• Bacillus subtilis natto: A non-toxic source of poly-γ-glutamic acid that could be used as a cryoprotectant for probiotic bacteria

      Bhat, AR; Irorere, VU; Bartlett, T; Hill, D; Kedia, G; Morris, MR; Charalampopoulos, D; Radecka, I; University of Wolverhampton, Wolverhampton, UK. i.radecka@wlv.ac.uk. (Springer Science and Business Media LLC, 2013-07-05)
      It is common practice to freeze dry probiotic bacteria to improve their shelf life. However, the freeze drying process itself can be detrimental to their viability. The viability of probiotics could be maintained if they are administered within a microbially produced biodegradable polymer - poly-γ-glutamic acid (γ-PGA) - matrix. Although the antifreeze activity of γ-PGA is well known, it has not been used for maintaining the viability of probiotic bacteria during freeze drying. The aim of this study was to test the effect of γ-PGA (produced by B. subtilis natto ATCC 15245) on the viability of probiotic bacteria during freeze drying and to test the toxigenic potential of B. subtilis natto. 10% γ-PGA was found to protect Lactobacillus paracasei significantly better than 10% sucrose, whereas it showed comparable cryoprotectant activity to sucrose when it was used to protect Bifidobacterium breve and Bifidobacterium longum. Although γ-PGA is known to be non-toxic, it is crucial to ascertain the toxigenic potential of its source, B. subtilis natto. Presence of six genes that are known to encode for toxins were investigated: three component hemolysin (hbl D/A), three component non-haemolytic enterotoxin (nheB), B. cereus enterotoxin T (bceT), enterotoxin FM (entFM), sphingomyelinase (sph) and phosphatidylcholine-specific phospholipase (piplc). From our investigations, none of these six genes were present in B. subtilis natto. Moreover, haemolytic and lecithinase activities were found to be absent. Our work contributes a biodegradable polymer from a non-toxic source for the cryoprotection of probiotic bacteria, thus improving their survival during the manufacturing process. © 2013 Bel-Rhlid et al.
    • Bacterial production of poly-γ-glutamic acid and evaluation of its effect on the viability of probiotic microorganisms

      Radecka, Iza Dr; Bhat, Aditya (University of Wolverhampton, 2012-04)
      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.
    • Synthesis of hydrogels made of poly-γ-glutamic acid (γ-PGA) for potential applications as probiotic-delivery vehicles

      Kwiecien, Iwona; Niewolik, Daria; Gupta, Abhishek; Itohowo Ekere, Anabel; Radecka, Izabela (MDPI, 2020-04-17)
      Numerous hydrogels made of poly-γ-glutamic acid (γ-PGA) and various cross-linkers have been explored, but only limited data on hydrogels made of γ-PGA and poly(ethylene glycol) (PEG) are available. In this study, γ-PGA, a biodegradable and edible biopolyamide, was successfully cross-linked with selected PEGs to obtain a series of hydrogels. The swelling behaviour of these hydrogels was investigated under various pH conditions. It was also found that the structure of the cross-linker (linear or branched) affected the hydrogels’ swelling behaviour. In addition, in disc diffusion assay, hydrogel discs loaded with antibiotic were tested against Staphylococcus aureus and Escherichia coli. Prolonged activity of hydrogels loaded with antibiotics in comparison to paper discs containing antibiotics was observed. Moreover, the protective effect of hydrogels on entrapped probiotic cells subjected to low pH was investigated. The hydrogel swelling ratio and amount influenced the survival rate of the protected bacteria. Considering potential biomedical applications of hydrogels, cytotoxicity was evaluated towards two cell lines, MSTO and PANC 1.