Optimal Extraction Of Polysaccharides From Bee Collected Pollen Of Chinese Wolfberry And Exploration Of Its In Vitro And In Vitro Immunomodulatory Activity

Bee pollen is a kind of "super food",a nutritional food with high biological value ingredients.The bee pollen of Chinese wolfberry is a pollen mass formed by bees adding some nectar and saliva during the process of collecting pollen from wolfberry,and its functional research on nutritional components is rarely reported.In recent years,natural polysaccharides have received more and more attention due to their multiple biological functions,and have become a popular dietary nutrient.Therefore,it is significant to discuss the polysaccharides from bee collected pollen of Chinese wolfberry(WBPPS).Previously,our laboratory has carried out a systematic study on the isolation and purification,physical and chemical properties,in vitro simulated digestive properties,intestinal beneficial life,and antioxidant activity of the WBPPS.In this study,the optimized extraction and the in vitro and in vivo immunomodulatory activities of the WBPPS have been studied in detail,which can lay a certain theoretical foundation for the development and utilization of the polysaccharides from bee collected pollen of Chinese wolfberry.The main results are as follows:1.Optimal extraction of WBPPS and its rheological propertiesIn this section,the temperature difference-ultrasonic wall broken method is used to optimize the extraction of the polysaccharides from bee collected pollen of Chinese wolfberry(WBPPS)which based on the existing extraction methods.Effects of extraction parameters including the temperature difference of the wall broken,the power of the ultrasound,and the time of the ultrasound on the yields of WBPPS were investigated by single-factor tests.A three-level,three-variable Box-Behnken design(BBD)was applied to determine the optimal levels of extraction variables for the extraction of WBPPS.By using the software of Design Expert version 8.0.6,the optimum extraction parameters were obtained as follows:temperature difference of wall broken 90℃,ultrasonic power 400W,ultrasonic time 40min,here Under the conditions,the experimental value was 2.73%,which was more than doubled before optimization.The effects of concentration,temperature,pH,sucrose and NaCl on the static rheological properties of WBPPS were investigated.The results showed that in the concentration range of 0.5%-6%,the apparent viscosity of WBPPS increased with the increase in the MLP concentration,indicating that the WBPPS is a pseudoplastic fluid.In addition,the viscosity of the solution of WBPPS showed a trend of rising first and then decreasing with the increase of the pH value.The addition of sucrose made an increase of the viscosity,which was positively correlated with the concentration of sucrose.The apparent viscosity of WBPPS did not obviously changed with addition of low concentration of NaCl,and gradually increased with further increase of the NaCl concentration2.Immune activity of WBPPS on mouse macrophage RAW264.7The cell model was used to evaluate the in vitro immune activity of crude WBPPS and purified components WBPPS-1,WBPPS-2,and WBPPS-3.The results show that WBPPS,WBPPS-1,WBPPS-2,WBPPS-3 can effectively promote the proliferation of RAW264.7 macrophage cells;it can also effectively increase the release of RAW264.7 NO and cytokines TNF-α,IL-1β and IL-6.At the same time,it also promoted the mRNA expression levels of TNF-α,IL-1β and IL-6.In addition,compared with WBPPS,WBPPS-1 and WBPPS-2,WBPPS-3 showed greater beneficial immune stimulating activity.Therfore,WBPPS-3 has the potential to be explored as a promising candidate as antioxidant and immunomodulator for human consumption.3.Immunoenhancement of WBPPS on immunosuppressed mice and its regulating effect on intestinal microorganismsIn this section,the effects of WBPPS on immunoregulation and gut microbiota dysbiosis in cyclophosphamide(CTX)-induced mice were investigated to illuminate whether the attenuation of immunosuppression being related to the modulation of gut microbiota.The results showed that WBPPS promoted the immune function and regulated the diversity and composition of gut microbiota in CTX-treated immunosuppressed mice.WBPPS could modulate gut microbiota by restoring the relative abundances of Family_ⅩⅢ,Saccharimonadaceae,Ruminococcaceae,and as well as decreases of Tannerellaceae,Rikenellaceae in CTX-treated mice.Furthermore,WBPPS could repair the thymus and spleen indices and stimulate the production of TNF-α,IgA,IL-10 and TGF-β1 in serum.WBPPS improved intestinal barrier and activated the colonic TLR4/MyD88/NF-κB p65 pathways.In addition,the administration of WBPPS also effectively prevented the hepatotoxicity induced by CTX through anti-oxidant activities.On the basis of these findings,WBPPS could potentially serve as an effective immunomodulator.