*2.6. Water Activity Tests (aw) and Moisture Content (MC)*

While the statistical analysis of the water activity tests showed significant differences between samples (*p* < 0.001), the data indicate narrow values between 0.3562 and 0.3915. However, our results are below the critical values where microbiological spoilage can occur. Majumdar et al. (2018) summarized in a review that a minimum water activity value of 0.6 is required to initiate the growth of microorganisms [36]. These data are also consistent with more in-depth research by Beuchat (1983), which concluded that below 0.61 a<sup>w</sup> there can be no microbial growth, between 0.61 and 0.85 a<sup>w</sup> food spoilage starts with mold and yeast formation, and above 0.85 a<sup>w</sup> bacteria start to grow [37].

Another important parameter in food packaging applications is that the moisture content varied between 10.19 and 14.39%. Moisture content of films is closely related to the total amount of water molecules in the network microstructure of the composite films [38]. Abdalrazeq et al. (2019) stated that a high MC considerably limits the use of coatings for packaging materials and found the highest moisture value of 33.27% in film samples with 50% of glycerin prepared at pH 7 [39].

### *2.7. Color and Opacity*

Consumer acceptance is influenced by two parameters of the film appearance: Color and opacity. The opacity and L, a\*, b\* values presented in Table 2 were significantly different. The two main factors that varied in the film-forming solution, β-glucan and bilberry juice, have been evaluated in Table 5 for their influence on the opacity, brightness characteristics of L value (between 0 and 100), redness/greenness (a\* value), and yellowness/blueness (b\* value).

**Table 5.** The influence of the β-glucan and bilberry juice on opacity and color of the films.



**Table 5.** *Cont.* b\* 1.36 (0.24) <sup>b</sup> 1.61 (0.19) <sup>a</sup> 6.48 \*\*

ns: Not significant; \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001. a,b Different letters in the same rows indicate significant differences. ns: Not significant; \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001. a,b Different letters in the same rows indicate significant differences.

The results in Table 5 showed that a high amount of β-glucan is reflected by an increased brightness, a tendency to blueness, and an opaquer film, while the a\* parameter does not show a significant variation (*p* > 0.05). Regarding bilberry juice, a significant variation (*p* < 0.001) can be observed between the addition of 10 and 20 g to the red color of the samples (a\* value). L and b\* parameters for bilberry juice are not statistically significant (*p* > 0.05), while the opacity increased significantly (*p* < 0.05). The results in Table 5 showed that a high amount of β-glucan is reflected by an increased brightness, a tendency to blueness, and an opaquer film, while the a\* parameter does not show a significant variation (*p* > 0.05). Regarding bilberry juice, a significant variation (*p* < 0.001) can be observed between the addition of 10 and 20 g to the red color of the samples (a\* value). L and b\* parameters for bilberry juice are not statistically significant (*p* > 0.05), while the opacity increased significantly (*p* < 0.05).

### *2.8. Scanning Electron Microscopy (SEM) 2.8. Scanning Electron Microscopy (SEM)*

Figure 1 shows the microstructure of the β-glucan/bilberry juice films in the cross section at 1 kx to analyze the differences between surfactant and non-surfactant samples and to observe potential microcracks. As can be seen, on the transverse section, the samples with 2% surfactant (Figure 1B,D,G) showed a more compact surface, while the remaining samples present porous surfaces with micropores. The presence of pores makes the film less efficient in the water vapor barrier performance. Figure 1 shows the microstructure of the β-glucan/bilberry juice films in the cross section at 1 kx to analyze the differences between surfactant and non-surfactant samples and to observe potential microcracks. As can be seen, on the transverse section, the samples with 2% surfactant (Figure 1B,D,G) showed a more compact surface, while the remaining samples present porous surfaces with micropores. The presence of pores makes the film less efficient in the water vapor barrier performance.

**Figure 1.** *Cont*.

**Figure 1.** β-glucan/bilberry juice images and SEM micrographs of the film samples. (**A**–**G**) indicate the film samples from 1–7. **Figure 1.** β-glucan/bilberry juice images and SEM micrographs of the film samples. (**A**–**G**) indicate the film samples from 1–7.

#### *2.9. FT-IR Spectroscopy 2.9. FT-IR Spectroscopy*

The ATR-FTIR spectra (Figure 2A) of the most abundant group of polyphenols from bilberry juice (rich in anthocyanins) are identified by peaks found near the wavenumbers of ∼1716.01 cm−1 (C=O stretching for aromatic nucleus) [40], ∼1652.15 cm−1 characteristic for benzene skeleton vibration in anthocyanins, while the 3317.61 and 2924.44 cm−1 are assigned to O–H stretching vibration of water and CH, CH2, and CH<sup>3</sup> groups, respectively [41], and ∼1417.07 cm−1 corresponds to the C–H deformation [42]. Figure 2B showed that the incorporation of bilberry juice in films preserves characteristic peaks in the fingerprint region of the BJ, which indicates that the heat treatment of 15 min from the addition of bilberry juice to obtain the film-forming solution did not affect the bioactive compounds in the obtained β-glucan/bilberry film. Moreover, peaks near the wavenumbers of ∼1149.20, 1024.65, and 920.90 cm−1 are characteristic for yeast β-glycosidic configuration, sodium alginate (carboxyl stretching bands), and glycerin, respectively [43–45]. The ATR-FTIR spectra (Figure 2A) of the most abundant group of polyphenols from bilberry juice (rich in anthocyanins) are identified by peaks found near the wavenumbers of <sup>∼</sup>1716.01 cm−<sup>1</sup> (C=O stretching for aromatic nucleus) [40], <sup>∼</sup>1652.15 cm−<sup>1</sup> characteristic for benzene skeleton vibration in anthocyanins, while the 3317.61 and 2924.44 cm−<sup>1</sup> are assigned to O–H stretching vibration of water and CH, CH2, and CH<sup>3</sup> groups, respectively [41], and <sup>∼</sup>1417.07 cm−<sup>1</sup> corresponds to the C–H deformation [42]. Figure 2B showed that the incorporation of bilberry juice in films preserves characteristic peaks in the fingerprint region of the BJ, which indicates that the heat treatment of 15 min from the addition of bilberry juice to obtain the film-forming solution did not affect the bioactive compounds in the obtained β-glucan/bilberry film. Moreover, peaks near the wavenumbers of ∼1149.20, 1024.65, and 920.90 cm−<sup>1</sup> are characteristic for yeast β-glycosidic configuration, sodium alginate (carboxyl stretching bands), and glycerin, respectively [43–45].

(**A**) **Figure 2.** *Cont*.

**Figure 2.** ATR−FTIR spectra: (**A**) Individual FT−IR spectra for pure samples of BG, BJ, GLY, SA; (**B**) FT−IR spectra of the <sup>β</sup>-glucan/bilberry juice film samples (4000−650 cm−<sup>1</sup> ).

### **3. Discussion**

In this study, fast dissolving films from β-glucans and bilberry juice were successfully prepared. In addition to the role of plasticizer and sodium alginate, which confers vital primary film structure characteristics for a rapid dissolution, the addition of surfactant significantly reduces the dissolution time by improving the solubility of the β-glucans/bilberry juice films. In the case of fast dissolving films, this becomes very important, particularly when dispersing compounds, such as β-glucans or bioactive compounds from bilberry juice with antidiabetic properties, such as anthocyanins that attenuate the glycemic response. Our experimental data showed that the dissolution time of the films was halved by adding 2% surfactant. The film with the best dissolution properties compared with an increased content of 20 g of bilberry juice and 1.5 g of β-glucan is represented by Sample 7, which dissolves in 50.33 ± 1.52 s.

Moreover, we can conclude that in all of the samples, the water vapor barrier properties are remarkably low. Furthermore, the low water absorption rate (WVTR) between 3.2562 and 7.1111 g × h <sup>−</sup><sup>1</sup> <sup>×</sup> <sup>m</sup>−<sup>2</sup> makes <sup>β</sup>-glucans/bilberry juice films suitable for packaging dry powdered pharmaceuticals. Ultimately, of course, these values are up to 32 times higher than a plastic film (e.g., LDPE). However, for a fast dissolving bioactive film that is intended for edible packaging, the values are outstanding. Water activity tests and moisture content proved the film stability by values below 0.6 a<sup>w</sup> and MC up to 14.39%, which will not affect the quality of the packaged products. Since the film-forming solution contains a high amount of β-glucan and bilberry juice (rich in anthocyanidins), a considerably more opaque and reddish film will form after drying. Images of β-glucan/bilberry juice films and laboratory tests had detected the influence of these changes on different levels of significance, while SEM micrographs identified porous and compact structures on the cross section depending on the analyzed samples. FT-IR spectra revealed all of the compounds in film samples. Therefore, no degradation was found for β-glucans and bilberry juice.

The bioactive films developed in the present work could be used for packaging materials along with the bioactive delivering system of releasing compounds in aqueous solutions, which is necessary for people with a special medical condition, such as diabetes. Dispersion of the packaged product will be carried out simultaneously with the dissolution of bioactive film.

### **4. Materials and Methods**

### *4.1. Chemicals*

Bilberry juice (*Vaccinium myrtillus* L.) was purchased from a local market (distributed by SC. Deco Italia SRL, Suceagu, Cluj, Romania). According to the manufacturer, it is a 100% natural juice from bilberry fruit. The determined dry weight of the clear juice was 11.5% (*w*/*w*) with a pH value of 3.3. β-glucan was extracted from spent brewer's yeast provided by the SC. Bermas SA. brewery (Suceava, Romania). Other film components were: Sodium alginate, Product No. 9180.1 (Carl Roth, Karlsruhe, Germany), Glycerin, Product No. G7893 (Sigma-Aldrich, ACS reagent ≥ 99.5%, St. Louis, MO, USA), and Soybean oil (oil of genetically unmodified soybeans, Dachim SRL, Cluj, Romania).

### 4.1.1. β-Glucan Isolation

β-glucan, especially from spent brewer's yeast, is known to have particular potential in the inducement of innate immune response due to the triple helix structure of the insoluble polysaccharide conformation [46]. The most reliable method used for yeast β-glucan isolation is the alkaline-acid process [47]. Briefly, yeast slurry was purified and debittered at 50 ◦C with NaOH 2 N (up to pH 10) for 10 min according to [48]. Yeast cells were autolyzed at 55 ◦C/24 h and then were subjected to an alkaline extraction with NaOH 1.5 N at 90 ◦C/2 h in a ratio of 1.5 (*w*/*v*) according to [49], followed by an acid treatment with HCl solution for 2 h at 75 ◦C. The wet extract was washed three times and labeled as yeast β-glucan since it contains this polysaccharide as the principal component [19]. The FT-IR spectra showed characteristic bands for β-1,3 configuration at the wavenumbers of 1153.43 and 1104.87 cm−<sup>1</sup> , while the β-1,6 glucan specific for yeast glucan was found at 889.33 cm−<sup>1</sup> [43,50].

### 4.1.2. Film Preparation and Casting

The film-forming solution was prepared from the isolated β-glucan in different proportions and the addition of sodium alginate into each beaker. Therefore, bilberry juice with a dry weight of 11.5% (*w*/*w*) has been measured and prepared for each sample. Glycerin was added as plasticizer as 25% related to the dry weight of the solids of β-glucan, sodium alginate, and bilberry juice. Two percent (*w*/*w*) soybean oil of the total solid weight was added as a surfactant to samples 2, 4, and 7 in order to observe whether there are significant changes between the physical chemical parameters analyzed. Distilled water was added up to a total volume of 150 mL. The mixture was subjected to heating at 80 ◦C under continuous stirring (900 rpm) for 15 min. After 15 min of stirring in the homogeneous solution, the measured bilberry juice was added in each sample and stirred continuously for another 15 min for incorporation and for slight sterilization (30 min in total).

β-glucan/bilberry juice films were prepared by the casting technique as reported by [51]. Accordingly, equal suspensions from the film-forming solution were poured onto plastic petri dishes and dried at 40 ◦C for 48 h. The dried films were stored at room temperature prior to the analysis.

### *4.2. Methods*

### 4.2.1. Determination of Thickness

The film thickness was determined with the thickness gauge PosiTector 6000 (DeFelsko, Ogdensburg, NY, USA) with an accuracy of 0.1 µm. Measurements were taken at 10 different points, and the average was used to calculate the film properties. Thickness of the films was expressed in µm.

### 4.2.2. Determination of Water Vapor Transmission Rate (WVTR)

Water vapor transmission rates were measured using the described standard ASTM E96/96M method [52]. The dry cup method involved sealing films horizontally on a petri dish containing about 10 g CaCl<sup>2</sup> as desiccant to create 0% RH inside the cups. Samples with desiccant were placed in an environmental chamber with a NaCl solution, which provides 75% RH. The WVTR of the films was calculated by dividing the slope to the area of exposed film using the following equation:

$$\text{WVTR} = \frac{\Delta \mathcal{W}}{\Delta t \times A} \left( \text{g} \times \text{h}^{-1} \times \text{m}^{-2} \right) \tag{1}$$

where ∆*W*/∆*t* is the amount of water gained in the unit of time (g/h) and *A* is the area exposed to the water vapor diffusion (m<sup>2</sup> ).

### 4.2.3. Determination of Water Vapor Permeability (WVP)

The permeation characteristic of the β-glucan/bilberry juice films was investigated by dividing the *WVTR* values to the water vapor partial pressure across the film and multiplying by the film thickness (in mm) as described by [53]. The *WVP* was expressed by the following equation:

$$WVP = \frac{WVT \times L}{\Delta p} \left(\text{g} \times \text{mm} \times \text{kPa}^{-1} \times \text{h}^{-1} \times \text{m}^{-2}\right) \tag{2}$$

where *WVTR* is the water vapor transmission rate (g × h <sup>−</sup><sup>1</sup> <sup>×</sup> <sup>m</sup>−<sup>2</sup> ), *L* is the thickness of the film (mm), and ∆*p* is the water vapor partial pressure across the film (kPa) calculated according to the formula:

$$
\Delta p = S \times (R\_1 - R\_2) \text{ (kPa)}\tag{3}
$$

where *S* is the saturated vapor pressure of water (3.1687 kPa at 25 ◦C [54]) and the moisture gradients *R*<sup>1</sup> and *R*<sup>2</sup> are 0.75 and 0, respectively.

### 4.2.4. Dissolution Time

The film samples were cut into squares of 2 × 2 cm, immersed in 50 mL of distilled water, and then vigorously shaken until dissolution. The dissolution time (s) was recorded using a chronometer.

### 4.2.5. Determination of Color and Opacity

Film opacity was determined by measuring the absorbance at 600 nm and dividing by the film thickness [55]. The absorbance was acquired in UV-VIS-NIR Shimadzu 3600 spectrophotometer (Tokyo, Japan). The following equation for *opacity* is shown below:

$$Opacity = Abs\_{600nm}/L\tag{4}$$

where *Abs*600*nm* is the absorbance (600 nm) and *L* is the film thickness (mm).

The color was quantified according to the CIELab color space (lightness (L), redness (a\*), and yellowness (b\*)) using a portable chromameter CR-400 (Konica Minolta, Tokyo, Japan).

### 4.2.6. Water Activity Tests and Moisture Content

The water activity was measured with a water activity analyzer AquaLab 4TE (Meter Group, Inc., Pullman, WA, USA). With the use of chilled-mirror dew point technology, the instrument was able to determine a<sup>w</sup> values with a resolution of 10−<sup>4</sup> between 0.03 and 1.

Moisture content was determined gravimetrically according to [56], samples were weighed before and after drying at 105 ◦C for 24 h, and the difference in weight loss was expressed as the moisture content in films, according to the following equation:

$$\text{MC} = \frac{W\_0 - W\_1}{W\_0} \times 100 \,\text{(\%)}\tag{5}$$

where *W*<sup>0</sup> is the initial weight of the sample and *W*<sup>1</sup> is the final weight of the dried film.

### 4.2.7. FT-IR Spectroscopy

FT-IR spectra with attenuated total reflectance unit (ATR) analysis were achieved using Nicolet iS-20 FT-IR spectrometer (Thermo Scientific™, Karlsruhe, Dieselstraße, Germany). Measurements were conducted by placing samples directly on the ZnSe crystal plate. The spectra were collected in the region of 4000–650 cm−<sup>1</sup> by 32 scans per spectrum at a resolution of 4 cm−<sup>1</sup> .

### 4.2.8. Scanning Electron Microscopy (SEM)

The cross-section morphology of the films was observed using a scanning electron microscope VEGA II LMU (Tescan, Brno, Czech Republic) under HighVac conditions using a secondary electron (SE) detector operated at an accelerating voltage of 30 kV and a magnification of 1 kx without preliminary coating on the investigated surface.

### 4.2.9. Dry Weight Determination (*w*/*w*)

Dry weight was determined by weighing about 10 g of juice, solids and dry in an oven at 105 ◦C to constant weight. Samples were transferred into a desiccator to prevent moisture uptake. The measurements for dry weight were made according to the following equation:

$$DW = \frac{w\_2 - w\_3}{w\_2 - w\_1} \times 100 \ (\% \, w/w) \tag{6}$$

where *w*<sup>1</sup> is the weight of crucible; *w*<sup>2</sup> is the initial weight of crucible with sample, g; and *w*<sup>3</sup> is the final weight of crucible with sample after drying, g.

### 4.2.10. Statistical Analysis of the Results

A one-way ANOVA test was used to determine whether there was a statistically significant difference between the means of the independent groups. Table 2 summarized the mean values and standard deviation of the physicochemical test results on a 95% confidence level. All of the tests, except for thickness, which require a mean of minimum 10 data points were expressed as the average and standard deviation of triplicates.

### **5. Conclusions**

This study demonstrated good compatibility between yeast β-glucans and bilberry juice in the development of fast dissolving films. The addition of bilberry juice in films is of particular importance in the management of metabolic disorders, such as diabetes. Considering the results, films based on β-glucans/bilberry juice with improved fast dissolving time and good water vapor barrier properties can be a potential novel film for packaging dry powdered pharmaceuticals.

**Author Contributions:** Conceptualization, S.A. and I.A.; methodology, I.A. and S.A.; software, I.A.; validation, S.A. and I.A.; formal analysis, I.A.; investigation, I.A.; resources, S.A.; writing—original draft preparation, I.A.; writing—review and editing, I.A.; visualization, I.A.; supervision, S.A.; project administration, S.A.; funding acquisition, S.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by the Ministry of Research, Innovation and Digitalization within Program 1—Development of national research and development system, Subprogram 1.2—Institutional Performance–RDI excellence funding projects, under contract no. 10PFE/2021.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

