| Bone resources are very rich in China, but there is a great waste in the utilization ofbone resources. Enzymatic hydrolysis of bone protein is an effective way to improve theutilization of animal bone resources, which produces high quality proteinpeptone.Peptone is a mixture of nitrogen source composed by the proteose, peptone,polypeptide, amino acid that formed after enzyme hydrolysis. It can provide the Nsource for microbial growth.In this study, low temperature protease was used to hydrolyze bovine protein.Thesingle factor experiments were considered according to pH, temperature, enzymeaddition and reaction time to optimize the condition for bovine-bone peptone hydrolysis.Based on the results of signle factor optimization,3important factors were selectedwhich affect ammonia nitrogen significantly, pH,temperature and reaction time.Theoptimum hydrolysis conditon was obtained by the response surface methodology (RSM)with temperature38℃, pH7.1, reaction time77min,enzyme addition150U/mL. Theammonia nitrogen content reached to4.55%,.To further investigate the interaction of factors in the process of the the bovine bonehydrolysis by low-temperature protease, The hydrolytic kinetic experiment wasdesigned, the dynamics equation of protease hydrolysis based on experimental dataand formula:dh=aexp(-bh)=38.8194e01.17h The Michaelis constantKm was0.7g/L, indicating that the affinity of the enzyme and substrate was relativelylarge. The maximum reaction rate Vmax was0.541mol/L.min, which meant the speedof hydrolysis reaction was fast. Activation energy Ea4.012KJ/mol, lower activationenergy showed that low-temperature protease was more sensitive to temperature. Hightemperature makes the enzyme lost activity easily.The results of enzymatic kineticsexperiment can provide parameters for the fermentation in the future, the hydrolysis temperature of low-temperature protease is generally lower than the temperature ofmedium-temperature enzyme, while the hydrolysis time is very short.The quality of peptone products normally is test as the content of ammonia nitrogenand total nitrogen according to factory standard. The size and distribution of the peptidefragment as nitrogen source have a great influence on microorganism growth andfermentation. SDS-PAGE gel electrophoresis was used to primarily determine thedistribution of peptide fragments,the results showed that the hydrolysis of peptide hadcontinuous distribution from the200KD to6.5KDa.With the extension of hydrolysistime, peptides of large molecular were hydrolyzed into low molecule peptides or freeamino acids. To further determine the distribution of peptide, we selected three types ofgel chromatography with different MW ranges, Sephadex G-100, Sephadex G-50, andSephadex G-25. The results indicated that peptide of45KDa to150KDa accounted for14.83%of the total amount of cattle bone peptone,11.7KDa to45KDa accounted for30.37%,1KDa to11.7KDa accounted for34.16%and smaller than1KDa accounted for10.74%of the total protein. The content of amino acid is an important standard todetermine the peptone quality. According to amino acid autoanalyzer,the total contentof free amino acids in bovine-bone peptone after hydrolysis is60.21%higher than thatbefore hydrolysis,among which the essential amino acids accounted for26.72%, halfessential amino acids accounted for5.02%. Flavor amino acid accounted for46.3%,bitter taste amino acids accounted for34.57%.Compared with commodity peptone(domestic) and tryptone(import), bovine-bonepeptone we obtained showed higher content of ammonia nitrogen, but Gelchromatography showed that commodity peptone and tryptone peptone contented morepeptide of smaller molecular. By amino acid analysis, this conflict was proved to bedue to a large number of bone protein broken down into free amino acids in thehydrolysis. The total amino acids and free amino acids in bovine bone protein weresignificantly higher than those in commodity peptone and tryptone. |
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