Assessing synergistic effect of Jerusalem Artichoke juice and antioxidant compounds on enhanced viability and persistence of Bifidobacterium species, palatability, and shelf life

Abstract Commercial vegetable and fruit juices with probiotics are new functional type of beverages; however, limitations including persistence and impact of probiotic bacteria on palatability and shelf life may prevent their industrial development. This study evaluated the effect of antioxidant compounds (ascorbic acid, astaxanthin, and ginseng) on viability and persistence of Bifidobacterium spp. in Jerusalem Artichoke (JA) juice; and determine the impact of these antioxidants on the sensory (color, texture, flavor, acidity) properties, free reducing sugar (inulin and fructose), and shelf life in the fortified JA juice. Overall, the JA juice fortified with ascorbic acid showed a significant impact on the rate of persistence of two targeted bifidobacterial strains from 1 to 28 days at 5°C. Both strains produced slight acidity in ascorbic acid fortified JA juice as compared to other tested samples. Similarly, the JA juice fortified with ascorbic acid showed a significantly high increase in the total number of bifidobacterial cells of both species, enhanced palatability, and shelf life as compared to astaxanthin and ginseng extract. The quadratic model indicated a strong association between ascorbic acid, ginseng extract, and astaxanthin with a bifidobacterial cell concentration in the fortified JA juices. The Box–Behnken design was considered a feasible analysis for describing fortified JA juice and the rate of viability and persistence of bifidobacteria during 28 days of storage at 5°C in all trials. In conclusion, JA juice fortified with ascorbic acid showed a significant impact on improving the cell viability and persistence of probiotic bacteria, enhanced palatability, and shelf life as compared to other compounds tested.

can directly influence the hormones that regulate appetite. Inulin enhances glucose consumption and raises glycogen synthesis with the key factor in preserving natural glucose content. It also has the function of helping liver to remove contaminants (Alsharafani et al., 2007;Roberfroid, 2007).
Commercial juices with probiotics have, recently, gained popularity among health and wellness circles; however, certain limitations such as persistence of probiotics and shelf life may prevent their industrial development. In recent years, consumers have raised their consumption of nondairy probiotic products such as fermented soy products and vegetable and fruit juices (Acevedo-Martínez et al., 2018). The consumption of these type of products have been substantially increased since they are a high source of enzymes, vitamins, fibers, and minerals as well as the bioactive compounds may enhance their absorption as compared to the raw or fresh plant tissues (Havas et al., 2014;Towviriyakul et al., 2012). Therefore, maintaining the viability of probiotic bacteria is an essential requirement to mitigate adverse effects in these fresh food products (Nualkaekul et al., 2011;Perricone et al., 2015). Adding Bifidobacterium spp. to food is restricted due to their susceptibility to aerobic and acidic conditions. Moreover, low pH and aerobic environment inhibit viability and persistence of bifidobacteria, particularly in vegetable and fruit matrices. The incorporation of ascorbic acid in food can, however, reduce the amount of oxygen stress for the Bifidobacterium activities (Talwalkar & Kailasapathy, 2004). Similarly, ginseng (Panax ginseng root c.a. Mayer) contains many bioactive compounds (e.g., ginsenosides). The ginsenoside sugar chains are strongly connected to their functionality. The pharmacologically fermented ginseng with bifidobacteria and lactic acid bacteria has been identified in the biotransformation of ginsenosides (Hong et al., 2011;Lu et al., 2009).
Moreover, astaxanthin has a specific molecular structure characterized by the presence of oxygen as a hydroxyl group that creates a powerful antioxidant (Bolin et al., 2010). Considering the benefits of ascorbic acid, ginseng, and astaxanthin, JA juice fortified with these antioxidant compounds and probiotic bacteria with no added sugars would be a delicious healthy option over low-calorie (e.g., orange) juices (Serpen, 2012). Similarly, fermented milk beverages made from JA inulin could be a better alternative drink.
Considering the health benefits of JA, the present study was designed to evaluate the effect of three antioxidant compounds, including ascorbic acid, astaxanthin, and ginseng extract on the viability and persistence of Bifidobacterium breve and B. longum subsp.
longum in JA juice under aerobic and low pH conditions to maintain and improve sensory properties and shelf life at 5ºC. Secondly, assessing the impact of these antioxidants on acidity, inulin, and fructose in the fortified JA juice. Box-Behnken experimental design for response surface methods applied to explain the study design which scales up the innovative JA juice in the food industry that cannot be applied in conventional analyses at laboratory-scale conditions.

| Preparation of JA juice
Juice from fresh Jerusalem Artichoke (H. tuberosus) tubers, obtained from the local market, was extracted using the following procedure: 2 kg of tubers was washed, cleaned, and boiled in 1-L water at 85°C for 15 min for blanching of the tuber fruit. The cooked tubers were sliced into small pieces (1-2 cm 3 ) and mashed in a blender 600w bl-60phnmy (Toshiba). The smoothie was filtered and pressed according to the procedure described by Kays and Nottingham (2008) for removing dregs. The total soluble solids were 15° Brix after the juice was diluted to a 1:1 ratio (v/v) with sterile water. The final juice was sterilized at 85°C for 15 min, cooled at 25°C, and stored at 5°C for further analysis (Bampidis et al., 2019;Lee et al., 2018).

| Ginseng extract preparation
Ginseng extract was prepared from commercial Korean ginseng powder (P. ginseng C. A. Meyer) (NaturaHouse) as described by Bolin et al. (2010). Ethanol 70% was used as an extraction solvent for heat reflux extraction. The ginseng extract (GE) was collected after 6 h during the heat reflux process when the temperature reached at 80°C. The GE was separated from the extraction solvent with a rotary evaporator vacuum (RV-3, IKA ® -Werke) and dried at 45°C in the vacuum dryer (Kambic-Vakuumtrockner-CiK Solutions). The dried GE was milled and the powder was kept at −18°C until used for the assay.

| Fortified JA juice preparations
According to daily required intake (Bampidis et al., 2019;Lee et al., 2018), ascorbic acid, astaxanthin, and ginseng extract were prepared at various concentrations (15, 12, and 100 mg/L). Seven combinations of JA juice with individual or multiple antioxidants including JA juice only with ascorbic acid, astaxanthin, or ginseng extract, JA juice with ascorbic acid and astaxanthin extracts, JA juice with ascorbic acid and ginseng extract, JA juice with astaxanthin and ginseng extract, and JA juice with ascorbic acid, astaxanthin, and ginseng extract. In addition, JA juice without an antioxidant compound was used as a control.

| Total carbohydrate analysis
Total carbohydrates as free reducing sugar (fructose) and inulin, in the JA juice, were determined using a colorimetric method. Major carbohydrates (Inulin and fructose) in the JA juice were determined using the free reducing sugars method with 3.5-dinitrosalicylic acid (DNS) as described previously (AOAC, 1980;Chaplin & Kennedy, 1994).
The optical absorption value was measured at 540 nm by SPUV-19-Spectrophotometer (SCO-Tech). The standard curve of fructose was determined using the following formula: where , absorbance; x, concentration; R 2 = .9924: coefficient of determination.
To determine the inulin concentration (g/L), the juice was hydrolyzed to release inulin in the form of fructose (Alsharafani, 2006).
Briefly, hydrochloric acid solution (5%) was added to 100 ml juice to obtain 1% final concentration followed by boiling for 50 min. The hydrolyzed solution was neutralized with NaOH (1N), to obtain reduced sugar formed as fructose equivalent, which contains carbohydrate expressed as inulin. Inulin concentration (g/L) was estimated by calculating the difference of fructose concentration before and after hydrolysis as described previously (AOAC, 1980;Chaplin & Kennedy, 1994) using the following equation: where f: fructose equivalently formed; 1.2: conversion factor.

| Sensory criteria and assessment
The sensory properties of the fortified JA juice after adding bifidobacterial strains were assessed as previously described by Poste (1991). Based on preliminary satisfactory sensory acceptance from nine experienced panelists, the sensory evaluation criteria including taste, consistency, color, and smell of the juice mixtures, and the tested juices were graded as: 4.1-5.0 very good; 3.1-4.0: good; 2.1-3.0: satisfactory; 1.1-2.0: sufficient; and 0.0-1.0: not acceptable.
The sensory evaluation process was carried out by expert panelists from the Department of Food Sciences, University of Baghdad.
The panelists followed standardized sensory criteria, measurements and made objective decisions without any personal preferences.
Each panelist detected, recognized, and agreed upon common procedure and exact connotation of each descriptive term by using evaluation techniques for odor, appearance, flavor, and texture criteria. The samples were provided in plastic cups at 20°C. The taste, color, quality, consistency, and general acceptability of the food were taken into consideration while making the final decision.

| Preparation of fortified juices with bifidobacterial strains
The B. breve M4A and B. longum subsp. longum FA1 reference strains with functional properties previously reported (Alsharafani et al., 2016) were subcultured in MRS medium broth (Merck KGaA) supplemented with l-cysteine (0.5 g/L) (Merck KGaA) incubated at 37°C under anaerobic conditions for 24 h using anaerobe Jar and AnaeroGen GasPak System (Thermo Scientific™ Oxoid).
The broth was centrifuged at 2500 g for 15 min at 4°C (Allegra 64R, Refrigerated). The pellet was washed twice with phosphatebuffered saline (PBS) (0.1 mol/L pH 7.4) and resuspended in PBS. The cell suspensions 10% (v/v), form cell density (~10 9 CFU/ml), of both bifidobacterial species were prepared and mixed with six different combinations of JA juice mixtures, incubated at 37°C for 12 h, and transferred to 5°C for 4 weeks (Alsharafni et al., 2015(Alsharafni et al., , 2016. The viable cell concentration of each bifidobacterial species was measured from day one to last day of storage on MRS agar supplemented with l-cysteine (0.5 g/L) by incubating at 37°C under anaerobic condition for 72 h using 10-fold serial dilution assay. The bifidobacterial cells were further confirmed and counted based on colony morphology and microscopic analysis after incubation of Nutrient agar plates at 5, 27, and 37°C under aerobic and anerobic conditions, respectively.

| Experimental design
Two levels and three-factor full factorial experiments were established using the Box-Behnken design (BBD). Based on 14 factorial points (Center point run) and three replicates (bacterial cell concentration) at the central point, full factorial BBD was carried out for three independent variables (ascorbic acid, astaxanthin, and ginseng extract) (Anderson & Whitcomb, 2017). The response surface method (RSM) was applied to assess the optimum juice supplementation for improved viability and persistence of bacterial cells in the fortified JA juices (Anderson et al., 2015(Anderson et al., , 2018. The independent and multiple variables were symbolized as: ascorbic acid: X 1 ; astaxanthin: X 2 ; ginseng extract: X 3 ; ascorbic acid and astaxanthin: X 1 X 2 ; ascorbic acid and ginseng extract: X 1 X 3 ; astaxanthin and ginseng (1) = 0.304x + 0.0324 (2) Inulin = f × 1.2 extract: X 2 X 3 ; and ascorbic acid, astaxanthin, and ginseng extract: longum FA1 strain was used as a dependent variable. The RSM was fitted to each of the response (or dependent Y 1 and Y 2 ) variables according to Design Expert (version 12; Stat-Ease Inc.) using the following equation: where Y: Dependent response variable (expressed as log 10 ); β 0 , β 1 , β 13 , and β 23 : Estimated regression coefficients, where β 0 is an intercept; β 1 , β 2 , and β 3 : Linear effects; β 11 , β 22 , and β 33 : Quadratic effects; β 12 , β 13 , and β 23 : Interaction effects and the residue (ε).
For developing prediction models for each dependent variable, regression analysis was applied to calculate the coefficient of determination (R 2 ). Overall statistical data analysis was performed using Design Expert (version 12; Stat-Ease Inc.). Generally, a BBD was employed using Design-Expert software according to the selected variables. The practical range and level of the identified factors were outlined. The statistical analysis was performed based on the concentration of the predicted factors coded as −1: low; 0: medium; and +1: high levels. According to a second-order BBD, 17 experiments including 14 factorial and three center point runs as replicates for experimental error estimation with three independent variables were performed. The experimental design in the coded and actual levels is described in Table 1.

| Fitness model and statistical analyses
The evaluation of RSM model was performed by using statistical analysis of BBD. Three main tests, including the significance of terms, regression model, and lack-of-fit, were applied to assess the fitness and reliability of a model (Anderson & Whitcomb, 2017). A lack-of-fit test augmented to screen described the model design to a response surface design to better model the relationship between the factors and the response. Based on the experimental data for obtaining the regression equation, multiple polynomial fitness models including linear, two factorial, quadratic, and cubic models were applied using ANOVA test. Based on the significance of terms, f-and p-values were used to determine the most effective factor in the response of interest. Higher f-value along with lower p-value (normally ˂0.05) of the model terms were considered as a crucial effect on the response. The lack-of-fit test was used to identify an unsuccessful model where the data were excluded from the regression equation and assess the fitness and reliability of the model. The significant model suggests that the residual error did not exceed pure error, and consequently the model was significant. To verify the adequacy of the model, determination coefficient R 2 , adjusted R 2 , and predicted R 2 were applied. The R 2 value represents the proportion of total variations in experimentation, whereas adjusted R 2 was the variation of mean presented by the model in which a number of variables of data set was taken into account. Typically, a good model should have an R 2 value close to one, and this indicates that better fitness between existing data and the empirical model is achieved.
Additionally, power calculations were performed using response type "Continuous" at a 5% α-level to detect the specified signal/ noise ratio of the difference to detect Delta (δ signal) and estimated standard deviation Sigma (noise). A factorial effect alias was not found for the performed model. The reproducibility of the model was tested using the coefficient of variation (CV). The effect of individual variables and their interaction effect on the response were additionally investigated using three-dimensional response surface plots. Finally, the proposed model was used to define and verify the appropriate conditions of each supplement to obtain the best viable cell concentration.
The carbohydrate and pH data obtained from triplicate runs and duplicate analysis were statistically analyzed using ordinary oneway analysis of ANOVA as well as Tukey's multiple comparisons tests. The mean and standard deviation (SD) data are presented in this study. The GraphPad Prism program (v8.0 software, USA) was used for data processing, where p < .05 was considered as statistically significant.

| Fortified Jerusalem Artichoke: a promising juice
The natural amount of sugar per serving of the Jerusalem Artichoke (JA) juice is slightly less than the natural sugar in grape and orange juice (Serpen, 2012;Towviriyakul et al., 2012). Most dietary fiber in JA juice comes from inulin and fructooligosaccharides which act as probiotics and are considered as an alternative diet for people who are at risk for less dietary fiber consumption, especially the elderly community (Towviriyakul et al., 2012). In the present study, the JA juice was prepared as a novel beverage from the tubers of the plant. The new JA juice fortified with ascorbic acid successfully improved bacterial viability maintained bacterial cell concentration of B. breve strain M4A and B. longum subsps. longum strain FA1 and enhanced palatability. Preheating treatment of the JA tubers inhibited the browning reaction enzymes that hindered the development of color pigments during the process of extracting JA juice. The preheating treatment of JA tubers also helps in maintaining the color during JA juice production (Bach et al., 2013;Jeong et al., 2011;Kim et al., 2010). Additionally, the JA tubers were subjected to the preheating treatment to obtain a healthy and flavorful drink, preserving distinctive freshness as well as better sensory properties. This also substantially impacts the shelf life of juice (Cassano et al., 2007).
Most vitamins degrade while heating that leads to decrease effectiveness. The heat often helps to prevent off-flavors (Braddock & Goodrich, 2002). The JA freshness is due to volatile organic compounds known as ethyl acetate, ethyl butyrate, and terpene thiols (3) where terpene is the most common compound, and β-bisabolene is one of the dominant compounds of a terpene. In addition, hexanal is the highest and aldehydes is the second most abundant compound (Jung & Shin, 2017).

| Carbohydrate fermentation analysis
Fiber has numerous health benefits where weight regulation is the most common since inulin enhances glucose consumption and raises glycogen synthesis; therefore, inulin can directly influence the hormones and regulate appetite. However, a slow-burning carbohydrate can help in the ability to regulate appetite without the need for complex and often difficult diet adjustment. Overall, fructose can act as a great source of energy where inulin aids in the production of protein, bile acids, and cholesterol as well as it has function to help in removal of contaminants from liver with regard to health benefit that may impact on improving human health (Alsharafani et al., 2007;Alsharafani & Al-Nouri, 2009;Roberfroid, 2007).
Previously, Milani et al. (2015) reported that Bifidobacterium strains induce a high rate of fermentation in sour cherry and orange juice.
Therefore, in the present study, fortified JA juice samples either with single or multiple antioxidant compounds (X 1 , X 2 , or X 3 ) showed a high rate of carbohydrate assimilation during fermentation than the control sample. Our results are in congruence with a previous study where a significant increase in cell concentration (ranging from 6.2 × 10 6 to 1.8 × 10 9 CFU/ml) of B. longum strain Bb-46 in orange, carrot, and tomato juices was observed after 24 h of fermentation (Havas et al., 2014). In contrary, the growth rate of B. bifidum NCFB 1454 in sour cherry juice and orange juice was not significant in contrast to tomato juice samples fermented with B. longum Bb-46, B. bifidum B3.2, and B. longum A4.8 strains (Goderska et al., 2007).
Our study results are supported by other studies where pre-and probiotic bacterial (Lactobacillus acidophilus and Bifidobacterium animalis subsp. Lactis) species were used for fermentation in food products that could be applied to the industrial scale (Shafi et al., 2019;Zacarias et al., 2020). 5.5 up to 5.44 for X 2 , X 2 X 3 , X 3 , X 1 , X 1 X 2 , X 1 X 3 , and X 1 X 2 X 3 , respectively ( Figure 2). Likewise, the initial pH value of fortified JA juice samples spiked with B. longum subsp. longum FA1 strain was 6.35

| Acidic production
which is decreased to pH 5. 94, 5.86, 5.74, 5.52, 5.5, and 5.45 up to 5.44 for fortified JA samples X 2 , X 2 X 3 , X 3 , X 1 X 2 , X 1 X 3 , and X 1 X 2 X 3 , respectively. During the storage period, changes in the pH values of JA juice were detected ( Figure 2 and Lactobacillus bulgaricus (Kun et al., 2008); however, other study findings have shown that the fermented carrot juice is a suitable medium for probiotic lactobacilli but not bifidobacteria although it may change lactobacilli phenotypic characteristics (Tamminen et al., 2013).

| Application of regression model
The statistical analyses were used to perform the experimental data regression analysis and to draw the surface plot of the response.

| Analysis of fortified JA juice for sensory assessment
The appearance of fortified JA juice was turbid with light brown color similar to the color of turbid apple juice showing a semi-thick texture. The taste of fortified JA juice revealed sweet to slightly acidic savor and astringent with a characteristic aroma, which indicates the (4) presence of different aromatic compounds in the JA tubers. The sensory scoring results of each type of fortified JA juice sample were measured by panelists as illustrated in Table 3. The results showed that ascorbic acid had the highest score followed by ginseng extract with organoleptic production that fermented with probiotic strains as compared to the astaxanthin (Table 3).
Overall, a combination of fortified JA juice X 1 X 2 X 3 showed a significantly (p < .05) high acceptance as compared to the control JA juice simulated with B. breve M4Astrain. The highest mean color grade 3.55 (fair to good) was observed in the X 2 and X 2 X 3 fortified JA juice as compared to the control (2.55). The texture score of the fortified JA juice samples had a similar rate of high acceptance. The flavor was graded 3.44 in the combination of X 1 X 2 X 3 fortified JA juice sample, which presented the highest value as compared to the control sample. In contrast, there was no significant differences in the overall acceptance and texture scores for all fortified JA juice samples. The highest mean color was 3.55 observed in individual (X 2 ) and combination (X 2 X 3 ) as compared to control (2.55) sample. The mean flavor (3.44) was significantly (p ˂ .05) different in X 1 as compared to the control (2.22) sample.
The sensory evaluations showed no significant differences among various fortified JA juice samples. The improvement of sensory properties was enhanced by the addition of ascorbic acid, astaxanthin, and ginseng extract. The previous study has shown that the probiotics have minimal effect on the overall acceptance of juices (Perricone et al., 2015). An overall acceptability of fortified JA juices simulated with both bifidobacterial strains was observed with the benefit of the bifidobacterial cultures in JA juice that could add value in terms of health aspects (Alsharafani, 2006). Overall, the results indicate that there is suitable potential of supplements in the development of flavors for future use.

| Development of regression model
The added factors (ascorbic acid, astaxanthin, and ginseng extract) were correctly described by the quadratic model of fortified JA juices on the response. The cell concentrations of both strains predicted by the ANOVA test formed a regression curve in actual versus predicted response (Figure 3a,b). Similarly, Figure 3c,d shows the internally studentized residuals versus predicted strain cell con- can be used as a natural preservative in fruit juices since probiotics showed resistance to biocitro; moreover, biocitro may also improve the biocontrolling action of L. plantarum against Zygosaccharomyces bailii. However, probiotics showed no effect on sensory properties (Bevilacqua et al., 2013). Table 2  Similarly, the effect of the combination of X 1 X 2 , X 1 X 3 , and X 2 X 3 fortified JA juices on bacterial cell concentration was significantly high as compared to the control JA juice spiked with B. breve M4A strain ( Figure 4; models a-c). However, the bacterial cell concentration was not significantly affected by combinations X 1 X 2 and X 2 X 3 of fortified JA samples spiked with B. longum subsps. longum FA1 strain ( Figure 4; models d-f). Two-dimensional contour plots demonstrated the interaction of components in the combination of X 1 X 2 X 3 fortified JA juice which has the most effective combination for gaining high bacterial cell concentration in JA juice ( Figure 5). showed a significant curvature by the points that are sticking above the surface plot ( Figure 4). Design Expert v.12 provides special treatment for center points, which show the lack-of-fit is not significant.

| Assessment of regression model
Therefore, Design-Expert by a special handling of center points provides a much more enlightening view of the design (Anderson et al., 2015).
According to the ANOVA test analysis, the results of the obtained model were facilitated to study the effects of fortified factors of the three-dimensional response surfaces plot. A positive association of X 1 and X 2 with B. breve M4A cell concentration has been observed ( Figure 4a). However, a significantly high impact of X 1 and X 3 on the cell concentration can be explained as higher both strains' cell concentrations can be obtained in comparison to the control. In addition, the effect of adding X 2 and X 3 on the bacterial cell concentration is illustrated in Figure 4c. It can be summarized that both X 2 and X 3 are synergized and positively related to the growth of B. breve M4A and B. longum subsp. longum FA1 strains.

TA B L E 3
Sensory data analysis of Jerusalem Artichoke juice fortified with ascorbic acid (X 1 ), astaxanthin (X 2 ), and ginseng extract (X 3 ) after 4-week storage at 5°C  Similarly, a synergistic relationship between ascorbate and inulin on cell viability for eliminating the stress of remaining oxygen in treated juices that contained ascorbic acid was observed (Shah et al., 2010). The addition of ascorbic acid and l-cysteine-HCl showed a significant impact on the growth of B. bifidum BB01 and B. bifidum BB03 strains. Adding 0.8 g/L of ascorbic acid or 0.6 g/L of l-cysteine-HCl has a significant impact on the growth of bifidobacterial strains (Shu et al., 2013). The cellular uptake of astaxanthin nanodispersions in skimmed milk was significantly higher than astaxanthin nanodispersions in orange juice and deionized water. High in vitro cellular uptake of astaxanthin from the prepared astaxanthin nanodispersions can be achieved by the incorporation of protein-based foods such as milk (Anarjan & Tan, 2013;Mezquita et al., 2014).

| Interaction effect of variables on cell concentration and viability
The findings provided evidence that when the X 1 is 15 g/L, in the mixture supplement, the CFU/ml of Bifidobacterium sp. was one log higher than strain in the X 2 and the control JA juice samples. The interaction effect of X 1 and X 3 with the Bifidobacterium sp. cell concentration (CFU/ml) is presented in Figure 4 (models a and b). The total number of cells (CFU/ml) of both bifidobacterial strains remained unchanged in X 2 JA juice samples, in comparison with the control sample, which may be explained by a limited bioavailability of X 2 during the storage period.
The combination of X 1 , X 2 , and X 3 fortified JA juice samples showed no effect on the cell concentration (CFU/ml) of B. longum subsp. longum FA1 (Figure 4; models d-f). The interaction effect of the combination X 1 X 2 X 3 on the bifidobacterial cell concentration (CFU/ml) is illustrated in Table 1. The significant impact of X 1 , X 2 , and X 3 on the bacterial concentration (CFU/ml) where a higher bacterial cells concentration than the control sample was observed. On the other hand, the cell concentration of B. longum subsp. longum FA1was significantly (p ˂ .05) higher in the combination of X 1 , X 3 , X 1 X 3 , and X 1 X 2 X 3 fortified JA juices than X 1 X 2 and X 2 X 3 after 28 days at 5ºC ( has the potential to be used as a probiotic component because it can provide great stability to probiotics and acid production (Elaheh et al., 2016). However, probiotic viability should be at least 10 7 CFU/ ml in the product at the end of the shelf life (Nualkaekul et al., 2011).
Previous study used microwave freeze-dried at 25°C and microwave

Vit-C (mg)
Vit-C (mg) vacuum storage methods to enhance persistence of B. animalis subsp.
lactis INL1 (Zacarias et al., 2020). The results suggest that dehydration method can also be applied in the food industry.
The survivability of probiotic bacteria in juices depends on various factors such as storage temperature during refrigeration that can prolong survival rate. For combating harmful effect of high temperature, various strategies including microencapsulation and reduce acidification methods have been investigated (Nag & Das, 2013;Sohail et al., 2012). Also, antioxidant molecules could also be a useful strategy for limiting the harmful effects of oxygen exposure by using plant-based extracts (Nag & Das, 2013).

| Optimization of JA juice with three factors
The two-dimensional contour plot of factors X 1 versus X 2 including six center points is indicated by the dot in the middle of the contour plot of the B. breve M4A and B. longum subsp. longum FA1 strains ( Figure 5a,b). As a result of two-dimensional overviews of the contour plot, a contour plot of conversion as a function of X 1 and X 2 at a mid-level slice of the coded level of X 3 , where X 3 did not currently assign to axes or plotted on the graphs, whereas the third-factor effect of X 3 at a higher coded level (+1) on the contour plot of the B. breve M4A and B. longum subsp. longum FA1 strains at higher response levels (Figure 5c,d). This result is consistent with the regression analysis results, where X 1 , X 2 , and X 3 had the highest regression coefficient.
The most interaction limitation in the combination of X 1 X 2 and X 2 X 3 fortified JA juice was observed against B. longum subsp. longum

| CON CLUS ION
The study has provided data on the persistence of both Bifidobacterium species that remained ˃10 7 (CFU/ml) up to 28 days at 5ºC and produced a functional drink as compared to nonfortified JA juice. JA juice fortified with ascorbic acid (X 1 ) showed a significantly high rate of persistence of probiotic bacteria (CFU/ml). The benefits (taste and shelf life) of B. breve M4A and B. longum subsps. longum FA1 had resulted in increasing their incorporation into JA juice supplemented with X 1 , X 2 , and X 3 extracts to produce functional drinks.
In the case of the examined JA juice supplemented with X 1 and X 3 served as appropriate supplements, whereas X 2 was strain dependent. The findings of the surface response analysis were obtained on a laboratory scale and can eventually be used to various production volumes. The results would help food industry in producing JA juice fortified with antioxidant compounds that can be an alternative to dairy-based probiotics and provide essential human nutrition.
Studies are underway to further determine the effects of JA beverage fortified with ascorbic acid, astaxanthin, and ginseng extract on human health.

ACK N OWLED G M ENTS
We thank co-op students for their laboratory assistance and data collection.

CO N FLI C T O F I NTE R E S T
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The study data can be shared upon request from the corresponding author. The data are not publicly available due to privacy restrictions.