Bacterial production of poly-γ-glutamic acid and evaluation of its effect on the viability of probiotic microorganisms

Abstract

Poly-γ-glutamic acid (γ-PGA) is a naturally occurring biopolymer made up of repeating units of glutamic acid and can be potentially used for multiple applications. This study compared the production of γ-PGA by eight bacteria (B. subtilis 23856, B. subtilis 23857, B. subtilis 23858 B. subtilis 23859, B. subtilis natto, B. licheniformis 1525, B. licheniformis 6816 and B. licheniformis 9945a) in GS and E media. B. subtilis natto and B. licheniformis 9945a have been investigated extensively for γ-PGA production, however, the remaining six have not previously been used. Using the eight bacteria, yields of up to 22.3 g/l were achieved in shake flasks. On characterization, it was observed that γ-PGA with different properties (crystallinity, acid/salt form and molecular weights ranging from 3,000 Da to 871,000 Da) was produced. Production of γ-PGA by B. subtilis natto in GS medium was scaled up using a fermenter and was tested for novel probiotic applications. The survival of probiotics during freeze drying, storage and ingestion was improved by combining them with a γ-PGA matrix. For L. paracasei, 10% γ-PGA protected the cells significantly better (P < 0.05) than 10% sucrose during freeze drying, whereas for B. longum and B. breve, it showed comparable cryoprotectant activity (P > 0.05) to 10% sucrose. This study also demonstrated the potential use of a non-dairy foodstuff (orange juice) for delivery of probiotics. Two Bifidobacteria strains protected with γ-PGA survived significantly better (P < 0.05) in orange juice for 39 days, with a log reduction in viability of less than 2.99 CFU/ml, when compared to unprotected cells, which showed complete loss in viability by day 20. In addition, γ-PGA protection improved survival of Bifidobacteria in a solution mimicking the environment of the stomach. γ-PGA-protected Bifidobacteria showed little (< 0.47 log CFU/ml) or no loss in viability when stored in simulated gastric juice (pH 2.0) for four hours, whereas unprotected cells died within two hours.