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Probiotic Innovation in Ice Cream: Quality during Storage with the Addition of Encapsulated Lactiplantibacillus plantarum SN13T

S
Siti Auliarasulina1
https://orcid.org/0000-0002-7271-0955
S
Sri Melia2,*
https://orcid.org/0000-0003-2855-7794
I
Indri Juliyarsi2
https://orcid.org/0009-0001-3557-4309
1Magister Program of Animal Science Faculty, Universitas Andalas, Padang, West Sumatra, 25163, Indonesia.
2Department of Animal Product Technology, Faculty of Animal Science, Universitas Andalas, Padang, West Sumatra, 25163, Indonesia.

ABSTRACT

Background: This study examines the quality of probiotic ice cream containing encapsulated and non-encapsulated Lactiplantibacillus plantarum SN13T over a 60-day period, as probiotic survival and sensory attributes may alter during storage, despite the health benefits and diversification of livestock-based foods it provides in preventing degenerative diseases.

Methods: The treatments included two types of probiotics (A), without encapsulation (A1) and with encapsulation (A2) and various storage times (B) of 1, 20, 40 and 60 days.

Result: The results show that encapsulated L. plantarum SN13T ice cream contained 5.15% fat, 9.01% protein, 66.62% water, pH 5.71, 10.03 Log CFU/mL total lactic acid bacteria, 80.75% simulated gastric juice resistance and 81.04% simulated intestinal juice resistance. The encapsulation effectively maintained the viability and resistance of L. plantarum SN13T to simulated digestive conditions, while also maintaining the physicochemical and sensory quality of the ice cream for up to 60 days of storage.

INTRODUCTION

Lifestyle changes have led to less physical activity and a shift towards less healthy food options as technology and society have evolved. Lifestyle changes may decrease quality of life and complicate illness prevention, increasing the risk of degenerative diseases such as obesity, diabetes, cardiovascular disease and metabolic disorders. Preventing degenerative illnesses is needed to solve this problem. Suciati and Safitri (2021) recommend consuming functional foods. According to the European Commission (2010), functional foods support fetal growth and development, maintain overall health and reduce the risk of degenerative and chronic diseases.
       
Probiotics are living bacteria that confer health benefits when consumed in sufficient quantities (Hill et al., 2014). They contribute to gut health, synthesise vitamins, aid cholesterol regulation and inhibit the effects of harmful chemicals (Aini et al., 2021). One probiotic that can be used is Lactiplantibacillus plantarum SN13T (LP SN13T), a starter culture of lactic acid bacteria (LAB) isolated from stingless honeybees from West Sumatra. According to Ahmad et al., (2022), one of the most significant LAB species is L. plantarum, which exhibits antagonistic activity against many pathogens and has been shown to confer health benefits. This isolate LP SN13T has an acid tolerance of 82.75%, a bile salt tolerance of 94.44% and the highest antimicrobial activity against pathogenic bacteria (Melia et al., 2022).
       
Pure cultures of LP SN13T have been applied to several fermented products (Auliarasulina et al., 2025; Syahnara et al., 2025). However, to maintain probiotic stability during processing (for example, in ice cream), an encapsulation technique using alginate and bengkuang flour (Pachyrhizus erosus) as a coating material is required. Alginate has good gel-forming properties but limited water-absorption capacity, which can affect stability during storage (Subaryano, 2010). Bengkuang flour functions as a natural coating agent suitable for probiotic applications (Wang et al., 2018). Meanwhile, because of its high starch content, cassava flour may be developed as an encapsulating material. According to Yeni et al., (2018), cassava flour contains 73.47% starch, 70.4% amylopectin and 29.6% amylose. Because of its biocompatibility, capacity to create a stable matrix, ease of gel formation and water binding and efficacy in boosting the survival of encapsulated microorganisms, starch is utilised in encapsulation (Lopes et al., 2024). Ice cream has the potential to serve as a probiotic carrier and is very popular among consumers (Adil et al., 2022).
       
This study aimed to determine the quality and viability of probiotics in ice cream containing encapsulated LP SN13T cells compared to non-encapsulated colonies during storage. The concept is that encapsulation will increase probiotic viability and maintain the ice cream’s taste quality.

MATERIALS AND METHODS

Materials
 
The L. plantarum SN13T bacteria were obtained from the Animal Product Technology laboratory. Bengkuang flour was produced in the laboratory using commodities sourced from the local market in Padang City, Indonesia. The Animal Product Technology Laboratory, Faculty of Animal Husbandry andalas University, conducted this study from May to September 2025.
       
This research design is an experiment with a 2 x 4 factorial randomized block design (RBD) with 3 replications. The treatments used were LP SN13T probiotics: Unencapsulated (A1) and encapsulated (A2). Then storage was carried out for 1 day (B1), 20 days (B2), 40 days (B3) and 60 days (B4).
 
Preparation of encapsulation
 
Following the extrusion technique outlined by Sultana et al., (2022), LP SN13T was encapsulated by initially combining bengkuang flour with 100 mL of sterile water while agitating at 400 rpm. Subsequently, 1% alginate and 1% bengkuang flour were incorporated to achieve a uniform mixture. Then, 1% probiotic biomass was homogenized into this blend for 5 minutes. The resulting solution was subsequently dripped into a 1.5% calcium chloride bath (agitated at 200 rpm) to form microcapsules, which were allowed to solidify for 20-30 minutes. The microcapsules were then rinsed with physiological saline and stored at 4oC.
 
Ice cream making
 
The ice cream preparation process described by Akalin et al., (2018) is as follows: After measuring all components, 500 mL of milk was combined with skim milk, sugar, eggs and carboxymethyl cellulose until a soft consistency was achieved; a second 500 mL of milk was mixed with whipped cream and beaten until fluffy; the two mixtures were subsequently mixed and homogenized, after which LP SN13T cultures (either free (A1) or encapsulated (A2)) were incorporated, the mixture was incubated at 37oC for 3.5 hours and finally, the product was packaged and stored in a freezer for 0, 20, 40 and 60 days.
 
Proximate analysis
 
The AOAC (2005) procedure was used to determine the moisture, protein and fat levels. Measurements for these components were taken after storage durations of 1, 20, 40 and 60 days, with each assessment performed three times.
 
pH and total lactic acid bacteria colony count 
 
The sample’s acidity was measured using a pH meter according to the AOAC (2005) method. Microbial growth was evaluated using the method developed by Szołtysik and colleagues (2020). Samples were serially diluted and then cultured on MRS Agar medium. Bacteria were incubated under anaerobic conditions at 37oC for 48 hours and the number of colonies formed was counted at the end of the incubation period. The total number of colonies was calculated using the CFU (colony forming unit) calculation formula:
 
  
 
Simulated gastric juice
 
Following the protocol of de Andrade et al., (2019), with adjustments, the endurance test of encapsulated probiotic bacteria in artificial stomach fluid was conducted. A pepsin solution (3 g in 1 L sterile water with 0.2% NaCl, pH 2) was prepared. Subsequently, 10 mL of sterile simulated intestinal fluid containing 1 g of encapsulated bacteria was added, vortex-mixed and incubated at 37oC for 5 h; viability was quantified as the percentage of cells surviving after the incubation.
 
Simulated intestinal juice
 
A solution of 0.05 M KHPO4, which has been autoclaved, is mixed with 0.6% bile salt to create SIJ. For each sample, 0.1 g of microcapsules is combined with 9.9 mL of the SIJ solution and incubated at 37oC for 5 hours. Following incubation, the suspension is diluted to 10-6, plated on MRS agar using the spread-plate technique and incubated at 37oC for 48 hours. The remaining bacteria are quantified as colony-forming units (CFU) and the variation in cell counts before and after incubation is documented as viability (Sun et al., 2022).
 
Organoleptic
 
A sensory evaluation was conducted by 50 panelists, all of whom were accustomed to consuming ice cream and comprised 25 males and 25 females aged 20 to 45. All samples were served in plastic cups and labeled with randomly assigned three-digit codes to ensure neutrality. Water was provided between each serving to cleanse the palate. Ice cream was evaluated on color, taste, texture and flavor (Souza et al., 2019), using a hedonic scale from 1 (“strongly dislike”) to 5 (“strongly like”).
 
Statistical analysis
 
The statistical analysis used one-way ANOVA with IBM SPSS Statistics 26 (2016). Significant differences were defined as p<0.05.

RESULTS AND DISCUSSION

Proximate analysis of ice cream
 
During storage, the fat and protein content of probiotic ice cream declined gradually in both treatments. The decrease in fat content was more pronounced in the unencapsulated treatment, especially after day 20, while the encapsulated treatment maintained the fat content until day 60. Protein content also decreased with increasing storage time, but the decrease was slower in the encapsulated treatment than in the unencapsulated treatment. In contrast, the ice cream’s water content remained relatively stable throughout storage.
       
The fat content was significantly affected by probiotic type (A) and storage period (B) (P<0.05) (Table 1). Independent of encapsulation, LP SN13T significantly decreased fat by day 40. The non-encapsulated sample decreased by 4.79% in fat after 60 days, while the encapsulated one retained 5.25%. The gel matrix prevents the breakdown of enzymes and lipids (Kailasapathy, 2009). High-viscosity alginate beads decrease lactic acid bacteria fermentation and preserve fat (Hernández-Gallegos  et al., 2023). In Table 1, LP SN13T addition and storage duration had a significant impact on protein content (P<0.05). LP SN13T-encapsulated ice cream lost little protein after 60 days because the beads prevented bacteria from directly interacting with the substrate, limiting fermentation and proteolysis. The addition of probiotics and storage duration significantly affected water content (P<0.05) (Table 1). The encapsulated LP SN13T lost little moisture, as the hygroscopic alginate gel retained moisture until day 60. The probiotic without encapsulation lowered moisture levels faster, indicating direct bacterial action and the effects of the stabilizer and carbohydrates (Jain and Rai, 2018). Ice cream with alginate-based beads maintains its fat, protein and moisture content, thereby improving stability and quality.

Table 1: Proximate content of Ice cream (%) with the addition of L. plantarum SN13T during storage.


 
pH and total lactic acid bacteria
 
The pH value of ice cream decreased gradually during storage. On day 0, the pH remained relatively high, then decreased on days 20 and 40 and reached its lowest value on day 60. The decrease in pH was more pronounced with the unencapsulated treatment, while the encapsulated treatment showed a slower, more stable decrease. The total number of LAB also decreased gradually during storage. On day 0, the highest LAB count was observed in both treatments, then decreased on days 20 and 40 and decreased further on day 60.
       
The analysis of variance revealed a highly significant effect (P<0.05) of incorporating LP SN13T (A) and storage period (B) on the pH of the ice cream (Table 2). The pH decreased during ice cream storage after the addition of encapsulated LP SN13T, but the change was not significant, indicating improved stability.  The increased acidity is caused by the production of lactic acid by bacterial fermentation of lactose in dairy products (Bajad et al., 2016). The alginate-based capsule protects the bacteria from temperature and pH variations, establishing a diffusion barrier that restricts the release of acidic metabolites (Anal and Singh, 2007).  Žadeikë  et al. (2024) similarly observed that the protective gel matrix inhibits LAB activity, leading to gradual acidification. In contrast, unencapsulated LP SN13T significantly lowers pH through lactose fermentation and organic acid production, a phenomenon explained by Prasertsiriphan and Kusump (2015), who related direct substrate access with increased fermentative activity in the product overall.

Table 2: PH values and total lactid acid bacteria colony count of ice cream with the addition of L. plantarum SN13T during storage.


       
The study found that adding LP SN13T and increasing storage time both had a substantial effect on the total LAB count. There was no significant change in the LAB count in encapsulated samples on days 1, 20 and 40. However, by day 60, there was a slight decrease to 10.03 Log CFU/ml, which was an 18.95% reduction compared to the non-encap- sulated control. This suggests that the alginate-based encapsulation matrix acts as a barrier, protecting the probiotic cells and reducing damage during freezing, thereby maintaining a more stable LAB population. This was supported by Sedefoğlu et al., (2022) and Gullo and Zotta (2022). In contrast, unencapsulated L. plantarum exhibited a 42.97% decrease in LAB after 60 days, as the free cells are more susceptible to ice-crystal formation and other stressors, as described by Homayouni et al., (2008). Therefore, encapsulation is a more effective method for maintaining the minimum 106 CFU mL-1 required by CODEX (2003) during ice cream storage, thereby ensuring consumer safety.
 
Simulated gastric juice (SGJ)
 
The resistance of ice cream to SGJ decreased significantly (P<0.05) during storage in both treatments. On day 0, the resistance to SGJ showed the highest value, then decreased on days 20 and 40 and reached the lowest value on day 60. The incorporation of encapsulating LP SN13T over a duration of 60 days (A2B4) resulted in a 6.7% reduction of SGJ (Fig 1). Encapsulated LP SN13T safeguards bacteria against gastric acid during storage. Various defensive mechanisms render encapsulation efficacious. Starch protects probiotic organisms from gastric acid, which can reach pH 1.5 (Kim et al., 2017). Starch and calcium alginate provide superior acid protection compared to unencapsulated probiotics (Ngov, 2014). Probiotics in ice cream require this safeguarding. A1B4 exhibits a 17.17% reduction in resilience to stomach-like acid after 60 days with LP SN13T without encapsulation. Bacteria are subjected to acidic conditions without encapsulation. According to Lee (2010), probiotic bacteria such as Lactobacillus acidophilus exhibit increased mortality in the presence of strong acid. Prolonged storage diminishes resistance to gastric acid. Bilang et al., (2018) also reported that unencapsulated LAB experienced a greater decrease in viability compared to encapsulated LAB. Unencapsulated Lactobacillus plantarum and Streptococcus thermophilus decreased by 31% and 38%, respectively, while in the encapsulated treatment, the decreases were lower, namely 24% and 37%.

Fig 1: Graph of resistance to SGJ ice cream (%) with the addition of L. plantarum SN13T during 60 days of storage.


 
Simulated intestinal juice (SIJ)
 
The resistance of ice cream to SIJ decreased significantly (P<0.05) with increasing storage time in both treatments. On day 0, the SIJ resistance value was still relatively high, then decreased on days 20 and 40, reaching its lowest value on day 60. Adding encapsulated LP SN13T had no significant impact on the survival of the bacteria in SIJ after 60 days of storage (A2B4, Fig 2). Still, the same strain without encapsulation had a 14.80% loss (A1B4, Fig 2). The microencapsulation made of starch creates a physical barrier that protects probiotic cells from the bile salts in simulated gut juice. Numerous studies demonstrate that encapsulating bacteria in starch is more effective than encapsulating them in alginate alone. For example, Lactobacillus acidophilus LA1, which is encapsulated in starch, survives higher bile salt concentrations better than free cells (Sabikhi et al., 2010). Therefore, starch microen-capsulation effectively maintains the stability and activity of probiotics in ice cream products during storage, making them more appealing to customers. Bilang et al., (2018) also reported that unencapsulated LAB experienced a greater decrease in viability than encapsulated LAB. Unencapsulated Lactobacillus plantarum and Streptococcus thermophilus experienced a decrease of 30-32%, while encapsulated LAB only experienced a decrease of 21-26%.

​

Fig 2: Graph of resistance to SGJ ice cream (%) with the addition of L. plantarum SN13T during 60 days of storage.


 
Organoleptics 
 
The organoleptic properties of LP-SN13T ice cream, both without encapsulation and with encapsulation, significantly affect (P<0.05) flavor, color, taste and texture during 60 days of storage. Panelists favored non-encapsulated (direct-addition) samples, which maintained a constant hue from day 20 to 60, likely due to the formulation’s natural ingredients and lack of coloring (Fig 3). Flavor is the most important positive attribute of ice cream. However, flavor stability can decline during processing and storage (Vanathi and Dorai, 2020). Diez-Libreros  et al. (2025) found that adding probiotics directly did not modify the color or taste of the ice cream. Non-encapsulated samples preferred flavor throughout storage, while encapsulated samples decreased significantly (P<0.05) on days 20 and 40 but improved by day 60 (Fig 4). The encapsulated ice cream initially fluctuated, while the non-encapsulated version had more stable texture scores. The findings support prior research: Fávaro-Trindade  et al. (2016) observed that bead encapsulation changes sensory profiles, while Syed et al., (2018) and English et al., (2023) found that additives and encapsulation affect texture and quality.

​

Fig 3: Organoleptic value chart of probiotic ice cream with the addition of L. plantarum SN13T without encapsulation during storage.



​

Fig 4: Organoleptic value chart of probiotic ice cream with the addition of L. plantarum SN13T with encapsulation during storage.

CONCLUSION

Both encapsulated and non-encapsulated LP SN13T had a major effect on ice cream quality over a 60-day period, according to the study. The non-encapsulated LP SN13T ice cream has a pH of 5.16, 4.79% fat, 8.11% protein, 65.84% water, 10.01 Log CFU/mL total LAB, 71.76% SGJ resistance and 72.16% SIJ resistance. The results of organoleptic tests were 3.76 for colour, 3.78 for taste, 3.78 for texture and 3.82 for flavour. The nutritional content, bacterial viability and sensory attributes of encapsulated LP SN13T ice cream were all preserved. Additionally, encapsulated LP SN13T demonstrated higher SGJ and SIJ resistance. These results suggest that encapsulation technology may be used in the production of probiotic ice cream on an industrial scale to create stable, functional goods for cold storage and distribution.

ACKNOWLEDGEMENT

The present study was supported by the Research and Community Service Institute of Universitas Andalas, which funded this research under the Research Contract Master’s Thesis Research Scheme Batch I, Number: 181/UN16.19/PT.01.03/PTM/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 has passed the ethical review by the Faculty of Pharmacy Commission Team andalas University, with Number: 46/UN.16.10.D.KEPK-FF/2025.

CONFLICT OF INTEREST

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

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    Full Research Article

    Probiotic Innovation in Ice Cream: Quality during Storage with the Addition of Encapsulated Lactiplantibacillus plantarum SN13T

    S
    Siti Auliarasulina1
    https://orcid.org/0000-0002-7271-0955
    S
    Sri Melia2,*
    https://orcid.org/0000-0003-2855-7794
    I
    Indri Juliyarsi2
    https://orcid.org/0009-0001-3557-4309
    1Magister Program of Animal Science Faculty, Universitas Andalas, Padang, West Sumatra, 25163, Indonesia.
    2Department of Animal Product Technology, Faculty of Animal Science, Universitas Andalas, Padang, West Sumatra, 25163, Indonesia.

    ABSTRACT

    Background: This study examines the quality of probiotic ice cream containing encapsulated and non-encapsulated Lactiplantibacillus plantarum SN13T over a 60-day period, as probiotic survival and sensory attributes may alter during storage, despite the health benefits and diversification of livestock-based foods it provides in preventing degenerative diseases.

    Methods: The treatments included two types of probiotics (A), without encapsulation (A1) and with encapsulation (A2) and various storage times (B) of 1, 20, 40 and 60 days.

    Result: The results show that encapsulated L. plantarum SN13T ice cream contained 5.15% fat, 9.01% protein, 66.62% water, pH 5.71, 10.03 Log CFU/mL total lactic acid bacteria, 80.75% simulated gastric juice resistance and 81.04% simulated intestinal juice resistance. The encapsulation effectively maintained the viability and resistance of L. plantarum SN13T to simulated digestive conditions, while also maintaining the physicochemical and sensory quality of the ice cream for up to 60 days of storage.

    INTRODUCTION

    Lifestyle changes have led to less physical activity and a shift towards less healthy food options as technology and society have evolved. Lifestyle changes may decrease quality of life and complicate illness prevention, increasing the risk of degenerative diseases such as obesity, diabetes, cardiovascular disease and metabolic disorders. Preventing degenerative illnesses is needed to solve this problem. Suciati and Safitri (2021) recommend consuming functional foods. According to the European Commission (2010), functional foods support fetal growth and development, maintain overall health and reduce the risk of degenerative and chronic diseases.
           
    Probiotics are living bacteria that confer health benefits when consumed in sufficient quantities (Hill et al., 2014). They contribute to gut health, synthesise vitamins, aid cholesterol regulation and inhibit the effects of harmful chemicals (Aini et al., 2021). One probiotic that can be used is Lactiplantibacillus plantarum SN13T (LP SN13T), a starter culture of lactic acid bacteria (LAB) isolated from stingless honeybees from West Sumatra. According to Ahmad et al., (2022), one of the most significant LAB species is L. plantarum, which exhibits antagonistic activity against many pathogens and has been shown to confer health benefits. This isolate LP SN13T has an acid tolerance of 82.75%, a bile salt tolerance of 94.44% and the highest antimicrobial activity against pathogenic bacteria (Melia et al., 2022).
           
    Pure cultures of LP SN13T have been applied to several fermented products (Auliarasulina et al., 2025; Syahnara et al., 2025). However, to maintain probiotic stability during processing (for example, in ice cream), an encapsulation technique using alginate and bengkuang flour (Pachyrhizus erosus) as a coating material is required. Alginate has good gel-forming properties but limited water-absorption capacity, which can affect stability during storage (Subaryano, 2010). Bengkuang flour functions as a natural coating agent suitable for probiotic applications (Wang et al., 2018). Meanwhile, because of its high starch content, cassava flour may be developed as an encapsulating material. According to Yeni et al., (2018), cassava flour contains 73.47% starch, 70.4% amylopectin and 29.6% amylose. Because of its biocompatibility, capacity to create a stable matrix, ease of gel formation and water binding and efficacy in boosting the survival of encapsulated microorganisms, starch is utilised in encapsulation (Lopes et al., 2024). Ice cream has the potential to serve as a probiotic carrier and is very popular among consumers (Adil et al., 2022).
           
    This study aimed to determine the quality and viability of probiotics in ice cream containing encapsulated LP SN13T cells compared to non-encapsulated colonies during storage. The concept is that encapsulation will increase probiotic viability and maintain the ice cream’s taste quality.

    MATERIALS AND METHODS

    Materials
     
    The L. plantarum SN13T bacteria were obtained from the Animal Product Technology laboratory. Bengkuang flour was produced in the laboratory using commodities sourced from the local market in Padang City, Indonesia. The Animal Product Technology Laboratory, Faculty of Animal Husbandry andalas University, conducted this study from May to September 2025.
           
    This research design is an experiment with a 2 x 4 factorial randomized block design (RBD) with 3 replications. The treatments used were LP SN13T probiotics: Unencapsulated (A1) and encapsulated (A2). Then storage was carried out for 1 day (B1), 20 days (B2), 40 days (B3) and 60 days (B4).
     
    Preparation of encapsulation
     
    Following the extrusion technique outlined by Sultana et al., (2022), LP SN13T was encapsulated by initially combining bengkuang flour with 100 mL of sterile water while agitating at 400 rpm. Subsequently, 1% alginate and 1% bengkuang flour were incorporated to achieve a uniform mixture. Then, 1% probiotic biomass was homogenized into this blend for 5 minutes. The resulting solution was subsequently dripped into a 1.5% calcium chloride bath (agitated at 200 rpm) to form microcapsules, which were allowed to solidify for 20-30 minutes. The microcapsules were then rinsed with physiological saline and stored at 4oC.
     
    Ice cream making
     
    The ice cream preparation process described by Akalin et al., (2018) is as follows: After measuring all components, 500 mL of milk was combined with skim milk, sugar, eggs and carboxymethyl cellulose until a soft consistency was achieved; a second 500 mL of milk was mixed with whipped cream and beaten until fluffy; the two mixtures were subsequently mixed and homogenized, after which LP SN13T cultures (either free (A1) or encapsulated (A2)) were incorporated, the mixture was incubated at 37oC for 3.5 hours and finally, the product was packaged and stored in a freezer for 0, 20, 40 and 60 days.
     
    Proximate analysis
     
    The AOAC (2005) procedure was used to determine the moisture, protein and fat levels. Measurements for these components were taken after storage durations of 1, 20, 40 and 60 days, with each assessment performed three times.
     
    pH and total lactic acid bacteria colony count 
     
    The sample’s acidity was measured using a pH meter according to the AOAC (2005) method. Microbial growth was evaluated using the method developed by Szołtysik and colleagues (2020). Samples were serially diluted and then cultured on MRS Agar medium. Bacteria were incubated under anaerobic conditions at 37oC for 48 hours and the number of colonies formed was counted at the end of the incubation period. The total number of colonies was calculated using the CFU (colony forming unit) calculation formula:
     
      
     
    Simulated gastric juice
     
    Following the protocol of de Andrade et al., (2019), with adjustments, the endurance test of encapsulated probiotic bacteria in artificial stomach fluid was conducted. A pepsin solution (3 g in 1 L sterile water with 0.2% NaCl, pH 2) was prepared. Subsequently, 10 mL of sterile simulated intestinal fluid containing 1 g of encapsulated bacteria was added, vortex-mixed and incubated at 37oC for 5 h; viability was quantified as the percentage of cells surviving after the incubation.
     
    Simulated intestinal juice
     
    A solution of 0.05 M KHPO4, which has been autoclaved, is mixed with 0.6% bile salt to create SIJ. For each sample, 0.1 g of microcapsules is combined with 9.9 mL of the SIJ solution and incubated at 37oC for 5 hours. Following incubation, the suspension is diluted to 10-6, plated on MRS agar using the spread-plate technique and incubated at 37oC for 48 hours. The remaining bacteria are quantified as colony-forming units (CFU) and the variation in cell counts before and after incubation is documented as viability (Sun et al., 2022).
     
    Organoleptic
     
    A sensory evaluation was conducted by 50 panelists, all of whom were accustomed to consuming ice cream and comprised 25 males and 25 females aged 20 to 45. All samples were served in plastic cups and labeled with randomly assigned three-digit codes to ensure neutrality. Water was provided between each serving to cleanse the palate. Ice cream was evaluated on color, taste, texture and flavor (Souza et al., 2019), using a hedonic scale from 1 (“strongly dislike”) to 5 (“strongly like”).
     
    Statistical analysis
     
    The statistical analysis used one-way ANOVA with IBM SPSS Statistics 26 (2016). Significant differences were defined as p<0.05.

    RESULTS AND DISCUSSION

    Proximate analysis of ice cream
     
    During storage, the fat and protein content of probiotic ice cream declined gradually in both treatments. The decrease in fat content was more pronounced in the unencapsulated treatment, especially after day 20, while the encapsulated treatment maintained the fat content until day 60. Protein content also decreased with increasing storage time, but the decrease was slower in the encapsulated treatment than in the unencapsulated treatment. In contrast, the ice cream’s water content remained relatively stable throughout storage.
           
    The fat content was significantly affected by probiotic type (A) and storage period (B) (P<0.05) (Table 1). Independent of encapsulation, LP SN13T significantly decreased fat by day 40. The non-encapsulated sample decreased by 4.79% in fat after 60 days, while the encapsulated one retained 5.25%. The gel matrix prevents the breakdown of enzymes and lipids (Kailasapathy, 2009). High-viscosity alginate beads decrease lactic acid bacteria fermentation and preserve fat (Hernández-Gallegos  et al., 2023). In Table 1, LP SN13T addition and storage duration had a significant impact on protein content (P<0.05). LP SN13T-encapsulated ice cream lost little protein after 60 days because the beads prevented bacteria from directly interacting with the substrate, limiting fermentation and proteolysis. The addition of probiotics and storage duration significantly affected water content (P<0.05) (Table 1). The encapsulated LP SN13T lost little moisture, as the hygroscopic alginate gel retained moisture until day 60. The probiotic without encapsulation lowered moisture levels faster, indicating direct bacterial action and the effects of the stabilizer and carbohydrates (Jain and Rai, 2018). Ice cream with alginate-based beads maintains its fat, protein and moisture content, thereby improving stability and quality.

    Table 1: Proximate content of Ice cream (%) with the addition of L. plantarum SN13T during storage.


     
    pH and total lactic acid bacteria
     
    The pH value of ice cream decreased gradually during storage. On day 0, the pH remained relatively high, then decreased on days 20 and 40 and reached its lowest value on day 60. The decrease in pH was more pronounced with the unencapsulated treatment, while the encapsulated treatment showed a slower, more stable decrease. The total number of LAB also decreased gradually during storage. On day 0, the highest LAB count was observed in both treatments, then decreased on days 20 and 40 and decreased further on day 60.
           
    The analysis of variance revealed a highly significant effect (P<0.05) of incorporating LP SN13T (A) and storage period (B) on the pH of the ice cream (Table 2). The pH decreased during ice cream storage after the addition of encapsulated LP SN13T, but the change was not significant, indicating improved stability.  The increased acidity is caused by the production of lactic acid by bacterial fermentation of lactose in dairy products (Bajad et al., 2016). The alginate-based capsule protects the bacteria from temperature and pH variations, establishing a diffusion barrier that restricts the release of acidic metabolites (Anal and Singh, 2007).  Žadeikë  et al. (2024) similarly observed that the protective gel matrix inhibits LAB activity, leading to gradual acidification. In contrast, unencapsulated LP SN13T significantly lowers pH through lactose fermentation and organic acid production, a phenomenon explained by Prasertsiriphan and Kusump (2015), who related direct substrate access with increased fermentative activity in the product overall.

    Table 2: PH values and total lactid acid bacteria colony count of ice cream with the addition of L. plantarum SN13T during storage.


           
    The study found that adding LP SN13T and increasing storage time both had a substantial effect on the total LAB count. There was no significant change in the LAB count in encapsulated samples on days 1, 20 and 40. However, by day 60, there was a slight decrease to 10.03 Log CFU/ml, which was an 18.95% reduction compared to the non-encap- sulated control. This suggests that the alginate-based encapsulation matrix acts as a barrier, protecting the probiotic cells and reducing damage during freezing, thereby maintaining a more stable LAB population. This was supported by Sedefoğlu et al., (2022) and Gullo and Zotta (2022). In contrast, unencapsulated L. plantarum exhibited a 42.97% decrease in LAB after 60 days, as the free cells are more susceptible to ice-crystal formation and other stressors, as described by Homayouni et al., (2008). Therefore, encapsulation is a more effective method for maintaining the minimum 106 CFU mL-1 required by CODEX (2003) during ice cream storage, thereby ensuring consumer safety.
     
    Simulated gastric juice (SGJ)
     
    The resistance of ice cream to SGJ decreased significantly (P<0.05) during storage in both treatments. On day 0, the resistance to SGJ showed the highest value, then decreased on days 20 and 40 and reached the lowest value on day 60. The incorporation of encapsulating LP SN13T over a duration of 60 days (A2B4) resulted in a 6.7% reduction of SGJ (Fig 1). Encapsulated LP SN13T safeguards bacteria against gastric acid during storage. Various defensive mechanisms render encapsulation efficacious. Starch protects probiotic organisms from gastric acid, which can reach pH 1.5 (Kim et al., 2017). Starch and calcium alginate provide superior acid protection compared to unencapsulated probiotics (Ngov, 2014). Probiotics in ice cream require this safeguarding. A1B4 exhibits a 17.17% reduction in resilience to stomach-like acid after 60 days with LP SN13T without encapsulation. Bacteria are subjected to acidic conditions without encapsulation. According to Lee (2010), probiotic bacteria such as Lactobacillus acidophilus exhibit increased mortality in the presence of strong acid. Prolonged storage diminishes resistance to gastric acid. Bilang et al., (2018) also reported that unencapsulated LAB experienced a greater decrease in viability compared to encapsulated LAB. Unencapsulated Lactobacillus plantarum and Streptococcus thermophilus decreased by 31% and 38%, respectively, while in the encapsulated treatment, the decreases were lower, namely 24% and 37%.

    Fig 1: Graph of resistance to SGJ ice cream (%) with the addition of L. plantarum SN13T during 60 days of storage.


     
    Simulated intestinal juice (SIJ)
     
    The resistance of ice cream to SIJ decreased significantly (P<0.05) with increasing storage time in both treatments. On day 0, the SIJ resistance value was still relatively high, then decreased on days 20 and 40, reaching its lowest value on day 60. Adding encapsulated LP SN13T had no significant impact on the survival of the bacteria in SIJ after 60 days of storage (A2B4, Fig 2). Still, the same strain without encapsulation had a 14.80% loss (A1B4, Fig 2). The microencapsulation made of starch creates a physical barrier that protects probiotic cells from the bile salts in simulated gut juice. Numerous studies demonstrate that encapsulating bacteria in starch is more effective than encapsulating them in alginate alone. For example, Lactobacillus acidophilus LA1, which is encapsulated in starch, survives higher bile salt concentrations better than free cells (Sabikhi et al., 2010). Therefore, starch microen-capsulation effectively maintains the stability and activity of probiotics in ice cream products during storage, making them more appealing to customers. Bilang et al., (2018) also reported that unencapsulated LAB experienced a greater decrease in viability than encapsulated LAB. Unencapsulated Lactobacillus plantarum and Streptococcus thermophilus experienced a decrease of 30-32%, while encapsulated LAB only experienced a decrease of 21-26%.

    ​

    Fig 2: Graph of resistance to SGJ ice cream (%) with the addition of L. plantarum SN13T during 60 days of storage.


     
    Organoleptics 
     
    The organoleptic properties of LP-SN13T ice cream, both without encapsulation and with encapsulation, significantly affect (P<0.05) flavor, color, taste and texture during 60 days of storage. Panelists favored non-encapsulated (direct-addition) samples, which maintained a constant hue from day 20 to 60, likely due to the formulation’s natural ingredients and lack of coloring (Fig 3). Flavor is the most important positive attribute of ice cream. However, flavor stability can decline during processing and storage (Vanathi and Dorai, 2020). Diez-Libreros  et al. (2025) found that adding probiotics directly did not modify the color or taste of the ice cream. Non-encapsulated samples preferred flavor throughout storage, while encapsulated samples decreased significantly (P<0.05) on days 20 and 40 but improved by day 60 (Fig 4). The encapsulated ice cream initially fluctuated, while the non-encapsulated version had more stable texture scores. The findings support prior research: Fávaro-Trindade  et al. (2016) observed that bead encapsulation changes sensory profiles, while Syed et al., (2018) and English et al., (2023) found that additives and encapsulation affect texture and quality.

    ​

    Fig 3: Organoleptic value chart of probiotic ice cream with the addition of L. plantarum SN13T without encapsulation during storage.



    ​

    Fig 4: Organoleptic value chart of probiotic ice cream with the addition of L. plantarum SN13T with encapsulation during storage.

    CONCLUSION

    Both encapsulated and non-encapsulated LP SN13T had a major effect on ice cream quality over a 60-day period, according to the study. The non-encapsulated LP SN13T ice cream has a pH of 5.16, 4.79% fat, 8.11% protein, 65.84% water, 10.01 Log CFU/mL total LAB, 71.76% SGJ resistance and 72.16% SIJ resistance. The results of organoleptic tests were 3.76 for colour, 3.78 for taste, 3.78 for texture and 3.82 for flavour. The nutritional content, bacterial viability and sensory attributes of encapsulated LP SN13T ice cream were all preserved. Additionally, encapsulated LP SN13T demonstrated higher SGJ and SIJ resistance. These results suggest that encapsulation technology may be used in the production of probiotic ice cream on an industrial scale to create stable, functional goods for cold storage and distribution.

    ACKNOWLEDGEMENT

    The present study was supported by the Research and Community Service Institute of Universitas Andalas, which funded this research under the Research Contract Master’s Thesis Research Scheme Batch I, Number: 181/UN16.19/PT.01.03/PTM/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 has passed the ethical review by the Faculty of Pharmacy Commission Team andalas University, with Number: 46/UN.16.10.D.KEPK-FF/2025.

    CONFLICT OF INTEREST

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

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