Studies on survivability, storage stability of encapsulated spray dried probiotic powder.

Awareness about probiotic food and their health benefits is increasing tremendously. However, probiotics have to withstand the harsh conditions that come across during their processing, handling, storage, and gastrointestinal conditions. Encapsulating technologies can be used to protect the probiotics during their passage through the gastrointestinal system of the human gut. Probiotics as an ingredient in dry powder form can be easily handled, stored, and used in developing the probiotic functional products. In the present study, probiotic cells (Lactobacillus acidophilus) were encapsulated by spray drying technology to produce a probiotic powder using 20% of maltodextrin and varied concentrations of gum arabic. The effect of processing conditions such as inlet air temperature (130-150 °C) and gum arabic concentration (0-10%) on the encapsulation efficiency and physical properties were studied. Further, the free and encapsulated probiotic cells were exposed to the simulated-gastric intestinal (SGI) fluid conditions and different storage conditions for their viability. For all the tested formula, moisture content, water activity, encapsulation efficiency, hygroscopicity, and wettability obtained were in the range of 4.59-9.05% (w.b.), 0.33-0.52, 65-89.15%, 12-21.15 g H2O/100g dry weight, and 116 s-353 s, respectively. The Fourier transform infrared (FTIR) results have shown that gum arabic and maltodextrin have structural stability during spray drying. The encapsulated probiotic cells have shown a positive effect and exhibited better viability after exposure to a SGI solution at different pH levels and duration compared to free cells. The viability of encapsulated cells stored at refrigerated condition (4 °C) was found to be higher than the viability of cells stored at room temperature (25 °C).

Introduction

Good health is maintained by the useful bacteria that are present inbuilt in the gut system. Gut microbial system gets disturbed due to changes in the food intake. To restore the gut microbial homeostasis, consumption of probiotics is highly essential. “Probiotics” is defined as “live microbes when consumed in adequate quantity confer a health benefit on the host” (FAO/WHO, 2001). This led to the development of probiotic foods comprising 60%–70% of the total functional foods (Soccol et al., 2014). It is recommended that probiotic food should contain a minimum of 108 colony forming units (CFU) per milliliter or per gram at the time of consumption (Ganguly et al., 2011).

Among various probiotic species, Lactobacillus are robust probiotic microbes and greatly used in developing the probiotic functional foods due to their wider attributes. As a probiotic, L. acidophilus has been investigated for its efficacy in the prevention of traveler's diarrhea and various GI disorders, reduces the risk of tumor development (Gomes and Malcata, 1999; Arepally et al., 2019), and stimulate the immune system with better digestion (Urmann et al., 2016).

Most of the probiotics are sensitive and lose their viability on exposure to harsh environmental conditions. The probiotics are undeniably encapsulated with the protective matrix to resist against the harsh conditions and to improve their viability. Among the several encapsulating techniques such as emulsion, freeze-drying, extrusion, spray drying, coacervation, fluidized bed drying; spray drying is commonly used method due to its low energy consumption, less cost, higher production efficiency, storage and flexibility in developing the functional food (Tao et al., 2019). Moreover, the final product obtained using this technology is in the powder form with many merits such as convenience for consumption, a longer shelf life, and secure handling. Spray drying method atomizes the liquid feed at high velocity into the fine droplets of size 10–150 μm to produce dry powders by injecting a hot and dry air usually at a temperature of 150–200 °C (Reddy et al., 2014).

However, for the usage of the spray drying technology to produce probiotic powder, challenges like heat, oxidative, osmotic, and desiccation stresses has to overcome, especially with the sensitive probiotic strains. During spray-drying process, the membrane of probiotic cells gets damaged, and as a result, they are prone to heat and dehydration. Moreover, process parameters such as inlet and outlet air temperature, feed rate, drying time, and drying medium results in inactivation thus, have a significant impact on the probiotics viability (Peighambardoust et al., 2011). Among the various process variables, inlet air temperature is a critical variable for the encapsulation of probiotics. Various probiotic food products are produced using spray drying technique including yogurt, orange juice powder, chocolate (Kearney et al., 2009; Alves et al., 2016; Klindt-Toldam et al., 2016). In a study reported by Liu et al. (2016) found that the encapsulation of Lactobacillus zeae LB1 using sodium caseinate and gum arabic by spray drying had greatly improved the cell viability during gastrointestinal conditions and storage.

From the past 20 years, abundant research was accomplished on the encapsulation of different strains of probiotics. Regarding the encapsulation matrices, different food-grade polymer like carbohydrates, proteins, lipids, and their combination have been used. Among the different polymers used for encapsulation, carbohydrates like maltodextrin are used because of its ability to prevent thermal stress due to its high activation energy and prevents oxidative stress during storage. On the other hand, maltodextrin is commonly used as a wall material, since it can be easily absorbed and digested. Besides, its advantages such as high solubility, non-toxicity, low viscosity, and cost are significant factors for its extensive use as an encapsulating material.

Nevertheless, maltodextrin is favoured to use in combination with other wall material due to its low emulsifying capacity. One such wall material is gum arabic (a natural carbohydrate polymer attained from Acacia Senegal (L.)) (Premi and Sharma, 2017). Gum arabic at a low level of 1 and 10% acts as a stabilizer, mouth feel booster, film former (Meance, 2004), hinders the dehydration of cell components and preserves microbial cells during drying and storage. With the numerous intense properties, it is used as an additive for the manufacturing of beverages, confectionery, bakery products, emulsions, brewing, and flavour encapsulations (Sanchez et al., 2018). Moreover, the combination of maltodextrin and gum arabic provides controlled release, stability against oxidation, easy handling, better solubility, and extended shelf-life (Igual et al., 2014). Concurrently, gum arabic helps in increasing the glass transition temperature, reduces the hygroscopicity, caking, and improve the physicochemical properties of the obtained powder (Zhang et al., 2019). However, encapsulation of probiotics at a low level of gum arabic in producing the probiotic powder is scanty. Therefore, in the present study, spray drying technology was employed for the encapsulation of probiotics using maltodextrin and varied concentration levels of gum arabic as wall materials. Additionally, survivability of encapsulated probiotics was evaluated during simulated gastric, intestinal conditions, and its storage period.

Material and methods

Materials

Lyophilized L. acidophilus NCDC 016 was purchased from the National Dairy Research Institute (NDRI), Karnal, Haryana, India. The wall materials (maltodextrin and gum arabic), De Mann, Regosa, Sharpe (MRS) broth and MRS agar used in culturing of probiotics were acquired from Hi-Media Laboratories Private Limited, Mumbai, India. The chemicals used during the period of experimentation were of analytical grade. All the glassware were autoclaved at a temperature of 121 °C for 15 min.

Cell culture preparation

Initially, the freeze-dried probiotic culture was revived by incubating in 10 mL MRS broth for one week in order to activate the culture. L. acidophilus NCDC 016 was propagated three times in MRS broth at 37 °C for 24 h before the experiments were carried out. For further use, the activated strain was mixed with 80% glycerol in 1:1 ratio and stored at −20 °C. The fresh culture was obtained by inoculating the 1% (v/v) of inoculum in a fresh sterilized MRS broth and incubating at 37 °C for 24 h. The inoculum was prepared according to the method followed by Arepally and Goswami (2019). The final inoculum concentration obtained was almost in the range of 10–11 log cfu/mL. The cell suspension was obtained by centrifugation (7000 g) at a temperature of 4 °C for 10 min and further suspending in 0.85% (w/v) sterile saline solution twice. The obtained supernatant was discarded, and probiotic cell suspension settled down in the test tubes was stored at 4 °C and used on the same day.

Production of probiotic powder

Probiotics were encapsulated using a different concentrations of gum arabic, and at different inlet air temperature. For spray drying operation, the feed solution with 20–30% solid content is generally considered to be optimum for ensuring high viability of bacterial cells (Jantzen et al., 2013). Therefore, the concentration of maltodextrin was taken as 20% (w/v) for different concentration levels of gum arabic. The wall material solution was dispersed in hot distilled water for complete dissolution and hydration at a temperature of 50 ± 3 °C for about 30 min under magnetic stirrer. Before conducting the treatments, the spray dryer was operated to reach the desired process temperature. The polymer solution was spray-dried in a co-current laboratory spray dryer (Jay Instruments and Systems Private Limited, India). The cell suspension was introduced into the feed solution just before the spray drying. During atomization, the feed solution was kept agitated through a magnetic stirrer at 300 rpm. The feed flow rate was maintained at 8 mL/min. During the period of drying operation, the outlet air temperature was recorded to be 55 ± 3 °C. Finally, the obtained powder was transferred to a sterilized glass vial and immediately stored in a desiccator.

Enumeration and encapsulation yield

The viability of probiotic cells were determined by enumerating the encapsulated probiotic powder on MRS agar by pour plate method. The sample powder was suspended in 0.85% (w/v) sterile saline solution in 1:10 dilution. The sample powder was completely dissolved in the saline solution by operating the vortex spinner for 5–10 min at room temperature. The serial dilution of the initial and encapsulated suspension was done and plated in sterile petri dishes and incubated at a temperature of 37 °C under anaerobic conditions for 48 h. Plates containing 30–300 colonies were chosen for the enumeration.

The encapsulation yield was done as explained by Arepally and Goswami (2019).Encapsulation yield = N/NWhere N is log cfu/g after encapsulation and No is log cfu/mL before encapsulation.

Moisture content

The encapsulated probiotic powder (2 g) was placed in an aluminum sample holder and placed in a conventional hot air oven at 105 °C. It is exposed to the drying condition till it reaches a constant weight.

Water activity

The water activity of obtained powder was determined by using a water activity meter (Hygrolab 3-HP23, Rotronic) at room temperature. The water activity meter was initially adjusted by calibrating with distilled water for its accuracy.

Hygroscopicity

The hygroscopicity was measured according to the method followed by Fritzen-Freire et al. (2012). 1 g of encapsulated powder sample was taken in known weight of petri dish and was placed in an airtight glass desiccator containing NaCl saturated solution of 75% RH and stored for 1 week at a room temperature of 25 °C. At the end of the week, the sample was weighed, and hygroscopicity was calculated from the below formula as

Where mf and mi are the weight of samples before and after the storage period, respectively.

Wettability

The wettability time was evaluated by static wetting test method (Hammes et al., 2015). One gram of encapsulated probiotic powder was sprinkled over the surface of 100 mL distilled water at room temperature without agitation. Time taken for the powder particles to sediment, submerse and disappear from the water surface was recorded.

Fourier transform infrared (FTIR) measurement

The spectra of microencapsulated probiotic cells were measured by a Fourier transform infrared spectrometer (Model – NICOLET 6700, M/S Thermo Fisher Scientific Instruments, Madison) using potassium bromide disk method. The sample was mixed with potassium bromide containing 1% by weight of the sample and then pressed into a pellet and placed in a sample holder. The spectra of the samples were obtained from 400 to 4000 cm−1 wave number range.

Survival of free and encapsulated L. acidophilus in simulated gastro-intestinal fluid conditions

The simulated gastric fluid (SGF) was prepared, according to Rajam et al. (2015) with slight modifications. Briefly, 100 mL of MRS broth solution was prepared and 1 M HCl was added in order to adjust it to a pH of 2. This solution was autoclaved at 121 °C for 15 min for sterilization. Simultaneously, Pepsin (1:3000 μ/g, extra pure) was filtered through a sterile membrane filter of pore size 0.45 μm and added to sterile MRS broth to a final concentration of 0.3% (v/v). The prepared SGF solution was kept in an incubator to warm the solution to 37 °C. Then, the 9 mL of SGF solution was transferred into the sterile test tubes. The samples of free cells (1 mL) and encapsulated cells (1 g) were added to these different test tubes separately and incubated at 37 °C. The encapsulated cells produced at the inlet air temperature of 150 °C and gum arabic concentration of 8.51% were used. Similarly, the SGF solution of pH 1 and 1.5 were prepared by adjusting the MRS broth solution with 1M HCl, and the above mentioned procedure was followed. Enumeration of encapsulated and free cells were done at the time intervals of 0, 1, 2, and 3 h for different pH levels and survivability was represented as a number of viable cells (log cfu/g). The simulated intestinal fluid (SIF) was prepared by dissolving the 2% (w/w) of bile salt in 1000 mL of MRS broth, and the pH was adjusted to 6.8 using NaOH solution. The prepared solution was autoclaved. After 3 h, the test tubes containing SGF solution of pH 2 with free cells and encapsulated cells were centrifuged at 5000 g for 15 min. The sediment was suspended with 9 mL of SIF solution and incubated at 37 °C as done for the SGF test. The aliquot sample (1 mL) of free and encapsulated cells were taken at 0, 1, 2, and 3 h and evaluated for cell survivability.

Survivability of encapsulated L. acidophilus during storage

The encapsulated probiotic powder produced by spray-drying was filled in a sterile glass vial and stored at different storage conditions (4 °C and 25 °C) for a storage period of 12 weeks. One gram of probiotic powder was withdrawn from the glass vials for every week of storage. The sample (1g of probiotic powder) stored at a temperature of 4 and 25 °C was dissolved in 0.85% (w/v) saline solution, diluted, plated and incubated at 37 °C for 24 h to enumerate the viability of cells.

Statistical analysis

Experiments on the encapsulation of probiotics were repeated in triplicate except for the enumeration of probiotics which was repeated five times for better accuracy of the enumerated results. Results were reported as mean ± standard deviation. IBM SPSS Statistics 25 software was used for statistical analysis. One way ANOVA with Tukey's post hoc test was used to determine the level of statistical significance (p < 0.05).

Results and discussion

Moisture content and water activity of probiotic powder

The moisture content of produced probiotic powder for all the treatments is in the range of 4.59%–9.05% (wet basis (w.b)). From Fig. 1, it is illustrated that the moisture content of encapsulated probiotic powder gets decreased with an increase in inlet air temperature for all the gum arabic concentrations. The difference in temperatures of the drying air and feed solution prompted in faster heat transfer for water removal. Under high inlet air temperatures, the heat transfer rates within the particles are much faster due to a large temperature gradient causing the higher driving forces for moisture evaporation and thereby eventually producing the powder with low moisture content. Mishra and Athmaselvi (2016) reported that the moisture content of encapsulated Lactobacillus rhamnosus powder as 7.49, 7.10, and 6.51% (w.b) for the inlet air temperature of 130, 140, and 150 °C respectively. The maximum moisture content of 9.05% at an inlet temperature of 130 °C was due to the outlet temperature of 55 ± 2 °C in the current study. Similarly, Riveros et al. (2009) in his study obtained a moisture content of less than 10% when the outlet air temperature was less than 60 °C. As the concentration of gum arabic was increased from 0% to 10% with an interval of 2.5%, a typical trend was observed. Initially, the moisture content was observed to be increased for the gum arabic concentration of 2.5 and 5%, and then it gets decreased from 7.5% to 10%. This initial increase might be related to the presence of proteins in gum arabic (Liu et al., 2016). Moreover, higher gum arabic concentration results in a higher emulsion viscosity that hinders the diffusion rate of water during spray drying ultimately leading to higher moisture content than the moisture content at 0% GA concentration. The decrease in moisture content from 7.5 to 10% of gum arabic concentration might be due to the reduction in total moisture diffusion because of an increase in feed solids (Frascareli et al., 2012).

Fig. 1

Moisture content of encapsulated probiotic powder.

In general, food powders with a water activity of <0.6 are considered to be safe. The average water activity of probiotic powder was seemed to be 0.42, indicating the stability of powder against detrimental chemical and microbiological reactions. The highest water activity of 0.52 was obtained for the conditions of 0% gum arabic concentration and inlet air temperature of 130 °C, whereas, the lowest water activity value of 0.33 was observed for the conditions (gum arabic%/inlet air temperature: 10%/150 °C). From Fig. 2, it was found that the water activity gets decreased with an increase in inlet air temperature. Similar results were confirmed for kefir powder (Atalar and Dervisoglu, 2015), goat milk powder (Reddy et al., 2014). De Medeiros et al. (2014) produced the probiotics containing goat yogurt powder. They observed a water activity of 0.19, 0.18, and 0.15 at an inlet air temperature of 130, 150, and 170 °C, respectively. In the present study, increasing the concentration of gum arabic caused a decrease in the water activity of probiotic powder being between 0.33 and 0.52 for different inlet air temperatures (130, 140, and 150 °C). For the gum arabic concentration of 0%–2.5% and 5%, the drastic decrease of water activity was observed from 0.52 to 0.49 and 0.44 respectively at the inlet air temperature of 130 °C. This decrease in water activity was elucidated by the water-binding capacity of gum arabic which in turn probably due to the highly branched characteristics of carbohydrate component of gum arabic that prevented the water from being released from its three-dimensional structure (Arepally and Goswami, 2019).

Fig. 2

Water activity of encapsulated probiotic powder.

Encapsulation yield of probiotic powder

The most important factor in the microencapsulation of probiotics is the bacterial viability. Encapsulation yield is affected by the inlet air temperature and wall material formulations. The viability of bacterial cells in the feed solutions prepared with different concentration levels of gum arabic before spray drying process was found to be similar in the study and falls in the range of 10.81–11.36 log cfu/g. For any health benefits, a minimum concentration of 106 cfu/g in the product at the time of consumption is recommended (Tripathi and Giri, 2014). The concentration of viable bacterial cells obtained in probiotic powder was comprised between 7.3 and 9.97 log cfu/g and was satisfying. An increase in an inlet air temperature got decreased the encapsulation yield whereas it was increased with increasing the gum arabic concentration, as shown in Fig. 3. The decrease in survival of bacterial cells with the increase in inlet air temperature is mainly attributed to the thermal and dehydration inactivation of cells. At high temperatures, cellular components like DNA, RNA, proteins, and ribosomes get damaged (Lee and Kaletunç, 2002). In general, water molecules contribute to the stability of bacterial proteins, lipids, DNA, and cell structure. However, during spray drying, the cells get dried up at such high temperatures resulting in the rupture and collapse of cytoplasmic membranes leading to cellular injuries and loss of intracellular components.

Fig. 3

Encapsulation efficiency of spray dried probiotic powder.

Additionally, rupture of the cell wall leads to the direct contact of cells with surrounding air resulting in the lipid oxidation (Peighambardoust et al., 2011). During dehydration, components like nucleic acid and ribosomes might also get injured due to the escape of Mg2+ from the cell membranes. Similar results were observed for L. acidophilus and S. boulardii with gum arabic as wall material (Arslan et al., 2015). In another study, L. acidophilus NRRL B-4495 and L. rhamnosus NRRL B-442 were encapsulated at the inlet air temperatures of 100, 115, and 130 °C using maltodextrin and obtained a maximum survival% of 81.17% at 100 °C (Anekella and Orsat, 2013). This lesser encapsulation yield obtained in their study at 100 °C was due to the outlet temperature, which was maintained at 80 °C–90 °C. Previous reports indicated that the outlet temperature ranging from 40 to 60 °C yielded satisfactory cell survivability after spray drying (Fu and Chen, 2011). The encapsulation yield observed in the present study was found to be higher even at 130 °C because of the milder outlet temperatures of 55 ± 2 °C that was maintained in conducting the experiments.

On increasing the gum arabic concentration, the encapsulation yield was found to be increased. This could be attributed to the formation of a protective layer on the microbial cell wall because of the presence of proteins in the gum arabic. Furthermore, the presence of hydrophobic amino acids residues such as glycine, isoleucine, valine, leucine, phenylalanine in the gum arabic structure could have reduced the heat transfer further increasing the encapsulation yield (Sanchez et al., 2018). This is supported by the study of Arslan et al. (2015) who encapsulated the Saccharomyces cerevisiae var. boulardii using the gelatin as a wall material. They reported that the encapsulation yield was increased due to the hydrophobic components of gelatin.

Moreover, during drying, the presence of fiber in gum arabic might have provided partial replacement of sites of water molecules in the cells and prevented the disruption of the cell membrane; ultimately increased the encapsulation yield. Concurrently, it was presumed that the proteins of the gum arabic might have contributed to the stability of the intracellular proteins of the probiotic cells during the drying process (Arepally and Goswami, 2019). The results are in agreement with the Rajam and Anandharamakrishnan (2015) who encapsulated the L. plantarum (MTCC 5422) with whey protein and fructo-oligosaccharide. Similarly, Pereira et al. (2014) reported a higher encapsulation yield of 77–82% using maltodextrin and gum arabic as a carrier material. Gul and Atalar (2019) in their study, stated that the gum arabic as a best protective carrier in encapsulating the Lactobacillus casei.

The wettability time for the spray dried probiotic powder was found to be in the range of 116 s–353 s for all the treatments carried out at different inlet air temperatures and gum arabic concentration and is shown in Fig. 4. Higher wettability time was found to be observed for higher temperatures. A rigid surface formed at higher temperature could prevent the penetration of water through the particle. Moreover, this could be due to lower moisture content attained at higher inlet air temperatures. The wetting time of the probiotic powder encapsulated with maltodextrin alone was found to be lower because of the hydrophilic nature of the maltodextrin that facilitates the penetration of water into the powder (Bae and Lee, 2008). On increasing the gum arabic concentration, the wettability time gets increased for inlet air temperatures of 130–150 °C. Similar results were claimed by the Ghosal et al. (2010) for gum added powder.

Fig. 4

Wettability of encapsulated probiotic powder.

The hygroscopicity of probiotic powder under different inlet temperatures and gum arabic concentration was depicted in Fig. 5. The hygroscopicity of powder varied from 12 to 21.15 g/100g dry weight. The lowest hygroscopicity was obtained at the temperature and gum arabic concentration of 130 °C and 5%, respectively. For all the different concentration levels of gum arabic, it was observed that the hygroscopicity of probiotic powder was increased with increasing the inlet air temperature. Hygroscopicity is mainly affected by the composition, type, and concentration of carrier material used for the encapsulation. On increasing the gum arabic concentration, the hygroscopicity was increased. This could be ascribed to the faster water adsorption of gum arabic that affects the balance of the hydrophilic and hydrophobic sites of powder sample (Suhag et al., 2016). Presence of proteins contributes to the adsorption of water molecules due to their hydrophilic nature. Suhag et al. (2016) confirmed similar results for the encapsulated honey powder produced at different gum arabic concentration of 35–45% and inlet air temperature of 160–180 °C.

Fig. 5

Hygroscopicity of encapsulated probiotic powder.

Fourier transform infrared (FTIR) analysis

FTIR spectroscopy is used to find the extent of interactions between polymers such as maltodextrin and gum arabic and their chemical stability during encapsulation by determining any change in their original structure. The FTIR spectra were analyzed for the probiotic powder produced at the inlet air temperature of 150 °C for different concentration levels of gum arabic assuming that the wall materials retain their originality at the inlet air temperatures of 130 and 140 °C if they were stable regarding their structure at 150 °C. The individual spectra of control maltodextrin (CMD) and control gum arabic (CGA) samples were also shown in Fig. 6.

Fig. 6

FTIR spectra of control maltodextrin (CMD), control gum arabic (CGA), encapsulated probiotic powder.

In the spectrum obtained for control gum arabic (CGA), a broad absorption band was observed around 3000–3600 cm−1 representing the characteristics of the glucosidic ring and for –OH stretching (Dai et al., 2018), 3000 - 2800 cm−1 for –CH stretching of methyl groups, 1613 cm−1 for –C=O stretching and –NH bending, and 1100 - 1070 cm−1 for –COC stretching, vibrations. The results were supported by previous studies (Williams et al., 2006; Kang et al., 2019). The absorption at wave number 887 cm−1 and 891 cm−1 corresponds to the β-D-manopyranose units. For maltodextrin, the band around 3330 cm −1 corresponds to OH stretching vibration, 2929 and 2852 cm−1 corresponds to asymmetric and symmetric lipid CH2 stretching vibration respectively. The area between 800 and 1200 cm−1 is called fingerprint region for carbohydrates. The main infrared regions of maltodextrin are below 1000 cm−1 and characterize the stretching vibrations of anhydroglucose ring. Concurrent peaks were found in the spectra of probiotic powder at different concentration levels of gum arabic without any significant deviation indicating that there was no interaction between the wall materials and probiotics and retained their originality even after treating at such high temperature. This observation was supported by the similar results obtained by Nayak et al. (2012).

Survivability in simulated gastric and intestinal fluid conditions

The survival of probiotic bacteria in free and encapsulated form at different pH values of simulated gastric and intestinal fluid condition was evaluated and shown in Table 1. It was determined that exposure time to simulated gastric fluid solution has a significant effect on the viability of both free and encapsulated cells. At the pH 1 of SGF solution, free cells reduced to 1.43 log CFU/g after 3 h of exposure time whereas the viability of encapsulated cells was found to be 5.61 log CFU/g at the end of the analysis. Similarly, at pH 1.5 and 2, the viability of encapsulated cells was found to be 6.15 and 6.46 log CFU/g respectively. However, the reduction in survival was slightly lesser at the pH 2 compared to pH 1 and 1.5. The reduction of survivability of free cells was found to be almost double compared to the reduction of encapsulated cells. This indicates that the encapsulated cells were able to maintain their viability in gastric conditions. Moreover, the survivability of probiotics against the detrimental effects of the extremely acidic conditions was provided by the wall material maltodextrin and gum arabic at pH of 1, 1.5, and 2. It could be mainly due to the impermeable structure of microcapsules produced at high temperature.

Table 1

Survivability of encapsulated probiotic cells and free cells under simulated gastrointestinal conditions.

SGF conditions							SIF conditions
Time, h	pH-1		pH-1.5		pH-2		pH-6.8
	Encapsulated cells	Free cells	Encapsulated cells	Free cells	Encapsulated cells	Free cells	Encapsulated cells	Free cells
0	8.98 ± 0.08a	9.05 ± 0.13a	8.98 ± 0.17a	9.05 ± 0.09a	8.98 ± 0.06a	9.05 ± 0.05a	6.46 ± 0.07a	1.94 ± 0.07a
1	8.11 ± 0.11b	5.20 ± 0.07b	8.15 ± 0.14b	5.53 ± 0.07b	8.34 ± 0.07b	5.8 ± 0.07b	6.11 ± 0.09b	1.36 ± 0.06b
2	6.86 ± 0.12c	2.74 ± 0.14c	7.2 ± 0.15c	2.91 ± 0.1c	7.5 ± 0.1c	3.16 ± 0.11c	5.98 ± 0.11c	1.26 ± 0.09c
3	5.61 ± 0.07d	1.43 ± 0.11d	6.15 ± 0.12d	1.68 ± 0.06d	6.46 ± 0.11d	1.94 ± 0.08d	5.76 ± 0.10d	1.11 ± 0.07d

Note: values are expressed as mean ± standard deviation. Different superscript letters in the same column indicates the significant difference (p ≤ 0.05).

Moreover, the protein structure of gum arabic, which have less hydroxyl polar groups could have kept the acid effect out of the core material. Concurrently, the thermo protective polymer such as maltodextrin used as a carrier material in the encapsulation of probiotics could have resulted in a powder containing probiotic cells with slight injuries; thus able to withstand the SGF conditions (Yonekura et al., 2014). The survivability was found to be reduced for lower pH value and increasing the exposure time. Similar results were obtained to the Arslan et al. (2015) on the survivability of encapsulated S. boulardii in the simulated gastric conditions at different pH levels (1, 1.5, and 2). In another study, Lian et al. (2002) spray-dried B. longum B6 using gum arabic, whey, and soluble starch and found a higher survivability for the cells encapsulated with gum arabic and gelatin at a pH 2. Under simulated intestinal fluid conditions, the viability of 1.11 log CFU/g was observed in free cells, and encapsulated cells maintained their viability to 5.76 log CFU/g at the end of 3 h of exposure time. The SIF solution in our study has a minor effect on the survival of encapsulated probiotic cells compared to free cells. The physical embedment of the probiotic cells in the compact gum arabic matrix might have protected them from the harsh gastrointestinal conditions (Zhang et al., 2018).

Viability of encapsulated probiotics under different storage conditions

The probiotic cells protected in an encapsulating matrix has to withstand against the harsh conditions, including the environment, gastrointestinal tract and also has to maintain their viability during storage. Plenty of studies have focused on the maintenance of probiotic cell viability during storage. The cell viability during the storage period is chiefly dependent on the storage temperature. Moreover, at higher temperatures such as 37 °C results in more significant loss of cell viability (Fu and Chen, 2011). In our study, the free L. acidophilus cells and encapsulated cells were stored at room (25 °C) and refrigerated (4 °C) temperatures. Free and encapsulated cells were tested for viability for every week up to 12 weeks. The viability of both free and encapsulated cells from the starting day of storage period were presented in Fig. 7, Fig. 8. It was found that the viability of free cells stored at 4 and 25 °C was less than 6 log CFU/g after 12 weeks.

Fig. 7

Survivability of encapsulated cells (EC) and free cells (FC) during storage period at 4 °C.

Fig. 8

Survivability of encapsulated cells (EC) and free cells (FC) during storage period at 25 °C.

However, the encapsulated capsules stored at 4 °C achieved the highest viability among free and encapsulated cells stored at 25 °C and free cells stored at 4 °C. The cells stored at temperatures close to above 0 °C reduces the rate of detrimental chemical reactions (Heidebach et al., 2010). The loss of viability of free and encapsulated cells at 25 °C is probably due to the oxidation of membrane lipids and denaturation of proteins that lead to the degradation of macromolecules in bacterial cells (Fu and Chen, 2011). However, the viability of encapsulated cells was higher compared to the free cells. The reason could be because the probiotic cells encapsulated in protective carriers stabilizes the cellular structures and thus reduce the environmental stresses by restricting the molecular movement. Desmond et al. (2002) also observed the higher viability of probiotic cells encapsulated in the gum arabic and maltodextrin matrix than the free cells. The viability of encapsulated cells stored at 4 °C maintained the minimum requirement of 106 log CFU/g even after 12 weeks of storage period.

Conclusions

Currently, the probiotic-containing functional foods are dragging attention in the market. In this research, the probiotics were encapsulated using spray drying technology to produce a probiotic powder that can be used as a functional ingredient in the food. The inlet air temperature and gum arabic concentration affected the encapsulation efficiency, moisture content, and water activity. The FTIR results showed the stability of the material during the spray drying conditions. The encapsulated cells were produced at the inlet air temperature of 150 °C and gum arabic concentration of 8.51%. The viability of encapsulated probiotic cells was found to be higher compared to the free cells in the gastrointestinal conditions. This provided proof for the protecting ability of encapsulation using maltodextrin and gum arabic against the harsh conditions. Moreover, the encapsulated cells stored at refrigerated condition have shown better viability compared to the cells stored at room temperature.

Credit author statement

Divyasree Arepally: Data curation, Writing- Original draft preparation, Reviewing and Editing.

Ravula Sudharshan Reddy: Methodology, Software, Reviewing and Editing.

Tridib Kumar Goswami: Conceptualization, Supervision.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.