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Physicochemical Properties of Fermented Cow Milk Enriched with Different Microencapsulated Levilactobacillus brevis

S
Sri Melia1,*
https://orcid.org/0000-0002-7271-0955
I
Indri Juliyarsi1
https://orcid.org/0000-0003-2855-7794
R
Rizki Dwi Setiawan1
https://orcid.org/0009-0005-1703-6688
I
Iceu Agustinisari2
https://orcid.org/0000-0002-4905-9631
Z
Zaky Syahputra1
https://orcid.org/0009-0005-3135-3137
1Department of Animal Products Technology, Faculty of Animal Science, Universitas Andalas, Padang 25163, Indonesia.
2Research Center for Agroindustry, National Research and Innovation Agency, Jl. Raya Serpong, Muncul, Kec. Setu, Kota Tangerang Selatan, Banten 15314, Indonesia.

ABSTRACT

Background: Fermented cow milk is generated when lactic acid bacteria transform lactose into lactic acid, enhancing its nutritional quality, texture and longevity. Microencapsulated probiotic cultures provide a contemporary method to enhance bacterial viability and preserve physical and chemical stability. This study examines the effect of various wall materials on the physicochemical properties of microencapsulated Levilactobacillus brevis in fermented milk.

Methods: This research utilized some treatments with the addition of 1% L. brevis starter with various types of wall materials using the freeze-drying method with the combination of 1% Alginate (A), 1% Alginate + 1% Glucomannan (B), 1% Alginate + 5% WPI (C) and 1% Alginate + 5% WPI + 1% Glucomannan (D).

Result: The combination treatment of 1% alginate + 1% glucomannan + 5% whey protein isolate (WPI) (D) produced the best results of total lactic acid bacteria (18.45 x 109 CFU/mL), pH value of 4.85, total titratable acidity of 1.14% and moisture content of 88.17%. It showed a high protein content of 4.45% with a relatively lower fat content of 2.33% in fermented milk. The combination of wall materials in microencapsulated products serving as a dry starter for fermented milk can be applied in the processing of fermented milk products that are beneficial for health.

INTRODUCTION

Fermented milk, a type of functional food, has experienced a surge in global demand due to increased consumer awareness of digestive health and immune system support. Fermented cow milk is generated when lactic acid bacteria transform lactose into lactic acid, enhancing its nutritional quality, texture and longevity. In comparison to fresh milk, it possesses a denser viscosity, a more pronounced taste and improved digestion. According to Elango (2024), research and development activities in the functional food sector have been heavily influenced by products targeting gut health. By preserving the healthy balance of the gut microbiota, probiotics are increasingly being added to foods and drinks to enhance general health. There are several opportunities to effectively and simply add probiotics to dairy products, particularly beverages. Typical examples include yogurt, kefir and buttermilk, esteemed for their health benefits and culinary versatility. In a study by Soni et al., (2021), Lactobacillus species were detected in yogurt from Gujarat, suggesting their potential as probiotics. Lactic acid bacteria (LAB) are pivotal among helpful germs. Levilactobacillus brevis, recognized for its ability to ferment diverse substrates and influence gut microbiota (Banerjee et al., 2021), is a promising strain. This research utilizes L. brevis isolated from dadih, a traditional Minangkabau fermented milk from West Sumatra, Indonesia, which is naturally fermented in bamboo (Puspawati et al., 2018). Metagenomic investigations have validated the predominance of LAB in dadih from areas such as Bukittinggi and Solok; however, comprehensive strain characterization remains insufficient (Venema et al., 2019; Fatdillah et al., 2021). Several previous studies have mentioned that several strains of L. brevis from various sources have been found to be probiotic (Song et al., 2020; Ait Chait et al., 2020; Somashekaraiah et al., 2021).

LAB-based fermented products struggle to maintain probiotic viability during processing, storage and digestion. Encapsulating probiotic cells in protective substances enhances their survivability in harsh environments, including low pH, digestive enzymes and high temperatures (Razavi et al., 2021). Microencapsulation has demonstrated efficacy in enhancing nutritional stability, inhibiting interactions and preventing the degradation of raw materials, as the coated matrix effectively segregates particles and prevents their contact (Adeyeye et al., 2025). Microencapsulated products exhibit stability when integrated into actual food matrices. Food products shouldn’t be adversely affected by this integration, particularly their sensory qualities, which may change due to the addition of microencapsulated substances (Dias et al., 2017).

This approach utilizes wall components such as sodium alginate, glucomannan and WPI, either alone or in combination. Ionic gelation using CaCl2 at ambient temperature creates gel beads from alginate, preventing thermal degradation. This protects sensitive bioactive molecules and improves stability. Alginate and glucomannan reinforce the gel matrix, whereas whey protein isolate (WPI) improves encapsulation and structural integrity (Tonon et al., 2011).


Active compounds are synergistically protected from oxidation, enzymes and stomach acid by a three-component matrix consisting of alginate, glucomannan and WPI. Alginate and glucomannan stabilize the capsule in gastric conditions, allowing for regulated release in the digestive tract, while WPI creates an oxygen barrier. These wall materials influence the physicochemical characteristics of fermented goods, including moisture, pH, acidity, protein and fat content and they also promote the viability of probiotics (Kowalska et al., 2022).

Previous studies on fermented milk have employed pure cultures free of microencapsulation (Melia et al., 2020; Melia et al., 2022). Fan et al., (2023) employed Levilactobacillus brevis CGMCC1.5954 as a starter culture in yogurt production. This study evaluates the effect of combining various wall materials with alginate on the microencapsulation of Levilactobacillus brevis as a dry starter on the physicochemical quality of fermented milk.

MATERIALS AND METHODS

Materials
 
The materials used in this study were cow’s milk (full-cream milk) and L. brevis isolated from dadih, obtained from the Laboratory of Animal Product Technology and Processing at the Faculty of Animal Science Universitas Andalas.
 
Methods
 
This research used the experimental method with a completely randomized design. The treatments applied in this research were the addition of 1% L. brevis starter with various types of wall materials of microencapsulation: 1% Alginate (A), 1% Alginate + 1% Glucomannan (B), 1% Alginate + 5% WPI (C) and 1% Alginate + 5% WPI + 1% Glucomannan (D). This research was conducted at the Animal Product Technology Laboratory, Faculty of Animal Science Universitas Andalas, from April to September 2025.
 
Making fermented milk
 
Milk was pasteurized at 85°C for 15 minutes, then cooled to 37°C. After the previous stages, 1% of the starter L. brevis was inoculated according to the treatment and incubated at 37°C for 18 hours in an incubator (Melia et al., 2024).
 
Preparation of microencapsulated product
 
L. brevis probiotics were revived with 30 mL of isolate into 270 mL of MRS Broth, then incubated for 24 hours at 37°C. The culture was then centrifuged at 3000 rpm for 10 minutes at 25°C. The pellet was then washed with 5 mL of sterile distilled water solution and centrifuged again to obtain probiotic bacterial cells. 200 mL of sterile distilled water was stirred using a magnetic stirrer to dissolve the wall material (alginate, glucomannan and WPI). The pellet (bacterial cells) was then resuspended in the solution. Add sterile distilled water to 15% (weight/volume). Homogenize for 5 minutes before drying (Rajam et al., 2012).
 
Measurement of total lactic acid bacteria
 
Microbial growth was analyzed using the method described by Szołtysik et al., (2020). The sample was dissolved in a serial dilution and poured onto MRS Agar medium. Bacterial cells were incubated under anaerobic conditions at 37°C for 48 hours. Bacterial colonies were counted at the end of the cultivation period.
 
Measurement of pH and titratable acidity (TA)
 
AOAC (2005) pH guidelines were followed with a calibrated pH meter. The electrode was immersed in a 30 mL sample until a stable reading was achieved. The Yenrina technique (2015) measured titratable acidity. Five drops of phenolphthalein in 5 mL were titrated with 0.1 N NaOH until a persistent pink color developed. Total fermented milk acidity was calculated using NaOH volume.
 
Proximate analysis
 
The AOAC (2005) methodologies were used to determine the moisture, protein and fat content. A 5 g sample was oven-dried at 105°C for 8 hours to determine the moisture content. It was then cooled in a desiccator and weighed to determine weight loss. The Kjeldahl method, which involves acid digestion with H2SO4 and selenium, alkaline distillation and titration of the distillate with standard NaOH using methyl red as an indicator with a blank for correction, was used to determine the protein concentration. Soxhlet extraction was used to assess the fat content. A 1 g dried sample was extracted with an organic solvent for 5-6 hours, the solvent was eliminated by distillation and the recovered fat was dried in an oven at 100-105°C before being weighed.
 
Morphology of microencapsulated analysis
 
The microencapsulated morphology was examined using a Scanning Electron Microscope (SEM) (Leo 435 VP, Leo Electronic System, Cambridge, UK), following the procedure outlined by Rajam et al., (2012). Samples that had been freeze-dried were placed on specimen holders, covered with gold for two minutes at a pressure of 2 mbar and then examined at 15 kV and a vacuum of 9.75 x 10-5 Torr.

RESULTS AND DISCUSSION

Total lactic acid bacteria
 
The statistical analysis in Table 1 indicates that the wall material has a significant impact on the lactic acid bacteria (LAB) count (P<0.05). Fermented milk containing 1% alginate, 1% glucomannan and 5% WPI (D) had the highest LAB count, surpassing A, B and C. Glucomannan and alginate (B) enhanced viability above alginate alone (A), but WPI alone (C) lowered LAB populations. Treatment D works better since all three components work together. Alginate’s calcium gel matrix protects microorganisms from acidity. As with glucomannan, gel density and viscosity decrease oxygen transport and protect cells from oxidative stress.

Table 1: Characteristics of fermented milk with the addition of microencapsulated L. brevis products from several types of wall material.



Hayuningtyas (2018) asserts that glucomannan enhances the viscosity of emulsion systems in milk, hence increasing stability. WPI creates an acid-resistant protective layer, enhances the microcapsule structure and provides nitrogen for the metabolism of LAB. It additionally stabilizes pH, facilitating bacterial viability during fermentation (Picot and Lacroix, 2004). Studies have demonstrated that the combination of proteins and polysaccharides enhances the stability of microencapsulation (Liu et al., 2023; Puttarat et al., 2021). The formulation in treatment D provides structural strengthening, environmental protection and nutritional support, markedly enhancing LAB viability.
 
pH
 
The pH of fermented milk is significantly influenced by wall material type (P<0.05). Treatment D (1% alginate + 1% glucomannan + 5% WPI) had the lowest pH (4.85±0.057), indicating effective acid production and increased LAB activity. Alginate’s protective gel, glucomannan’s viscosity and prebiotic characteristics and WPI’s nitrogen stimulation of bacterial metabolism may explain this. Treatment C (1% alginate + 5% WPI) had the highest pH (5.86±0.021), indicating slower fermentation. The compact WPI-alginate matrix could prevent LAB growth by preventing nutrition and oxygen transfer. The buffering capacity of WPI stabilizes pH. Treatment D increases LAB activity by increasing viability and acid generation. These findings align with prior studies on the pH of fermented milk and yogurt (Rossi et al., 2021; Falah, 2021). The pH in this study was almost the same as that reported by Narayanan et al., (2020), who used microencapsulation of L. acidophilus and carrot juice in yogurt, which was 4.65.
 
Titratable acidity (TA)
 
The wall material used for microencapsulated L. brevis significantly affected the titratable acidity (TA) of fermented milk (P<0.05), as indicated in Table 1. Treatment D (1% alginate, 1% glucomannan, 5% WPI) had the highest TA, indicating substantial metabolic activity due to its low pH and high LAB count. Alginate forms a protective gel, glucomannan strengthens matrix porosity and nutrient diffusion due to its hydrophilic nature and WPI stabilizes cell membranes. Glucomannan-free Treatment C (1% alginate, 5% WPI) had the lowest TA, highest pH and lowest LAB due to substrate penetration and a denser matrix. Protein walls, such as WPI, can buffer acids and bind water, thereby slowing acidification and altering microbial activity. Polysaccharides promote the viability and metabolism of LAB, resulting in the production of acid. However, excessive encapsulation may reduce mass transfer, nutrient availability and fermentation efficiency. These findings support Quintero et al., (2012), emphasizing balanced microencapsulation design for best performance.
 
Moisture, protein and fat content
 
The moisture content in fermented milk was significantly affected (P<0.05) by the type of microencapsulation wall applied (Table 2). Treatment D fermented milk enhanced with a composite of 1% alginate, 1% glucomannan and 5% WPI yielded the greatest moisture content. This result aligned with the highest overall bacterial count observed in this treatment, indicating improved probiotic viability and metabolic activity. Several investigations have shown that the carboxyl groups of alginate residues rapidly bind to metal ions, including Ca2+, forming the “egg-box” structure, a three-dimensional gel network (Zan et al., 2023), effectively retaining water. In contrast, as a soluble dietary fiber, glucomannan becomes a viscous fiber with high viscosity after absorbing water (Behera and Ray, 2016). It maintains the ability to absorb and store substantial quantities of water, thereby enhancing the gel-like consistency and moisture retention. Treatment C exhibited the lowest moisture content, indicating that WPI alone is insufficient for effective water retention without the addition of other hydrophilic polymers. This discovery aligns with previous research, which indicates that the moisture content in fermented dairy products ranges from 81% to 85% (Meliaet_al2020).

Table 2: Proximate analysis of fermented milk with the addition of microencapsulated L. brevis products from various types of wall material.



Treatment C demonstrated the highest protein content, owing to its elevated concentration of whey protein isolate, a premium and highly refined milk protein source. Treatment B, utilizing just alginate (devoid of protein), produced the minimal protein content, thus affirming that non-protein substances do not augment protein levels. Despite treatment D exhibiting a decreased concentration of WPI compared to treatment C, it yet preserved elevated protein levels owing to the inclusion of WPI in the wall formulation (Table 2). The results align with previous studies, which indicate that protein content ranges from 3% to 6% (Melia et al., 2020; Susmiati et al., 2022).

According to a fat content analysis, treatment D had the lowest fat levels (Table 2). This was probably because glucomannan and WPI worked in concert to promote lipid binding during fermentation and lower the availability of free fat (Zhu et al., 2024). The elevated water content in treatment D dispersed the total solids, hence decreasing fat concentration. This result supports the development of low-fat fermented milk rich in probiotics and protein. The results of this study aligned with the fat content in fermented milk reported in previous studies, which was approximately 3% (Melia et al., 2022).
 
The morphology of microencapsulation L. brevis
 
Scanning electron microscopy (SEM) revealed the unique morphological characteristics of L. brevis microcapsules, which are based on the composition of the encapsulating wall (Fig 1). Microcapsules composed solely of alginate had spherical forms with smooth surfaces, but they frequently appeared linked and layered. The majority of particles measured less than 2 µm, with bigger aggregates presumably resulting from agglomeration, aligning with previous research on alginate-based microcapsules < 15 µm (Pupa et al., 2021). Upon the addition of glucomannan, the particles assumed a more cubic morphology, remaining agglomerated and stacked, with sizes varying from <0.5 µm to 10 µm, a pattern corroborated by de Etchepare et al., (2016) in alginate-Hi-maize systems. The surfaces remained unblemished, devoid of cracks or pores, consistent with prior findings on konjac glucomannan microcapsules (Mu et al., 2018).

Fig 1: Scanning electron microscope (SEM) morphology of microencapsulated probiotic Levilactobacillus brevis. (10K magnification (A1, B1, C1, D1), 20K magnification (A2, B2, C2, D2) and 30K magnification (A3, B3, C3, D3).



Alginate molecular mass, the ratio of mannuronic acid to guluronic acid, pH, Ca2+, or the addition of transglutaminase can all affect the hydrogels formed by the strong interaction between alginate and whey proteins. Alginate and whey protein hydrogels in the shapes of beads, microparticles, microcapsules and nanocapsules  (Pedrali et al., 2023), with previous research corroborating the formation of deflated and adhesive structures in WPI-based microcapsules (Sompach et al., 2022).  The triple combination of alginate, glucomannan and whey protein isolate produced irregular, spherical particles measuring less than 2 µm, along with substantial agglomerates exceeding 10 µm, incorporating characteristics from both preceding systems. Structural collapse and surface adhesion were observed, possibly due to interactions between hydrophilic polymers and protein-induced desiccation pressures.  The desiccation pressure resulting from water evaporation causes matrix contraction; when proteins form a rigid layer on the surface, while the interior still undergoes shrinkage, deformation and structural collapse occur. Thus, this three-component system reflects a compromise between good micro-particle formation and the tendency toward agglomeration and post-drying instability.

CONCLUSION

This study shows that the L. brevis microencapsulation wall material affects the physicochemical properties of fermented milk. Using 1% alginate, 1% glucomannan and 5% whey protein isolate (WPI) (D) resulted in optimal outcomes, including 18.45 x 109  CFU/mL, pH 4.85, total titratable acidity 1.14% and moisture content 88.17%. Fermented milk also had 4.45% protein and 2.33% fat. Thus, a wall formulation that combines hydrophilic components and functional proteins improves the stability and quality of fermented milk as a probiotic product, a healthy functional meal.
 

ACKNOWLEDGEMENT

The present study was supported by Universitas Andalas, in accordance with the Research Contract Scheme Penelitian Unggulan Jalur Kepakaran Batch I Number: 401/UN16.19/PT.01.03/PUJK/2025.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
This research did not involve animals.

CONFLICT OF INTEREST

There are no conflicts of interest regarding the publication of this article.

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    Full Research Article

    Physicochemical Properties of Fermented Cow Milk Enriched with Different Microencapsulated Levilactobacillus brevis

    S
    Sri Melia1,*
    https://orcid.org/0000-0002-7271-0955
    I
    Indri Juliyarsi1
    https://orcid.org/0000-0003-2855-7794
    R
    Rizki Dwi Setiawan1
    https://orcid.org/0009-0005-1703-6688
    I
    Iceu Agustinisari2
    https://orcid.org/0000-0002-4905-9631
    Z
    Zaky Syahputra1
    https://orcid.org/0009-0005-3135-3137
    1Department of Animal Products Technology, Faculty of Animal Science, Universitas Andalas, Padang 25163, Indonesia.
    2Research Center for Agroindustry, National Research and Innovation Agency, Jl. Raya Serpong, Muncul, Kec. Setu, Kota Tangerang Selatan, Banten 15314, Indonesia.

    ABSTRACT

    Background: Fermented cow milk is generated when lactic acid bacteria transform lactose into lactic acid, enhancing its nutritional quality, texture and longevity. Microencapsulated probiotic cultures provide a contemporary method to enhance bacterial viability and preserve physical and chemical stability. This study examines the effect of various wall materials on the physicochemical properties of microencapsulated Levilactobacillus brevis in fermented milk.

    Methods: This research utilized some treatments with the addition of 1% L. brevis starter with various types of wall materials using the freeze-drying method with the combination of 1% Alginate (A), 1% Alginate + 1% Glucomannan (B), 1% Alginate + 5% WPI (C) and 1% Alginate + 5% WPI + 1% Glucomannan (D).

    Result: The combination treatment of 1% alginate + 1% glucomannan + 5% whey protein isolate (WPI) (D) produced the best results of total lactic acid bacteria (18.45 x 109 CFU/mL), pH value of 4.85, total titratable acidity of 1.14% and moisture content of 88.17%. It showed a high protein content of 4.45% with a relatively lower fat content of 2.33% in fermented milk. The combination of wall materials in microencapsulated products serving as a dry starter for fermented milk can be applied in the processing of fermented milk products that are beneficial for health.

    INTRODUCTION

    Fermented milk, a type of functional food, has experienced a surge in global demand due to increased consumer awareness of digestive health and immune system support. Fermented cow milk is generated when lactic acid bacteria transform lactose into lactic acid, enhancing its nutritional quality, texture and longevity. In comparison to fresh milk, it possesses a denser viscosity, a more pronounced taste and improved digestion. According to Elango (2024), research and development activities in the functional food sector have been heavily influenced by products targeting gut health. By preserving the healthy balance of the gut microbiota, probiotics are increasingly being added to foods and drinks to enhance general health. There are several opportunities to effectively and simply add probiotics to dairy products, particularly beverages. Typical examples include yogurt, kefir and buttermilk, esteemed for their health benefits and culinary versatility. In a study by Soni et al., (2021), Lactobacillus species were detected in yogurt from Gujarat, suggesting their potential as probiotics. Lactic acid bacteria (LAB) are pivotal among helpful germs. Levilactobacillus brevis, recognized for its ability to ferment diverse substrates and influence gut microbiota (Banerjee et al., 2021), is a promising strain. This research utilizes L. brevis isolated from dadih, a traditional Minangkabau fermented milk from West Sumatra, Indonesia, which is naturally fermented in bamboo (Puspawati et al., 2018). Metagenomic investigations have validated the predominance of LAB in dadih from areas such as Bukittinggi and Solok; however, comprehensive strain characterization remains insufficient (Venema et al., 2019; Fatdillah et al., 2021). Several previous studies have mentioned that several strains of L. brevis from various sources have been found to be probiotic (Song et al., 2020; Ait Chait et al., 2020; Somashekaraiah et al., 2021).

    LAB-based fermented products struggle to maintain probiotic viability during processing, storage and digestion. Encapsulating probiotic cells in protective substances enhances their survivability in harsh environments, including low pH, digestive enzymes and high temperatures (Razavi et al., 2021). Microencapsulation has demonstrated efficacy in enhancing nutritional stability, inhibiting interactions and preventing the degradation of raw materials, as the coated matrix effectively segregates particles and prevents their contact (Adeyeye et al., 2025). Microencapsulated products exhibit stability when integrated into actual food matrices. Food products shouldn’t be adversely affected by this integration, particularly their sensory qualities, which may change due to the addition of microencapsulated substances (Dias et al., 2017).

    This approach utilizes wall components such as sodium alginate, glucomannan and WPI, either alone or in combination. Ionic gelation using CaCl2 at ambient temperature creates gel beads from alginate, preventing thermal degradation. This protects sensitive bioactive molecules and improves stability. Alginate and glucomannan reinforce the gel matrix, whereas whey protein isolate (WPI) improves encapsulation and structural integrity (Tonon et al., 2011).


    Active compounds are synergistically protected from oxidation, enzymes and stomach acid by a three-component matrix consisting of alginate, glucomannan and WPI. Alginate and glucomannan stabilize the capsule in gastric conditions, allowing for regulated release in the digestive tract, while WPI creates an oxygen barrier. These wall materials influence the physicochemical characteristics of fermented goods, including moisture, pH, acidity, protein and fat content and they also promote the viability of probiotics (Kowalska et al., 2022).

    Previous studies on fermented milk have employed pure cultures free of microencapsulation (Melia et al., 2020; Melia et al., 2022). Fan et al., (2023) employed Levilactobacillus brevis CGMCC1.5954 as a starter culture in yogurt production. This study evaluates the effect of combining various wall materials with alginate on the microencapsulation of Levilactobacillus brevis as a dry starter on the physicochemical quality of fermented milk.

    MATERIALS AND METHODS

    Materials
     
    The materials used in this study were cow’s milk (full-cream milk) and L. brevis isolated from dadih, obtained from the Laboratory of Animal Product Technology and Processing at the Faculty of Animal Science Universitas Andalas.
     
    Methods
     
    This research used the experimental method with a completely randomized design. The treatments applied in this research were the addition of 1% L. brevis starter with various types of wall materials of microencapsulation: 1% Alginate (A), 1% Alginate + 1% Glucomannan (B), 1% Alginate + 5% WPI (C) and 1% Alginate + 5% WPI + 1% Glucomannan (D). This research was conducted at the Animal Product Technology Laboratory, Faculty of Animal Science Universitas Andalas, from April to September 2025.
     
    Making fermented milk
     
    Milk was pasteurized at 85°C for 15 minutes, then cooled to 37°C. After the previous stages, 1% of the starter L. brevis was inoculated according to the treatment and incubated at 37°C for 18 hours in an incubator (Melia et al., 2024).
     
    Preparation of microencapsulated product
     
    L. brevis probiotics were revived with 30 mL of isolate into 270 mL of MRS Broth, then incubated for 24 hours at 37°C. The culture was then centrifuged at 3000 rpm for 10 minutes at 25°C. The pellet was then washed with 5 mL of sterile distilled water solution and centrifuged again to obtain probiotic bacterial cells. 200 mL of sterile distilled water was stirred using a magnetic stirrer to dissolve the wall material (alginate, glucomannan and WPI). The pellet (bacterial cells) was then resuspended in the solution. Add sterile distilled water to 15% (weight/volume). Homogenize for 5 minutes before drying (Rajam et al., 2012).
     
    Measurement of total lactic acid bacteria
     
    Microbial growth was analyzed using the method described by Szołtysik et al., (2020). The sample was dissolved in a serial dilution and poured onto MRS Agar medium. Bacterial cells were incubated under anaerobic conditions at 37°C for 48 hours. Bacterial colonies were counted at the end of the cultivation period.
     
    Measurement of pH and titratable acidity (TA)
     
    AOAC (2005) pH guidelines were followed with a calibrated pH meter. The electrode was immersed in a 30 mL sample until a stable reading was achieved. The Yenrina technique (2015) measured titratable acidity. Five drops of phenolphthalein in 5 mL were titrated with 0.1 N NaOH until a persistent pink color developed. Total fermented milk acidity was calculated using NaOH volume.
     
    Proximate analysis
     
    The AOAC (2005) methodologies were used to determine the moisture, protein and fat content. A 5 g sample was oven-dried at 105°C for 8 hours to determine the moisture content. It was then cooled in a desiccator and weighed to determine weight loss. The Kjeldahl method, which involves acid digestion with H2SO4 and selenium, alkaline distillation and titration of the distillate with standard NaOH using methyl red as an indicator with a blank for correction, was used to determine the protein concentration. Soxhlet extraction was used to assess the fat content. A 1 g dried sample was extracted with an organic solvent for 5-6 hours, the solvent was eliminated by distillation and the recovered fat was dried in an oven at 100-105°C before being weighed.
     
    Morphology of microencapsulated analysis
     
    The microencapsulated morphology was examined using a Scanning Electron Microscope (SEM) (Leo 435 VP, Leo Electronic System, Cambridge, UK), following the procedure outlined by Rajam et al., (2012). Samples that had been freeze-dried were placed on specimen holders, covered with gold for two minutes at a pressure of 2 mbar and then examined at 15 kV and a vacuum of 9.75 x 10-5 Torr.

    RESULTS AND DISCUSSION

    Total lactic acid bacteria
     
    The statistical analysis in Table 1 indicates that the wall material has a significant impact on the lactic acid bacteria (LAB) count (P<0.05). Fermented milk containing 1% alginate, 1% glucomannan and 5% WPI (D) had the highest LAB count, surpassing A, B and C. Glucomannan and alginate (B) enhanced viability above alginate alone (A), but WPI alone (C) lowered LAB populations. Treatment D works better since all three components work together. Alginate’s calcium gel matrix protects microorganisms from acidity. As with glucomannan, gel density and viscosity decrease oxygen transport and protect cells from oxidative stress.

    Table 1: Characteristics of fermented milk with the addition of microencapsulated L. brevis products from several types of wall material.



    Hayuningtyas (2018) asserts that glucomannan enhances the viscosity of emulsion systems in milk, hence increasing stability. WPI creates an acid-resistant protective layer, enhances the microcapsule structure and provides nitrogen for the metabolism of LAB. It additionally stabilizes pH, facilitating bacterial viability during fermentation (Picot and Lacroix, 2004). Studies have demonstrated that the combination of proteins and polysaccharides enhances the stability of microencapsulation (Liu et al., 2023; Puttarat et al., 2021). The formulation in treatment D provides structural strengthening, environmental protection and nutritional support, markedly enhancing LAB viability.
     
    pH
     
    The pH of fermented milk is significantly influenced by wall material type (P<0.05). Treatment D (1% alginate + 1% glucomannan + 5% WPI) had the lowest pH (4.85±0.057), indicating effective acid production and increased LAB activity. Alginate’s protective gel, glucomannan’s viscosity and prebiotic characteristics and WPI’s nitrogen stimulation of bacterial metabolism may explain this. Treatment C (1% alginate + 5% WPI) had the highest pH (5.86±0.021), indicating slower fermentation. The compact WPI-alginate matrix could prevent LAB growth by preventing nutrition and oxygen transfer. The buffering capacity of WPI stabilizes pH. Treatment D increases LAB activity by increasing viability and acid generation. These findings align with prior studies on the pH of fermented milk and yogurt (Rossi et al., 2021; Falah, 2021). The pH in this study was almost the same as that reported by Narayanan et al., (2020), who used microencapsulation of L. acidophilus and carrot juice in yogurt, which was 4.65.
     
    Titratable acidity (TA)
     
    The wall material used for microencapsulated L. brevis significantly affected the titratable acidity (TA) of fermented milk (P<0.05), as indicated in Table 1. Treatment D (1% alginate, 1% glucomannan, 5% WPI) had the highest TA, indicating substantial metabolic activity due to its low pH and high LAB count. Alginate forms a protective gel, glucomannan strengthens matrix porosity and nutrient diffusion due to its hydrophilic nature and WPI stabilizes cell membranes. Glucomannan-free Treatment C (1% alginate, 5% WPI) had the lowest TA, highest pH and lowest LAB due to substrate penetration and a denser matrix. Protein walls, such as WPI, can buffer acids and bind water, thereby slowing acidification and altering microbial activity. Polysaccharides promote the viability and metabolism of LAB, resulting in the production of acid. However, excessive encapsulation may reduce mass transfer, nutrient availability and fermentation efficiency. These findings support Quintero et al., (2012), emphasizing balanced microencapsulation design for best performance.
     
    Moisture, protein and fat content
     
    The moisture content in fermented milk was significantly affected (P<0.05) by the type of microencapsulation wall applied (Table 2). Treatment D fermented milk enhanced with a composite of 1% alginate, 1% glucomannan and 5% WPI yielded the greatest moisture content. This result aligned with the highest overall bacterial count observed in this treatment, indicating improved probiotic viability and metabolic activity. Several investigations have shown that the carboxyl groups of alginate residues rapidly bind to metal ions, including Ca2+, forming the “egg-box” structure, a three-dimensional gel network (Zan et al., 2023), effectively retaining water. In contrast, as a soluble dietary fiber, glucomannan becomes a viscous fiber with high viscosity after absorbing water (Behera and Ray, 2016). It maintains the ability to absorb and store substantial quantities of water, thereby enhancing the gel-like consistency and moisture retention. Treatment C exhibited the lowest moisture content, indicating that WPI alone is insufficient for effective water retention without the addition of other hydrophilic polymers. This discovery aligns with previous research, which indicates that the moisture content in fermented dairy products ranges from 81% to 85% (Meliaet_al2020).

    Table 2: Proximate analysis of fermented milk with the addition of microencapsulated L. brevis products from various types of wall material.



    Treatment C demonstrated the highest protein content, owing to its elevated concentration of whey protein isolate, a premium and highly refined milk protein source. Treatment B, utilizing just alginate (devoid of protein), produced the minimal protein content, thus affirming that non-protein substances do not augment protein levels. Despite treatment D exhibiting a decreased concentration of WPI compared to treatment C, it yet preserved elevated protein levels owing to the inclusion of WPI in the wall formulation (Table 2). The results align with previous studies, which indicate that protein content ranges from 3% to 6% (Melia et al., 2020; Susmiati et al., 2022).

    According to a fat content analysis, treatment D had the lowest fat levels (Table 2). This was probably because glucomannan and WPI worked in concert to promote lipid binding during fermentation and lower the availability of free fat (Zhu et al., 2024). The elevated water content in treatment D dispersed the total solids, hence decreasing fat concentration. This result supports the development of low-fat fermented milk rich in probiotics and protein. The results of this study aligned with the fat content in fermented milk reported in previous studies, which was approximately 3% (Melia et al., 2022).
     
    The morphology of microencapsulation L. brevis
     
    Scanning electron microscopy (SEM) revealed the unique morphological characteristics of L. brevis microcapsules, which are based on the composition of the encapsulating wall (Fig 1). Microcapsules composed solely of alginate had spherical forms with smooth surfaces, but they frequently appeared linked and layered. The majority of particles measured less than 2 µm, with bigger aggregates presumably resulting from agglomeration, aligning with previous research on alginate-based microcapsules < 15 µm (Pupa et al., 2021). Upon the addition of glucomannan, the particles assumed a more cubic morphology, remaining agglomerated and stacked, with sizes varying from <0.5 µm to 10 µm, a pattern corroborated by de Etchepare et al., (2016) in alginate-Hi-maize systems. The surfaces remained unblemished, devoid of cracks or pores, consistent with prior findings on konjac glucomannan microcapsules (Mu et al., 2018).

    Fig 1: Scanning electron microscope (SEM) morphology of microencapsulated probiotic Levilactobacillus brevis. (10K magnification (A1, B1, C1, D1), 20K magnification (A2, B2, C2, D2) and 30K magnification (A3, B3, C3, D3).



    Alginate molecular mass, the ratio of mannuronic acid to guluronic acid, pH, Ca2+, or the addition of transglutaminase can all affect the hydrogels formed by the strong interaction between alginate and whey proteins. Alginate and whey protein hydrogels in the shapes of beads, microparticles, microcapsules and nanocapsules  (Pedrali et al., 2023), with previous research corroborating the formation of deflated and adhesive structures in WPI-based microcapsules (Sompach et al., 2022).  The triple combination of alginate, glucomannan and whey protein isolate produced irregular, spherical particles measuring less than 2 µm, along with substantial agglomerates exceeding 10 µm, incorporating characteristics from both preceding systems. Structural collapse and surface adhesion were observed, possibly due to interactions between hydrophilic polymers and protein-induced desiccation pressures.  The desiccation pressure resulting from water evaporation causes matrix contraction; when proteins form a rigid layer on the surface, while the interior still undergoes shrinkage, deformation and structural collapse occur. Thus, this three-component system reflects a compromise between good micro-particle formation and the tendency toward agglomeration and post-drying instability.

    CONCLUSION

    This study shows that the L. brevis microencapsulation wall material affects the physicochemical properties of fermented milk. Using 1% alginate, 1% glucomannan and 5% whey protein isolate (WPI) (D) resulted in optimal outcomes, including 18.45 x 109  CFU/mL, pH 4.85, total titratable acidity 1.14% and moisture content 88.17%. Fermented milk also had 4.45% protein and 2.33% fat. Thus, a wall formulation that combines hydrophilic components and functional proteins improves the stability and quality of fermented milk as a probiotic product, a healthy functional meal.
     

    ACKNOWLEDGEMENT

    The present study was supported by Universitas Andalas, in accordance with the Research Contract Scheme Penelitian Unggulan Jalur Kepakaran Batch I Number: 401/UN16.19/PT.01.03/PUJK/2025.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Informed consent
     
    This research did not involve animals.

    CONFLICT OF INTEREST

    There are no conflicts of interest regarding the publication of this article.

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      Asian Journal of Dairy and Food Research

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