Studies On Isolation, Purification, Idenfication And Immune Functions Of Polysaccharopeptide From The Natto

Natto is a traditional fermented food made by fermenting boiled soy beans withBacillus natto. Natto exerts several health-promoting effects, such as thrombus dissolving,anti-tumor, anti-hypertensive, estrogen regulation and osteoporosis prevention. Severalbioactive components responsible for these pharmacological effects have been isolated.These compounds include Nattokinase, vitamin K2, surfactin isoflavone andpoly-γ-glutamic acid, et al. However, despite numerous studies on Natto, limitedinformation on the biological activities of glycoconjugates is available. This study takesdegreased natto powders as raw material, optimizes the extractive craft and produces twohigh purified Bacillus natto polysaccharopeptide complexes (BNP) through a series ofmodern separation and purification technology from crud BNP. Their structure andimmunomodulatory effects on splenocyte proliferation and cytokine expression were alsodiscussed. The main contents and results of experiments are as following.1、Response surface methodology (RSM) was used to optimize technological parametersfor ultrasonic-assisted extraction of glycopeptide from natto. Single factor experienceswere carried out to investigate the effects of liquid/material ratio, ultrasonic power andultrasonic time on extraction yield of crude BNP. Further, the process parameters wereoptimized using a Box-Behnken experimental design coupled with response surfacemethodology. The results show: liquid/material ratio (mL/g)24:1, ratio of NaClconcentration0.15mol/L, ultrasonic time41min, under this optimal conditions, the highestextraction yield of crude BNP was1.89%.2、Two novel polysaccharide-protein complexes, termed BNP-1-b and BNP-2-a, wereextracted and purifed from Natto via ion-exchange and gel fltration chromatography. Themolecular weights (Mw) of the complexes, determined using high-performance gelpermeation chromatography, were26.93and357.6kDa, respectively. The two complexeswere further characterized using FT-IR, UV-visible spectrophotometry, monosaccharideand amino acid composition analyses. Our results showed that BNP-1-b contains anO-glycosidase while BNP-2-a contains an N-glycosidase. BNP-1-b was principallycomposed of Man, Rha and Ara, with molar percentages of67.45%,25.67%and2.44%,respectively. However, BNP-2-a was mainly composed of Glc, Man, Xyl, Gal and little Ara,with contents of36.64%,21.71%,10.35%,9.00%and5.17%, respectively. Seventeenamino acids were identified in both complexes.3、Immunosuppressed model mice were established by ip with Cyclophosphamide for threetimes. After ig administration of BNP for30d, the effects on their immune organ weightindex, hemolysin content, lymphocyte transformation capability, cytokines secretion andspleen lymphocyte proliferation response in vitro were examined to investigate its immunomodulatory effects. The results showed that the spleen index and medianhemolytic dose (HC50) in middle and high BNP groups were significantly increased thanthat in CTX group (P <0.05, P <0.01), so does the thymus index in high BNP group; Thelymphocyte transformation capability in three BNP groups were significantly stronger thanthat in CTX group (P <0.05, P <0.01); Serum IL-2in middle, high BNP groups and IFN-γin high BNP group were significantly enhanced than that in CTX group (all P <0.05),while serum IL-10in both three BNP groups were much decreased (P <0.05, P <0.01). Inaddition, BNP-1-b (100~200μg/mL) and BNP-2-a (12.5~200μg/mL) can significantstimulated the proliferation activities of splenocyte in vitro, the effects were also observedin ConA-induced splenocyte proliferation activities (P <0.05, P <0.01).