| China has the highest output of aquatic products in the world. In China, the production of aquiculture is more than that of fishing. Freshwater fish is the richest resource in culture and catfish is a good kind of freshwater fish. With the improvement of living standards, the fresh fish can not meet the demands of people's daily life anymore. Thus the processed fish products with convenient, nutritive and health function have been generated. Currently, catfish has been processed into a variety of products, which are one of the export processed products. During this process a lot of byproducts including bones, skins, viscera and heads are produced, which account for more than half of the total fish weight, especially fish head and backbone. Therefore, the objective of this study was to investigate the bone as raw materials. The physico-chemical properties of catfish bone, optimization of enzymatic hydrolysis of the bone and the enzymatic hydrolysates from it were studied, especially, the antimicrobial activity, antimicrobial mechanism and application in foods of the enzymatic hydrolysates were systematically investigated.Fitstly, the physico-chemical property and the microstructure of catfish bone were studied. The main chemical compositions of catfish bone were protein and ash, accounting for 30.6% and 50.4% of the total weight respectively, with the ratio of calcium and phosphorus being 1.84. The glycine was 4.917% which was the richest amino acid and its ratio to the total amino acids was 23.23%. This amino acid composition was in conformity with that in collagen. The infrared characteristic peak of bone tallied with the peak of carbonated hydroxyapatite, except for many small peaks existed in the area of amide. With atomic force microscope, the obvious hole-like structure and the mineralized collagen fibre released from the holes were was observed in the surface of bone.According to the physico-chemical properties, catfish bones were hydrolyzed with one of five proteases (alcalase, neutrase, papain, pepsin and trypsin) in order to generate antimicrobial agents. The antimicrobial activity of hydrolysates recovered through enzyme hydrolysis was tested by radial diffusion assay (RDA). Pepsin hydrolysate was found to have the greatest antimicrobial activity. Thus, the conditions of hydrolysis with pepsin were further optimized by response surface methodology (RSM). After screening and optimization, a quadratic model was proposed. The model predicted the optimum antimicrobial activity with a hydrolysis condition of pH 3.5, reaction temperature of 40℃, enzyme-substrate ratio of 1.97/100 (g/g), substrate concentration of 0.15 g/ml and reaction time of 4 h.The antimicrobial activity of catfish bone hydrolysate was evaluated. The results showed that the hydrolysate could inhibit the growth of E. coli,B. subtilis and M. luteus. Antimicrobial properties of the hydrolysate were stable under temperature, storage and ultraviolet radiation treatment. However, with the increase of the pH and time of freeze-thaw, the antimicrobial properties reduced quickly, although NaCl could increase the antimicrobial property greatly.Determination of growth curve and membrane permeability, together with application of SDS-PAGE and electron microscopy were used to investigate the antimicrobial mechanism of bone hydrolysate. The results of growth curve indicated that the hydrolysate could inhibit the logarithmic growth of E. coli. The conductivity of E. coli treated with hydrolysate increased with time while conductivity of the control group hardly changed. OD260 value of the E. coli treated with hydrolysate increased significantly with time while for the control group it was stable. Glucose levels of the E. coli treated with hydrolysate increased significantly whereas the control had a descending trend. Bacterial protein was analyzed by SDS-PAGE. Obviously, the band of E. coli treated with hydrolysate turned lighter or even disappeared while in the control it was stable. All these results indicated that the hydrolysate changed the bacterial membrane permeability. It made the cells leaky. After being treated with hydrolysate, E. coli was observed by transmission electron microscope. When the bacteria were treated for 0.5 h, some bacteria were found to shrink a bit, and their cytoplasm reduced, cell membrane damaged and nucleus blurred. In contrast, after being treated for 6 h, the bacteria shrank completely, cytoplasm reduced further, some bacteria even lost all of their cytoplasm, cell membrane disappeared or only a shell remained. All these evidence indicated that the inhibitory mechanism of the hydrolysate could be summarized as follows. First, the hydrolysate contacted the surface containing a lot of negatively-charged groups, then caused damages to cell membrane, resulting in the leak of the material in the cell.At last the hydrolysate was applied to sausage and yogurt production. For catfish sausage, the adding amount of 1.5% was the best for its preservation while adding 0.15% of hydrolysate facilitated the yogurt production. The data suggested that catfish bone hydrolysate could be applied in food processing.
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