MATRIZ EFE
1. Cambio de hábitos de los consumidores 2 Competencia actual
To quantify the response to the DETA-NONOate treatment, the number of viable bacteria and spore counts in the milk effluent during 20 h of the trial were periodically determined. Various concentrations of DETA-NONOate were used, including much higher concentrations in flask culture compared to flow-through experiments and including levels that have been reported to disperse either growing or non-growing biofilms of pseudomonads (Barraud et al., 2015). The treatments applied, however, were found inadequate to disperse G. stearothermophilus biofilms in milk (Figures 5.4 and 5.5).
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Figure 5. 4 (Upper). Changes in TVC and spore counts in milk that has passed through a flow reactor initially inoculated with a low level of G. stearothermophilus in milk for ~1 h, before being replaced by reconstituted skim milk without added G. stearothermophilus. This experiment represents a ‘control’, conducted at 65°C. (Right) Added NaOH (needed, in other experiments, to dissolve the DETA-NONOate) in all milk used in the trial. (Lower). Counts obtained from RSM broth inoculated with 100 CFU/mL G. stearothermophilus W14 spores and exposed to varied (DETA)-NONOate treatments in the flow through reactor. NO application times are indicated by the dashed lines.
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Figure 5. 5 Vegetative cell counts recovered on TSA over time from A) RSM, and B) BHIB inoculated with G. stearothermophilus W14 in flask (static culture) experiments at 65 °C for 10 h and exposed to 0.1 mM and 1 mM DETA-NONOate concentrations.
Responses represented in Figure 5.4 and Figure 5.5 suggest that there is no systematic effect on dispersal since, e.g., there was no evidence of any sudden increases in counts in the milk effluent coinciding with application of milk dosed with NO.
Figure 5.4 shows the growth of G. stearothermophilus W14 in the flow through reactor; using sequential sampling of total viable cell and spore counts every 1 h over 20 h. it also shows the growth of untreated controls and controls that include 0.01 mM NaOH (the solvent needed for solutions
of
DETA-NONOate. A G. stearothermophilus W14 spore inoculum was fed into the system during the first hour to assess the bacterial attachment (and potential detachment) during the run. Over the period of the assay, from hours151
3 to 20, the microbial density in the effluent rose exponentially. Differences in the microbial density in the effluent over time, between DETA-NONOate and controls, were used as a measure of the effect of DETA-NONOate application at specified times.
G. stearothermophilus W14 cultures and flow-through experiments were treated with pulses of 0.5, 0.1, and 0.01 mM DETA-NONOate at various times. As seen in Figure 5.4, no systematic effects of DETA-NONOate on cell densities in milk effluents were observed. Log reduction counts in the effluent were suggested at hour 10, but the rate of regrowth was similar to that of the control. Importantly, if detachment had occurred, an increase in cells, or spores, in the effluent would have been expected rather than a decrease. In the presence of DETA-NONOate, generation time of the treatments were not systematically different for the 0.01, 0.1 mM and 0.5 mM concentrations (data are not shown).
Total viable cell and spore counts shown in Figure 5.4 and Figure 5.5 indicate that cell and spore numbers did not show consistent or systematic differences in response to the various NO treatments applied.
It was not possible to assess spores and vegetative cells on the internal surfaces during the experiment, since it was hard to open the chamber and to do so would have caused disruption to the milk flow. However internal surfaces of the chamber were swabbed at the end of experiment. A 1 log lower total viable count and 2 log reduction of spore counts for 0.5 mM NO
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addition were observed compared with the control (Figure 5.6), though it was not possible to quantify the statistical significance of these apparent differences due to the difficulty and expense of conducting replicated experiments. However, enumeration of biofilm densities at the completion of trials (Figure 5.6) refutes this assessment, with no systematic differences in vegetative cell or endospore loads on different parts of the flow-through system, after treatments including DETA-NONOate, compared to treatments not involving DETA-NONOate.
Enumeration of biofilm densities on contact surfaces at the completion of flow-through trials (Figure 5.6) reinforces the above observations, with no systematic differences in vegetative cell or endospore loads on different parts of the flow-through system, after treatments including DETA-NONOate, compared to treatments not involving DETA-NONOate.
The response to NO could be complex in G. stearothermophilus since, like many bacilli, it possesses the means to generate NO via nitric oxide synthase (NOS) and reduce NO via nitric oxide reductases (H-NOX). NOS synthesises NO from L-arginine and oxygen (O2) requiring NADPH (Kunst et al., 1997).
Proposed functions for bacterial NOS proteins include protection against oxidative stress and metabolic nitration activities (Sudhamsu and Crane, 2009; Schreiber et al. 2011).
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Figure 5. 6 Vegetative cell (‘TVC’) and spore densities (per cm2) at various sites inside the flow through system. Milk containing various concentrations of DETA-NONOate in 10mM NaOH and control treatments (no additions, or NaOH only), was passed through the system after the standard pulsed G. stearothermophilus W14 inoculation procedure (~1 h), and 20 mL/min flow rate at 65 °C. Samples were taken after 20-24 h of milk flow.
H-NOX proteins seem to have a role in regulating bacterial biofilm formation by controlling NO levels or regulatory switches involving adaptation between
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oxic and anoxic states (Schreiber et al. 2011; Derbyshire and Marletta, 2012) and influencing the physiological adaptations to anoxic conditions. H-NOX- converts NO to N2O (nitrous oxide), which takes part in regulating anaerobic
respiration of nitrate and nitrite (Rodionov et al., 2005; Lundberg et al., 2009). G. stearothermophilus also possess a NO-sensitive transcriptional repressor similar to that of NsrR in Bacillus subtilis that could act as a master regulator for the above responses including managing oxidative stress (Henares et al., 2013)
Triggers for dispersion of cells from a biofilm involve oxygen and/or nutrient limitations. Moreover, G. stearothermophilus W14 attached to stainless steel may be influenced by factors that allow this microorganism to survive within biofilms (Parkar et al., 2001). Although the mechanistic understanding of how NO effects biofilm formation by Pseudomonas aeruginosa (Barraud et al., 2006) is very preliminary, NO is known to be involved as a signalling molecule in biological systems. Thus its influence in signalling processes regulating biofilm formation could result in reduction of early biofilm formation.
Nitric oxide is a common chemical signal molecule in biological systems. It exists under standard conditions as a colourless gas. As such, it is not expected to leave residues in milk powder if it were proposed to use it to retard biofilm formation. DETA-NONOate is commonly used as a NO donor compound (Feelisch, 1998; Yamamoto and Bing, 2000; Wang et al., 2002). The exposure times the NO donor was applied in this study were compatible with typical milk production plant production times between cleaning. In this
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study various doses of NO were used to attempt to induce dispersal of biofilm cells at (at least) one time during the development of the biofilm using the total vegetative cell count and the spore count to monitor biofilm dispersal into the milk flow, i.e., assuming that more cells in the effluent suggests that more cells have attached, and also detached. A pulse at 4 to 6 h enabled assessment of whether the NO application induced dispersal, or may have delayed attachment. Since biofilm development, or delay of biofilm development, was not evident within four hours of seeding with cells of G. stearothermophilus W14, a second treatment was applied later in the process (e.g., at 9 to 10 h; see Figure 5.5).
Schreiber et al. (2011) investigated the role of NO in biofilm formation and dispersal of cells of a strain of Bacillus subtilis (3610) that exhibits swarming motility and develops complex biofilms. B. subtilis 3610 may represent an appropriate model system for exploring the role of NO dispersal of a Gram-positive, endospore-forming, bacterium. Zeigler (2014) reports, however, that the thermophilic spore-forming bacteria have considerably reduced genomes and proteomes compared to their mesophilic relatives. Nonetheless, the observation of Schreiber et al. (2011) provides insights into the apparent lack of effect of NO against G. stearothermophilus biofilms. Based on their rigorous investigations Schreiber et al. (2011) concluded that, rather than biofilm dispersal, endogenous NO derived from expression and activity of the enzyme nitric-oxide-synthase accelerated endospore formation and retention of cells in the biofilm. Furthermore, they noted that endospores are not dispersal cell phenotypes in Bacillus spp., but that motile vegetative
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cells are the dispersal cell phenotype. They further reported that NO did not induce germination of spores, i.e., that endospores were not precursors to dispersal cells. The role of spores is perhaps better understood as a bet-hedging phenomenon (Veening et al., 2008) under different sets of controls and evolutionary objectives.
Schreiber et al. (2011) concluded that “the effect of NO on dispersal is a species-specific phenomenon with different bacteria using NO for opposing dispersal strategies”. They also observed that self-produced D-amino acids are known to insert into cell walls of Gram-positive biofilm bacteria and to disrupt the bonding between cells and the EPS, thereby promoting cell dispersal.
Taken together, the results from relevant literature suggest that exogenous NO is not likely to produce significant extension of powder plant run times. This is particularly true because the growth rates of thermophilic spore-formers under normal operating conditions in powder plants are rapid, with generation times at 65 °C in the range 15 -20 min (see Chapter 2). As such, as 50% reduction in cell numbers could only be expected to extend run times by 15 – 20 min, a 75% reduction might be expected to extend run times by about 45 min. To extend run-times by two hours would require reductions of biofilms load of 90 – 99%, far in excess of any reductions reported in the literature. Coupled with the costs of the reagents, and that the milk powder processing system is based on continuous flow of milk, including milk flowing past any developing biofilms in the system, it is difficult to envisage how
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exogenous NO could be applied practically to extend run times.
5.4 Conclusions
These experiments were done to assess whether NO addition can induce G. stearothermophilus biofilm dispersal in industrial contexts. It was proposed that dispersal could contribute to control of biofilm growth, and thus use it as an aid for cleaning and the prevention of biofilms. The study indicated NO does not cause dispersal of G. stearothermophilus W14 biofilms based on TVC and SC numbers and also suggested by SEM imaging. Further research may be required to confirm that this is the case, but there is little evidence for it in the studies reported here or in the more recent published literature.
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