Enzymatic Hydrolysis Of Earthworm Protein And Production Amino Acid Chelate-salt And Its Bioeffects Characteristics

The utilization of earthworm protein has switched to research the development of multifunctional products from original utilization as feedstuff .The preparation of amino acid chelates is a systematic process which refered to the production of amino acid, composition of chelate and bioassay of production. Chelate salt have been widely used in chemical and agrochemical industry, such as micronutrient fertilizers, liquid fertilizers, animal feed additives and seed coating agents. This research used worm protein as amino acid donor , was compared the changes in hydrolyzing degree of autolysis and outer protease treatments, and also the response surface methodology (RSM) was introduced to optimize hydrolyzing conditions, aimed at the optimum condition of making copper chelates and zinc chelates. Wheat germination rate and bud sensitivity experiment were conducted as bioassay methods to test the bioeffects of three kinds of iron salts (FeSO4,EDTA-Fe,AA-Fe )and evaluated target position action of iron salt on wheat bud region and function. Main results were as followed:1. In the whole process, the concentration of amino nitrogen in outer protease treatment was obviously higher than autolysis, which had significant difference. From 3h to 8h, the concentration of amino nitrogen in outer treatment was significant increased from 12.7% to 22%, while autolysis treatment was increased from 6.62% to 16.05%, but increased ratio of hydrolyzing degree decreased from 91.8% to 37.1%.2. Non-linear regression analysis was made between hydrolysis time(X) and amino nitrogen concentration(Y).Outer protease and autolysis treatments was simulated by exponential equations, Y=0.432×exp (0.130X), Y=0.260×exp (0.143X), correlation coefficients were 0.9383* and 0.9928**, respectively. The error test of fitted value indicated that, fitted level of autolysis treatment was lower than outer protease treatment. The result also showed that the autolysis of earthworm was an unstable reaction process.3. Storage methods of material in production were important to hydrolysis process, and storage time and temperature (4℃and 25℃) had significant effects on hydrolyzing degree. The hydrolyzing degree of worm pulp at 4℃was higher than at 25℃under same storage time, which reached very significant level. So 4℃was beneficial to hydrolysis process and storage time should not exceed 4h.4. Three codes temperature (A), protease concentration (B), time(C), and three levels were included in RSM design. Design Expert 7.1 regression analysis was used to obtain regression between predicted value of hydrolyzing degree (Y),and code independent variable A,B,C: Y=22.86+1.66A+1.83B+ 0.92C+1.23AB-0.70AC-0.55BC+1.58A²+0.24B²-0.5C², after coefficient T–test, model was modified into: Y=22.86+1.66A+1.83B+1.58A2, the optimum condition were calculated: temperature= 50℃, concentration = 5%, time=4.43h.5. The optimum condition of copper chelate: temperature 40℃, time 30min, mass ratio 2:1; the optimum condition of zinc chelate : temperature 30℃,time 50min,mass ratio 2:1.6. Germination rate was a common method for bioassay, at 10—100 mg·L^-1, AA-Fe had no marked stimulation and inhibition effect on wheat germination rate, but the inhibition ratio of AA-Fe at 250 mg·L^-1was greater than FeSO4 and EDTA-Fe. Types of iron salt had different inhibition effects on length of wheat root, concentration overruned 100 mg·L^-1, the length and biomass were restrained by three iron salts, the greatest inhibition treatment was AA-Fe.7. Curve regression model, between concentration of iron salt and germination index (GI), was an effective way to analyze and estimate the bioeffects of iron salt. Three models were lnY=ln(99.6378)-0.01627X, Y=97.16-42.2889Lgx+8.588(lgx)2 , Y=99.46-40.47 lgx, respectively(where X is concentration of iron salt; Y is germination index).8. Wheat shoot were especially sensitive to addition of outer chemical substance and the effect can be observed in length and biomass of shoot and root. Root morphology and growth was more sensitive than shoot. Concentration overruned 100 mg·L^-1, different inhibition effect of three kinds of iron salt on the length and weight of root and shoot were observed, and AA-Fe was the greatest, inhibition rate on root growth were: AA-Fe >FeSO4>EDTA-Fe. 9. Through analyzed the effect of iron salt on wheat photosynthetic system, the beginning inhibition concentration of AA-Fe was between 50-100 mg·L^-1, while FeSO4 and EDTA-Fe between 100-250 mg·L^-1.The inhibition of chlorophyll was : AA-Fe >FeSO4>EDTA-Fe.10. The malonaldehyde (MDA) concentration were changed with types and concentration of iron salt. In FeSO4 and EDTA-Fe treatments, MDA concentration in shoot and root increased with the increment of concentration of iron salt, and then decreased slightly. The change bound of MDA in shoot was -41.32%—172% and 24.31%—148.96%, in root MDA change between 75%—571% and 90.78%—285.82%, respectively. Increment of AA-Fe concentration increased MDA in shoot and root, and MDA increased by 26.04%—103.82% and 21%—652. 48%, respectively. The change bound of MDA in root was wider than in shoot.11. For response degree, inhibition rate index was greatest, followed by lipid per-oxidation index, and chlorophyll index least; besides, growth index was higher sensitive than physiology index. Action target position of FeSO4 fixed in shoots, and AA-Fe and EDTA-Fe fixed in roots. The root lipid per-oxidation was more sensitive to FeSO4 and AA-Fe, and shoot was to EDTA-Fe.