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

Date Published: July 5, 2013

Publisher: Springer

Author(s): Aditya R Bhat, Victor U Irorere, Terry Bartlett, David Hill, Gopal Kedia, Mark R Morris, Dimitris Charalampopoulos, Iza Radecka.


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.

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Over the years, extensive research has been done to determine the efficacy of probiotic foods in controlling and alleviating disorders/diseases (de Moreno de LeBlanc et al. 2007 Falagas et al. 2007 Garrait et al. 2009 Lara-Villoslada et al. 2007). Probiotic bacteria are helpful in maintaining a healthy gut and have been used for controlling several types of gastrointestinal infections (Anukam et al. 2008 Benchimol and Mack 2004 Kligler et al. 2007 Lara-Villoslada et al. 2007 Park et al. 2007 Pochapin 2000 Szymanski et al. 2006). Some lactic acid bacteria have been shown to have antitumor activity (de LeBlanc et al. 2005). Research has also shown a marked reduction in total serum cholesterol in human volunteers after ingestion of Enterococcus faecium (EF) M-74 enriched with selenium (Hlivak et al. 2005). Furthermore, strains of Lactobacillus and Bifidobacterium have been shown to cure dental disorders (Allaker and Douglas 2009). Because of these beneficial effects, probiotic microorganisms have been introduced into a variety of food and drink products for administration to humans or animals. Various strains of Lactobacillus and Bifidobacterium are used commonly as probiotic bacteria to benefit the health of the host (Benno and Mitsuoka 1992 de LeBlanc et al. 2005 De Simone et al. 1992 Kailasapathy and Rybka 1997).

The antifreeze activity of γ-PGA has been assessed previously (Mitsuiki et al. 1998 Mizuno et al. 1997 Shih et al. 2003). However, it has never been used to maintain and protect the viability of bacteria. This study is the first to assess the effect of γ-PGA on the viability of probiotic bacteria during freeze drying.

The authors declare that they have no competing interests.




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