Improved synbiotic formulation and its evaluation of stability during storage and simulated gastric ph - Amarender R Donthidi Probiotics Range is 1 to 1 1 cells/gram Probiotic bacteria lose viability upon storage Need large number of probiotic bacteria because high percentages are killed by acid in stomach (where ph is 1. -.) Bacteria need protection from the above conditions
How probiotics are used Yogurts Fermented milks (Kefir, Calpis, Yakult etc) Fermented soy (Natto) Fruit based drinks Dry infant foods Powders (freeze dried or microencapsulated) Tablets or capsule form Methods of encapsulation Extrusion technique Supporting material Emulsion technique Continuous phase(vegetable oils) Supporting material Spray Drying Enteric polymer coated capsule
Extrusion technique This method is the most popular due to its ease simplicity low cost and gentle formulation conditions ensuring high retention of cell viability (high entrapment efficiency) Preparation of feed suspensions Starch was gelatinised After cooling to room temperature, required concentrations of additives were added 1% freshly prepared cell suspension of Lactobacillus casei was added Table 1: Composition of air dried beads S. No. Code Conc. of sodium alginate Probiotic Conc. of starch Conc. of Lecithin 1 A % w/v sodium alginate L. casei - - A/S % w/v sodium alginate L. casei % w/v rice starch - 3 A/S/L % w/v sodium alginate L. casei % w/v rice starch 1% w/v lecithin
Preparation of air-dried beads Feed suspensions were extruded with a peristaltic pump through the solvent resist tubes (internal diameter. mm) into.1m CaCl solution. Stabilised in the CaCl solution for 3 minutes. Salt solution was drained off and the beads were washed twice with sterile deionised water Transferred into sterile Petri plates and (1) dried at C temperature overnight and () dried at 35 C for hours in air convection oven. Sealed and stored at and 5 C Studies Moisture content Storage stability Stability in 1.5pH HCl buffer (USP) Stability in % and % bile solutions
Moisture content Table 1: Moisture content in the beads (Type A) dried at C for 15 hours Composition of beads Alginate Alginate and starch Alginate, starch and lecithin Moisture content (%) 1. ±. 1.1 ±. 15.1 ±.15 Table : Moisture content in the beads (Type B) dried at 35 C for hours Composition of beads Alginate Alginate and starch Alginate, starch and lecithin Moisture content (%) 15. ±.1 1.3 ±.3 13.17 ±. Storage stability at 5 C CFU/ml (Log) y = -.357x + 7.7 y = -.53x + 7.9 y = -.591x + 7.99 Storage duration (in weeks) Alginate Alginate & starch Alginate, starch & lecithin Figure 1: Type A beads Rate of decrease in viability of L. casei in the beads with lecithin was low (significantly) compared to rate of decrease in viability in other beads CFU/ml(Log) y = -.177x + 7. y = -.19x + Storage duration (in weeks) y = -.9x +.5 Alginate Alginate & starch Alginate, starch & lecithin Li (Al i t Figure : Type B beads Rate of decrease in viability of L. casei in the beads with lecithin was low compared to rate of decrease in viability in other beads The order of storage viability was A/S/L>A/S>A
Simulated gastric conditions (ph 1.5) CFU/ml (Log) 7 5 3 Alginate beads beads lecithin beads Figure 3: Type A beads matrix offered significantly less protection to the bacteria against HCl buffer. 1 3 9 1 Time (min) CFU/ml(Log) 7 5 3 Alginate beads beads lecithin beads Figure : Type B beads Significant difference in the viability in the beads with lecithin when compared to the viability in beads without lecithin. 3 9 1 Time (min) Viability of L. casei (encapsulated in the beads) with respect to time in HCl buffer was A/S/L>A>A/S Stability in bile solutions 1 Free cells Figure 5a: Type B beads in % bile solution CFU/ml (log) Alginate beads beads lecithin beads 1 3 Time (hrs) Figure 5b: Type B beads in % bile solution CFU/ml (log) 1 Free cells Alginate beads beads lecithin beads 1 3 Time (hrs) No significant difference in the viability of L. casei in beads at different bile concentrations
Conclusions Lecithin along with gelatinised starch and alginate in the beads increased the storage stability of the probiotic bacteria in the formulations. Bacteria encapsulated in the beads with lecithin were more stable (viability was significantly different from the beads without lecithin) in simulated gastric acid solution Moisture content influenced the viability of L. casei in the beads during storage and simulated gastric studies Encapsulated cells were more stable in the bile solutions than the free cells Commercial applications These beads may be - mixed in yogurts or ice creams. - encapsulated in hard gelatine capsules for oral administration. - compressed into tablets.
Acknowledgements Prof. Richard Tester Prof. Kofi Aidoo Dr. Farage Al-Ghazzewi