Studies on microencapsulation of Lactobacillus acidophilus NCIM 5306 and evaluation of matrix material efficiency in pomegranate juice

The present study was conducted on the microencapsulation of Lactobacillus acidophilus NCIM 5306 probiotic lactic acid bacteria. Sodium alginate, sodium alginate + 0.4% chitosan, sodium alginate with 2% inulin, and sodium alginate with 2% inulin + 0.4% chitosan coating were used as microencapsulated matrix material. The extrusion method was used for the formation of various micro encapsulates. The probiotic pomegranate juice was formulated using the obtained beads. The freshly prepared beads were first subjected to viability assay and then inoculated in pasteurized pomegranate juice at 1% quantity. The juice was stored at 4 o C and further subjected to analysis for cell viability and sensory analysis. Response for cell viability was found discrete in various types of beads. The obtained results showed the use of 2% inulin as prebiotic in microencapsulation results in the prolonged period survival of the L. acidophilus NCIM 5306 up to six weeks.


Introduction
Probiotics are the 'live microorganisms that, when administered in adequate amounts, confer a health benefit to the host' (Hill et al., 2014) These lactic acid bacteria are characterized by their ability to ferment carbohydrates into lactic acid, a weak acid conducive to the conservation and improvement of the organoleptic quality of food. Lactic acid bacteria are Gram-positive and non-spore-forming microorganisms. The ability to convert some sugars into lactate and so, acidify the surrounding environment is the main reason for the growing interest in the application of Lactic Acid bacteria (LAB) in the food industry. The lactic acid bacteria produced as commercial starter cultures are pure cultures or a mix belonging to the genera Streptococcus, Lactobacillus, Enterococcus, Leuconostoc, Aerococcus Lactococcus, Streptococcus, Pediococcus, and Bifidobacterium (Luquet and Corrieu et al.,2005). Extensive research in light of the selection of proper probiotic strains has revealed the use of the bacteria from genera Bifidobacteria and Lactobacillus as effective probiotic strains. Bifidobacteria are commonly used in the manufacture of fermented dairy products (Ranadheera et al., 2010). Consumption of products containing probiotic Bifidobacteria has related to the numerous health benefits about lowering serum cholesterol levels, enhancing immune function, alleviating diarrhoea, reducing lactose intolerance, modulation of the gut microbiota, and prevention of allergy (Prasanna et al., 2014). However, a significant number of bacterial cells die during the processing and storage of the food products, and subsequently during passage through the gastrointestinal tract (Champagne et al., 2005) survival of probiotic bacteria in food products used as delivery vehicles is of major concern. The consumption of the required quantity (10 8 -10 9 cells per day) only may lead to confer their health benefits to the host (Champagne et al., 2008).
Microencapsulation is one of the effective methods to enhance probiotic survival in the food systems process among the available techniques (Martın et al., 2015). In this method, the live cells are entrapped within an encapsulating matrix. Microencapsulation of probiotics has been investigated for improving their viability in food products and the intestinal tract (Rao et al., 1989). Sodium alginate is a commonly used material for probiotic encapsulation. However, this material is disintegrated very easily at low pH leading to the release of microorganisms entrapped in beads into the environment (Krasaekoopt et al., 2004). Therefore, alginate is mixed with other materials to improve the stability of alginate capsules in food systems. Various eISSN: 2550-2166 © 2022 The Authors. Published by Rynnye Lyan Resources FULL PAPER polymers are reported, which can be used as encapsulation materials including alginate, pectin, kcarrageenan, xanthan gum, gellan gum, starch derivatives, cellulose acetate phthalate, casein, whey proteins, and gelatin; among these alginate is the most studied and commonly used encapsulation material. Alginate is an anionic copolymer of 1,4-linkeddmannuronic acid and l-guluronic acid residues which form a gel through the cross-linking of the guluronic acid blocks by the calcium ions, resulting in an "egg-box" structure (Gombotz and Wee, 1998). The concentration of alginate commonly used to form the gel ranges from 0.6% to 3%, whereas that of calcium chloride from 0.05 to 1.5 M (Krasaekoopt et al., 2003). Chitosan, is another coating material that can be used with sodium alginate and which has proven to increase in survival of probiotics in simulated gastric and intestinal juices compared to uncoated alginate beads. Chitosan is the partly acetylated (1-4)-2-amino-2-deoxy-D-glucan obtained from chitin (Chavarri et al., 2010;Muzzarelli et al., 2012). The negatively charged alginate forms a semipermeable membrane with the positively charged chitosan and as a result, the capsule has a smoother surface and is less permeable to water-soluble molecules (Krasaekoopt et al., 2004).
The survival of probiotics, such as Lactobacilli and Bifidobacteria, has already been studied in a variety of fruit juices during refrigerated storage. It was shown that the free cells survived well during storage for up to 6 weeks (cell decrease <1 log) in certain fruit juices with high pH (orange, apple, grapefruit, blackcurrant, pineapple) (Champagne et al., 2008;. In other juices, however, such as pomegranate, strawberry, and cranberry, the probiotic cells died very quickly, within 1-4 weeks. The reason for this was most likely the very low pH of these juices (pH ≤ 3) and the high total phenol concentration . The pomegranate is acknowledged as a rich source of bioactive compounds such as phenols, (ellagic acid, punicalagin, punicalin, and flavonoids), anthocyanin, ascorbic acid, vitamins and minerals (potassium, calcium, phosphorous, magnesium, sodium) as well the complex polysaccharides and organic acids emphasized pomegranate to be the outstanding functional food (Mirdehghan and Rahemi 2007). To develop the pomegranate juice as effective functional food under the category of probiotic food, in the present study attempt was made to develop a suitable microencapsulation system (proper coating materials) using coating material along with prebiotic as an effective microencapsulation system which could deliver through the pomegranate juice. The present investigation was carried out aiming to investigate the potential protective effect of uncoated as well as single and double chitosan, inulin and chitosan and inulin coated alginate beads on the survival of a model L. acidophilus strain in pomegranate juice, a highly acidic juice.

Preparation of the bacterial suspension
A volume of 100 µL of Lactobacillus acidophilus NCIM 5306 was inoculated in 10 mL of sterile MRS broth and incubated at 37°C for 24 hrs under aerobic conditions. The probiotic cells were harvested by centrifugation at 3000 rpm and 4°C for 5 mins. The cells were washed with sterile 0.1% peptone solution and then successively dispensed to microencapsulation (Krasaekoopt et al., 2004).

Microencapsulation using sodium alginate and inulin
For the preparation of sodium alginate beads, the extrusion technique was used (Krasaekoopt et al., 2004). The 3% sodium alginate, 2% prebiotic inulin, bacterial cell culture (approximately 10 12 CFU/mL) were kept for 30 mins hardening in 0.15 M calcium chloride solution. The cell suspension/sodium alginate and cell suspension/ sodium alginate and inulin mixture were extruded through a nozzle having a diameter of 200 microns into the 0.15 M calcium chloride. The droplets formed gel spheres. The distance between the nozzle and calcium chloride (CaCl 2 ) solution was 25 cm (Mirzaei et al., 2012). For hardening beads were allowed to stand for 30 mins then beads were harvested using Whatman filter paper no. 4. The whole procedure was carried out under aseptic conditions by using a horizontal laminar airflow cabinet (Darjani et al., 2016)

Coating with chitosan
Two-stage methods were used for the coating of sodium alginate beads with chitosan (Krasaekoopt et al., 2004). For encapsulation low molecular weight chitosan was used.0.4 g of chitosan was dissolved in 90 mL of distilled water and acidified with 0.4 mL of glacial acetic acid (0.1M) to achieve 0.4% final chitosan concentration. After dissolution, pH was adjusted to 5.5 -6.0 by adding 1 M NaOH. The solution was filtered through a Whatman filter paper no.4 and then the volume was adjusted to 100 mL. About 25 g of Alginate and alginate inulin beads were washed with the distilled water placed in 100mL of chitosan solution and kept on an orbital shaker at 100 rpm for 20 mins. The chitosan-coated beads were harvested using Whatman filter paper no. 4 and washed with sterile peptone solution (Darjani et al., 2016

Assessment of viability of free and encapsulated bacteria
Determination of cell viability of microencapsulated strains of probiotic in the juice was done by releasing the entrapped cells from the capsules. The released culture was enumerated using the MRS Agar media at 37°C for 24-48 hrs (Hruyia et al., 2018).

Determination of encapsulation yield of capsules
The encapsulation yield (EY) of the encapsulated cell with different matrices were determined as described previously using the following formula (Prasanna and Charalampopoulos, 2018). EY = (Number of cells released from microcapsules)/ (Number of cells added to the respective alginate-based microsphere formulation) × 100.

Scanning electron microscopic (SEM) analysis of microcapsules
Dehydration of capsules was carried out sequentially in a series of ethanol solutions (30, 50, 70, 80, 90, and 100%). For this purpose, capsules were soaked for 15 min in each solution. Thereafter, a critical point dryer (Balzers CPD030, Liechtenstein, Germany) with liquid carbon dioxide was used to dry capsules. Coating of samples and morphology of microcapsules were measured using a scanning electron microscope (FEI, Quanta 600 F, Thermo Scientific, Oregon USA) were carried out as described earlier (Prasanna and Charalampopoulos 2018).

Effect of probiotic microencapsulation on bioactive compounds
Total phenolic compounds were determined by Folin -Ciocalteu (FC) colourimetric method at 765 nm as described by Singleton and Rossi (1965). The results were expressed as mg of Gallic acid equivalent (GAE) per litre of the sample. Total anthocyanin contents were estimated by a pH-differential method using two buffer systems, 0.025 M potassium chloride buffer at pH 1.0 and 0.4 M sodium acetate buffer at pH 4.5 using a spectrophotometric method at 510 and 700 nm wavelengths (Wrolstad et al., 2005). The antioxidant activity was determined by performing FRAP assay reported by (Benzie and Strain 1996) with slight modifications. Free radical scavenging activity using DPPH assay was determined as like (Siddhuraju et al., 2002) and DPPH% inhibition was calculated.

Statistical analysis
Results were presented as a mean ± standard deviation of three independent determinations. Experimental data were analysed for analysis of variance (ANOVA) and differences between means were assessed by Duncan's new multiple range tests at the significance defined p≤ 0.001using SAS 9.3 software.

Effect of different coating materials on cell survival
The freshly prepared beads with different combinations of coating materials as mentioned in Table  1 are first subjected to viability assay and then inoculated in pasteurized pomegranate juice at 1% quantity. The juice was stored at 4°C and further subject to analysis for cell viability the different patterns in the cell survival were obtained with respect to different types of the bead.
The obtained values of bacterial survival are presented in Table 1.
The cell viability in the sample inoculated with live probiotic bacteria was found to be decreasing till at the end of 5 weeks, while no growth was detected at the end of six weeks. Our results are in agreement with the studies of Yoon et al. (2004), in their study, the viable cell population of L. plantarum and L. delbruekii remained at an acceptable level (10 6 CFU/mL) after 1 week of cold storage, but their microbial population decreased below the minimum accepted after 2 weeks (Yoon et al. 2005). These results are in accordance with the previous studies performed by Mousavi et al., (2013) they used L. plantarum, L. delbrueckii, L. paracasei and L. acidophilus for the fermentation of pomegranate juice. Their results demonstrated that L. plantarum and L. delbrueckii had higher viability levels during fermentation and storage time compared to the other LAB (2.8×10 5 CFU/mL and 1.5×10 5 CFU/mL after the second week of storage, respectively). The probiotic viability was obtained for up to two weeks and then cell viability decreases thereafter.
The effect of 2% inulin addition during bead formation was studied with chitosan coating and uncoating. The obtained results were very promising, no FULL PAPER reduction in bacteria numbers in both the sample was detected at the end of 3 rd week when juice was stored at 4°C. After 3 rd week till the end of six weeks, the inulin containing beads were shown the highest cell viability as compared to inulin containing chitosan-coated beads. In fact, the uncoated beads containing inulin and chitosancoated beads containing inulin was the first time studied for the survival of Lactobacillus acidophilus in pomegranate juice in the present study.

Scanning electron micrographs
The surface morphology of the microcapsules was investigated using SEM micrographs. Figure 2 shows the surface of different microcapsules at a magnification of 10000. Porous microcapsules were observed with sodium alginate Figure 2(A). Furthermore, Sodium alginate microcapsules had cracks on their surface and could not protect entrapped cells from acidity, P H and other adverse effects on cell survival. Similarly, Li et al. (2009) also noted the porous structure of alginate microcapsules in their study. Modification of alginate with the addition of 2% inulin resulted in the microcapsules with denser surface morphology Figure 2 (B). In addition, these microcapsules did not have cracks that could ensure high protection for encapsulated cells from adverse conditions. Chitosan coated microcapsules (Figure 2(C) and 2(D) showed irregular surface morphology with porous nature which could not give better protection for entrapped cells than that of alginate and inulin containing alginate beads.
Scanning electron microscopy showed that the shape of all microcapsules was generally spherical and uniform and starch granules were present on the surface of the capsules without coating (Figure 2). Chitosan coating changed the size of the alginate capsules increased with the addition of chitosan coating. The diameter of chitosan-coated microcapsules was reported significantly higher than that of uncoated microcapsules (Table 2). Obtained results are in agreement with chitosan coating with alginate beads and their findings with respect to an increase in size and viability with Nualkaekul et al. (2012). This study also reveals the addition of 2% inulin in sodium alginate also increases the size of capsules than that of normal alginate beads. The encapsulation efficiency of each type of bead was also calculated and the average size was presented in Table 2 The type of encapsulating matrices had no significant (p>0.05) effect on the encapsulation yield (

Effect of different beads on bioactive compounds of pomegranate juice
The microencapsulated beads of Lactobacillus acidophilus formed using different encapsulated and coating material was inoculated in the pasteurized juice and preserved at 4°C for six weeks. Effect on the major bioactive compounds of juices such as total phenolic content and antioxidant activity were evaluated and the obtained results were presented in Figure 3. The total phenolic content of fresh pomegranate juice was found to be high as compared to the few pomegranate varieties studied by Gözlekçi et al. (2011). In other studies, the total phenolic contents of various pomegranate cultivars showed significant differences and were 208-344 mg/100 g (Çam et al., 2009), 2015-5186 mg/L (Fischer et al., 2011). When the TPC content of free and immobilized L. acidophilus inoculated in pomegranate juice at different storage times was compared the 17.4%, 31%, 25.19%, 28.23% and 17.11% TPC content were found in juice sample of free culture, sodium alginate beads, chitosan-coated sodium alginate beads, inulin containing beads and inulin containing chitosan-coated beads respectively at the end of six weeks storage. This indicates the highest loss of TPC in the free cell samples while less TPC reduction in all immobilized cells except inulin containing chitosan beads.
When the antioxidant content of free and immobilized L. acidophilus inoculated in pomegranate juice at different storage time was compared the rise in FRAP antioxidant activity at the end of six weeks storage was found to be 36%, 37%, 43% and 39% in the juice sample of free culture, sodium alginate beads, chitosan-coated sodium alginate beads, inulin containing beads and inulin containing chitosan-coated beads respectively, which indicate high amount of antioxidant formation in pomegranate juice containing immobilized cells as compare to free cells. However, DPPH% inhibition was found to be increasing by the end of three weeks storage period while it is depleted at the end of six weeks storage. In the present study, the reduced TPC did not show a positive correlation with the enhanced antioxidant activity (FRAP and DPPH% inhibition) of microencapsulated pomegranate juice.

Effect of different coating materials on cell survival
The decrease in cell viability of inoculated probiotic culture might be due to the high acidity (pH 3.5) of the pomegranate juice . Moreover, studies by Srisukchayakula et al. (2018) on the survival of probiotics in acidic juices including pomegranate, cranberry, lemon and lime have revealed less survival in pomegranate juice as compared to lime and lemon may be because of high phenolic compounds (Gil et al., 2000). The reason could be addressed to the lack of their ability to survive in the stressful condition of low pH and high acidity of the pomegranate juice and also the relatively low temperature of the environment (4°C). As Sheehan et al. (2007) reported, low pH fruit juices, with a range of pH The prebiotic effect of inulin could be the reason for getting the survival constant for 6 weeks. A similar protective effect of inulin on probiotic viability was recorded by Haghshenas et al. (2015) when studying the effect of the addition of inulin and fenugreek on the survival of microencapsulated Enterococcus durans 39°C in alginate-psyllium polymeric blends in simulated digestive system and yoghurt. In the present study addition of inulin may improve the strength of the matrix and reduce the dissolution of capsules, consequently protecting the probiotic cells within the matrix. Better protection for bacterial survival is provided due to the more compact structure of inulin beads as shown in scanning electron micrographs Figure 2B than other types of beads (Pradeep Prasanna, and Charalampopoulos, 2019). Further addition of inulin causes the limited potential of growth-inhibiting substances passing the capsule wall, resulting during the fermentation process, which includes acids and hydrogen peroxide, as reported by Krasaekoopt and Watcharapoka (2014). Furthermore, it was observed that capsules containing prebiotics could provide the carbon and nitrogen sources for encapsulated probiotics leading to higher survival rates for Bifidobacterium sp. and Lactobacillus sp. in milk in the study of Chen et al. (2005).
Microencapsulation using sodium alginate without the coating of chitosan was already studied by so many researchers, in the present study the survival rate is more than free cell survival and chitosan coating beads but less than inulin containing beads. At the end of the 3 rd week, 4 log reductions of probiotic cells were observed in pomegranate juice and the cell viability was reported of the cell at the end of six weeks of juice preservation. This is because sodium alginate is capable of forming a highly versatile matrix, biocompatible and non-toxic for the protection of cells, as probiotic microorganisms are sensible to heat, pH, oxygen, and other processing and storage factors (Goh et al., 2012). Due to ease of handling, non-toxic nature and low cost, besides increasing the viability of probiotic bacteria when exposed to different conditions and when compared with non-encapsulated bacteria the polymer is mainly preferred for encapsulation study (Burgain et al., 2011). Similar findings were reported by Ozer et al. (2009) they have added L. acidophilus microencapsulated in 2% of alginate gel by the extrusion technique in white cheese and analysed it throughout 90 days of storage, at 4°C and obtained the counting above 10 6 CFU/mL. Sodium alginate gel presents porosity and sensibility to extreme pH, which can interfere both with the release and protection of the compounds (Mortazavian et al., 2007). Hence in order to improve the stability of microorganisms coating the particles with ionic gelling with biopolymers through electrostatic interactions (Patil et al. 2010) the addition of prebiotics in the capsule formulation (Chen et al., 2005) are the preferred way. Therefore, in the present study, the attempt was made to quote the sodium alginate beads with chitosan and also the addition of inulin in beads and its coating with chitosan were attempted to select the better combination of encapsulated matrix on survival of probiotic strain. The effect of chitosan coating on bacterial survival is also reported positive as compared to uncoated sodium alginate beads. 3 log reductions in chitosan-coated beads at the end of 3 rd week when juice is stored at 4 o C, while 4 log reductions in uncoated beads of sodium alginate under similar storage conditions were reported in the present study. This might be due to that coating alginate beads with chitosan develops a complexation of chitosan with alginate resulting in several important properties, such as alginate beads with reduced porosity, reduced linkage of encapsulated bacteria and stability at various P H ranges. Moreover, the protective effect could be due to the fact that the polysaccharides might be acting as a buffer against acids as they penetrate the bead by binding to the protons, thus raising the pH inside the matrix to a level that is less harmful to the bacteria. The development of a semipermeable membrane occurs when negatively charged alginate is in contact with positively charged chitosan resulting in a capsule with a smoother surface with reduced permeability properties (Krasaekoopt et al., 2004). Moreover, protection in simulated gastrointestinal conditions was also reported with the chitosan-coated beads (Morales and Ruiz 2016). In the study by Varankovich et al. (2017) they used extrusion technique was used for the development of novel pea protein-alginate microcapsules coated with chitosan. These microcapsules when tested for immobilization and survivability of L. rhamnosus R0011 and L. helveticus R0052 during storage and exposure to in vitro GI conditions increased cell viability was reported. Four weeks of storage at room temperature, significantly improved the microcapsule performance when compared to non-chitosan coated microcapsules. Similar findings were reported by Nualkaekul et al. (2012), who investigated the potential protective effect of uncoated as well as single and double chitosan-coated alginate beads on the survival of a model L. plantarum strain in simulated gastric solution and pomegranate juice. In their study, the enhanced protection to probiotics in the case of the single and double coated beads with chitosan compared to the uncoated beads was most likely due to the even increased ability of the polyelectrolyte matrix to buffer acidic compounds as eISSN: 2550-2166 © 2022 The Authors. Published by Rynnye Lyan Resources FULL PAPER they penetrate the beads, which was probably reflected by the thicker membrane observed in the images of their beads. Surprisingly, in the present study after 4 weeks till the end of six weeks, the results show a decrease in cell viability in chitosan-coated beads as compared to uncoated beads. This indicates the matrix material combination needs to be optimized to achieve better survival

Scanning electron micrograph
The results clearly showed that there was a very low loss of cell viability during the encapsulation which was due to the mild conditions used. In conclusion, the extrusion method of microencapsulation is when used with hydrocolloids offers higher encapsulation yield (Krasaekoopt et al., 2003). In this technique, the capsules are formed in micron range size furthermore, several reports have stated the capsules with micron range size could deliver soft texture when they are added to food products (Fahimdanesh et al., 2012).

Bioactive compounds
Different amounts of total phenolic content in various studies can be attributed to analytical methods, cultivar, maturity stage, and environmental conditions and also due to overestimation of the total individual phenolic compounds by Folin-Ciocalteu reagent due to interference with other reducing factors (Fischer et al., 2011).TPC decrease might be due to conversion of simple phenolic compounds and the depolymerization of high molecular weight phenolic compounds due to the slow metabolic activity of microencapsulated probiotics in pomegranate juice (Mousavi et al., 2013). The decrease in soluble phenolic content could also result from the polymerization of phenolic compounds. LAB has a range of enzymes such as β-glucosidase, pcoumaric acid decarboxylase, decarboxylase, which may help in degrading certain phenolic compounds (Towo et al., 2006). The obtained results are in accordance with Li et al. (2009). These results are highlighting the significance of microencapsulation in preserving the health beneficial properties of pomegranate juice. Because the slower metabolic activity of lactic acid bacteria in microencapsulated beads might have depleted the available glucose molecule in the phenolic compounds, resulting in the production of free aglycones with a higher number of hydroxyl groups or lower steric hindrance to hydroxyl groups (Cai et al., 2006). Which led to some metabolites with higher antioxidant activity being produced during the storage of pomegranate juice containing microencapsulated probiotics (Mousavi et al., 2013). The studies of Ankolekar et al. (2012) based on L. acidophilus fermentation of pear juice also concluded the decrease in total phenolic with the increase in DPPH linked antioxidant activity (Figure 3).

Conclusion
In the present research, the efficiency of different matrix materials for microencapsulation of L. acidophilus NCIM 5306 was evaluated in pomegranate juice. Among the different matrix materials used for the microencapsulation of L. acidophilus NCIM 5306, inulin containing beads showed a significant survival rate. Inulin being prebiotic for the L. acidophilus NCIM 5306 could have favoured the long-term growth of bacteria. The study also provided information on a gradual increase in bead size because of inulin incorporation in sodium alginate. Scanning electron micrographs studies revealed compact interior structural characteristics of inulin containing beads. The present investigation also underlined the pomegranate juice as a suitable medium for the development of functional food/ juice using microencapsulated L. acidophilus NCIM 5306. However no significant effect was determined on the efficiency of microencapsulation with various matrix martial beads Furthermore, bioavailability and inaccessibility of the transformed phenolic compounds, antioxidants etc. due to free and immobilized L. acidophilus NCIM 5306 cells needs to be deliberate to quote the health benefits of probiotic pomegranate juice.

Conflict of interest
The author declares no conflict of interest (2012)