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

Abstract

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.

Figure 1

FT-IR Spectroscopy of (a) unsterilised γ-PGA and (b) sterilised γ-PGA showing relevant peaks.

Figure 2

Effect of γ-PGA and sucrose on viability of probiotic bacteria during freeze drying. Cells were freeze dried at −40°C and 5 mbar pressure and viability was measured before and after freeze drying on BSM agar. Experiments were conducted in triplicate (n = 3).

Figure 3

SEM image of a) Freeze dried B. longum cells with no γ-PGA protection (EHT = 20.00 kV; Signal A = SE1; WD = 4.0 mm) b) Freeze dried B. longum protected with γ-PGA (EHT = 20.00 kV; Signal A = SE1; WD = 4.5 mm). SEM analysis was performed using Zeiss EVO50, U.K. and photographs were analysed using the software provided by Zeiss EVO50.

Figure 4

PCR patterns of B. subtilis and B. cereus for screening of genes coding for toxins. A - hbl- D/A, B – nheB, C – bceT, D – entFM, E – sph, F – piplc, G – pfkA, W = Water, S = B. subtilis, C = B. cereus.

Figure 5

Physiological analysis of the haemolytic and lecithinase activities of B. subtilis natto and B. cereus: a – B. cereus haemolytic activity showing halo around the cells, b – B. subtilis natto haemolytic activity with no halo observed around the cells, c: B. cereus lecithinase activity with halo indicating the presence of lecithinase action, d – B. subtilis natto lecithinase activity test without halo, indicating the absence of lecithinase activity.