La ley natural y su formulación primigenia

The probiotic species: B. bifidum, BB-12^® and LA-5^® showed poor growth in CMM but very good growth in CMMg. Figure 3.1 shows the power-time curves of 3 repeats of LA-5^® inoculated to a density of 10⁶ CFU/mL in CMM unsupplemented. The power-time curves of 10⁶ CFU/mL repeats of pure cultures of the probiotic species and the other species inoculated into CMMg are shown in Figures 3.2, 3.3, 3.4, 3.5, 3.6 and 3.7.

The power-time curves of the different species were characteristic in CMMg even though the curves lacked the complexity/structure demonstrated in CMM or NB in Chapter 2. The cumulative heat outputs associated with the growth of LA-5^® and most of the species were significantly greater (P<0.05) in CMMg than in unsupplemented CMM.

Figure 3.1. Power-time curves of 3 repeats of L. acidophilus, LA-5^® in CMM inoculated to 10⁶ CFU/mL

Figure 3.2. Power-time curves of 3 repeats of L. acidophilus LA-5^® in CMMg inoculated to 10⁶ CFU/mL

Figure 3.3. Power-time curves of 3 repeats of B. lactis BB-12^® in CMMg inoculated to 10⁶ CFU/mL

Figure 3.4. Power-time curves of 3 repeats of B. bifidum in CMMg inoculated to density of 10⁶ CFU/mL

Figure 3.5. Power-time curves of 3 repeats of P. aeruginosa in CMMg inoculated to density of 10⁶ CFU/mL

Figure 3.6. Power-time curves of 3 repeats of S. aureus in CMMg inoculated to density of 10⁶ CFU/mL

Figure 3.7. Power-time curves of 3 repeats of E. coli in CMMg inoculated to density of 10⁶ CFU/mL

It should be noted that most of the members of the Lactobacillus genus are facultative anaerobic organisms (Schleifer, 2009). Optimal growth is however achieved in microaerophilic or anaerobic conditions for some members (Schleifer, 2009). Members of the Bifidobacterium genus are however obligate anaerobes. S. aureus and E. coli on the other hand are facultative anaerobes (Schleifer and Bell, 2009, Scheutz and Strockbine, 2005) and P. aeruginosa, an aerobe, but sometimes considered a facultative anaerobe because it is known to survive in anaerobic environments using nitrate or nitrite if available as terminal electron carriers or arginine in the absence of nitrate or nitrite (Eschbach et al., 2004, Garrity et al., 2005). Growth is however generally favoured in aerobic environment for P. aeruginosa (Garrity et al., 2005) also demonstrated by the results in Chapter 2. Thus the probiotic species, in particular, the bifidobacteria species can only be grown in oxygen-free environments which is achieved by the utilisation of natural medium containing reducing substances or by the addition of reducing substances for example glucose, sodium thioglycollate, ascorbic acid, cysteine hydrochloride etc. to some media (Charteris et al., 1997, Champagne et al., 2011). They are easily killed when exposed to oxygen because of their lack or limited activity of superoxide dismutase and or catalase (Brioukhanov et al., 2002). On the other hand, the other species in particular P. aeruginosa, require oxygen for growth and has limited growth in anaerobic environment, hence the ampoule oxygenation or deoxygenation was

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compared to the other species, the probiotic species are fastidious and metabolise by fermentation. For instance, LA-5^®, which grew optimally in microaerophilic or anaerobic environment is extremely fastidious and is adapted to complex organic substrates. Apart from requiring carbohydrates as energy and carbon sources, these species also require nucleotides, amino acids, and vitamins (Schleifer, 2009) for growth.

The various requirements for essential nutrients of these species are however normally met when the media contains fermentable carbohydrate, peptone, meat and yeast extracts (Harrigan, 1998).

CMM consists of minced meat in nutrient broth (Harrigan, 1998). The minced meat naturally contains reducing substances, which produce and maintain anaerobic conditions in the medium (Harrigan, 1998). As has been stated in Chapter 2 of this thesis, CMM is a useful media for the culture of both aerobic and anaerobic organisms.

By heating the medium, dissolved oxygen is removed and anaerobic environment can be maintained by incubation in an oxygen-free environment. Similarly, aerobic environment can be achieved when the medium is incubated uncapped or with loosed cap. Aerobic organisms grow at the top of the medium whilst anaerobic grow deeper in the medium. The addition of 2% w/v glucose to CMM to reduce the medium further for optimum growth of the probiotic species notably changed the power-time curves of the other species as well. Its addition in a naturally reduced medium kept the concentration of oxygen extremely low in the sealed ampoules. Hence, the characteristic aerobic peaks of all the facultative species were completely lost. Presumably, only anaerobic or also possibly microaerophilic growth occurred in the CMMg filled calorimetric ampoules.

The species also metabolised more energetically and for longer time likely because of the substrate potential of the additional glucose. This is well illustrated by LA-5^®, S.

aureus and E. coli, which produced mean heat outputs of 34.52 ± 1.2 J in CMMg compared to 0.70 ± 0.03 J in unsupplemented CMM; 13.63 ± 0.66 J compared to 2.23 ± 0.32 J and 14.34 ± 1.08 J compared to 3.63 ± 0.30 J respectively.

Comparing all of the species in CMMg (Figure 3.8), the power-time and heat curves suggest that BB-12^® and B. bifidum are slower growing species relative to the other species. The peak of the growth curves occurred at ca. 10 h for BB-12^® and 11 h for B.

bifidum; whilst it occurred at 7 h for LA-5^®; 4.5 h for P. aeruginosa; 6.5 h for S. aureus and 5 h for E. coli. Amongst the species, LA-5^® showed maximum growth in the

medium generating the greatest heat outputs and reducing the pH of the medium the most (from 7.20 ± 0.2 to 4.0 ± 0.13).

Figure 3.8. Comparison of the power-time curves [A] and heat curves [B] of pure cultures of LA-5^®, BB-12^®, B. bifidum, P. aeruginosa, S. aureus and E. coli in CMMg at densities of 10⁶ CFU/mL of each. The curves show differences in the growth peaks and lag time duration respectively for the different species

P. aeruginosa on the contrary showed the least growth, with heat output of 2.17 ± 0.31 J and changing the pH of the medium only slightly. The superior growth of LA-5^® in the

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generated in the ampoule suited its growth the most. P. aeruginosa on the other hand showed least growth in this reduced medium probably due to its inability to ferment available substrates in the medium for growth like the others. To illuminate further, it has been previously shown by Eschbach et al., (2004) that apart from denitrification and arginine fermentation, P. aeruginosa is also capable of fermenting pyruvate; but while this fermentation has been shown to afford it a capacity for long-term survival in anaerobic environment, it has been shown not to sustain significant anaerobic growth (Eschbach et al., 2004).