Research Of The Construction And Application Of Phytase Fingerprinting Database By Near Infrared Spectroscopy

With the rapid development of biology and fermentation engineering, the biotechnological enzymes which possessing high efficiency and specifically physiological function have become a research focus of new feed additives, and the production and application of the biotechnological enzymes are growing continuously. Due to the risk of biotechnological products, which always have been with a long incubation period and the serious damage, how to ensure and improve the safety level of feed enzyme, and how to scientifically utilize the super-advantage of feed enzymes in feed industry, has become a serious problem. This paper had collected 134 spray drying phytase and 121 adsorption drying phytase samples from the same manufacturers, the samples were scanned at the near infrared reflectance spectroscopy (NIRS) region of 950-1650nm. With constructing phytase MR fingerprint database, the product authenticity discrimination model, the phytase enzyme activity and water quantitative determination model, we exploited to introduce the MR fingerprint database into the phytase quality and safety management, and we found that it not only benefited the producers to improve the quality stability and protect the product brands, but also helped managers to establish an efficient phytase traceability system. Meanwhile, the paper also utilized high performance liquid chromatography, differential scanning calorimetry, spray drying experiments, and constant temperature and humidity storage experiments to investigate the composition and function characteristics of different phytase products, to further explore the relationship between the near infrared spectral information and the key indicators of quality. Specific results were as follows:(1) We investigated and established the phytase product MR fingerprint database which could clearly distinguish the phytase products from different manufacturers, different form. The phytase product MR fingerprint database could provide scientific and rational methods for authenticity identification and brand protection.(2) With the Principal Component Analysis (PCA) to construct two-dimensional map, we extracted the near-infrared diffuse reflectance spectra principal components from the phytase of different manufacturers at the MR range of 950-1650nm. The results showed that the different models, the cumulative contribution rate which reflecting the sample information was over 90% from the first two principal components. The same manufacturers, the same pattern of phytase products could gather into one group; an established NIR qualitative model used to discriminate 15 unknown samples, the accuracy rate was 100%, this showed that principal component analysis could be used to establish a qualitative model for evaluating the authenticity of phytase products. With Mahalanobis distance and corresponding threshold, it could also make further discrimination for their quality and stability.(3) Multiplicative Scatter Correction (MSC) was chosen as scatter correction, and the first derivative and smooth combination chosen as spectral pretreatment methods. In the optimal wave scope, using internal cross-validation, based on Root Mean Square Error of Cross Validation (RMSECV), the optimal factor was determined. The calibration models to predict the phytase activity and moisture content were developed using Partial Least Squares (PLS-1) technique. The coefficient of determination in calibration (R~2) of moisture, activity in phytase were all over 0.90, the Relative Standard Deviation (RSD) all less than 10%, and the Relative Predictive Determination (RPD) all over 3; the R~2of spray drying phytase was 0.937, the RSD and RPD of spray drying phytase were 5.23% and 3.64 respectively; the R~2 of adsorption drying phytase was 0.926, the RSD and RPD of adsorption drying phytase were 6.21% and 3.45 respectively. The results showed that NIRS analysis technique could be adopted to correctly measure the activity in spray drying phytase and adsorption drying phytase.(4) We also exploited to establish a NIR method for fast-tracking the quality changing of the spray drying and storage process of phytase products.