Study On Sterilization And Increasing Immune Activity Of Cordyceps Militaris Fresh Medicine By High Pressure

Fresh Chinese herbal medicine has some unique curative effects due to the varieties of active ingredients. Making fresh medicine with fresh herbs juicing attracted widespread attention. The sterilization and maintenance of medicinal activity will be key to the fresh medicine processing and utilization. Cordyceps militaris has extensive pharmacological effects, hence it has high medicinal value.In order to effectively kill microorganisms in C. militaris fresh juice and improve the pharmacological activity, with the aim of exploiting fresh medicine of C. militaris, immunosuppressed mice model was used as pharmacological index in this article. We firstly evaluated the differences in immune activity of fresh and dry C. militaris, then studied the contribution of all polysaccharides and cordycepin to immune activity. The immune activity was associated with chemical information using multivariable correlation analysis to study the the material basis of differences in immune activity of fresh and dry C. militaris, which was used as pharmaceutical chemistry index. We processed the C. militaris fresh juice using ultra-high pressure to study the effective sterilizing technology and the better technology for keeping activities. The two technologies were optimized to get the optimal, and then investigated the impacts of the optimal technology on immune activities of fresh medicine. We selected the appropriate enzyme to combine the technology of sterilization and keeping activity to process the C. militaris fresh juice, and study the effects of ultra-high pressure on release of active ingredients and immune activity. Then the technology of increasing dissolution rate of active ingredients in C. militaris fresh juice by enzyme combined with ultra-high pressure with response surface method. The results are as follows:1) The immune-enhancing activities of fresh C. militaris was stronger than dry C. militaris, the material basis of which was the higher contents of cordycepin, total polysaccharides and the polysaccharides of averaging molecular weight 300 kDa,250 kDa in fresh C. militaris.Compared to dry C. militaris, the aqueous extract of fresh C. militaris had stronger effects on enhancing the organ index, phagocytic activity of macrophages, spleen lymphocyte proliferation, IL-2 and INF-y levels in mice with cyclophosphamide (Cy)-induced immunosuppression (.P<0.05). Multivariable correlation analysis of efficacy and chemical fractions showed that cordycepin and total polysaccharides were closely related to differences in immune activityies of fresh and dry C. militaris, and the contents of which in fresh C. militaris was 252.95% and 30.99% higher than in dry C. militaris.Total polysaccharides of fresh and dry C. militaris, and components’molecular weight were analyzed using high performance liquid chromatography. The efficacy of polysaccharide components and chemical composition were associated using multivariable correlation analysis. The results showed that the main chemical compositions of differences in immune activity of total polysaccharides in fresh and dry C. militaris were the polysaccharides of averaging molecular weight 300 kDa and 250 kDa, the contents of which in fresh C. militaris were 104.65% and 123.71% higher than in dry C. militaris.2) The better ultra-high pressure processing of effective sterilization and keeping activity was: 420 MPa,12 min, solid-liquid ratio 1:3 and pressure for 1 time.The effective ultra-high pressure processing of sterilization was:261 MPa,14 min, solid-liquid ratio 1:2 and pressure for 1 time. The effective ultra-high pressure processing of keeping activity was: 420 MPa,12 min, solid-liquid ratio 3:1 and pressure for 1 time. The two processes were optimized to get the optimal processing of sterilization and keep activity for fresh C. militaris juice:420 MPa,12 min, solid-liquid ratio 1:3 and pressure for 1 time. Under the processing, the clones of bacteria, yeasts, molds and Escherichia coli by the processing were 728,67,51 and 0 cfu every 1 g extract, which significantly lower than the limited value of oral medicine preparation defined by the "People’s Republic of China Pharmacopoeia".Compared to ultrasonication (control), the process improved the contents of cordycepin and polysaccharides by 36.79% and 23.34%, also promoted the immune-enhancing activities of C. militaris juice. HPLC detection showed that the process effectively hold the polysaccharide of averaging molecular weight 300 kDa,250 kDa.3) Cellulose combined with ultra-high pressure processing dramatically improved the immune activity of C. militaris juice.The effects of ultra-high pressure processing on the activities of pectinase and celluase were studied, and the results showed that 300-500 MPa could inhibit the activities of pectinase, while promoted the activities of celluase.C. militaris juice was processed by cellulase combined with ultra-high pressure to study the processing technique of further improving active ingredients and enhancing immune activities in C. militaris fresh medicine. Compared to the processing of sterilization and keeping activity, the processing of cellulase combined with ultra-high pressure further enhanced the contents of total polysaccharides, cordycepin, and the polysaccharides of 300 kDa and 250 kDa by 23.2%,6.786%, 69.56% and 49.17%. Also the immune-promoting activities were further enhanced.The ultra-high pressure processeing combined with cellulose was optimizated with response surface method. The results showed that the best processing of enhancing activity was:410 MPa,14 min, solid-liquid ratio 1:2.5, enzyme adding amounts 2.5% and pressure for 1 time, which further enhanced the contents of total polysaccharides, cordycepin, and the polysaccharides of 300 kDa and 250 kDa by 24.21%,7.45%,75% and 48.89%, respectively. Microbiological detection showed the clones of bacteria, yeasts, molds and E. coli were 654,41,44 and 0 cfu/g every 1 g extract, which was significantly lower than the limited value of oral medicine preparation defined by the "People’s Republic of China Pharmacopoeia".In conclusion, the process of ultra-high pressure was able to effectively kill the microorganism in C. militaris fresh juice, promote the dissolution of active ingredient, and improve the immune-enhancing activities of C. militaris fresh juice.