Survival and behavior of free and encapsulated probiotic bacteria under simulated human gastrointestinal and technological conditions

Abstract The present study was designed with the objective to compare the viability and stability of free and encapsulated probiotics under simulated technological and human gastrointestinal conditions. L. acidophilus was encapsulated using two wall materials (sodium alginate, soy protein isolate, and SA‐SPI) by extrusion method for enhanced viability under stressed conditions. Free and encapsulated probiotics were subjected to some simulated technological and gastrointestinal conditions. Furthermore, free and encapsulated probiotics were also incorporated in dairy dessert to evaluate the viability and stability during storage. Encapsulation using sodium alginate and SPI as a coating materials significantly (p < .05) improved the survival of probiotics under simulated gastrointestinal and thermal conditions. The buffering effect of microbeads prolonged their survival and stability of under simulated conditions. The number of surviving probiotic cells encapsulated with sodium alginate, SPI, and SA‐SPI over 120 days of product storage was 7.85 ± 0.39, 7.45 ± 0.37, and 8.50 ± 0.43 cfu/ml, respectively. In case of free cells, the surviving cells were just 3.5 ± 0.18 cfu/ml over the period of storage. In short, the study depicted that encapsulation provides protection during exposure to various hostile conditions.


| INTRODUC TI ON
Probiotics are described as "live beneficial micro-organisms that, when ingested in sufficient quantities boost up host's immunity against intestinal pathogens and prevent an array of gastrointestinal disorders" (Hill et al., 2014). Various compounds like organic acids (lactic and acetic acids), produced by probiotics bacteria decrease the pH of growth medium thereby inhibiting the pathogen's growth. Lactobacilli represent a substantial part of intestinal microflora, and their relationship with the general state of human health is still under rigorous investigation phase. The genus Lactobacillus is one of the major groups of lactic acid bacteria used in food fermentation and is thus of great economic importance. (Pyar & Peh, 2014).
In a wide range of food and beverage products such as fruit juices, yoghurt and sour milk probiotics exhibit plentiful health benefits to the human such as improving intestinal microbial stability, by producing antimicrobial substances inhibiting pathogenic growth, simulating and modulating the innate immune systems, exhibiting antimutagenic activities, and preventing carcinogenesis. The genera Lactobacillus and Bifidobacterium are the most important probiotic micro-organisms commonly related with gastrointestinal tract.
Probiotics used in different products should optimally accomplish all of the following measures: remain viable during industrial production and processes; retain viable under harsh storage conditions as well as survive in host gastrointestinal environment to deliver the actual health benefits to the consumer. However, most of the probiotics incorporated in food and beverage are sensitive to processing and environmental factors including low pH and heat. Stability and viability of probiotics during processing and gastrointestinal transit (GIT) can be improved by encapsulation (Praepanitchai, Noomhorm, & Anal, 2019).
Encapsulation of probiotics controls the discharge of active molecules and improves the organism viability by resisting the unfavorable conditions like variation in pH, moisture, and oxygen availability. (Dubey, Shami, & Bhasker, 2009). The main aim of employing encapsulation on commercial basis in food applications is to enhance the probiotic stability to improve the bioavailability and functionality (Milanovic et al., 2010;Shi et al., 2013). Sodium alginate (SA), is being extensively used for the encapsulation of probiotics due to its excellent pH-responsive properties (Feng et al., 2020). Probiotics are highly sensitive to the food handling operations, digestive enzymes, pH, and mechanical strain in the stomach. Encapsulation can cowl the bitter taste of a few meals by means of inhibiting reactions with different additives, such as water and oxygen in adverse conditions (Nedovic, Kalusevic, Manojlovic, Levic, & Bugarski, 2011;Rescigno, Rotta, Valzasina, & Riccardi-Castagnoli, 2001).
Ice cream is a frozen dessert that comprises of air cells scattered in a watery framework (Muse & Hartel, 2004). The three basic principle parts of frozen ice cream are air cells, ice crystals, and fat globules, which are dispersed in a continuous aqueous phase (serum).
The uniform air distribution is a key factor in deciding the product melting resistance and mouth feel. Fermented food products have been extensively utilized as probiotic carrier, but the current study has been exclusively designed to probe the effect of encapsulation on the viability and stability of probiotics in nonfermented frozen desserts and under simulated conditions.

| Procurements
Probiotic Lactobacillus acidophilus (ATCC-4356) was obtained from NIFSAT, University of Agriculture Faisalabad Pakistan. Milk was purchased from local dairy farm. Food additives, media, wall materials (sodium alginate and soy protein isolate), ringer solution, sodium chloride, hydrochloric acid, distilled water, calcium chloride, and porcine bile extract (Sigma-Aldrich) were purchased from local scientific market.

| Culture activation
Probiotic cells were activated by inoculating them in MRS (Man Rogosa Sharpe) broth at 37°C for 24 hr. Afterward, the cells were harvested by centrifugation (Thermo Scientific Megafuge 8R) at 1,960 g for 10 min at 4°C. The obtained beads were washed twice using phosphate buffer (pH 7.0).

| Encapsulation of probiotic bacteria
Lactobacillus acidophilus was encapsulated by the method as described by (Gul & Dervisoglu, 2017). Briefly, solution of sodium alginate (2%), soy protein isolate (2%), and sodium alginate-soy protein isolate (1:1) % w/w was dissolved in distilled water. The prepared solutions were sterilized in autoclave (121°C for 15 min). After cooling, prepared solutions were mixed with culture (10 10 log cfu/ml) suspended in 0.1% sterile peptone at 9:1 (v/v) ratio. The encapsulation plan is shown in Table 1. For extrusion method, obtained mixtures were homogenized with ultra-turret at 1,960 g for 2 min and the suspensions were injected drop wise through a syringe into 0.2 M CaCl 2 solution with gentle stirring. The formed hydrogel beads were shaken at 300 rpm for 30 min in CaCl 2 for hardening. They were then filtered with sterile filter, washed twice with sterile distilled water, kept in sterile Petri dishes, and stored at 4°C.

| Measurement of bead size and morphology of beads
The shape of the capsules obtained by extrusion method was observed using an optical microscope equipped with a digital camera.
The size of capsules obtained by extrusion was measured using a digital micrometer. procedure. The EE (%) was determined by digestion method as described by Afzaal, Khan, et al. (2019), Afzaal, Saeed, Arshad, et al. (2019),  with slight modifications.

| Encapsulation efficiency
The EE (%) for the probiotic bacteria was calculated as follows: where N is the number of viable cells entrapped in capsules, and N 0 is the total number of cells added in solution.

| Survival of free and encapsulated probiotics under heat treatment
The stability and viability of Lactobacillus acidophilus-encapsulated beads were exposed to heat treatment by following the method described by Fang et al. (2012)

| Survival and stability of free and encapsulated probiotics under acidic conditions
Under acidic conditions, the survival of the Lactobacillus acidophilus probiotics encapsulated in the beads was evaluated through the protocol earlier described by Praepanitchai et al. (2019) with slight modification. The viability of encapsulated and nonencapsulated Lactobacillus acidophilus probiotics under acidic environment was evaluated at pH 6.5, 3.0, and 2.0. Lactobacillus acidophilus as free cell and encapsulated were added to test tubes containing 9 ml of MRS medium adjusted to the preferred pH with 5 M HCl or 1 M NaOH).
Incubation was done at 37ºC for 3 hr, and then centrifugation of samples at 1,960 g for 10 min at 4ºC was carried out. The viability of the free and encapsulated Lactobacillus acidophilus probiotics was assessed by standard plate count method. (2019). The simulated gastric juice (SGJ) was prepared using the regents (sodium chloride, 0.5%, KCl, 0.2%, pepsin 0.3%, NaHCO3, 0.1%, and 0.022% (w/v) CaCl 2 ) and the desired pH (2.5) was adjusted with (0.1M) HCl. Free and encapsulated cells of sodium alginate, soy protein isolate, and sodium alginate-soy protein isolate were added to the test tubes and incubated at 37°C. The viability of free and encapsulated cells was recorded at 0, 25, 50, 75, 100 min. Simulated intestinal juice was prepared with (pancreatic, NaCl, KCl, NaHCO 3 , and bile salts) and required pH was adjusted to 7.5. Similarly, the cell survival and stability in simulated intestinal conditions was enumerated.

| Product development
The ice cream was prepared by following a standard recipe containing 11% fat, 12.5% nonfat solids, 14.5% sugar, mango flavor and color, 0.4% emulsifier and stabilizers, and 0.2% starch. The treatment plan for preparation of dairy dessert is given in

| Enumeration of probiotics in ice cream during storage
The survival of entrapped and free cells counts in ice cream during storage was determined by method as described by (Gul & Dervisoglu, 2017 and then dropped into sterile stomacher bag. After homogenization for 15 min, 1 ml of homogenate sample was serially diluted with 9 ml of ringer solution and samples were plated on MRS agar. The plates were incubated under anaerobic conditions at 37°C for 48 hr. All enumerating plates of probiotics were incubated at 37°C for 3 days, and the results were recorded in colony-forming units per g (cfu/g).

| pH
The pH of the each sample of ice cream was determined using HANNA pH meter. The pH meter was calibrated using buffered solution of pH 4.00 and 7.00.

| Sensory evaluation of ice cream
A panel of twenty expert judges of Government college university Faisalabad took part in the sensory evaluation of ice cream (Meilgaard, Civille, & Carr, 2007). The panelists evaluated the ice cream samples for the attributes like color, flavor, body, texture, and overall acceptability on 9-point hedonic scale. The sensory analysis was carried out at an interval of 0, 30, 60, 90, and 120 days for all four treatments.

| Statistical analysis
All the data were directly subjected to ANOVA (analysis of variance) to observe the significant difference (p < .05) between different treatments. The results were stated as the mean values from the three replicates.

| Measurement of bead size
The polynomic polymer solution of sodium alginate and soy protein isolate in aqueous solution containing a suitable divalent counter cation like Ca 2+ has the tendency to form hydrogel beads.

| Encapsulation efficiency
The encapsulation yield or the encapsulation efficiency is affected by the type of the hydrogel materials and the method used for encap-

| Thermal stability of free and encapsulated probiotics
Probiotics must survive the recommended pasteurization temperatures to be useful and remain viable in food and beverage products. Survival of probiotic cells in free cells, encapsulated with sodium alginate, encapsulated with soy protein isolate, and encapsulated with sodium alginatesoy protein isolate hydrogel beads, was evaluated under different heat treatment at 72°C, 63°C, and 50°C and results shown in

SA-SPI hydrogel beads provided better protection on the viability of
Lactobacillus acidophilus more than free, SA-, and SPI-encapsulated cells.
The result of the study is also in line with the experiments conducted by results were promising and corroborate with those obtained by Rather, Akhter, Masoodi, Gani, and Wani (2017) who reported that encapsulation maintain higher cell count during exposure to heat treatment.

| Evaluation of survival of encapsulated probiotics in acidic solutions
The data on the survival of encapsulated probiotics in acidic solution are demonstrated in the showed viability 6.10-7.75 log cfu/ml, but the maximum viability was shown by C4 (Encapsulated with Sodium Alginate-Soy Protein Isolate) 7.75 ± 0.39 cfu/ml. The same trend was found at pH 3 and 6.5. Maximum trend was found at pH 6.5 of C4 (Encapsulated with Sodium Alginate-Soy Protein Isolate), which was 8.6 ± 0.43 cfu/ ml. At pH 6.5, the results of all four treatments were between 7.1 and 8.6 log cfu/ml because bacteria can survive better at pH 6.5, so total viable counts were observed. Soy protein isolate-sodium alginate combined seems to exert a synergetic effect on the survival of encapsulated probiotics. Ding and Shah (2009)

| Viability and stability of encapsulated probiotics in simulated gastric conditions
The viability and stability of probiotic bacteria are very important in GIT. Feasibility of probiotic cells is vital in stomach and intestinal conditions so that the desired benefits of probiotics can be achieved. The probiotic cells (nonencapsulated and encapsulated) were subjected in gastric juice results are shown in Table 7. A rapid log reduction was observed for nonencapsulated bacteria in contrast to encapsulated probiotic cells. Encapsulation of SA-SPI results better for the survival of probiotics as compared to SA and SPI as shown in Table 7. The results confirmed that encapsulation has a shielding effect toward probiotics in simulated gastric conditions. De Prisco, Maresca, Ongeng, & Mauriello, 2015 also found that in simulated gastric conditions probiotic survive better when encapsulated with different materials. Yasmin, Saeed, Pasha, and Zia (2019) found that the use of whey proteins as wall materials provided protection in various stressed conditions.

| Stability and viability of encapsulated probiotics in intestinal conditions
Wall materials that are dissimilar showed a shielding result on probiotics after they were exposed to the intestinal conditions. Current study showed probiotics in free (un-encapsulated) and encapsulated form were added in artificial simulated intestinal solution for a defined time period. A sudden drop in probiotics which were without encapsulation was observed in contrast to the encapsulated cells at pH 7.5 as shown in Table 8. The C2 (Sodium Alginate), C3 (Soy Protein Isolate), and C4 (Encapsulated with Sodium Alginate-Soy Protein Isolate)cells showed a gentle log reduction in comparison with nonencapsulated probiotics as showed in Table 8.

| Probiotic viability and stability in dessert during storage
The data on the total viable count in ice cream are demonstrated in the

| pH
The result regarding the pH of ice cream with storage is given in study are also in line with the findings of Hekmat, & McMAHON, (1992) who reported that probiotics produce fast acid in ice cream mix.

| Sensory evaluation
The consumer's response to product sensory evaluation is very important. The results regarding sensory evaluation are shown in Beads). However, ice cream with free cells observed very poor sensory evaluation of product with storage that is due to free cells produce more acidity and have poor texture. Gul et al. (2017) also found that dessert with encapsulation has exceptional sensory evaluation rather than dessert with free cells and without probiotic.

| CON CLUS ION
Encapsulating wall materials (sodium alginate, soy proteins isolate, and sodium alginate-soy proteins isolate) were found to be effective for augmenting the viability and stability of probiotics under different stressed conditions. Encapsulation with SA-SPI combination showed best results in terms of encapsulation efficiency and viability. Encapsulated probiotic bacteria showed more thermal stability compared with free cells.
Additionally, the incorporation of free and encapsulated probiotics affected the physiochemical and sensorial parameters of carrier food.

ACK N OWLED G M ENTS
The authors are thankful for the Government college University and NIFSAT for technical support.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interests.