Hydrogel beads from sugar cane bagasse and palm kernel cake, and the viability of encapsulated Lactobacillus acidophilus

2.1 Materials

Sugar cane bagasse was collected from the night market in Taman Connaught, Cheras, in Kuala Lumpur, Malaysia, after the sugar cane juice was extracted. The sugar cane sold at the night market was cultivated in Hulu Langat, Selangor. Palm kernel cake was collected from a palm oil factory in Klang, Selangor, after crushing palm kernels to extract their oil.

2.2 Cellulose pulp

After oven-drying at 80°C for 3 h, SCB was blended and sieved with a 0.5-mm-mesh flour strainer. Moisture loss after drying was calculated. SCB (100 g) was bleached thrice in 200 ml of 0.5% acetic acid and 1% sodium chlorite in a stoppered conical flask to ensure the complete removal of lignin. In a well-ventilated hood, the mixture was heated in a shaking water bath at 80°C for 2 h. The cellulose pulp was then filtered, washed with 3 l of deionised water and oven-dried at 80°C. Cellulose yield was calculated as A/B×100%, where A is the weight of the pulp (g) and B is the weight of the dried plant material (g).

2.3 Carboxymethyl cellulose

The procedure used in the preparation of CMC from sago pulp (5, 6) was adapted. SCB and PKC cellulose pulp (20 g) was immersed in 400 ml of isopropyl alcohol and 100 ml of 30% NaOH. The mixture was allowed to stand for 1 h in a shaking water bath at 60°C to alkalise the cellulose. Etherification was initiated by adding 30 g of SMCA, and the reaction was maintained for 3 h at 60°C. The residue was filtered and suspended in 500 ml of methanol. The pH of the mixture was adjusted to pH 7 using 10% acetic acid before storage overnight. The purpose of this step was to neutralise any NaOH left in the mixture (23). The following day, the mixture was filtered, washed with methanol and the residual alcohol was then removed by oven-drying at 80°C for 3 h. CMC yield was expressed in grams per 20 g of pulp.

2.4 Degree of substitution

The DS of CMC was determined by potentiometric titration (5, 6). Prior to titration, the material had to be acid washed to ensure that its carboxyl groups were completely ion free. CMC (5 g) was suspended in 60 ml of methanol and stirred in a 250-ml beaker. Subsequently, 10 ml of nitric acid (2 mol/l) was added and the mixture was heated in a water bath until boiling. The mixture was then swirled in an orbital shaker for 15 min and allowed to cool. After the solution had settled, the suspended CMC was filtered and washed with 80% methanol at 60°C to remove all traces of nitric acid. The washed CMC was transferred to a beaker, oven-dried at 105°C for 3 h and the resultant weight obtained.

For DS analysis, 0.5 g of CMC was transferred to a 250-ml conical flask, dissolved in 100 ml of NaOH (0.1 mol/l) and brought to a boil for 15 min on a hot plate. After cooling for 30 min, the solution was titrated with 0.3 mol/l of HCl using phenolphthalein as an indicator. The colour change of phenolphthalein from dark pink to colourless indicated the end point of the titration. The CM content (%) and DS of CMC were calculated as [(V_o−V_n)×0.15×58/1000×100]/M and (162×%CM)/[100× 58–(58–1)×%CM], respectively, where V_o is the amount (ml) of HCl used to titrate blank, V_n is the amount (ml) of HCl used to titrate samples, 0.15 is the normality of HCl used, 58 is the molecular weight of the CM group, M is the amount (g) of the sample and 162 is the molecular weight of the anhydro-glucose unit (24).

2.5 CMC hydrogel beads

CMC hydrogel beads were prepared by ionotropic gelation (6). CMC solution (20% w/v) and a CL solution comprising 200 ml of 5% AlCl₃ (w/v) were prepared. The CL reaction was carried out with continuous agitation using a magnetic stirrer. The CMC solution (50 ml) was transferred drop-wise to the CL solution through a plastic syringe without the needle. The height of the syringe was fixed at 10 cm above the CL solution using a retort stand. Hydrogel beads, formed when the CMC polymer bound with the multivalent Al³⁺ cations, were allowed to settle in the solution for 15 min (Figure 1). The beads were then washed thrice with 50 ml of deionised water before freeze-drying overnight.

Figure 1:

Hydrogel beads in cross-linking solution (left), after washing (middle) and after encapsulation (right).

2.6 Cross-linking and bead size

The CL efficiency of the hydrogel beads was calculated as A/B×100%, where A is the weight of the hydrogel beads produced and B is the amount of CMC used (6). To determine the bead size, the diameters of 10 randomly selected beads were measured with a Vernier calliper under a dissecting scope.

2.7 Swelling characteristics

The swelling characteristics of the hydrogel beads at different pH levels were determined by immersing 1 g of hydrogel beads in 10 ml of simulated juice for a period of time. The experiment was conducted at room temperature in simulated gastric juice (SGJ) at pH 2 for 1.5 and 3 h, in simulated intestinal juice (SIJ) at pH 7.5 for 24 h and in phosphate buffer at pH 10 for 24 h. After hydration for 24 h, the hydrated hydrogel beads were weighed after excess surface water was blotted away with a filter paper. The percentage swelling was calculated as A/B×100%, where A is the difference in mass between the swollen hydrogel beads at time t and the dry hydrogel beads at time 0, and B is the mass of dry hydrogel beads at time 0 (6).

2.8 Simulated gastrointestinal juices

The preparation of simulated gastrointestinal juices followed earlier described procedures (25). Simulated gastric juice was prepared by mixing 3.5 ml of HCl and 1 g of NaCl in 500 ml of distilled water. Adjusted to pH 2 using a calibrated pH meter, the solution was sterilised at 121°C for 15 min and cooled to room temperature before adding 1.6 g of pepsin. Simulated intestinal juice was prepared by dissolving 3.4 g of potassium dihydrogen phosphate in 125 ml of distilled water and mixing with 95 ml of NaOH before topping up with distilled water till 500 ml. The solution was adjusted to pH 7.5, sterilised at 121°C for 15 min and cooled to room temperature before adding 3 g of bile salt. Pepsin and bile were added subsequently owing to degradation during autoclaving.

2.9 Activation of Lactobacillus acidophilus

Frozen stock culture of LA-5 (Nu-Trish^®) was purchased from Chr. Hansen (Chicago, IL, USA) and stored in the UCSI University microbiology laboratory. With the use of a sterile spatula, 0.5 g of LA-5 was introduced into a universal bottle containing 10 ml of deMan, Rogosa and Sharpe (MRS) broth, and incubated at 37°C for 24 h. The bacterial cells were centrifuged (4500 rpm) at 4°C for 15 min, harvested and suspended in sterile 0.1% peptone solution to yield 5 ml of suspension.

2.10 Cell encapsulation and viability

The extrusion technique was used for the encapsulation of LA-5 in hydrogel beads (26). The coating material and CL solution used were 20% CMC and 5% AlCl₃, respectively. The bacterial cell suspension (5 ml) was added to 50 ml of 20% sterile CMC solution and mixed thoroughly. The mixture was then transferred drop-wise to the CL solution through a pipette with a 3-mm-wide tip. No surfactants were added to reduce the surface tension of the solution. The gelling reaction was carried out with continuous agitation using a magnetic stirrer to prevent overcrowding of the beads at the drop zone. The height of the tip was fixed at 10 cm above the CL solution using a retort stand. After the beads had formed, they were left to stand in the solution for 15 min to allow them to harden. The beads were filtered and washed twice with 0.1% peptone solution. Alginate hydrogel beads, prepared with a similar technique, were also used as a coating material for encapsulation to compare with the CMC hydrogel beads.

To enumerate the encapsulated cells, freshly prepared beads (1 g) were washed twice with 0.1% peptone solution. The beads were then dissolved in 9 ml of sodium citrate and homogenised in a stomacher (Interscience, France) for 30 s. This step was crucial to break the beads and to release the entrapped LA-5 cells. The solution was then serially diluted from 10³- to 10⁸-fold using phosphate buffer solution. Each diluent of 0.1 ml was pipetted onto MRS agar, and spread plate counting was performed. To test the viability of the encapsulated cells, hydrogel beads (1 g) were incubated at 37°C in universal bottles containing 9 ml of SGJ for 1.5 and 3 h before transferring them to 9 ml of SIJ for 24 h. The beads were recovered via filtration and washed twice with 0.1% peptone solution before homogenisation in the stomacher for enumeration of the viable cells.

To enumerate the free cells after incubation in SGJ and SIJ, the cells were recovered by centrifugation, suspended in 10 ml of 0.1% peptone solution and serially diluted with phosphate buffer. To test the viability of the free cells, an overnight culture of LA-5 was prepared and the cells were centrifuged for suspension in 10 ml of SGJ for 1.5 and 3 h, and centrifuged again for re-suspension in 10 ml of SIJ for 24 h.

A plate count was carried out to determine the viability of bacterial cells on MRS agar. Agar plates with 25–250 colonies were considered for bacterial enumeration. The cell count was calculated and expressed in colony forming units (CFU) per millilitre.

2.11 Experimental design and statistical method

The experimental design of this study comprised four phases, i.e. preparation and characterisation of CMC from SCB and PKC, preparation and characterisation of CMC hydrogels, encapsulation of LA-50 in hydrogels and determination of cell viability in SGJ and SIJ. All analyses were conducted in triplicate, and the results were presented as mean±standard deviation. Data were analyzed using the Tukey honestly significant difference (HSD) one-way analysis of variance (ANOVA), with the significant difference set at p<0.05.

3.1 Sugar cane bagasse and palm kernel cake

The moisture content and cellulose yield of SCB were 52.8% and 42.8%, respectively. The values for PKC were 3.2% and 26.0%, respectively, which were significantly lower. Earlier studies on SCB reported values of 46–52% for moisture content (27) and 27–54% for cellulose content (28). The CMC yield from 20 g of cellulose pulp of SCB and PKC was 27.3 and 14.3 g, respectively. The addition of CM groups to the cellulose increased the mass of CMC as in SCB. An earlier study on CMC from SCB reported that the addition of 10% and 20% NaOH to 5.0 g of SCB cellulose resulted in 5.7 and 7.3 g of CMC, with yields of 113% and 145%, respectively (29). The increase in CMC yield with increasing NaOH concentration was attributed to the reaction of the cellulose with monochloroacetic acid under alkaline condition. The substitution of the hydroxyl groups of cellulose molecules with the CM groups led to a higher mass. For PKC, the lower cellulose content and the presence of non-cellulosic material in the pulp might have contributed to the decrease in CMC yield.

3.2 Carboxymethyl cellulose

CMC from SCB had a CM content of 12.4% and a DS of 0.40, while the CMC from PKC had a higher CM content of 19.2% and a DS of 0.70 (Table 1). Higher values of 24.7% and 0.91 were observed in the low-viscosity commercial CMC, and 32.1% and 1.31 in the high-viscosity commercial CMC. The DS of CMC produced from SCB has been reported to be 0.7 (30), and commercial CMC has a DS value in the range of 0.4–1.4 (31).

3.3 CMC hydrogel beads

From Table 2, the CL efficiency of SCB hydrogel beads (15.7%) was significantly greater than that of PKC (11.0%). As CL and DS were determined by the cellulose content of the pulp materials, those with higher cellulose content produced hydrogel beads with higher CL efficiency and DS. The bead size of SCB hydrogels (3.92 mm) was comparable to that of PKC hydrogels (3.96 mm). The bead size for both hydrogel beads has been reported to be in the range of 2–5 mm (26).

Maximum swelling of SCB hydrogel beads was observed at pH 7.5 (1970%) followed by pH 2 (524% and 561%). For PKC hydrogel beads, maximum swelling was observed at pH 2 (780% and 800%) followed by pH 7.5 (690%). In phosphate buffer at pH 10, both hydrogel beads completely disintegrated. It is likely that the disintegration of the beads was due to the excessive influx of solution into the beads, causing them to burst. Similar to the swelling characteristics of SCB hydrogels, sago hydrogel beads also displayed maximum swelling at pH 7.4, with less swelling at lower pH (6).

In aqueous solution, hydrogels swell by absorbing water and bind various ions to the polymer chains consisting of acidic or basic groups (32). To balance the charge, dissociation of the functional groups will cause an influx of counterions, resulting in higher concentration of ions in the hydrogels. The difference in osmotic pressure causes the hydrogels to swell. At low pH, the carboxylic acid groups are protonated, anion-anion repulsive forces are weaker and swelling is less (33). At high pH, the carboxylic groups are ionised and the electrostatic repulsive forces between the charged sites (COO^–) cause swelling to increase.

3.4 Cell encapsulation and viability

The free cell count of LA-5 was 7.9 log CFU/ml. There was no survival of LA-5 after 1.5 and 3 h in SGJ and after 24 h in SIJ. This indicated that LA-5 is highly susceptible to the low pH of juices in the human stomach and colon.

In the stomach, the acidic environment is deleterious to probiotics and poses a powerful barrier to their entry into the colon (34). Many articles have reported that free probiotics are easily damaged by stomach acid. The initial viable count of L. acidophilus was reduced from 9.3 to 3.8 log CFU/ml in pH 2.0 after 90 min (18) and was completely inactivated after 1 h of incubation at pH 1.2 (35). In the small intestine, bile acids can inhibit bacterial growth by their antimicrobial activity and this leads to the paucity of microbes (36). Gram-positive Bacillus and Lactobacillus are rapidly inactivated by bile (37).

After encapsulation, the viability of LA-5 was enumerated to find out how well the probiotic bacteria were entrapped in the hydrogel beads. From an initial count of 8.04 log CFU/ml, 7.03 log CFU/ml of LA-5 was entrapped (Table 3). The encapsulation efficiency of SCB hydrogel beads (87.4%) was higher than that of alginate hydrogel beads (75.0%) used as a comparison. It was also higher than that of PKC hydrogel beads (80.9%).

Encapsulated SCB and PKC hydrogel beads were exposed to two different pH regimes, i.e. 1.5 h in SGJ followed by 24 h in SIJ, and 3 h in SGJ followed by 24 h in SIJ (Figure 2). After 1.5 h in SGJ, the viability of LA-5 was 6.14 log CFU/ml (87%) for SCB hydrogel beads and 6.03 log CFU/ml (94%) for PKC hydrogel beads, whereas after 3 h in SGJ, it was 4.15 log CFU/ml (59%) and 4.97 log CFU/ml (78%) for SCB hydrogel beads and PKC hydrogel beads, respectively. In SGJ, none of the LA-5 encapsulated in alginate hydrogel beads survived. There was no survival after 1.5 and 3 h in SGJ. There was no survival of LA-5 after 24 h in SIJ for all three hydrogel beads.

Figure 2:

Hydrogel beads after 1.5 h in SGJ followed by 24 h in SIJ (left) and after 3 h in SGJ followed by 24 h in SIJ (right). SGJ, simulated gastric juice (pH 2); SIJ, simulated intestinal juice (pH 7.5).

These results indicated that encapsulation with SCB and PKC hydrogel beads was more effective than that with alginate hydrogel beads in protecting probiotic LA-5 from the low pH of the stomach. In a related study, the survival of L. acidophilus encapsulated in alginate micro-beads was 4.3 log CFU/ml after 90 min of acidic condition (18). Alginate macro-beads provided better protection, with a survival count of 5.5 log CFU/ml. Chitosan-coated alginate micro-beads afforded comparable protection as alginate macro-beads. Another study reported that Lactobacillus gasseri and Bifidobacterium bifidum encapsulated in calcium alginate hydrogel with chitosan coating were resistant to simulated gastric conditions (pH 2) and 3% bile solution after 2 h, with significantly better survival compared to free bacteria (25).

In this study, no survival of LA-5 was observed after 24 h of incubation in SIJ. LA-5 did not survive the prolonged exposure to higher pH and bile salt of the SIJ because the micro-aerophilic conditions of the intestine could not be accurately replicated. As an intestinal targeted delivery vehicle, the CMC hydrogel coating was designed to degrade at high pH and release the encapsulated probiotic cells. At pH 7.5, the beads swelled and disintegrated, releasing the probiotic cells into the SIJ.