Study On Calcium Ionization And Collagen Polypeptide Extraction Optimization From Bovine Bone By PEF

In recent years, with the meat consumption increased, a large number of animal bones have also increased. Especially consumption of beef was the largest and fastest. Bovine bone is a by-product of meat production, and it is a waste, but also a resource. Taken Ji-Lin (china) Haoyue Islamic Meat Industry for example, currently available slaughtering capacity has reached 500,000 cattle for a year, and the daily slaughtering was 1500. By-products were brought tremendously. Every day 75 tons of bone was generated, so bovine bone resources was very rich. But in the current bone consumer market, with the exception of ribs and the cavity bone can be used directly in diet and greater demand, the bones of poor sales, combined with low prices, storage inconvenience, they often were abandoned, or processed into bone meal added to feed, causing enormous waste and pollution.Therefore, the full use of bone resources and vigorously develop the nutrition of animal bones has become the primary issues that need to be solved in the food industry, especially the meat processing enterprises. As the bone has a special structure, made of bone nutrition could not be digested and absorbed. It must be processed before being used. However, bone processing technology was draggled in our country, and refining and full-hydrolysis processing was almost blank, failed to fully achieve the bone resource's utilization and value-added. The high nutritional value of bovine bone, in which the natural calcium content is very large, and 1kg of bone which can be extracted more than 200g natural compound calcium. According to another report of the Chinese Nutrition Society, the content of bovine protein is more than 14.8%, the report showed that if the resources of Chinese meat bone protein fully utilized, the equivalent to Chinese industry does not put any cost, and meat protein production was nearly doubled. This is the country's huge economic impact.PEF technology has the advantages of non-thermal, reaction speed quick, efficient, easy to operate and low cost. More importantly, its application scope is very abroad, such as in the industrial, agricultural, medical, clinical, and many basic subject research. The application prospect of PEF is very impressive. At present, domestic and foreign reports on the PEF was particularly prevalent in the sterilization and blunt enzyme. While the reports of the application of speeding up chemical reactions is completely blank, and the study of enzyme activation has been rarely reported. The innovation of this study was the first time to use the chemical reaction and enzyme activation by PEF. It provides a new idea for PEF theoretical research and application. Therefore, this study was not only of great value, but also more important theoretical innovation.In this paper, the discarded bovine bone was chosen for the raw materials, through PEF and biotechnology matching methods, a complete set of comprehensive development processing of bovine bone PEF technology was explored. The goal of full use of bone resources, comprehensive development and utilization of bone calcium and collagen active ingredients was achieved. And the products were made with high calcium, high-nutrition and high value-added, attained to bovine bone value-added, solve environmental pollution and increase corporate profitability. This method has an advantage of short processing time, low temperature of samples and high efficiency. If it was put into actual production, it will significantly reduce production costs, increase productivity and make a solid foundation of the leap of modern enterprise economy.Calcium ionization and collagen polypeptide extraction optimization were studied by PEF on bovine bone. It includes:(1) Calcium fast ionization by PEF was studied systematically. It was found that PEF is a highly efficient, non-thermal, continuous operation technology. Ionic calcium could be dissolved from bovine bone by PEF. Under the conditions of solubilization of calcium ionic, PEF method was compared with traditional methods. It was found that the content of calcium ionic by PEF was the highest. It was 8.53 times than boiling, 3.06 times than microwave. So PEF was chosen for calcium ionization on bovine bone. Three factors of calcium ionization from bovine bone by PEF were studied systematically, and the most content of ionic calcium can achieved at 4.324.mg/mL by PEF on the conditions of 1.25% citric acid, 70kv/cm field strength, and 12 pulse numbers.Calcium ionization by mixed acid from bovine bone was studied. Through the single factor test and ternary quadratic regression rotation test to draw significant levels ofα=0.05 off field strength x1, pulses number x2, mixed acid x3 mathematical regression model: Y= 9.002+ 1.097 x1+1.204x2-2.504x3-2.812x32, The optimization conditions were: field strength 23kV/cm, pulses number 12, mixed acid 3.881g (two molar ratio of 1:1.5, bovine bone 10g), feed water ratio 1:10, and the maximum content of ionic calcium 12.90mg/mL. Under optimization conditions of calcium ionic, PEF method was validated and compared with traditional methods. The content of calcium ionic was 5.68 times than boiling, 2.85 times than microwave. So PEF is the most effective way for calcium ionization.The extraction-rate of calcium by PEF three treatments was studied, and the total extraction-rate could reach 98.57%. Calcium ionization effect is very ideal.(2) Practical application of ionic calcium was studied by PEF. High calcium commercialization product was prepared——high calcium oral solution, high calcium beverage and high calcium chelate protein powder.The processing technologies were:The optimum processing technology of high calcium beverage: The centrifugal supernatant liquid ionic calcium 20% by PEF→added 6% sucrose, whisked 7min by magnetic stirring blender→0.15% APM, adequate spices and a little water dissolved and filtered→added remaining water→filling→121℃sterilization 10min→finished product.The optimum processing technology of high calcium oral solution: The centrifugal supernatant liquid ionic calcium by PEF→added calcium gluconate 4mg/mL, 65℃stirred and dissolved→filtered insolubles impurities by filter cloth and got mixture A→added 8% sucrose, whisked 7min by magnetic stirring blender and got solution B→0.05% APM, 0.2% calcium lactate, 0.4% lactic acid, 0.2% PGA and solution B dissolved and filtered→adjusted pH with edible sodium hydroxide to 4.7→filling→63℃, sterilization 30min→finished product.The optimum processing technology of high calcium chelate protein powder:The centrifugal sedimentation by PEF→added contents intraday tile, thickness 10mm→pre-refrigerator at low temperatures 2h→freeze vacuum freeze-drying vacuum 0.025Mpa→high calcium chelate protein powder.The quality of products of high calcium product was studied. It was found that high calcium products are very good traits; physical and chemical indicators, microbial indicator are up to grade; storage stability was ideal, the shelf-life of high calcium beverage and high calcium oral solution was at least 247d. At the base of analytical hierarchy process, the weight of each factor was fixed. And then, with fuzzy comprehensive evaluation, the quality of sensory was analyzed. The comprehensive evaluation of high calcium beverage and high calcium oral solution were 93.0840, 92.1874, and they have achieved excellence.(3) Bovine collagen was enzymolysis by PEF and collagen polypeptide was prepared after purification. It solves the problems of temperature and short-term heating difficult to utilizing collagen. It was found that pepsin was activated by PEF, and the soluble protein content was increased 92.22%; while trypsin and papain were inhibited by PEF, and the soluble protein content were reduced to 5.56%, 9.84% respectively. So pepsin was chosen for bovine bone enzymolysis. Collagen polypeptide extraction optimization from bovine bone by PEF was studied through the Center Box-Behnken design and response surface method, and the Optimization conditions were: substrate concentration 1g/100mL, pepsin concentration of 3%, pH 2.5, field strength 21.98kV/cm, pulses number 7, Under these conditions, the soluble protein content was as high as 16.21mg/mL.The rough collagen solution was purified, and ethanol was chosen as collagen polypeptide purification preferred organic solvents, its purification time 8h, and adding 70%. The purified collagen polypeptide indicators were analyzed: SDS-PAGE electrophoresis results showed that the collagen has been broken down into polypeptide. An electrophoretic pattern of strip was clear, and the few miscellaneous shows a higher purity. Analysis of amino acid automatic analyzer results showed that the content of glycine, glutamic acid, alanine and proline are very high in the collagen polypeptide samples. It does not contain tryptophan, and the content of cysteine was rarely, in line with the characteristics of collagen. Collagen polypeptide was test by UV, and identified that it was the pure collagen polypeptide and has no other impurity protein.In order to verify the effect of high calcium products, the experiments of animal absorption was studied before the test of human body. Compared with calcium gluconate in the market, it was found that mice calcium absorption and utilization of very good with high calcium products, and the products were of high biological activity, and can effectively complement the body of calcium.The growth and development of the mice was very good through the experimental period. It was found that each group has significant changes by monitoring the mouse weight with the time. High calcium oral solution low-dose group> high calcium oral solution middle-dose group> high calcium beverage>high calcium oral solution high-dose group>high calcium chelate protein powder group>positive control group> blank control groupⅡ> blank control groupⅠ. The increase weight of high calcium oral solution low-dose group was the most significant, and it was higher than the blank control group 12%, positive control group 10%.The high calcium products in apparent absorption rate of mice are superior to the positive control group and blank control group; they have the significant impact on the thighbone of mice, especially the high calcium low-dose oral solution showed the superiority of the positive control group and the blank control group. It suggested that the high calcium products could delay the aging bones to a certain extent, and the effect of calcium supplementation was significant.The activity of TRAP, ALP and the content of Ca and P in blood serum were detected. It was found that the high calcium products have a positive role on the indicators of serum, and the effects are better than calcium gluconate oral solution. So the three high calcium products could be used as a good source of calcium in the body, and could be widely used in calcium supplements, food additives et al.(5) A theoretical analysis of PEF mechanism modeling was studied. Dynamic models were established and it was validated for the solid theoretical foundation for the future of the widely used of PEF.The basic principle of calcium ionization from bovine bone was analyzed systematically from the perspective of the chemical reaction, and chemical reaction dynamics model was established. The increment of activation energy was negative at all time, that is, PEF technology reduces the chemical reaction activation energy, and the chemical reaction was promoted. So the reason of the speed of chemical reaction increased rapid was explained by PEF, and the mechanism of the calcium fast ionization by PEF was illustrated too. At the same time, the confirmatory tests of chemical reaction were completed:①Chemical reaction of chiti deacetylation reaction under alkaline conditions was studied. The content of chitosan was 3.45 times as many as heating and 1.72 times as many as microwave. The speed of PEF was 210 times as fast as heating, 3.75 times as fast as microwave.②Starchy ether reaction of epoxy propyl methyl ammonium chloride and starch was studied. The positive ion groups replace degree of the starch was more 0.41% than the traditional wet method, 2.86% than microwave wet method. Reaction speed of PEF was 120 million times as fast as the traditional wet method, 2.5 times as fast as microwave wet method.The enzymolysis activated dynamic model was established. The dynamic equation of the rate of pepsin activation and field strength by PEF was:Y = 29.910 ? 6.865 x + 0.987 x 2 ? 0.030x3(R2=1,P<0.01),and the error of the model and experimental was only 0.69%; The dynamic equation of the rate of pepsin activation and pulses number by PEF was:Y = 12.852 x ? 1.590 x 2 ? 0.051x 3? 0.656(R2=1,P<0.01),and the error of the model and experimental was only 0.78%. So the suitability of enzymolysis activated dynamic model was validated, and a good reference and basis were provided for the PEF enzymolysis theoretical research and industrialization design.In summary, a new way of calcium and collagen polypeptide from bovine bone was explored. PEF technology has the advantages of non-thermal, reaction speed quick, efficient, easy to operate and low cost, which makes bovine bone resources greatly increase the value-added benefits, at the same time, environmental pollution and corporate profitability problems were solved. It has the very significant social benefit and economic benefit.