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    Effect of bacteriophage control and artificial neural networks prediction in the inactivation of Listeria monocytogenes on fresh produce
    (2017) Oladunjoye, Adebola Olubukola; Ijabadeniyi, Oluwatosin Ademola; Singh, Suren
    There has been a global increase in fresh produce consumption, due to its attendant nutritional a nd health benefits. On the other hand, increase in the outbreak of diseases, accompanied with health and economic implications, have been traced to this deve lopment. A good number of pathogenic contaminants along the food chain have been identified as causative agent s with Listeria monocytogenes identified as one of such. Among other control strategies, the use of bacteriophage, was recommended as a palliative measure. Furthermore, the a ppli cation of artificial neural networks (ANN) in food safety remains an emerging concept in risk assessment study. Therefore, the aim of this research is to investigate the effect of bacteriophage or phage control and artificial neural network prediction in the inactivation of L. monocytogenes ATCC 7644 on fresh produce. Fresh-cut tomato and carrot were artificially inoculated with L. monocytogenes (108 CFU/ml) and subjected to antimicrobial treatment of Listex P100 bacteriophage (108 PFU/ml), sucrose monolaurate (SML at 100, 250 and 400 ppm), with chlorine (sodium hypochlorite at 200 ppm) used as control. Also, application of ANN to predict the risk effect of antimicrobial treatments of bacteriophage, sucrose monolaurate and chlorine was evaluated on the fresh-cut produce. Mathematical models were developed using a linear regression and sigmoid (hyperbolic and logistic) activation function-(120). Data sets were trained using Back propagation ANN, containing one hidden layer with four hidden neurons. Furthermore, carbon utilization profile of phage-treated L. monocytogenes using phenotypic micro array method was evaluated. In the first phase, susceptibility of L. monocytogenes subjected to certain stress-adapted conditions (acid,-adapted AA, chlorine-adapted CA, heat-adapted HA) and non-adapted-NA to phage treatment inoculated on the fresh-cut produce stored for 10 days at 4, 10 and 25oC was evaluated. The second phase investigated the combination of bacteriophage and sucrose monolaurate (using chlorine at 200 ppm as control) to inhibit the L. monocytogenes growth on the fresh-cut produce stored for 6 days at 4, 10 and 25oC. Physicochemical properties (pH, titratable acid-TTA, total soluble solids-TSS, and colour values-CIE L* a* b*) of the fresh produce after treatment were evaluated. In the third phase, ANN as a predictive tool was used to evaluate the risk involved in the relationship among the initial bacterial load, fresh-produce type, antimicrobial concentration and residual bacteria. In the final phase, 100 µL of phage-treated L. monocytogenes was introduced into a 96-micro well plate impregnated with a tetrazolium dye. The Carbon utilization profile was evaluated at intervals of 4 hours for 48 hours using a biolog micro station. Generally, L. monocytogenes grew on both fresh-cut produce and the storage temperature did not adversely affect the lytic ability of the phage treatment. Antimicrobial treatment of phage and sucrose monolaurate had minimal variations on the physicochemical properties of both fresh-cut samples. All stress-adapted and non-adapted L. monocytogenes were (p ≤ 0.05) susceptible to bacteriophage control. Phage treatment reduced non-adapted, acid adapted, chlorine-adapted, and heat-adapted L. monocytogenes population by 0.57, 0.81, 0.86 and 0.95 log CFU/ml in fresh-cut tomato, and 2.26, 2.41, 2.49 and 2.54 log CFU/ml in fresh cut carrot respectively. Furthermore, the additive effect of SML at 100 and 250 ppm had no significant effect on phage lysis. However, combination of phage with SML at 400 ppm significantly (p ≤ 0.05) resulted in 1 and 3 fold reductions in tomato and carrot respectively. Control treatment with chlorine resulted in 1-2 log reductions on both fresh produce. Algorithm data set trained using ANN gave 100% accuracy. Prediction with logistic activation function showed the highest positive correlation relationship between predicted and observed values with ~ 0.99 R2-value and MSE of 0.0831. Carbon utilization profile showed hexose and pentose sugars-ribose, glucose, fructose and sugars were maximally utilized while oligosaccharide sugars of sucrose, cellobiose and gentiobiose were similarly observed to be utilized. Notably, utilization of glucose-6-phosphate which determines L. monocytogenes pathogenicity was not very pronounced in the carbon profile. Bacteriophage application in the inactivation of L. monocytogenes contamination of fresh produce provides a safe means of control. Its perceived limitation however, can be overcome by combining with other antimicrobials. Similarly, the use of artificial neural networks prediction, remains an improved approach to harness the potential risk that could occur through this method.
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    Strategies to control bacteriophage infection in a threonine bioprocess
    (2009) Cele, Nolwazi; Permaul, Kugen; Snyman, Francisca
    Production of numerous biotechnologically-important products such as threonine is based on cultivation of bacterial cultures. Infection of these bacterial cultures by bacteriophages has a detrimental effect in the production of these bioproducts. Despite this, most people controlling these bioprocesses do not recognize the early signs of bacteriophage infection. SA Bioproducts (Ply) Ltd was no exception and has suffered tremendous loss of production time after bacteriophages infected threonine producing E. coli strain B. This study was aimed at developing assays to control and prevent bacteriophage infection at this company. These included determining the source of phages by monitoring the process plant environment, optimising the detection and enumeration methods so as to monitor the levels of bacteriophages in the environment, identification of bacteriophages in order to determine the number of bacteriophages capable of infection threonine producing E. coli strain B, treatment and of phages, and possible prevention of phage infection. Adam's DAL method was very efficient at detecting phages in the samples collected at various areas (sumps, odour scrubber, process water, and soil) around the plant for 16 weeks. High levels of phages were found in the sumps and this was identified as the source of infection. Samples collected were grouped together according to their source. The samples were enriched and purified in order to characterise them. The prevalent phage in all samples was identified as a T1-like phage. Bacterial strains that grew on the plate in the presence of phages were assumed to be resistant to phages or contained lysogenic phages which would explain the new lytic cycles that were observed whenever these resistant strains were used for production. UV light, green v indicator plates, and a mutagen (Mitomycin C) were used to detect Iysogens. Mitomycin C at 1 IJg/ml was found to be most effective in detecting lysogenic phages. This was shown by new plaque forming units that were visible on the DAL plates. Temperature (heat), chemicals, and inhibitors (vitamins) were investigated as strategies for prevention and treatment of bacteriophage infection. Bacteriophage samples were exposed to 70, 80, 100, and 120°C. At these temperatures pfu counts in the samples were reduced significantly. At 120°C there was a complete inactivation of bacteriophages within 30 minutes. Chemicals investigated such as sodium hydroxide and Albrom 100T were capable of complete deactivation of bacteriophages at a very low concentration (0.1%). Therefore, these chemicals can be used to clean the plant area and sumps. Vitamins C, K and E solutions were investigated to determine their inhibitory effect on bacteriophages. Vitamin C, K and E reduced pfu counts by 3, 2, and 4 logs, respectively. Therefore vitamin C and E solutions were mixed and to determine if mixing them would enhance their inactivation capabilities. This resulted in a reduction greater than 9 logs of phage in the sample (from 7.7 x 109 to 3 pfu/ml). The host bacterium was also exposed to this mixture to determine effect of the vitamin mixture on its growth. It was found that there was no effect exerted by this mixture on the host bacteria. This proved to be an ideal mixture for combating phages during fermentation. However, vitamin E is not cost effective for co-feeding in 200 m' fermenters, and therefore vitamin C solution was a cost-effective alternative. It was concluded that bacteriophage contaminated bioprocessing plant should be properly cleaned using a combination of heat and chemicals. Bacteriophage infection should be prevented by employing inhibitors.