| As a natural macromolecular substance in food,polysaccharides not only have high nutritional value,but also can be widely involved in the regulation of various important life activities and physiological processes such as cell recognition,cell differentiation,signal transduction and immune response.Diversity of polysaccharide structure gives it different functions and bioactivities,and different processing methods have significant effects on the molecular structure and solution chain conformation of polysaccharides.How to control the molecular structure and solution chain conformation of polysaccharides to produce highly active polysaccharides has become the focus in multiple areas.Subcritical water extraction has shown its unique advantages and good application prospects in polysaccharides extraction because of its advantages such as high efficiency,non-toxicity,environmental protection and economy.However,the research on the extraction of polysaccharides with subcritical water is still based on the basic information such as the structure and functional characteristics of the final product,and the basic theory of structure,chain conformation and biological activity changes of polysaccharides caused by parameter changes during the extraction process has not been reported.Due to the relative lack of these basic information,the development of subcritical water extraction technology is relatively lagging behind.Based on this,this paper intended to use Lentinus edodes polysaccharides as the research object,and took the microenvironmental changes of subcritical water extraction as the starting point.Through physical,chemical,and biological research methods combined with the polymer solution conformation theory,the effect of subcritical water extraction temperature on the chemical structure,chain conformation,and biological activity of Lentinus edodes polysaccharides was studied.The structure and biological activity changes with the change of microenvironment are revealed,which may lay a theoretical foundation for the research on the preparation process with subcritical water extraction of highly active polysaccharides.The main contents and results are as follows:（1）Based on the single factor test,the extraction rate was as the response value,the optimal process conditions for subcritical water extraction of Lentinus edodes polysaccharides were obtained by using response surface test optimization:extraction temperature 150℃,extraction time 20 min,and liquid-to-material ratio 30:1 mL/g,the maximum extraction rate under this condition is 12.01±0.27%,which is 111.1%higher than the traditional method,and the contribution of different extraction parameters to the extraction rate is:extraction temperature>extraction time>liquid-material ratio.Multiple stepwise regression analysis verified that the correlation between extraction temperature and extraction rate was closer.Pearson correlation analysis showed that the Pearson correlation coefficient between the subcritical water extraction temperature and the extraction rate is 0.692,and the two-sided test value is 0.002<0.01,which showed a significant correlation between subcritical water extraction temperature and yield.However,there was no correlation between extraction time/liquid-to-material ratio and extraction rate.Temperature is the most important factor when using subcritical water to extract Lentinus edodes polysaccharides.（2）A Lentinus edodes polysaccharide with a relatively uniform molecular weight was prepared by an alkaline extraction method with content of 98%.It was a white flocculent solid that did not contain monosaccharides,polyphenols,uronic acid,proteins,nucleic acids and other substances.This polysaccharide was as the research object,and the effect of subcritical water extraction temperature on the chemical structure and its changing rules were studied.The results showed that the Lentinus edodes polysaccharides treated with subcritical water at 100-160℃were allβ-glucans.With the increase of extraction temperature,the molecular weight of polysaccharides gradually decreased from 7.79×105 Da to 8.721×104 Da.Each polysaccharide is composed ofβ-1-D-Glcp,β-1,3-D-Glcp,β-1,6-D-Glcp,β-1,3,6-D-Glcp residues.As the extraction temperature increased,the proportion of 1→3 or 1→3,6 sugar residues gradually increased,while the proportion of 1→or 1→6 sugar residues gradually decreased.（3）The Congo red experiment,viscosity method,and polymer dilute solution theory were used to study the chain conformational transition of Lentinus edodes polysaccharides extracted with subcritical water at different temperatures.The results of Congo red experiments and viscosity method showed that the polysaccharides treated at 100-150℃had a triple-helix conformation,while the polysaccharides treated at 160℃were not triple-helix conformation.[η]and SEC-MALLS were used to determine the Mark-Houwink equation（[η]=kMwα）of polysaccharides treated at different subcritical water temperature.The indexαof Lentinus edodes polysaccharides treated at 100-150℃was between 1.16 and 1.47,while the value ofαat 160℃was0.66.In the dependence of<S2>z1/2 and Mw of<S2>z1/2=KMwa,the a of Lentinus edodes polysaccharide treated at 100-150℃was greater than 0.6,while a at 160℃was 0.54.The indexesαand a indicated that the Lentinus edodes polysaccharides treated at 100-150℃has a rigid chain structure,while the Lentinus edodes polysaccharide treated at160℃is a compliant polymer.The results of atomic force microscopy and scanning electron microscopy have further proved that Lentinus edodes polysaccharide treated with subcritical water at 100-150℃is rigidly triple helix conformation,while Lentinus edodes polysaccharide treated at 160℃is a linear compliant polymer aggregate.During subcritical water extraction process,150℃was determined as the chain conformational transformation temperature.（4）The chemical system and in vitro cell model were used to study the antioxidant activity,immunomodulatory activity and antitumor activity of Lentinus edodes polysaccharides extracted with subcritical water at different temperatures.The results showed that the Lentinus edodes polysaccharides treated at 110-130℃has stronger ability to scavenge DPPH free radicals,ABTS free radicals and reducing power than the Lentinus edodes polysaccharides treated at 150-160℃,and it has a stronger ability to protect H2O2-induced RAW264.7 cell damage（p<0.01）,and promoted ROS level and SOD activity（p<0.01）,showed higher antioxidant activity in vitro.The Lentinus edodes polysaccharides treated at 120-140℃can more effectively promote the proliferation and phagocytosis of macrophages,and more effectively promote the secretion of cytokines such as NO,TNF-α,IFN-γ,and IL-2,which exhibit higher immune activity in vitro.The Lentinus edodes polysaccharides treated at 120-130℃can better inhibit the proliferation of tumor cells HepG2 and MCF-7 cells,showed higher antitumor activity,but the relative inhibition rate is different.（5）Slit divergent ultrasonic-assisted wet heat treatment was used to prepare LNT-SPI conjugates.The effects of ultrasound on the structural properties,functional characteristics and emulsion stability of the conjugates were studied.The results showed that the slit divergent ultrasound can significantly improve the glycosylation efficiency of LNT（treated with subcritical water at 130℃）and SPI.Among them,the grafting degree of LNT-SPI conjugates prepared by ultrasonic-assisted wet heat treatment for 40 min was as high as 26.48%,which was 1.91 times higher than the traditional wet heat method.The ultrasonic-assisted wet heat method significantly changed the secondary structure,fluorescence intensity,surface hydrophobicity,and surface morphology of the LNT-SPI conjugates,and further significantly improved the solubility,foamability,emulsification,thermal stability,and apparent viscosity（p<0.01）.In addition,the oil-in-water emulsion of LNT-SPI conjugates has strong stability against environmental stresses（pH,heat treatment,and ionic strength）. |
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