Natural Gum from Flaxseed By-Product as a Potential Stabilizing and Thickening Agent for Acid Whey Fermented Beverages

Abstract

The valorization of food industry by-products is still a major challenge. Here, we report the production of acid whey fermented beverages stabilized with flaxseed gum (derived from oil industry by-product). Four variants of drinks were prepared: (1) fermented whey (W), (2) fermented whey with milk powder added (5% w/v) (WMP), (3) fermented whey with flaxseed gum added (0.5% w/v) (WFG1) and (4) fermented whey with flaxseed gum added (1.0 % w/v) (WFG2). The beverages were kept in refrigerated conditions (5 ± 1 °C) for 28 days. Alterations in lactic acid bacteria population, pH, titratable acidity, water activity, syneresis, viscosity, acetaldehyde content, color, consumer acceptance, bio-active compounds and antioxidant activity were identified. The findings revealed that flaxseed gum addition significantly enhanced bacteria survivability and improved the viscosity of acid whey at a level comparable with milk powder, meeting consumer acceptance criteria. The beverages were characterized by normative physicochemical properties and showed high antioxidant activity and free amino acids level. The use of valuable by-products from the dairy and oil industries opens up a promising route for the production of innovative beverages, which is in accordance with the principles of circular economy and the idea of zero waste.

1. Introduction

Whey is a by-product of the dairy industry, produced in vast quantities and obtained during the production of rennet cheeses, cottage and acid curd cheeses (tvorogs) as well as milk protein preparations [1,2,3,4]. It is estimated that the processing of 1 kg of cheese produces ca. 9 L of whey [1,2,5,6,7]. It is a yellow-greenish liquid, which contains about 45–55% milk solids, including lactose, whey proteins and mineral compounds [6,7]. The fat content of whey is low, and the casein content, which forms the cheese curd, is negligible. The total solid content in whey is approximately 6–7% [1,2,3,6]. Whey comprises a significant number of nutritional values and innumerable health benefits, as it is a source of valuable proteins, bioactive peptides, amino acids, vitamins (such as folic acid, cobalamin and riboflavin), as well as minerals [1,6,8]. This product has a high biochemical and chemical oxygen demand, so discharging whey with wastewater directly into the water bodies degrades natural ecosystems. It must be disposed of and processed. The processing of whey consists mainly of the use of membrane processes, such as reverse osmosis, nanofiltration and ultrafiltration, and drying processes, in order to obtain the concentrates and isolates of whey proteins, as well as powdered whey, which are used in many branches of the food industry. These products are obtained primarily from the so-called “sweet whey” obtained when rennet cheeses are produced. The management of acid whey (AW), which is a by-product in the production of acid curd cheese (tvorog), is much more difficult due to its low pH, lower lactose content, higher mineral content and a more sour and distinct taste compared to sweet whey [1,2,6,9]. AW presents less favorable processing properties compared with rennet whey, and it presents a sustainability challenge to the dairy industry [1,6,9,10]. Unprocessed whey is also not attractive to consumers because of its sensory properties [1,6]; therefore, it seems promising to use it as a raw material for the production of unfermented and fermented beverages [1,2,11]. Various whey-based beverages are being successfully developed around the world, which include fermented, unfermented, alcoholic, non-alcoholic, carbonated, plain and fruit-flavored products [10,11,12,13]. Whey fermentation leads to advantages, such as a partial hydrolysis of whey protein (which may cause allergies), increase in shelf life (due to lactic acid production) and the production of volatile compounds that improve the sensory features [2,8]. On the other hand, the production of whey-based fermented beverages becomes a challenge due to the high water content, and therefore, the low dry matter content, and consequently, the watery consistency of the final product [2,5,14,15]. The texture and mouthfeel of fermented whey beverages (FWB) tend to be weak and watery compared to fermented milk, since liquid whey contains a low percentage of total solids (ca. 6 g/100 g) [16]. Until now, the evaporation, centrifugation or ultrafiltration processes have been used to increase the dry matter of milk intended for the production of fermented milk. An alternative to the above-mentioned processes is adding, among others, milk powder, whey powder, whey protein concentrates, casein and caseinates. The addition of milk powder or whey, however, significantly increases the lactose content in the product, which is not well perceived by consumers who have problems with this sugar intolerance [17]. Additives of non-dairy origin were also used in the past, e.g., modified starch. The current trends in creating new products are directed toward “clean label”. Therefore, the most commonly used structure-forming compounds are substances of natural origin. This has created a requirement for using exopolysaccharide-producing starter cultures or the addition of hydrocolloids [12]. Hydrocolloids are defined as hydrophilic molecules, which have a high molecular weight. Their role in food products is the formation of texture, water and oil binding, as well as stabilization [18].

Against this background, more attention has been paid to the by-products and co-products of flaxseed (Linum usitatissimum L.) [19]. Due to its high content of bioactive compounds, it has been categorized as a functional or bioactive food [20]. The mucilage obtained from flaxseed seeds contains L-galactose, D-xylose, L-arabinose, L-rhamnose and D-galacturonic acid, and it demonstrates a high ability to hold water and bind oil, which is important when stabilizing the food systems [13,21,22,23,24,25]. The polysaccharides that make up the mucilage are responsible for the formation of a multi-form structure and better resistance to environmental stresses. From a nutritional perspective, flaxseed mucilage is a fine source of dietary fiber and has many health benefits, such as the prevention of diabetes, obesity or colon cancer, decreasing blood cholesterol levels and improving insulin sensitivity. Flaxseed polysaccharides are called flaxseed gum (FG), as the main component of soluble dietary fiber in flaxseed (3–9 wt% of the flaxseed) [16,22]. FG is composed of 80% neutral and acidic polysaccharides and 9% protein. Neutral monosaccharides consist of xylose, glucose, arabinose and galactose, while acidic monosaccharides consist of rhamnose, galactose, fucose and galacturonic acid [16,20,25]. The protein in FG is predominantly conlinin [23]. FG is most commonly used as a thickener, stabilizer, gelling agent and emulsifier [16,22,23,25]. From a functional point of view, FG more closely resembles gum Arabic or guar gums than other commonly used gums and can be used to replace most non-gelling gums in food and non-food applications due to its “weak gel”-like properties and remarkable ability to retain water [16,20,25,26,27,28,29]. FG is a promising new dairy ingredient, which acts, for instance, as a natural stabilizer in stirred yogurts [24]. However, very little is understood regarding its effect when added to dairy by-products. Moreover, FG also has the advantage of relatively low cost compared with most commercial gums [28]. Hence, based on the technological characteristics of FG, it can be considered that it can potentially be used as a stabilizer and thickener for acid whey. It was reported that FG can be obtained from flaxseed oil cake (FOCE)—a cheap by-product from flaxseed oil production [25,30]. This is particularly meaningful, since a large amount of press cakes and residues are available, and their management meets the criteria of a “zero waste” economy [21].

To the best of our knowledge, there are no reports of studies on the use of FG as a natural stabilizer for fermented beverages based on acid whey. We hypothesized that the application of FG as a natural stabilizer would allow obtaining new products with high added value. Hence, the goal of the research was to produce fermented beverages from acid whey with various FG concentrations and evaluate their microbiological, physicochemical properties, as well as bioactivity and sensory acceptance during refrigerated storage (5 ± 1 °C) for 28 days.

2. Materials and Methods

2.1. Materials and Reagents

Flaxseed oil cake (FOC) was procured from ACS Sp. z o.o. (Bydgoszcz, Poland). Sodium hydroxide, disodium phosphate, monosodium phosphate, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), potassium persulphate, methanol, ethanol, hydrochloric acid, phenolphthalein, sodium chloride, 3,5-dinitrosalicylic acid, sodium tartate tetrahydrate, glucose, iron chloride, glycine, ninhydrin, glacial acetic acid, sodium acetate and cadmium chloride were purchased from Merck (Darmstad, Germany). Sulphuric acid 96%, dimethylsulfoxide, acetone, sodium tungstate dihydrate and 3-methyl-2-benzothiazolinone hydrazinone hydrochloride hydrate were obtained from P.P.H. Stanlab Sp. J. (Lublin, Poland). All reagents were of analytical grade. MRS (de Man, Rogosa and Sharpe) agar was obtained from Merck (Darmstad, Germany).

2.2. Acid Whey Production

Acid whey (AW) was derived from the production of acid curd cheese (ACC). Full-fat pasteurized (85 °C, 15 s) and homogenized (15 MPa, 55 °C) cow’s milk (containing 3.2% fat, 3.1% protein and 4.5% lactose) was procured at a local market. ACC was produced with traditional technology [31], using a freeze-dried DVS starter to direct the inoculation of milk containing Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris (Lactoferm MSO Cheese-Tek^®, Biochem s.r.l., Monterotondo (Rome), Italy). The production of ACC in laboratory conditions started with heating the milk to 23 °C followed by the addition of 2.5% (v/v) of the activated starter. The inoculated milk was incubated (23 °C, 12 h) until the curd reached pH 4.5. The curd was gently heated to separate it from the walls of the cheese tub; then, it was cut into cuboidal shapes with dimensions of approx. 120 × 120 mm. The curd was then gently stirred and gradually heated (1 °C/10 min) to 40 °C in order to intensify the separation of whey. The curd mass was divided into disposable, polyethylene cheese cloths and allowed to drain. Subsequently, the obtained ACCs were pressed with a laboratory press for 45 min (1 kg per 1 kg of cheese) [31].

2.3. Flaxseed Gum Preparation

Flaxseed gum (FG) was obtained from the flaxseed oil cake extract (FOCE) produced from FOC, as described elsewhere [26]. In the first step, flaxseed protein was precipitated by 0.1 M HCl from FOCE at flaxseed protein isoelectric point (4.2); then, the crude flaxseed gum was extracted from FOCE with 96% ethanol at a ratio 1:1. The obtained pellet was washed with distilled water at a ratio 1:1 and centrifuged for 10 min at 5000 rpm. The supernatant was carefully decanted, and FG was dried for 12 h at 55 °C, then pulverized.

2.4. Fermented Whey Beverages Production

The fermented whey beverages (FWB) were produced on the day of AW production. Whey was submitted to a thermal treatment at 72 °C for 10 min and cooled to 20 °C. Four variants of FWB were prepared: (i) fermented whey (W), (ii) fermented whey with milk powder added (5% w/v) (WMP), (iii) fermented whey with flaxseed gum added (0.5% w/v) (WFG1) and (iv) fermented whey with flaxseed gum added (1.0% w/v) (WFG2). The levels of FG addition were based on preliminary trials, and the intention was to obtain FWB with a consistency similar to a drinkable yogurt. To increase the dry matter content in the WMP variant, skim milk powder (MP) with 0.2% fat, 35% protein and 96% total solids was used (SM Gostyń, Gostyń, Poland). In the case of the WFG1 and WFG2 variants, the previously prepared FG was added to the whey in amounts of 0.5% and 1.0%, respectively. Whey (W), whey with milk powder (WMP) and whey with both concentrations of FG (WFG1 and WFG2) were heated to 40 °C and inoculated with 0.6 g/L of direct vat set starter cultures of Streptococcus salivarius ssp. thermophilus, Lactobacillus delbrueckii ssp. bulgaricus (Lactoferm YO 122 YogurtTek^®, Biochem s.r.l., Italy). Then, the samples were poured into sterile, low-density polyethylene cups (50 mL capacity), tightly sealed and incubated at 42 ± 1 °C for 7 h. After incubation, the samples were cooled and stored at 5 ± 1 °C in the dark for 28 days.

2.5. Fermented Whey Beverages Characterization

2.5.1. Microbial Analyses

Microbial analyses were performed as described elsewhere [32]. Briefly, a series of dilutions of the samples were prepared with physiological saline (0.85%), and the lactic acid bacteria counts were enumerated in triplicate on MRS medium (Merck, Darmstad, Germany) after incubation at 37 °C under anaerobic conditions for 72 h. The viable bacteria counts were expressed as CFU/mL of the samples.

2.5.2. Determination of Titratable Acidity, pH, Water Activity and Acetaldehyde Content

The following physicochemical properties of the FWB were evaluated: titratable acidity (% w/w lactic acid) was assessed by titration with 0.25 N NaOH using phenolphthalein as an indicator [33,34]; pH was determined using a pH meter (CP-411, Elmetron, Zabrze, Poland); water activity (a_w) was measured at a temperature of 23 °C with a HygroLab C1 hygrometer (Rotronic, Bassersdorf, Switzerland). Acetaldehyde content was determined using a diffusive method [2].

2.5.3. Viscosity, Syneresis and Particle Size Measurements

Viscosity measurements were carried out with a rheometer (AR G2, TA Instruments Ltd., Lukens Drive, New Castle, USA). The temperature was kept at 20 °C during the measurements. Flow experiments were performed with a stainless-steel cone plate of 62 mm diameter at 60° for 50 s⁻¹. Data from rheological measurements were collected using TA Rheology Advantage Data Analysis equipment software V 5.4.7.

Syneresis was examined for all variants during the storage according to Yu et al. [35], with some modifications. A 10 mL aliquot of the sample was poured into 15 mL Falcon tubes and centrifuged at 890 rpm for 10 min at 4 °C, followed by reading the amount of the clear supernatant. Syneresis (%) was expressed as the percentage volume of the supernatant over the initial volume of the sample.

Particle size distribution measurements of the samples were performed using a Mastersizer 2000 (Malvern Instrument Ltd., Worcestershire, UK) [15]. The samples were dispersed in distilled water (stirring speed—2000 rpm) until an obscuration rate of 10% was obtained. The optical properties of the sample were defined as follows: refractive index 1.500 and absorption 1.00.

2.5.4. Color Analysis

The color of FWB was assessed with the objective method using colorimeter WR 18 (FRU^®, Shenzhen Wave Optoelectronics Technology Co., Ltd.) [36] based on the white standard plate (L* = 92.4; a* = −0.04; b* = +1/9) and CIE L*a*b*, illuminant D65, observer 10°, illumination mode d/8 and caliber 8 mm. The total color difference (ΔE), hue (h) and (C*) chroma of the color were calculated based on the following formulae:

$$C^{\ast }=\sqrt{a^2+b^2}$$

$$H^0=ta{n}^{-1}\left(\frac{b}{a}\right)$$

$$\Delta E={\left[{\left(L_{standard }-L_{sample}\right)}^{2 }+{\left(a_{standard}-a_{sample}\right)}^2+{\left(b_{standard}-b_{sample}\right)}^2\right]}^{0.5}$$

2.5.5. Sensory Evaluation

Ten panelists (male and female) of different age groups, tested for taste sensitivity and experienced in sensory evaluation of dairy products, participated in the sensory evaluation. The panelists were asked to indicate how much they liked or disliked each variant of FWB on a 5-point hedonic scale (5 = like extremely; 1 = dislike extremely). The appearance, consistency, smell and taste were evaluated according to standards [37,38]. The samples used for the analysis were selected randomly. The evaluation was carried out in a room that was free of any foreign odors; each panelist had a separate test stand and distilled water to rinse the mouth. The sensory analysis was performed by the same group of panelists each time. The results for each descriptor were added together and were expressed as an arithmetic mean. The mean scores for each attribute were used to calculate the overall sensory quality.

2.5.6. Preparation of Supernatants

To obtain clear supernatants for analyses, the samples were prepared based on a methodology described elsewhere [32]. Samples were transferred to 1.5 mL Eppendorf tubes and centrifuged at 14,000× g rpm for 10 min at 20 °C (Centrifuge 5418 Eppendorf, Warsaw, Poland). After centrifugation, the supernatants were filtered through 0.22 µm nylon membrane filters (Merck, Darmstad, Germany) and used for further determinations.

2.5.7. Determination of Reducing Sugars Content and Total Free Amino Acids Level

The reducing sugars content (RSC) was determined by the DNS (3,5-dinitrosalicylic acid) method, as described in a previous study [32]. A total of 10 g of DNS was dissolved in 200 mL of distilled water by continuous stirring; then, 16 g of NaOH (first dissolved in 150 mL of distilled H₂O) was slowly added. The mixture was incubated at 50 °C with stirring to obtain a clear solution. Then, 403 g of potassium sodium tartrate tetrahydrate was added in small portions. The mixture was filtered using a paper filter, and the volume was raised to 1000 mL with distilled water. A volume of 1 mL of the supernatant was mixed with 1 mL of 0.05 m acetate buffer (pH 4.8), and 3 mL of the DNS reagent was added, then vigorously shaken. The mixtures were incubated in boiled water for 5 min, then cooled at room temperature. The absorbance was measured at 540 nm (UV-Vis Thermo Scientific Evolution 220 spectrophotometer). Glucose in acetate buffer was used for the calibration curve.

Total free amino acids level (TFAAL) was analyzed based on a methodology described elsewhere [32]. A quantity of 1 mL of the supernatants was mixed with 2 mL of a Cd-ninhydrin reagent (0.8 g ninhydrin was dissolved in a mixture of 80 mL ethanol and 10 mL glacial acetic acid, followed by the addition of 1 g CdCl₂ dissolved in 1 mL of distilled water). The mixtures were shaken and heated at 84 °C for 5 min and cooled in ice water; then, the absorbance was measured at 507 nm. The results were expressed as milligram Gly per mL of the sample by reference to a standard curve, which was first prepared using glycine at various concentrations.

2.5.8. Determination of DPPH and ABTS Radicals Scavenging Activity

The scavenging activity against DPPH and ABTS radicals was determined as described elsewhere [32]. Briefly, the DPPH radical scavenging activity was determined by mixing 1 mL of the supernatants with 1 mL of 0.01 mM DPPH methanolic solution and incubation for 30 min. Subsequently, the absorbance was measured at 517 nm. A volume of 3 mL of ABTS^+· solution was mixed with 50 µL of the supernatants, and the absorbance was measured at 734 nm after 6 min of incubation.

2.6. Statistical Analysis

All analyses were performed at least in triplicate. All data were expressed as mean ± standard deviation (SD). The results were subjected to a statistical analysis carried out using Statistica software (StatSoft Polska, Kraków, Poland), using the Tukey test and a two-factor analysis of variance with repetition (ANOVA). All the tests were performed at a significance level of p = 0.05.

3. Results and Discussion

3.1. The Lactic Acid Bacteria (LAB) Survivability during Cold Storage

One of the most pivotal factors that affect the quality of fermented beverages is the number of live cells of lactic acid bacteria (LAB) [2]. LAB counts, as well as their survivability during refrigerated storage, are summarized in

Table 1. The lactic acid bacteria (LAB) survivability during storage time.

Sample	Time of Storage (Days)
	1	7	14	21	28
LAB (CFU/mL)
W	7.65 × 106 ± 1.05 Aa	5.05 × 106 ± 0.71 Aa	4.86 × 106 ± 0.79 Aa	4.50 × 105 ± 2.15 Ba	4.60 × 105 ± 0.85 Ba
WMP	6.40 × 107 ± 1.55 Ab	2.47 × 107 ± 0.20 BCb	5.30 × 107 ± 0.05 ABb	1.49 × 107 ± 0.01 Cb	3.32 × 106 ± 0.09 Cb
WFG1	2.65 × 108 ± 0.07 Ac	7.25 × 107 ± 2.33 Bc	1.15 × 108 ± 0.07 Cc	7.50 × 107 ± 0.71 Bc	1.49 × 107 ± 0.32 Ec
WFG2	2.75 × 108 ± 1.63 Av	1.18 × 108 ± 0.06 Bd	1.37 × 108 ± 0.14 Cc	4.67 × 107 ± 0.40 Dd	7.90 × 107 ± 0.99 Ed

W—fermented whey, WMP—fermented whey with milk powder added (5% w/v), WFG1—fermented whey with flaxseed gum added (0.5% w/v), WFG2—fermented whey with flaxseed gum added (1.0% w/v). Values are means ± standard deviation of triplicate determinations. Means with different lowercase letters in the same column are significantly different at p < 0.05. Means with different uppercase letters in the same row are significantly different at p < 0.05.

. Generally, whey has been reported to be a good medium for the growth of yogurt bacteria [2]. The minimum level of bacteria required for a functional product was reported to be at least 10⁶ CFU/mL, and it cannot be lower during storage, until expiration date [2]. After fermentation, the lowest bacterial concentration was noted for sample W (7.65 × 10⁶ ± 1.05 CFU/mL), and the highest LAB counts were observed for samples with FG (2.65 × 10⁸ ± 0.07 CFU/mL and 2.75 × 10⁸ ± 1.63 CFU/mL, for samples WFG1 and WFG2, respectively). There is a lack of reports regarding the effect of the addition of FG to whey on LAB growth. However, the survival rate of LAB in fermented milk with the addition of mucilage and whey-based beverages containing ingredients of plant origin was examined [39,40]. According to the results described in the literature, LAB were characterized by high survival rates in whey and in whey-based beverages [39,41]. For instance, Sady et al. [41] found a high survival rate of probiotic bacteria in drinks produced from whey combined with orange, apple and blackcurrant juice. The results of their research, as well as those of other authors, confirm that the addition of plant-based ingredients may create favorable conditions for the growth of LAB [3,39]. Additionally, Madhavi and Shah [42] observed high survivability of Lactobacilli counts in symbiotic whey drinks during refrigerated storage for 28 days. Hadi Nezhad et al. [43] showed that soluble dietary fiber from flaxseed functions as a good prebiotic, promoting LAB growth in a kefir model. Basiri et al. [44] reported that flaxseed mucilage enhances the starter bacterial counts of stirred yogurt. It can be assumed that, also in our studies, the significantly higher survival of LAB in samples with FG during refrigerated storage (p < 0.05) compared to W and WMP variants resulted from the presence of FG presumably acting as a prebiotic.

3.2. Titratable Acidity and pH

The changes in titratable acidity (TA) and pH are listed in

Table 2. pH and titratable acidity of the samples during storage time.

Sample	Time of Storage (Days)
	1	7	14	21	28
pH
W	4.40 ± 0.01 Ab	4.39 ± 0.02 Aab	4.42 ± 0.03 Aa	4.43 ± 0.02 Aac	4.48 ± 0.03 Ba
WMP	4.51 ± 0.02 Aa	4.52 ± 0.02 Ad	4.52 ± 0.01 Ab	4.52 ± 0.01 Ab	4.55 ± 0.03 ABb
WFG1	4.32 ± 0.02 Ac	4.37 ± 0.02 Bbc	4.40 ± 0.02 Cac	4.41 ± 0.02 Cacd	4.44 ± 0.01 Cc
WFG2	4.26 ± 0.02 Ad	4.34 ± 0.02 Bc	4.38 ± 0.01 Cc	4.39 ± 0.01 Ccd	4.41 ± 0.07 Cc
TA (%)
W	0.86 ± 0.00 Aa	0.88 ± 0.03 ABa	0.89 ± 0.01 ABa	0.90 ± 0.01 Ba	0.82 ± 0.05 Ca
WMP	0.65 ± 0.01 Ab	0.64 ± 0.01 Ab	0.63 ± 0.01 Ab	0.60 ± 0.01 Bb	0.60 ± 0.01 Bb
WFG1	0.94 ± 0.01 Ac	0.95 ± 0.01 Abc	0.96 ± 0.01 Abc	0.97 ± 0.01 Ac	0.96 ± 0.04 Abc
WFG2	1.00 ± 0.01 ABd	0.99 ± 0.01 Ad	1.03 ± 0.01 BCd	1.04 ± 0.00 Cd	0.97± 0.02 Dd

W—fermented whey, WMP—fermented whey with milk powder added (5% w/v), WFG1—fermented whey with flaxseed gum added (0.5% w/v), WFG2—fermented whey with flaxseed gum added (1.0% w/v), TA—titratable acidity. Values are means ± standard deviation of triplicate determinations. Means with different lowercase letters in the same column are significantly different at p < 0.05. Means with different uppercase letters in the same row are significantly different at p < 0.05.

. The addition of FG significantly increased the content of lactic acid and the active acidity (pH) of the samples (p < 0.05). Generally, the WMP sample had the lowest titratable acidity during the storage period, which may be attributed to the buffering capacity of milk powder [45]. The pH of the W sample did not change until the last week of storage, when an increase in pH was observed (p < 0.05). This is partially consistent with the results of the study conducted by Terpou et al. [46] who did not find any significant differences concerning the acidity of whey beverages during 30 days of storage at 4 °C. A significant increase in pH (p < 0.05) was observed in the first half of the research period and a stabilization in the second half of the cycle for variants with the addition of FG (WFG1 and WFG2). An overall increase in pH was observed for these samples. The highest acidity during storage was observed for the WFG2 sample (p < 0.05). Generally, according to the information in the literature, the cooling storage of WFB was associated with an increase, decrease or stabilization of the acidity of the product, depending on the type of gum used and the additives. Khoshdouni Farahani et al. [47] and Tabibloghmany and Ehsandoost [48] noted that the presence of gums in the beverage medium can increase pH and decrease acidity. Additionally, Aamer and El-Kholy [49] observed a gradual change in titratable acidity during a monthly storage of WFB. In a study on whey-based mango herbal beverage, Alane et al. [50] found that the pH of the beverage decreased during the storage period; the lowest pH value was recorded on the last day of the study and was 4.15. The acidity of the beverages changed during storage and was found to increase from an initial value of 0.24% citric acid to a final value of 0.32% after 30 days. These results of pH and acidity are in line with those reported by other authors [51]. The pH of the WFB containing fruit juices during storage, according to Sady et al. [41], was in the range of 3.89–4.17, while the content of citric acid was 0.42–0.61%. The value of both parameters was stable during storage. The high acidity of WFB compared to values reported in the present study was due to the high acidity of the whey as well as the fruit juices as flavoring agents. In their work, Pescuma et al. [8] tested the pH of functional fermented whey-based beverages produced with lactic acid bacteria (L. delbrueckii subsp. bulgaricus, S. thermophilus and L. acidophilus) and reported on the stabilization, decrease or increase in the pH of beverages during storage (28 days) depending on the experimental variant used. The fluctuation of pH values could be connected to the buffering capacity of whey protein and dairy products. It is noted that this effect could be increased by milk products’ treatment, such as heating, high-pressure treatment or salt addition [52]. The same effect was observed in the present study because the samples’ pH was no lower than 4.26 ± 0.02 and no higher than 4.55 ± 0.03. On the other hand, flaxseed gum also consists of a protein fraction with a possible buffering effect [53].

3.3. Water Activity, Syneresis, Viscosity and Particle Sizes

The water activity, syneresis, viscosity and particle sizes of the samples are summarized in

Table 3. Water activity, syneresis, viscosity and particle sizes of the samples during storage time.

Sample	Time of Storage (Days)
	1	7	14	21	28
aw (-)
W	0.98 ± 0.01 Aa	0.98 ± 0.00 ABa	0.98 ± 0.00 BCa	0.97 ± 0.00 Ca	0.97 ± 0.01 CDa
WMP	0.97 ± 0.00 Ab	0.97 ± 0.01 Ab	0.97 ± 0.01 Aa	0.97 ± 0.03 Bb	0.96 ± 0.00 Bb
WFG1	0.96 ± 0.00 Ac	0.96 ± 0.01 ABc	0.95 ± 0.00 ABb	0.95 ± 0.02 BCc	0.95 ± 0.01 Cc
WFG2	0.96 ± 0.03 Ac	0.95 ± 0.00 BCd	0.95 ± 0.00 BCb	0.95 ± 0.02 Bc	0.95 ± 0.02 ACc
Syneresis (%)
W	94.83 ± 0.30 Aa	94.67 ± 0.30 Aa	95.47 ± 0.50 Aa	94.77 ± 0.40 Aa	94.30 ± 1.10 Aa
WMP	74.50 ± 0.50 Ab	74.50 ± 0.50 Ab	74.67 ± 2.10 Ab	72.67 ± 2.50 BCb	73.99 ± 1.60 ABb
WFG1	74.67 ± 0.60 Ab	74.70 ± 0.30 Ab	75.57 ± 1.30 Ab	74.83 ± 0.20 Ac	74.67 ± 0.70 Ab
WFG2	67.00 ± 1.00 Ac	65.67 ± 0.80 Ac	65.03 ± 0.30 Bb	64.00 ± 0.40 Cbc	61.82 ± 1.50 Dc
Viscosity (Pa·s)
W	125.31 ± 0.05 Aa	122.30 ± 0.03 Aa	89.21 ± 0.10 Ba	41.48 ± 0.03 Ca	33.52 ± 0.10 Da
WMP	604.30 ± 0.02 Ab	749.52 ± 0.02 Bb	425.10 ± 0.05 Cb	159.32 ± 0.02 Db	89.30 ± 0.12 Eb
WFG1	1038.22 ± 0.10 Ac	1012.32 ± 0.01 Bc	989.22 ± 0.02 Cd	975.13 ± 0.02 Dc	911.34 ± 0.05 Ec
WFG2	1800.21 ± 0.05 Ad	1740.90 ± 0.02 Bd	1534.00 ± 0.01 Ce	1522.23 ± 0.09 Dd	1501.24 ± 0.06 Ed
D4,3 (µm)
W	279.48 ± 1.14 Aa	130.52 ± 0.85 Ba	124.63 ± 0.36 Ca	132.04 ± 0.59 Da	133.22 ± 0.35 Ea
WMP	261. 44 ± 1.12 Ab	259.17 ± 0.48 Bb	262.24 ± 0.79 Cb	252.49 ± 0.10 Db	257.12 ± 0.77 Eb
WFG1	110.48 ± 0.46 Ac	115.18 ± 0.58 Bc	114.16 ± 0.41 Cc	115.97 ± 0.19 Dc	116.79 ± 0.39 Ec
WFG2	81.20 ± 0.36 Ad	70.76 ± 0.39 Bd	69.47 ± 0.35 Cd	68.86 ± 0.37 Dd	70.92 ± 0.36 Bd
D3,2 (µm)
W	38.86 ± 1.18 Aa	22.52 ± 0.92 Ba	21.48 ± 0.47 Ca	22.58 ± 0.68 Ba	21.00 ± 0.47 Da
WMP	79.04 ± 1.21 Ab	81.52 ± 0.58 Bb	79.63 ± 0.79 Ab	71.93 ± 0.11 Cb	75.81 ± 0.78 Db
WFG1	14.42 ± 0.66 Ac	17.25 ± 0.71 Bc	14.97 ± 0.61 Cc	16.32 ± 0.13 Dc	14.99 ± 0.61 Cc
WFG2	16.89 ± 0.53 Ad	14.85 ± 0.57 Bd	15.29 ± 0.53 Cd	14.52 ± 0.56 Bd	14.76 ± 0.56 Bc

W—fermented whey, WMP—fermented whey with milk powder added (5% w/v), WFG1—fermented whey with flaxseed gum added (0.5% w/v), WFG2—fermented whey with flaxseed gum added (1.0% w/v). Values are means ± standard deviation of triplicate determinations. Means with different lowercase letters in the same column are significantly different at p < 0.05. Means with different uppercase letters in the same row are significantly different at p < 0.05.

. Water activity (a_w), which is a thermodynamic measure of the chemical potential of water, affects the durability and quality of products, influencing the potential of microorganisms to develop [54]. Fermented whey (W) had the highest a_w during storage, whereas the lowest values were observed for the WFG2 sample. A significant decrease in the a_w of all samples during storage was found (p < 0.05), and the greatest decrease was observed for the W sample. The use of FG decreased the a_w of FWB, but the amount of FG did not significantly differentiate the WFG1 and WFG2 samples (p > 0.05). The lowest syneresis (serum release from the gel matrix) during refrigerated storage was observed for the sample containing 1% addition of FG (WFG2) and the highest for fermented whey (W) (p < 0.05). The addition of 0.5% FG limited the syneresis of the samples to a level similar to the addition of milk powder (MP). Skryplonek and Jasińska [55,56] reported that FWB are characterized by a significant whey leakage, amounting to one-third of the sample volume, which results in a low assessment of the appearance of this type of product. According to Dehghan et al. [57], the main problem in the production of FWB is precisely their colloidal instability caused by protein aggregation, mainly β-lactoglobulin. Changes in colloidal stability negatively affect the viscosity and appearance of the product. The results of studies by other authors also confirm the positive effect of FG on the release of whey from fermented beverages. Hashemi et al. [58] found that the addition of hydrocolloids positively influences the sensory characteristics of FWB. Zendeboodi et al. [59] found an increase in the apparent viscosity of the beverages, and thus the lowest release of whey, and recommended the use of gum in the industrial production of FWB. This is consistent with the finding that all adsorbent and non-adsorbent hydrocolloids can stabilize beverages and prevent phase separation by increasing the consistency of the continuous phase and chemically interacting with proteins [59]. The reduction in syneresis observed in our studies after adding FG to the samples is consistent with the results of Basiri et al. [44] who reported reduced syneresis of mixed yogurt supplemented with FG. The decreased hydrodynamic properties, as well as syneresis, can be linked to the tendency of FG biopolymers to associate due to the intermolecular associations via hydrogen bonding, leading to pseudogel behavior and the formation of complexes with proteins such as conlinin [60].

In dairy beverages, viscosity is an important characteristic for consumer acceptance, and it depends on the solids content, the type and concentration of the additives, and the fermentation conditions, such as time, temperature and the LAB strains used. The addition of some natural ingredients allows the design of the viscosity of the products [4]. AW has a low total solids content when compared to milk [2,13,21]. The addition of FG could fix this problem and may improve the texture parameters because biopolymer complexes formed by protein–polysaccharide interaction can serve as food texture modifiers in food products [61]. Cui and Mazza [62] as well as Chen et al. [63] found that under acidic conditions, the FG gel strength decreases as the pH value decreases, and under basic conditions, the gel strength decreases as the pH value increases. In our study, the addition of FG, despite the acidic environment, clearly improved the viscosity of the tested samples. As can be seen in Table 3, the viscosity of fermented whey significantly increased when MP was added (p < 0.05). Moreover, a significant increase in viscosity (8-fold and over 14-fold for samples WFG1 and WFG2, respectively) was noted (p < 0.05). However, a viscosity decrease was observed for all samples during cold storage, which can be attributed to proteins and carbohydrates hydrolysis. It should be pointed out that on day 28, the viscosity of the WFG2 sample (1501.24 ± 0.06 Pa·s) was approximately 45-fold higher than the viscosity of whey (33.52 ± 0.10 Pa·s) (p < 0.05). The largest particle size during storage was noted for the WMP sample, followed by the W sample. As can be seen in Table 3, the addition of FG caused a significant decrease in particle size (p < 0.05). The enhanced viscosity of the samples can be linked to FG’s “weak gel-like” properties and the ability to form networks [23,60]. Moreover, it was reported that FG plays the role of a thickening agent, which is able to increase the viscosity and decrease the particle size of emulsions [26]. Electrostatic interactions produce profound effects on biopolymer interactions between the charged species [60]. In the case of a protein–anionic polysaccharide biopolymer system, electrostatic interactions can be attractive or repulsive. When the pH of the solution is greater than the pI of the protein, repulsive interactions will dominate, since both biopolymers have negative charges. However, attractive electrostatic interactions occur when the pKa of the anionic polysaccharide is less than the pH of the solution, and the pH, in turn, is less than the pI of the protein [60]. As reported, whey protein’s isoelectric point is approximately at pH = 5.2, and below this value, it will be positively charged, whereas polysaccharide (FG) is negatively charged [27,64]. In an acidic environment, FG possesses a negative charge [27]. Thus, presumably, FG and whey proteins interacted and formed complexes, resulting in enhanced textural properties, syneresis and decreased hydrodynamic properties.

3.4. Changes in Acetaldehyde Content and Sensory Evaluation Results

The acetaldehyde content and sensory scores are presented in

Table 4. Changes in acetaldehyde content and sensory evaluation results.

Sample	Time of Storage (Days)
	1	7	14	21	28
Acetaldehyde (mg/dm3)
W	0.075 ± 0.00 Ac	0.393 ± 0.03 Bb	0.491 ± 0.06 Cd	0.475 ± 0.06 Cd	1.343 ± 0.04 Dc
WMP	0.088 ± 0.01 Ab	0.923 ± 0.03 Ba	0.984 ± 0.01 Bc	0.964 ± 0.03 Bc	1.558 ± 0.08 Cb
WFG1	0.441 ± 0.10 Aa	0.952 ± 0.03 Ba	1.389 ± 0.10 Ca	1.346 ± 0.04 Cb	1.645 ± 0.04 Da
WFG2	0.364 ± 0.01 Ab	0.449 ± 0.01 Bb	1.184 ± 0.03 Cb	1.613 ± 0.02 Da	1.638 ± 0.04 Da
Consistency (points)
W	2.20 ± 0.30 Aa	2.30 ± 0.30 Aa	2.20 ± 0.30 Aa	2.20 ± 0.30 Aa	2.00 ± 0.00 Ba
WMP	2.70 ± 0.30 Aa	3.20 ± 0.30 Bb	3.30 ± 0.30 Bb	3.20 ± 0.30 Bb	3.50 ± 0.50 BCb
WFG1	4.00 ± 0.60 Ab	4.20 ± 0.60 Abc	4.30 ± 0.30 BCc	4.30 ± 0.30 BCc	4.50 ± 0.00 Cc
WFG2	4.80 ± 0.30 ABc	4.80 ± 0.30 ABd	5.00 ± 0.00 Bd	5.00 ± 0.00 Bd	5.00 ± 0.00 Bc
Appearance (points)
W	3.20 ± 0.30 ABa	3.00 ± 0.00 Aa	3.20 ± 0.30 ABa	3.20 ± 0.30 ABa	3.50 ± 0.50 Ba
WMP	3.70 ± 0.30 Ab	3.80 ± 0.30 Ab	4.00 ± 0.00 Ab	4.00 ± 0.00 Ab	3.80 ± 0.30 Ab
WFG1	4.00 ± 0.50 Abc	4.20 ± 0.30 Bbc	4.2 ± 0.30 Bb	4.20 ± 0.30 Bb	4.30 ± 0.30 Bc
WFG2	4.30 ± 0.30 ABc	4.50 ± 0.00 BCc	4.70 ± 0.30 Cc	4.70 ± 0.30 Cc	4.70 ± 0.30 Cc
Taste (points)
W	3.80 ± 0.30 Aa	3.80 ± 0.30 Aa	3.80 ± 0.30 Aa	3.40 ± 0.50 Ba	3.40 ± 0.50 Ba
WMP	3.80 ± 0.30 Aa	3.80 ± 0.30 Aa	3.80 ± 0.30 Aa	4.20 ± 0.30 Bb	4.20 ±0.30 Bb
WFG1	3.80 ± 0.30 Aa	3.80 ± 0.30 Aa	3.80 ± 0.30 Aa	3.80 ± 0.30 Ac	3.80 ± 0.30 Ac
WFG2	3.20 ± 0.30 Ab	3.30 ± 0.30 Ab	3.20 ± 0.30 Ab	3.30 ± 0.30 Ad	3.30 ± 0.30 Ad
Smell (points)
W	4.80 ± 0.30 Aa	5.00 ± 0.00 Ba	5.00 ± 0.00 Ba	5.00 ± 0.00 Ba	5.00 ± 0.00 Ba
WMP	5.00 ± 0.00 Ab	5.00 ± 0.00 Aa	5.00 ± 0.00 Aa	5.00 ± 0.00 Aa	5.00 ± 0.00 Aa
WFG1	4.30 ± 0.30 Ac	4.20 ± 0.30 Ab	4.00 ± 0.00 Bb	3.80 ± 0.30 Cb	3.80 ± 0.30 Cb
WFG2	4.20 ± 0.30 Ac	4.20 ± 0.30 Ab	3.80 ± 0.30 Bc	3.70 ± 0.30 Bb	3.70 ± 0.30 Bb
Overall sensory quality (points)
W	3.50 ± 1.12 Aa	3.54 ± 1.15 Aa	3.54 ± 1.19 Aa	3.44 ± 1.17 Aa	3.48 ± 1.23 Aab
WMP	3.79 ± 0.96 Aa	3.96 ± 0.76 ABa	4.04 ± 0.70 Ba	4.08 ± 0.75 Ba	4.13 ± 0.64 Ba
WFG1	4.04 ± 0.21 Aa	4.08 ± 0.17 Aa	4.08 ± 0.22 Aa	4.04 ± 0.25 ABa	4.13 ± 0.34 Bb
WFG2	4.13 ± 0.70 Aa	4.21 ± 0.64 Aa	4.17 ± 0.83 Aa	4.17 ± 0.79 Aa	4.17 ± 0.79 Aab

W—fermented whey, WMP—fermented whey with milk powder added (5% w/v), WFG1—fermented whey with flaxseed gum added (0.5% w/v), WFG2—fermented whey with flaxseed gum added (1.0% w/v). Values are means ± standard deviation of triplicate determinations. Means with different lowercase letters in the same column are significantly different at p < 0.05. Means with different uppercase letters in the same row are significantly different at p < 0.05.

. As can be seen, the highest acetaldehyde content at the beginning and at the end of the storage was determined in the WFG1 variant (0.44 ± 0.10 mg/dm³ and 1.65 ± 0.04 mg/dm³, respectively), whereas the lowest content was noted for the fermented whey (W) (0.07 ± 0.00 mg/dm³ and 1.34 ± 0.04 mg/dm³, respectively). A significant increase (p < 0.05) in acetaldehyde content was observed during storage in all the FWB variants. The highest increase was found for the WMP (1.470 mg/dm³). The addition of FG generally resulted in an increase in acetaldehyde content. Acetaldehyde is the main compound shaping the aroma of fermented milk, synthesized from lactose or amino acid threonine [2]. As reported in the literature, the acetaldehyde content in fermented milk varies, and in yogurt, it can be as high as 10 ÷ 15 mg/dm³, and the threshold of its detectability by the human sense of smell is 0.415 mg/dm³ [65]. The values determined are comparable with those reported for sour milk, kefir, quark or crème fraiche but lower than those reported for ayran or yogurt [65].

From the perspective of the consumer, beverages based on by-products should be visually as homogeneous as dairy products [14,15]. Consumers pay attention to their sensory features and stability during storage. The most important sensory characteristics of fermented milk that determine their choice are texture, taste and aroma [66,67]. The highest overall sensory FWB rating was assigned to the WFG2 sample during storage, whereas the lowest was noted for the W sample (Table 4). The overall sensory scores of the WFG1 and WFG2 samples are comparable to those reported for whey drinks with fruit concentrates [68]. The WFG2 variant received the highest score for consistency (4.80–5.00) and appearance (4.30–4.70) but the lowest score for taste due to straw and grassy aftertaste (3.20–3.30). Chen et al. [63] reported that a creamy mouthfeel was noticed in whey-based beverages when xanthan gum was added. The lowest scores for appearance (3.20–3.50) and consistency (2.00–2.20) were given to fermented whey (W). The consistency of the beverage containing 0.5% FG (WFG1) was similar to that of the beverage produced with the addition of milk powder (WMP). The maximum score for smell was given to the W and WMP samples. The addition of FG lowered the score for smell, as the panelists emphasized the perceptible vegetable note, unusual for dairy products. Basiri et al. [44] reported that an increase in acidity and firmness of yogurt prevents the release of aromatic compounds. Similarly, Gallardo-Escamilla et al. [14] showed that the yogurt aroma of samples containing hydrocolloids (such as carboxymethyl cellulose and propylene glycol alginate) was perceived as less intense. Hydrocolloids can be used to increase the viscosity of whey-based dairy beverages, although they may mask the typical beverage flavor when using a yogurt culture [58]. Thus, it is reasonable to conclude that FG influenced the release of acetaldehyde, despite the highest content in samples with FG added, resulting in a lower score for the smell. Presumably, this can also be linked to the formation of FG–whey protein complexes. The highest rated taste was noted for the fermented whey containing the addition of milk powder (WMP). Hydrocolloids can be applied to enhance the viscosity of whey-based dairy beverages, although they may mask the typical beverage flavor when a yogurt culture is involved [58]. The effect of gum on the sensory characteristics of fermented whey beverages, based on the information available in the literature, may depend on the type of gum. Terpou et al. [46], studying the functional whey drinks containing Chios mastic gum, noted that all tasters could detect the taste and aroma of mastic gum in the whey drinks; however, they sensed no difference in smoothness compared to the control drinks. In fact, all whey drinks with Chios mastic gum were characterized as cooling beverages with a pleasant distinct aroma. The industry should focus on increasing the flavor acceptance of FWB with gums, as the flavor is directly related to the overall acceptance and should also develop the attributes that are linked to the most accepted samples [46].

3.5. Color Changes

The results of color determination are summarized in

Table 5. Color changes of the samples during storage time.

Sample	Time of Storage (Days)
	1	7	14	21	28
L*
W	48.82 ± 2.80 Aa	50.13 ± 0.80 Aa	49.92 ± 0.40 Aa	48.51 ± 0.40 Aa	49.78 ± 1.00 Aa
WMP	65.50 ± 0.40 Ab	66.67 ± 3.00 Ab	65.27 ± 0.50 Bb	61.85 ± 2.90 Cb	57.81 ± 1.50 Db
WFG1	69.42 ± 0.50 Ac	67.79 ± 1.60 Bb	67.40 ± 0.70 Ac	66.50 ± 6.10 Ac	60.20 ± 1.20 Cb
WFG2	70.72 ± 0.00 Acd	71.35 ± 2.20 Ac	69.50 ± 1.10 Ac	69.78 ± 1.20 Ac	70.52 ± 0.40 Ac
a*
W	2.92 ± 0.09 Aa	2.90 ± 0.11 Aa	3.07 ± 0.05 Aa	2.83 ± 0.06 Aa	2.89 ± 0.07 Aa
WMP	3.57 ± 0.34 Ab	3.83 ± 0.16 BCbc	3.54 ± 0.08 Ab	3.49 ± 0.44 Ab	3.67 ± 0.06 ABb
WFG1	4.06 ± 0.03 Abc	4.08 ± 0.04 Abc	4.07 ± 0.08 Abc	4.04 ± 0.00 Bc	4.28 ± 0.18 ACc
WFG2	4.19 ± 0.05 ABcd	4.17 ± 0.21ABcd	4.18 ± 0.10 ABc	4.36 ± 0.16 Bd	3.92 ± 0.04 Cb
b*
W	2.32 ± 0.11 Aa	2.50 ± 0.13Aa	2.38 ± 0.06 Aa	2.38 ± 0.05 Aa	3.00 ± 0.03 Ba
WMP	5.68 ± 0.36 ABb	5.72 ± 0.17ABb	5.24 ± 0.08 Cb	5.83 ± 0.10 Db	4.40 ± 0.11 Ab
WFG1	5.27 ± 0.15 Ac	5.10 ± 0.21Abc	5.76 ± 0.39 Cc	5.06 ± 0.07 Abc	4.53 ± 0.19 Dbc
WFG2	4.73 ± 0.11 ABd	5.50 ± 0.08Dd	4.86 ± 0.17 ACd	4.91 ± 0.03 ACc	4.74 ± 0.40 ABc
C*
W	3.73 ± 0.07 Aa	3.83 ± 0.06Aa	3.88 ± 0.05 ABa	3.70 ± 0.07 Aa	4.25 ± 0.03 Ba
WMP	6.71 ± 0.39 Ab	6.88 ± 0.18Ab	6.32 ± 0.03 Bb	6.79 ± 0.30 Ab	6.60 ± 0.12 Cb
WFG1	6.65 ± 0.13 Ab	6.53 ± 0.14Ac	7.05 ± 0.36 Bc	6.47 ± 0.05 Ac	6.73 ± 0.26 Cb
WFG2	6.32 ± 0.10 ABc	6.90 ± 0.19Cd	6.41 ± 0.08 ABb	6.57 ± 0.09 Ac	6.21 ± 0.33 Bc
h°
W	0.67 ± 0.03 ABa	0.71 ± 0.04Ca	0.66 ± 0.01 Aa	0.70 ± 0.01 BCa	0.79 ± 0.02 Da
WMP	1.01 ± 0.04 ABb	0.98 ± 0.02ACb	0.98 ± 0.02 ACb	1.03 ± 0.05 Bb	0.84 ± 0.01 Dc
WFG1	0.92 ± 0.01 Ac	0.90 ± 0.02Abc	0.96 ± 0.03 Cb	0.90 ± 0.01 ABc	0.83 ± 0.00 Db
WFG2	0.85 ± 0.01 Ad	0.92 ± 0.02Bc	0.86 ± 0.03 Ac	0.84 ± 0.02 Ad	0.70 ± 0.04 Cc
ΔE
W	used as standard	2.19 ± 0.76Aa	2.48 ± 0.65 Aa	2.35 ± 1.42 Ba	2.92 ± 0.65 Aa
WMP	16.59 ± 2.48 Aa	16.47 ± 3.15Ab	15.26 ± 0.62 Ab	13.23 ± 2.46 Ab	7.98 ± 0.47 Bb
WFG1	22.17 ± 0.62 Aa	17.19 ± 1.33Bb	17.41 ± 1.07 Bb	17.95 ± 5.71 Abc	10.44 ± 0.57 Cc
WFG2	23.49 ± 0.17 Aa	20.75 ± 0.85Bc	19.54 ± 1.41 Cc	21.27 ± 1.65 Bc	20.72 ± 1.15 Bd

W—fermented whey, WMP—fermented whey with milk powder added (5% w/v), WFG1—fermented whey with flaxseed gum added (0.5% w/v), WFG2—fermented whey with flaxseed gum added (1.0% w/v). Values are means ± standard deviation of triplicate determinations. Means with different lowercase letters in the same column are significantly different at p < 0.05. Means with different uppercase letters in the same row are significantly different at p < 0.05.

. As can be noted, the addition of FG had a significant effect on the color parameters, i.e., L*, a*, b*, while it did not significantly affect the color saturation (C) and the hue (h). The addition of FG as well as MP caused a significant increase in the L* parameter (brightness) (p < 0.05). The cooling storage of FWB resulted in a decrease in brightness for WMP and WFG1 and no significant changes in this parameter for the fermented whey (W) and the sample containing 1% flaxseed gum (WFG2). The redness/greenness parameter (a*) of the W sample did not change during storage (p > 0.05). In the case of the WMP variant, fluctuations in the a* parameter during storage were generally statistically insignificant (p < 0.05). When analyzing the color of the WFG1 and WFG2 samples, an initial stabilization of this parameter was observed until the third week and second week of storage, respectively, and a subsequent increase in the value of a*, respectively, by 0.49 and 0.56 units. The W and WMP samples differed significantly in the a* parameter, and the addition of FG increased its value in the WFG1 and WFG2 samples (p < 0.05). The b* parameter (yellowness/blueness) during cold storage of FWB ranged from 2.32 ± 0.11 (sample W on day 1) to 5.76 ± 0.39 (sample WFG2 on day 14). The highest value of parameter b* was noted for the WMP sample and the lowest for sample W. Yellow-greenish coloration is characteristic for whey due to the presence of riboflavin [1,2,13]. All the samples differed significantly in the b* parameter until the second week of storage; in the following two, there was no significant difference in the b* parameter between the samples containing the addition of FG (WFG1 and WFG2). The addition of FG caused a significant color change toward yellow (p < 0.05). The increase in the L* parameter in samples with FG can be linked to lower particle size, thus higher light scattering [22,26]. On the first day, the color differences (ΔE) between the fermented whey (W) and the other variants of FWB, i.e., WMP, WFG1 and WFG2, were 16.59 ± 2.48, 22.17 ± 0.62 and 23.49 ± 0.17, respectively. The ΔE for the samples containing the addition of FG (WFG1 and WFG2) and WMP was higher than 1 (which is considered as perceptible for the human eye), indicating a clear color difference between the samples [28]. The highest color saturation (C) on the first day was found in the WMP sample (6.71 ± 0.39), whereas the lowest was noted for the W sample (3.73 ± 0.07). Significant fluctuations of the C value during storage and an overall decrease for the WMP and WFG2 samples, at the same level (by 0.11), were found. The color saturation in the W sample did not change significantly (p > 0.05) during the 3 weeks of storage, but on day 28, a significant increase was observed (0.52) (p < 0.05) when compared to the initial value. The overall increase in color saturation (by 0.08) was also found in the WFG1 sample (p < 0.05). The refrigerated storage of the experimental samples was associated with an increase in the h parameter for the W sample (by 0.12) and a decrease for the WMP, WFG1 and WFG2 samples by 0.17, 0.09 and 0.15, respectively.

The phenomena observed in our research are confirmed in the available literature. Keshtkaran et al. [69] found that gum concentration correlated with color parameters. Similarly, Behbahani and Abbasi [70] claimed that the yellowness–blueness index (b*) of the drink samples stabilized with Iranian native hydrocolloids (Persian gum and tragacanth gum) was significantly higher than that of the sample without hydrocolloids. This may be due to the color characteristics of hydrocolloids, their concentrations and possible interactions with salts and sugars [28].

3.6. Reducing Sugars Content, Total Free Amino Acids Level and Free Radicals Scavenging Activity

The changes of reducing sugars content (RSC), total free amino acids level (TFAAL) and free radicals scavenging activity during storage are presented in

Table 6. Free radicals scavenging activity, reducing sugars content (RSC) and total free amino acids level (TFAAL) of the samples during storage time.

Sample	Time of Storage (Days)
	0	1	7	14	21	28
DPPH (%)
W	96.17 ± 0.11 ABab	96.22 ± 0.11 Ba	95.99 ± 0.35 Aa	95.67 ± 0.11 Ca	95.71 ± 0.10 Ca	95.53 ± 0.21 Ca
WMP	95.90 ± 0.17 ABEa	95.86 ± 0.05 ACEb	96.25 ± 0.23 Ca	96.10 ± 0.14 Bb	95.63 ± 0.26 Da	95.81 ± 0.11 DEab
WFG1	96.32 ± 0.08 Ab	96.29 ± 0.04 ACa	96.01 ± 0.14 Ba	96.10 ± 0.16A BCb	96.31 ± 0.06 ACb	95.59 ± 0.17 Da
WFG2	96.16 ± 0.12 ABab	96.91 ± 0.19 Cc	95.54 ± 0.54 Db	96.71 ± 0.07 Cc	96.32 ± 0.15 Ab	95.95 ± 0.17 Bb
ABTS (%)
W	69.84 ± 1.70 Aa	62.78 ± 2.10 BCa	64.34 ± 2.30 CDa	61.72 ± 1.20 Ba	67.02 ± 1.90 Ea	65.40 ± 2.20 DEa
WMP	73.02 ± 1.80 Aabc	71.61 ± 1.40 Ab	72.95 ± 1.10 ABb	72.39 ± 3.90 Ab	72.67 ± 2.30 ABb	74.79 ± 2.30 Bb
WFG1	76.77 ± 2.30 Ab	75.56 ± 1.20 ABb	72.39 ± 0.10 Db	70.06 ± 1.40 Cb	72.25 ± 2.50 CDb	73.94 ± 2.10 BDbc
WFG2	77.54 ± 0.40 Ac	72.46 ± 2.20 Bb	75.07 ± 1.50 Cb	71.89 ± 2.40 CDb	73.52 ± 3.50 BCb	72.46 ± 1.20 Cbc
RSC (mg/mL)
W	141.46 ± 1.52 Aa	133.46 ± 2.50 Ba	150.35 ± 3.32 Ca	162.15 ± 3.05 Da	133.27 ± 1.03 Ba	106.38 ± 2.18 Ea
WMP	188.73 ± 3.86 Ab	203.54 ± 2.39 Bb	222.85 ± 5.87 Cb	253.23 ± 5.66 Db	244.85 ± 2.72 Db	217.46 ± 8.81 Bb
WFG1	248.32 ± 1.23 Ac	232.69 ± 12.29 Bc	224.45 ± 2.21 Cc	222.96 ± 0.82 Cb	234.35 ± 3.97 Bc	216.08 ± 4.79 Dc
WFG2	271.04 ± 5.60 Ad	247.69 ± 5.33 Bd	274.81 ± 4.51 Ad	224.31 ± 0.65 Cc	247.23 ± 2.39 Bc	211.19 ± 2.23 Dd
TFAAL (mg/mL)
W	1.51 ± 0.12ABa	1.60 ± 0.18ABa	1.72 ± 0.11Aa	1.63 ± 0.15 Aa	1.32 ± 0.14 Ba	0.92 ± 0.00 Ca
WMP	2.22 ± 0.15Ab	2.48 ± 0.27ABb	2.66 ± 0.19Bb	2.68 ± 0.23 Bb	2.23 ± 0.20 Ab	2.31 ± 0.19 Ab
WFG1	2.59 ± 0.22ABbc	2.31 ± 0.24ACb	2.82 ± 0.27BDb	3.10 ± 0.30 Db	2.21 ± 0.18 Cb	3.83 ± 0.80 Ec
WFG2	3.03 ± 0.31Ac	3.26 ± 0.46Ac	3.92 ± 0.32Bc	1.77 ± 0.11 Ca	1.81 ± 0.18 Cab	1.59 ± 0.15 Cd

W—fermented whey, WMP—fermented whey with milk powder added (5% w/v), WFG1—fermented whey with flaxseed gum added (0.5% w/v), WFG2—fermented whey with flaxseed gum added (1.0% w/v). Values are means ± standard deviation of triplicate determinations. Means with different lowercase letters in the same column are significantly different at p < 0.05. Means with different uppercase letters in the same row are significantly different at p < 0.05.

. As can be seen, the addition of MP caused a significant increase in RSC and TFAAL due to the presence of lactose and proteins, respectively. Interestingly, the addition of FG also caused a significant increase in TFAAL, as well as RSC (p < 0.05). Some fluctuations were observed during storage; however, on day 28, the highest TFAAL was noted for the WFG1 sample (3.83 ± 0.80 mg/mL) and was over four-fold higher than that observed for the W sample (0.92 ± 0.00 mg/mL), whereas the RSC of the WFG1 and WFG2 samples was comparable to that of the sample with MP addition. However, a significant decrease in TFAAL was observed from day 14 in the WFG2 sample, which can presumably be linked to the consumption of the released amino acids by LAB [8]. The results for the RSC are comparable to those reported by Alane et al. [50]. Although there is no clear evidence, this result was presumably linked to the prebiotic effect of FG, enhancing microbial activity, causing hydrolysis of polysaccharide (FG) as well as proteins [3,13]. Whey protein has a high biological value, mainly due to its high content of branched-chain essential amino acids (isoleucine, leucine and valine) [1,13,21]. Moreover, the generation of high TFAAL can also be linked to the production of bioactive peptides as intermediates [4]. The decrease in TFAAL in the W variant could be an effect of the lower content of nutrients for the bacteria. According to Neis et al., amino acids could also be used as an energy source for fermentation [71]. On the other hand, by introducing WMP, which is a source of protein, into the system, as well as FG, which also contains a protein fraction, it is thus possible to potentially increase the amino acids level [43]. However, some reports indicated that FG has a prebiotic potential; thus, presumably, it could have a stimulating effect on the growth of LAB as well as on their metabolic activity, including both amino acid production and proteolytic activity. However, this requires further in-depth analysis. The presence of bioactive peptides in beverages contributes not only to their physicochemical stability but also to health-promoting properties, i.e., cardiovascular, antihypertensive, antithrombotic, antioxidant and antimicrobial [4,8,13]. Generally, the addition of FG caused an increase in antioxidant activity (p < 0.05), which was more clearly seen in the case of the ABTS radical. This observation is consistent with the findings of Kadyan et al. [3]. Ansari et al. [72] also reported an increased antioxidant activity of whey drink fermented with L. acidophilus LA5 [72]. Whey proteins show an antioxidant potential by chelating transition metal ions through lactoferrin or scavenging free radicals through sulfhydryl-containing amino acids. Fermentation by starter cultures increases the release of reductants, such as cysteine, present in the peptide chains of whey protein, thus neutralizing the free radicals present in the system [3,73]. The enhanced antioxidant activity in FG samples is presumably multifactorial and can be attributed to the metabolic antioxidant capacity of the bacteria (production of antioxidant metabolites, such as glutathione, butyrate, etc., or chelation of metal ions) as well as the production of amino acids [3,13,73].

4. Conclusions

The findings of the study indicated that acid whey can be successfully supplemented with flaxseed gum in a novel functional dairy beverage. With the aim to evaluate the properties of the novel beverage, several parameters, such as microbial survivability, physicochemical properties, color changes, viscosity, sensory profile and antioxidant potential, were analyzed. The addition of flaxseed gum as a natural stabilizer and thickening agent improved the viscosity of acid whey to a level comparable with milk powder, meeting the consumer acceptance criteria. Moreover, the added value of using FG as a whey additive was the high antioxidant activity and free amino acids level of the products. It should be emphasized that the consumption of the drinks may exert a potentially beneficial action on human organisms due to the highly beneficial microflora (lactic acid bacteria) viability, antiradical activity and amino acids level. However, further tests should be carried out focusing on proteolytic changes and the production of bioactive peptides, amino acids, as well as an in vivo analysis of potential health benefits for the functioning of the human body. Research should also focus on increasing the taste acceptance of FWB with gums, as taste is directly related to the overall acceptance of beverages to consumers. The use of valuable by-products from the dairy and oil industries opens up a promising direction for the production of innovative food products, which is in line with the principles of circular economy and the idea of zero waste.