Encapsulation and dispersion of Lactobacillus acidophilus in a chocolate coating as a strategy for maintaining cell viability in cereal bars

Probiotic encapsulation efficiency

Preliminary tests evaluating the concentrations of sodium alginate (0.5%, 1%, 2% and 3%, w/v) in the preparation of the hydrogel showed that 2% (w/v) of sodium alginate resulted in uniform and resistant spheres, and allowed adequate flow in the apparatus used for extrusion (data not shown). In order to obtain hydrogel spheres of high cell density, a suspension of 15.5 log CFU mL⁻¹ L. acidophilus LA02 ID 1688 was added to the alginate solution in the encapsulation process. This strategy was used to circumvent possible reduction in cell viability during the crosslinking step of the alginate in calcium chloride solution. Actually, this approach resulted in a 1.63 log CFU decrease in the number of viable cells in the hydrogel spheres compared to the probiotic cells added to the cell suspension prior to alginate bead formation. The reduction is associated with the detachment and non-entrapment of cells during the extrusion process, as well as the loss of viability during the lyophilization process of the spheres. It is important to mention that during the immobilization process, high concentrations of calcium chloride (0.68 mol L⁻¹) were used for the crosslinking of sodium alginate, which may have contributed to cell injuries due to conditions of high osmolality. The freezing of the spheres prior to lyophilization may also have contributed to cell death.

Although cell death occurred, a high number of viable bacteria survived when entrapped in the hydrogel matrix (13.87 log CFU), which corresponded to an average encapsulation efficiency (EE) of 89.4%. Close values (82.80%) of efficiency were reported by Arenales-Sierra et al.¹³ when encapsulating Lactobacillus casei in calcium alginate spheres containing magnesium hydroxide, and following cell entrapment conditions similar to the present study. On the otherhand, lower values of EE were reported by Rather et al.¹⁶ when encapsulating Lactobacillus plantarum (EE: 72.48%) and Lactobacillus casei (EE: 62.54%) using the technique of double-layered spheres of calcium alginate.

It is important to note that different encapsulation techniques can promote different values for cell efficiency and viability. The cell viability of entrapped cells in hydrogels is associated with factors, such as osmotic stress conditions in the hydrogel crosslinking stage; the possibility of plasmolysis due to the formation of ice crystals during the freezing step prior to lyophilization; changes in the physical state of the membrane lipids and structures of proteins sensitive to lyophilization; changes in protein conformation during water removal; and cellular adaptability to the lyophilization process²¹. Other factors that can affect the probiotic encapsulation yield include the dimensions of the capsule, the nature of the wall material used, the microbial cell load, and the curing time in calcium chloride solution²².

Resistance of free and encapsulated probiotic cells to simulated gastric and intestinal conditions, and to heat treatment

Probiotics having beneficial health effects must reach the region of the large intestine (colon) where they will colonize, resisting the adverse conditions of acidic pH of the stomach and the bile salts of the upper portion of the intestine. The survival of the probiotic bacteria (Table 1) as free cells after exposure to simulated gastric juice (SGJ) for between 5 and 120 min, ranged from 92.55 to 47.4% (decrease of 4.87 log CFU), while the survival rate for the encapsulated probiotic cells ranged from 96.76 to 88.68% (decrease of 1.57 log CFU).

The cell encapsulation process contributed to the protection of the probiotic bacteria against the acidic conditions of the simulated gastric juice, maintaining close to 90% of viable cells over the 120 min of testing. It is interesting to note that, after one hour of exposure to SGJ, there was a reduction of only 4.6% in the viability of the encapsulated cells, in contrast to a 41% reduction in the viability of the free cells. Similar cell viability observations were reported by Arenales-Sierra et al.¹³ in gastric simulation tests of L. casei as free cells, and cells encapsulated in calcium alginate gel containing added magnesium hydroxide. They emphasized that the presence of magnesium hydroxide in the preparation of calcium alginate spheres contributed to the maintenance of a neutral pH inside the spheres, counteracting the acidic pH of the stomach, and hence increasing the viability of the probiotic cells.

Gu et al.²³, in a study on the encapsulation of Bifidobacterium pseudocatenulatum G7, reported that free cells became unviable after 2 h under simulated gastric conditions. In the same study, when magnesium hydroxide was added to sodium alginate, this resulted in enhanced cell protection and promoted a low reduction in cell viability. Similarly, they reported that the free cells showed less resistance to simulated intestinal juice. On exposure to SIJ for 120 min there was a marked reduction in cell viability of around 53% (from 9.26 to 4.38 log CFU). On the other hand, the encapsulated cells showed greater resistance (82.05% viability) with a 17.95% reduction in cell viability (from 13.87 to 11.38 CFU) after 120 min.

The survival rate (47.3%) of the free probiotic cells observed in our study after subjection to SIJ for 120 min was considered low (Table 1). To obtain cell counts considered of probiotic value in a food product containing Lactobacillus (10⁹ CFU, in Brazil), it would be necessary to use very high counts of free probiotic cells (> 10¹⁸ CFU g⁻¹) in the formulation of a cereal bar, considering the survival rate of the free bacteria. The data obtained are in agreement with those observed by Rather et al., (2017), who reported a very low survival rate of free cells of Lactobacillus plantarum NCDC201 when incubated in simulated intestinal juice, which was attributable to the low resistance of the cells to bile salts. The authors emphasized that microencapsulation of the probiotic cells contributed significantly to improve their survival on simulation to gastrointestinal conditions. Considering the reduction of cell viability in the free-form of probiotic cells along with gastric and intestinal simulation, a total reduction in the viable cell count of 7.16 log CFU was verified. This value corresponds to a reduction of 77% in cell viability, which would confer a load of viable cells with a cell count of approximately 2 × 10² CFU. The above observations would indicate that L. acidophilus LA02 ID 1688 cells in the free-form would not reach the intestine in adequate probiotic quantities. On the otherhand, after gastric and intestinal simulations the encapsulated cells maintained a viability of 72.8% (1.26 10¹⁰ CFU).

The thermal resistance of probiotic cells is an important quality parameter, as several technological food-processing stages involve the application of heating. In the present study, the encapsulated probiotic cells showed high resistance to heat (Table 1). After a 10-min exposure to heating, the encapsulated cells showed viabilities above 94% at 55 ºC, and 70% at 65 ºC. The free probiotic cells, by contrast, proved to be more sensitive to heat, which is clearly observed in the tests at 55 ºC for 10 min, and at 75 ºC for one minute. While heat treatment at 55 ºC/10 min. resulted in a reduction of 5.3% (0.74 log CFU) in cell viability, a 15% loss of viability (1.41 log CFU, three times higher) was found in the tests with the free cells. Similarly, heating to 75 ºC for 1 min, demonstrated that encapsulation of the probiotic cells provided 2.3 times greater protection, with a 17.95% reduction in cell viability, in contrast to a reduction of 40.6% exhibited by the free cells. Even under the most drastic condition of heat treatments (75 ºC/10 min.), encapsulation contributed to maintaining 32.6% of viable cells, while free cells showed only 11.9% viability. It is interesting to note that by this heat treatment condition, a count in the range of 10⁴ log CFU of encapsulated L. acidophilus LA02 ID 1688 cells was found.

Morphological aspects and FT-IR spectroscopy of the encapsulated probiotic cells

The lyophilized calcium alginate capsules containing the entrapped probiotic cells presented an average diameter of 1.73 ± 0.16 mm (p value > 0.05, p = 0.141), with no significant statistical variation between the particle’s dimensions, which indicates that the encapsulation technique employed promoted obtaining spheres of a regular size. The images of optical microscopy and scanning electron microscopy (Fig. 1) reveal a compact structure, free from apparent cracks or breaks and a rough surface with grooves. The roughness and grooves formed were due to the loss of water by sublimation in the process of lyophilization of the spheres, which promoted a deformation of the spherical structure obtained immediately after the process of crosslinking sodium alginate in the calcium chloride solution.

According to Bassani et al.²⁴ the appearance of grooves in alginate spheres can be attributed to the drying process. Dolly et al.²⁵ described that during the freeze-drying process of polysaccharide hydrogel spheres, ice crystals occur due to the low temperatures to which these spheres are subjected in their preparation for lyophilization. After sublimation of these crystals under reduced pressure, a dry and porous matrix similar to a sponge is formed. Fareez et al.²⁶ related the irregularity of the surface on the spheres to a higher concentration of polymer in specific areas of the encapsulated spheres.

The absence of cracks or ruptures on the external surface of the encapsulated spheres can contribute to greater protection of the internalized probiotic cells against permeability of gases, liquids or other materials, such as bile salts and stomach acid, as these features are directly related to loss of cell viability during the passage through the gastrointestinal tract. Figure 1g shows that the capsules have a macroporous internal structure, which is essential for trapping a larger number of probiotic cells. The formation of larger pores and cavities may be related to the use of magnesium hydroxide in the process to obtain hydrogel particles. Arenales-Sierra et al.¹³ reported that the use of magnesium hydroxide led to the production of spheres with larger pores and a less rigid alginate network, indicating that the presence of magnesium hydroxide affects the crosslinking of sodium alginate by calcium chloride.

The probiotic cells were entrapped inside the pores of the freeze-dried capsules as best seen at the 600× (Fig. 1h) and 1200× (Fig. 1i) magnifications. Apparently not all the pores of the macroporous structure of the calcium alginate matrix were filled with the probiotic cells, as can be seen in Fig. 1g. This aspect is possibly associated with the encapsulation efficiency of the hydrogel (89.4%) which, although high, many cells detached from the capsules during the encapsulation process, and therefore some pores were not filled with the probiotic cells.

It is noteworthy that a possible strategy for a better use of the macroporous structure of the matrix would be to cultivate the capsules containing the probiotic cells in MRS broth for cell proliferation within the pores before lyophilization of the capsules. This procedure could possibly allow obtaining capsules with a higher cell density.

The FT-IR spectra of hydrogel capsules with the entrapped probiotic cells (Fig. 2) showed typical polysaccharide vibrational bands with alginate characteristic bands in the regions of 3225 cm⁻¹, 1593 cm⁻¹, 1417 cm⁻¹, 1012 cm⁻¹ and 816 cm⁻¹. The band in the region of 3225 cm⁻¹ is attributed to the stretching vibration of the hydroxyl groups (–OH) and the strong intensity of this band is due to the presence of many hydroxyl groups in the alginate structure. The weak intensity band in the region of 2905 cm⁻¹ is attributed to the asymmetric stretching vibration of the CH₃ and CH₂ groups¹⁶. The band at 1593 cm⁻¹ is related to the asymmetric stretching vibration of the connection between C–O of the COO– alginate group²⁷. The intense absorption at 1593 cm⁻¹ is also related to amide band I (stretching vibration C=O) of the functional groups of endogenous proteins ¹⁶, and vibration of amide II (C–N–H angular deformation in the plane and C–N stretch of probiotic cell proteins)¹³. The band at 1417 cm⁻¹ is related to the symmetrical stretching vibration of the carboxyl group (C(=O)OH) (Nissola et al.²⁸). The band at 1012 cm⁻¹ is related to the symmetrical and asymmetric stretching of the C–O and C–O–C groups^13,16,29. The absorptions in the regions between 1200 and 900 cm⁻¹ can be attributed to the symmetric phosphorus-oxygen (P-O) stretching vibrations of the phosphodioxy group (PO₂⁻) found in nucleic acids, and the vibration of C–O–C deformation of the polysaccharides belonging to cell membrane glycoproteins and lipopolysaccharides of the probiotic cells^16,30. The band at 816 cm⁻¹ is in a region (900 to 700 cm⁻¹) called the true fingerprint region and contains very specific and weak spectral patterns of aromatic ring vibrations of aromatic amino acids (tyrosine, tryptophan, phenylalanine) and nucleotides³⁰. Absorptions in this region are related to the presence of cellular material present in the encapsulated probiotic cells.

Proximal and nutritional composition of Pereskia aculeata leaf flour and the formulated cereal bar

High contents of proteins (21.4%), dietary fiber (39%) and minerals (16.1%) were present in the flour of P. aculeata leaf (Table 2). P. aculeata leaves have been mentioned in the scientific literature as an interesting nutritional source, as they contain protein amounts (26% w/w) far higher than other vegetables commonly used as foods, such as beans, corn and cabbage³¹. The proteins of P. aculeata leaf flour are considered of good quality, presenting 85% digestibility^32,33. However, according to Zem et al.³³, P. aculeata flour provided as a single source of protein is inadequate for growth, although it is relevant for the maintenance of protein metabolism. According to these authors, it is a source of good quality protein due to the presence of few limiting essential amino acids, and meets the requirements of a diet for humans as recommended by FAO/WHO. Considerable levels of essential amino acids were present in the flour studied, viz., leucine (65.42 mg g⁻¹), valine (48.83 mg g⁻¹), lysine (48.13 mg g⁻¹) threonine (44.86 mg g⁻¹) isoleucine (37.15 mg g⁻¹), tryptophan (18.93 mg g⁻¹) and histidine (18.69 mg g⁻¹) (Table 2).

The amino acid composition and the chemical score (CS) were compared for essential amino acids recommended by FAO/WHO for children aged between 6 months to 3 years³⁴. The findings indicated that the sulfur amino acids (methionine and cystine, CS: 52.6) were the limiting amino acids. By contrast, tryptophan (CS: 222.6) was the essential amino acid with the highest CS and leucine (65.42 mg g⁻¹), an essential amino acid was present in higher amounts.

Leucine and tryptophan are amino acids that can influence weight reduction, since they can act on controlling appetite^35,36,37. Another positive aspect that should be mentioned is the low-fat content (1.65 g 100 g⁻¹), with 66.1% of the total fat content corresponding to unsaturated fatty acids (Table 2).

P. aculeata flour was used as an ingredient in the cereal bar formulation with the main purpose of nutritional enrichment of the product, due to its high content of good quality protein and minerals. Most of the cereal bars available on the market are products designed to provide energy due to their carbohydrate content, and satiety due to their high fiber content. However, in recent years, the market has demanded more nutritionally complete products and, in this sense, the use of ingredients rich in proteins has been a good option.

Thus, P. aculeata flour was used as a proposal for a new low-cost protein ingredient in a high value-added product. There are still few reports in the literature about the industrial use of this unconventional edible plant that has great potential for use.

The formulated cereal bar showed high levels of carbohydrates (71.81 g 100 g⁻¹), dietary fiber (19 g 100 g⁻¹) and lipids (12.63 g 100 g⁻¹), as well as a relevant protein content (5.44 g 100 g⁻¹) (Table 3).

Carbohydrates were the components present in greater amounts in the formulated cereal bar, which arises from the addition of the agglutinating syrup used (cane molasses and brown sugar) (Table 3). Other sources of carbohydrates such as cereals (rice and oats) that contain β-glucans and hemicelluloses, and flour from P. aculeata and green banana (a source of resistant starch), as well as the chocolate coating also contributed to the total carbohydrate content in the formulation. In this context, it is worth mentioning cereal bars are products that are frequently consumed as sources of energy.

The fiber content present (19 g 100 g⁻¹) characterizes the cereal bar as a product with an increased content of dietary fiber according to Brazilian legislation^38,39. The 20-g portion of the product (i.e., one cereal bar) corresponds to 15.20% of the daily reference value (DRV) of dietary fiber intake based on a 2000 kcal diet.

The cereal bar showed a total fat content of 12.63 g 100 g⁻¹, which corresponds to a DVR of 3.89% per 20 g portion based upon a 2000 kcal diet. The fats present in the bar comes from the chocolate coating (72% cocoa), as well as from the nuts used as an ingredient in the formulation. In addition to the nutritional quality, the probiotic potential of the product developed should be highlighted. The viability of the probiotic cells incorporated into the cereal bars was monitored over a period of 120 days storage at 25 ºC, and the results are presented in the Table 1 (see Supplementary Table S1 online).

The association of the probiotic encapsulation technique and the covering of the food bars with dark chocolate (72% cocoa) contributed to the maintenance of the viability of the probiotic cells during the storage period. In this context, Hossain et al.⁴⁰ pointed out that the association of a chocolate coating with microencapsulated probiotic strains can be an excellent solution to protect the probiotic cells from environmental stresses, as our work also demonstrated.

A high percentage of cell survival was observed throughout the storage period (see Supplementary Table S1 online). Shortly after the incorporation of probiotic into the chocolate topping, a small reduction in cell viability (2%) was observed, which is possibly associated with possible physiological stress, which may be related to the temperature of the chocolate coating syrup (45 ºC to 50 ºC). However, throughout the storage period, the probiotic cells maintained high viabilities. After 60 and 90 days of storage, cell viability was maintained at levels close to 95% (10¹³ CFU) of the initial content of the probiotic cells incorporated into the product. At the end of the 4^th month (120 days), 90% of the probiotic cells added to the cereal bar was observed to remain viable, conferring a high probiotic potential on this food product (10¹² CFU).

Instrumental texture profile of the cereal bar

In the shear test an average force of 61.43 N was recorded, which represents the stress/force exerted when cutting the sample. As shown in Table 4, during the application of the shear force there were variations of intensity between 53.28 N and 77.25 N. Such variations are possibly associated with the composition of the product, which contains ingredients with different textures and, therefore, possess different resistances to cutting.

The instrumental test of hardness in food expresses the maximum compression force applied until the sample is deformed and/or ruptured. The maximum deformation force verified in the studied cereal bar was 161.02 N. Similar values of hardness were reported by Munhoz et al.⁴¹ in a cereal bar containing fruit pulp and bocaiuva almonds. These authors mentioned that products prepared with a high fiber content tend to result in denser and harder products, which we did not observe in the present work.

Adhesiveness represents the energy needed for food to separate from other materials. The cereal bar produced presented adhesion values that ranged from −1.80 to −0.77 N mm, with an average value of − 1.25 N mm. The low adhesion values may be associated with the chocolate that coated the product, as well as the presence of flours from green banana and P. aculeata that absorbed part of the agglutination syrup.

The elasticity parameter corresponds to the percentage of product recovery when deformed, i.e., the ability of a product to return to its original state after compression by the teeth⁴². The food bar had an average elasticity of 39.84 mm, with variations between 12.90 and 70.73 mm, which can be justified by the diversity of ingredients present in the formulation. The cohesiveness of a material represents the extent to which a material can be deformed before it breaks. This is related to the degree of compressibility that a material resists to the breaking point^42,43. The cohesiveness value of the studied food matrix remained in the range of 0.03 to 0.08, indicating that the cereal bar did not present a high deformation capacity before rupture. This behavior may be associated with the presence of the flours (P. aculeate and green banana), which absorbed part of the agglutination syrup, making the food matrix more compact. The high fiber content also possibly contributed to a lower cohesiveness of the sample.

Chewability and gumminess correspond to the energy needed to perform the process of mastication and disintegrating a solid or semi-solid food, respectively, reducing its consistency to levels suitable for ingestion⁴³. The chewiness and gumminess values were 3.94 N and 8.11 N, respectively. Values were lower than those reported by Muniz et al.⁴⁴.