Development of a Functional Yogurt Containing Probiotics and Phenolic Compounds of Coffee Encapsulated in Alginate Beads

Abstract

Probiotics and phenolic compounds provide benefits to humans when they are consumed in adequate amounts. However, these materials are not very stable and can easily be degraded during processing and storage; so, they must be protected. This study evaluated the encapsulation of Lactiplantibacillus fabifermentans BAL-27 ITTG and phenolic compounds from coffee husks via alginate beads. The research considered variables such as alginate concentration (1.5% and 3%), crosslinking time (8 and 20 min), and the inclusion of chitosan. A 2³ factorial design was employed, and the effects were analyzed via ANOVA (p < 0.05). The encapsulation efficiency of the probiotic exceeded 80%, and its viability following gastrointestinal simulation ranged from 73.65% to 85.34%. The phenolic compounds achieved encapsulation efficiencies of up to 20%. In yogurt, the alginate beads maintained probiotic viability at approximately 9 Log₁₀ CFU/g and preserved the stability of the antioxidant compounds over 28 days. Moreover, the incorporation of beads did not adversely affect the physicochemical properties or sensory acceptance of the yogurt, supporting their potential application in functional foods.

1. Introduction

Currently, interest in functional foods and beverages has increased significantly because of the importance of proper nutrition and health [1]. Among these foods, the roles of probiotics and phenolic compounds have been widely studied, as both have demonstrated health benefits when they are consumed in adequate amounts [2]. However, the incorporation of these bioactive compounds into food matrices may alter their physicochemical and organoleptic properties or reduce their bioactivity during processing [3]. To address these challenges, researchers have implemented microencapsulation techniques to protect and stabilize these compounds in a way that preserves both the sensory characteristics of the food and the functional properties of the encapsulated ingredients during storage [2]. Over the years, various technologies have been developed to extend the shelf-life of these bioactive compounds, with encapsulation emerging as one of the most employed strategies [4,5,6].

For the encapsulation of lactic acid bacteria, spray drying, lyophilization, and encapsulation in alginate beads are among the most prominent techniques. However, probiotics present significant challenges, as their viability tends to decline rapidly during storage [4,5,6]. This decrease in viability is often attributed to the physicochemical properties of the beads and their water activity (Aw). Another critical factor is the ability of probiotics to withstand encapsulation conditions, prompting ongoing efforts to identify more resilient strains [7]. In our laboratories, Lactiplantibacillus fabifermentans BAL-27 ITTG has shown promising probiotic potential [7], suggesting its suitability for food applications.

Another group of important bioactive components includes phenolic compounds (antioxidants), which are highly susceptible to degradation by high temperatures, light, and oxygen during processing and storage. The antioxidant capacity of these compounds is associated with various health benefits, including anti-inflammatory, anticancer, and antiatherosclerotic properties, and improved gut microbiota health [8]. Several studies have reported promising encapsulation efficiencies for phenolic compounds, which are distinct from those reported for probiotics. For example, Belščak et al. [9] and Li et al. [10] reported encapsulation efficiencies exceeding 57% for caffeine and phenolic compounds in green tea. These studies highlighted the crucial role of chitosan in achieving high encapsulation efficiency. Given these favorable outcomes, researchers are actively exploring new sources of phenolic compounds, particularly from agricultural byproducts.

Coffee cherry has long been utilized; however, owing to its nutritional potential, its byproducts have attracted increasing interest worldwide. One such byproduct is the pericarp (outer skin), which contains phenolic compounds at concentrations ranging from 9.2 to 13 mg GAE/g, as reported by Ballesteros et al. [11], Heeger et al. [12], and Ribeiro et al. [13]. These phenolic compounds are particularly valued for their antimicrobial and antioxidant properties, making their extraction and application especially appealing.

Despite numerous reports on the individual encapsulation of probiotics and phenolic compounds, to our knowledge, there is limited information on the coencapsulation of probiotics and phenolic compounds. This is largely due to the antimicrobial nature of phenolic compounds, which may inhibit beneficial microorganisms. Therefore, in the present study, Lactiplantibacillus fabifermentans BAL-27 ITTG was coencapsulated with phenolic compounds extracted from the coffee pericarp via alginate beads. The purpose of this study was to assess the effects of chitosan, alginate concentration, and crosslinking time on Lactiplantibacillus fabifermentans BAL-27 ITTG viability and phenolic compound encapsulation. Additionally, probiotic survival, survival in gastrointestinal simulations, phenolic compounds, and the physicochemical properties of yogurt were evaluated during storage.

2. Materials and Methods

2.1. Materials and Microorganisms

Lactiplantibacillus fabifermentans BAL-27 ITTG was provided by the National Technological Institute of Mexico/Technological Institute of Tuxtla Gutiérrez, Chiapas, Mexico. The microorganism was reactivated in de Man, Rogosa, and Sharpe, MRS (Difco Laboratories, Franklin Lakes, NJ, USA) broth and incubated at 37 °C for 8 h. Subsequently, the culture was centrifuged at 2490× g and 4 °C for 30 min. The resulting cell pellet was washed with 0.9% (w/v) saline solution and centrifuged again under the same conditions. Finally, the cell pellet was stored at 4 °C until further use. All unspecified reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.2. Extraction of Phenolic Compounds

Ripe coffee cherries (Coffea arabica) were supplied by Rilly de Lievano Co. (Chiapas, Mexico). The cherries were washed with soap and water, disinfected by immersion in chlorinated water (manually stirred), rinsed thoroughly, and shade-dried to remove the residual surface moisture at room temperature (approximately 1 h). The pericarp was manually separated from undamaged, fully ripe cherries. For the extraction of phenolic compounds, the pericarp was immersed in distilled water at a 1:5 (w/v) ratio and stirred at 60 °C for 24 h. The resulting mixture was filtered through Whatman No. 4 paper, and the filtrate was lyophilized via a freeze dryer (Labconco, Cole-Parmer, Vernon Hills, IL, USA) at −40 °C and 0.03 mbar of pressure [14]. The resulting coffee cherry pericarp extract (hereafter referred to as “coffee extract”) was stored in the dark at −18 °C until further use.

2.3. Encapsulation of Bioactive Compounds

Alginate beads were obtained via the ionic gelation method using two concentrations of sodium alginate (1.5 and 3% w/v), two crosslinking times (8 and 20 min), and with or without a chitosan coating (molecular weight: 100,000–300,000) (Sigma-Aldrich, USA).

To prepare the beads, hydrated alginate (12 h under refrigeration) was mixed with the cell pellet of Lactiplantibacillus fabifermentans BAL-27 ITTG, and the lyophilized coffee extract was resuspended to final concentrations of 10 Log₁₀ CFU/mL and 2% (w/v). The mixture was homogenized at 123× g for 10 min and then added dropwise to a sterile 5% (w/v) calcium chloride solution. The beads were subsequently immersed in the CaCl₂ solution for 8 or 20 min, according to the experimental design. A batch of beads was subsequently coated by immersion in a 0.4% (w/v) chitosan solution according to the methodology described by Krasaekoopt et al. [15]. Previously, low-molecular-weight chitosan was dissolved in 1% (v/w) acetic acid. The pH of the solution was subsequently adjusted to 5.7 ± 0.3 with 1 M NaOH. The mixture was filtered with filter paper (Whatman # 4) and sterilized at 121 °C for 15 min prior to immersion of the beads. Fifteen grams of beads were immersed in 100 mL of the chitosan solution and stirred with the help of a magnetic stirrer for 40 min. Finally, the beads were removed from the chitosan solution, washed twice with sterile distilled water, and stored under refrigeration until use. Importantly, the beads were used for no more than 5 h for future determinations.

2.4. Determination of the Viability of Lactiplantibacillus fabifermentans BAL-27 ITTG

To evaluate the viability of the microorganisms, 1 g of beads was mixed with 5 mL of 2% sodium citrate (p/v). The mixture was homogenized at 1260× g for 5 min using an Ultra-Turrax homogenizer IKA T25 (Wilmington, NC, USA) following the protocol described by De Prisco et al. [5]. The viable cell concentration was determined by plating on MRS agar according to Ramírez-Pérez et al. [7]. The encapsulation efficiency (MEE) was determined via Equation (1):

MEE(%) = (Log₁₀ Ni/Log No) × 100

(1)

where Ni is the concentration (CFU/g) of cells encapsulated in the beads and No is the concentration (CFU/g) of cells in the encapsulation mixture.

2.5. Gastrointestinal Simulation

The gastrointestinal simulation was performed according to Ramírez-Pérez et al. [7]. Briefly, the tolerance of the microorganisms to acidic conditions was evaluated by exposing them to a pH of 1.9 for 30 min. The passage through the small intestine was subsequently simulated via intestinal juices (pH 7.5), and the beads were incubated for 6 h. The resistance gastrointestinal simulation (RGS) was calculated via Equation (2).

RGS (%) = (Log₁₀ Ni/Log₁₀ No) × 100

(2)

where Ni represents the cell concentration present at the end of the intestinal stage and No represents the initial cell concentration.

2.6. Quantification of Phenolic Compounds in the Beads

The phenolic compound content in the alginate beads was quantified by dissolving 1 g of the beads, as described in Section 2.3. The concentration of phenolic compounds was determined via the Folin-Ciocalteu spectrophotometric method [16]. Briefly, 1 mL of the sample was mixed with 4.2 mL of distilled water, followed by the addition of 0.5 mL of 2 N Folin–Ciocalteu reagent and 1 mL of 20% (w/v) sodium carbonate. The mixture was incubated at room temperature for 2 h. The absorbance was measured at 765 nm via a spectrophotometer (Cole-Parmer UV-2100, USA). A standard calibration curve was prepared using gallic acid (GA). The results are expressed in mg gallic acid equivalents per gram of bead (mg GAE/g).

The encapsulation efficiency of phenolic compounds (PCEE) was determined via Equation (3):

PCEE (%) = (CFi/CFo) × 100

(3)

where CFi represents the concentration of phenolic compounds encapsulated in the beads and CFo corresponds to the initial concentration of phenolic compounds in the mixture before encapsulation.

2.7. Preparation of Yogurt

A commercial starter culture from the BIOPROX brand (Mexico), containing Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, was used, activated in sterile distilled water, and incubated at 37 °C for 1 h according to the manufacturer’s instructions. For inoculum preparation, whole milk powder was reconstituted to a concentration of 12% (w/v) and sterilized at 121 °C for 15 min. The starter culture was subsequently inoculated at 1% (v/v) and incubated at 35 °C for 8 h [17].

The yogurt preparation was conducted following the methodology described by Morales-Ruiz [17], with slight modifications. The milk was standardized by dissolving 6 g of powdered whole milk (Nestlé, Ocotlan, Jal, Mexico) and 7 g of sugar in 100 mL of liquid whole milk (Pradel, Berriozabal, Chiapas, Mexico). The mixture was subjected to low-temperature pasteurization (73 °C for 20 s), followed by inoculation with the activated culture at 10% (v/v). The mixture was stirred for 20 min, and 30 mL aliquots were transferred into 40 mL polyethylene containers containing alginate beads at a 1:10 (w/v) ratio. Fermentation was carried out at 40 °C for 2 h, after which the samples were refrigerated (approximately 5 °C) to allow for gelation (yogurt control without beads). For yogurt with beads, 3 g of beads formulated previously were added to 30 mL of milk to be fermented in 40 mL plastic cups. The starter culture was subsequently added to the fermentation of the milk as mentioned and maintained at 40 °C for 2 h. Finally, the yogurt was refrigerated to allow gelation. After 2 h, the pH of the yogurt was approximately 4.3.

The yogurt was stored at 5 °C for 28 days, and samples were collected every 7 days to assess the viability of the microorganisms, as well as the physicochemical and sensory properties of the product. In addition, the phenolic compound content and release of Lactiplantibacillus fabifermentans BAL-27 ITTG under simulated gastrointestinal conditions were quantified at the end of the storage period.

To demonstrate the functionality of yogurt as a functional food, ensuring the viability of the encapsulated probiotic during storage is essential. Therefore, the viability of the probiotic and the stability of the phenolic compounds encapsulated within the beads incorporated into the yogurt were evaluated.

2.7.1. Viability of Lactiplantibacillus fabifermentans BAL-27 ITTG in Yogurt

To determine the viability of the probiotic on the beads within the yogurt, the beads were removed, carefully washed with sterile water to remove excess yogurt, and analyzed weekly over a 28-day storage period. The determination of the viability of microorganisms was then carried out as described in Section 2.4.

2.7.2. Physicochemical Analysis of Yogurt

Syneresis

The syneresis of the yogurt was determined according to Zainoldin and Hi Baba [18]. For this purpose, 20 g of yogurt was weighed in 50 mL tubes and centrifuged at 2490× g for 20 min at 4 °C. The supernatant was subsequently weighed, and the percentage of syneresis was calculated via Equation (4).

Syneresis (%) = ((weight of the supernatant)/(weight of the sample)) ∗ 100

(4)

pH

The pH of the yogurt was measured during storage via a digital pH meter (Mettler Toledo, Cd. De Mexico, Mexico).

Titratable Acidity

The titratable acidity, expressed as a percentage of lactic acid, was determined according to NOM-243-SSA1-2010 [19]. Eighteen grams of yogurt was mixed with thirty-six g of distilled water. Ten drops of 1% phenolphthalein (w/v) were added and titrated with 0.1 N NaOH. The acidity was calculated via Equation (5).

Acidity (%) = (V ∗ N ∗ 9)/M

(5)

where V is the volume (mL) of NaOH used in the titration, N is the normality of NaOH (0.1 N), and M is the weight of the sample.

Sensory Analysis

The degree of global acceptance, as well as the properties of smell, color, flavor, and texture, was evaluated via a structured seven-point hedonic test (1: dislike extremely; 7: like extremely). The evaluation was performed by a panel of 60 untrained judges (aged 18–24 years) at random. Assessment was conducted at the beginning and end (28 days) of the yogurt shelf-life. The sensory test was performed in two sessions to avoid panel fatigue.

2.8. Experimental Design and Statistical Analysis

A completely randomized 2³ factorial experimental design was implemented with three replicates. A total of eight treatments were randomly performed in different sessions. The following factors were evaluated: alginate concentration (1.5% and 3%), immersion time (8 and 20 min), and the presence or absence of chitosan. All the data were analyzed via an analysis of variance (ANOVA) at a significance level of p < 0.05. Mean comparisons were performed via Tukey’s test with the aid of Statgraphics Centurion XVI software (Version 16.1.03). For the sensory analysis, yogurt without alginate beads was used as the control.

3. Results

3.1. Encapsulation of Lactiplantibacillus fabifermentans BAL-27 ITTG and Phenolic Compounds

3.1.1. Viability of Encapsulated Lactiplantibacillus fabifermentans BAL-27 ITTG

The viability of Lactiplantibacillus fabifermentans BAL-27 ITTG microencapsulated with coffee extract ranged from 8.83 to 9.47 Log₁₀ CFU/g of beads (

Table 1. Viability and MEE of Lactiplantibacillus fabifermentans BAL-27 ITTG and phenolic compounds (PCs), PCEE, and RGS of probiotics encapsulated in beads.

Treatment	Viability (Log10 CFU/g Bead)	MEE (%)	PC (mg GAE/g Bead)	PCEE (%)	RGS (%)
T1	8.83 c	82.97 b	0.48 a	14.22 c	84.38 ab
T2	9.09 abc	82.86 b	0.49 a	17.02 bc	83.82 b
T3	9.45 a	84.70 a	0.47 a	19.75 ab	85.34 a
T4	9.47 a	84.84 a	0.51 a	20.99 a	73.65 e
T5	8.91 bc	81.00 c	0.48 a	20.86 a	75.51 d
T6	9.00 abc	81.79 c	0.52 a	19.12 ab	84.08 ab
T7	9.29 abc	83.47 b	0.52 a	19.06 ab	77.94 c
T8	9.38 ab	83.31 b	0.52 a	20.89 a	76.11 d
Tukey	0.532	1.01	0.07	3.03	1.50

MEE = microbial encapsulation efficiency. PC = phenolic compound. PCEE = phenolic compound encapsulation efficiency. RGS = resistance gastrointestinal simulation. T1 = 1.5% (w/v) alginate, 8 min, without chitosan. T2 = 3.0% (w/v) alginate, 8 min, without chitosan. T3 = 3.0% (w/v) alginate, 20 min, without chitosan. T4 = 3.0% (w/v) alginate, 20 min, with chitosan. T5 = 1.5% (w/v) alginate, 8 min, with chitosan. T6 = 1.5% (w/v) alginate, 20 min, without chitosan. T7 = 3.0% (w/v) alginate, 8 min, with chitosan. T8 = 1.5% (w/v) alginate, 20 min, with chitosan. Similar lowercase letters in the same column indicate no significant difference between treatments (p > 0.05).

). The highest alginate concentration (3%) combined with the longest crosslinking time (20 min) resulted in the greatest probiotic viability (9.45 and 9.47 Log₁₀ CFU/g), regardless of the presence or absence of a chitosan coating. In contrast, treatments T1 and T5 presented the lowest content of viable microorganisms in the beads (8.83 and 8.91 Log₁₀ CFU/g, respectively). This is related to the low alginate concentration (1.5% w/v) and short crosslinking time (8 min), regardless of the presence or absence of chitosan.

Although achieving high cell viability within beads is one of the main goals of encapsulation, ensuring high microbial encapsulation efficiency (MEE) is also essential. The T3 and T4 treatments presented the highest MEE values, at 84.70% and 84.84%, respectively (Table 1). This outcome may be influenced by the alginate concentration and crosslinking time, as illustrated in the Pareto diagram (Figure 1A). The diagram indicates that both factors had a positive effect on the MEE, whereas the addition of chitosan had no significant influence. The MEE increased with the increase in the alginate concentration and crosslinking time. In contrast, the T5 and T6 treatments presented the lowest MEE values, at 81.00% and 81.79%, respectively (Table 1).

3.1.2. Encapsulated Phenolic Compounds

The encapsulation of phenolic compounds is another important criterion, as it directly affects their stability, bioavailability, and biological activity in the consumer. These compounds play crucial roles in preventing oxidative stress, as their consumption is associated with reduced cellular damage, enhanced immune defense, and a lower risk of developing diseases related to free radicals [20]. The concentration of phenolic compounds in the beads ranged from 0.47 to 0.51 mg GAE/g across all the treatments (Table 1), corresponding to phenolic compound encapsulation efficiencies (PCEEs) between 14% and 20%.

The analysis of variance revealed that, while the factors studied did not significantly affect the concentration of encapsulated phenolic compounds, they did have a significant influence on the PCEE (Figure 1B). The lowest PCEE values were observed in treatments T1 and T2 (14.22% and 17.02%, respectively), both of which used an 8-minute crosslinking time. Conversely, the addition of chitosan contributed to higher PCEE values, even under short crosslinking conditions, as observed in treatments T1 and T2 (Table 1).

3.1.3. Viability of Lactiplantibacillus fabifermentans BAL-27 ITTG During Gastrointestinal Simulation

The resistance of a probiotic to gastrointestinal simulation is key to ensuring its arrival in the large intestine and ability to colonize it. The highest resistance to gastrointestinal simulation was 85.34% for treatment T3, compared with treatments T5 and T8, with values of 75.51 and 76.11%, respectively (Table 1). This may be because treatment T3 contained a higher concentration of alginate (3%) without the addition of chitosan than did treatments T5 and T8, which presented the lowest concentration of alginate (1.5%) supplemented with chitosan in both treatments.

3.2. Evaluation of the Quality of Alginate Beads During Yogurt Storage

3.2.1. Viability of Lactiplantibacillus fabifermentans BAL-27 ITTG

Beads prepared with 1.5% (w/v) sodium alginate and crosslinked for 8 or 20 min, both with and without chitosan, maintained a probiotic concentration of approximately 9 Log₁₀ CFU/g throughout the 28-day storage period. The viability kinetics (Figure 2A) revealed that, from week 0 to week 3, the concentration of viable microorganisms remained stable. However, a significant increase in viability was observed between weeks 3 and 4. A similar trend was observed with higher alginate concentrations (3%), as shown in Figure 2B.

3.2.2. Phenolic Compounds in Beads During Storage

At the beginning of the storage period, no significant differences were observed in the phenolic compound content among the treatments (p > 0.05). However, by the end of the storage period, treatments T1, T2, T3, and T6 presented significant differences (p < 0.05), indicating that storage time had a measurable effect on these treatments (

Table 2. Concentration of phenolic compounds (mg EAG/g bead) in alginate beads during yogurt storage.

Treatment	Storage Time (Days)		Tukey
	0	28
T1	0.48 Aa	0.43 Bb	0.04
T2	0.49 Aa	0.45 Bb	0.04
T3	0.47 Aa	0.41 Babc	0.08
T4	0.51 Aa	0.52 Aa	0.07
T5	0.48 Aa	0.47 Aabc	0.13
T6	0.52 Aa	0.48 Babc	0.02
T7	0.52 Aa	0.52 Aa	0.04
T8	0.52 Aa	0.50 Aab	0.02
Tukey	0.07	0.04

T1 = 1.5% (w/v) alginate, 8 min, without chitosan. T2 = 3.0% (w/v) alginate, 8 min, without chitosan. T3 = 3.0% (w/v) alginate, 20 min, without chitosan. T4 = 3.0% (w/v) alginate, 20 min, with chitosan. T5 = 1.5% (w/v) alginate, 8 min, with chitosan. T6 = 1.5% (w/v) alginate, 20 min, without chitosan. T7 = 3.0% (w/v) alginate, 8 min, with chitosan. T8 = 1.5% (w/v) alginate, 20 min, with chitosan. Similar lowercase letters in the same column indicate no significant difference between treatments (p > 0.05). Similar capital letter in the same row indicates no significant difference between 0 and 28 days of storage (p > 0.05).

).

The treatments with chitosan (T4, T5, T7, and T8) did not significantly differ (p < 0.05) during storage, which suggests that the chitosan layer also functions as a protective barrier, limiting the release and diffusion of phenolic compounds.

3.2.3. Viability of Lactiplantibacillus fabifermentans BAL-27 ITTG from Alginate Beads During Gastrointestinal Simulation after Four Weeks of Yogurt Storage

After four weeks of yogurt storage, the viability of Lactiplantibacillus fabifermentans BAL-27 ITTG under simulated gastrointestinal conditions ranged from 71% to 89% (

Table 3. Survival of Lactiplantibacillus fabifermentans BAL-27 ITTG on alginate beads during gastrointestinal simulation after 28 days of storage.

Treatment	RGS (%)
T1	89.82 a
T2	83.82 c
T3	84.22 c
T4	79.86 d
T5	73.25 f
T6	88.09 b
T7	77.23 e
T8	71.88 g
Tukey	2.8104

RGS = Resistance gastrointestinal simulation. T1 = 1.5% (w/v) alginate, 8 min, without chitosan. T2 = 3.0% (w/v) alginate, 8 min, without chitosan. T3 = 3.0% (w/v) alginate, 20 min, without chitosan. T4 = 3.0% (w/v) alginate, 20 min, with chitosan. T5 = 1.5% (w/v) alginate, 8 min, with chitosan. T6 = 1.5% (w/v) alginate, 20 min, without chitosan. T7 = 3.0% (w/v) alginate, 8 min, with chitosan. T8 = 1.5% (w/v) alginate, 20 min, with chitosan. Similar lowercase letters in the same column indicate no significant difference between treatments (p > 0.05).

). The highest survival rate was observed in treatment T1, which used 1.5% alginate, an 8-minute immersion time, and no chitosan coating. In contrast, the lowest survival was recorded in the T8 treatment, which involved 1.5% alginate, a 20-minute immersion time, and a chitosan coating.

3.3. Effects of the Addition of Lactiplantibacillus fabifermentans BAL-27 ITTG and Encapsulated Phenolic Compounds on the Physicochemical and Sensory Properties of Yogurt

Once the yogurt was confirmed to function as a functional food, it was necessary to evaluate whether its physicochemical and sensory properties were affected. For the analyses of syneresis, pH, and titratable acidity, the encapsulated beads were removed from the yogurt prior to measurement. However, for the sensory evaluation, the beads were left intact.

3.3.1. Determination of the Physicochemical and Microbiological Properties of Yogurt

Yogurt Syneresis

Syneresis is a key physical parameter used to assess yogurt quality, as it reflects the instability of the gel network and its inability to retain the serum phase within the matrix [22]. Elevated levels of syneresis are generally undesirable from a consumer perspective. The incorporation of alginate beads significantly increased (p < 0.05) the syneresis of the yogurt compared with the control sample (yogurt without beads) (

Table 4. Syneresis (%) of yogurt without beads (control) and with alginate beads stored for 28 days under refrigeration.

	Time (Days)
Treatment	0	7	14	21	28	Tukey
T1	20.65 Ac	23.40 Bc	25.52 Cc	25.29 Cc	28.48 Dc	1.42
T2	21.03 Acd	24.74 Bc	24.74 Bc	24.91 Bc	25.09 Bb	0.97
T3	21.84 Ade	24.1 Bc	24.21 Bc	25.41 Cc	25.91 Cb	1.01
T4	29.70 Ag	31.37 Ad	34.57 Bf	39.26 Cf	39.93 Cf	1.88
T5	22.42 Ae	24.45 Bc	27.37 Cd	29.91 Dd	35.45 Ed	1.50
T6	17.60 Ab	18.84 Ab	21.33 Bb	23.36 Cb	25.56 Db	1.88
T7	23.73 Af	27.69 Bd	29.65 Ce	30.46 Cd	38.33 De	1.90
T8	23.06 Af	25.65 Bcd	28.52 Cde	32.86 De	37.54 Ee	0.86
Control	13.63 Aa	15.29 Ba	17.71 Ca	20.14 Da	20.58 Da	1.33
Tukey	1.00	2.77	1.95	1.04	1.02

T1 = 1.5% (w/v) alginate, 8 min, without chitosan. T2 = 3.0% (w/v) alginate, 8 min, without chitosan. T3 = 3.0% (w/v) alginate, 20 min, without chitosan. T4 = 3.0% (w/v) alginate, 20 min, with chitosan. T5 = 1.5% (w/v) alginate, 8 min, with chitosan. T6 = 1.5% (w/v) alginate, 20 min, without chitosan. T7 = 3.0% (w/v) alginate, 8 min, with chitosan. T8 = 1.5% (w/v) alginate, 20 min, with chitosan. Similar lowercase letters in the same column indicate no significant difference between treatments (p > 0.05). Similar capital letters in the same row indicate no significant difference between storage times (p > 0.05).

). The highest percentages of syneresis (22–29%) were observed in treatments T4, T5, T7, and T8, all of which included chitosan.

On the other hand, the T1, T2, T3, and T6 treatments, which did not contain chitosan, also resulted in significantly greater syneresis (p < 0.05) than the control yogurt. The presence of beads in yogurt from the beginning of fermentation disrupted the homogeneity of the product, as illustrated in Figure 3. This disruption likely interfered with the formation of a uniform casein network, thereby contributing to the increased syneresis observed. The syneresis of the yogurt during storage (Table 4) increased even for the yogurt without beads. Table 4 shows that the chitosan treatments presented the highest percentage of syneresis, which was above 35%.

Titratable pH and Acidity

The initial pH of the yogurt ranged from 4.26 to 4.34 (

Table 5. pH of yogurt without beads (control) and with alginate beads stored for 28 days under refrigeration.

	Time (Days)
Treatment	0	7	14	21	28	Tukey
T1	4.34 Aa	4.09 Bd	4.00 BCc	3.97 Cb	3.97 Ccd	0.08
T2	4.32 Aab	4.09 Bd	4.03 Bc	3.96 Cb	3.93 Cd	0.06
T3	4.26 Aabc	4.13 Bcd	4.02 Cc	3.97 CDb	3.96 Dcd	0.05
T4	4.29 Aabc	4.15 Bbc	4.09 Bb	4.00 Cab	4.00 Cabc	0.08
T5	4.26 Aabc	4.17 Bbc	4.09 Cb	4.01 Dab	4.00 Dabc	0.04
T6	4.26 Aabc	4.18 ABb	4.09 BCb	4.03 Cab	3.98 Cbcd	0.13
T7	4.27 Aabc	4.17 Bbc	4.10 Cb	4.04 Dab	4.04 Dab	0.05
T8	4.27 Aabc	4.15 Bbc	4.10 Cb	4.04 Dab	4.00 Eabc	0.03
Control	4.34 Aa	4.27 Ba	4.20 Ca	4.11 Da	4.05 Ea	0.03
Tukey	0.08	0.04	0.05	0.12	0.06

T1 = 1.5% (w/v) alginate, 8 min, without chitosan. T2 = 3.0% (w/v) alginate, 8 min, without chitosan. T3 = 3.0% (w/v) alginate, 20 min, without chitosan. T4 = 3.0% (w/v) alginate, 20 min, with chitosan. T5 = 1.5% (w/v) alginate, 8 min, with chitosan. T6 = 1.5% (w/v) alginate, 20 min, without chitosan. T7 = 3.0% (w/v) alginate, 8 min, with chitosan. T8 = 1.5% (w/v) alginate, 20 min, with chitosan. Similar lowercase letters in the same column indicate no significant difference between treatments (p > 0.05). Similar capital letters in the same row indicate no significant difference between storage times (p > 0.05).

), values that are close to those of the control yogurt, which did not contain beads, and are within the maximum allowable limit (pH~4.50) according to NOM-243-SSA1-2010 [19]. During yogurt production, the initial milk pH of 6.2 decreases due to the activity of the starter culture, as approximately 20–40% of the lactose is converted into lactic acid, as reported by Cheng [23]. Throughout storage, the pH of the yogurt decreased, with statistically significant differences (p < 0.05) observed between the treatments. At the end of storage, the pH of the yogurt ranged from 3.93 to 4.0, with a significant difference (p < 0.05) between the control yogurt and the yogurt stored without chitosan (treatments T1, T2, T3, and T6). This variation could be attributed to the potential release of L. fabifermentans BAL-27 ITTG into the yogurt. The titratable acidity of yogurt supplemented with alginate beads increased slightly during storage, which accounts for the observed decrease in pH (

Table 6. Titratable acidity (% lactic acid) of yogurt without beads (control) and with alginate beads stored for 28 days under refrigeration.

Treatment	Time (Days)
	0	7	14	21	28	Tukey
1	0.96 Abc	0.98 Aab	0.99 Aab	1.04 Bab	1.11 Cabc	0.03
2	0.94 Aab	0.96 Aa	1.04 Bab	1.09 BCab	1.12 Cbc	0.06
3	0.94 Aab	1.06 Bc	1.06 Bb	1.14 Cb	1.14 Cc	0.02
4	0.94 Aab	0.99 ABab	1.01 ABab	1.03 ABa	1.08 Ba	0.10
5	0.92 Aa	0.95 Ba	0.98 Ca	1.05 Dab	1.09 Eab	0.01
6	0.96 Abc	1.03 ABbc	1.05 BCb	1.12 Cab	1.14 Cc	0.08
7	0.95 Abc	1.02 Bbc	1.06 Cb	1.10 Dab	1.12 Eab	0.02
8	0.98 Ac	1.03 ABbc	1.06 BCb	1.11 Cab	1.09 BCab	0.07
Control	0.92 Aa	0.96 ABa	1.00 Bab	1.05 Cab	1.09 Cab	0.04
Tukey	0.02	0.05	0.02	0.05	0.04

T1 = 1.5% (w/v) alginate, 8 min, without chitosan. T2 = 3.0% (w/v) alginate, 8 min, without chitosan. T3 = 3.0% (w/v) alginate, 20 min, without chitosan. T4 = 3.0% (w/v) alginate, 20 min, with chitosan. T5 = 1.5% (w/v) alginate, 8 min, with chitosan. T6 = 1.5% (w/v) alginate, 20 min, without chitosan. T7 = 3.0% (w/v) alginate, 8 min, with chitosan. T8 = 1.5% (w/v) alginate, 20 min, with chitosan. Similar lowercase letters in the same column indicate no significant difference between treatments (p > 0.05). Similar capital letters in the same row indicate no significant difference between storage times (p > 0.05).

). A similar decrease in pH was also observed in the control yogurt without beads. According to NOM-243-SSA1-2010 [19], the minimum allowable titratable acidity is 0.50%, and all the treatments met this standard.

To demonstrate the potential release of Lactiplantibacillus fabifermentans BAL-27 ITTG from the beads, the concentration of Lactiplantibacillus fabifermentans BAL-27 ITTG in the yogurt was determined (

Table 7. Concentration of Lactiplantibacillus fabifermentans BAL-27 ITTG and starter culture in yogurt at the end of storage (28 days).

Treatment	L. fabifermentans (CFU/g Yogurt)	Starter Culture (CFU/g Yogurt)
T1	~104	~109
T2	~103	~109
T3	~104	~109
T4	ND	~109
T5	ND	~109
T6	~103	~109
T7	ND	~109
T8	ND	~109

ND = NOT DETECTED.

). The concentration of L. fabifermentans was extremely low (between ~10³ and ~10⁴ CFU/g) in treatments T1, T2, T3, and T6, which did not contain chitosan. This value was negligible compared with the concentration of the starter culture (9 Log₁₀ CFU/g yogurt).

Sensory Analysis of Yogurt

A hedonic test was conducted to assess the palatability of the yogurt containing beads. Compared with those of the control yogurt (without beads), the sensory attributes were slightly affected (

Table 8. Results of the seven-point hedonic test performed on the yogurt at 0 and 28 days of storage.

Descriptor	Time (Days)	Treatment
		Control	T1	T2	T3	T4	T5	T6	T7	T8	Tukey
Color	0	6.26 Aa	5.25 cdA	5.08 dA	5.56 bcdA	6.05 abA	5.38 cdA	5.66 bcdA	6.25 abA	5.80 abcA	0.56
	28	6.50 Aa	5.46 cA	5.57 cA	5.89 abcA	6.39 abA	6.16 abcA	5.7 bcA	6.36 abA	6.40 abA	0.77
Odor	0	6.05 aA	5.36 bA	5.41 bA	5.71 abA	5.48 abA	5.18 bA	5.78 abA	5.80 abA	5.50 abA	0.63
	28	6.14 Aa	5.46 aA	5.67 aA	5.82 aA	5.82 aA	5.56 aA	5.50 aA	5.70 aA	5.86 aA	0.90
Flavor	0	6.29 Aa	4.73 dA	5.21 cdA	5.3 bcdA	5.38 bcdA	5.70 abcA	5.55 bcA	5.91 abA	5.51 bcA	0.69
	28	6.28 aA	5.35 abA	5.42 abA	5.67 abA	6.0 abA	5.60 abA	5.16 abA	5.66 abA	6.16 aA	0.98
Texture	0	5.85 aA	4.6 cA	4.91 bcA	5.15 abcA	5.16 abcA	4.56 cA	4.98 bcA	5.43 abA	5.11 bcA	0.84
	28	6.17 aA	5.07 bcA	4.28 cA	5.10 bcA	5.75 abcA	5.33 abA	5.33 abcA	5.76 abA	5.86 abA	0.95
Global Appearance	0	6.25 aA	5.01 bA	5.2 cA	5.40 bcA	5.41 bcA	5.50 bcA	5.45 bcA	5.95 abA	5.46 bcA	0.11
	28	6.42 aA	5.42 abcA	5.17 cdA	5.5 bcdA	5.96 abcA	5.73 abcdA	5.10 dA	5.76 abcdA	6.06 abA	0.84

T1 = 1.5% (w/v) alginate, 8 min, without chitosan. T2 = 3.0% (w/v) alginate, 8 min, without chitosan. T3 = 3.0% (w/v) alginate, 20 min, without chitosan. T4 = 3.0% (w/v) alginate, 20 min, with chitosan. T5 = 1.5% (w/v) alginate, 8 min, with chitosan. T6 = 1.5% (w/v) alginate, 20 min, without chitosan. T7 = 3.0% (w/v) alginate, 8 min, with chitosan. T8 = 1.5% (w/v) alginate, 20 min, with chitosan. Control = yogurt without beads. Similar lowercase letters in the same row indicate no significant difference between treatments for each descriptor (p > 0.05). Similar capital letters at 0 and 28 days for each descriptor indicate no significant difference (p > 0.05).

) by the addition of beads. However, the observed decrease was minimal, corresponding to only one point on the hedonic scale for each attribute. Although this comparison was made, it does not imply that the product is undesirable, as the products are inherently different.

The color of a product is a crucial indicator, as it is the first visual cue for consumers when selecting a product [24]. At the beginning of storage, color (Table 8) was significantly different (p < 0.05) between the treated yogurt and the control yogurt. However, the yogurt containing chitosan beads did not significantly differ (p > 0.05) from the other yogurt samples, indicating that this attribute was favorable. The chitosan beads afforded the yogurt a shinier appearance, which was considered by the panelists to be an improvement. Odor is another key property that distinguishes yogurt. On day zero, the acceptance scores were in the range of 5.18–5.80 across the treatments (Table 8). By 28 days, no significant difference (p > 0.05) was detected between the yogurt from the treatment groups containing beads and the yogurt from the control group. These results are favorable, suggesting that the addition of beads did not negatively impact the odor of the product and that the yogurt remained acceptable at the end of storage. Similarly, the flavor did not significantly differ (p > 0.05) between the treated yogurt and the control yogurt at the end of storage (Table 8).

Furthermore, although the addition of beads to the yogurt matrix can affect its texture, the yogurt with added beads showed no significant change in texture at either the beginning or the end of storage (Table 8). Finally, in terms of general acceptance at the end of storage, the T1, T4, T5, T7, and T8 treatments did not significantly differ (p > 0.05) from the control yogurt (Table 8). Therefore, the addition of alginate beads to yogurt did not affect its overall acceptance. In general, the sensory attributes of flavor, odor, texture, and general acceptance were not adversely affected by the incorporation of alginate beads. These results suggest that the decrease in pH during storage and the increase in acidity did not influence the sensory outcomes, probably because of the low concentration of lactic acid.

4. Discussion

A key aspect of this study was the evaluation of the survival of Lactiplantibacillus fabifermentans BAL-27 ITTG and the encapsulation efficiency of phenolic compounds under different formulation conditions. The survival of Lactiplantibacillus fabifermentans BAL-27 ITTG ranged from 8.83 to 9.47 Log₁₀ CFU/g, while the encapsulation efficiency of phenolic compounds (PCEE) was approximately 20%. An increase in the alginate concentration (3%) and crosslinking time (20 min) led to improved encapsulation efficiency. However, chitosan did not significantly influence the MEE (Figure 1A). This could be because chitosan was applied as a coating on the bead, where the probiotic and phenolic compounds were already protected. These findings are consistent with those reported by Abbaszadeh et al. [25], who demonstrated that higher alginate concentrations enhanced the encapsulation efficiency of Lactobacillus rhamnosus. This behavior is attributed to the increased rigidity provided by the alginate matrix, which limits the release of microorganisms during the hardening and coating stages [25,26]. The MEE values are particularly noteworthy considering the report by Khochapong et al. [14], which indicated that aqueous coffee husk extract inhibits the growth of microorganisms belonging to the genus Lactobacillus. Despite this, the viability of Lactiplantibacillus fabifermentans BAL-27 ITTG remained above 6 Log₁₀ CFU/g, the minimum threshold recommended by the FAO/WHO [21] for probiotics to confer health benefits through food consumption. Therefore, the successful coencapsulation of Lactiplantibacillus fabifermentans BAL-27 ITTG with phenolic compounds from coffee husk supports the potential development of a novel functional food product.

With respect to the crosslinking time, longer durations favor enhanced intermolecular interactions between the guluronic acid residues in sodium alginate, leading to a more cohesive gel network [26,27]. In contrast, shorter crosslinking times may result in incomplete gelation and reduce the probiotic MEE [26]. Additionally, during the gelation process, alginate forms a more stable crosslinked matrix in the presence of calcium ions, thereby supporting probiotic survival [28].

Nevertheless, prior to gelation, alginate could interact with both the phenolic compounds from the coffee extract and the probiotic cell wall, creating potential competition for calcium ion (Ca²⁺) binding. Caffeine and chlorogenic acid—two of the principal phenolic compounds identified in coffee husks [2]—could engage in hydrogen bonding with alginate. Specifically, interactions could occur between the hydroxyl groups of chlorogenic acid, the carbonyl groups of caffeine, and the carboxyl groups of alginate. Such interactions could influence the structure and functionality of the encapsulation matrix. These mechanisms may significantly influence the stability and encapsulation efficiency of phenolic compounds within the alginate matrix, as reported by Machado et al. [29]. Additionally, the low PCEE observed in this study could be attributed to the diffusion of phenolic compounds into the calcium chloride solution during the crosslinking process, thereby reducing their retention within the encapsulating matrix. The physical entrapment of these compounds in the “egg-box” structure formed by calcium–alginate interactions could also be insufficient to ensure complete encapsulation, ultimately compromising both stability and efficiency.

The PCEE values obtained in this study are comparable to those reported by other authors. For example, Arriola et al. [30] reported a PCEE of 20% in alginate beads containing dandelion extract, whereas Flamminii et al. [31] reported a PCEE of 21% when encapsulating olive leaf extract with a combination of alginate and pectin. However, our encapsulation efficiencies were lower than those reported by Belščak et al. [9], who achieved 68.94% encapsulation efficiency for caffeine using an alginate–chitosan matrix, and those reported by Li et al. [10], who obtained 57.76% efficiency for phenolic compounds from green tea via the same encapsulation system.

These discrepancies may be attributed primarily to methodological differences among the studies. In the present work, beads containing coffee extract were immersed in a chitosan solution after gelation, whereas in previous studies, chitosan was incorporated directly into a mixture of phenolic compounds and CaCl₂ prior to the crosslinking process. This alternative strategy may increase the encapsulation efficiency by promoting earlier interaction and stabilization of the phenolic compounds within the polymer matrix.

Although the Pareto diagram (Figure 1B) indicates that both the addition of chitosan and extended crosslinking time had a statistically significant and positive effect on the PCEE, the magnitude of this increase appears to be constrained by the nature of intermolecular interactions among phenolic compounds, sodium alginate, microbial cell wall components, and calcium chloride during gelation. Given the hydrophilic nature of phenolic compounds, they are more likely to remain in the aqueous phase, thereby reducing their entrapment within the polymer matrix and subsequently limiting the PCEE [31].

The observed increase in the PCEE may be attributed to the cationic nature of chitosan, which can form an external membrane around alginate beads by establishing electrostatic interactions with the carboxyl groups of alginate. These interactions help to stabilize the matrix and reduce the diffusion and loss of phenolic compounds during the encapsulation process [32].

During gastrointestinal simulation, the encapsulated probiotic demonstrated a certain degree of protection. Under simulated gastric conditions (pH 1.9), the chitosan layer may have been partially solubilized, contributing to the degradation of the polymeric network. As a result, the probiotic was exposed to gastric fluid containing chitosan, which may have exerted a synergistic antimicrobial effect and reduced probiotic viability. Indeed, Barbosa et al. [33] reported the antimicrobial activity of chitosan under acidic conditions.

In contrast, Ramírez-Pérez et al. [7] reported a viability of 90% for Lactiplantibacillus fabifermentans BAL-27 ITTG in gastrointestinal simulations when no encapsulation system was applied. Although a greater viability may be observed in the absence of encapsulation under short-term conditions, in the long term, encapsulation remains essential to protect microorganisms from environmental stressors and to ensure their stability during prolonged storage.

On the other hand, during the initial stages of gastric simulation, the volume of the alginate beads gradually increased, allowing for the controlled release of Lactiplantibacillus fabifermentans BAL-27 ITTG. At the end of the gastric phase and throughout the intestinal simulation, the beads continued to swell without undergoing structural disintegration. This volumetric expansion is likely related to the increase in internal pH caused by exposure to intestinal fluid, which promotes bead swelling. Under acidic conditions, the amino groups of chitosan are ionized, while the hydroxyl groups of phenolic compounds become deprotonated, leading to matrix expansion and facilitating the release of encapsulated microorganisms [32,34].

The survival rates observed in this study are consistent with those reported by Azam et al. [35], who reported approximately 80% viability of L. rhamnosus encapsulated in sodium alginate beads. According to these authors, the internal structure of alginate beads contains intramolecular voids that enable bile salt diffusion, which in turn contributes to the increased bead volume during intestinal simulation.

Throughout the storage period, Lactiplantibacillus fabifermentans BAL-27 ITTG maintained its viability within the encapsulation system. The “egg-box” structure of the calcium–alginate matrix likely played a crucial role in retaining both the microorganisms and phenolic compounds while simultaneously reducing water availability within the beads. This decrease in water activity (Aw) likely limited microbial metabolism, rendering the probiotic less susceptible to environmental stress. The survival of Lactiplantibacillus fabifermentans BAL-27 ITTG in the bead during 28 days of storage could be explained by the fact that coffee husk contains approximately 12% sugars [36], which may serve as a carbon source supporting minimal growth and survival over the period, despite the limited nutrient and water mobility inside the bead. Similarly, Machado et al. [37] demonstrated that sugars extracted from coffee husks promoted the growth of L. paracasei, suggesting that these carbohydrates exert a beneficial effect on probiotic viability. These findings are particularly encouraging, as they confirm the sustained viability of the encapsulated probiotic in yogurt. In contrast, previous studies by Silva et al. [4], De Prisco et al. [5], and Chaikha [38] reported a decline of approximately three logarithmic cycles in the viability of encapsulated probiotics during storage. Similarly, Kumar and Kumar [6] reported a decrease in Lactobacillus rhamnosus viability from 8.8 to 4.35 Log₁₀ CFU/g over a 15-day period when beads composed of 2% alginate and 2% CaCl₂ were used.

The retention of the gastrointestinal survival (RGS) of probiotics after four weeks of storage is particularly relevant; however, these values decreased over time. This reduction in probiotic viability may be attributed to the prolonged storage of the beads in yogurt. As storage progressed, the acidity of the yogurt increased, potentially leading to the partial dissolution of the chitosan-coated beads under acidic conditions. This process facilitates the release of the encapsulated probiotic. Once released, chitosan may interact with the positively charged free amino groups present in the probiotic cell wall, causing structural damage and, consequently, cell death [39]. In contrast, beads composed solely of alginate as the encapsulating agent did not exhibit this detrimental effect, thereby allowing for higher survival rates of the probiotic throughout the four-week storage period.

A decrease in the concentration of phenolic compounds was observed at the end of the storage period. This reduction may be attributed to the diffusion of phenolic compounds into the yogurt matrix. As previously described by Chan et al. [40] and Gombotz and Wee [41], water-soluble bioactive compounds can readily diffuse from the encapsulation matrix into the surrounding medium, driven by a concentration gradient.

Nevertheless, the presence of chitosan appeared to mitigate this diffusion, functioning as a protective barrier. The addition of chitosan likely reduced the release of phenolic compounds through electrostatic interactions between the carboxyl groups of alginate and the amino groups of chitosan. These interactions lead to the development of a semipermeable membrane around the beads, which restricts the outward diffusion of the encapsulated compounds [42].

At this stage, we confirmed both the high survival rate of Lactiplantibacillus fabifermentans BAL-27 ITTG and the retention of phenolic compounds within the encapsulated beads. However, it was also necessary to assess the impact of these beads on the yogurt matrix. Syneresis was evaluated, and higher levels were observed in formulations containing chitosan-coated beads. This phenomenon may be attributed to the behavior of the beads during milk fermentation, wherein the chitosan-coated beads tended to sink to the bottom of the container, as illustrated in Figure 3.

During fermentation, milk acidification leads to the formation of a gel, altering its density and allowing denser chitosan-coated beads to settle. As fermentation proceeds, this downward movement may disrupt the structural integrity of the gel, contributing to water release and consequently increasing syneresis. The sinking behavior of the beads may also be due to the formation of a polyelectrolyte complex between the amino groups of chitosan and the carboxylate groups of alginate, which results in a denser polymeric network [43] than that of uncoated beads. Moreover, increased syneresis may be partially explained by the acid-induced contraction of the protein network. Higher acidity promotes protein hydration and gel shrinkage, ultimately leading to enhanced syneresis [44]. Among the physicochemical parameters, pH and acidity are essential indicators of yogurt quality. The initial drop in pH is primarily due to lactic acid production by the starter culture. However, the continued decrease in pH during storage can be attributed to the gradual release of Lactiplantibacillus fabifermentans BAL-27 ITTG from the beads, which ferment lactose into organic acids, mainly lactic and acetic acids [7,45].

Several studies have examined the incorporation of alginate beads into yogurt and their influence on the food matrix. For example, Pourjafar et al. [46] and Budianto et al. [47] reported that the addition of alginate beads did not significantly alter the pH of the yogurt compared with that of control formulations and that the pH remained stable throughout storage. These findings contrast with our results, where a noticeable decrease in pH was observed during storage, presumably due to probiotic release and metabolic activity.

Considering the slight acidification observed, a sensory evaluation was conducted via a hedonic test to assess consumer acceptability. The results indicate that none of the treatments were negatively affected by the addition of beads. These findings align with those of Kailasapathy [48], who reported that the use of encapsulated beads in yogurt did not alter color, acidity, or flavor properties. During fermentation, milk protein proteolysis generates peptides and amino acids, which contribute to the formation of flavor compounds [49], and short-chain fatty acids are also produced [7]. Even at the end of the storage period, the yogurt was rated as acceptable by the panelists. Notably, despite the release of Lactiplantibacillus fabifermentans BAL-27 ITTG and phenolic compounds, the sensory attributes of the yogurt—such as odor, texture, and appearance—remained unchanged. This finding contrasts with previous observations by Rahmani et al. [3], who reported that the direct addition of free phenolic compounds could negatively impact sensory properties. Nonetheless, the overall acceptability in their study remained above the threshold of consumer acceptance.

5. Conclusions

An increase in the concentration of sodium alginate and chitosan, as well as the gelation time during ionic gelation, positively influenced the encapsulation efficiency of the coffee extract and the viability of Lactiplantibacillus fabifermentans BAL-27 ITTG in alginate beads. This ionic gelation process enabled the encapsulation of phenolic compounds and L. fabifermentans BAL-27 ITTG in stable gel beads, which were successfully maintained in yogurt for 28 days of storage. The concentration of phenolic compounds and the viability of Lactiplantibacillus fabifermentans BAL-27 ITTG in chitosan-coated beads remained stable throughout the four-week yogurt storage period. Although the physicochemical properties of yogurt supplemented with alginate beads were significantly different from those of the control yogurt at the end of storage, these changes did not negatively impact consumer acceptance, as assessed through sensory evaluation. Yogurt containing L. fabifermentans BAL-27 ITTG and coffee extract encapsulated in sodium alginate beads exhibited acceptable physicochemical and organoleptic properties, making it a promising functional food product. Given its beneficial characteristics, this yogurt formulation has potential applications within the food industry.