| As people's living standard continue to increase, the consumption of aquatic products are increasing, the requirement for its freshness is higher and higher. The main reason of causing fishery foods deterioration is bacterial growth and metabolism. Because of natural, safe and small poisonous effect of biological perservatives, it is gradually replacing conventional synthetic chemical preservatives to use in fishery foods widely. Chitosan, tea polyphenol, nisin and other natural preservatives have been widely used to reduce the extent of deterioration of fish.Egg yolk immunoglobulin, also called IgY, is one of three principal classes immunoglobulins in avian sera. Its preparation is simple, suitable for mass production, and it has good thermal stability and strong acid and alkaline resistance, resistance to ionic strength and certain enzyme-resistant ability. Moreover, IgY also has a very important property is its remarkable inhibitory effect to its antigen bacteria. At present, IgY has been used widely in immune diagnostic testing, animal disease immune control and food additive and cosmetic fields.In this research, 4℃chilled Paralichthys olivaceus was chosen as object, the main spoilage organisms were qualitatively and quantitatively investigated in cultured Paralichthys olivaceus at the end of shelf stage at 4℃. Then, four kinds of egg yolk antibodies (IgY) against main spoilage bacteria were generated respectively, and the antibacterial efficiency of specific IgY in liquid and solid medium were studied. On the basis, sensory, microbiological and chemical (pH,TVBN,TBA) indices were comprehensive analyzed to develop the studies which IgY antibodies were applied to the Paralichthys olivaceus samples to inhibit bacteria and prevent corruption. Main results are listed as following:1. The main spoilage organisms in cultured Paralichthys olivaceus during chillesd storage at 4℃were analyzed. Based on the colony morphology, Gram stain, biochemical and physiological characteristics as well as nutritional properties, bacteria strains were isolated at the end of shelf life and identified with VITEK and BIOLOG bacteria identification systems. Result demonstrated that Shewanella putrefaciens was dominated spoilage bacteria, which accounted for about 58.4% of all bacteria at the end of shelf stage at 4℃. Pseudomonas which were common spoilage bacteria in aquatic products included Pseudomonas fluorescens, Pseudomonas taetrolens and Pseudomonas marginalis,with a corresponding proportion of 63.2%,29.2%,7.6%, respectively.2. Using whole cells as antigens, four kinds of specific egg yolk antibodies (IgY) were generated respectively against Shewanella putrefaciens, Pseudomonas fluorescens and Pseudomonas taetrolens, three main spoilage bacteria. The antibacterial efficiency of specific IgY in liquid medium were studied. The isolated specific IgY demonstrated a concentration-dependent inhibitory effect on the antigen bacteria, the density is higher, the inhibitory effect is more obvious. After incubation for 8h in liquid culture at 25℃, the cell number of S. putrefaciens was reduced approximately 84.7% with 200 mg/mL prepared IgY against S. putrefaciens, the cell number of P. fluorescens was reduced approximately 88.1% with 200 mg/mL prepared IgY against P. fluorescens. The inhibition ratio of two kinds of 100 mg/mL mixed IgY (M1-IgY and M2-IgY) against S. putrefaciens could respectively attain 61.6% and 67.2%. The inhibition ratio of two kinds of 100 mg/mL mixed IgY (M1-IgY and M2-IgY) against P. fluorescens could respectively attain 69.2% and 77.8%. After incubation for 40 h in liquid culture at 4℃, the cell number of S. putrefaciens was reduced approximately 90% with 100 mg/mL prepared IgY against S. putrefaciens, the cell number of P. fluorescens was reduced approximately 66.6% with 100 mg/mL prepared IgY against P. fluorescens.3. In solid culture, after incubation for 24h, the cell number of S. putrefaciens was reduced approximately 52.38% with 100 mg/mL prepared IgY against S. putrefaciens, the cell number of P. fluorescens was reduced approximately 62.55% with 100 mg/mL prepared IgY against P. fluorescens. The inhibition ratio of two kinds of 100 mg/mL mixed IgY (M1-IgY and M2-IgY) against S. putrefaciens could respectively attain 27.8 % and 38.3 %,the inhibition ratio of two kinds of 100 mg/mL mixed IgY (M1-IgY and M2-IgY) against P. fluorescens could respectively attain 31.1 % and 40.0 %.4. Comprehensive indices such as pH, TVBN, total colonies and sensory evaluation in chilled Paralichthys olivaceus samples were analyzed and the results demonstrated, the keep freshness efficiency of specific IgY processed samples was significantly superior in comparsion to controls. Therefore, specific IgY can effectively extend the shelf life of chilled Paralichthys olivaceus at 4℃. The shelf life of samples could be extended approximately 3 days after pretreatments with 100 mg/mL P-IgY,the shelf life of samples could be extended approximately 5～6 days after pretreatments with 100 mg/mL S-IgY,the shelf life of samples could be extended approximately 3 days after pretreatments with 100 mg/mL M1-IgY or M2-IgY. |
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