3.1 Viability and pH of free and microencapsulated L. lactis R7 in different food matrices during storage
In view of improving the stability of probiotic bacteria, microencapsulation is presented as one of the most efficient solutions, not only for maintaining cell viability during processing and storage, but also to guarantee its activity in the digestive tract . The microcapsule used in this study has previously been characterized , presenting adequate size (12.73µm) and morphology for application in food.
The results show that the acidity of blueberry juice (pH 3.0 ± 0.1) (Figure 1 a) had damaging effects on free cells, resulting in viability loss below the minimum value to be considered probiotic  after 14 days of storage at 4 °C, as well as a marked reduction in cell viability (3 log CFU.mL-1) observed at 7 days of storage.
When the microcapsules added to the blueberry juice were analyzed, the concentration of viable cells was 5.00 ± 0.50 log CFU.mL-1, indicating that the cell content was exposed from the loss of capsule integrity. This effect is perceived up to 14° days of storage at 4 °C when it presents a cell concentration of 10.30 ± 0.25 log CFU.mL-1, with loss of viability only at 21 days.
Thus, this demonstrates that the exposure time to the acidity of blueberry juice is determinant for the loss of microcapsule integrity and microorganism exposure, corroborating the results presented by Rosolen et al. . Miranda et al.  showed that microencapsulation of Lactobacillus casei with sodium alginate had no protective effect on orange juice (pH 3.74-3.92) when compared to free cells.
The pH variations were observed (Figure 1a) and no statistical difference was noted during the analyzed period, indicating that the presence of metabolizable substrates in the blueberry juice was not sufficient to guarantee the viability of L. lactis R7 free cells in an acidic environment over the long term. It needs to be emphasized that the viability of probiotics in fruit juices is affected by strain, microbial culture preparation method, inoculated cell status, storage temperature, oxygen level and fiber presence . Mokhtari et al.  evaluated pH changes in grape juice and found that Lactobacillus acidophilus-free cells had greatest reduction in pH during 60 days of storage at 4 °C decreased significantly from 3.8 to 3.21 (p<0.05).
Regarding free cells incorporated into milk (Figure 1b), there was a reduction in cell concentration of 3.08 log CFU.mL-1 at the end of 28 days of storage, while for microcapsules it remained stable (p < 0.05). The physicochemical characteristics of milk associated with pH near neutrality were sufficient to promote the maintenance of microcapsules in the storage. Shi et al.  obtained similar results for storing Lactobacillus bulgaricus microcapsules in milk at 4 °C, with complete preservation for 30 days. A reduction of free cells was reported from 10 log CFU.mL-1 to 6.88 log CFU.mL-1 over the period, showing that microencapsulation significantly increases bacterial stability in refrigerated systems.
Free cells promoted higher acidification (p < 0.05) in milk when compared to microcapsules, demonstrating the ability of L. lactis R7 to use naturally occurring disaccharides in milk . The lower acidification of dairy products containing microencapsulated microorganisms suggests that the encapsulation process was effective in physically trapping the material of interest [11, 26].
Due to the high fat content, the milk cream (Figure 1c) proved to be an adequate delivery vehicle for probiotic bacteria, given the high content of total solids, which helps to maintain cell viability. Free cells reduced 1.00 log CFU.mL-1 over the first 7 days and were stable at 8.70 log CFU.mL-1 (p > 0.05) during 28 days of storage.
Microcapsules incorporated into the milk cream had a cell concentration of 8.48 log CFU.mL-1 from day 7, remaining stable during 28 days of storage. Considering that there are no reports in the literature using the matrix of the present study, the research was compared to the research for Vasile et al.  which used as a food matrix for soft cheese. For this, the cell viability of Lactobacillus casei 461 was evaluated for storage at 4 °C and a reduction of 2 cycles logarithmic (5.47 log CFU.g-1) was observed at the end of 14 days, not having minimum probiotic viability (<6 log CFU.g-1). The microcapsules showed an increased 0.5 cycles at the end of the same period indicating concentrations lower than the present study, which demonstrates that the combination of the materials used (inulin and serum) associated with the technique spray drying was effective in protecting L. lactis R7.
The pH near the neutrality of the milk cream, as well as the milk, was not sufficient for the total loss of microcapsule integrity and exposure of L. lactis R7 in the matrix. The pH values of free and microencapsulated cells were significantly different (p < 0.05), being 1.68 and 0.94, respectively.
It is worth highlighting that viability data for individual food matrices are available for some probiotic strains [7, 28–30]. However, comparing multiple matrices with respect to storage-free cell viability [17, 31] under similar conditions is less frequent  and, the occurrence is even lower for microencapsulated cells under the same conditions .
3.2 Survival assessment of free and microencapsulated L. lactis R7 applied in blueberry juice, milk, and milk cream when exposed to simulated gastric fluid
Different behaviors were observed among L. lactis R7 free and microencapsulated cells when added to blueberry juice, to milk, and to milk cream matrices and then submitted to gastric simulation, as shown in Figure 2.
When added to blueberry juice (Figure 2a), free cells do not show probiotic viability  after 60 min of gastric simulation at pH 2.0 and 2.5, not being detected at all conditions from 120 min on, suggesting their sensitivity to HCl and pepsin in gastric juice . However, the microcapsules showed high cell concentration after 120 min of gastric juice, evidencing the protection conferred by the encapsulating material. Different studies report that whey and milk proteins have technological properties such as buffering capacity, good emulsification and ability to form networks even at low concentrations, ensuring good survival during digestion [19, 34]. Added to this is the presence of inulin as an encapsulating material, which due to its low solubility in water may result in a longer time for powder rehydration and consequently a slower release of encapsulated bacterial cells . Amakiri and Thantsha  noted that the addition of inulin as encapsulating material improved Bifidobacterium longum Bb46 performance during gastric fluid exposure to free cells.
Free and microencapsulated L. lactis R7 cells inserted in milk (Figure 2b) showed similar behavior (p > 0.05) when evaluating resistance at 30 min of gastric simulation at all conditions. However, after 120 min, there was loss of capsule integrity and microorganism exposure (> 10 log CFU.mL-1) were observed at all pH levels, demonstrating that stress conditions (pH and enzymes) promote loss of microcapsule integrity.
The ability to tolerate digestive stress is one of the important properties for incorporating probiotics into food matrices, as food can protect the microorganism from gastric fluids . With the milk cream (Figure 2c) as carrier matrix, it was observed that the microcapsule showed high viability (> 11.65 log CFU.ml-1) when compared to the free cell (> 7.00 log CFU.ml-1) (p<0.05) at all times and pH values analyzed. Values below that found in the present study were reported by Martins et al.  who analyzed the viability of the passage to the gastrointestinal tract in vitro of free cells Lb rhamnosus applied to goat cheese. The authors observed that in 7 days of storage at 4 °C the cell concentration reduced 4.8 log CFU.g-1 after 120 minutes (pH 2.33) no minimum probiotic count.
The results support the hypothesis that the application of microencapsulated probiotics in food matrices may represent a strategy for the promotion of acid pH tolerance during gastric tract passage .
3.3 Survival assessment of free and microencapsulated L. lactis R7 exposed to simulated intestinal fluid
The survival of L. lactis R7 free and microencapsulated cells in the different food matrices was analyzed during 4 h of exposure to intestinal fluids in the absence (Figure 3a) and presence of bile salts (Figure 3b).
Free cells presented the lowest cell concentrations when compared to microencapsulated cells in all food matrices (p < 0.05), showing the highest sensitivity in the presence of bile salts. The antimicrobial nature of bile salts is related to its detergent property, which dissolves microorganism membranes, and its amphiphilic nature makes its strong inhibitory for the gastrointestinal tract .
Still, exhibited the best performance as a carrier for free L. lactis R7, with cell concentration in the absence and presence of bile salts of 9.3 ± 0.30 and 8.18 ± 0.18 log CFU.mL-1 respectively. The other matrices had lower viability as free cell carriers, but still had minimum probiotic value (< 6 log CFU.mL-1). Different studies have shown that food matrix has significant influence on in vitro gastrointestinal tolerance of different probiotics exposed to low pH and bile salts [19, 39].
The microencapsulated microorganism when submitted to intestinal fluids in the absence of bile salts presents lower cell concentrations when compared to the presence of bile salts, especially when applied in blueberry juice. The presence of pancreatin and bile salts are determinant for the loss of capsule integrity and exposure of L. lactis R7.
The action of enzymes and bile salts is controlled by the ability to identify emulsion interfaces, which is controlled by the size of the emulsion and interfacial composition, that is, its structure, thus impacting the type of food matrix in which the probiotic is inserted and its viability .
Although L. lactis R7 did not have intestinal origin, the high survival can be explained by the potential of some bacteria as antagonists to specific adverse environments. Bacteria can respond to changes in the environment via metabolic reprogramming, leading to increased resistance . The findings of the study showed that microencapsulation with whey and inulin may increase the protection of L. lactis R7 when submitted to gastric and intestinal fluid survival tests, as well as ensure better cell protection regardless of the physicochemical characteristics of the carrier matrix .
3.4 Evaluation of the thermal resistance of free and microencapsulated L. lactis R7 in milk cream, milk and blueberry juice
For probiotic cells to be effective and remain viable in food and beverages, they must withstand the recommended pasteurization temperatures and/or other industrial processing parameters . The search for suitable materials that increase the thermal resistance of probiotics is also considered important, in order to facilitate their incorporation in food matrices. In addition, the developed encapsulation system should act as an isolation environment for probiotic cells .
Microencapsulation use in the protection of probiotics applied to heat-treated food matrices has been described by different authors as an indispensable technique for application in the food industry [20, 41].
Regarding the formulation and/or preparation of food products using heat, the thermotolerance of microencapsulated L. lactis R7 was evaluated  according to the Table 1. Results showed the protective effect of microencapsulation when analyzing the thermal resistance, independent of the food matrix, compared to free cells. Free cells showed a reduction in viable cell count of 3.28 log cycles for blueberry juice and milk and of 2.98 log cycles for milk cream after 15 min treatment at 60 °C. After 30 min, viability was less than 6 log, showing no probiotic effect. Same behavior was observed at 65 °C, however no survival was observed after 10 minutes of exposure. It is noteworthy that L. lactis R7 free, at 70 °C, did not present viability at all times analyzed. This corroborates the results obtained by Pinto et al. , who found a decrease in Bifidobacterium BB-12 free cells by 2.57 log cycles after 5 min of heat treatment at 60 °C. The excessive heat affects the structure of macromolecules such as proteins and nucleic acids of bacterial cells, causing the breakdown of the bond between monomeric units and destruction of monomers, leading to cell death .
When applied to L. lactis R7 microcapsules in blueberry juice, after 30 min of heat treatment at different temperatures, increased cell viability was observed, demonstrating that the time/temperature binomial was effective in disintegrating encapsuling material and exposure of the microorganism, but without cellular damage. For milk, it was observed that at 60 °C there was no disruption and total exposure of the microorganism after 30 min, since the concentration of viable cells increased as a function of exposure time. At 65 °C high cell viability was observed after 30 min, unlike the temperature of 70 °C, at which cell viability decreased as exposure time increased.
In milk cream, high cell concentration was observed after 30 min at 65 °C and after 15 min at 70 °C. As far as we know there are no reports in the literature of microencapsulated probiotics applied to food matrix and tested for thermal resistance. For comparative purposes the study by Malmo et al.  microencapsulated Lactobacillus reuteri DSM17938 by spray drying and used alginate and chitosan as wall materials, applying free and microencapsulated cells in chocolate soufflé. The authors observed when submitting the matrix to cooking at 180 °C by 10 min (80 °C inside the dough), a survival rate of 10% of microencapsulated cells, thus not obtaining a probiotic product. The same authors report that the disintegration/collapse of microcapsules after treatment at 80 °C led to the release of cells with their consequent cell death. Unlike the present study in which the highest temperature evaluated was not able to promote total disruption of the microcapsule and exposure of the microorganism since high probiotic concentrations were observed in juice and milk cream.
Some specific genes and/or proteins are related to the tolerance of heat probiotics. It has been reported in the literature to improve this tolerance with treatments that expose these microorganisms to moderate heat, as it occurs with the microencapsulation technique [43–45]. The results obtained in the present study indicate the need for L. lactis R7 microencapsulation when the food is subjected to heat treatment, such as the pasteurization process applied to various foods. According to Tárrega et al. , the high polymerization inulin used in the present study is thermally stable and poorly soluble in water, thus offering greater protection to heat treatment.
In conclusion, food matrices such as blueberry juice, reconstituted milk, and milk cream are suitable for maintaining the viability of microencapsulated L. lactis R7, using inulin and whey encapsulants stored at 4 °C. Furthermore, regardless of the food matrix, the encapsulating material influenced the protection of microcapsules under the conditions of the simulated gastrointestinal tract and thermal resistance. Therefore, the results show that L. lactis R7 microcapsules have potential for application in different matrices and in the development of new probiotic products using thermal processing.