Functional quality evaluation and shelf life study of synbiotic yacon juice

Abstract The research aimed to utilize the prebiotic potential of yacon fruit by preparation of synbiotic yacon juice containing probiotic microorganism Lactobacillus acidophilus (La5) and studying the changes in physicochemical properties during storage. The fresh juice extracted was added with probiotic microorganism and was subjected for storage at refrigerated (4°C) and room condition (25°C) along with control (nonfermented) juice. The viability of probiotic strains in the juice was found to be at the satisfactory level (106 CFU/ml) for 27 and 15 days, respectively, in refrigerated and room condition. Total soluble solid, pH, acidity, bioactive compounds (ascorbic acid, carotenoid content, and total phenol), and antioxidant capacity of the products were evaluated. Total soluble solid of the prepared synbiotic juice stored at refrigerated and nonrefrigerated conditions decreased from 7.8 ± 0.05 to 5.5 ± 0.26 and 5.3 ± 0.10 while the acidity increased from 0.067 ± 0.05 to 0.80 ± 0.06 and 1.02 ± 0.04 after 27 and 15 days of storage, respectively. Similarly, yeast and mold count, total plate count, and the coliform count were determined during the storage period. The low amount of yeast and mold count and absence of coliform in the synbiotic juice even after 27 and 15 days of storage indicated that the synbiotic juice can be continued to be consumed as a normal juice even after the viability decreased below the satisfactory level. Ascorbic acid, phenolic content, and antioxidant activity were higher in fermented juice when compared to control juice stored at the refrigerated conditions for the same period (p < .05). High therapeutic properties and numerous health benefits of yacon, when combined with the health benefits of probiotic bacteria, could lead to the development of commercial beverages with high health and nutritional values.


| INTRODUC TI ON
Probiotics can be defined as live microorganisms that when administered in adequate amounts confer a health benefit on the host (Hill et al., 2014), while prebiotics are defined as "selectively fermented ingredients that allow specific changes both in composition and/or activity in the GI microflora that confer benefits upon host well-being and health" (Gibson et al., 2017). Prebiotic can be added to food or combined with a probiotic to make synbiotic (Hamilton-miller, 2004). A synbiotic can be defined as the supplement which contains both prebiotic and probiotic factors that work together and improve the "friendly flora" of the human intestine (Thakur, 2016).
Dairy products such as yogurts, fermented sour milk, and cheese are the major probiotic foods produced and used at present (Ranadheera, Naumovski, & Ajlouni, 2018). However, the increase in the number of vegetarian consumers has increased the demand for nondairy probiotic products (Vasudha & Mishra, 2013). Lactose intolerance, allergies to milk protein, and high cholesterol are other major drawbacks related to fermented dairy products (Granato, Branco, Nazzaro, Cruz, & Faria, 2010;Martins et al., 2013).
Yacon (Smallanthus sonchifolius) is a native Andean plant, grown for its juicy tuberous roots in South America, on the eastern slope of Andes from Venezuela to northwest Argentina (Valentova, Cvak, Muck, Ulrichova, & Simanek, 2003;Zardini, 1991). The presence of nondigestible oligosaccharides (NDOs) such as inulin and fructo-oligosaccharides as well as the high amount of phenolic compounds has increased the global interest on yacon (Choque Delgado, da Silva Cunha Tamashiro, Maróstica Junior, & Pastore, 2013). Yacon stores FOS instead of starch, which is considered prebiotic as these compounds are fermented selectively by bacteria (bifidobacteria and lactobacilli) in the bowel but are neither absorbed nor hydrolyzed in the upper part of the gastrointestinal tract (Ojansivu, Ferreira, & Salminen, 2011;Pedreschi, Campos, Noratto, Chirinos, & Cisneros-Zevallos, 2003). Besides, yacon roots have numerous health-promoting properties including prebiotic, antidiabetic, antioxidative, and antimicrobial effects (Ojansivu et al., 2011). A study on yacon flour and its effect on intestinal microbiota and gut microflora using mice model showed stimulation in the growth of bifidobacteria and lactobacilli. The study also showed improvement in the intestinal immune system with an increase in IgA and different cytokines (Bibas Bonet et al., 2010).
The product prepared from yacon (prebiotic source) can act as a perfect alternative to the dairy probiotic products. As there is a high demand for functional food across the globe, the inclusion of yacon as a source of fructan-type prebiotics in the production of synbiotic beverage for human use represents a great opportunity for both innovation and adding value in the functional food industry.
The objective of this research work was to prepare a yacon-based synbiotic juice and to access the viability of probiotic microorganism in the produced beverage. The research also aims for the comparative study of bioactive compounds and antioxidant activity of fermented and nonfermented juice stored at refrigeration temperature.

| Chemicals and reagents
2,2-diphenyl-1-picrylhydrazyl (DPPH) was purchased from Sigma-Aldrich, Germany. Folin-Ciocalteau reagent was purchased from Merck Specialities Private Limited, India. Gallic acid was purchased from LOBA Chemie, India. Methanol was purchased from Fisher scientific, India. PCA agar, PDA agar, MRS agar, and VRBA agar media were purchased from Hi-Media Laboratories, India. The spectrophotometer used was of Model GENESYSTM 10S Vis spectrophotometer from Thermo Scientific TM, Germany.

| Preparation of probiotic yacon juice
Matured yacon fruit (10 days after harvesting stored at normal room temperature) was taken. Yacon fruit was peeled and cut into chunks of small size after washing in running tap water. The juice was extracted from blanched chunks (95°C for 3 min) (Reina et al., 2015). The blanching step was done to inactivate polyphenol oxidase enzyme as well as to reduce the number of indigenous yeast (Reina et al., 2015). The juice was extracted with the addition of water (0.5 times the weight of fruit), and the resulting juice was filtered through muslin clothes. The juice was then pasteurized at 85°C for 10 min for the preparation of pasteurized yacon juice.

| Physicochemical analysis of control and fermented product
Total soluble solid, pH, and acidity were measured by standard techniques (Ranganna, 2007).
Lane and Eynon method using carrez solutions as a clarifying agent was used for the determination of total sugar and reducing sugar (AOAC, 2005). Clarity was measured by measuring transmittance at a wavelength of 590 nm using a UV-Vis spectrophotometer (Singh, Kumar, & Sharma, 2012).

| Microbial analysis
For microbial analysis, 1 ml of pasteurized and fermented sample was diluted with 10 ml of saline solution and filtered through ster- were used for enumeration of fungi, lactobacilli, coliform, and total plate count, respectively. Three samples were used and surface plates were made in triplicates in the appropriate selective media.
Pour plate method and serial dilution technique were used. For computation, the average number per plate was divided by sample volume and is expressed as CFU/ml (Sharma, 2006).

| Extract preparation
For ascorbic acid, 10-20 ml of the sample was taken and volume was made to 100 ml using 3% HPO 3 followed by filtration.

RCY NCY
Change in Yeast and mold count during storage Antioxidant activity was determined by DPPH radical scavenging method as determined by Sochor et al., 2010 andStajcic et al., 2012.

%DPPH radical scavenging activities = [(A
where A 0 is the absorbance of the control (DPPH).
A 1 is the absorbance of the sample.

| Sensory analysis
Ten untrained panelists (college students and staff) were involved to assess the sensory properties according to 9 point hedonic ranking

| Statistical analysis
The analysis was carried out in triplicate. The data were statistically analyzed using SPSS Edition 20 and Microsoft excel 2007. Data were analyzed for significant difference by ANOVA at 5% level of significance using Tukey test.

| Experimental design
The experiment is carried out in a completely randomized design with two treatments and three replicates for chemical and microbial analysis.

| Changes during fermentation and storage
The results showed decrease in pH while acidity increased during fermentation at 37°C for 24 hr (

| Physicochemical changes upon storage
The change in total soluble solid (TSS), pH, and acidity during storage was monitored in the space of 3 days and shown in Figure 1a-c, respectively.
The decrease in pH and increase in acidity were significant in probiotic juice as compared to nonfermented juice in both refrigerated and nonrefrigerated conditions. For fermented juice RPY, and NPY, the pH decreased from 4.58 to 4.02 ± 0.02 and 3.48 ± 0.02 after 27 and 15 days of storage, respectively. However, for nonfermented sample pH remained quite stable and decreased from 6.78 ± 0.02 to 6.03 ± 0.02 and 5.32 ± 0.02, respectively, after

| Microbial changes upon storage
The change in Lactobacillus count, TPC and yeast, and mold during storage was monitored in the space of 3 days and shown in Similarly, total plate count (TPC) of the nonfermented sample was 1.47 log CFU/ml which increased to 2.57 and 3.17 log CFU/ml after storing for 27 and 15 days. However, TPC of the fermented sample was higher initially 9.54 log CFU/ml and decreased to 7.3 and 7.8 log CFU/ml during the storage time.
There was no growth of yeast and mold in probiotic juice and for nonprobiotic, and the growth was in the minimal count and hence expressed in CFU/ml. Lack of oxygen during storage along with blanching

| Physicochemical analysis of yacon juice
The mean score for the physicochemical properties of all the samples and statistical analysis (ANOVA) for the values obtained were presented in Table 2.
The significant reduction of sugar content in probiotic added juice is due to the fermentation process. During fermentation homofermentative, Lactobacillus is responsible for the conversion of fermentable sugar into lactic acid resulting in the reduction of the reducing sugar.
Surprisingly, for normal juice, the reducing sugar value showed a slight increase over the storage period. Wong, 2003 andBibas Bonet et al.., 2010 studied the effect of storage on FOS of yacon and the result showed the conversion of FOS to glucose, fructose, and sucrose at a temperature higher and lower than 10°C which might be the reason for the increase in reducing sugar value.

| Bioactive compounds and antioxidant property
The change in the amount of ascorbic acid, carotenoid content, phenol content, and antioxidant activity from the freshly prepared juice to fermented and control juice stored in refrigerated condition for 27 days is shown in Figure 3a-d, respectively.
The bioactive components were higher in the freshly extracted initial juice. Upon storage, the bioactive components decreased.
However, the decrease was less severe for fermented juice as compared to nonfermented juice except for carotenoid value. Similar results were also seen during the storage of fermented (Lactobacillus plantarum) and nonfermented pomegranate juice stored for 30 days (Filannino et al., 2013).

| Sensory analysis
The sensory analysis result is shown in Figure 5.
A comparative sensory analysis between INI (fresh juice), RCY, and RPY (stored for 25 days) showed no significant difference at 5% level of significance. The acceptance for all the juice was very good even after 25 days of storage.

| CON CLUS ION
The result concludes that yacon can act as a substrate for the growth of probiotic microorganisms and the storage of yacon as synbiotic juice is beneficial in both sensory and nutritional aspects. The low yeast and mold count indicated synbiotic juice can be continued to be consumed as a normal juice even after the viability of the lactic organism decreased.
The fermentation provided a good preservative effect on phenolic content and antioxidant activity of yacon juice which confers the nutritional benefits of the functional food prepared. Research Council, for providing the support for completion of this project.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

I N FO R M E D CO N S E NT
Written informed consent was obtained from all participants.