Development of a Ready-to-Drink (RTD) Beverage Formulated with Whey and Beetroot Juice

Abstract

Whey, a large by-product of cheese manufacturing, is nutritionally valuable, mainly because of its whey proteins. Beetroot juice, purple–red in colour, has numerous benefits for human health; if used as an ingredient in a whey-based RTD beverage, it could mask whey’s less pleasant organoleptic characteristics. This research aimed to develop such a beverage by mixing whey with beetroot juice in a ratio that would yield a nutritionally valuable product with acceptable sensory characteristics. To this end, three RTD beverage formulations, prepared with beetroot juice and whey in different ratios (10:90, 15:85, and 20:80 v/v), were subjected to sensory analysis. The one with a 20:80 v/v ratio of beetroot juice and whey was found to be preferred by consumers, registering the highest overall score (7.8). Next, this formula was prepared with (RTD20:80a) and without citric acid (RTD20:80a) and analysed physiochemically and microbiologically. RTD20:80a was also sensory tested and proved to be the optimum one (with an overall score of 8.1); it showed a content of 1.18 g/100 mL fat, 1.07 g/100 mL protein, 0.56 g/100 mL ash, 9.90 g/100 mL carbohydrate, total soluble solids of 11.69 °Brix, an energy value of 54.44 kcal/100 mL, titratable acidity of 0.77% lactic acid, pH of 3.75, content of 1.16 mg/mL betacyanins, 0.86 mg/mL betaxanthins, and 0.36 mg GAE/mL total phenolics. Based on the microbiological results, RTD20:80a revealed a shelf-life of 14 days. By producing this beverage, a dairy plant could reduce its whey-related losses while increasing its profitability through selling it.

1. Introduction

Whey is the liquid that results after milk is curdled and strained to produce cheese [1]. Indeed, 80–90% of the milk that enters cheese production facilities becomes whey [2]. From 1988 to 2023, the world’s milk production grew by 59%, reaching 944 million tonnes; this surge led to an annual by-product of 200 million tonnes of whey [3]. There are two kinds of cheese whey: sweet and acid; sweet whey is derived from the production of ripened cheeses (pH 5.8–6.6), whereas acid whey is derived from that of fresh cheeses (pH 3.6–5.1) [4]. Due to the large production volume and nutritional composition, whey is of relative importance to the dairy industry [5]. Hence, it may be better for the environment and a sustainable economy to recover whey components and use whey as a fermentation medium or food ingredient [6,7].

This by-product of the cheese industry contains about 6% dry matter, representing half the amount in whole milk; it includes whey protein, lactose, water-soluble vitamins, and mineral substances. Therefore, whey is a valuable ingredient with various applications in the food and pharmaceutical industries [5]. Due to these properties, it is used as a raw material to formulate food products or dietary supplements, such as whey powder, hydrolysed whey protein, whey protein concentrate, whey protein isolate, or whey-based beverages [1,8].

Whey beverages represent a category of dairy products that offer superior nutritional benefits, thus making them appropriate for various consumers, including children and older people [9]. Whey proteins, through their amino acid composition, have a beneficial impact on regular physical activity. In addition, consuming dairy products based on whey proteins is important for lipid metabolism and protein and glucose homeostasis, respectively; this allows for more effective weight control, lowering serum cholesterol and triglycerides, reducing fat deposition, and increasing insulin sensitivity [10]. Consumers are becoming more concerned about their health and more aware of the products they consume; many seek food products with high nutritional value but attractive organoleptic characteristics [11]. Therefore, producers in the food industry are interested in finding ways to meet such demands and attract more customers.

Beetroot juice has recently received attention in the scientific literature, especially due to its nitrate content [12]. Nitrates from food are converted into nitrites by anaerobic bacteria in saliva, and later, into nitric oxide in the stomach, thus completing the endogenous synthesis of nitric oxide from L-arginine [13]; in the human body, nitric oxide causes blood pressure to drop by dilating blood vessels [14]. Beetroot juice also contains phenolic compounds, ascorbic acid, as well as carotenoid and betalain pigments; the latter, which include betacyanin and betaxanthin, possess strong antioxidant and anti-inflammatory activity [15]. Given the biologically active compounds contained in it, beetroot juice could contribute to the overall benefits of a whey beverage if it were used as an ingredient in a certain ratio; it possesses some potential to improve the organoleptic characteristics of such a beverage, especially the appearance and colour, thus positively influencing its purchase intention.

Numerous studies have been published on developing whey-based beverages with fruit juice, such as cactus [16,17], guava [18], watermelon [19], banana [20], pineapple [21,22], orange [22,23,24], papaya [25], and passion fruit [26]. Still, only a few have been published regarding the use of vegetable juice: tomato [27], spinach [28], broccoli [29], and carrot [30]. Therefore, in this study, we aimed to develop a nutritionally valuable RTD beverage with acceptable sensory characteristics by mixing dealbuminated whey with filtered beetroot juice; three ratios of whey and juice (10:90, 15:75, and 20:80 v/v) have been tested to this end. Although other researchers have tested the combination of whey and beetroot to obtain a whey-based beverage, they used extracts from beetroot pulp [31] and peel [32]. Therefore, this study proposed the preparation of such a beverage by blending filtered beetroot juice and dealbuminated whey in a ratio of 10:90, 15:85, and 20:80 (v/v), 0.15% (m/v of the blend) pectin stabiliser, 0.001% (m/v of the blend) mint essential oil, and acidifier for pH 3.8, respectively, via the Patent Application RO134175A2 from 20/11/2018.

2. Materials and Methods

2.1. Preparation of RTD Beverage and Experimental Design

Preparation of beetroot juice. Beetroot (m₁ = 9.32 kg) was bought from a local supermarket (Mega Image, Cluj-Napoca, Romania) to prepare the juice. It was washed, peeled (m₂ = 7.86 kg), sliced, and introduced into a juicer machine (HE-541; China) to extract the juice. The liquid thus obtained (m₃ = 3.73 kg; V₁ = 3.5 L) was filtered using cheesecloth folded in two (m₄ = 3.44 kg; V₂ = 3.2 L) to be clarified.

Preparation of dealbuminised whey. The whey used to prepare the RTD beverage formulations, a by-product of manufacturing a Gouda-type cheese, was received from SC Bonas Import-Export SRL (Dezmir, Romania); it was transported on the same day (under refrigerated conditions) from the cheese factory to the Dairy Pilot Plant of the University of Agricultural Sciences and Veterinary Medicine of Cluj-Napoca. For the dealbuminisation, the whey was heated at 72 °C for 10 min and then filtered through cheesecloth.

Preparation of RTD beverage formulations. The dealbuminised whey was divided into three stainless steel containers to prepare the experimental variants of the RTD beverage: 5.4 L for RTD10:90 (RTD beverage containing a mixture of beetroot juice and whey in a 10:90 (v/v) ratio), 5.1 L for RTD15:85 (RTD beverage containing a mixture of beetroot juice and whey in a 15:85 (v/v) ratio), and 4.8 L for RTD20:80 (RTD beverage containing a mixture of beetroot juice and whey in a 20:80 (v/v) ratio). The containers were filled with 0.6, 0.9, and 1.2 L of beetroot juice (prepared as described above), respectively, and their content was mixed using a multifunction robot (RS-20; RS Tecnology s.r.l., Castello d’Argile, Italy) until homogenisation. Next, 420 g of table sweetener based on Stevia extract and erythritol (SC Sly Nutritia SRL, Buzău, Romania), 9 g of pectin (Sigma-Aldrich, Saint Louis, MO, USA), and 57 µL of mint essential oil (Adams Vision SRL, Târgu Mureș, Romania) were added to each container and mixed until dissolved.

The three RTD beverage formulations were bottled in 250 mL glass containers and pasteurised at 65 °C for 30 min (WNB7; Memmert GmbH & Co. KG, Schwabach, Germany). After cooling to room temperature, they were stored in a chilled room at 4 °C for 70 days.

Experimental design. Samples were analysed on the next day to determine the proximate composition, energy value, titratable acidity, and pH (Table 1; whey, beetroot juice, and RTD beverage formulations), content of betalain pigments and total phenolics (Table 4; beetroot juice and RTD beverage formulations). The RTD beverage formulations were subjected to sensory assessment to identify the one preferred by consumers (hedonic test in Section 2.3; Table 2) and their willingness to buy (purchase intention test in Section 2.3; Table 3). Although the panellists preferred the RTD20:80 formulation, they mentioned in the comments section that it was too sweet. Therefore, we prepared another formulation with 20 volumes of beetroot juice (BJa) and 80 of whey (Wa) that was acidified (RTD20:80a) with a 50% (m/v) citric acid solution (VWR Chemicals, Solon, OH, USA) up to a pH of 3.8 (InoLab^® Multi 9310 IDS; WTW, Weilheim, Germany) before bottling; it was analysed like the others.

To estimate the shelf-life of RTD20:80 and RTD20:80a, their titratable acidity (Figure 1a), pH (Figure 1b), total viable count (Figure 2a), as well as total yeast and mould count (Figure 2b) were monitored for 70 days (at intervals of 7 days, starting with the 7th storage day).

2.2. Determination of Proximate Composition, Total Soluble Solids, Energy Value, Titratable Acidity, and pH

The whey, beetroot juice, and RTD beverage formulations underwent proximate analysis for fat [33], protein [34], and moisture [35] using standardised methods. The ash content was determined by incinerating the dried sample at 600 °C for 6 h using a muffle furnace (L3/11/B170; Nabertherm GmbH, Bremen, Germany). The results were given in g/100 mL. Their total carbohydrate content and energy value were calculated with the following Equations (1) and (2) as in Fogarasi et al. [36]:

Total carbohydrate (g/100 mL) = 100 − (g/100 mL moisture − g/100 mL protein − g/100 mL fat − g/100 mL ash)

(1)

Energy value (kcal/100 mL) = 4 × (g protein + g carbohydrate) + 9 × g fat

(2)

The titratable acidity of the whey, beetroot juice, and RTD beverage formulations was determined as described by Baljeet et al. [21], consisting of neutralising the total amount of acids present in a sample. Twenty-five millilitres of the sample was weighed and titrated with a 0.1 M sodium hydroxide volumetric solution using a phenolphthalein solution (1% in 96% ethanol) as an indicator until the pH reached 8.3. The result, expressed in percentages of lactic acid for the whey and beverage or citric acid for the beetroot juice, was calculated with the following Equation (3):

$$\mathrm{T}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{a}\mathrm{b}\mathrm{l}\mathrm{e}\mathrm{a}\mathrm{c}\mathrm{i}\mathrm{d}\mathrm{i}\mathrm{t}\mathrm{y}\left(\%\mathrm{l}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{c}\mathrm{i}\mathrm{d}\mathrm{o}\mathrm{r}\mathrm{c}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{c}\mathrm{i}\mathrm{d}\right)=\left({\displaystyle \frac{c_c\times V}{w}}\right)\times 100$$

(3)

where c_c is the conversion coefficient of lactic acid (0.009) or citric acid (0.0064), V is the sample volume, and w is the sample weight.

The total soluble solids (°Brix) in the beetroot juice and RTD beverage formulations were measured with a digital refractometer (HI 96801; Hanna Instruments, Bedford, UK) and the pH using a digital multi-parameter meter (InoLab^® Multi 9310 IDS; WTW, Weilheim, Germany).

All the above determinations were carried out in triplicate.

2.3. Sensory Evaluation

Sensory evaluation of the RTD beverage formulations was carried out by applying the hedonic test and the purchase intention test, as Socaciu et al. described [37]. It was conducted in the Sensory Analysis Laboratory with volunteers from the Faculty of Food Science and Technology from 30 October to 30 November 2018: undergraduate, graduate, and postgraduate students, staff members, researchers, and other university employees.

After being randomly assigned three-digit codes, the RTD beverage formulations were served to participants in clear glass beakers, 100 mL of each sample. They were advised to rinse their mouths with water before sampling and refrain from eating, drinking, and smoking for at least 1 h before the assessment. The sensory tests were performed in individual cabins at 20 °C (air conditioning) under natural light.

Eighty-seven participants, with an average age of 24 years, 57 women and 30 men, evaluated the RTD beverage formulations on a scale from 1–dislike extremely to 9–like extremely, based on their appearance and colour, odour, taste, consistency, and overall acceptability. The overall score was calculated as the mean score received by a formulation following the hedonic test. Each RTD beverage formulation’s acceptance rate was calculated by dividing the mean sensory score by the maximum sensory score granted by the panellists and multiplied by 100.

For the purchase intention test, participants were asked to score the RTD beverage formulations using a 5-point scale, where one meant “definitely will buy” while five meant “definitely will not buy”. The sample RTD20:80a was analysed under the same conditions by the same participants but on another day.

2.4. Determination of Betalain Pigments

Quantification of the betalain pigments (betacyanins and betaxanthins) in the beetroot juice and RTD beverage formulations was carried out as described by Porto et al. [38], with minor modifications. Here, 15 mL of the test sample was introduced into a 15 mL Falcon tube and centrifuged (Hettich Mikro 22R; Andreas Hettich GmbH & Co. KG, Tuttlingen, Germany) at 6006× g (7360 rpm) for 20 min to 25 °C to separate the suspended particles. The absorbance value of the supernatant was read at 476 and 538 nm using a single-beam UV-Vis spectrophotometer (UV-1280; Shimadzu Corporation, Kyoto, Japan); a reading at 600 nm was also performed to correct any possible impurities (turbidity). Suppose the absorption value of betacyanins or betaxanthins exceeds 1.0. In that case, the sample must be diluted with distilled water to reach 0.8 ≤ A ≤ 1.0 [16], and the dilution factor is considered when calculating the results. Each sample was analysed in triplicate. The results were given in mg betacyanins/mL and mg betaxanthins/mL, being calculated with the following Equations (4) and (5):

$$\mathrm{B}\mathrm{e}\mathrm{t}\mathrm{a}\mathrm{c}\mathrm{y}\mathrm{a}\mathrm{n}\mathrm{i}\mathrm{n}\mathrm{s}(\mathrm{m}\mathrm{g}/\mathrm{m}\mathrm{L})={\displaystyle \frac{A_{bc}\times DF\times {MW}_{bc}\times 100}{{\epsilon }_{bc}\times l}}$$

(4)

where A_bc is the difference between the maximum absorption of betacyanins (476 nm) and the correction absorption (600 nm), DF is the dilution factor, MW_bc is the molecular weight for betacyanins (550 g/mol), ε_bc is the molar extinction coefficient (60,000 L/mol × cm) for betacyanins, and l is the path length (cm).

$$\mathrm{e}\mathrm{t}\mathrm{a}\mathrm{x}\mathrm{a}\mathrm{n}\mathrm{t}\mathrm{h}\mathrm{i}\mathrm{n}\mathrm{s}(\mathrm{m}\mathrm{g}/\mathrm{m}\mathrm{L})={\displaystyle \frac{A_{bx}\times DF\times {MW}_{bx}\times 100}{{\epsilon }_{bx}\times l}}$$

(5)

where A_bx is the difference between the maximum absorption of betaxanthins (538 nm) and the correction absorption (600 nm), DF is the dilution factor, MW_bx is the molecular weight for betaxanthins (308 g/mol), ε_bx is the molar extinction coefficient (48,000 L/mol × cm) for betaxanthins, and l is the path length (cm).

2.5. Determination of Total Phenolic Content

The procedure outlined by Semeniuc et al. [39] was used to determine the total phenolic content in the beetroot juice and RTD beverage formulations. Here, 15 mL of the test sample was introduced into a 15 mL Falcon tube and centrifuged (Hettich Mikro 22R; Andreas Hettich GmbH & Co. KG, Tuttlingen, Germany) at 8981× g (9000 rpm) at 4 °C for 15 min to separate the suspended particles. One hundred microliters of supernatant were transferred into a 16 mL glass bottle with a rubber stopper. Next, 6 mL of distilled water and 0.5 mL of 2 N Folin–Ciocalteu’s phenol reagent were added and vortexed (6776; Corning Life Sciences, Monterrey, Mexico). After 4 min of rest, 1.5 mL of 0.71 M sodium carbonate solution and 1.9 mL distilled water were added to the mixture. The test sample was then incubated in the dark at room temperature for 2 h. The absorbance value was read at 750 nm against a blank sample using a double-beam UV-VIS spectrophotometer (UV-1900i; Shimadzu Scientific Instruments, Inc., Columbia, MD, USA). The blank was prepared with distilled water and treated identically to the test sample. Each sample was analysed in triplicate. The result was given in mg gallic acid equivalents (GAE)/mL sample.

2.6. Determination of Total Viable Count and Total Yeast and Mould Count

First, 1 mL of the beverage sample was added to 9 mL of 0.85% (w/v) sodium chloride solution (VWR Chemicals, Leuven, Belgium) and vortexed (6776; Corning Life Sciences, Monterrey, Mexico). The subsequent ten-fold serial dilutions (10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, and 10⁻⁶) were prepared by transferring 1 mL from the previous dilution into 9 mL of saline solution and mixing well.

The total viable aerobic microorganisms were counted using the pour plate method, according to the ISO 4833-1:2013/Amd 1:2022 method [40]. One millilitre of each dilution was aseptically transferred into a sterile Petri dish. Next, 15 mL of plate count agar (VWR Chemicals Prolabo, Leuven, Belgium) was poured, and the medium was homogenised with the inoculum by gentle rotational movements and left to solidify. Each sample was prepared in triplicate, and the plates were maintained for 72 h at 30 °C under aerobic conditions; the colonies formed were counted with a colony counter (Colony Star 8500, Funke Gerber, Berlin, Germany).

The total yeast and mould count were counted according to the ISO 21527-1:2008 method [41]; 0.1 millilitres of each dilution, prepared as mentioned above, was aseptically transferred into a sterile Petri dish containing Dichloran–Rose-Bengal–Chloramphenicol agar (Oxoid, Basingstoke, UK) and spread using a Drigalsky spatula. Each sample was prepared in triplicate, and the plates were kept for five days at 25 °C. Typical colonies of yeasts and moulds were counted with a colony counter (Colony Star 8500, Funke Gerber, Berlin, Germany).

2.7. Statistical Analysis

The influence of the whey partial replacement with beetroot juice in the recipe for an RTD beverage on its proximate composition, total soluble solids, energy value, titratable acidity, pH, sensory attributes, betalain pigments, total phenolic content, and microbial load was evaluated using the one-way ANOVA (with Tuckey’s post hoc test at a confidence level (p < 0.05) of 95%); the Minitab statistical software (version 19.1.1; LEAD Technologies, Inc., Charlotte, NC, USA) was employed for this analysis.

3. Results and Discussion

The proximate composition of the whey (W) and beetroot juice (BJ), respectively, RTD10:90, RTD15:85, and RTD20:80 formulations is shown in Table 1. The BJ was characterised by a high content of water (90.35 g/100 mL) and carbohydrate (given that it had total soluble solids of 10.99 °Brix), an average ash content (0.42 g/100 mL), and traces of fat and protein, as was also reported by Ansari et al. [42]. Kale et al. [43] found comparable total soluble solids (9.0 °Brix) and water content (87.4 g/100 mL) in beetroot juice, but a higher ash content (1.4 g/100 mL) as it was not filtered; also, a 1.35 g/100 mL protein content and 0.3 g/100 mL fat content.

The W had moisture of 93.05 g/100 mL and contents of 0.55 g/100 mL fat, 0.82 g/100 mL protein, 0.50 g/100 mL ash, and 5.09 g/100 mL carbohydrate. Given the composition of the BJ, as more was used in the preparation of the RTD beverage, the moisture content decreased significantly (from 87.42 g/100 mL in RTD10:90 to 85.99 g/100 mL in RTD20:80), as did the fat (from 0.50 g/100 mL in RTD10:90 to 0.40 g/100 mL in RTD20:80) and protein (from 0.97 g/100 mL in RTD10:90 to 0.88 g/100 mL in RTD20:80). Devi et al. [44] also noticed a downward trend for the same compositional parameters in their whey-based beverage with the increasing concentration of pineapple juice used in its formulating.

The total dry matter content of the RTD beverage increased instead with its amount of BJ, being influenced mainly by the increase in the carbohydrate content (from 10.70 g/100 mL in RTD10:90 to 12.22 g/100 mL in RTD20:80) but also by that of ash (from 0.38 g/100 mL in RTD10:90 to 0.51 g/100 mL in RTD20:80); the total soluble solids also increased significantly, from 12.55 °Brix in RTD10:90 to 14.04 °Brix in RTD20:80. The total soluble solids also increased significantly, from 12.55 °Brix in RTD10:90 to 14.04 °Brix in RTD20:80. This is an important quality parameter that indicates a beverage’s sweetness and includes the content of sugars and acids as well as small amounts of dissolved vitamins, fructans, proteins, pigments, phenolic compounds, and minerals [45]. Aly et al. [17] also found an increase in the total soluble solids with the amount of cactus pear juice used to formulate a whey-based beverage. All these changes caused by using BJ to prepare the whey-based RTD beverage determined a significant increase in the energy value, from 51.18 kcal/100 mL at RTD10:90 to 55.98 kcal/100 mL at RTD20:80. Silva et al. [26] also reported an increase in the energy value of a whey-based beverage with the amount of passion fruit juice used to formulate it.

RTD20:80a had a different proximate composition than the non-acidified formulation, as it was prepared from Wa and BJa. The proximate composition of whey depends on that of the milk from which it is derived, which in turn is influenced by the climate, lactation stage, feeding, breeding, and individual differences in animals [46], while that of juice is influenced by the beetroot growing and harvesting conditions alike [47]. Therefore, the fat (1.18 g/100 mL), protein (1.07 g/100 mL), and ash (0.56 g/100 mL) contents of RTD20:80a were slightly higher than those of RTD20:80, while the carbohydrate content (9.90 g/100 mL) and total soluble solids (11.69 °Brix) were lower.

The titratable acidity levels are not comparable between the samples of whey (W-0.10% lactic acid, Wa-0.12% lactic acid) and those of beetroot juice (BJ-0.11% citric acid, BJa-0.09% citric acid), as the former are expressed in percentages of lactic acid and the latter in percentages of citric acid; however, BJ (6.02) and BJa (6.25) showed lower pH values than W (6.49) and Wa (6.34). The use of BJ in a concentration of up to 20% from the mixture of BJ and W did not significantly influence the titratable acidity of the RTD beverage, although a slight increase was noticed (from 0.11% lactic acid in RTD10:90 to 0.12% lactic acid in RTD20:80); instead, it led to a significant decrease in the RTD beverage’s pH, from 6.17 in RTD10:90 to 6.08 in RTD20:80. Panghal et al. [25] did not find any significant differences between their whey-based beverages prepared with different concentrations (25 and 50%) of papaya juice in terms of the acidity.

A lower pH increases a beverage’s storage stability by slowing the growth of unwanted microorganisms, including coliforms [24]. Naik et al. [19] reported lower pH values than those in the present study for a whey-based beverage prepared with 15% watermelon juice (5.1), and Chatterjee et al. [23] for one prepared with a mixture of 60 mL concentrated whey and 40 mL orange juice (4.78). Dhamsaniya et al. [20] also recorded a decrease in the pH of a whey-based beverage (from 4.74 to 4.37) when increasing the concentration of banana juice used in its preparation (from 5 to 15 mL juice per 100 mL beverage).

Table 1. Physicochemical properties of the whey, beetroot juice, and RTD formulations.

Sample	Fat (g/100 mL)	Protein (g/100 mL)	Moisture (g/100 mL)	Ash (g/100 mL)	Total Carbohydrate (g/100 mL)	Total Soluble Solids (°Brix)	Energy Value (kcal/100 mL)	Titratable Acidity (% Lactic Acid or Citric Acid)	pH
W	0.55 ± 0.0	0.82 ± 0.007	93.05 ± 0.071	0.50 ± 0.0	5.09 ± 0.064	-	28.55 ± 0.283	0.10 ± 0.002	6.49 ± 0.007
Wa	1.90 ± 0.0	0.91 ± 0.014	92.75 ± 0.071	0.51 ± 0.007	3.93 ± 0.049	-	36.48 ± 0.255	0.12 ± 0.001	6.34 ± 0.014
BJ	-	-	90.35 ± 0.071	0.42 ± 0.028	-	10.99 ± 0.0	-	0.11 ± 0.003 #	6.02 ± 0.028
BJa	-	-	89.55 ± 0.071	0.76 ± 0.014	-	10.94 ± 0.0	-	0.08 ± 0.0 #	6.25 ± 0.014
RTD10:90	0.50 ± 0.0 a	0.97 ± 0.0 a	87.42 ± 0.071 a	0.38 ± 0.014 b	10.70 ± 0.057 c	12.55 ± 0.0 c	51.18 ± 0.226 c	0.11 ± 0.003 a	6.17 ± 0.007 a
RTD15:85	0.45 ± 0.0 b	0.92 ± 0.014 b	86.70 ± 0.0 b	0.47 ± 0.028 a	11.46 ± 0.042 b	13.34 ± 0.0 b	5357 ± 0.113 cb	0.11 ± 0.0 a	6.16 ± 0.007 a
RTD20:80	0.40 ± 0.0 c	0.88 ± 0.007 c	85.99 ± 0.0 c	0.51 ± 0.007 a	12.22 ± 0.0 a	14.04 ± 0.0 a	55.98 ± 0.028 ca	0.12 ± 0.002 a	6.08 ± 0.0 b
p	0.0	0.004	0.0	0.014	0.0	0.0	0.0	0.092	0.001
RTD20:80a	1.18 ± 0.035	1.07 ± 0.007	87.30 ± 0.0	0.56 ± 0.028	9.90 ± 0.014	11.69 ± 0.0	54.44 ± 0.290	0.77 ± 0.002	3.75 ± 0.014

^#, percentage of citric acid. Values are expressed as the mean ± standard deviation of three replicates. Means in the same column with the same letter are not significantly different (p ≥ 0.05, Tukey’s test).

Table 1. Physicochemical properties of the whey, beetroot juice, and RTD formulations.

Sample	Fat (g/100 mL)	Protein (g/100 mL)	Moisture (g/100 mL)	Ash (g/100 mL)	Total Carbohydrate (g/100 mL)	Total Soluble Solids (°Brix)	Energy Value (kcal/100 mL)	Titratable Acidity (% Lactic Acid or Citric Acid)	pH
W	0.55 ± 0.0	0.82 ± 0.007	93.05 ± 0.071	0.50 ± 0.0	5.09 ± 0.064	-	28.55 ± 0.283	0.10 ± 0.002	6.49 ± 0.007
Wa	1.90 ± 0.0	0.91 ± 0.014	92.75 ± 0.071	0.51 ± 0.007	3.93 ± 0.049	-	36.48 ± 0.255	0.12 ± 0.001	6.34 ± 0.014
BJ	-	-	90.35 ± 0.071	0.42 ± 0.028	-	10.99 ± 0.0	-	0.11 ± 0.003 #	6.02 ± 0.028
BJa	-	-	89.55 ± 0.071	0.76 ± 0.014	-	10.94 ± 0.0	-	0.08 ± 0.0 #	6.25 ± 0.014
RTD10:90	0.50 ± 0.0 a	0.97 ± 0.0 a	87.42 ± 0.071 a	0.38 ± 0.014 b	10.70 ± 0.057 c	12.55 ± 0.0 c	51.18 ± 0.226 c	0.11 ± 0.003 a	6.17 ± 0.007 a
RTD15:85	0.45 ± 0.0 b	0.92 ± 0.014 b	86.70 ± 0.0 b	0.47 ± 0.028 a	11.46 ± 0.042 b	13.34 ± 0.0 b	5357 ± 0.113 cb	0.11 ± 0.0 a	6.16 ± 0.007 a
RTD20:80	0.40 ± 0.0 c	0.88 ± 0.007 c	85.99 ± 0.0 c	0.51 ± 0.007 a	12.22 ± 0.0 a	14.04 ± 0.0 a	55.98 ± 0.028 ca	0.12 ± 0.002 a	6.08 ± 0.0 b
p	0.0	0.004	0.0	0.014	0.0	0.0	0.0	0.092	0.001
RTD20:80a	1.18 ± 0.035	1.07 ± 0.007	87.30 ± 0.0	0.56 ± 0.028	9.90 ± 0.014	11.69 ± 0.0	54.44 ± 0.290	0.77 ± 0.002	3.75 ± 0.014

^#, percentage of citric acid. Values are expressed as the mean ± standard deviation of three replicates. Means in the same column with the same letter are not significantly different (p ≥ 0.05, Tukey’s test).

Table 2 shows the hedonic scores of the sensory characteristics for the three RTD beverage formulations. Following the sensory evaluation of these, it emerged that the RTD20:80 sample was preferred by consumers, receiving an overall score of 7.8, although not significantly different from those of the RTD10:90 (7.6) and RTD15:85 samples (7.7); this was because there were no significant differences between their scores in terms of the appearance and colour, odour, taste, consistency, and overall acceptability. In the study by Kamate et al. [31], the formulation prepared with 20% beetroot extract also received the highest overall score (8.51) among the three tested (with 10, 20, and 25% beetroot pulp extract). In 2022, Abdo et al. [32] developed a beverage by mixing whey protein isolate (5%) with different levels of beetroot peel water extract (1, 2.5, and 5%) and flavoured with strawberry puree (5%); the formulation with 2.5% extract was found to be the most sensory-preferred.

However, increasing the amount of beetroot juice used in preparing the whey-based RTD beverage led to an increase in its acceptance rate, from 85% in RTD10:90 to 86% in RTD15:85 and RTD20:80. The acidified sample (RTD20:80a) received slightly higher scores for all the sensory attributes evaluated; therefore, it had the highest overall score (8.1) and acceptance rate (90%). Since an acceptance rate of ≤70% is considered reasonable [36], any of these formulations could be marketed. Devi et al.’s [22] study showed that beverages with a whey–water mixture (70:30) and 20% orange juice or pineapple juice, respectively, were the most sensory-appreciated of the fifteen blends tested.

Table 2. Hedonic scores for the sensory attributes of the RTD formulations and their acceptance rates.

Sample	Appearance and Colour	Odour	Taste	Consistency	Overall Acceptability	Overall Score	Acceptance Rate (%)
RTD10:90	8.0 ± 0.833 a	7.6 ± 1.028 a	7.2 ± 1.608 a	7.8 ± 0.825 a	7.5 ± 1.129 a	7.6 ± 0.830 a	85
RTD15:85	8.1 ± 0.993 a	7.7 ± 0.883 a	7.3 ± 1.421 a	7.9 ± 0.947 a	7.6 ± 1.055 a	7.7 ± 0.798 a	86
RTD20:80	8.3 ± 0.712 a	7.6 ± 0.804 a	7.1 ± 1.640 a	8.1 ± 0.717 a	7.7 ± 1.033 a	7.8 ± 0.675 a	86
p	0.137	0.847	0.769	0.255	0.660	0.590	-
RTD20:80a	8.6 ± 0.616	7.7 ± 0.694	8.0 ± 0.829	8.3 ± 0.798	8.1 ± 0.761	8.1 ± 0.740	90

Values are expressed as the mean ± standard deviation of eighty-seven responses. Means in the same column with the same letter are not significantly different (p ≥ 0.05, Tukey’s test).

Table 2. Hedonic scores for the sensory attributes of the RTD formulations and their acceptance rates.

Sample	Appearance and Colour	Odour	Taste	Consistency	Overall Acceptability	Overall Score	Acceptance Rate (%)
RTD10:90	8.0 ± 0.833 a	7.6 ± 1.028 a	7.2 ± 1.608 a	7.8 ± 0.825 a	7.5 ± 1.129 a	7.6 ± 0.830 a	85
RTD15:85	8.1 ± 0.993 a	7.7 ± 0.883 a	7.3 ± 1.421 a	7.9 ± 0.947 a	7.6 ± 1.055 a	7.7 ± 0.798 a	86
RTD20:80	8.3 ± 0.712 a	7.6 ± 0.804 a	7.1 ± 1.640 a	8.1 ± 0.717 a	7.7 ± 1.033 a	7.8 ± 0.675 a	86
p	0.137	0.847	0.769	0.255	0.660	0.590	-
RTD20:80a	8.6 ± 0.616	7.7 ± 0.694	8.0 ± 0.829	8.3 ± 0.798	8.1 ± 0.761	8.1 ± 0.740	90

Values are expressed as the mean ± standard deviation of eighty-seven responses. Means in the same column with the same letter are not significantly different (p ≥ 0.05, Tukey’s test).

Table 3. Response rates (%) for the purchase intention of the RTD formulations.

Sample	Definitely Will Buy	Probably Will Buy	May or May Not Buy	Probably Will Not Buy	Definitely Will Not Buy
RTD10:90	10	29	31	22	8
RTD15:85	14	29	35	20	2
RTD20:80	10	33	25	22	10
RTD20:80a	17	46	25	13	0

reports the response rates for consumers’ purchase intention of the RTD formulations prepared in this study. Between 10 and 17% of participants declared that they definitely would buy a whey-based beverage with beetroot juice, between 29 and 46% probably would buy it, and between 25 and 31% were undecided. The rest of the panellists were not interested in such a product. The RTD20:80a sample revealed the highest likelihood of being purchased, receiving 17% “definitely will buy” and 46% “probably will buy” answers; consequently, it had the lowest response rates for “may or may not buy” (25%), “probably will not buy” (13%), and “definitely will not buy” (0%). Devi et al. [22] reported that beverages with a whey–water mixture (70:30) and 20% orange or pineapple juice were the most sensory-appreciated of the fifteen blends tested. Singh et al. [18] instead showed a ratio of 67.5:20 whey and guava juice as being the sensory optimum when testing nine such blends.

Table 4 shows the results regarding the determination of the betalain pigments (betacyanins and betaxanthins) and total phenols from the non-thermally treated beetroot juices (BJ and BJa) and whey-based RTD beverages (RTD20:80 and RTD20:80a). The content of betacyanins (10.88 mg/mL), betaxanthins (9.45 mg/mL), and total phenols (1.15 mg GAE/mL) in BJ was significantly higher than that in BJa (of 3.6 times for betacyanins (3.06 mg GAE/mL) and betaxanthins (2.59 mg GAE/mL) and of 1.6 times for total phenols (0.69 mg GAE/mL)). Other authors reported lower levels of betacyanins (1.87 mg/mL) and betaxanthins (0.82 mg/mL) [48] as well as total phenols (0.52 mg GAE/mL) [38] in fresh beetroot juice.

As expected, the content of betacyanins (1.26 mg/mL), betaxanthins (1.43 mg/mL), and total phenols (0.38 mg GAE/mL) in RTD20:80 was also higher than that in RTD20: 80a (1.16 mg/mL, 0.86 mg/mL, and 0.36 mg GAE/mL respectively); however, these compounds’ content differences between the beverages were much lower (1.1 times for betacyanins, 1.7 for betaxanthins, and 1.1 for total phenols) than between the juices. Given that both RTD beverage formulations were prepared with the same concentration of beetroot juice and subjected to the same heat treatment, the unexpectedly low content of betalain pigments in RTD20:80 was mainly due to the beverage’s pH, even though they are relatively stable in the pH range of 3–7 [49]; regarding the heat sensitivity, these pigments are known to degrade at temperatures higher than 60 °C, especially after prolonged exposure [50], as is the case for the pasteurisation in the current study.

Table 4. Content of betalain pigments and total phenolics in the beetroot juice and RTD formulations.

Sample	Betacyanins (mg/mL)	Betaxanthins (mg/mL)	Total Phenolic Content (mg GAE/mL)
BJ	10.88 ± 0.049 a	9.45 ± 0.028 a	1.15 ± 0.014 a
BJa	3.06 ± 0.021 b	2.59 ± 0.021 b	0.69 ± 0.006 b
p	0.0	0.0	0.0
RTD20:80	1.26 ± 0.021 a	1.43 ± 0.014 a	0.38 ± 0.004 a
RTD20:80a	1.16 ± 0.0 b	0.86 ± 0.007 b	0.36 ± 0.008 b
p	0.024	0.0	0.024

Values are expressed as the mean ± standard deviation of three replicates. Means in the same column with the same letter are not significantly different (p ≥ 0.05, Tukey’s test).

Table 4. Content of betalain pigments and total phenolics in the beetroot juice and RTD formulations.

Sample	Betacyanins (mg/mL)	Betaxanthins (mg/mL)	Total Phenolic Content (mg GAE/mL)
BJ	10.88 ± 0.049 a	9.45 ± 0.028 a	1.15 ± 0.014 a
BJa	3.06 ± 0.021 b	2.59 ± 0.021 b	0.69 ± 0.006 b
p	0.0	0.0	0.0
RTD20:80	1.26 ± 0.021 a	1.43 ± 0.014 a	0.38 ± 0.004 a
RTD20:80a	1.16 ± 0.0 b	0.86 ± 0.007 b	0.36 ± 0.008 b
p	0.024	0.0	0.024

Values are expressed as the mean ± standard deviation of three replicates. Means in the same column with the same letter are not significantly different (p ≥ 0.05, Tukey’s test).

The highest sensory-rated whey-based RTD beverages (RTD20:80 and RTD20:80a) were used for the shelf-life study, which consisted of periodic monitoring (every 7 days) of the titratable acidity (Figure 1a), pH (Figure 1b), total viable count (Figure 2a), and total yeast and mould count (Figure 2b) in these two formulations over 70 days of storage at 4 °C.

As can be observed from Figure 1a,b, the titratable acidity increased significantly during storage, reaching a level of 0.17% lactic acid for RTD20:80 and 0.84% lactic acid for RTD20:80a on storage day 70; at the same time, the pH significantly decreased to 5.64 for RTD20:80 and 3.50 for RTD20:80a from the initial values mentioned above in the paragraph discussing the Table 2 results. These changes in acidity and pH during storage are explained by the conversion of lactose from whey to lactic acid [51] and proteins to amino acids [52]. The sweetener’s acidic nature may also contribute to the increase in the acidity and decrease in the pH during storage, which was also noticed by Thakkar et al. [24] in their whey-based beverage with orange juice.

Figure 1. (a) Titratable acidity of the RTD formulations during the storage period. Values are expressed as the mean ± standard deviation of three replicates. Means in the same row with the same letter are not significantly different (p ≥ 0.05, Tukey’s test). (b) pH of the RTD formulations during the storage period. Values are expressed as the mean ± standard deviation of three replicates. Means in the same row with the same letter are not significantly different (p ≥ 0.05, Tukey’s test).

Figure 1. (a) Titratable acidity of the RTD formulations during the storage period. Values are expressed as the mean ± standard deviation of three replicates. Means in the same row with the same letter are not significantly different (p ≥ 0.05, Tukey’s test). (b) pH of the RTD formulations during the storage period. Values are expressed as the mean ± standard deviation of three replicates. Means in the same row with the same letter are not significantly different (p ≥ 0.05, Tukey’s test).

The European legislation on the microbiological criteria for food products [53] does not stipulate limits regarding the total viable count or the total yeast and mould count in pasteurised RTD beverages to be able to establish a shelf-life for them. However, in 2020, the Food Safety Authority of Ireland developed a guidance note on testing ready-to-eat foods [54], which contains indicative microbiological limits for this category of products. According to this document, the result of determining the total viable count in an RTD beverage can be interpreted, depending on the number of microorganisms found, as satisfactory (less than 10⁴ cfu/mL (9.2 log CFU/mL)), borderline (between 10⁴ cfu/mL (9.2 log CFU/mL) and less than 10⁷ cfu/mL (16.1 log CFU/mL)), or unsatisfactory (greater than or equal to 10⁷ cfu/mL (16.1 log CFU/mL)); a borderline result means the food sample meets the guideline limit but may be approaching unsatisfactory.

In the current study, a satisfactory level of viable aerobic microorganisms (<9.2 log CFU/mL) was maintained in both beverage formulations until day 14 of storage (from 3.6 log CFU/mL in RTD20:80 and 3.7 log CFU/mL in RTD20:80a on day 7), after which a borderline level until day 70 of storage (that was lower in the acidified beverage (5.6 log CFU/mL) than the other (6.3 log CFU/mL) despite the fact their levels were close until day 63 of storage). Considering the limit for a satisfactory microbiological result, both RTD20:80 and RTD20:80a had a 14-day shelf-life under the pasteurisation and storage temperatures tested here. Abdo et al. [32] reported a total viable count between 25.0 and 1.9 log CFU/mL on the 14th day of storage in their beverages formulated with whey protein isolate and increasing concentrations of beetroot peel extract, with the extract showing an antibacterial effect.

Panghal et al. [25] reported a higher shelf-life (60 days) for a whey-based beverage prepared with papaya juice and kept under refrigeration conditions; still, their sample was subjected to a higher pasteurisation temperature (80 °C, 10 min). Silva et al.’s [26] whey-based beverage prepared with passion fruit juice, pasteurised at 85 °C for 30 sec, and stored at 7 °C reached a shelf-life of 56 days. Although the whey-based RTD beverages with beetroot juice tested in this study contained mint essential oil, whose antimicrobial properties are well known, the short shelf-life was due to their low-temperature pasteurisation; it could be extended if a more aggressive heat treatment is used.

The total yeast and mould count remained lower in RTD20:80a than in RTD20:80 until day 35 of storage, after which, on day 42, the levels of the two formulations came near. From this moment, it increased in RTD20:80 until day 70, reaching a level of 6.3 log CFU/mL from 4.3 log CFU/mL on day 7 of storage; in RTD20:80a, however, it was maintained until day 49 of storage, after which it increased to 6.4 log CFU/mL (day 70) from 4.0 log CFU/mL on day 7. Abdo et al. [32] found much higher total yeast and mould counts (9.6–3.2 log CFU/mL) in their beverages based on whey protein isolate and beetroot peel extract on the 14th day of storage. The beverage of Silva et al. [26], prepared with whey and passion fruit juice, remained microbiologically stable during storage, with a mean total yeast and mould count of 2.0 log CFU/mL.

Figure 2. (a) Total viable count in the RTD formulations during the storage period. Values are expressed as the mean ± standard deviation of three replicates. Means in the same row with the same letter are not significantly different (p ≥ 0.05, Tukey’s test). (b) Total yeast and mould count in the RTD formulations during the storage period. Values are expressed as the mean ± standard deviation of three replicates. Means in the same row with the same letter are not significantly different (p ≥ 0.05, Tukey’s test).

Figure 2. (a) Total viable count in the RTD formulations during the storage period. Values are expressed as the mean ± standard deviation of three replicates. Means in the same row with the same letter are not significantly different (p ≥ 0.05, Tukey’s test). (b) Total yeast and mould count in the RTD formulations during the storage period. Values are expressed as the mean ± standard deviation of three replicates. Means in the same row with the same letter are not significantly different (p ≥ 0.05, Tukey’s test).

4. Conclusions

Following this study, a whey-based RTD beverage with beetroot juice was successfully developed. The formulation prepared with a beetroot juice–whey mixture of 20:80 (v/v) and acidified with citric acid was found to be the optimum one among those tested due to its enhanced sensory attributes, superior nutritional composition, and higher energy value, having a shelf-life of 14 days at 4 °C. Variations in the composition of the whey and beetroot juice could be an issue when preparing the RTD beverage, but this can be avoided by standardising it through technological operations. Placing such a beverage on the market would reduce the losses related to whey in a dairy plant; moreover, it would increase its profitability through assortment diversification. This beverage is easy to digest and can be consumed by both children and adults. It is also convenient, as it does not require preparation before drinking.

5. Patents

Patent Application RO134175A2 from 20 November 2018. Nutritive ready-to-drink beverage based on whey and beetroot juice and process for preparing the same. https://patentscope.wipo.int/search/pt/detail.jsf;jsessionid=6EC1B372A98903D28B6F49EF8AEFEBB1.wapp2nC?docId=RO298652589&_cid=P22-KPKH8B-15133-14 (accessed on 14 September 2024).