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Article

Enhancing Soy Yogurt with Microencapsulated Limosilactobacillus reuteri: Viability and Sensory Acceptability

by
Ricardo H. Hernández-Figueroa
,
Yani D. Ramírez
,
Aurelio López-Malo
and
Emma Mani-López
*
Departamento de Ingeniería Química, Alimentos y Ambiental, Universidad de las Américas Puebla, Santa Catarina Mártir S/N, San Andrés Cholula, Puebla 72810, Mexico
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(8), 423; https://doi.org/10.3390/fermentation11080423
Submission received: 17 June 2025 / Revised: 11 July 2025 / Accepted: 17 July 2025 / Published: 22 July 2025
(This article belongs to the Special Issue Emerging Microbial Technologies in Fermentation: Innovations in Food, Environmental, and Health Bioprocesses)
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Abstract

This study aimed to microencapsulate Limosilactobacillus reuteri DSM 17938 to enrich soy yogurt flavored with peach jam. The effect of three concentrations of alginate and coating chitosan were evaluated in terms of probiotic viability, and the physicochemical and sensory properties of soy yogurt. Lim. reuteri was microencapsulated in alginate (1, 2, and 3%) and coated with chitosan (0, 0.4, and 0.8%). Soymilk was fermented using Lactobacillus bulgaricus and Streptococcus thermophilus. Soy yogurt was combined with probiotic beads and peach jam and stored for 27 days at 4 °C. The pH, titratable acidity, and probiotic viability of probiotic peach soy yogurt (PPSY) were determined during storage. Alginate at 3% and alginate (2%) coated with 0.4% chitosan maintained probiotic counts at 8 and 7.5 log CFU/g after 27 days. The pH of PPSY decreases rapidly and drastically during storage when probiotic-free cells are added. The PPSY containing alginate (3%) beads, alginate (2%) coated with chitosan (0.4%), and probiotic-free cells had a similar level of acceptance in color, texture, and odor (p > 0.05), while flavor and overall acceptability were significantly higher (p < 0.05) in PPSY with probiotic beads. These findings support the use of microencapsulation strategies in developing functional plant-based probiotic foods.

1. Introduction

Limosilactobacillus reuteri, previously classified as Lactobacillus reuteri, is a lactic acid bacterium (LAB) commonly found in the gastrointestinal tract of humans and various animals, including sheep, chickens, and rodents [1]. One of its distinguishing features is the production of reuterin, a broad-spectrum antimicrobial compound synthesized during glycerol fermentation, which provides a competitive advantage within the gut microbiota. Among probiotic species, Lim. reuteri stands out due to its well-documented ability to adhere to intestinal epithelial cells, facilitating colonization and sustained activity in the host [2]. Selected strains of Lim. reuteri have been recognized as probiotics due to their demonstrated ability to support gut health, modulate the immune response, lower serum cholesterol levels, enhance epithelial regeneration, improve intestinal barrier function, modulate the gut microbiota and their metabolites, and attenuate inflammation [1,2,3]. These functional attributes have encouraged the incorporation of Lim. reuteri into various functional food products [4]. However, the viability of Lim. reuteri can be compromised when they are incorporated into food matrices, especially during processing, storage, and gastrointestinal transit.
To address this challenge, microencapsulation has emerged as an effective technique for protecting probiotic cells and enhancing their stability in food systems [5]. Various encapsulation materials, including sodium alginate, chitosan, starch, and protein-based matrices, have been explored for their protective capacity. For example, Naklong et al. [6] reported that encapsulating Bifidobacterium breve in green soybean milk using alginate and calcium lactate significantly enhanced both encapsulation efficiency (99.8% ± 0.07%) and cell survival under simulated gastrointestinal conditions. Similarly, Fu et al. [7] demonstrated an improvement in the stability of Lim. reuteri when they were encapsulated in matrices derived from Maillard reaction products of soy protein isolate and lactose. These matrices also enhanced functional characteristics such as emulsification and gel strength. Various reports [6,8] confirm that combining alginate with polysaccharides or proteins, such as starch, locust bean gum, or whey protein isolate, improves probiotic survival during processing and in gastric conditions. These findings highlight the importance of optimizing encapsulation systems, particularly for plant-based matrices such as soymilk, to ensure the viable delivery of probiotics to consumers. Sodium alginate remains one of the most widely used encapsulating materials for probiotics due to its biocompatibility, flexibility, low cost, and capacity to maintain probiotic viability under adverse conditions [9,10]. Alginate beads can enhance the survival rates of probiotics by up to 80–95% under gastrointestinal stress [6]. Higher concentrations and larger bead sizes further enhance cell retention [11]. The combination of alginate and chitosan in encapsulation systems offers improved probiotic protection compared with using either biopolymer alone. Alginate provides a mild, biocompatible matrix that supports initial cell entrapment; however, its structure can be compromised at a low pH. When coated with chitosan, a polyelectrolyte complex is formed, significantly enhancing the bead’s resistance to gastric acid and providing a controlled-release profile. Moreover, chitosan’s properties may further enhance the delivery and function of probiotics [12]. Several studies have demonstrated that alginate–chitosan microcapsules exhibit greater structural integrity, higher encapsulation efficiency, and improved viability of probiotics compared with alginate or chitosan alone [13,14,15].
Soybeans (Glycine max) are rich in proteins, lipids, carbohydrates, vitamins, minerals, and biologically active compounds such as isoflavones, soyasaponins, phenolic acids, and sterols [16,17]. They also contain anti-nutritional factors, such as phytates, trypsin inhibitors, and lectins [18]. Fortunately, fermentation with LAB can significantly reduce or eliminate these components, thereby improving the nutritional and sensory quality of soy-based products [18,19]. Consequently, fermented soy products, such as soy yogurt, offer a promising vehicle for probiotic delivery, particularly for individuals who avoid dairy due to lactose intolerance, milk protein allergies, or personal dietary preferences [6].
Traditionally, dairy products have been the primary carriers of probiotics, but there is a growing interest in using them in other products as well [20]. This includes non-dairy probiotic products, such as soy-based foods, cereals, and bars [20]. Numerous studies have explored the performance of probiotics in soy-based matrices. Champagne et al. [21] investigated the fermentation of soy beverages by various lactic acid bacteria strains. They found that the performance of probiotic cultures, such as Lactobacillus helveticus and Bifidobacterium longum, was influenced by co-culturing with Streptococcus thermophilus and fermentation conditions. Additionally, Gu et al. [22] demonstrated that co-fermentation of glycerol and fructose by Lactobacillus reuteri (currently Lim. reuteri) in soy yogurt enhanced vitamin B12 biosynthesis, addressing a common nutritional limitation in plant-based diets. Other probiotics were also used as cultures or co-cultures in soy yogurt fermentation, including Lactobacillus acidophilus LAFTI L10, Lactobacillus casei LAFTI L26, and Bifidobacterium animalis subsp. lactis LAFTI B94 [23]; L. casei Zhang, B. animalis ssp. lactis V9, L. acidophilus NCFM, Lactobacillus rhamnosus GG, B. animalis Bb12, and L. casei Shirota [24]; L. acidophilus NCDC 15 and B. bifidum NCDC 235 [25]; Lactiplantibacillus plantarum ATCC 14917, Lacticaseibacillus casei DSM 20011, L. acidophilus ATCC 20552, Lactococcus thermophilus DSM 20259, and Bifidobacterium longum B41409 [26]; and Lacticaseibacillus rhamnosus GG [27]. Recently, Jiang et al. [28] and Nanyondo et al. [29] have developed soy yogurt formulations with favorable sensory, physicochemical, and probiotic profiles using different strains and flavoring agents such as soursop or added sugars. Lim. reuteri DSM 17938 is a well-studied probiotic strain (formerly L. reuteri ATCC 55730) [30,31]. It survives gastrointestinal transit, adheres to intestinal cells, and modulates immune responses, as proven (in vitro and in vivo) [32]. It also demonstrates good viability in various food matrices, including dairy, fermented vegetables, and beverages. Its tolerance to processing and storage stability makes it ideal for functional foods [1,2]. To our knowledge, no previous studies have reported on the incorporation of the encapsulated probiotic Lim. reuteri into soy yogurt. Previous research has investigated other encapsulated probiotics such as B. breve TISTR 2130 microencapsulated in sodium alginate + calcium lactate to improve its viability in green soybean yogurt [6]; two vaginal probiotics (Lactobacillus crispatus BC4 and Lactobacillus gasseri BC9) were encapsulated in soy beverages by spray-drying and added into soymilk to produce fermented soy beverages [33]; and B. animals BB-12 encapsulated in sodium alginate + xanthan gum to supplement grape-flavored soy beverages [34].
Given the increasing consumer demand for functional and plant-based foods, particularly those enriched with probiotics, soy yogurt is a promising matrix for innovation [35]. Incorporating microencapsulated probiotics into soy-based yogurt formulations may thus represent a viable strategy for developing high-quality, plant-based functional foods with health-promoting benefits. Microencapsulated Lim. reuteri aims to improve probiotic viability and offers potential for enhancing the final product’s physicochemical stability and sensory appeal. Additionally, fruit-based flavorings, such as peach jam, may improve consumer acceptability of soy yogurt. Therefore, in this study, a probiotic soy yogurt enriched with microencapsulated Lim. reuteri DSM 17938 was developed and evaluated. The effects of different concentrations of alginate–chitosan as encapsulating agents on probiotic viability, the physicochemical properties of soy yogurt, and the sensory acceptance of probiotic peach soy yogurt were assessed.

2. Materials and Methods

2.1. Materials

Commercial powdered soymilk (Symkën®, lot 73720), sugar (Potrero SA, lot F-0178174), and preservative-free peach jam (peach, agave syrup, agave inulin, and citric acid) were purchased in a local supermarket in Puebla, Puebla, Mexico. Freeze-dried starter culture “YO-MIX” containing a mixture of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (Danisco Culture, D-25889) of direct vat was used. The probiotic Lim. reuteri DSM 17938 was obtained from a chewable tablet (BioGaia, Lund, Sweden). Sodium alginate was obtained from FMC BioPolymer (Haugesund, Norway), medium-molecular-weight chitosan (Sigma-Aldrich, St. Louis, MO, USA), trisodium citrate dihydrate (Jungbunzlauer, Basel, Switzerland), and calcium chloride (CaCl2, RBM, Puebla, Mexico).

2.2. Proximal Analysis and Physicochemical Properties of Soymilk

The proximal composition of reconstituted soymilk (9 g of powder dissolved in 91 g of water according to the manufacturer’s recommendations) was determined following AOAC [36] methods 927.05 for moisture, 930.29 for protein, 945.46 for ash, and 989.05 for fat. Total carbohydrates were calculated by difference. The pH was measured by immersion of the electrode coupled to a pH meter (Hanna Instruments HI 2210, Woonsocket, RI, USA). The °Brix was analyzed using a portable refractometer 0–90 °Brix scale (Atago, Tokyo, Japan). For TA (%), 10 mL was titrated with 0.1 N NaOH, and the acidity was expressed as a percentage of oxalic acid. Oxalic acid is the major acid in fresh soymilk [37]; thus, it was chosen to calculate the TA.

2.3. Lim. reuteri Encapsulation

The chewable tablet was suspended in de Man Rogosa Sharpe (MRS) broth (Difco, BD, Sparks, MD, USA) and incubated for 18 h at 37 °C. Afterwards, it was routinely subcultured in MRS broth at 37 °C for 18 h. Bacterial cells from 400 mL of MRS broth were obtained by centrifugation at 8000× g for 10 min at 4 °C (Marathon 21 K/R, Fisher, Pittsburgh, PA, USA) and washed twice with phosphate buffer (pH 7.0). Wet cells were refrigerated and used for encapsulation.
To investigate the optimal conditions for alginate encapsulation and chitosan coating to maintain the viability of Lim. reuteri during soy yogurt storage, a factorial design 23 was tested. Table 1 shows the levels of alginate and chitosan evaluated for the viability of Lim. reuteri beads. For each condition, 100 mL of water was heated (~75 °C) to dissolve the alginate. The alginate was added slowly to avoid the formation of lumps and achieve complete suspension. Then, sodium citrate (0.5%) was added, and the temperature was maintained with vigorous stirring for 5 min. The mixture was cooled to 40 °C to prevent affecting the viability of the microorganisms, and the wet cells were added at a concentration of 109 CFU/mL. Approximately 3 g of the mixture was loaded into a 10 mL syringe using a 32 mm length needle with an internal diameter of 0.9 mm. The syringe was adapted to a piston pump (Cole-Parmer, Vernon Hills, IL, USA) and was pumped into a 30 mL CaCl2 solution (1 M) at a flow rate of 0.1 mL/min as previously described [38]. The dropping height was 5 cm. The beads remained in CaCl2 for 30 min for curation, after which they were recovered, rinsed twice with distilled water, and excess water was removed with paper tissue.
A chitosan suspension was prepared using 100 mL of sterile distilled water; it contained 0.4 or 0.8 mL of glacial acetic acid and 0.6 mL of olive oil. The mixture was homogenized for 15 min, and the pH was adjusted to 5.7–6.0 with 1.0 M NaOH. Afterwards, alginate beads were coated with chitosan using a bath of alginate beads prepared as described above. Alginate beads were added to the chitosan suspension and stirred for 40 min at room temperature (22 ± 2 °C). Later, the beads were drained, washed twice with sterile water, and placed on paper tissue to remove excess water. The experiments were performed in triplicate.
The size of the beads was determined by taking twenty-five beads of each encapsulated combination. A digital micrometer (Mitutoyo Corporation, Kanagawa, Japan) was used to measure them. The encapsulation yield was determined by analyzing the probiotic’s viability before and after encapsulation. One gram of dispersion (free cells + alginate suspension) and one gram of alginate and alginate coated with chitosan were put into 9 mL of sterilized sodium citrate solution (1%) to dissolve the beads. Adequate 10-fold dilutions were cultured on MRS agar (Difco, BD, Sparks, MD, USA). Plates were incubated anaerobically at 37 °C for 72 h, and colonies were counted. Encapsulation yield was calculated as follows: % EY = (N/N0) × 100, where N is the count after encapsulation and N0 is the count of the alginate cell suspension.

2.4. Soy Yogurt Preparation and Analysis During Fermentation

The starter culture was reactivated in 100 mL of soymilk containing 5% sucrose. This mixture was heated to 85 °C for 15 min and cooled to 42 °C. Then, 0.3% (w/v) starter culture was added and homogenized thoroughly. The mixture was incubated at 42 °C for ~6 h; after that, it was refrigerated for later use, but for no longer than 4 h. Afterwards, soy yogurt was prepared using the following ingredients: purified water (1000 mL), powdered soymilk (180 g), sugar (40 g), and 30 g of activated starter culture. The powdered soymilk and sugar were added to the purified water and heated to ~45 °C. When the soymilk was completely suspended, the activated starter culture was added and mixed until it was completely homogenized. The mixture was incubated at 42 °C for 4–5 h until it reached a pH of ~4.5. Then, the product was refrigerated at 4 °C for 12 h. The pH and TA were measured every 30 min during the fermentation process. The pH was measured as described in Section 2.2. For TA, 5 g of soymilk and 5 mL of distilled water were mixed and titrated with 0.1 N NaOH; the acidity was expressed as a percentage of lactic acid (the primary organic acid produced during fermentation). All analyses were performed in triplicate.
The probiotic peach soy yogurt (PPSY) was prepared by combining 10 g of peach jam, 3 g of probiotic beads (approximately 60 to 90 beads), and 100 g of soy yogurt in sterile glass containers. The mixture was stirred until a homogeneous blend was obtained, and then it was stored at 4 °C for ~30 days. We prepared yogurts containing alginate–chitosan beads, free cells, and without beads or free cells as controls.

2.5. Microbial Quality of Probiotic Peach Soy Yogurt After Manufacturing

PPSY at the beginning of the storage was microbiologically analyzed for total coliforms following the method of NOM-113-SSA1-1994 [39] using violet red bile agar (Bioxon, BD, Estado de Mexico, Mexico), and plates were incubated at 37 °C for 24 h. Yeast and mold were counted using the method of NOM-111-SSA1-1994 [40], utilizing the potato dextrose agar (Bioxon, BD, Estado de Mexico, Mexico) acidified (1.4 mL/100 mL agar from a 10 g/100 g of tartaric acid solution), and plates were incubated at 25 °C for 72 h.

2.6. Viability of Lim. reuteri During the Refrigerated Storage of PPSY

The viability of the probiotic microorganism was determined in PPSY supplemented with different types of beads every 72 h for 27 days of refrigerated storage. For the analysis, beads were separated from PPSY, rinsed with sterile water, weighed 1 g, and put into 9 mL of sterilized sodium citrate solution (1%) to dissolve the beads and release the probiotic. Afterwards, tenfold dilutions were performed, and the adequate dilutions were cultured on MRS agar. The plates were incubated at 37 °C for 72 h under anaerobic conditions. In the control PPSY, free cells of Lim. reuteri were added. For Lim. reuteri count in control PPSY, 1 g of soy yogurt was diluted ten-fold and cultured in agar MRS supplemented with 0.1% of bile salts to suppress L. bulgaricus growth as previously described by Mani-López et al. [41]. The Petri dishes were incubated at 37 °C for 72 h under anerobic conditions. All analyses were performed in triplicate.

2.7. pH and Titratable Acidity of Probiotic Peach Soy Yogurt During Storage

Every 7 days, the pH and total acidity (TA) of soy yogurt were analyzed. The pH was determined as described in Section 2.2. For TA, 5 g of soy yogurt and 10 mL of distilled water were mixed and titrated with 0.1 N NaOH until the pH reached 8.3; the acidity was expressed as a percentage of lactic acid. All analyses were performed in triplicate.

2.8. Probiotic Peach Soy Yogurt Sensory Evaluation

PPSY underwent sensory evaluation after 14 days of storage using a 9-point hedonic scale to assess the level of acceptance, where 9 represents “I like it very much” and 1 represents “I dislike it very much”. The evaluation was conducted with 30 untrained panelists, consisting of postgraduate students and faculty members. The criteria for selecting participants included their consumption of yogurt, familiarity with flavored yogurts, and the absence of dietary restrictions. The panelists ranged in age from 20 to 45 years, with a gender distribution of 55% female and 45% male. Their prior frequency of soy product consumption ranged from occasional to moderate use. These characteristics were considered to ensure that panelists had sufficient consumer experience with soy-based fermented products. They rate five attributes: texture, flavor, color, odor, and overall acceptability. Four PPSYs were selected for the sensory evaluation: PPSY containing alginate (3%) beads, alginate (2%) beads coated with 0.4% chitosan, with probiotic free-cells, and without beads or probiotic free cells (negative control).
Participants in the sensory evaluation tests provided informed consent by acknowledging the following statement: “I understand that my responses are confidential, and I consent to participate in this sensory evaluation.” Only those who agreed to the statement could participate and the participants were informed they could withdraw at any time without giving a reason. We explicitly stated, “The products being tested are safe for consumption.” We emphasized to participants that their data would remain confidential and only be shared with explicit consent.

2.9. Statistical Analysis

The results are presented as mean values along with their standard deviations. The data was statistically analyzed using Analysis of Variance (ANOVA). Pairwise comparisons of the mean values were conducted using Tukey’s test (p < 0.05) with Minitab v. 20 statistical software (Minitab LLC, State College, PA, USA). Before conducting the statistical analysis, the normality of the data and the homogeneity of the variances were assessed. Additionally, the experimental design was analyzed (Minitab) to assess the effects of alginate and chitosan on Lim. reuteri viability during storage and their impact on soy yogurt pH and TA.

3. Results and Discussion

3.1. Soymilk Characterization

Due to the nutritional properties of soybeans, particularly their high protein content, significant scientific and technological developments have occurred in recent decades with the aim of facilitating the comprehensive utilization of soy [42]. For this reason, soy-based products can serve as an alternative to the existing deficiency of conventional proteins, such as milk, meat, and eggs [43,44,45]. Soy yogurt’s popularity increases due to its lactose-free nature and its low levels of cholesterol and saturated fat [46]. Depending on the lactic acid bacteria strain(s) used to ferment soymilk, various health benefits are associated, including antioxidant activity, anti-proliferative activity, anti-diabetic activity, immunomodulatory properties, anti-inflammatory activity, antilipidemic activity, and promoting skin health, among others [47].
Table 2 contains a proximal analysis and the physicochemical properties of reconstituted soymilk. The yield-of-reconstitution ratio (1:10) is consistent with typical industry practices for soy-based beverages. The pH (6.93 ± 0.01) aligns with values reported for fresh or reconstituted soymilk, which typically range between 6.7 and 7.0 [48]. The protein content (4.81 ± 0.62%) is slightly higher than that of most commercial soymilks, which typically contain around 3–4% protein [39,40], indicating a formulation designed to enhance nutritional value. The fat content (3.65 ± 0.75%) also appears higher than in many commercial low-fat soy beverages, which typically range from 1.5% to 3% fat [17,49]. The ash content and TA fall within the expected ranges for soymilk products [50,51]. Moisture content is similar to that reported for soymilk (88.25%) by Basharat et al. [52]. The composition of reconstituted soymilk suggests a high-quality product with nutritional characteristics comparable to those of commercial formulations.

3.2. Soymilk Fermentation

The changes in pH and TA are shown in Figure 1. Figure 1 illustrates the dynamic changes in pH and TA (%) during soymilk fermentation with S. thermophilus and L. bulgaricus, the key starter cultures traditionally used in dairy yogurt production. Over 4.5 h, the pH decreased from 6.93 to 4.44, while the TA increased from 0.02% to 0.11%. This inverse relationship is typical of lactic acid fermentation, where the bacteria’s metabolic activity converts sugars (primarily sugars) into lactic acid, thereby acidifying the medium and causing the pH to drop. The observed fermentation profile aligns well with patterns reported in the literature [53,54,55]. According to Liu et al. [56] and Yang et al. [57], soymilk fermented with S. thermophilus and L. bulgaricus typically reaches a final pH between 4.4 and 4.7 within 4 to 6 h, with corresponding TA values ranging from 0.10% to 0.15%. Similar results were reported by Hati et al. [58], who documented a pH decline from 6.8 to around 4.6 during soy yogurt fermentation, with TA values rising to approximately 0.12%. The slight differences in acidification rate and final acidity values in various studies are often attributed to variations in soymilk composition (protein, sugar, and buffer capacity), inoculum size, fermentation temperature, and the specific strains used.
The acidification kinetics in this case suggest effective symbiotic activity between the two bacterial species: S. thermophilus initiates fermentation with sucrose metabolism (sucrose is the primary fermentable sugar that is derived from soybeans [51] and is also added to the formulation) and early acid production, which supports the growth of L. bulgaricus, which is known for its high acid tolerance and ability to produce lactic acid more robustly as fermentation progresses [58,59,60]. Lactic acid bacteria can metabolize sucrose, raffinose, and stachyose in soy milk, resulting in a decrease in pH and an increase in TA [61]. The final pH of 4.44 falls within the ideal range for achieving a desirable soy yogurt consistency and ensuring microbial safety, while also favoring protein coagulation due to the formation of globular proteins, which contribute to a firm and smooth gel and enhance the texture and stability. Several variables influence the properties of these products, including heating, protein content, acidity achieved during fermentation, and the type of culture used. Once the desired pH and acidity were reached, the yogurt was refrigerated (4 °C) for 12 h to reduce the fermentation activity.
The product obtained met the physicochemical characteristics of pH and percentage acidity, making it possible to incorporate Lim. reuteri microcapsules and peach jam, thereby obtaining a product that benefits the consumer’s health. Regarding microbial quality, soy yogurt fulfilled the requirements in terms of indicator microorganisms, mold and yeast (<10 CFU/g), and total coliforms (<10 CFU/g).

3.3. Probiotic Beads Characterization

The encapsulation yield (EY) of Lim. reuteri was 88.9 ± 1.19% for E1, E2, E3, and E9; 83.6 ± 0.5% for E4, E5, and E6; and 84.5 ± 0.9% for E7 and E8. In general, the probiotic exhibited good survivability due to the biocompatibility of alginate [9], demonstrating the expected protective effects of encapsulation. The EY obtained in this work was aligned with previous reports for Lactobacillus fermentum (68.7 to 91.9%) encapsulated in mango nectar + alginate (1%) [62]; B. brevis TISTR 2130 (92.6–99.8%) entrapped in sodium alginate + calcium lactate [6]; and for Bifidobacterium animalis BB-12 (50–88%) encapsulated in mixtures of alginate + xanthan gum [34].
Bead sizes are shown in Table 1. An increasing bead size was observed with the rising alginate concentration due to the increase in viscosity. The alginate bead sizes at different concentrations are similar to or smaller than those reported for alginate beads at 3% (2.10 mm; [14]); flavored beads of alginate at 1% (1.63 mm; [62]); and alginate beads at 1.5–2.5% (2.84 to 3.68 mm; [6]). Alginate beads coated with chitosan were smaller than alginate beads owing to acetic acid shrinkage of the beads during chitosan coating. Similar results have been reported by [63] (1.85 mm for beads of alginate (2%) + chitosan) and [14] (1.93 mm for beads 3% alginate + 1% chitosan). Despite the beads’ dimensions not being identical, the size differences were imperceptible to the human eye. Moreover, in the soy yogurt mixture (soy yogurt plus peach jam), the beads mimic peach jam (fruit pieces). Probiotic beads reached the bottom of the soy yogurt during storage, as was the case with fruit pieces. However, this type of product should be mixed or shaken before intake to homogenize the fruit and the beads. Probiotic beads remain distributed in the soy yogurt after mixing.

3.4. pH and TA of PPSY During the Storage

Figure 2 shows the pH and the TA during the refrigerated storage of the PPSY. In general, for the nine PPSYs, the pH decreases from ~4.5 to 4.2 ± 0.1 within the first week, and this decrease is more pronounced in the control with free cells. TA increases in all PPSYs from 1.24% to a maximum of 1.75%; this increase becomes more evident in the control soy yogurt, indicating that the microorganisms are producing lactic acid. This can be attributed to residual microbial activity during storage, which led to the acidification of the medium, as reported in previous studies on yogurts with different probiotics [41] and soy yogurts [26,64].
The control soy yogurt exhibited a steady increase in TA (1.3% to 1.8%), reflecting ongoing lactic acid production by the bacteria. In contrast, most PPSY with probiotic beads showed slower acidification, particularly those with 0.8% chitosan (TA stabilized at 1.4–1.5%). This suggests that alginate–chitosan beads effectively modulate Lim. reuteri metabolism, likely by slowing bacterial release and metabolic activity [65,66]. Similar TA stabilization was reported in chitosan-coated L. acidophilus LA-5 yogurts [67], attributed to the barrier properties of chitosan. Notably, higher alginate concentrations (3%) without chitosan had minimal impact on TA, whereas chitosan inclusion (0.4 or 0.8%) further suppressed acidification. At low pH, composite hydrogel beads containing chitosan interact with protons by their amine groups, resulting in swelling beads [68,69]. The stability of chitosan complexes is associated with electrostatic interactions, hydrogen bonds, and hydrophobic effects, resulting in a good acidic barrier [69]. This aligns with studies showing that chitosan’s cationic nature restricts proton diffusion, delaying pH drop [70].
The control’s pH declined sharply to 4.0 by week 5 (p < 0.05), while PPSY containing beads (3% alginate and chitosan) maintained a pH of 4.1–4.3. The encapsulation role in pH stabilization is critical, as pH remained higher than in soy yogurts containing free cells due to their lower bacterial activity [71,72]. The alginate beads, or those coated with chitosan, optimized pH stability without sensory trade-offs, suggesting their suitability for functional soy yogurts. This is advantageous for probiotic delivery, as a pH greater than 4.0 enhances the survival of Lim. reuteri [73].

3.5. Lim. reuteri Viability During the Storage Incorporated into PPSY

The viability of Lim. reuteri (Table 3) decreased over time in all treatments, including encapsulated and free-cell forms. Encapsulation significantly improved survival compared with free cells during storage. After 9 days, all beads preserved probiotic viability, achieving a count between 6.7 log CFU/g and 7.7 log CFU/g. Alginate (1, 2, and 3%) beads maintain the viability of Lim. reuteri to the greatest extent. Beads with 2% alginate and 0.4% chitosan also exert a protective effect on probiotics. On day 27, free cells showed a decline from 9.0 to 4.4 log CFU/g (Figure 3), dropping sharply until very low counts were reached. An increasing alginate concentration (Table 3 and Figure 3) slightly improved probiotic survival. For example, 3% alginate remained at 7.9 log CFU/g on day 27, higher than 1% and 2%, which maintained 7.6 and 7.8 log CFU/g, respectively. These results suggest that higher alginate concentrations may improve matrix density and protective capability. This aligns with the findings of Anal and Singh [74], who reported that thicker alginate matrices provide better barriers against acid diffusion and oxygen exposure. Mandal et al. [64] observed an increase in the survival of L. casei with higher alginate concentrations, and Rodklongtan et al. [65] reported a similar trend for Pediococcus pentosaceus.
Figure 3 shows the effect of the beads’ formulation (alginate–chitosan) on the reduction in the viability of Lim. reuteri after 27 days in PPSY. Beads formed with 2% alginate + 0.4% chitosan showed the highest viability, maintaining 7.4 log CFU/g on day 27, while the 1% alginate and 3% alginate groups reached 6.9 and 6.8 log CFU/g, respectively. Increasing the chitosan concentration to 0.8% did not improve survival and, in some cases, was slightly less effective (Figure 4). For example, beads containing 3% alginate and 0.8% chitosan retained 6.5 log CFU/g by day 27. This reduction in viability may be due to excessive chitosan compacting the bead matrix, which can limit nutrient diffusion and metabolite exchange. Furthermore, at higher concentrations, chitosan may exert mild antimicrobial effects on the probiotic [75,76], possibly contributing to reduced survival during storage. Although no microstructural analysis of the beads was conducted in this study, the observed differences in probiotic viability among formulations, especially those with higher chitosan concentrations, align with previous findings in the literature [77,78,79]. Previous studies have shown that increased chitosan levels can lead to a denser and more compact matrix, which may limit nutrient diffusion or exert mild antimicrobial effects on encapsulated cells [76,80]. These mechanisms may help explain the reduced viability observed in some formulations. This suggests that 2% alginate might offer an optimal balance between porosity and mechanical strength when combined with chitosan (0.4%). In contrast, the quantity of free cells fell to 4.4 log CFU/g, reinforcing the value of encapsulation. Without a protective matrix, Lim. reuteri is highly susceptible to acid stress, low temperatures, and metabolic byproducts from starter cultures during soy fermentation. In this regard, Rodrigues et al. [81] demonstrated that the encapsulation of Lim. reuteri in alginate–konjac gum, –cassia tora gum, or –psyllium mucilage enhances its survival during prolonged refrigerated storage (20–60 days). The results of the present study agree with earlier works on Lim. reuteri viability of free cells and encapsulated (whey proteins + Arabic gum) added to sheep’s milk yogurt; after 21 days of storage; free cells lost 27% of their viability and encapsulated only 1% [82]. Encapsulation demonstrated that probiotics maintain significantly higher viability in fermented dairy and plant-based matrices. Other studies show contrary data on probiotic viability, probably due to the soy matrix or encapsulation type. For example, Roy et al. [83] reported that microencapsulation (alginate–ε-poly-lysine–alginate) did not offer any extra protection compared with the free L. reuteri probiotic when added to a soy beverage. The study found a decrease in viability of less than 1 log CFU/mL after 8 weeks of storage at 4 °C and 8 °C. D’Alessandro et al. [33] recorded similar viability between free cells and encapsulated (soy milk as a wall material and encapsulated by spray drying) of two potential probiotics (L. crispatus BC4 and L. gasseri BC9) during soy yogurt fermentation and its storage at 4 °C. After 24 h of fermentation, bacterial counts ranged between 6.95 and 7.39 log CFU/g, while 28 days later, survivals oscillated 6.71 to 7.14 log CFU/g.

3.6. Sensory Evaluation of PPSY

The beany flavor of soy is known to be predominant even when aqueous extraction is carried out to obtain soymilk; thus, the addition of peach jam is intended to mask the flavor of the raw material. In addition, beads could mimic the texture of peach jam, allowing consumers to overlook them during the product’s sensory evaluation. The acceptance test was conducted to observe the acceptability of PPSY with Lim. reuteri alginate beads with and without chitosan, as well as soy yogurt without beads, and soy yogurt with Lim. reuteri free cells. Table 4 presents the average scores for the attributes of PPSY.
The color of PPSY containing alginate beads coated with chitosan scored slightly higher (7.38) than the PPSY containing alginate beads (7.24). Chitosan films can enhance opacity and whiteness [84], thereby improving the visual appeal of dairy-like products. The PPSY containing alginate beads had a marginally higher texture score (6.95) than the PPSY with alginate–chitosan beads (6.76); however, the difference was not statistically significant (p > 0.05). Chitosan can introduce a firmer or slightly grittier mouthfeel [80,85], which may not be preferred in a product like yogurt. The flavor of PPSY with alginate–chitosan beads (6.86) obtained similar scores to PPSY containing alginate beads (6.88); thus, they were not significantly different (p > 0.05). Chitosan and alginate may help mask bitterness from probiotics [86] or reduce the release of acidic metabolites that affect flavor. The odor scores of PPSY containing beads were similar (~6.4), suggesting that chitosan and alginate did not significantly alter odor perception [87]. For overall acceptability, the PPSY containing alginate–chitosan beads had a minimally higher score (7.00) than the PPSY containing alginate beads (6.98). The chitosan coating did not have a negative impact (p > 0.05) on consumers’ enjoyment of the product and may have even slightly improved the color. However, the PPSY containing free cells received a significantly lower score for flavor and acceptability (p < 0.05). Despite sensory evaluation not taking place at the end of the storage period, PPSY maintained gel stability and an acceptable texture after 14 days, which was comparable with soy yogurt containing or not containing probiotic-free cells (p < 0.05). Overall, visible syneresis or gel rupture was not perceived in the PPSY with or without probiotic beads.
Alginate is commonly used for probiotic encapsulation due to its gentle gelation and protection against stomach acid [71]. Adding chitosan as a secondary layer can enhance gastric resistance [84]; however, it may slightly alter the product’s texture. Adding probiotic beads to yogurt suggests that encapsulation improves bacteria’s survival without major sensory trade-offs, as described by Krasaekoopt and Tandhanskul [88] and Atta et al. [82]. Chitosan can sometimes introduce a slight astringency or firmness [86], which aligns with this study’s slightly lower texture score. However, its film-forming properties can improve stability and mask off-flavors [87], supporting the marginally better flavor and acceptability scores. The alginate–chitosan beads performed comparably to or slightly better than the alginate beads in terms of color, flavor, and acceptability, but exhibited a minor textural drawback. Future research could investigate combining alginate with other biopolymers to improve texture while retaining its benefits. A growing trend is emerging in the use of sustainable plant-based proteins as dairy alternatives in yogurts [89]. Still, further study is needed to enhance product properties, particularly flavor and texture, which are essential for sensory appeal.
The sensory results indicate that chitosan-coated alginate beads containing Lim. reuteri do not significantly diminish the acceptability of PPSY but may slightly improve their color and flavor. Based on the statistical analysis carried out, no significant difference was observed between the different attributes of both PPSY (p > 0.05), indicating that the consumer does not perceive a difference in the beads treated with chitosan; the coating does not influence sensory attributes. Soy yogurts have a moderate degree of acceptance based on the ratings obtained, as they are perceived to have an aftertaste and a smell reminiscent of soybeans.

4. Conclusions

PPSY was formulated from soymilk and demonstrated acceptable physicochemical characteristics regarding its pH and TA percentage. To enhance its functional value, beads containing Lim. reuteri were incorporated. Lim. reuteri encapsulated with alginate (2% and 3%) without a chitosan coating effectively maintained its viability during refrigerated storage of PPSY. For alginate coating beads, the optimal concentrations of alginate and chitosan were 2% and 0.4%, respectively, ensuring counts of probiotics were maintained at > 107 CFU/g after 27 days of storage. The pH drop of PPSY was notably higher when probiotic-free cells were added, whereas PPSYs with probiotic beads were only reduced by 0.2 units of pH. The PPSY containing alginate (3%) or alginate (2%)-coated chitosan (0.4%) beads and probiotic-free cells had similar sensory acceptance in terms of their color, texture, and odor. In contrast, the flavor and overall acceptability scored significantly higher (p < 0.05) in PPSY with beads. Further research could include instrumental measurements of texture, color, and aroma profiles to understand the properties of PPSY better. Also, microstructural analysis by electron microscopy (SEM and TEM) that provides detailed data on coating beads and microbial distribution within the beads should be included.

Author Contributions

R.H.H.-F., methodology, data curation, formal analysis, writing—review and editing. Y.D.R., investigation, methodology, data curation, formal analysis. A.L.-M., supervision, resources, visualization, formal analysis, writing—original draft, writing—review and editing. E.M.-L., methodology, formal analysis, visualization, writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

The Research and Ethics Committee on Sensory Evaluation of Foods of the doctoral program in Food Science of the Universidad de las Américas Puebla approved the protocol for the sensory evaluation carried out in this work on 12 September 2024 (document number SEDCL-2024/038).

Informed Consent Statement

For the sensory evaluation tests performed in our research, the participants gave informed consent via the following statement: “I am aware that my responses are confidential, and I agree to participate in this sensory evaluation of probiotic peach soy yogurt.” The participants who provided an affirmative reply could participate. They were also informed that they could withdraw from the test at any time without giving a reason. We explicitly stated, “The tested products are safe for consumption; they were prepared under strict standard conditions and are microbiologically safe for consumption”. The data of the participants will not be disclosed without their knowledge.

Data Availability Statement

The original contributions presented in the study are included in the article; further inquiries can be directed to the corresponding author.

Acknowledgments

The Authors acknowledge Universidad de las Americas Puebla (UDLAP).

Conflicts of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Fermentation 11 00423 g001
Figure 1. pH and titratable acidity (TA) of soymilk during fermentation with Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.
Figure 1. pH and titratable acidity (TA) of soymilk during fermentation with Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.
Fermentation 11 00423 g001
Fermentation 11 00423 g002
Figure 2. pH and titratable acidity of probiotic peach soy yogurt during storage at 4 °C. Closed symbols and continuous lines represent pH; open symbols and dotted lines represent titratable acidity. Probiotic free cells of Limosilactobacillus reuteri DSM 17938 (blue, ■, □); alginate (1%) beads (red, ●, ○); alginate (2%) beads (orange, ▲, △); and alginate (3%) beads (purple, ◆, ◇). (a) Alginate beads without chitosan coating; (b) alginate beads coated with 0.4% chitosan; and (c) alginate beads coated with 0.8% chitosan.
Figure 2. pH and titratable acidity of probiotic peach soy yogurt during storage at 4 °C. Closed symbols and continuous lines represent pH; open symbols and dotted lines represent titratable acidity. Probiotic free cells of Limosilactobacillus reuteri DSM 17938 (blue, ■, □); alginate (1%) beads (red, ●, ○); alginate (2%) beads (orange, ▲, △); and alginate (3%) beads (purple, ◆, ◇). (a) Alginate beads without chitosan coating; (b) alginate beads coated with 0.4% chitosan; and (c) alginate beads coated with 0.8% chitosan.
Fermentation 11 00423 g002
Fermentation 11 00423 g003
Figure 3. Viability losses (Log N/N0) of encapsulated Limosilactobacillus reuteri DSM 17938 and free cells into probiotic peach soy yogurt after 27 days of storage at 4 °C (labels on the x-axis, E1–E9, correspond to the formulations shown in Table 1). Mean values that do not share a letter are significantly different (p < 0.05).
Figure 3. Viability losses (Log N/N0) of encapsulated Limosilactobacillus reuteri DSM 17938 and free cells into probiotic peach soy yogurt after 27 days of storage at 4 °C (labels on the x-axis, E1–E9, correspond to the formulations shown in Table 1). Mean values that do not share a letter are significantly different (p < 0.05).
Fermentation 11 00423 g003
Fermentation 11 00423 g004
Figure 4. Contour plot of Limosilactobacillus reuteri DSM 17938 viability (V, Log N) encapsulated in alginate–chitosan and added into peach soy yogurt after 27 days of storage at 4 °C.
Figure 4. Contour plot of Limosilactobacillus reuteri DSM 17938 viability (V, Log N) encapsulated in alginate–chitosan and added into peach soy yogurt after 27 days of storage at 4 °C.
Fermentation 11 00423 g004
Table 1. Sodium alginate and chitosan levels to encapsulate Limosilactobacillus reuteri DSM 17938 and bead size.
Table 1. Sodium alginate and chitosan levels to encapsulate Limosilactobacillus reuteri DSM 17938 and bead size.
Encapsulation CombinationAlginate (%)Chitosan (%)Bead Size (mm) *
E1101.65 ± 0.04 d
E2202.10 ± 0.05 b
E3302.35 ± 0.09 a
E410.41.51 ± 0.06 de
E520.41.91 ± 0.04 c
E630.42.25 ± 0.03 a
E710.81.45 ± 0.04 e
E820.81.85 ± 0.04 c
E930.82.10 ± 0.05 b
Control 100---
1 The control is soy yogurt with Lim. reuteri free cells. * Means that do not share a letter are significantly different (p < 0.05).
Table 2. Characterization of reconstituted soymilk (Glycine max) powder.
Table 2. Characterization of reconstituted soymilk (Glycine max) powder.
Parameter/ComponentReconstituted Soymilk
pH6.93 ± 0.01
Titratable acidity 1 (%)0.18 ± 0.06
°Brix8.51 ± 0.17
Moisture (g/100 g)89.40 ± 0.25
Fat (g/100 g)3.65 ± 0.75
Protein (g/100 g)4.81 ± 0.62
Ash (g/100 g)0.61 ± 0.04
Carbohydrates (g/100 g)1.50 ± 0.14
1 g oxalic acid/100 g.
Table 3. Encapsulated * Limosilactobacillus reuteri DSM 17938 and free cells’ viability in probiotic peach soy yogurt during storage at 4 °C.
Table 3. Encapsulated * Limosilactobacillus reuteri DSM 17938 and free cells’ viability in probiotic peach soy yogurt during storage at 4 °C.
DayE1E2E3E4E5E6E7E8E9Free Cells
08.0 ± 0.2 aB8.0 ± 0.2 aB8.0 ± 0.2 aB7.5 ± 0.2 aC7.5 ± 0.2
C		7.5 ± 0.2 aC7.5 ± 0.2 aC7.5 ± 0.2 aC8.0 ± 0.2 aB9.0 ± 0.2
aA
37.4 ± 0.1 bcBC7.2 ± 0.1 bcC7.4 ± 0.1 bcBC7.3 ± 0.1 abC7.2 ± 0.1
C		7.2 ± 0.1 abcC7.4 ± 0.1 abBC7.4 ± 0.1 aBC7.8 ± 0.2 aB8.9 ± 0.2
aA
67.2 ± 0.1 cC7.0 ± 0.1 cC7.3 ± 0.1 cBC7.0 ± 0.1 bcC7.4 ± 0.1 aBC7.1 ± 0.1 abcdC7.0 ± 0.1 bcC7.0 ± 0.1 bC7.7 ± 0.2 aAB8.1 ± 0.2
bA
97.7 ± 0.2 abA7.6 ± 0.2 abAB7.7 ± 0.2 abcA7.2 ± 0.1 abBCD7.5 ± 0.2 aABC7.3 ± 0.1 abABDC6.9 ± 0.1 cDE6.7 ± 0.1 bcE7.0 ± 0.1 bDE7.1 ± 0.1
cCDE
127.6 ± 0.2 abcAB7.8 ± 0.2 aA7.6 ± 0.2 abcAB6.7 ± 0.1 cdeC7.3 ± 0.1
aB		6.8 ± 0.1 cdeC6.8 ± 0.1 cC6.4 ± 0.1 cdC6.6 ± 0.1 bcC6.8 ± 0.1
cC
157.6 ± 0.2 abcAB7.9 ± 0.2 aA7.6 ± 0.2 abcAB6.5 ± 0.1 defC7.2 ± 0.1
aB		6.7 ± 0.1 deC6.7 ± 0.1 cC6.3 ± 0.1 dC6.5 ± 0.1 cC6.4 ± 0.1
dC
187.5 ± 0.2 bcAB7.8 ± 0.2 aA7.6 ± 0.2 abcAB6.3 ± 0.1 fDE7.2 ± 0.1
aB		6.6 ± 0.1 eCD6.8 ± 0.1 cC6.2 ± 0.1 dE6.4 ± 0.1 cDE5.7 ± 0.1
eF
217.5 ± 0.2 bcAB7.8 ± 0.2 aA7.7 ± 0.2 abcA6.2 ± 0.1 fD7.3 ± 0.1
aB		6.7 ± 0.1 deC6.8 ± 0.1 cC6.3 ± 0.1 dD6.5 ± 0.1 cCD5.0 ± 0.1
fE
247.6 ± 0.2 abcAB7.9 ± 0.2 aA7.8 ± 0.2 abAB6.4 ± 0.1 efD7.5 ± 0.2
aB		6.9 ± 0.1 bcdeC6.9 ± 0.1 cC6.2 ± 0.1 dD6.5 ± 0.1 cD4.8 ± 0.1
fgE
277.6 ± 0.2 abcAB7.8 ± 0.2 aA7.9 ± 0.2 aA6.8 ± 0.1 cdCD7.4 ± 0.1 aBC6.8 ± 0.1 cdeCD6.9 ± 0.1 cC6.2 ± 0.1 dE6.5 ± 0.1 cDE4.4 ± 0.1
gF
* E1–E9 correspond to the formulations shown in Table 1. In the same column, equal lowercase letters indicate no significant difference (p > 0.05). In the same row, equal capital letters indicate no significant difference (p > 0.05).
Table 4. Sensory evaluation scores of probiotic peach soy yogurt with or without Limosilactobacillus reuteri DSM 17938 beads and free cells after 14 days of storage at 5 °C.
Table 4. Sensory evaluation scores of probiotic peach soy yogurt with or without Limosilactobacillus reuteri DSM 17938 beads and free cells after 14 days of storage at 5 °C.
Peach Soy YogurtColorTextureFlavorOdorAcceptability
Alginate (3%) beads/without chitosan7.24 ± 0.35 a6.95 ± 0.29 a6.88 ± 0.47 a6.43 ± 0.34 a6.98 ± 0.37 a
Alginate (2%) beads/with chitosan (0.4%)7.38 ± 0.38 a6.76 ± 0.35 a6.86 ± 0.42 a6.33 ± 0.72 a7.00 ± 0.39 a
Without beads7.14 ± 0.85 a6.96 ± 0.89 a6.58 ± 0.37 a,b6.53 ± 0.36 a6.90 ± 0.31 a
With free cells6.90 ± 0.78 a6.06 ± 0.48 a5.80 ± 0.45 b6.03 ± 0.49 a6.01 ± 0.49 b
Different lowercase letters in each column indicate significant differences (p < 0.05).
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Hernández-Figueroa, R.H.; Ramírez, Y.D.; López-Malo, A.; Mani-López, E. Enhancing Soy Yogurt with Microencapsulated Limosilactobacillus reuteri: Viability and Sensory Acceptability. Fermentation 2025, 11, 423. https://doi.org/10.3390/fermentation11080423

AMA Style

Hernández-Figueroa RH, Ramírez YD, López-Malo A, Mani-López E. Enhancing Soy Yogurt with Microencapsulated Limosilactobacillus reuteri: Viability and Sensory Acceptability. Fermentation. 2025; 11(8):423. https://doi.org/10.3390/fermentation11080423

Chicago/Turabian Style

Hernández-Figueroa, Ricardo H., Yani D. Ramírez, Aurelio López-Malo, and Emma Mani-López. 2025. "Enhancing Soy Yogurt with Microencapsulated Limosilactobacillus reuteri: Viability and Sensory Acceptability" Fermentation 11, no. 8: 423. https://doi.org/10.3390/fermentation11080423

APA Style

Hernández-Figueroa, R. H., Ramírez, Y. D., López-Malo, A., & Mani-López, E. (2025). Enhancing Soy Yogurt with Microencapsulated Limosilactobacillus reuteri: Viability and Sensory Acceptability. Fermentation, 11(8), 423. https://doi.org/10.3390/fermentation11080423

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