| Traditional Chinese medicine(TCM)has the characteristics of multiple components,multiple targets,multiple effects,and multiple variables and factors in the production process.In traditional methods,process parameters(such as extraction time and times)are usually fixed to determine the end point of a single process,while process analysis technology(PAT)determines the end of a process by determining whether the content of active components reaches the maximum value.The quality of TCM by design.It is necessary to introduce safe,scientific and effective PAT and measure the content of key indicators in the process to ensure the stable transmission of active ingredient quality in the process to further control the quality of the final product.Near-infrared spectroscopy(NIRS)analysis technique is one of the PAT,widely used in TCM in process quality control,has the characteristics of fast,no damage to the sample,the production unit can feedback in time,enhancing the quality control,ensure the quality of TCM homogeneity and stability,solve the problem of accurate and rapid detection of the active ingredients of TCM,improve the production efficiency and promote the modernization research of TCM.Fermented Cordyceps sinensis preparations are mainly a kind of products produced by Jiangxi Jimin kexin Jinshuibao Pharmaceutical Company for treating deficiency of the lungs and kidneys.Based on the previous research of the research group using TCM fingerprint,quantitative analysis of multicomponents(QAMS),principal component analysis(PCA)and cluster analysis(CA)for quality control methods of nucleosides,polysaccharides,amino acids,and sterols in fermented Cordyceps sinensis preparation products.In this study,PAT was introduced into the production process of fermented Cordyceps sinensis(strain culture,strain passage,strain fermentation,ferment filtration,ferment drying,ferment pulverizing,ferment mixing),NIRS combined with stoichiometry was used to establish a rapid determination model based on the TCM fingerprint.The contents of nucleosides and sterols in different operation units were measured by high performance liquid chromatography(HPLC),the contents of total polysaccharides were determined by phenol-sulfuric acid method.The quantitative analysis model of the components in the production process of fermented Cordyceps sinensis was established by combining NIRS with PLSR,and the spectral modeling model,spectral pretreatment method,wave number range,the absolute error between the chemical measured value and predicted value of the model,the accuracy,precision,repeatability and stability of the model were investigated.The optimal quantitative analysis model was screened by the model root-mean-square error of cross-validation(RMSECV),correlation coefficient and principal component number,and carried out qualitative discriminant analysis for different operation units in the range of each ingredient absorption wavenumber.In addition,compare nucleosides,polysaccharides and sterols content differences in each production node(pre-membrane sample,plate-and-frame sample,fermentation sample,post-membrane filtrate solution,post-membrane concentrated solution,plate and frame filtrate solution,plate and frame concentrated solution,fermentation filtrate solution,clear solution,and post-digestion solution)under different filtration equipment(plate and frame filter with ceramic membrane filtration),aim to control the quality and improve the production efficiency of fermented Cordyceps sinensis.The specific research contents and results are shown in the following aspects:1.Determination of the content of fermented Cordyceps sinensis in the production process Fermented Cordyceps sinensis production process were taken as the research object,and the qualitative and quantitative analysis of nucleosides and sterols ingredients were conducted by HPLC combined with TCM fingerprint similarity analysis.The similarity between different operation units and batches reached above 0.9.The average content of total nucleosides in the production process were 11.88 ±0.50mg/g,10.47±0.45mg/g,14.66±0.56mg/g,14.26±0.30mg/g,12.34±0.53mg/g,11.82 ±0.44 mg/g,11.27±0.62 mg/g,respectively;The content of daucosterol were 0.38 ± 0.03 mg/g,0.37± 0.03 mg/g,0.38±0.03 mg/g,0.39±0.03 mg/g,0.38±0.03 mg/g,0.38 ±0.03 mg/g,0.38±0.03 mg/g,respectively;The content of ergosterol were 1.69±0.31 mg/g,2.01±0.19 mg/g,1.98±0.25mg/g,1.96±0.25mg/g,2.13±0.18mg/g,1.83±0.19 mg/g,1.87±0.12 mg/g;The content of stigmasterol were 0.18±0.01mg/g,0.17±0.01 mg/g,0.18±0.01mg/g,0.18±0.01mg/g,0.18±0.01mg/g,0.18±0.01mg/g,0.18±0.01 mg/g;The content of β-sitosterol were 0.16±0.01mg/g,0.15±0.01mg/g,0.16±0.01 mg/g,0.16±0.01mg/g,0.16±0.01 mg/g,0.16±0.01mg/g,0.16±0.01mg/g.The results showed that nucleosides and sterols in fermented Cordyceps sinensis production process with high process stability and consistency among batches.Total polysaccharides contents were determined by phenol-sulfuric acid method.The results of external standard method showed that the average total polysaccharides contents among different batches in the production process were 70.11±11.18%,64.26 ±9.69%,67.04±10.91%,66.23±9.17%,65.69±8.15%,67.49±8.98%,and 66.62 ± 5.83%,respectively.The results showed that the quality of total polysaccharides in fermented Cordyceps sinensisthe production process were relatively uniform and stable.2.Near-infrared spectroscopy(NIRS)combined with partial least squares regression(PLSR)method to establish a quantitative analysis model The discriminant analysis was used to qualitatively analyze fermented Cordyceps sinensis in different processes,and the results showed that the discriminant distances of different nucleosides,polysaccharides and sterols were relatively consistent,mostly within the range of 0.5-1.5,indicating that the similarity of fermented Cordyceps sinensis in different processes were relatively high.Based on the contents determined by HPLC and phenol-sulfuric acid methods,and introduced into NIRS,compare different model parameters difference(root-mean square error,correlation coefficient,and principal component number)under different pretreatment methods(first derivative,second derivative and S-G smoothing)and compare the quantitative analysis model predicted values difference of nucleoside extract and fermented Cordyceps sinensis,the results showed that there were no significant difference on the two models predicted values.However,compared with the NIRS of the fermented Cordyceps sinensis,the NIRS of the extract is relatively noisy and hydrogen bonding in water may interfere with the establishment of the model.Finally,the original spectrum of the fermented Cordyceps sinensis was selected to establish the PLSR quantitative analysis model for each component.The absolute error of total nucleosides between the model predicted value and the chemical measurement value was within 0.33 mg/g,the absolute error of total polysaccharides was within 0.08 %,and the absolute error of ergosterol was within 0.13 mg/g,indicating that the established model could be used to accurately and rapidly measure the content of active ingredients in fermented Cordyceps sinensis production process.3.Process optimization of ferment filtration and application of near-infrared spectroscopy(NIRS)quantitative analysis model Taking the water content,the yield of strain,and the degree of filter membrane thickening as indicators,the membrane diameter,filtration pressure,filtration temperature,filtration time and circulation time in the ceramic membrane filtration system were investigated.Contrastive analysis the difference between plate and frame filtration and ceramic membrane filtration based on the measured content of each ingredient.The results show that the ceramic membrane filtration uses a dynamic "cross-flow filtration" method to achieve the purpose of separating,concentrating and purifying the strain solution,which is beneficial to improving the production environment,reducing waste water discharge,reducing environmental protection pressure,and improving the quality and yield of the wet mycelium of fermented Cordyceps sinensis,better than plate and frame filtration,and more in line with GMP requirements.The model verification results confirmed the feasibility of the established near-infrared PLSR quantitative analysis model,which can be used to determine the content of each ingredient.The above research results show that there is no significant difference in batch quality during fermented Cordyceps sinensis production process,the similarity is higher,and the quality is more uniform and stable;The quantitative analysis model established by NIRS combined with the PLSR method can accurately and quickly measure the content of total nucleosides,total polysaccharides,and sterols.It can be used for process control and improve the process quality measure speed of fermented Cordyceps sinensis preparations;Ceramic membrane filtration is better than traditional plate and frame filtration,which can save the time of secondary filtration and improve the utilization rate of filtrate,it can be used as a new process for the automatic upgrading and transformation of production processes. |
