Probiotic with gluten reduction property and its encapsulation in synbiotic aloe vera gel-alginate capsules with banana powder as prebiotic

Abstract

This study aims to hydrolyze the immunogenic gluten peptides by probiotic bacteria, Lactococcus lactis G01. It was isolated from curd and isolation was done based on ability to hydrolyze gluten. It was also tested for probiotic properties such as survival in gastric juice, bile salts, acid resistance, antibiotic sensitivity, antioxidant potential, sodium chloride tolerance, and antimicrobial activity. Lactococcus lactis G01 exhibited potential probiotic properties also, hence it was selected for microencapsulation. Probiotic was encapsulated in sodium alginate beads using banana powder as prebiotic and aloe vera as the adsorbent. The bead morphology was studied using scanning electron microscopy and transmission electron microscopy. The chemical composition of the bead was confirmed by FTIR. It was observed that 99% of the encapsulated probiotic cells were released into the simulated intestinal fluid in 90 min. Storage study was conducted for encapsulated probiotic and after four weeks of storage, the probiotic count in microcapsules was 7.82 log₁₀ CFU/g. The formulated synbiotic capsules are suggested to incorporate in porridge for celiac patients since the probiotic has gluten reduction property.

Graphical abstract

Introduction

Celiac disease (CD) results from the triggering of the immune system because of gluten content in wheat (Nilsen et al. 1998). According to the University of Chicago Medicine celiac center, it was estimated that about three million people in western countries are affected by this disease, among which women and children are most prone to this disease. The celiac disease foundation was established in 1990 by Elaine Monarch to improve the quality of life of people who are allergic to gluten through research and advocacy initiatives. In their 2020 reports, they said that there is a steady increase in Celiac disease incidence. The diagnosis at the initial stage is essential to reduce its effect on the body. Beyond 20, the chance of developing the autoimmune condition would be about 34%. Classic symptoms of CD include gastrointestinal problems such as anemia, loss of appetite, diarrhea, malabsorption, and failure to grow generally among small children. The damage to villi, a part of the small intestine, is of great concern as this would decrease the number of nutrients absorbed into the body (Cenit et al. 2015).

Celiac disease leads to long-term effects and complications in human health. The complications include type one diabetes, multiple sclerosis, dermatitis, osteoporosis, lactose intolerance, and infertility. In addition, it can cause harmful effects on the body, leading to flattened mucosa and the development of an imperfect absorption of nutrients, and issues related to deficiencies discussed in (Slot et al. 2016). There are strategies listed in the literature to minimize Celiac disease wherein the wheat flour fermented with Lactobacilli and fungal proteases reduce the concentration of gluten (Greco et al. 2011). The lactic acid bacteria, such as Lactobacillus and Bifidobacterium, are probiotics because they prevent mucosa inflammation. The excellent origin for probiotic organisms is fermented dairy products, including curd and yogurt. Probiotics are living microorganisms ingested in adequate amounts, as required to maintain a healthy body. The lactic cultures are considered "Generally regarded as safe" according to the guidelines proposed by FAO/ WHO (2002).

Encapsulation is the technique that will protect the probiotic organism from adverse conditions in the gastrointestinal tract of the body, such as acidic nature and the presence of oxygen. One of the encapsulation methods for incorporating probiotic bacteria is using the sodium alginate matrix (Lopez et al. 2017). In the present study, the sodium alginate aloe vera gel was used to enhance the encapsulation efficiency. The banana powder was used as a prebiotic for the probiotic organism, encapsulated, and included in the gluten-free diet. Aloe vera gel is used to treat gastrointestinal diseases, and Aloe vera alleviates ulcerative colitis by improving colon mucous barrier function (Shi et al. 2021). It is also used as an adsorbent for the microorganism. In the present study, the isolated probiotic with gluten peptide breakdown property was encapsulated in banana powder aloe vera sodium alginate beads to incorporate into the gluten-free diet.

The main objective of this work was to isolate the organism from curd and then grow it in a new gluten media to find out bacterial isolate with gluten utilization properties. Acidity resistance to the bile salts and the survival rate in gastric juice are the properties that will determine the competence of the probiotic organism getting adjusted to the gastrointestinal tract of the body (Shokryazdan et al. 2014). Hence, those properties of this organism were also evaluated. The study also includes the encapsulation of probiotics to protect from adverse gastric conditions and its survivability study during storage.

Materials and methods

Isolation of organism using gluten media

The curd was chosen as the source for isolating the probiotic organism. Curd (1 g) was added to sterile saline water (9 mL) and vortexed in a test tube, then serially diluted under the aseptic condition and then inoculated into gluten broth.

The gluten broth was prepared with 2%gluten as the nitrogen source,0.5%yeast extract, 2.5%glucose, 0.6% sodium acetate trihydrate, 0.2% polysorbate 80, 0.02% magnesium sulphate heptahydrate, and 0.005% magnesium sulphate. Then the pH of the broth was adjusted to 6.5 and filter sterilized. After that, 2% of melted agar was added to prepare agar plates. The spread plate technique was employed for the purification of isolated bacteria in gluten broth. Bacterial isolate grown in gluten broth (1 mL culture) was serially diluted and inoculated to agar plates with gluten media and incubated for 24 h at 37 °C with anaerobic conditions provided. After 24 h of incubation time, the isolated colonies were taken and used for further studies.

Estimation of the gluten utilization by probiotic isolate

Purified bacterial cultures were inoculated into gluten broth and incubated, and at regular intervals, one mL of culture was withdrawn and centrifuged at 10,000 rpm for 15 min at 4 °C. To the cell-free supernatant of culture (0.5 ml) obtained after centrifugation, Lowry’s C reagents were added, and total protein content was found out as described by Waterborg et al. (2009). The concentration change was noted down for seven days, which provided the gluten consumption by the microorganism.

Identification of the potential probiotic

The bacterial strains were cultured in the gluten media they were observed using a Fluorescence microscope (SP2, Germany). The principal component analysis applied to the fluorescence spectra showed that Fluorescence microscopy could identify the bacterial species with sensitivity and specificity greater than 90% by the differences that occur within the spectra with excitation of 410 nm and 430 nm reported (Krithika and Preetha 2019). As described by Rashmi and Gayathri (2017), PCR amplification of 16S rDNA was done for molecular characterization of a selected isolate of probiotic. The comparison of 16S rDNA sequences was made using the GenBank database available at NCBI (National Center for Biotechnology Information databases) using the BLASTN program.

Probiotic properties of the isolated organism

Resistance to gastric acidity and bile salts

The bacterial (probiotic) biomass at the log phase was collected after centrifugation (5000 rpm for 15 min), and it was investigated to know their ability to withstand gastric acidity (Ammor and Mayo 2007). Biomass of probiotic was suspended in 10 ml of MRS broth at three different pH (pH 1, pH 3, and pH 5), then pH was adjusted with hydrochloric acid (0.1 N). At regular intervals (after each hour, up to four hours), probiotic suspensions from the flask were serially diluted and plated onto MRS agar plates by the spread plate method. The plates were kept in the incubator for 24 h at 37 °C. The total viable cells were counted after 24 h.

The viable plate count method was used to determine the potential of probiotic isolates to withstand the bile salt condition, as reported by Azat et al. (2016). Bacterial isolates were cultured at 37 °C for overnight, and the bacterial cells were collected after centrifugation (10 min, 10,000 rpm, 4 °C), bacterial biomass obtained was rinsed two times with PBS with pH 7.2. The bacterial cells were then resuspended in PBS solution with 0.3% bile salts. At regular interval (0, 1, 2, 3, and 4 h) from the above bacterial suspension the bacterial cells were enumerated on MRS agar and incubated at 37 °C. Then, the survival rate was evaluated according to Azat et al. (2016) using the following equation:

$$\text{Survival Rate}=\left(\frac{{\text{n}}_{\text{f}}}{{\text{n}}_{\text{i}}}\right)\times 100\%$$

Where n_f and n_i are the total viable count of the strains used in the study after treatment and before treatment respectively (Azat et al. 2016).

Tolerance to simulated gastric juice

Probiotic cultures with an initial concentration of 10⁵ CFU/ml were centrifuged at a speed of 5000 rpm at 4 °C for 15 min. The pellets (biomass) were washed in phosphate buffer. Cell biomass was resuspended in sterile simulated gastric juice (3.5 g of pepsin,2 g of NaCl,7 ml of HCL made up to 1000 ml of distilled water) of pH 2.5. 1 ml of this solution was then serially diluted at different concentrations and then was plated onto MRS medium. The same procedure was carried out at regular intervals (after each hour up to four hours). The plates after inoculation were incubated for 24 h at 37 °C. The colonies were enumerated based on colony-forming units per milliliter. (CFU/ml) as explained by Azat et al. (2016).

NaCl tolerance test at different concentrations

Different concentrations (0.1 g, 0.2 g, and 0.3 g of NaCl) of sterile sodium chloride solution were prepared and then inoculated with washed biomass collected by centrifugation of 1 ml overnight bacterial culture. At regular intervals, the probiotic suspension was plated onto MRS medium and was kept for incubation temperature of 37 °C. Then the number of colonies was found out.

Antioxidant activity

The cells were lysed and centrifuged following the procedure reported by Lin and Yen (1999). The supernatant obtained after the centrifugation (10,000 rpm, 15 min, 4 °C) was used to find out free radicle scavenging activity of extracellular metabolites of probiotic.

The DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity was calculated by the modified protocol (Kao and Chen 2006). The reaction mixture was prepared by adding 1 ml of cell-free supernatant and 3 ml DPPH (40 µM), then it was incubated in the darkroom for 30 min at 37 °C. The triplicate absorbance value was measured spectrophotometrically at 517 nm. The DPPH scavenging activity was calculated using the following equation.

$$\text{DPPH scavenging activity}=\frac{\left({\text{A}}_{\text{b}}-{\text{A}}_{\text{s}}\right)}{{\text{A}}_{\text{b}}}\times 100$$

Where A_b denotes absorbance reading of blank (methanol solution of DPPH) and A_s denotes absorbance reading of sample (Jancy et al. 2020; Namratha et al. 2020).

Antibiotic susceptibility test

The sensitivity test for the isolated probiotic bacteria towards the antibiotic was estimated by the disc diffusion method. Erythromycin, Chloramphenicol, Ciprofloxacin, and Tetracycline (Siscon Research Laboratories Pvt. Ltd., Chennai, India) were used in the antibiotic susceptibility test. First, 100 µl of culture was inoculated onto the Muller Hinton agar plates by spread plating. After drying, the antibiotic discs were laid on the Muller Hinton agar plates. These plates were kept for 30 min for the diffusion of the antibiotic; then the plates were incubated at 37 °C for 24 h. The sensitivity of the organism towards the antibiotic was calculated by noting down the diameter of inhibition, and the inhibition zones diameter was measured using a scale (expressed in mm) as described earlier (Shruthy and Preetha 2019).

Antimicrobial activity

The antimicrobial activity of probiotics against Escherichia coli and Salmonella typhimurium was tested. The probiotic microorganisms were grown in the MRS broth, then centrifuged at 8000 rpm/min for 5 min and incubated for 24 h at 37 °C. Culture supernatant obtained after centrifugation was filter sterilized. Sterile Whattmann filter paper discs were placed on Muller Hinton agar plates seeded with pathogens, Escherichia coli, and Salmonella typhimurium, and filter paper discs were then loaded with 100 µl of cell-free supernatant of probiotic. Plates were incubated for 24 h at 37 °C and then the inhibition zones were measured in millimeters (Preetha et al. 2007; Shruthy and Preetha 2019).

Statistical analysis

All the information was written down as mean ± standard deviation, and ANOVA analysis was carried out using one-way analysis of variance (ANOVA) using Microsoft excel (MS-Office 2010). The differences were viewed as significant at the level of p < 0.05.

Encapsulation of probiotic bacteria

Encapsulation of probiotic

For the encapsulation of the probiotic organism, 10 ml of probiotic grown in gluten broth was centrifuged at 5000 rpm for 5 min. The pellets obtained were further washed in saline solution. Then, the aloe vera gel was extracted.0.6 g of sodium alginate was added to 20 ml of water and mixed it very well. After that, 4 g of aloe vera gel and 0.2 g of banana powder was added to this solution. The banana powder was used as a prebiotic for the probiotic organism. This mixture was then stirred at 1000 rpm in the magnetic stirrer for 15 min. It was later expelled via syringe into calcium chloride solution of 5% concentration with slow stirring at 37 °C. Beads obtained were made to settle for some time, and then it was separated by filtration using Whattmann filter paper.

Viable cell count

For finding out viable cell count, 0.2 g of bead was dissolved in a 1.5% sodium citrate solution at regular intervals, which was alleviated serially using aseptic saline water. Then 100 µL of the above samples was poured over the MRS agar, and plates were kept for incubation at 37 °C for 24 h. Then, the rate of encapsulation was determined using the following formula.

$$\text{Encapsulation efficiency}=\text{N}/{\text{N}}_0\times 100.$$

N is the formed number of colonies from prepared beads, and N₀ is the number of colonies formed from bacterial suspension.

Characterization of beads

Size and morphology of beads

Size measurements of the beads were done by OLM Vision Measuring system (OLM 3020) with a magnification of 30X-180X and field of view of 8.2 mm to 1.2 mm photographed using a color CCD camera. Randomly ten beads were taken, and their diameter was calculated. The morphology of the inner matrix of these beads was studied by using the scanning electron spectroscopy (Sultana et al. 2000). The alginate beads were dried and placed on aluminum supports coated with gold. The internal appearance of the encapsulated beads was viewed by transmission electron microscopy with a magnification of 25000x.

Confirmation of ingredients using Fourier transform infrared spectroscopy

The infrared spectra of probiotic beads were taken using the FTIR instrument (8400S, SHIMADZU, Europe). The instrument collected the data within a range of 500 cm⁻¹to 4000 cm⁻¹.

Release of encapsulated cells into simulated intestinal fluid

Padhmavathi et al. (2021) described the effective liberation of probiotic bacteria into the intestine. The beads were transferred in 10 ml of SIF (pH 6.8 50 mM KH₂PO₄) incubated at 37ºC at a predominant time interval such as 0, 30, 60, 90, 120, 150, 180 min. Then, 1 ml was taken and plated onto MRS agar. The viable counts were enumerated for both the free and encapsulated probiotic bacteria as a part of the comparison study.

Viability of encapsulated organism during storage

The beads of Lactococcus lactis G01 were kept at 8 °C. The survival rate of probiotics was estimated by measuring the viable cell counts at regular intervals up to 35 days using the standard plate count method (Krithika and Preetha 2019).

Results and discussion

Growth of bacterial culture

The bacteria isolated from curd proliferated in the new gluten media. As a result, the cell numbers increased rapidly, and cell count reached 9 Log₁₀ CFU/mL within 24 h.

Estimation of the gluten utilization by probiotic isolate

Lowry's test determined the gluten concentration reduction since gluten is a protein. A standard BSA solution was prepared, and its concentration was varied. The standard graph of absorbance vs. concentration was plotted (Fig. 1). From the standard graph (Fig. 1), the slope was calculated. The daily change in the concentration of gluten was deduced from the graph. The concentration of gluten on the first day was 1.40 mg/ml, then it decreased considerably, and later it became 0.32 mg/ml on the 7th day (Fig. 1). This result proves the isolated microorganism is utilizing gluten. Similarly, in work done by Rashmi and Gayathri (2017), eight gluten hydrolyzing bacteria were isolated from wheat sourdough and curd samples.

Fig. 1.

Decrease in the concentration of gluten on each day

Identification of microorganism and morphological study by fluorescence spectroscopy

Probiotic properties

As described by Rashmi and Gayathri (2017), the molecular characterization of a selected probiotic isolate was done by the PCR amplification of 16S rDNA. Their gene sequences were compared with available 16S rRNA gene sequences from GenBank using the BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine an approximate phylogenetic affiliation. G01 isolate showed a 96% similarity with Lactococcus lactis.

Figure 2 shows G01 cultured in the new gluten media. The findings from the figure reveal that the G01 forms a coccus-like structure when identified within spectra with an excitation of 410 nm and 430 nm. In a previous study, a microscopic fluorescence study was used to confirm the presence of live probiotic E. faecium in the inulin incorporated synbiotic nano-emulsion (Krithika and Preetha 2019).

Fig. 2.

.The Fluorescence spectroscopy images of the probiotic organism

Resistance to gastric acidity and bile salts

The data shows the survival of the isolated strain in the acidic medium. The probiotic bacteria resist varying pH ranges (pH 1, pH 3, pH 5). The growth of isolated microorganisms was more in the control medium with pH 7 (2.98 ± 0.05 log₁₀ CFU/ml), and then the initial number of cells began to increase at low pH levels. The survival rate of bacteria decreased from (2.99 ± 0.04) to (2.68 ± 0.04) log₁₀ CFU/ml after 4 h of incubation at pH 5. There was a significant difference in the survival rate of the bacteria between pH 1and pH 5. The survival of the microorganism decreases by decreasing the pH of the medium. When incubated for 4 h, at pH 1 survival rate was dropped from 2.98 ± 0.05 to 2.6 ± 0.0173 log₁₀ CFU/ml. However, there was a significant difference between neutral pH and pH 5 (p < 0.05). Thus, the survival rate of the organism in the acidic medium is high compared to neutral pH, which is evident from Table 1. Shokryazdan et al. (2014), reported that all the nine Lactobacillus strains used in their study exhibited good resistance to pH 3. However, there was variation in the tolerance level between the strains. Lactobacillus strains had significantly (p < 0.05) high acid tolerance compared to L. casei with cell viability loss of 0.34 log₁₀ unit and the reference strain L. casei Shirota with 0.37 log₁₀ unit viability loss.

Table 1.

Rate of Survival of probiotic organism (Log₁₀ CFU/ml) towards bile, different pH, Simulated Gastric Juice (SGJ) and different concentrations of NaCl for period of 4 h

	Time (Hours)
	1	2	3	4
Rate of Survival of probiotic organism (Log10 CFU/ml)
Control	5.56 ± 0.50	5.61 ± 0.00	5.75 ± 0.00	5.78 ± 0.50
With Bile	6.12 ± 0.50	5.98 ± 0.00	5.86 ± 0.00	5.78 ± 0.50
pH 7 (Control)	2.98 ± 0.05	2.69 ± 0.00	2.70 ± 0.00	2.71 ± 0.05
PH 1	2.98 ± 0.50	2.71 ± 0.075	2.65 ± 0.00	2.6 ± 0.0173
pH 3	3.00 ± 0.0 0	2.75 ± 0.05	2.68 ± 0.05	2.61 ± 0.075
pH 5	2.99 ± 0.04	2.78 ± 0.05	2.7 ± 0.05	2.68 ± 0.04
Control	6.09 ± 0.00	6.14 ± 0.06	6.18 ± 0.05	6.20 ± 0.00
With SGJ	7.34 ± 0.05	7.09 ± 0.075	6.34 ± 0.05	5.13 ± 0.00
Control	1.02 ± 0.05	1.25 ± 0.00	1.3 ± 0.00	1.4 ± 0.05
0.1 g NaCl	2.65 ± 0.05	2.48 ± 0.05	2.35 ± 0.00	2.10 ± 0.073
0.2 NaCl	2.71 ± 0.05	2.68 ± 0.05	2.50 ± 0.00	2.25 ± 0.00
`0.3 g NaCl	3.68 ± 0.04	3.50 ± 0.00	3.29 ± 0.05	3.00 ± 0.00

The selected probiotic microorganisms had potential survival capability in the medium supplemented with the bile salts. The potential to overcome 0.3% bile salt has physiological importance as it is normally encountered in the human intestine. However, several studies have reported that bile concentration is unpredictable and variable and keeps varying with respect to diet composition. It is also influenced by the secretion of enzymes from the pancreas (Shokryazdan et al. 2014). In the study by Shokryazdan et al. (2014), all the nine strains of Lactobacillus used exhibited good tolerance to 0.3% bile. It is evident from Table 1 that there is a considerable difference in growth seen when the medium is provided with bile salt by comparing it with a medium not supplemented with bile salt. A steady increase was observed in the growth of bacteria inside the control medium (without bile salt) for 4 h. There was a survival rate of 6.12 ± 0.05 log₁₀ CFU/ml initially.

Nevertheless, there was a gradual decrease in the survival rate of the bacteria during 4 h of incubation. It is noted that the survival rate of the bacteria decreased to 5.18 ± 0.05 log₁₀ CFU/ml at the end of 4 h of incubation time, proving that the bacteria isolated were tolerant towards the bile salts. In work performed by Shokryazdan et al. (2014), it was observed that the extent of tolerance towards bile changed noticeably depending upon the bacterial strains. Thus, the tolerance of bacterial strains towards bile was strain-specific and was not affected by the environment from which the bacteria was isolated.

Tolerance to simulated gastric juice

The potential of the probiotic organism to survive in gastric juice is an essential criterion to be checked. The results in Table 1 show the capability of the isolated organism to survive in the simulated gastric juice for 4 h. After 1 h, bacterial isolates exhibited an excellent survival rate of 7.34 + 0.05 log₁₀ CFU/ ml in SGJ compared to the control sample (6.09 log₁₀ CFU/ml), which shows that it can exist in the simulated gastric juice environment. At the same time, the cell count of bacterial isolates in SGJ was reduced to (5.13 ± 0) log₁₀ CFU/ml after 4 h of incubation. These results prove that Lactococcus lactis G01 can also sustain in a low pH. In a study by Padhmavathi et al. 2017, L. rhamnosus encapsulated in chitosan-coated skim milk alginate microsphere had a survival rate of 13.2 log₁₀ CFU/ ml at a pH of 2.5 when incubated in an artificial gastric fluid environment.

Growth at different NaCl concentrations

The isolates show an excellent tolerance over the NaCl concentration ranges, which were supplemented in MRS broth. The isolated bacteria showed the highest growth in the salt concentration ranging from 0.1 to 0.3 g. A significant difference was observed between broth containing 0.3 g of NaCl and without NaCl (as control), proving that the organism can thrive in salt conditions. For the broth containing the 0.1 g NaCl, the survival rate of the organism was 2.65 ± 0.05 log₁₀ CFU/ml, which decreased to 2.1 ± 0.073 log₁₀ CFU/ml. As the salt concentration was increased to 0.3 g in the broth, the growth was considerably increased (3.68 ± 0.04 log₁₀ CFU/ml). However, as the salt concentration was increased, the growth of the organism also increased due to the utilization of NaCl for its survival (Table 1). It was reported that L. sakei could grow at a salt concentration of 6.5% NaCl and pH of 4.2, and a meat-borne LAB could grow in 2% NaCl at a rate of 0.55 generations per hour (Ammor and Mayo 2007). Lavanya et al. (2021) reported that all the bile tolerant bacterial isolates from dosa, jangri, and kallapam batter used in their study survived in 3% NaCl concentration.

Antioxidant activity

The DPPH radical scavenging activities of the isolated culture filtrate were analyzed, and it was found to be about 47% compared to the synthetic antioxidant (ascorbic acid). Thus, it is clear from the experiment that the isolated microorganism has antioxidant activity. Azat et al. (2016) reported that all tested six strains showed excellent DPPH scavenging activity in their work. Among the tested six strains, the intact cells of L. rhamnosus R4 proved to be most powerful in removing DPPH radicals (53.78%) (Azat et al. 2016).

Antibiotic susceptibility and antimicrobial activity

The collected cell-free supernatant was tested safe for consumption by finding the antibiotic susceptibility. The results show the sensitivity of the isolated cultures towards selected antibiotics. The diameter of the zone of inhibition obtained for Chloramphenicol, Ampicillin, and Tetracycline were 20 ± 0.03, 16 ± 0.05, and 6 ± 0.04 mm, respectively.

The antimicrobial activity of the isolated probiotic was also found. The diameter of the zone of inhibition for E. coli and Salmonella typhi were 20 ± 0.05 and 16 ± 1.23 mm, respectively. In work done by Shokryazdan et al. (2014), L. casei exhibited a zone of inhibition of 11.7 mm for E. coli (ATCC 29,181), and L. acidophilus exhibited 11.8 mm for E. coli (ATCC 29,181). In the study conducted by Azat et al. (2016), one strain of Lactobacillus rhamnosus, one strain of Lactobacillus helveticus, and four strains of Enterococcus hirae demonstrated antimicrobial activity in opposition to E. coli (with a zone of inhibition of 6.3 to 10.5 mm) and Salmonella typhimurium (with a zone of inhibition of 2.5 to 8.5 mm). Antimicrobial activity is an important feature for probiotic microorganisms to prevent gastrointestinal diseases (Azat et al. 2016).

Viable cell count and encapsulation efficiency

The results show that the concentration of viable cells encapsulated in banana powder, aloe vera, and sodium alginate beads was 8.2 log₁₀ CFU/ml. This method of preparation obtained an encapsulation efficiency of as much as 98.18%. Pan et al. (2013), effectively made alginate skim milk microspheres containing 9.93 log₁₀CFU/g L. bulgaricus using the extrusion technique. One of the objectives of encapsulation is the effectual liberation of metabolically active viable cells in the intestine. In work done by Shi et al. (2013), the extrusion technique was used to encapsulate L. bulgaricus within alginate–milk microspheres with an encapsulation yield of around 100%.

Characterization of the beads: size, scanning electron microscopy, and transmission electron microscopy

The banana aloe vera gel sodium alginate beads prepared are given in Fig. 3a. The beads have a diameter of 4.24 ± 0.12 mm. Thus, the cross-sectional view of the bead indicates that the isolated microorganism is entrapped in it. Scanning electron microscopy images for prepared beads show gave an idea about the bead's external and internal morphology. The beads obtained appeared to be somewhat irregular in shape and not homogenous. (Fig. 3c and d). Sultana et al. (2000) made microcapsules of 0.5 to 1 mm using alginate and Hi-maize starch, the microcapsules were mostly spherical, and a few of them were elliptical. The bacteria and Hi-maize starch grains were located in the alginate matrix and within the cavities, respectively (Sultana et al. 2000).

Fig. 3.

Bead viewed under the OLM vision measuring system (a), SEM showing the external of beads (b), SEM showing internal morphology of beads (c), TEM images of the encapsulated bead (d)

In another study, beads made from 1% w/v alginate and 0.1% w/v gelatin had a size ranging from 1.53 to 1.90 mm, and the beads were spherical, white, and opaque. The bead cross-section depicted the integrity of the probiotic L. rhamnosus embedded inside the bead (Lopes et al. 2017).

The internal appearance of the beads is shown in Fig. 3c. The prepared banana aloe vera gel sodium alginate beads were observed under transmission electron spectroscopy with sizes ranging from 100 to 200 nm. The entrapment of the bacteria inside the prepared beads is shown in the TEM image of the beads (Fig. 3b).

Fourier transform infrared spectroscopy

Sodium alginate showed a band intensified at 1000 cm⁻¹ corresponding to the functional group O–H. The spectrum range between 1000 and 1500 cm⁻¹ shows C-H stretching symmetrically and asymmetrically. The peak viewed at 2150 cm⁻¹ indicates the C-O group, and then a rapid rise in the peak intensity at 1500 cm⁻¹ correlates to the symmetrical stretching of COO. Peaks other than the range above the spectrum 1000 cm⁻¹ are attributed to the vibrations caused by the C-H bond in the alginate (pyranose ring) ring (Bekhit et al. 2016). The band region between 3000 and 3500 cm⁻¹ indicates the strong stretching hydroxyl groups in the banana powder. The band at 2900 cm⁻¹ was allocated for –C–H stretching. In this region, between 1500 and 2000 cm⁻¹, the peak indicates the hemicellulose band carbonyl group stretching C = 0, and the downward peak in that region represents absorbed water. In Fig. 4, it is seen that the FTIR bands of the aloe vera gel show similar characteristics to that of banana powder. The findings from Fig. 4 suggest that the banana powder aloe vera gel sodium alginate bead showed an upward peak between the spectrum 1500 cm⁻¹ and 2000 cm⁻¹ range, indicating an interaction between the aloe vera, and banana powder, and sodium alginate used.

Fig. 4.

FTIR spectra of sodium alginate, banana powder, aloe vera, encapsulated bead

Release study of the probiotic organism in simulated intestinal fluid

The release pattern of the probiotic should be studied to know how well it will release into the intestine and determine its health benefits on human health (Chen et al. 2014). The encapsulated and free probiotic cells were tested for survival in simulated intestinal fluid with pH 6.8 (Fig. 5a). The number of encapsulated cells that got released at 30 min was about 2.5 log₁₀ CFU/ml, and then it started to get released faster. It was observed that within 90 min, 99%of of the cells got released into the simulated intestinal fluid (5.65 ± 0.05 log₁₀ CFU/ml). However, the viable count of the encapsulated probiotic microorganism began to become constant after 90 min, showing the complete release of the isolated organism into the acid environment.

Fig. 5.

Release of organism from banana aloe vera gel sodium alginate beads into SIF (a), Viable concentration of the encapsulated and free cells during storage life of 28 days (b)

Viability of encapsulated organism during storage

The viability of the encapsulated probiotic organism was found for four weeks when preserved at four degrees Celsius. The cell count of the enclosed organism in the bead was more than that of the free cells. The number of viable cells was 9 log₁₀ CFU/ml initially, then after one week, it was about 6.23 log₁₀ CFU/ml, and after four weeks, it was around 4.14 log₁₀ CFU/ml.

Meanwhile, the cell count of the encapsulated organism decreased only from 9 log₁₀ CFU/mL to 7.82 log₁₀ CFU/ml after four weeks of storage. The encapsulated probiotic organism showed an excellent survival rate for 4 weeks (Fig. 5b). The studies have revealed that the microorganisms encapsulated using the alginate-based microsphere have better survival capability than the non-encapsulated cells. Numerous studies have reported the encapsulation of bacteria in alginate-based microspheres (Pan et al. 2013; Shi et al. 2013). Kalpa et al. (2021) reported the microencapsulation of L. brevis with sodium alginate and oats and L. delbrueckii subsp. lactis with sodium alginate and oats bran, respectively, were found to be more stable in bile, simulated gastric fluid, and simulated intestinal fluid compared to free cells of L. brevis and L. delbrueckii subsp. lactis respectively.

Conclusion

In this study Lactococcus lactis G01, isolated from curd proved to be effective in degrading and decreasing gluten content. In the in vitro fermentation study, initial gluten of 1.40 mg/ml was reduced to 0.32 mg/ml on the 7th day. They also exhibited good probiotic properties such as bile and acid tolerance, NaCl tolerance, antimicrobial activity, and survivability in acidic pH. Lactococcus lactis G01 exhibited sensitivity to Chloramphenicol, Ampicillin, and Tetracyclin. Lactococcus lactis G01 was microencapsulated in beads made of banana powder aloe vera gel sodium alginate with an encapsulation efficiency of 98.18%. The characterization of these probiotic microorganisms microencapsulated in beads made of banana powder aloe vera gel sodium alginate was done. It was observed that within 90 min, 99%of of the cells got released into the simulated intestinal fluid. The microencapsulated Lactococcus lactis G01 showed better survivability with a cell count of 7.82 log₁₀ CFU/ml compared to free cells of Lactococcus lactis G01 with a cell count of 4.14 log₁₀ CFU/ml when stored at ambient conditions for four weeks. These Synbiotic capsules can be given to Celiac patients since the probiotic bacteria encapsulated in the bead could degrade the gluten when stored for 28 days.

Biological, chemical and microbiological

PBS

Phosphate buffered saline

MRS

De man, ragosa, and sharpe

CFU

Colony forming unit

NCBI

National center for biotechnology information

BLAST

Basic local alignment search tool

Instrumental techniques

SEM

Scanning electron microscope

PCR

Polymerase chain reaction

Author contributions

KA conceived, carried out the experiments, and wrote the original manuscript, VS wrote the original manuscript, edited the manuscript, did the formal analysis and statistical study, Conceptualization and supervision of the project work was done by Dr. RP and she also involved in editing of the manuscript. The first two authors have contributed equally.

Funding

No external funding for this work. Only SRM Institute of Science and Technology selective excellence funding was utilized for the project as mentioned in the acknowledgment.

Data availability

The research data is not shared.

Code availability

Not Applicable.

Declarations

Conflicts of interest

The authors declare no conflict of interest.

Ethics approval

Ethical approval is not required for this research.

Consent to participate

Not Applicable.

Consent for publication

This is an original work being done and it is not being submitted to any other journals for publication. The data provided including figures and table can be used for publishing in JFST and they are not being provided to any other journals.