Optimization of Parameters Using Response Surface Methodology to Develop a Novel Kefir-Like Functional Beverage from Cheese Whey Enriched with Myrtle Juice

Whey, liquid wastewater from cheese production, is one of the sources of dietary protein and lactose that are still largely unused for human consumption. It is only in recent years that it has aroused the interest of industries and sought as a valuable raw material and thus represents an opportunity for the manufacture of new products. ,e manufacture of fermented whey drink requires the mixing of whey with fruit juice or an aromatic plant to improve its organoleptic properties and acceptability. Myrtle, an aromatic medicinal plant, known for its health benefits is not well exploited formaking dairy products.,is is the first report on the development of kefir-myrtle beverage. ,ree factors were optimized (whey permeates (%), myrtle’s juice (%), and kefir grains as inoculum (%)) using a central composite design with response surface methodology. ,e analyses showed that the number of lactic acid bacteria (LAB) and yeast cells varied from 5.4 to 9.2 log10 CFU/mL and from 4.3 to 6.2 log10 CFU/mL, respectively. A decrease in pH and an increase in the total polyphenol content and antioxidant activity were observed. ,e analysis of variance indicated the goodness of fit of the model with R from 0.827 to 0.966. ,e absolute average deviation values of each model were low and ranged from 1.61% to 4.23%. ,e optimized fermented kefir whey beverage accomplished an overall acceptability of 5.41 (1 to 9 preference scale) and a high number of LAB cells (8.53 log10 CFU/mL).,e viability of LAB and yeast cell was maintained at 7.61 and 6.19 log10 CFU/mL, respectively, after 14 days of storage.


Introduction
e dairy industry in Tunisia is one of the important food industries in the country. is sector comprises 25 enterprises that processed daily about 3.8 million liters, and 13% of the total volume of produced milk is destined for cheese production [1]. In the past, whey was considered a byproduct, but now it is considered a coproduct. Its valorization is both an economic and ecological issue since it has a high chemical oxygen demand (COD) [2]. Indeed, when whey is discharged into rivers, it generates eutrophication problem and toxicity modifying the physicochemical properties of aquatic ecosystems [3]. e relatively new interest in this byproduct results mainly from its composition rich in proteins, lactose, and water-soluble vitamins and minerals. Furthermore, it represents a well-balanced source of essential amino acids [4].
ere are different processes to value whey. Among the most used technologies are drying, evaporation, reverse osmosis, nanofiltration, and ultrafiltration. By its biochemical composition, whey can be also considered an excellent culture medium for microorganisms.
Since lactose is the main component of whey solids, various biotechnological processes have been developed to use whey as a substrate to produce important industrial products having functional properties. For example, it can be converted into prebiotic such as galactooligosaccharides (GOS), lactulose, lactobionic acid, and tagatose [5]. In addition, lactic acid fermentation of whey makes it rich in GOS [6], which exerts a stimulating effect on the growth of probiotic bacteria [5]. Bioactive peptides manifesting antihypertensive, antioxidant, immunomodulatory, and antimicrobial activities can also be released during fermentation [7].
On the other hand, lactic acid fermentation was known as an important tool to increase the bioavailability of polyphenols in food. To date, kefir grains were largely exploited for dairy and nondairy beverages fermentation. Numerous studies described the health benefits of kefir including antihypertensive, antidiabetic, anti-inflammatory, anticancer, antioxidative, and antihypercholesterolemic properties reviewed by Azizi et al. [8]. ese therapeutic's aspects made kefir a suitable proposal for commercial intention.
However, to improve whey fermented beverages' flavor and increase their consumption among young people, they should be mixed with fruits juice [9][10][11][12]. In fact, fruit and vegetables are rich in nutrients and phytochemicals such as vitamins, minerals, and phenolic compounds [13]. Due to their various micronutrients, fruits and vegetables are often considered as "functional foods" helping to prevent various diseases such as cancer, obesity, and diabetes [14].
Myrtus communis L. is an aromatic and medicinal plant belonging to the family of Myrtaceae. It is widespread in the Mediterranean regions, such as North Africa and Southern Europe, and also found in South America, Australia, and in some areas of the Himalaya [15]. Myrtle berries have a long history of application in the pharmaceutical and food industries. ey contain many biologically active compounds such as phenolic compounds, flavonoids, and anthocyanins [16], which are thought to be responsible for their antioxidant proprieties. ey also have various positive effects on human health, and they are used as antiseptic, analgesic, cardiotonic, diuretic, anti-inflammatory, stomachic, nephroprotective, antidote, hemostatic, brain tonic, and antidiabetic properties [16].
Experimental designs can be used as a method for formulating food products. ey allow selecting the factors that influence the response, modeling the variations in the system response according to the fluctuations of the factors, and validating experimentally the model described by a mathematical equation. However, a suitable range for each factor is also an important consideration for the accuracy of the final model [17][18][19]. e aim of this study is to optimize the formula of a functional whey beverage. Optimization was done by a central composite design (CCD) to determine the optimum ratio of whey permeate, myrtle juice, and kefir inoculum on pH, lactic acid bacteria (LAB), and yeast viability, % radical scavenging activity, polyphenol content, and overall acceptability. From this design, the surface plots of considered responses with respective second-order polynomial models were obtained. Finally, the analysis of variance (ANOVA) was employed to judge the adequacy of the model, and the optimized formulation was validated experimentally. this study were collected from Tunisian households and  preserved by the laboratory of microbial ecology and microbial technology (LETMi, INSAT) [11,20]. e grains were cultured in sterile cow milk and renewed daily to maintain their viability. Kefir grain is polysaccharides and protein matrixes consisting of a symbiotic community were LAB (10 8 CFU/g) and yeasts (10 5 CFU/g). e predominant LAB in the used grains are related to Leuconostoc spp., Lactobacillus spp., and Lactococcus spp. Saccharomyces spp. and Zygosaccharomyces spp. are the dominant yeasts [21]. Kefir grains produce 0.6% lactic acid titratable acidity.
Lactose, total proteins, the total fat content, and the ash were determined using the HPLC method [22], the Kjeldahl method [23], and a solvent extraction [24] and by heating the samples at 550°C [25], respectively. e pH of the beverages was measured using a pH meter (Mettler-Toledo EL20).
Myrtle fruits (Myrtus communis) were collected from the area of Nefza (north-west of Tunisia, latitude 36°58′ 31″N, longitude 9°04′ 51″ E, altitude 500 m, far 147 km from Tunis, the capital), purchased from the local market in January 2019. e berries were washed and crushed with a mixer by adding distilled water (8°Brix). e mixture was filtered to obtain a juice that was pasteurized at 70°C for 15 min and used immediately.

Beverage Formulation Using a Response Surface Methodology (RSM).
e solution of cheese whey and whey permeate were sterilized for 20 min at 120°C and then mixed with myrtle juice according to the plan of RSM. Obtained beverages (400 mL) were placed in bottles and inoculated with kefir grains (Figure 1).
Central composite design (CCD) was used to develop a novel kefir-like functional beverage from cheese whey enriched by myrtle juice (MJ). e RSM is a combination of experiment design, statistics, empirical modeling, and mathematical optimization techniques. e number of experiments to be carried out was determined rationally, which avoids redundancies of information. In addition, the implementation of an optimization procedure allows the study of the interactions between the different factors. It is possible that a factor that apparently has no effect on the phenomenon being studied and influences this phenomenon indirectly through an interaction. e construction of the response surfaces is carried out following the adjustment of the model using mathematical functions such as polynomials [26]. e central composite design used for the developing of whey kefir-like beverage consists of nineteen experiments with five replicates at the central points. e independent variables (whey permeate X1, myrtle juice X2, and kefir grains inoculum X3) in the design were assigned into five levels, coded −1.68, −1, 0, 1, and 1.68 as described in Table 1. e responses were pH, lactic acid bacteria viability, yeast viability, total polyphenol content (TPC), antioxidant capacity (DPPH), and overall acceptability (OA). e results obtained from the CCD were used to fit a second-order polynomial equation: where Y is the predicted response; X 1 , X 2 , and X 3 are the independent variables; b 0 the model constant; b 1 , b 2 , and b 3 are the linear effect of variables; b 11 , b 22 , and b 33 are the squared effect of variables; and b 12 , b 13 , and b 23 are the interaction effect of variables. ese coefficients were calculated using software NEMROD-W (version 99901, LPRAI Company).

Lactic and Yeast Counts.
Lactic acid bacteria and yeasts were quantified using conventional culture techniques [27]. LAB were quantified on MRS agar (with cycloheximide 150 μg/mL) and yeasts on PDA supplemented with chloramphenicol (100 mg/L); they were incubated at 37°C and 30°C for 48 h, respectively. e results were expressed as CFU/mL. e numbers of LAB and yeasts were converted to log CFU/mL.

Polyphenol Content and Free Radical Scavenging Capacity (DPPH Assay).
e TPC was determined following the method of Folin-Ciocalteau [28], modified by Karaaslan et al. [29]. e mixture of each beverage (0.03 mL) and distilled water (2.730 mL) was added to the Folin-Ciocalteau reagent (0.15 mL). After adding sodium carbonate (20%), the mixture was left at room temperature for 30 min. e absorbance was measured at 750 nm (Jenway 63200 UV/Vis). e TPC was expressed as mg of gallic acid equivalent/mL of the sample. e correlation coefficient was R 2 � 0.991. e free radical scavenging activity of the samples was determined using DPPH (1,1-diphenyl-2-picrylhydrazyl) method according to the method proposed by Balakrishnan and Agrawal with some modifications [30]. Each sample (700 μL) was added to 700 μL DPPH methanolic solution (0.035 mol/L). e mixture was shaken and allowed to stand at room temperature for 30 min. Antioxidant capacity was measured by recording the absorbance at 517 nm using a spectrophotometer (Jenway 63200 UV/Vis). Methanol was used as the blank. All the determinations were performed in triplicate. A mixture of DPPH solution and methanol (instead of the sample) was used as the negative control for this assay. Percentage of DPPH radical scavenging activity was the result of antioxidant activity. e scavenging activity was calculated by the following equation:  Journal of Chemistry

Sensory Evaluation.
A total of 40 persons have contributed to a panel test; they were from the food technology department and the dairy industry (students and staff, ranging 24-56 ages). e evaluation was based on the hedonic sensory acceptance of beverage samples using a 9point hedonic scale. e drinking water was given to tasters to rinse their mouth between each sample. Beverages were evaluated for color, odor, sweetness, acidity, taste, and overall acceptability [31].

Statistical Analysis.
All experiments were carried out in triplicate. All data are reported as the mean ± standard deviation. Statistics were performed with the analysis of variance (ANOVA) procedure (STATIGRAPHICS 202 Centurion XVI software, Statpoint Technologies, Warrenton, USA). Differences were considered significant at p < 0.05.

Results and Discussion
Whey-based milk drinks can sometimes have unpleasant flavors. e improvement of their organoleptic characteristics can be obtained by the addition of fruit concentrates and/or by fermentation. To obtain a novel kefir-like functional beverage, the whey was fortified by WP, and myrtle juice was then fermented by kefir grains. e effects of whey permeate; myrtle juice and kefir grains' levels on the nutritional and organoleptic characteristics of the obtained beverages were investigated using a CCD. ese independent variables (WP (X 1 ), fruit juice (X 2 ), and percentage of inoculum (X 3 ) levels) were prescribed into five levels (−1.68, −1, 0, +1, and +1.68). Tables 2 and 3 present the experimental values of total polyphenol content (mg EGA/mL), radical scavenging activity, LAB and yeasts' viability, pH, and sensory evaluation for each run of the CCD. Results from the 19 runs were fitted to a second-order polynomial equation, and the removal of nonsignificant terms was assigned. When the values of R 2 and R 2 adj. are close to 1, the results indicate the adequacy of the fitted models. Yaakob et al. [32] mentioned that R 2 should be at least 80% and Cruz et al. [33] reported that the values of R 2 adj. should be over 70%. However, the calculation of R 2 and absolute average deviation values (AAD) together should be better to determine the accuracy of the model. R 2 must be close to 1.0, and the AAD between the predicted and observed data must be as small as possible [34]. AAD values of six predicted models were calculated; they were 4.23% for TPC, 1.61% for antioxidant activities, 2.75% for LAB number, 1.96% for yeasts counts, 2% for pH, and 4.13% for overall acceptability. erefore, the model illustrates the global variability; it is predictive (Supplementary Materials (available here)).

Effects of the Variables on Total Phenolic Content and
Antioxidant Activity. Myrtle is considered a preventive element against diseases related to oxidative stress. Indeed, it is rich in many chemical compounds including phenolic compounds and essential oils [35]. Many groups of phenolics were identified such as phenolic acids, hydrolyzable tannins (gallotannins), flavonoids, and anthocyanins [36]. Messaoud and Boussaid [37] reported that their antioxidant activities are due to their phenolic compounds. e TPC values of obtained beverages varied from 55.25 ± 0.68 to 90.45 ± 0.07 mg EGA/mL, and the DPPH radical scavenging from 65.25 ± 0.22 to 91.6 ± 0.29% (Table 2). R 2 and adj R 2 for TPC and DPPH radical scavenging activity are close to 1.
e AAD values were 4.23% and 1.61%, respectively. An increase in myrtle juice or whey permeate or kefir inoculum level improves the TPC and DPPH radical scavenging activity (p < 0.001; Table 4). It must be mentioned that a p value lower than 0.001 showed that the model is highly significant in the response. e coefficients for linear and quadratic models are also highly significant (p < 0.01; Table 4). e highest level of TPC and DPPH radical scavenging activity was obtained with 5.08% WP, 50.59 fruit juice (% w/v), and 5.04% inoculums (Figures 2  and 3).
With regard to interaction, myrtle juice and kefir inoculum's level affect positively the TPC and the antioxidant activity. ese results could be due to the metabolic activities of microorganisms in kefir grains. In fact, microbial enzymes break down polyphenol compounds and form aglycones [38]. ese latter can be also liberated from their corresponding glycosides, contributing to enhancing the bioavailability of polyphenol [39] and increasing their quantitative amount [40,41]. Sabokbar et al. [42] showed that the addition of kefir to apple juice enhanced both the total phenolic content and antioxidant activities. During the last years, several works have been interested in the action of lactic acid bacteria and kefir microflora on phenolic compounds [43][44][45][46]; they cited many enzymes, which are involved in the hydrolysis mechanisms and which are inducible as a specific stress response. e improvement of the antioxidant activity after fermentation (Table 5) is in line with other studies [11,[47][48][49][50][51]. e increase of DPPH radical scavenging of fermented beverages indicated that fermentation may produce metabolites with higher and better antioxidant activity. e release of bioaccessible phenolic compounds can be one of Journal of Chemistry the reasons, and their antioxidant capacity is always associated with their health-promoting properties. Hernández-Ledesma et al. [52] reported the antioxidant activity for some peptide chains in whey, which had higher radical scavenging activity than butylated hydroxyanisole (BHA). e antioxidant activity of kefir has also been shown, and it is due to some potential compounds like glutathione, organic acids, and kefiran [8,51,53]. e multiple coded equations in terms of coded factors generated for these responses are shown as follows:

Effects of the Variables on pH and LAB and Yeast Cell
Viability of Kefir Microorganisms. e effects of independent variables on LAB and yeast cell viability are shown in Table 3. eir number depends on whey permeate, fruit juice, and kefir inoculum level. e linear effects were significantly positive (p < 0.001; Table 4). e interaction effects of whey permeate-fruit juice and fruit-kefir grains were also significant (p < 0.01; Table 4). An increasing inoculum level allows enhancing LAB and yeasts' number. e same result was observed by Sabokbar and Khodaiyn [9] when they fermented a mixture of pomegranate juice and whey by kefir grains.
ey reported that the cell density of LAB increased by 1.3 Ulog when they increase the inoculum from 5% to 8%. However, for yeasts' number, nonsignificant difference was observed. M'hir et al. [11] obtained the same result when they prepared a fermented beverage from whey, whey permeate, and date syrup and when they prepared a kefir beverage made with carob, oat flour, and whey permeate [54]. is increase of LAB due to the increase of the level of juice can be explained by the probiotic effect of myrtle ( Figure 4). In fact, in the studies of Mangia et al. [55] andÖztürk et al. [56], they showed that myrtle juice exerts a positive effect on lactobacilli.    Figure 3: Response surface and contour plots representing the effect of MJ and KG on DPPH radical scavenging activity (%; X 2 : MJ (% w/v) and X 3 : KG (% w/v)).   Journal of Chemistry e positive interaction between myrtle juice and kefir inoculum on cell density was significant (p < 0.01; Table 4). Filannino et al. [45] showed that phenolic compounds can exert a positive effect on LAB growth.
As a consequence of kefir microflora growth, the pH decline during fermentation (pH ranging between 5.02 and 3.94; Table 3) due to lactic acid increase, which can inhibit spoilage and pathogenic bacteria development in the beverage [9]. e pH decrease was significantly related to whey permeate (p < 0.01), juice (p < 0.001), kefir grains (p < 0.001), and their quadratic effects (p < 0.01). However, the pH was not significantly influenced by their interaction (Table 4). e number of viable cells of yeast and LAB of the obtained beverage were in accordance with codex Standard FAO/WHO [57], suggesting at least 10 4 and 10 7 CFU/mL of bacteria and yeast counts, respectively. e multiple coded equations in terms of coded factors generated for these responses are shown as follows:

Effects of the Variables on Sensory Evaluation.
Sensory analysis is a real lever for the development of new food products. Indeed, the characterization of their organoleptic properties is essential to guarantee their acceptability by consumers.
Only myrtle juice linear and quadratic terms (b 2 and b 22 ) have relevant effects on the overall acceptability ( Table 4). As noticed in Figure 5, a significant interaction was detected between whey permeate (X 1 ) and myrtle juice (X 2 ). e OA scores ranged from 3.9 ± 0.1 to 5.6 ± 0.26 on a 9-point hedonic scale (Table 3). e most appreciated samples were the beverages from experiment 7 (1% WP, 42% myrtle juice, and 4.4% kefir grains) and 12 (2.5% WP, 50% myrtle juice, and 3.5% kefir grains). e acceptability ratings were highest in the samples containing a high amount of fruit juice. It can be explained by the fact that Myrtle's juice masked the unsavory taste of cheese whey. In the study ofÖztürk et al. [56], black and white myrtles improved the taste scores of probiotic goat milk ice cream samples by masking the low pH resulting from fermentation. Koksoy and Kilic [58] showed that acid odor was masked with the fruit aromas resulting in more acceptable drinks for consumption. e level of juice and whey used in many studies was different; it largely depends on the fruit matrix. For example, Islam et al. [12] suggested that the optimum formula was with 25% whey and 75% pineapple juice. However, Pereira et al. [59] added only 10% of mango fruit to liquid whey protein and concentrated permeates.
Phenolic compounds contained in myrtle juice have also an impact on the obtained beverages' sensory attributes. Indeed, they have an important effect on color, perceived taste, and flavor. Pinto and Vilela [60] reported that different color palettes may influence our taste and flavor perception. e polynomial model for OA is presented by the following equation:  Table 6 in which the number of LAB and yeasts, the antioxidant activity, TPC, and the OA were mentioned. TPC showed a lower value than the predicted ones. Taking into account the standard deviations of measured and calculated responses, the results obtained indicated that the experimental values were in good agreement with the predicted values. is suggested that the fitted model is satisfactory and accurate.

Microbial Viability of Kefir Culture and the Change in Physicochemical Parameters during Storage.
e results for the effects of storage temperature at 4°C on the counting of LAB and yeasts and on physicochemical parameters of the developed beverage are shown in Table 7. A reduction in the load of LAB was observed from the first day of storage. LAB were more sensitive to storage than yeasts. Indeed, the loss in the viability of LAB cells was more important than this of yeasts. A very little and nonsignificant decrease of yeasts counts was recorded after 14 days of storage at 4°C. However, the viability is maintained during the two weeks of storage at a high level for all tested microflora as recommended by FAO/WHO 2006 [57]. e number of LAB and yeast viable cells was 7.61 ± 0.14 and 6.19 ± 0.24, respectively, after 2 weeks of storage at 4°C. ese results were in agreement with the reports of previous investigators [59,61]. e microorganisms' viability largely depended both on medium composition and on storage temperature. e pH influence on cell viability has been widely mentioned [62]. Existing exopolysaccharides, as kefiran, might help improve the survival of microorganisms in an acidic or frozen medium by giving a protective envelop that may preserve them from stressful conditions. e resistance of LAB in acidic conditions is due to the action of the proton pump, the change in their cell membrane composition, and other mechanisms [63]. Nualkaekul and Charalampopoulos [64] suggested that Lactobacillus probably uses the energy generated through their metabolic activity in order to maintain their viability. During the storage time, the pH value decreased to 3.69 with the progress of the storage period. is acidification through storage could be caused by residual microbial activity. e same result was observed by Islam et al. [12]. e storage has a significant impact on polyphenol content and antioxidant activity. A significant decrease of TPC and of DPPH free radical scavenging activity (%) was observed during storage. e loss of phenolic compounds could be explained by their oxidation.
On day 1 of the postproduction, the sensory properties of the fermented beverage have been slightly improved and then decreased under refrigerated storage. e decrease in product quality was linked to the increase in the contents of organic acids. Indeed, a mild increase in acidity was observed from the second day of storage.

Conclusions
e supplementation of cheese whey allowed developing kefir-like beverage having functional proprieties such as high polyphenol content and good antioxidant capacity. e RSM was successfully employed to optimize the LAB and yeast cell counts, the TPC, the antioxidant activity, and the OA with the incorporation of different concentrations of WP, myrtle juice, and kefir grains. e beverage obtained by mixing (2.83% whey permeate and 3.71% kefir grains) gave acceptable sensorial properties. e enumeration of LAB and yeast cells showed that the obtained beverage fulfilled the criterion of probiotic beverage in an acceptable manner even after 14 days of storage at 4°C. Sensory attributes may be ameliorated with further investigations like using myrtle syrup and/or adding another carbon source.

Data Availability
e data used to support the findings of this study are included within the article (Tables 2-7).

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.