Effect Of Multi-mode Ultrasonic-assisted Extraction On Polysaccharide From Maca And In-situ Monitoring The Process And Evaluating The Immune Activity

Maca has been widely used as dietary supplements because of its high nutritional value.Maca is a high quality raw material for extracting polysaccharides because of the highest carbohydrate content of up to 59% in the basic components.The traditional extraction method doesn’t only has lower extraction rate but also needs longer extraction time.The ultrasound is widely used for the extraction of active ingredients in agricultural products as a new promising effective method.Many studies have shown that ultrasound can significantly improve the extraction rate and save time,it is green,safe and efficient technology.As extraction of continuous process,the key indicators of online real-time detection and the automatic judgment and decision on final implementation of the extraction process has the important value for the separation and extraction process of intelligent control.There is rarely reported on related research.In this paper,the extraction of Maca polysaccharides content by different ultrasonic extraction parameters and working modes was studied and optimized.The NIR was used to predict the polysaccharide content in the extraction process and determined the end point.The samples were applied to macrophages and determined the survival rate and the levels of pro-inflammatory cytokines(TNF-a and IL-6)to explore the toxicity and immune activity of polysaccharide.The main conclusions drawn by the research were as following:(1)Study on Ultrasound-assisted extraction of Maca polysaccharide:Taking the extraction of the polysaccharides content as the aim--the research was studied to find out the effect on polysaccharide content from different ultrasonic and working modes,and optimized the extraction parameters in the best working mode.The mid-infrared spectrum reveals whether ultrasound can changes the chemical bond of the polysaccharides.The experimental results showed that under the same treatment conditions,energy aggregation counter flow single-frequency ultrasound,energy aggregation counter flow dual-frequency ultrasound and divergence triple-frequency ultrasound can all significantly improve the extraction content of the polysaccharide,in which energy aggregation ultrasound was better than divergence ultrasound and alternating dual-frequency energy aggregation ultrasound was better than singlefrequency and synchronous dual-frequency energy aggregation ultrasound,the optimal mode was 20-35 kHz alternative dual-frequency ultrasound and the extraction time was 15 min.On the basis of this model,the optimum extraction condition were as follow: ultrasonic power per unit volume of 150W/L,ultrasound extraction time of 15 min,temperature at 50℃,the ratio of solid: liquid 1:10 g/ml,the on-time and off-time 4 s/3 s.Compared with traditional hot water extraction,the extraction rates of crude extract,crude polysaccharide and refined polysaccharide increased by 10.80%,29.40% and 44.90%,respectively.FTIR spectral analysis showed that the absorption peaks at 3226,2926,1026,923 cm-1 of ultrasound and without ultrasound extraction maintain consistency,indicating that the structure did not change.Further studies should be done on its function.(2)Study on on-line monitoring the extraction process of Maca polysaccharide:In-situ and real-time spectra were collected by near-infrared and used for monitoring the parameters of polysaccharide content during extraction process of Maca solution.The original spectra were pretreated by some different spectra data preprocessing methods,such as Centre,MSC,SG,SNV,1st,2nd,Centre and 1st,MSC and 1st,SG and 1st and SNV and 1st.According to the correlation coefficient R of calibration and prediction models,the root mean square error of cross-validation and root mean squared error of prediction,Si-PLS algorithm was used to set up calibrate regression model and selected the best spectral intervals.The experimental results showed that PLS model had the best effect after preprocessed on the original spectral.After the Si-PLS modeling,the optimal spectral intervals of polysaccharide were 1258.591-1316.735 nm,2027.121-2084.226 nm,2153.93-2210.888 nm and 2337.239-2387.696 nm.For the polysaccharide,the final model of Rc,Rp and RMSECV,RMSEP were 0.9621,0.9667 and 1.16%,1.37%,respectively.(3)Study on toxicity and immune activity evaluation:The polysaccharide samples after ultrasonic and hot water extraction were purified using DEAE-52 column,and then obtain fine polysaccharide which named MK-C and MK-R.UV spectroscopy was used to detect whether the protein was still present.The monosaccharide composition was identified by HPGPC.The mouse macrophages were cultured by the crude extraction of hot water or ultrasonic,MK-C and MK-R,and the cell activity was measured by MTT assay,cytokines existed in culture supernatant was measured by ELISA kit,in which to determine the toxicity and immune activity of polysaccharides in vitro.The results showed that MK-C and MK-R were purified by ultrasound and hot water polysaccharides from pure water,respectively;their extraction rate and purity were 10%,93.34% and 10%,95.6%;UV absorption spectra showed that there was only a peak at 200 nm which was the polysaccharide for the characteristic absorption peak,and there was no any absorption peaks in the wavelength 260 nm ~280 nm,indicating that MK-C and MK-R did not contain nucleic acid,protein,polypeptide and other impurities.The monosaccharide composition of MK-C and MK-R was arabinose,glucose,and glucose.Polysaccharide samples were not affected toxic on RAW 246.7 cells,on the contrary it has the role of promoting proliferation.Polysaccharides can significantly promoted the secretion of IL-6 and TNF-a by macrophages(p <0.05,p <0.01),and the MK-C and MK-R showed the best effect.The ultrasound-assisted extraction of polysaccharide promoted the release of IL-6 and TNFa on macrophages was better than hot water.