Survival and storage stability of encapsulated probiotic under simulated digestion conditions and on dried apple snacks

Abstract The objective of the current study was to explore the probiotics carrier potential of apple dried snacks and improve the survival of probiotics under simulated gastrointestinal conditions. Purposely, the probiotics were encapsulated using two hydrogel materials (sodium alginate and carrageenan) by using encapsulator. Briefly, slices of apple were immersed in solution containing free and encapsulated probiotics and then dried by conventional drying method. The dried apple snack was analyzed for different characteristics (physiochemical and microbiological) during storage. The viability of the free and encapsulated probiotics was accessed in apple snack and under simulated gastrointestinal conditions. Apple snack rich with encapsulated probiotics showed a significant result (p < .05) regarding the survival and stability. The encapsulated probiotics decreased from 9.5 log CFU/g to 8.83 log CFU/g as compared to free probiotics that decreased to 5.28 log CFU/g. Furthermore, encapsulated probiotics exhibited a better stability under simulated gastrointestinal conditions as compared to free. During storage, an increase in phenolic content and hardness was observed while decrease in pH was noted. Results of sensory parameters indicated apple snack as potential and acceptable probiotics carrier.


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AFZAAL et AL. report by WHO, it has been recommended that fruits and vegetables other than starchy tubers and potatoes help in retaining a healthy life style and prevent a number of diseases like diabetes, cancer, heart problems, and obesity. The consumption of fruits and vegetables also helps to meet certain deficiencies of nutrients in the body as well (WHO, 2004).
Along with the fresh fruits, they can be taken in frozen, juice, canned, or dried form as well (Keast & Jones, 2011). Among fruits, apples are considered as a healthy diet owing to good nutritional composition with high fiber and polyphenolic contents. The presence of high phenolic contents is helpful in prevention of different chronic diseases. These phenolic contents have potential anticarcinogenic and anti-inflammatory activities (Boyer & Liu, 2004;Gardner, White, McPhail, & Duthie, 2000;Lee, Kim, Kim, Lee, & Lee, 2003;Sun, Melton, O'Connor, Kilmartin, & Smith, 2008). Drying being the oldest food preservation method has an extensive use over centuries.
Drying not only prolongs the shelf life of fruits and vegetables, it also lowers the water activity and prevent microbial or enzymatic activity and also increases their nutritional properties, total energy, nutrient density, fiber content, and greater antioxidant activity due to removal of extra water (Doymaz, Karasu, & Baslar, 2016).
Dried food consumption has also been linked with better nutrient consumptions, higher overall diet quality scores, and lesser body weight/adiposity measures (Keast et al., 2011). An important way of consuming healthy foods is to include probiotics in the diet.
Probiotics are defined as living microorganisms that are beneficial for human body when consumed at sufficient levels (FAO, 2006;Hill et al., 2014). Consistent intake of viable probiotics can confer countless health benefits such as a antidiabetic properties (Devalaraja, Jain, & Yadav, 2011), improvement of lactose tolerance (Shah, 2007), and reduction of cholesterol (Nguyen, Kang, & Lee, 2007). Numerous efforts have been made for manufacture of probiotic enriched dried fruits and fermented probiotic fruit juices (Rein et al., 2013).
To achieve the most beneficial effects at target sites, probiotics reflect resistance toward hostile conditions in gastrointestinal tract (Rokka & Rantama, 2010). Protection of probiotics by microencapsulation improves their viability under adverse conditions in gastric conditions. Microencapsulation improves the resistance and survival of probiotics under various food processing conditions and can raise the probiotics level in human digestive system. Among other potential ingredients, alginate biopolymer is the most studied and examined material for encapsulation of probiotics (Zanjani, Ehsani, Tarzi, & Sharifan, 2018). Sodium alginate as a natural biopolymer has extensive application in encapsulation purpose owing to different attributes including economical, on-toxic, simplicity, biocompatibility, and biodegradability. Sodium alginate polymer has porous protective structure that help in diffusion (Chandy, Mooradian, & Rao, 1998;Devi & Kakati, 2013;Islan, De Verti, Marchetti, & Castro, 2012).
The core objective of the present study was to prepare dried apple-based innovative probiotic enriched snack with the addition of Bifidobacterium bifidum. The prepared product was analyzed for microbiological, physicochemical, and sensorial attributes over a period of 25 days of storage.

| MATERIAL S AND ME THODS
Fresh apples (Malus domestica) of good quality (Kala kulu) were purchased from Faisalabad Market. Apples were selected on the basis of their size, color, and absence of any physical harm. Probiotic Culture (Bifidobacterium bifidum) was obtained from NIFSAT, University of Agriculture Faisalabad. All general chemicals (Sigma-Aldrich GmbH, Sternheim, Germany) were purchased from scientific store. The research was carried out at Food Safety and Biotechnology laboratory, Government College University, Faisalabad. Sodium alginate having ratio of mannuronic (M) to guluronic (G) units (M/G = 65/35) and molar weight (MW = 90-180 KDa) was used in this study.

| Culture activation
Pure freeze-dried cells were activated by inoculating it in MRS (Man Rogosa Sharpe) broth for 24 hr at 37°C. Afterward, the cells were centrifuged in a centrifuge machine (750073276 EA, Thermo Fisher Scientific Inc. USA). The obtained probiotic cells were encapsulated as described below.

| Encapsulation process
The probiotic bacteria B. bifidum was encapsulated with k-carrageenan and sodium alginate microgels by following the method as

| Encapsulation yield
The encapsulation yield was calculated by using the method of Iqbal, Zahoor, Huma, Jamil, and Ünlü (2019). For this purpose, 20 microbeads were selected randomly from both type of encapsulated formulations. The selected beads were disintegrated using a stomacher bag containing a phosphate buffer solution and a solution of sodium citrate having a molarity of 0.1 M at pH of 6.3 by using a stomacher bag. The number of released cells from each type (-Sodium Alginate and kappa-carrageenan) was determined by using pour plate technique. The yield was calculated by using the following formula:

| Sample preparation
Apples were first washed with tap water, and all the dirt was removed by scrubbing it gently. After washing, the peel was removed with the help of a peeler and apples were cut in to disk-shaped slices (having diameter 15 mm and thickness 6 mm). For preventing apple slices from enzymatic browning throughout storage and drying, the slices of apple were heated in water bath at 80°C for 2-3 min.

| Probiotic Inoculation in apple slices
As B. bifidum was used as a probiotic which was inoculated in apple slices by following the method of Akman, Uysal, Ozkaya, Tornuk, and Durak (2019), with a slight modification. Inoculum in the range of 9-10 log CFU/g was added in peptone water (∼1%), and slices of apples were dipped in a solution (1:3 apple/liquid ratio w/v) for 8 min at an ambient temperature with moderate stirring. Afterward, the apple samples were kept in controlled environment for 15-20 min at ambient temperature to enable the probiotics attachment on apple slices. Control treatment was dipped in disinfected peptone water to avoid it from adverse environment conditions, without adding bacterial culture. Treatment plan for this research work is shown below (Table 1).

| Drying of apple slices
Apple slices were dried using conventional method of drying. Drying was done by using an oven dryer at 40-50°C until the moisture content left less than ≤12%. Normally, the drying interval was about 6h, respectively. After this drying process, the apple slices were packed in a closed polyethylene cups that were stored at 4°C for 25 days. All prepared snacks were evaluated after an interval of 5 days.

| Enumeration of B. bifidum
For enumeration of B. bifidum, 10 g slices of apple were assimilated with 90 ml of sterilized peptone water and standardized with a help a stomacher at an average speed for 2 min. serial dilution of ten-fold were prepared from the normalized samples with peptone water. Dilutions were inoculated in suitable form onto MRS Agar (Merck, Germany), and petri dishes were incubated for 48 h at 37°C.

| Determination of physicochemical properties
pH of the apple slices was determined by following AOAC (2006) method. pH was measured by a digital pH meter, and readings were noted as a mean of three replicates. The snack from each treatment was added in distilled water, and the pH was determined.
The value for dried apple slice hardness was determined as stress at the peak force. The stress at the peak force is associated with the hardness of the dried slices of apple. The experiments were performed by a texture analyzer. Experiments were performed in three replications, and the mean value was noted in grams (Albertos et al., 2016).

| Determination of phenolic components
The phenolic contents of all treatments were determined to investigate the impact of free and encapsulated probiotics enriched apple snacks. All samples were minced by using mixer to determine the total phenolic contents (TPC) in all dried apple snacks. Briefly, 2 g apple slices were combined with 35 ml of 85% methanol (v/v) and stirred at a temperature 37°C with the help of a normal mechanic stirrer at 160 rpm per minute. The extracts of methanol compounds were acquired by the filtration process. Now for extraction of phenolic content of dried apple snacks, the method of Akman et al. (2019) was followed with some modifications. Shortly, 1 ml of the prepared methanolic extract was homogenized with 3 ml of 0.2 N Folin-Ciocalteu's phenol reagent, which was then incubated for 5 min, then 2 ml of 6% Na 2 CO 3 was poured in it. The obtained solution was kept at for 30 min in dim light at an ambient temperature.
Afterward, the samples were measured for absorbance at 760 nm using a spectrophotometer (Shimadzu, U-1510, Japan) and numerical

| In vitro gastrointestinal assay
In vitro studies were carried out to investigate the survival of free and encapsulated probiotic bacteria in simulated gastrointestinal conditions. Purposely, simulated solutions were prepared by using analytical grade chemicals and aseptic conditions.

| Survival of free and encapsulated probiotic bacteria [Lactobacillus acidophilus] in simulated gastric juice [SGJ]
Free and encapsulated beads of sodium alginate and carrageenan were subjected to simulated gastric fluid/juice. The tolerance of free and encapsulated probiotic bacteria was determined by following method of Ahmed, Mudgil, and Maqsood (2019)  The survivability of probiotic in free and encapsulated was recorded with a time interval of 0, 30, 60, 90, and 120 min. The results were recorded in triplicate.

| Survival of free and encapsulated probiotic bacteria in simulated intestinal juice [SIJ]
The survival of probiotics is also important in the intestinal conditions after their passage through the stomach. The survivability of free and encapsulated probiotic bacteria was accessed by the method as de-

| Sensory evaluation
Evaluation of sensory parameters was achieved using nine pointhedonic scale with 15 inexpert panelists including 5 females and 10 males between 20 and 30 years of age. The volunteers examined the appearance, texture, flavor, taste, and general perception of the treatments by scoring from 1 to 9 (9 points, extremely good and 1 being extremely poor). All snacks samples (coded) were randomly presented to the panel for evaluation.

| Statistical analysis
Standard deviations and mean results of the values were obtained using SPSS software. Statistical examination was done by software Statix-8 with analysis of variance (ANOVA). Difference between the data was estimated by means of LSD and multiple comparison tests at a significance 95% level.

| Encapsulation yield
Constituents of wall medium have a direct effect on the yield of encapsulation. The encapsulation yields also affect the survival in simulated digestive conditions (Bora, Li, Zhu, & Du, 2018

| Measurement of hardness of dried apple snack
Hardness and permeable configuration are frequently used for the degree of crispness. Among the quality features of snack food, texture is an important factor. For snack, crispness is a well-known important parameter. The hardness of the ASWSA (dried apple snack encapsulated with sodium alginate) was significantly higher (p < .05) as compared to all other samples which was shown in Figure 2, possibly because little damage was induced in apple, resulting in an integrated but very thin cell wall. This thin cell wall had a poor capability to repel external factors, leading to reduced hardness (Jiang et al., 2017). Figure 3 shows the phenolic content of dried apple snack treat-

| Probiotic viability in dried apple snack
In the present study, dried apple slice snack was prepared by the addition B. bifidum dried by conventional drying method by using dying oven. As apples were kept in water bath with the intention of prevention of enzymatic browning, some microflora was deactivated due to heat. Figure 4 showed the initial microbial content of the probiotics was approximately 9.5 log CFU/g. The survival of probiotics is very important when subjected to different processing conditions.

| Survival of the encapsulated probiotic bacteria in simulated Intestinal Conditions
A rapid reduction in free cells was observed in as compared to the encapsulated probiotics at simulated intestinal fluid pH 7.5 ( Figure 6).
The encapsulation of the cells with either sodium alginate or carrageenan had statistically significant effect (p < .05) on cell survival.
The sodium alginate encapsulated beads showed a log reduction

| Sensory parameters
Results from the sensory evaluation are shown in Figure 7. The re- attributes of the snacks negatively of the results of the sensory evolution also indicated that the development of apple snack containing probiotic was liked by the sensory panelist and it should be considered as value addition for the food enterprise sector.

| CON CLUS IONS
The development and production of dried apple snacks containing the probiotic microorganism using convectional drying method were successful. Probiotics were added in encapsulated and free form.
When stored for 25 days at 4°C, snack with encapsulated bacteria showed more viability than that of free form which indicates the use of dried apple snack as an effective probiotic carrier. The findings of the study indicate that microencapsulation has a key role in improving the viability and stability of probiotics in stressed processing as well as in vitro digestion conditions. Microencapsulation ensured the endorsed level of probiotics under detrimental conditions for health benefits. The results of the study also indicated that the development of snack containing probiotic is an attractive approach to lift human health.

ACK N OWLED G EM ENTS
The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through research group no. RG-1441-405.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.