is broken down with an increase in fermentation time through enzymatic activity; to create smaller phenolic compounds such as aglycones, ellagic acids, and gallic acid which increase their bioavailability in the beverage.

Table II. TPC of Treatments Produced by Natural Fermentation (mg/L gallic acid)

Number of days
Treatment	Day 0	Day 5	Day 10	Day 21
T0	193.94 ± 0.14Bd	197.05 ± 0.12Bc	230.32 ± 0.29Bb	275.94 ± 0.61Ba
T1	216.79 ± 0.18Ad	241.45 ± 0.12Ac	261.08 ± 0.12Ab	297.27 ± 0.19Aa
T2	182.73 ± 0.29Cd	196.94 ± 0.18Cc	221.36 ± 0.30Cb	231.45 ± 0.12Ca
ANOVA Significance (p-values)	Treatments	0.000
	Fermentation days	0.000
	Interaction	0.000

Treatments T0, T1, and T2 did not show significant differences in the levels. However, there was a trend in the TPC level in each treatment with T1, showing a higher concentration. This could be associated with the levels of different ingredients used. Treatment T1 had a moderate mulberry to yacōn fruit ratio (2:1). This is in line with a study conducted by Mapelli-Brahm et al. [11], which revealed that fermentation of berries, such as elderberry and mulberry, has higher phenolic concentration than root tubers, such as yacōn. However, a combination of berries and root tubers has shown significant antioxidant abilities because of the combination of phenolic compounds in both fruits [20]. Moreover, de Souza et al. [10] indicated that yacōn fermented for 60 days had the highest phenolic compounds between days 15 and 25. This indicates that yacōn achieves a higher phenolic concentration at 15–25 during fermentation.

A statistically significant (p ≤ 0.05) interaction was observed between treatment and fermentation days. This is associated with biochemical reactions that break down complex phenolic compounds during fermentation. The process utilizes the substrate and breaks it down to simple molecules, such as lactic acid, which enhances the sensory and nutritional values of the beverage [21].

Determination of Total Flavonoid Concentration (TFC)

The total flavonoid concentration (TFC) of different samples are listed in Table III. There were no significant differences between the different treatments, but there were significant differences in the flavonoid concentration between the different days of fermentation. There was a significant increase in flavonoid concentration from day 0 to day 21 for all treatments. Treatment T0 ranged from 73.12 to 222.30, T1 ranged from 95.64 to 218.69, and T2 ranged from 79.15 to 190.93 (Table III). The increase in TFC with increasing fermentation time is associated with biochemical changes associated with the breakdown of intricate polyphenols into simple flavonoid compounds. This aligns with the results of Hu et al. [22] and Kwon et al. [16], who showed an increase in flavonoids with fermentation time. A study conducted by Adetuyi and Ibrahim [23] on the fermentation of okra seeds revealed that fermentation time has a positive influence on increasing the levels of flavonoids as the acidic value of the content increases.

Table III. TFC of Treatments Produced by Natural Fermentation (mg/LQE)

Number of Days
Treatment	Day 0	Day 5	Day 10	Day 21
T0	73.12 ± 0.14Cd	96.14 ± 0.14Cc	163.21 ± 0.08Bb	218.69 ± 0.08Ba
T1	95.64 ± 0.78 Ad	116.73 ± 0.44Ac	180.48 ± 0.21Ab	222.30 ± 0.13Aa
T2	79.15 ± 0.13Bd	96.68 ± 0.14Bc	136.59 ± 0.12Cb	190.93 ± 0.14Ca
ANOVA Significance (p-values)	Treatments	0.000
	Fermentation days	0.000
	Interaction	0.000

The formulation of the samples also influenced the TFC. This is evident from the results, where the T1 samples showed a higher level than T0 and T2. Treatment T1 had a proportion of 60% mulberry fruit and 30% yacōn. This combination results in a slightly higher TFC concentration. T0, which had a higher proportion of mulberry at 90% and 0% yacōn, did not yield higher concentrations of TFC. This confirmed that a combination of the two ingredients contributed to a higher flavonoid content. A study by Zhang et al. [24] indicated that mulberry is one of the berries with the highest levels of flavonoids compared to other berries. It also contains anthocyanins that produce chromogenic compounds that increase flavonoid levels [25], and some flavonoids, such as quercetin and kaempferol, are also found in yacōn in the lowest amounts.

A statistically significant interaction between the content of the treatments and fermentation days was identified at p ≤ 0.05. This interaction is linked to biochemical interactions between the substrate during fermentation and the microorganisms that convert the complex flavonoids into simple molecules, making them bioavailable. Tomar et al. [26] indicated that mutual interactions occur between the substrate composition and fermentation microorganisms, which improve the nutritional and sensory qualities of the beverage.

Antioxidant Properties of Mulberry Fruit-Yacōn Fermented Beverage

DPPH Activity

The DPPH antioxidant activity increased with fermentation time (Table IV). There was a statistically significant level for all samples across the days (0, 5, 10, and 21) of fermentation. For sample T0 (control), the DPPH levels increased from 50.82 to 70.55, for T1, from 59.05 to 81.26, and for T2, from 49.94 to 77.98 from day 0 to 21. The increase in DPPH levels with fermentation time was associated with high phenolic and flavonoid levels. This has been linked to the high antioxidant capacity and increased production of metabolic products, which causes the synthesis of polyphenols and phenolic compounds into active antioxidant forms [27], [23]. Moreover, the increase in antioxidant activity is also associated with increased enzymatic activity that produces β-glucosidase, which releases bound phenolic compounds into their free active forms [28].

Table IV. DPPH Level (%) for Treatments Fermented for Different Days

Number of days
Treatment	Day 0	Day 5	Day 10	Day 21
T0	50.82 ± 0.05Bc	63.17 ± 0.08Cb	67.68 ± 0.08Cb	70.55 ± 0.14Ca
T1	59.05 ± 0.02Ac	63.39 ± 0.16Bc	69.95 ± 0.11Bb	81.26 ± 0.11Aa
T2	49.94 ± 0.07Cd	65.33 ± 0.03Ac	73.23 ± 0.11Ab	77.95 ± 0.08Ba
ANOVA	Treatments	0.000
Significance	Fermentation days	0.000
(p-values)	Interaction	0.000

The DPPH levels were statistically significant in all samples based on the fruit formulation (mulberry, yacōn, and lemon). This trend was observed in all samples formulated across days. Statistical significance was associated with the ratio of mulberry fruit to yacōn fruit. The T0 sample had the lowest DPPH (70.55), which was statistically significantly different from T1 (81.26) and T2 (77.95). The results indicate that treatment with a moderate ratio of mulberry to yacōn T1 (60%:30%) had the highest DPPH levels. This indicates that mulberry fruits have higher antioxidant power than yacōn fruits [29]. A study conducted by Wang et al. [30] showed that fermented juice had a DPPH value of 73.64, which was lower than the highest score of 81.26, which is likely to be attributed to the addition of yacōn tubers to fermented mulberry fruits.

A statistically significant interaction was identified between treatment and fermentation days during DPPH activity. The interaction is linked to the ability of beneficial microorganisms to synthesize complex phenolic compounds with fermentation time and break them into simple compounds that enhance the quality of the beverage. This interaction enhances the flavor and improves the antioxidant capacity of the beverage [24].

Ferric Reducing Antioxidant Power (FRAP)

The antioxidant levels (ferric reducing antioxidant power) were measured based on the ability of the samples to reduce Fe³⁺ to Fe²⁺ and expressed in ascorbic acid equivalents. The results are presented in Table V. There were significant variations in FRAP levels in samples fermented for different times (p ≤ 0.05). The T0 sample show FRAP levels of 38.74 to 42.49. T1 samples ranged from 38.72 to 42.82, while T2 samples ranged from 35.77 to 42.13. FRAP levels increased with fermentation time, with the highest levels recorded on day 21 for all treatments. The significant increase in the FRAP levels of the samples with fermentation time was attributed to the breakdown of phenolic compounds that were synthesized as the fermentation time increased. Higher phenolic and flavonoid levels in the mixture were synthesized and broken down into simple molecules with an increase in fermentation time. These results are in line with those of Pande and Rai [31] and Chuah et al. [29], who showed an increase in the reducing power of yacōn roots and mulberry fruits, respectively, during fermentation. A study by Kim and Lee [20] shows that different mulberry species record 1.33 ± 0.12 to 82.87 ± 6.69 of FRAP levels depending on different species of mulberry, environmental, and climatic conditions. Tinrat [28] recorded higher FRAP levels ranging from 84.48 ± 0.02 to 134.45 ± 0.11 in the fermentation of mulberry wine, which would be associated with different procedures of fermentation, an extended time of fermentation to 24 days, and different species of mulberry fruit.

Table V. FRAP Level for Treatments Fermented for Different Days (Milligrams of Ascorbic Equivalent (AAE) per 100 Grams)

Number of days
Treatments	Day 0	Day 5	Day 10	Day 21
T0	38.74 ± 0.04Ac	39.73 ± 0.02Ad	41.38 ± 0.01Bb	42.49 ± 0.03Ba
T1	38.72 ± 0.01Ad	39.92 ± 0.02Ac	41.99 ± 0.03Ab	42.87 ± 0.01Aa
T2	35.77 ± 0.01Bd	37.89 ± 0.01Bc	39.25 ± 0.01Cb	42.13 ± 0.01Ca
ANOVA	Treatments	0.000
Significance	Fermentation days	0.000
(p-values)	Interaction	0.000

There were significant differences between the different treatments, with T2 showing the lowest FRAP levels compared to T0 and T1. This could be attributed to the variation in the number of mulberry and yacōn fruits, which affected the FRAP levels. Sample T0 (control) was statistically significantly different from sample T2, and not statistically significant from sample T1 on days 0 and 5. This was associated with a higher amount of mulberry fruit (90% and 60%) in T0 and T1, respectively, than in T2 (30%). Mulberry fruits have higher antioxidant levels than yacōn roots because they have higher levels of phenolics, flavonoids, and anthocyanins, which increase antioxidant activity [32]. There was statistical significance in all the samples on day 10, which was associated with the breakdown of the complex phenolic compounds present in the sample from both mulberry fruit and yacōn root. The same trend was observed on day 21. Sample T1 had the highest FRAP levels on days 10 and 21 because of the moderate levels of mulberry and yacōn fruits (60% and 30%), respectively. This indicates that a combination of yacōn and mulberry with fermentation time increases antioxidant power. A higher ratio of mulberry fruit to yacōn fruit results in higher antioxidant levels [33]. These results are consistent with those of Kim et al. [34], who reported higher antioxidant levels in mulberry leaves than in a mixture of mulberry leaves and yacōn fruit. Rohela et al. [35] also revealed that mulberry fruits have higher antioxidant properties than other berries and wild fruits.

There was a statistically significant interaction between the treatment content and fermentation days (p ≤ 0.05) for ferric reducing antioxidant power. This is linked to the breakdown of substrates during fermentation through biochemical reactions to simpler molecules such as alcohols [36].

Sensory Evaluation

Sensory evaluation was performed based on the sensory attributes of; appearance, color, taste, and aroma on a 9-hedonic scale. Fig. 1 shows the results of the sensory evaluation of the samples of a naturally fermented beverage for 21 days. The appearance of treatment T0 was not significantly different from that of treatment T1, but was significantly different from that of T2. Treatment T2 gave the lowest significance level for appearance because the lowest proportion of mulberry fruits, led to a pale color compared to the other treatments.

Fig. 1. Sensory evaluation data for treatments produced by natural fermentation.

The color of treatment T0 was the highest among the samples and was statistically significant compared to that of the other samples. This was associated with presence of 90% mulberry, which increased the concentration of color. The color of T1 was statistically significant in T2. This was associated with different levels of mulberry fruit and yacōn root slices. T2 had more yacōn than T1, which makes the color difference, as yacōn root has a pale white color; and mulberry fruit has a red-purple color. The color change among the samples would also be associated with longer fermentation times, causing only the sample with a higher concentration of mulberry (a dominant red-purple) color to remain throughout the fermentation period [37].

Note: Values are means ± standard deviation of three determinations. Values across the bars with different superscript letters indicate statistically significant differences at p ≤ 0.05. Abbreviation: T0: (90% mulberry fruit: 0% yacōn root: 10% lemon), T1 (60% mulberry fruit: 30% yacōn root: 10% lemon), and T2 (30% mulberry fruit: 60% yacōn root: 10% lemon)

The aroma of T0 was significantly higher than that of T1 and T2. This is attributed to the high content of mulberry fruit in the samples and the absence of yacōn roots in the treatment. This is supported by a study conducted by Reis et al. [38], which indicated that yacōn roots have few esters that influence the aroma, whereas mulberry fruit has many esters, such as terpenoids and alcohols, which create a fruity aroma [39]. Treatments T1 and T2 had some additions of yacōn roots in their formulations.

The taste results also showed that treatment T0 was significantly higher than that of T1 and T2. This is associated with the sour taste of sweet mulberries after fermentation [32]. Treatment T1 and T2 did not show any significant differences in the level of taste, indicating that the presence of yacōn in the formulation may have lowered the taste sensation, as it has a tangy taste [8].

T0 had the highest overall acceptability, whereas T2 had the lowest. This is because most participants were aware of mulberries as compared to yacōns. Because yacōn is a new product in the Kenyan market, only a few people have come across it or know it [40]

Conclusion

In the present study, it can be concluded that fermentation time influences the increase in the level of bioactive compounds. Increasing the fermentation time increase the level of total phenolic and flavonoid compounds. The different proportions of mulberry, yacon and lemon fruits in the formulations did not have any effects on the levels of total phenolic and flavonoids compounds. The increase in fermentation time increases the level of antioxidant activities of the samples. The different proportions of mulberry, yacon and lemon fruits in the formulations did not have any effects on the levels of antioxidant activities. The different proportions of mulberry, yacon and lemon fruits in the formulations influence the variations in the sensory varieties of the samples. The levels of appearance, aroma, taste, and overall acceptability were higher in samples with a higher composition of mulberry.

The present study was limited to microbial profilling and sensory bias. The study did not identify the specific microorganisms that aided fermentation but considered it from a general point. Future studies should focus in microbial profiling to identify specific micro organisms that support natural fermentation. The present study used only 50 panellists from the university; increasing the number of panelists in the future with a broader view of the different people from the society can reduce the sensory bias. The study did not do the shelf-life of the beverage and thus, a focus on shelf-life studies in the future may be beneficial for consumption patterns and safety. Moreover, there is need to study other fruits rich in bioactive and antioxidant properties and the different processing methods.

Acknowledgment

Marita Moraa Annah is grateful to the German Academic Exchange Service for funding her research. The authors are also thankful to the National Phototherapeutics Research Center and Dedan Kimathi University of Technology for granting access to the laboratory.

Conflict of Interest

The authors declare no conflict of interest.