The growing demand for functional foods, driven by a population increasingly aware of the relationship between diet and health, combined with public agency requirements for clearer and more accessible labelling, has directed researchers and industry towards the development of healthier food products (Fernandes et al., Reference Fernandes, Pitombo, Morais, Salvador, Rezende, Matias, Furquim and Pereira2017; Granato et al., Reference Granato, Barba, Kovačević, Lorenzo, Cruz and Putnik2020).
In this context, probiotic foods have gained prominence due to their numerous health benefits, which are widely recognised (Marco et al., Reference Marco, Heeney, Binda, Cordero, Hutkins, Martin and Sanders2021). Probiotics are live microbial supplements that beneficially affect the host by improving intestinal microbial balance (Fuller, Reference Fuller1992), are commonly associated with yoghurt. However, they are not essential for yoghurt production and are typically added to enable co-fermentation with starter cultures (Ng et al., Reference Ng, Yeung and Tong2011).
Since its discovery, yoghurt has been the focus of numerous technological innovations, resulting in a wide variety of product available in supermarkets. These products may serve as meal replacements, desserts or snacks and cater to children, adults and the elderly (Wang et al., Reference Wang, Wang, Wei, Xu, Cavender, Lin and Sun2024). Due to its widespread popularity and sensory appeal, yoghurt is extensively used as a vehicle for health-promoting agents through the addition of vitamins, probiotic bacteria, minerals and fibres, or by modifying its composition, such as lactose removal or fat reduction to meet dietary requirements (Kumar et al., Reference Kumar, Vijayendra and Reddy2015).
The development of a convenient yoghurt-based snack has been explored by Wang et al., (Reference Wang, Aalaei, Skibsted and Ahrné2020, Reference Wang, Munk, Skibsted and Ahrné2022). In these studies, the authors developed freeze-dried yoghurt-based snacks with a porous and brittle structure, intended for direct consumption as ready-to-eat products. The convenience of these products refers to their portability, extended shelf life and spoon-free consumption, demonstrating that such snacks can provide essential nutrients and energy without excess sugar and fat while being rich in calcium. However, these studies did not evaluate the textural properties of the products nor did consider the inclusion of probiotic bacteria. In addition, the freeze-drying process introduces an additional cost to product manufacture.
Elucidating the rheological properties and textural profile of a food product is crucial for new product development, as these parameters not only directly influence consumer acceptance but also provide insights into optimal transport and storage conditions (Rao, Reference Rao2007; Mohammadi et al., Reference Mohammadi, Shahidi, Rafe, Naghizadeh Raeisi and Ghorbani-hasansaraei2022).
These properties are largely governed by the ingredients used in the formulation, particularly those contributing to structural development. Gelatine, a partially hydrolysed form of collagen, is widely used due to its favourable functional properties (Gómez-Guillén et al., Reference Gómez-Guillén, Giménez, López-Caballero and Montero2019). It melts at body temperature, is thermoreversible, exhibits low allergenicity, and, as a natural protein, is suitable for clean-label products. Its technological functions include gelling to stabilising and thickening (Rao, Reference Rao2007).
Another key factor in product development is understanding the interactions among food matrix components, as these interaction may be either beneficial or detrimental. Maintaining the probiotics viability throughout the shelf life of yoghurt, for example, is a complex challenge influenced by factors such as the probiotic strain, interactions with starter cultures, lactic acid concentration, oxygen, hydrogen peroxide levels and the nature of substrate (Pinto et al., Reference Pinto, Fritzen-Freire, Dias and Amboni2019).
Microencapsulation of probiotics in hydrocolloid-based systems spheres has been investigated as a strategy to improve cell viability in food products and the during gastrointestinal transit (Amir et al., Reference Amir, Zuberi, Kamran, Imran and Ahmad2024). Additional benefits of microencapsulation include protection against environmental stress, improved survival during freeze-drying and freezing as well as and enhanced stability during storage (El Sayed and Mabrouk, Reference El Sayed and Mabrouk2023; Lin et al., Reference Lin, Si, Zhou, Hao, Zhang, Jiang and Cha2024).
The development of a yoghurt bar containing probiotic microcapsules represents a promising opportunity for the dairy industry, offering a palatable and convenient product with broad consumer appeal. This concept addresses the demand for spoon-free consumption and firmer food textures that enhance flavour perception through prolonged mastication (Laguna et al., Reference Laguna, Fiszman and Tárrega2021), while delivering the well-known benefits associated with probiotic intake (Mohammadi et al., Reference Mohammadi, Jazayeri, Khosravi-Darani, Solati, Mohammadpour, Asemi, Adab, Djalali, Tehrani-Doost, Hosseini and Eghtesadi2016; Chen et al., Reference Chen, Jiang, Yu, Liao, Wu, Fang, Chen, Wang, Luo, Long, Zhong, Liu, Li, Zhang and Zhu2021).
In this context, the present study aimed to develop a yoghurt bar containing passion fruit microcapsules with L. acidophilus NCFM®, to evaluate the effect of gelatine concentration as a gelling agent and to investigate its centesimal composition, microbiological properties, rheological behaviour and textural characteristics.
Materials and methods
Materials
Commercial UHT (ultra-high temperature) milk containing 3.0% fat, 3.0% protein and 4.6% carbohydrate (Embaré, Minas Gerais, Brazil), gelatine (Dr Oetker, São Paulo, Brazil), sucrose (Camil, São Paulo, Brazil) and yoghurt starter culture containing S. thermophilus and L. bulgaricus Bio Rich® (Chr. Hansen, Hoersholm, Denmark) were used for the production of the yoghurt bar. For microcapsules production, the following materials were used: the probiotic L. acidophilus NCFM® (Airela, Santa Catarina, Brazil), pasteurised passion fruit pulp (Frutpres, Minas Gerais, Brazil), calcium chloride (Dinâmica, São Paulo, Brazil) and sodium alginate (Êxodo científica, São Paulo, Brazil) which served as wall materials.
Encapsulation of passion fruit pulp and L. acidophilus NCFM® by ionic gelation
The ionic gelation process was carried out using the dripping technique and involved two solutions: Solution (1), consisting of an aqueous mixture containing 30% (w/w) passion fruit pulp, 2% w/w sodium alginate and sodium citrate for pH adjustment to 4.00; and Solution (2), consisting of a 2% (w/v) aqueous calcium chloride solution (Morais et al., Reference Morais, Xavier, Silva, Silva and Pagani2015). Passion fruit juice has a distinctive flavour and aroma and contains a wide range of nutrients, including vitamins, minerals, fibre and bioactive compounds. As such, it is a sought-after ingredient in formulated foods, particularly dairy products and beverages (Ning et al., Reference Ning, Luo, Chen, Zhou, Xie, Du and Wang2021).
Solution 1 was mixed using a homogeniser (Mixer Mallory Trikxer) for 2 min, after which L. acidophilus NCFM® (0.04%, w/v) was added and gently mixed using a spatula. The resulting dispersion was dripped using a 10 mL syringe fitted with a tip of 2 mm internal diameter into solution (2), with a distance of 15 cm between the syringe tip and the solution (2). The microcapsules were maintained in the CaCl2 solution for 15 min and subsequently washed with potable water.
After production, the microcapsules were stored in passion fruit juice under refrigeration (5 °C).
Counting of L. acidophilus NCFM® and encapsulating yield
Viable cell counts of L. acidophilus NCFM® in the microcapsules were determined 24 h after production. Enumeration was performed using the pour plate method on de Man, Rogosa and Sharpe (MRS) agar modified with maltose (1.0%, w/v) (MRS-M agar), as recommended by the International Dairy Federation (1995) for the selective enumeration of L. acidophilus in the presence of yoghurt starter cultures, based on this ability ferment maltose. Plates were incubated at 37 °C for 72 h under anaerobic conditions. Results were expressed as log CFU g−1.
The encapsulating yield (EY, %) was calculated according to Equation (1):
\begin{equation}EY{\text{ }}\left( \% \right) = \frac{N}{{N0}} \times 100\end{equation}where N is the number of viable cells (log CFU g−1) released from the microcapsules; and N0: number of free viable cells (log CFU g−1) in the solution (1) prior to the encapsulation process (Annan et al., Reference Annan, Borza and Hansen2008).
To release the probiotic cells from the microcapsules prior to enumeration, the beads were gently disintegrated in sterile sodium citrate solution (1.0%, w/v), which chelates calcium ions and dissolves the alginate matrix, as described by Annan et al. (Reference Annan, Borza and Hansen2008).
Manufacture of the yoghurt bar
UHT milk was standardised by the addition of skimmed milk powder (4%, w/v) to increase protein and total solids content, followed by homogenisation and heating to 43 °C prior to fermentation. The milk was then inoculated with the starter culture Bio Rich® (0.04% w/v) and incubated at 43 °C util a pH of 4.6 was reached after 3.5 h. After fermentation, the yoghurt was cooled to 5.0 °C and stored overnight. The yoghurt whey was subsequently drained to obtain Greek-style yoghurt by placing the yoghurt on filter paper for 20 h at 5 °C as described by Pinto et al. (Reference Pinto, Fritzen-Freire, Dias and Amboni2019). After production of the Greek-style yoghurt, sucrose 6.45% (w/w) was added, together with the appropriate volumes of the 12% (w/w) gelatine dispersion and filtered water for each formulation. The gelatine dispersion (12%, w/w) was prepared by gradually adding gelatine powder to filtered water under continuous stirring, followed by heating at 60 °C until complete dissolution. The dispersion was cooled to approximately 40 °C before incorporation into the yoghurt formulation to avoid thermal damage to probiotic cultures (Table 1).
Formulation of yoghurt bars
Table 1 Long description
Ingredient percentages are listed for three yoghurt bar formulations labeled YB6, YB9, and YB12. Greek-style yoghurt is constant at 64.50% in all three, sucrose is constant at 6.45%, and microcapsules are constant at 0.64%. Gelatine dispersion increases across the formulations, from 16.02% in YB6 to 22.43% in YB9 and 28.85% in YB12. Water decreases in the opposite direction, from 12.82% in YB6 to 6.45% in YB9 and none in YB12. Each formulation totals 100%, so the rise in gelatine dispersion is balanced mainly by reduced water rather than changes in yoghurt, sugar, or microcapsules.
YB6, yoghurt bar with a 6.66% gelatine dispersion; YB9, yoghurt bar with a 9.33% gelatine dispersion; YB12, yoghurt bar with a 12% gelatine dispersion.
Three different yoghurt bar formulations were prepared by varying the concentration of the gelling agent and water. In all formulations, the combined proportion of water and gelatine dispersion accounts for 28.85% (w/w) of the formulation. Therefore, when calculating gelatine concentration, and considering dilution by the added water, the following formulations were established: YB6, yoghurt bar with a 6.66% (w/w) gelatine dispersion; YB9, yoghurt bar with a 9.33% (w/w) gelatine dispersion; and YB12, yoghurt bar with a 12% (w/w) gelatine dispersion. These concentrations were defined based on preliminary tests aimed at achieve a texture suitable for a yoghurt bar.
The gelatine concentrations reported for formulations YB6, YB9 and YB12 refer to the concentration of gelatine in the gelatine dispersion added to the formulation, rather than to the final centesimal gelatine content of the yoghurt bars. This approach was adopted to describe the formulation strategy and avoid ambiguity regarding ingredient addition.
After production, the microcapsules were stored in passion fruit juice at 5 °C for 24 h prior to incorporation into the yoghurt bar formulation. Before use, the microcapsules were drained but not washed to preserve their integrity and minimise probiotic loss. Homogeneous dispersion within the yoghurt matrix was ensured by gently folding the microcapsules into the formulation immediately prior to moulding.
After homogenisation all the ingredients, the microcapsules were incorporated, and the mixture was poured into rectangular transparent acetate (PVC) moulds and refrigerated at 5 °C for 24 h to allow for gelation. Each yoghurt bar was then wrapped in aluminium foil and stored at 5 °C until analysis.
Characterisation of microcapsules
After 24 h of refrigerated storage at 5 °C in a sealed container immersed in passion fruit juice, the microcapsules were drained and characterised by weight determination using an analytical balance (Shimadzu AUY220) and size measurement with a digital micrometre (Mitutoyo 547–401) for characterization.
Technological characteristics of the yoghurt bar
Size, weight, pH and acidity
The size, weight, pH and acidity of the yoghurt bars were measured on the first day of storage. Size and weight results were obtained as the mean of ten replicates. pH and titratable acidity analyses were performed in triplicate. The pH of the samples was measured using a pH meter (MS TECNOPON model mPA-210). Titratable acidity, expressed as percentage of lactic acid, was determined by titrating up to pH 8.2 with 0.1 mol/L NaOH, according to AOAC (2016).
Chemical composition
Yoghurt bar samples were analysed on the first day of storage for moisture, protein, fat, ash, total carbohydrates, total sugars, calcium and sodium according to the methods described by AOAC (2016).
Counting of probiotic and starter cultures
Viable cell counts of the probiotic (L. acidophilus NCFM®) and starter cultures in the yoghurt bar samples were performed on days 1, 15 and 30 of storage at 5 °C. L. acidophilus NCFM® was enumerated as described in Section “Counting of L. acidophilus NCFM® and encapsulating yield”. L. delbrueckii subsp. Bulgaricus was enumerated using MRS agar under anaerobic conditions at 37 °C for 72 h. S. thermophilus was enumerated using M17 agar supplemented with lactose solution (10% w/v) under aerobic conditions at 37 °C for 48 h (International Dairy Federation, 2003).
The storage stability study was conducted exclusively through microbiological analyses, which were performed throughout a 30-day storage period. The remaining physical and chemical analyses were carried out only at the initial time point, with the aim of characterising the product.
Rheological properties
The rheological properties of the yoghurt bar were determined in dynamic mode using an oscillatory rheometer (HAAKE MARS, Modular Advanced Rheometer System, Thermo Electron Corp., Germany), equipped with a thermostatic bath (Phoenix 2C30P, Thermo Electron Corp., Germany). Measurements were performed using a plate-plate geometry (PP35/Ti) with a diameter 35 mm and a gap of 1 mm. The sample was placed at the centre of the lower plate, after which the upper plate was gradually lowered to the predetermined gap to minimize structural damage. Excess sample was carefully removed from the plate edge to reduce edge effects.
Sample temperature was precisely controlled at 10 °C and 25 ºC, using a thermostatic bath. Prior to testing, samples were equilibrated for at least 10 min to allow temperature stabilisation and structural rearrangement. A fresh sample was used for each measurement. The temperatures of 10 °C and 25 °C were selected to represent consumption temperature and oral temperature for semisolid foods, respectively (Tárrega and Costell, Reference Tárrega and Costell2007).
The linear viscoelastic region (LVR) was determined by performing a stress sweep from 0.1 to 1000 Pa, at a constant frequency of 1 Hz. The LVR limit was identified as the stress at which two consecutive values of the viscoelastic moduli differed by more than 3%, and the stress corresponding to the end of the LVR was defined as the yield stress (
${\tau _{y{\text{ }}}}$) (Yazar et al., Reference Yazar, Duvarci, Erturk, Kokini and Joyner2019). Subsequently, a frequency sweep was performed over the range of 0.01–10 Hz, with a constant shear stress of 10 Pa, which was within the LVR at both analysed temperatures. The results at 1 Hz were expressed as storage modulus (G’) and loss modulus (G”), and the loss tangent (δ) was calculated as (δ) = G”/G’.
Texture profile analysis
Texture profile analysis (TPA) of the yoghurt bars was performed using a universal testing machine (Instron Corporation, Norwood, MA, USA). The following parameters were obtained: firmness (the maximum force peak on the first compression cycle), cohesiveness (the ratio of the area under the first and second compression), gumminess (the hardness multiplied by cohesiveness), adhesiveness (measure of the force required to separate a test surface from a material after compression) and elasticity (calculated as the distance recovered by the sample between the end of the first compression and the start of the second compression cycle) (Sinthusamran et al., Reference Sinthusamran, Benjakul and Hemar2018).
A cylindrical probe with a diameter of 55 mm was moved perpendicularly through the sample which dimensions of 30 mm in length and 13 mm in height under the following conditions: load cell of 250 N, compression distance of 35% of the sample height, testing speed of 1 mm/s and two compression cycles. All analyses were performed in triplicate.
For comparative purposes, rheological (dynamic oscillatory tests) and TPAs were also conducted on two commercially available UHT-processed cheeses (“pocket cheese”) from the same brand: one full-fat (9.4% w/w protein, 5.2% w/w carbohydrates and 20% w/w fat) (QPI) and one light (12% w/w protein, 5.8% w/w carbohydrates and 11% w/w fat) (QPL). These products were selected because they share a similar physical appearance and market positioning with the yoghurt bar developed in this study.
TPA was performed after 24 h of storage at 5 °C. Measurements were conducted at 10 °C to simulate consumption conditions.
Statistical analysis
The experiment was conducted using a completely randomised design with two replications. All measurements were performed at least in duplicate, and results are expressed as mean ± standard deviations. Statistical analyses were carried out using R statistics software. One-way analysis of variance was applied to determine the significant differences among samples, and Tukey’s test was used for multiple comparisons at a 95% confidence level (p < 0.05).
Results and discussion
Encapsulation efficiency of L. acidophilus NCFM®
Evaluation of microencapsulation process showed an encapsulation efficiency of 93.46 ± 3.7% for L. acidophilus NCFM®, which is comparable to the values reported by Mahmoud et al. (Reference Mahmoud, Abdallah, El-Shafei, Tawfik and El-Sayed2020) at 96.27%, and Jouki et al. (Reference Jouki, Khazaei, Rezaei and Taghavian-Saeid2021) at 94.48%. In both studies, Lactobacillus plantarum was microencapsulated using a mixture of skimmed milk and 3% w/v sodium alginate at a 1:2 ratio. The high encapsulation efficiency observed in the present study indicates that most probiotic cells were successfully entrapped within the alginate matrix, demonstrating the effectiveness of the ionic gelation technique.
Characterisation of microcapsules
The passion fruit microcapsules containing L. acidophilus NCFM® developed in this study presented an external diameter of 2.45 ± 0.21 mm and a mean mass of 15.43 ± 1.16 mg (based on 10 beads). Ferreira et al. (Reference Ferreira, Xavier, Santos, Wartha and Pagani2021) reported a mass of 1.2 mg and a size of 3.88 mm for sodium alginate microcapsules containing beetroot. Fareez et al. (Reference Fareez, Lim, Mishra and Ramasamy2015) obtained sodium alginate microcapsules with a mean diameter of 1.33 ± 0.006 mm coated with chitosan. This variation in microcapsule size reported in the literature and in the present study can be attributed to several factors that directly influence bead formation, including solutions viscosity, nozzle diameter and the distance between the syringe tip and the calcium chloride solution (El Sayed and Mabrouk, Reference El Sayed and Mabrouk2023).
Size, weight, pH and acidity
The yoghurt bars were produced with dimensions of 2 × 4 × 1 cm. After production, the bars were weighed and analysed for pH and titratable acidity (Table 2).
Weight, pH and titratable acidity of the yoghurt bar samples
Table 2 Long description
Yoghurt bar formulations with three gelatine dispersion levels (YB6, YB9, YB12) are compared for weight, pH, and titratable acidity, reported as mean with standard deviation. Weights are similar: YB6 about 18.91 grams, YB9 about 20.64 grams, and YB12 about 19.20 grams, with no statistically detected differences among them. pH values are also similar across formulations, around 4.09 to 4.11, with no statistically detected differences. Titratable acidity differs: YB6 is higher at about 0.83 grams lactic acid per 100 grams, while YB9 and YB12 are lower at about 0.65 and do not differ from each other. Superscript letters indicate statistical groupings within each row, so only the acidity row shows a significant separation. Variability is small for most measures, but the pH standard deviation for YB9 is notably larger than the others and should be considered when comparing pH.
YB6, yoghurt bar with a 6.66% w/v gelatine dispersion; YB9, yoghurt bar with a 9.33% w/v gelatine dispersion; YB12, yoghurt bar with a 12% w/v gelatine dispersion.
Values are expressed as mean ± standard deviation. Means followed by the same superscript letter within a row do not differ statistically according to Tukey’s test (p < 0.05).
There was no significant difference (p > 0.05) in weight or pH among the yoghurt bar samples, which can be attributed to the mass balancing across the formulations. Lactic acid content ranged from 0.65% in YB9 and YB12 to 0.83% for YB6, meeting the requirements of IDF Standard No. 150 (International Dairy Federation, 1991), which establishes acceptable yoghurt acidity values between 0.6 and 1.5 g of lactic acid per 100 g.
The absence of pH variation among the formulations, despite the higher titratable acidity observed in YB6, can be explained by the difference in the principles underlying pH and titratable acidity measurement. While pH reflects the concentration of free H+ ions in solution, titratable acidity represents the total amount of acids present. In addition, the buffering capacity of the yoghurt matrix contributes to stabilisation pH values, even when total acid content varies. The higher gelatine content in YB9 and YB12 may have further limited acid availability for titration through matrix interactions, without significantly affecting pH, which depends primarily on free ion concentration (Fennema et al., Reference Fennema, Damodaran and Parkin2017).
Chemical composition
Quantification of chemical constituents of yoghurt bars is relevant due to their nutritional and health appeal. The chemical composition of the yoghurt bars is presented in Table 3.
Chemical composition of yoghurt bar formulations
Table 3 Long description
Chemical composition is compared across three yoghurt bar formulations (YB6, YB9, YB12) reported as mean with standard deviation. Moisture is similar across samples, decreasing slightly from 80.63 percent in YB6 to 79.39 percent in YB12, while total solids rise from 19.37 to 20.60 percent. Protein increases with formulation level, from 6.30 percent (YB6) to 7.65 percent (YB12), with YB12 higher than YB6 and YB9 intermediate. Fat remains about 4.00 percent in all three, and ash stays near 0.71 to 0.73 percent. Total carbohydrates and total sugars are also similar, with carbohydrates around 8.28 to 8.47 percent and sugars around 8.15 to 8.40 percent. Energy value increases from 94.76 kilocalories (YB6) to 100.27 kilocalories (YB12), with YB12 higher than YB6 and YB9 intermediate. Sodium rises from 54.50 to 57.50 milligrams per 100 grams, while calcium shows no clear pattern and has the widest variability, especially in YB9 and YB12. Superscript letters indicate which values differ statistically within each row, so small numeric differences without letter changes may not be meaningful.
YB6, yoghurt bar with a 6.66% w/v gelatine dispersion; YB9, yoghurt bar with a 9.33% w/v gelatine dispersion; YB12, yoghurt bar with a 12% w/v gelatine dispersion.
Values are expressed as mean ± standard deviation. Means followed by the same superscript letter within a row do not differ statistically according to Tukey’s test (p < 0.05).
Energy value (kcal) = (Protein (g) × 4 kcal/g) + (Carbohydrate (g) × 4 kcal/g) + (Fat (g) × 9 kcal/g).
After mass standardisation, significant differences (p < 0.05) among the three formulations were observed only in protein, sodium content and energy value. The yoghurt bar developed in this can be classified as high-moisture, high-total-solids product, with an average moisture content of approximately 80% and total solids of around 20%, composed mainly of proteins, carbohydrates, lipids and ash. Owing to these characteristics and the absence of additional preservatives, the product requires refrigerated (5–10 °C) to maintain its quality and stability.
An increase in gelatine concentration resulted in a higher protein content in the product. Gelatine is a protein-based hydrocolloid that forms a thermoreversible gel and is commonly used in yoghurt manufacture as a stabilizer to reduce syneresis and improve texture, particularly in low-fat yoghurt formulations (Wu et al., Reference Wu, Dai, Lin, Niu, Li, Chang, Jia, Zou, Jiang, FeiJin, Huang and Gao2023). In the present study, gelatine was incorporated to enhance the firmness of strained yoghurt, enabling its consumption in a bar form. Yoghurt-based snacks with a protein content above 5% have been reported as favourable options for weight control, as they contribute to reduce hunger, increase satiety and delayed subsequent food intake (Patel and Pinto, Reference Patel and Pinto2014).
With respect to fat, ash and carbohydrate contents, the yoghurt bar exhibited mean value of 4%, 0.72% and 8.4%, respectively. Comparable results were reported by Pinto et al. (Reference Pinto, Fritzen-Freire, Dias and Amboni2019) for a lactose-free Greek-style yoghurt containing Bifidobacterium lactis BB-12 microcapsules, which presented 22.68 ± 0.03% total solids, 6.68 ± 0.03 protein, 5.95 ± 0.03 fat and 0.78 ± 0.01 ash.
The development of new food product with improved health attributes, such as reduced sugar and/or salt content, represents a considerable challenge for both the food industry and researchers, since these ingredients contribute not only to flavour but also play important technological roles in food systems (Antunes et al., Reference Antunes, Gallina, Rodrigues, Cunha, Cruz, Ranadheera, Nazzaro and Mortazavian2022).
The slightly higher total sugar content compared with total carbohydrates observed in YB6 can be attributed to analytical and rounding variability. Total carbohydrates were calculated by difference, whereas total sugars were determined by direct analytical measurement, which may lead to small numerical discrepancies when values are close.
The average total sugar content of the yoghurt bars was 8.3%. Considering that sucrose addition accounted for 6.45% of the formulation (Table 1), the remaining 1.85% (8.3 − 6.45 = 1.85) can be attributed to residual lactose not metabolised during fermentation and to fructose originating from the passion fruit pulp microcapsules. Rosa and Alves (Reference Rosa and Alves2019), when evaluating six different brands of whole-milk yoghurt, reported lactose content ranging from 2.8% to 4.0%.
The three formulations differed significantly (p < 0.05) in sodium content, with higher gelatine concentration leading to increased sodium levels. This proportional relationship was explained by the presence of sodium in the gelatine formulation itself. Although YB12 exhibited the highest sodium content (57.5 mg 100 g−1), this value is approximately ten times lower than the threshold defined high in sodium for solid or semi-solid foods according to ANVISA Resolution RDC No 429 (2020).
Regarding calcium content, consuming 100 g of yoghurt bars (equivalent to 5 units) provides approximately 50% of the calcium found in a 200 mL serving of whole cow’s milk. This highlights yoghurt bars as a practical calcium source for individuals who do not habitually consume fluid milk, allowing incorporation as convenient snacks throughout the day. Similar calcium levels were reported by Fernandes et al. (Reference Fernandes, Pitombo, Morais, Salvador, Rezende, Matias, Furquim and Pereira2017) for a red fruit frozen yoghurt formulated with green banana biomass (103.33 mg 100 g−1).
Counting and viability of probiotic and starter cultures
L. bulgaricus and S. thermophilus are essential microorganisms in yoghurt manufacturing. During fermentation, these bacteria exhibit a well-established symbiotic relationship, and their growth behaviour is crucial for product quality, influencing aroma, flavour, texture and overall consumer acceptance (Aleman et al., Reference Aleman, Cedillos, Page, Olson and Aryana2023). After production and throughout 30 days of refrigerated storage, viable counts of the starter culture (S. thermophilus and L. bulgaricus) and the probiotic culture L. acidophilus NCFM® present in the yoghurt bar were determined to characterize microbial profile, as shown in Table 4.
Viable counts of starter cultures and the probiotic bacteria (log CFU g−1) in the different yoghurt bar formulations during storage at 5 °C
Table 4 Long description
Viable counts (log CFU per g, mean with standard deviation) are reported for L. bulgaricus, S. thermophilus, and L. acidophilus NCFM in three yoghurt bar formulations (YB6, YB9, YB12) stored at 5 degrees Celsius for 1, 15, and 30 days. For L. bulgaricus, counts rise from about 7.1 to 7.4 on day 1 to about 8.9 to 9.2 on day 15, then return to about 6.9 to 7.1 by day 30 across all formulations. For S. thermophilus, counts are highest overall: about 9.1 to 9.2 on day 1, peaking at about 9.3 to 10.0 on day 15 (highest value in YB9), then decreasing to about 8.7 to 8.9 by day 30. For L. acidophilus NCFM, counts start near 7.0 to 7.3 on day 1, increase to about 9.0 to 9.2 on day 15, and drop by day 30, with YB12 showing the lowest final value (about 6.3) compared with YB6 (about 7.1) and YB9 (about 6.8). Differences among formulations at the same day are generally small relative to the storage-time effect, with the clearest separation at day 30 for L. acidophilus where YB12 is lower. Letter annotations indicate statistical groupings within rows and columns, so apparent numeric differences may not be statistically meaningful unless the letters differ.
YB6, Yoghurt bar with a 6.66% w/v gelatine dispersion; YB9, Yoghurt bar with a 9.33% w/v gelatine dispersion; YB12, Yoghurt bar with a 12% w/v gelatine dispersion.
Values are expressed as mean ± standard deviation. Means followed by the same uppercase letter within a column and the same lowercase letters within a row, do not differ significantly according to Tukey’s test (p < 0.05).
Gelatine concentration did not significantly affect (p > 0.05) the counts of starter or probiotic bacteria among the formulations at any sampling points (within-row comparison in Table 4). This behaviour can be attributed to mass standardisation of all formulations, which ensured the same final yield and comparable matrix composition at the end of processing.
During storage, L. bulgaricus and S. thermophilus exhibited a similar growth pattern, characterised by a peak in viable counts after 15 days, followed by a reduction at 30 days, returning to values close to those observed at day 1. This trend suggests that prolonged storage may result in stabilisation or a decrease in starter culture viability, possibly due to nutrient depletion or changes in matrix conditions that initially supported microbial growth.
Similarly, L. acidophilus NCFM® did not show a significant difference (p > 0.05) in viable counts after 30 days of storage compared with day 1. However, unlike the starter culture, no significant change was observed after the first 15 days of storage. These results indicate that incorporation of probiotic culture into the yoghurt bar in alginate microcapsules provided a protective environment that enhanced cell survival throughout storage. García-Ceja et al. (Reference García-Ceja, Mani-López, Palou and López-Malo2015) reported a higher survival greater of L. acidophilus in blackberry jam set-style yoghurt when encapsulated in alginate beads (>107 CFU g−1), compared with free cells (105 CFU g−1). This protective effect is particularly relevant in yoghurt systems, where probiotic viability may be affected by factors such as strain characteristics, interactions with starter cultures, lactic acid concentration, oxygen and hydrogen peroxide levels and substrate composition (Pinto et al., Reference Pinto, Fritzen-Freire, Dias and Amboni2019).
According to the Codex Alimentarius (Codex Stan 243–2003) (Codex Alimentarius Commission, 2018), the total count of yoghurt starter microorganisms must not be lower than 7 log CFU g−1, and when additional if other microorganisms are added, their viable count must exceed 6 log CFU g−1. As shown in Table 4, both starter and the probiotic cultures in all yoghurt bar formulations remained above these minimum limits throughout storage. Similar findings were reported by Pinto et al. (Reference Pinto, Fritzen-Freire, Dias and Amboni2019) for lactose-free Greek-style yoghurt supplemented with Bifidobacterium animalis ssp. Lactis BB-12, who observed viable counts of 9.48 ± 0.10; 8.80 ± 0.10; e 6.85 ± 0.01 log CFU g−1 for S. thermophilus, L. bulgaricus and B. lactis BB-12, respectively.
Rheology and texture properties
Rheological characterization
The study of rheological properties is fundamental for understanding the stability, processing characteristics and quality control of yoghurt bars. In yoghurt systems, rheology describes how the gel matrix flows or deforms under applied normal and/or shear stresses (Wu et al., Reference Wu, Dai, Lin, Niu, Li, Chang, Jia, Zou, Jiang, FeiJin, Huang and Gao2023).
Foods can be classified rheologically as Newtonian fluid, viscoelastic fluid or viscoelastic solid (Zheng, Reference Zheng and Joyner2019). Yoghurt gels typically exhibit viscoelastic behaviour combined with weak shear-thinning (pseudoplastic) characteristics, in which apparent viscosity decreases with increasing shear rate or shear stress. Shear stress corresponds to the tangential force applied per unit area of the material (Clark et al., Reference Clark, Michael, Schmidt and Joyner2019). When stress is applied, viscoelastic materials store part of the deformation energy elastically within their structure, represented by the storage modulus (G′) while another portion is dissipate dissipated as viscous flow, represented by the loss modulus (G″). The evolution of G′ and G″ as a function of shear stress for yoghurt bars analysed at 10 °C and 25 °C can be visualized in Fig. 1.
Storage modulus (G′, filled symbols) and loss modulus (G″, open symbols) as a function of shear stress sweep for yoghurt bar formulations at (a) 10 °C and (b) 25 °C.
Figure 1 Long description
The graphs depict storage modulus (G′ with filled symbols) and loss modulus (G′′ with open symbols) as a function of stress (Pa) for yoghurt bar formulations. The x-axis is labeled ′Stress (Pa)′ ranging from 0.10 to 1000.00. The y-axis is labeled ′G′ or G′′ (Pa)′ ranging from 0.10 to 100000.00. In graph (a), the data is for 10 degrees Celsius. The curves for G′ and G′′ are shown for formulations GYB6, GYB9 and GYB12. The storage modulus remains relatively constant across the stress range, indicating a linear viscoelastic region (LVR), before decreasing at higher stresses. The loss modulus shows a similar trend. In graph (b), the data is for 25 degrees Celsius. The trends are similar to graph (a), with the storage and loss modulus remaining constant in the LVR before decreasing. The formulations GYB9 and GYB12 show a similar transition point, while GYB6 transitions at a different stress level. Transition points are marked with arrows labeled τf and τy.
As shown in Fig. 1, elastic behaviour predominated over viscous behaviour (G′ > G″) for all yoghurt bar formulations, indicating a solid-like structure. At the lowest applied shear stress (0.1 Pa), all samples exhibited a well-defined LVR, in which the moduli were independent of the applied stress. The lower critical stress observed at 25 °C reflects the increased molecular mobility and partial relaxation of the gelatine network at higher temperatures, resulting in an earlier deviation from linearity. The onset of non-linear behaviour occurred at a shear stress of approximately 88.70 Pa at 10 °C (Fig. 1a) and from 33.64 Pa at 25 °C (Fig. 1b).
The LVR represents the range of reversible elastic deformation, beyond which the material undergoes irreversible structural rearrangement and eventual rupture (Zheng, Reference Zheng and Joyner2019). The limit of the LVR was identified when two consecutive values of the viscoelastic moduli differed by more than 3%, and the stress was defined as the yield stress (
${\tau _{y}}$). The 
${\tau _{y}}$ represents the maximum shear stress that can be applied while maintaining the integrity of the gel network before structural breakdown occurs (Yazar et al., Reference Yazar, Duvarci, Erturk, Kokini and Joyner2019).
Figure 1a and 1b shows that the evolution of storage G′ and loss G′′ moduli as a function of shear stress was dependent on gelatine concentration at both 10 °C and 25 °C. Although all formulations exhibited a similar deformation pattern, YB6 was more susceptible to permanent deformation as indicated by its lower 
${\tau _{f}}$, whereas YB9 and YB12 showed greater resistant to deformation, reflecting a stronger gel network. This behaviour is associated with the higher gelatine content, which increases protein–protein interactions and reinforces the structural integrity of the yoghurt bar matrix.
Similar observations were reported by Nguyen et al. (Reference Nguyen, Ong, Kentish and Gras2015) who demonstrated that the rheological properties of buffalo yoghurt were strongly influenced by total solids content, particularly protein concentration and composition, which play a key role in determining gel strength and resistance to deformation.
A crossover between the storage and loss moduli may occur when the gel structure is sufficiently disrupted to allow the material to flow. This point is known as the flow stress 
$({\tau _{f}}$) (Yazar et al., Reference Yazar, Duvarci, Erturk, Kokini and Joyner2019). At 10 °C, the G′ and G″ values of YB6 began to decrease as the shear stress amplitude increase up to 1000 Pa (Fig. 1a), with the crossover occurring at 
${\tau _{f{\text{ }}}}$= 1000 Pa. For YB9 and YB12, G′ started to decrease while G″ increased as the shear stress amplitude increase up to 1000 Pa; however, no crossover was observed within the applied stress range (Fig. 1). At 25 °C, a crossover point was observed at 
${\tau _{f{\text{ }}}}$= 616.80 Pa for YB6, whereas for YB9 and YB12 the crossover only occurred at 
${\tau _{f{\text{ }}}}$= 1000 Pa. These results indicate a relatively stronger gel structure for YB9 and YB12 at 25 °C as well as greater structural stability when the product is stored at 10 °C.
The crossover phenomenon is associated with the breakdown of the gel network and the formation of aggregated structures. As shear stress increases, microstructural components present in yoghurt systems, such as lipid droplets, casein micelles coated with whey protein and microbial constituents dispersed in the serum phase, tend to align in the direction of flow, resulting in shear-thinning behaviour (Yazar et al., Reference Yazar, Duvarci, Erturk, Kokini and Joyner2019; Gilbert and Turgeon, Reference Gilbert and Turgeon2021).
In addition to the storage and loss moduli, the loss tangent (tan δ = G″/G′), also referred to as the phase angle (Fig. 2) can be used to characterise the viscoelastic stability (force) of yoghurt bars. This parameter is particularly useful when comparing different formulations, as G′ and G″ may present similar magnitudes while including distinct elastic and viscous contributions (Nguyen et al., Reference Nguyen, Kravchuk, Bhandari and Prakash2017).
Loss tangent (tan δ = G″/G′) of yoghurt bar formulations and commercial pocket ultrafiltrated cheeses obtained from frequency sweep tests at a constant shear stress of 10 Pa at (a) 10 °C and (b) 25 °C.
Figure 2 Long description
Line graph with the y-axis labeled Loss tangent (delta), ranging from 0.0 to 0.6. The x-axis is labeled Frequency (hertz), with tick labels 0.01, 0.10, 1.00 and 10.00. A legend lists YB6, YB9, YB12, QP1 and QPL. At 0.01 hertz, QPL is about 0.50, YB6 about 0.45, QP1 about 0.40, YB9 about 0.20 and YB12 about 0.15. From 0.01 to about 0.10 hertz, all series decrease, with QPL dropping to about 0.20, YB6 to about 0.15, QP1 to about 0.12, YB9 to about 0.10 and YB12 to about 0.08. From about 0.10 to 1.00 hertz, the lines are nearly flat around 0.10 to 0.16. From 1.00 to 10.00 hertz, all series rise. At 10.00 hertz, YB6 is about 0.25, QPL about 0.20, QP1 about 0.18, YB9 about 0.17 and YB12 about 0.16. Line graph with the y-axis labeled Loss tangent (delta), ranging from 0.0 to 0.7. The x-axis is labeled Frequency (hertz), with tick labels 0.01, 0.10, 1.00 and 10.00. A legend lists YB6, YB9, YB12, QP1 and QPL. At 0.01 hertz, QPL is about 0.60, YB6 about 0.30, QP1 about 0.25, YB9 about 0.20 and YB12 about 0.15. From 0.01 to about 0.10 hertz, QPL decreases to about 0.25, while the other series move toward about 0.12 to 0.18. From about 0.10 to 1.00 hertz, the lines are close together around 0.14 to 0.20. From 1.00 to 10.00 hertz, all series rise. At 10.00 hertz, QP1 is about 0.30, YB12 about 0.28, YB9 about 0.25, YB6 about 0.22 and QPL about 0.20.
The frequency sweep results presented in Fig. 2 demonstrate that all samples exhibited viscoelastic semi-solid behaviour at both evaluated temperatures, 10 °C (Fig. 2a) and 25 °C (Fig. 2b). This behaviour is confirmed by the predominance of elastic properties, as indicated by a phase angle (δ) of less than 1. Low δ values are characteristic of more solid rubbery gels, whereas higher δ values indicate weak and more fluid gels structure (Zheng, Reference Zheng and Joyner2019). At 10 °C (Fig. 2a), a clear relationship between gelatine concentration and gel strength was observed, with increasing gelatine concentrations resulting in lower tangent values. Conversely, at 25 °C (Fig. 2b), gelatine concentration did not significantly affect the tan δ values of the samples.
These results indicate that temperature plays a decisive role in the viscoelastic behaviour of yoghurt bars. Under refrigeration conditions (10 °C), formulations YB9 and YB12 exhibited greater elasticity and lower susceptibility to deformation, suggesting enhanced structural stability during handling and transportation compared with YB6, QPI and QPL. However, when evaluated at 25 °C, corresponding to oral temperature, all samples exhibited similar consistency. The reduction in viscoelastic moduli at higher temperatures can be attributed to partial melting of gelatine junction zones and increased molecular mobility, which weaken the gel network and reduce resistance to deformation.
For statistical comparison, the values of the storage modulus, loss modulus and loss tangent were obtained at a frequency of 1 Hz at both 10 °C and 25 °C, as presented in Table 5.
Viscoelastic parameters of different yoghurt bar formulations measured at a frequency of 1 Hz at 10 °C and 25 °C
Table 5 Long description
Viscoelastic properties were measured at 1 hertz for three yoghurt bars with increasing gelatine and two pocket cheeses, at 10 and 25 degrees Celsius. Storage modulus (stiffness) was much higher for pocket cheeses than yoghurt bars at both temperatures: at 10 degrees, about 360 to 410 millipascal for cheeses versus about 19 to 33 for yoghurt bars. At 25 degrees, storage modulus dropped strongly for every product, to about 0.80 to 2.47 for yoghurt bars and about 36 to 46 for cheeses. Loss modulus followed the same pattern: at 10 degrees, about 53 to 62 for cheeses versus about 2.6 to 2.9 for yoghurt bars, then at 25 degrees falling to about 6.6 to 8.3 for cheeses and about 0.14 to 0.44 for yoghurt bars. Within yoghurt bars at 10 degrees, higher gelatine corresponded to higher storage modulus, with YB12 highest and YB6 lowest; differences were smaller at 25 degrees. Tan delta values were low and similar across products, around 0.09 to 0.15 at 10 degrees and about 0.18 at 25 degrees, indicating comparable relative damping despite large modulus changes. Values are means with standard deviations, and letter superscripts indicate statistically significant differences within rows and columns.
YB6, Yoghurt bar with a 6.66% w/v gelatine dispersion; YB9, Yoghurt bar with a 9.33% w/v gelatine dispersion; YB12, Yoghurt bar with a 12% w/v gelatine dispersion, QPI, full-fat pocket cheese; QPL, light pocket cheese.
Values are expressed as mean ± standard deviation. Values within a row (lowercase) or column (uppercase) that do not share common superscripts are significantly different (p < 0.05) using Tukey.
1 Tangent of the phase angle (δ), determined by tan (δ) = (G″/G′).
Temperature exerts a significant influence (p < 0.05) on the elastic and viscous module, with the highest values observed at 10 °C. For both temperatures, the Cheese Pocket demonstrated higher values of G′ and G″ compared to the yoghurt bar samples, and a significant difference (p < 0.05) was observed between the cheese and yoghurt samples. Although the QPL presented a lower storage modulus than the QPI, this difference does not imply greater elasticity, as no significant variation was in the loss tangent (tan δ). The similarity in tan δ values among yoghurt bars and pocket cheeses indicates a comparable balance between elastic and viscous components, despite absolute differences in viscoelastic moduli.
Pocket cheeses were included as reference products due to their similar physical appearance, portability and handheld consumption characteristics. Although their viscoelastic moduli were higher, the comparable tan δ values support the positioning of yoghurt bars as structured, solid-like dairy snacks with viscoelastic behaviour analogous to commercially available portable dairy products.
Texture profile analysis
The instrumental TPA results for yoghurt bar formulations with different gelatine concentrations are presented in Table 6. Firmness, cohesiveness, gumminess, adhesiveness and elasticity were evaluated to characterise the mechanical behaviour and oral perception of the developed products.
Texture profile analysis (TPA) parameters of yoghurt bar formulations and commercial pocket cheeses
Table 6 Long description
Texture profile parameters are reported for three yoghurt bar formulations with increasing gelatine concentration and for full-fat and light pocket cheeses. Firmness rises across yoghurt bars from 3.40 newtons in YB6 to 23.87 newtons in YB12, and is highest in full-fat pocket cheese at 31.21 newtons; light pocket cheese is 22.89 newtons. Cohesiveness increases strongly with gelatine in yoghurt bars, from 0.11 in YB6 to 0.92 in YB12, while both pocket cheeses are lower than YB12 at 0.83 for full-fat and 0.79 for light. Gumminess follows a similar pattern, increasing from 2.50 newtons in YB6 to 19.07 newtons in YB12, with full-fat pocket cheese highest at 23.06 newtons and light pocket cheese at 16.11 newtons. Adhesiveness is near zero and similar for YB6, YB9, YB12, and full-fat pocket cheese, but light pocket cheese is more adhesive in magnitude at minus 0.0115 joules. Elasticity is highest for YB12 at 4.00 millimetres and lowest for the pocket cheeses at 2.78 millimetres for full-fat and 2.41 millimetres for light. Values are means with standard deviations, and letter superscripts indicate statistically significant differences within each column.
YB6, yoghurt bar with a 6.66% w/v gelatine dispersion; YB9, yoghurt bar with a 9.33% w/v gelatine dispersion; YB12, yoghurt bar with a 12% w/v gelatine dispersion, QPI, full-fat pocket cheese; QPL, light pocket cheese.
Values are expressed as mean ± standard deviation. Values within the same column that do not share common superscripts differ significantly according to Tukey’s test (p < 0.05).
An increase in gelatine concentration resulted in a significant increase (p < 0.05) in firmness, gumminess and elasticity of the yoghurt bars. This behaviour is expected, as gelatine contributes to the formation of a stronger and more cohesive three-dimensional gel network, enhancing resistance to deformation and elastic recovery. Adhesiveness, however, was not significantly affected by gelatine concentration, indicating that gelatine reinforcement did not promote excessive stickiness. The formulation with 12% (w/w) gelatine dispersion (YB12) exhibited the highest firmness and elasticity among yoghurt in bar, which is to obtain a yoghurt bar, producing a structure consistent with the intended product concept: a yoghurt-based snack suitable for handheld consumption, like a chocolate bar.
Compared with commercial pocket cheeses (QPI and QPL), yoghurt bars exhibited lower firmness and gumminess but higher elasticity. These differences reflect compositional and structural distinctions between products. Pocket cheeses are designed to withstand mastication and slicing, whereas yoghurt bars are structured to deform and partially melt during oral processing.
Cohesiveness, which describes the extent to which a product withstands deformation before rupture (Poiani and Montanuci, Reference Poiani and Montanuci2019), was higher in yoghurt bars than in pocket cheeses. This indicates that yoghurt bars require less mechanical force for disintegration and swallowing, contributing to a melting-in-the-mouth sensation. In contrast, pocket cheeses rely primarily on mastication for breakdown, requiring greater cutting force by the teeth.
Adhesiveness, defined as the work required to overcome the attractive forces between food and oral surfaces (Poiani and Montanuci, Reference Poiani and Montanuci2019), did not differ between yoghurt bars and full-fat pocket cheese. This result demonstrates that, despite their softer and more elastic structure, yoghurt bars do not leave a sticky residue in the mouth, which is a desirable sensory attribute.
Conclusion
This study developed an innovative yoghurt bar enriched with probiotic microcapsules, demonstrating its potential as a versatile and health-oriented functional food. The investigation revealed that gelatine concentration significantly influenced properties, such as texture and structural stability, without compromising the microbiological quality and probiotic viability. The evaluated bars maintained microbiological stability up to the 30 days of refrigerated storage for L. bulgaricus, S. thermophilus and L. acidophilus NCFM®. The yoghurt bars exhibited predominantly elastic behaviour and rheological properties dependent on both gelatine concentration and temperature. YB6 showed greater susceptibility to deformation compared to YB9 and YB12 under shear stress. Specifically, formulations with 9% and 12% gelatine dispersion achieved optimal balance in texture, protein content and storage stability, making them ideal for consumer-friendly, handheld consumption.
By expanding the scope of fermented dairy products, this research bridges a critical gap between consumer demand for convenience and the nutritional appeal of probiotics. The findings not only support the development of novel dairy-based bars but also provide a framework for enhancing the functional qualities of these products. Future applications of this work could extend to new innovations in probiotic delivery systems, catering to dietary preferences and health-conscious markets.
Acknowledgements
The authors acknowledge the financial support provided by CNPq, CAPES, FINEP, and FAPEMIG.
Competing interests
The authors declare that they have no competing interests.
