Research On Preparation Technology Of Compound Amino Acid Chelated Calcium And Antioxidant Properties

Tilapia has become to be the most prominent bulk trades products in China’s freshwater fish breeding product, but large scraps got from in the machining process, just for head and fish,it’s weight taken more than thirty-six percent of total amount of fish weight, which cheap saled as a feed raw materials ,or thrown away as waste, which leads to a little valueas before. Animals bones not just contains protein, fat and other nutrients, but also have a large amount of calcium, iron, and zinc, magnesium and phosphorus, bone collagen composition, which can to be i the good source of calcium product.It is studied on the amino acids compound chelating calcium which get from chelating action of zymolysing fluid and fish meal–prepared by compound enzyme enzymolysis technique,using tilapia fish fillet and fish head as raw material,through chelation、concentration、vacuum freeze-drying technology And the characterization of amino acids compound chelating calcium was analyzed through ultraviolet-visible spectroscopy analysis, infrared spectrum, X-ray powder diffraction method, finally studied oxidation resistance of compound amino acid chelated calcium .Through comparing the effect of enzymatic hydrolysis between Flavorzyme, complex protease, trypsin and papain in their best condition of hydrolysis ability on tilapia meat, the results showed that trypsin has the best effect, then, composite protease, flavorzyme, papain respectively. trypsin （inside cut enzyme）and flavorzyme （outside cut enzyme ） are choosed to composite double enzyme hydrolysis, obtaind the best process : pH6.5, liquid-solid ratio3:1, hydrolysis time 4h, enzyme concentration1.5%, temperature50℃, compound enzyme proportion1:3 （flavorzyme: trypsin）. It is applied to fish fillet, analyzed amino acid obtained by hydrolyzing technics. The results showed that the hydrolysate is rich in amino acids, especially the content of leucine, phenylalanine, lysine, arginine which is more than 1g/L .In the basis of above experimental conditions, compound zymolysing liquid as amino acids resourse and fish meal tankage liquid acid solution as source of calcium were used to chelating reaction , through Packett - Burman （PB） design, the steepest hill-climbing experiment and center combination design experiments to study amino acids chelating calcium chelating conditions and established the mathematical model.Meanwhile , The product was puried and analysised nature structure.First, PB experiment selection was used to evaluate the influence of five factors on chelating rate,and the results showed that pH value and amino nitrogen concentration has remarkable effect. Then, the steepest hill-climbing experiment was used to approximate biggest chelating rate area, and then the response surface center experiment was used to optimizate significant factors on pH value （X₁） and amino nitrogen （X₄）, which found that the optimal conditions pH7.8965, 3.6543 g/L respectively. minitab software was used to get fitting equation for:f （x） = 52.5360-7.9785 X₁ + 0.6392 X4 2.2530 X₁*X1-2.7430 X₄ * X₄ - 0.3950 X₁ * X₄, optimization chelating process parameters: pH value7.90, temperature50℃, chelating time 60min, amino nitrogen concentration is 3.66 g/l volume ratio1:20, theory of chelating rate 62.0415%, and the actual chelating rate 58.1912%, which explained actual throughput and forecast are not varied considerably. Amino acids varieties , contents of hydrolysis fluid and compound amino acid chelated calcium are comparative analyzed, the results showed that the gross amount of leucine, phenylalanine, lysine, arginine, threonine are accounted for more than half of all amino acids, and which explained the five kinds of amino acids are the main influence factors of chelating effect. Varieties and contents of amino acids in zymolysing fluid and chelating compound amino acid calcium were analysised and compared, which found that the amount of five kinds of amino acids contained leucine, phenylalanine, lysine, arginine, threonine accounted more than half of the total content in zymolysing fluid and chelating compound amino acid calcium, which explained the five kinds of amino acids are the main influence factors on chelating effect. At the same time, comparing these three zymolysing fluid chelating effect, the results showed that①zymolysing liquid free amino acids for 26.3901 mg/ml, chelating calcium in amino acids for 15.7061%, chelating rate is 45.49%;②zymolysing liquid free amino acids for 37.8528 mg/ml, chelating calcium in amino acids for 15.9279%, chelating rate is 46.33%;③zymolysing liquid free amino acids for 28.4020 mg/ml, chelating calcium in amino acids for 21.5325%, chelating rate is 55.47%.It indicated that when the amount of the calcium ion adding was in certain circumstances, chelating effect quality was not only relative with the free amino acid total, but also with all kinds of amino acid content concerned.It was Studied that the content of amino acid in③chelating calcium samples was around 5.6% more than①,②chelating calcium, including leucine, phenylalanine, lysine, arginine, threonine, valine, total to 12.9691%, while in①,②chelating calcium, the amount of the first six kinds of amino acids, total to 9.9772%、9.9432%, respectively, a difference of 3%, contribution to 60%, state-of-art l-methionine, isoleucine, glutamic acid, alanine, total to 4.6713%, while in①,②chelating calcium, he amount of the late four kinds of amino acid, total to 2.9784%、2.8903%, respectively a difference of 1.8%, contribution to 30% ,which explained that the ten kinds of amino acid concentration differences on chelating effects has significant affect. Choose leucine, phenylalanine, lysine, arginine, threonine, valine, state-of-art l-methi -onine, isoleucine, glutamic acid, alanine concentration for 14.16 mg/ml, 9/11 mg/ml, 13.44 mg/ml, 9.53 mg/ml, 6.62 mg/ml, 6.28 mg/ml, 5.99 for mg/ml, 5.15 mg/ml, 3.92 mg/ml, 377 mg/ml to configure amino acid compound respectively,and chelate with hydrochloric acid, lactic acid liquid acid solution obtained chelating compound amino acid calcium, calcium content of the product was 2.088%、2.770% ,respectively;chelating rate 69.44、86.94% respectively.The chelating compound amino acid calcium was puried, then analyzed in product characterization through ultraviolet-visible spectroscopy analysis, IR, X-ray powder diffraction method.The result showed that the 207nm, 257nm place absorption peaks in composite amino acids moving into 217nm, 261 nm place after chelating in uv-vis spectra; 2930cm^-1 （VC-H） and 1654cm^-1 （VC= O） characteristic absorption peaks disappeared, hydroxyl associating peak is abate, 1594cm^-1 and 1406cm^-1 appeared ring structures characteristic absorption peaks, 775cm^-1 and 668cm^-1 appeared Ca-N telescopic vibrating peaks and Ca-O telescopic vibrating peaks in the ir spectrum of chelating compound amino acid calcium; peak number and position of composite amino acids and chelating compound amino acid calcium had significant difference in X-ray diffraction ;which fully proved the existence of chelating calcium.Finally oxidation resistance of compound amino acid chelated calcium solution and solid are studied, evaluated chelating compound amino acid calcium total reductive ability, the oxygen free radicals （O2-）inhibition ratio, hydroxyl radical （OH-） clearance rate, and (1,1- diphenyl-2 - bitter benzene hydrazine free radicals （DPPH·） clearance rate. Experimental results showed that the total antioxidant ability remained at more than 50% of the original total antioxidant capacity, super oxygen free radical inhibiting rates maintained in 50% more than the original inhibiting rates, hydroxyl radicals clearance maintained at 80%, and DPPH free radicals clearance maintained in more than 50%, but its antioxidant capacity in vitro overall a gradually declines in the preservation of 3 months above.