Probiotic yogurt supplemented with nanopowdered eggshell: Shelf‐life stability, physicochemical, and sensory characteristics

Abstract The objectives of this study were to produce probiotic yogurt (5.0–7.0 log cfu/g) fortified with nanopowdered eggshell (NPES) at a rate of 0.02, 0.04, and 0.06 mg/ml, as well as, examine the effect of NPES on the physicochemical, microbial, sensory properties, and shelf‐life of probiotic yogurt. The NPES was prepared by milling preboiled dried eggshell using a mortar grinder. The size of the milled powder was measured to assure that the diameter of the powder is 27 ± 1.7 nm. Yogurt was manufactured by dividing the pasteurized milk into four aliquots portions. The first portion was utilized as control (T1), while the other three portions were supplemented with 0.02 (T2), 0.04 (T3), and 0.06 (T4) mg/ml NPES. All treatments were inoculated with 5.11 log cfu of Lactobacillus delbruckii ssp. bulgaricus (Lb) and Streptococcus thermophilus (St) combined and 5 log cfu of Bifidobacterium bifidum (Bb) per kg of milk at 40°C until the pH of 4.6 was reached. The acidity, sensory properties, Bb count, total bacterial count (TBC), yeast, and mold counts were examined. The results showed that the acidity was increasing during storage, however, increasing NPES resulted in low acid development (p < .05). The shelf‐life of control was ended after 8 d of storage at 4°C because molds were grown on the surface of the sample. The TBC significantly decreased (p < .05) as the concentration of NPES increased. Bb count in probiotic yogurt was also decreasing during storage. Yeast and molds were detected in control after 8 d; however, NPES did not result in molds even after 16 d of storage but yeast was exhibited. The NPES improved the sensory evaluation of probiotic yogurt slightly and increased the shelf‐life of probiotic yogurt as compared to control.

dried eggshell using a mortar grinder. The size of the milled powder was measured to assure that the diameter of the powder is 27 ± 1.7 nm. Yogurt was manufactured by dividing the pasteurized milk into four aliquots portions. The first portion was utilized as control (T1), while the other three portions were supplemented with 0.02 (T2), 0.04 (T3), and 0.06 (T4) mg/ml NPES. All treatments were inoculated with 5.11 log cfu of Lactobacillus delbruckii ssp. bulgaricus (Lb) and Streptococcus thermophilus (St) combined and 5 log cfu of Bifidobacterium bifidum (Bb) per kg of milk at 40°C until the pH of 4.6 was reached. The acidity, sensory properties, Bb count, total bacterial count (TBC), yeast, and mold counts were examined. The results showed that the acidity was increasing during storage, however, increasing NPES resulted in low acid development (p < .05). The shelf-life of control was ended after 8 d of storage at 4°C because molds were grown on the surface of the sample. The TBC significantly decreased (p < .05) as the concentration of NPES increased. Bb count in probiotic yogurt was also decreasing during storage. Yeast and molds were detected in control after 8 d; however, NPES did not result in molds even after 16 d of storage but yeast was exhibited. The NPES improved the sensory evaluation of probiotic yogurt slightly and increased the shelf-life of probiotic yogurt as compared to control.

K E Y W O R D S
functional properties, nanopowdered eggshell, probiotic yogurt, shelf-life Nanotechnology is a novel process that can be used to improve the physicochemical and biological properties of dairy products. Nano sizing particles play a significant role in increasing the bioavailability of the microcomponent, such as calcium (Hilty et al., 2011;Park et al., 2008;Seo et al., 2009). As a result, nanopowdered eggshell (NPES) can be used as nanoparticles in the manufacture of probiotic yogurt. NPES has a potential application in manufacture of yogurt using probiotics. It has been found that NPES did not show any negative effects on the characteristics of yogurt (Al Mijan et al., 2014).
In 2003, Codex mentioned that probiotic dairy products should contain at least 6-7 log cfu/g of at the time of consumption in quantity higher than 100 g per d to have at least 9 log cfu per d (FAO/ WHO, 2010). Probiotic bacteria, including Bifidobacterium bifidus, has several benefits, such as improving the gastrointestinal tract.
This can also reduce acute diarrhea and E. coli infections (Caballero et al., 2015). Additionally, it has been presented that the NPES can be utilized to improve the growth of probiotic bacteria in yogurt during storage (Al Mijan et al., 2014). The objectives of this work were to produce probiotic yogurt (5.0-7.0 log cfu/g) fortified with NPES at a rate of 0.02, 0.04, and 0.06 mg/ml, as well as, study the effect of NPES on the physicochemical, microbial, sensory properties, and shelf-life of probiotic yogurt. Ain Shams University, Egypt). The milk was divided into 4 aliquots portions. The first portion was utilized as control (T1; with no NPES) while 0.02, 0.04, and 0.06 mg/ml of NPES were added to the second (T2), third (T3), and fourth (T4) portions, respectively. All treatments were inoculated at 40°C until a pH of 4.6 was reached and this process took approximately 4 hr. Subsequently, the yogurt was cooled and stored at 4°C for 16 d. This experiment was repeated 3 times using 3 different batches of raw milk.

| Preparation of nanopowdered eggshell (NPES)
ES was collected from domestic sectors in Assiut governorate, Egypt.
ES was washed thoroughly with warm water and dried at room temperature for 2 d. After drying, it was kept in boiling water for 2 hr to remove the interior membranes as well as undesirable substances.
ES was then dried in the oven at 60°C for 6 hr. Further, the dried ES was milled using mortar grinding Fritsch Pulversitte 2, for one h.
The X-ray diffraction (XRD) of the milled powder was recorded using PW1700 X-ray diffractometer in the 2θ range from 20°C to 50°C.
The mean crystallite size D of the obtained NPES was calculated by using the Scherrer Equation (1): where λ is the CuK α x-rays (1.54056 A°), β is the full width at half maximum of the diffraction peak, Ɵ is the different angle. The mean crystallite size D was found to be 27 ± 1.7 nm.

| Sensory evaluation
Sensory evaluation of probiotic yogurt was also determined as described by Hamdy and others with some modifications (Hamdy et al., 2021). Samples were evaluated for color and appearance (15 points), flavor (45 points), acidity (10 points), body and texture (30 points) to have 100 points as a total. The sensory characteristics were determined at 0 and 16 d.

| Statistical analysis
Data were statistically analyzed using R software (R x64-3.3.3, 9,205 NW 101st St, Miami, Florida, United States) by ANOVA using a GLM for each variable to study the effect of NPES and time or their interaction on the characteristics of probiotic yogurt. Mean separation was done using the least significant difference (LSD) comparison test when significant differences were detected at p < .05.

| Particle size analysis
The morphology of NPES was observed by scanning electron microscopy (SEM; model JEOL JSM-5400 LV), as shown in Figure 1.
The SEM demonstrated that the average particle size for NPES was about 18 to 20 µ and there are on the nanoparticles (20 to 40 nm). Also, Figure 2 shows the XRD pattern of the NPES. Figure 2 indicates the nanocrystalline of the NPES. The average crystal size (D) was calculated by using Scherrer Equation (2), thus D = 27±1.7 nm:

| Titratable acidity (% lactic acid)
The changes in the acidity of probiotic yogurt supplemented with different concentrations of NPES during 16 d of storage at 4°C are presented in Figure 3. All probiotic yogurt samples gradually increased in acidity during the storage period. The acidity values in 0.02, 0.04 and 0.06 mg/ml NPES yogurt ranged from 0.85 to 1.14, 0.86 to 1.05, and 0.80 to 1.02%, respectively through the 16 d of storage, whereas it ranged from 0.80% to 0.92% after 8 d of storage in the control probiotic yogurt. However, increasing the concentrations of NPES in probiotic yogurts resulted in a decrease in the acidity value. From Figure 3, it shows that the average of acidity in T4 (0.06 mg/ml NPES) was lower than the acidity in T2 and T3. This can be due to the higher buffering capacity of calcium in NPES.
The obtained results are in agreement with the results obtained by Pirkul et al. (1997)

| Total bacterial count (TBC)
The TBC of probiotic yogurt made with NPES is shown in Figure 4.

| Bifidobacterium bifidum (Bb)
The Bb count in probiotic yogurt is presented in Figure 5. The Bb in control decreased from 7 to 5.36 log cfu/g after 8 d of storage (end of storage for control due to growth of molds). Also, this number lactic acid bacteria count in yogurt fortified with NPES was elevated during storage (Al Mijan et al., 2014). This trend can be due to the differences in the acidity level.

| Yeast and mold count
The yeast count of probiotic yogurt is presented in Figure 6.

| Sensory evaluation
The sensory evaluation of probiotic yogurt is presented in Table 1.
The NPES did not significantly change (p > .05) the sensory characteristics of yogurt during the storage time. On the first day, the acidity score decreased with increasing concentration of NPES.

| CON CLUS ION
NPES can be used to manufacture probiotic yogurt to improve the physicochemical, microbial properties, and shelf-life stability without any effects on the sensory properties. Therefore we conclude that the addition of 0.06 mg/ml NPES could be applicable to manufacture probiotic yogurt with an acceptable composition and quality as compared to control. The addition of NPES increased the shelf-life of probiotic yogurt as compared to control with a range of 5 to 7 log cfu/g of probiotic yogurt.