Date of Award

12-2010

Document Type

Thesis

Degree Name

Master of Science (MS)

Legacy Department

Food, Nutrition, and Culinary Science

Advisor

Jiang, Xiuping

Committee Member

Greene , Annel K

Committee Member

Dawson , Paul L

Abstract

In the United States, billions of pounds of animal by-products are generated by the food processing industry every year. Hydrogen sulfide producing bacteria (SPB) can utilize the sulfur-containing proteins and amino acids in the raw animal materials destined for the rendering process to produce harmful hydrogen sulfide (H2S) gas rapidly under the ambient conditions, resulting in hazardous working environments and inferior quality of finished products. In this study, the application of bacteriophage was explored as an effective solution for the elimination of H2S production in the rendering industry. The objectives of this study were to: 1) isolate and characterize strains of SPB and their specific bacteriophages, 2) to develop and optimize a bacteriophage cocktail specific for SPB, 3) to reduce the SPB population and H2S production in raw animal materials by administering phage cocktail under both laboratory condition and greenhouse environment.
Twenty two meat, chicken offal and feather samples collected from local supermarkets and rendering processing plants were tested for the presence of SPB. Hydrogen sulfide producing bacteria population ranged from 2 to 6 logs CFU/g. One hundred and forty two SPB were isolated and purified, and five predominant strains were identified to species level as Escherichia coli, Citrobacter freundii and Hafnia alvei. Bacteriophages (n=52) specific to SPB were successfully enriched from these samples using the isolated SPB strains as hosts. The host ranges of purified bacteriophages against 5 predominant strains of SPB (isolate S12, S201, S203, S183 and S211) were determined. Electron microscopy of nine phages selected for phage treatment revealed that bacteriophage isolates 211a, 214a, 214c, 217a, 218a and 12a belonged to the family of Siphoviridae, whereas isolates 213a, 214b and 201a were to Myoviridae. Restriction enzyme digestion analysis with endonuclease Dra I detected that phages 218a, 201a and 12a had identical patterns whereas phages 211a, 214a and 214c shared one pattern, and phages 213a, 217a and 214b shared another pattern. The cocktail of the nine selected phages was further tested for inhibiting the growth of five predominant SPB strains in the tryptic soy broth (TSB) medium at 20 and 30¡C. Phage treatment was able to prevent the growth of SPB up to 10 h with the multiplicity of infection (MOI) ratio of 0.1, 1, 2, 5 and 10 at 30¡C, but was less effective at 20¡C.
The phage cocktail was further tested for controlling H2S production by SPB in different raw poultry materials (chicken meat, chicken offal and chicken feathers). The multiplicity of infection (MOI) ratios of 1, 10 and 100 were compared. The amount of H2S production was determined using either test strips impregnated with 0.05, 0.5 or 10% lead acetate or a H2S monitor. The five predominant SPB strains were inoculated into fresh ground chicken meat at an initial population of 4 logs CFU/g, which produced H2S detectable by the test strip assay after 4 and 8 h of incubation at 37 and 30¡C, respectively. With the bacteriophage treatment, the H2S production was reduced up to 35 and 47% at 37 and 30¡C, respectively. At low temperature (20¡C), the detectable time for H2S production was extended to ca. 9 and 12 h with the initial SPB populations of 5 and 6 logs CFU SPB/g, respectively. The phage treatment reduced H2S production by 69 and 55% in fresh ground chicken meat inoculated with initial SPB populations of 5 and 6 logs CFU/g, respectively. To test the effectiveness of the bacteriophage cocktail against naturally occurring SPB, ground chicken meat was incubated at room temperature (ca. 22¡C) for 5 h, which allowed the indigenous SPB to grow to ca. 4 logs CFU/g. The bacteriophage treatment resulted in 25 and 57% reduction of H2S production at 37 and 30¡C, respectively. In blended chicken guts and feathers containing ca. 4 and 6 logs CFU/g of indigenous SPB, respectively, the bacteriophage treatment reduced H2S production by 56 and 62% at 30¡C, respectively. Among all phage treatments, the MOI of 100 exhibited the highest inhibitory activities against SPB on H2S production, but was not significantly different (p>0.05) from MOIs of 1 or 10.
Under the greenhouse condition, phage treatment with MOI of 1,000 achieved ca. 30~85% reduction of H2S production and 61% reduction in SPB population in chicken offal. As compared to maximum SPB reduction of 32% achieved by spraying phage cocktail (MOI of 100) on chicken feathers, the alternative phage treatment of adding phages with MOI of 100 to feather processing water reduced SPB population by 82%.
Overall, phage treatment with high MOI values of 100 and 1,000 achieved ca. 25~85% reduction of H2S generated by SPB in raw poultry materials. Several factors affecting lytic activities of bacteriophages were identified as initial SPB level, temperature, and contact efficacy between SPB cells and phages. Our results demonstrated our phage cocktail is effective to significantly reduce the production of H2S by SPB in raw poultry materials under certain conditions.

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