Study On The Structure And Immune Activity Of Extracellular Polysaccharide From Antarcticum Penicillium Chrysogenum MS-02

Exopolysaccharides(EPS)are water-soluble polysaccharides secreted extracellular by specific microorganisms during growth and metabolism.It can protect thalli from environmental stress,promote the absorption of nutrients and colonization of thalli.Fungal exopolysaccharides have biological activities such as improving immunity,anti-tumor and anti-radiation.In this study,Penicilliμm chrysogenum MS-02,a fungus isolated from the rot of Antarctic seaweed,was used as experimental material.First,we investigate the effect of temperature on its growth.Then a extracellular polysaccharide PPS2-A was isolated and purified from its fermentation broth,Last the structural characteristics and immune activity of PPS2-A were studied.The specific results are as follows:1.Effects of fermentation time and temperature on the growth and exopolysaccharide secretion of P.chysogenum MS-02.MS-02 belongs to the genus Penicillium chrysogenum.In liquid fermentation at 10℃,mycelia grew continuously with the extension of fermentation time.Before 120 h,mycelia growth increased exponentially,and then entered a stable period.The content of polysaccharide in fermentation liquid decreased gradually with the extension of fermentation time,increased from 96 h to 144 h,and then continued to decline.P.chrysogenum MS-02 can grow normally at 5℃ to 25℃.Between 5℃ and 15℃,the mycelial growth rate was positively correlated with temperature,while the contents of protein and polysaccharide were negatively correlated with temperature.In total,the optimal culture temperature and duration for EPS production were 10℃ and 144 h.2.Separation and purification of the exopolysaccharide from P.chrysogenum MS-02.Crude polysaccharide PPS were isolated from P.chrysogenum MS-02 fermentation solution.First,PPS were separated by SephacryL S-300 and obtained PPS 1 and PPS-2.PPS-2 was the main component of PPS.accounting for 71.4±1.68%,and then was purified by DEAE-Fast Flow.PPS2-A,PPS2-B and PPS2-C were obtained by elution with 0.0 M,0.1 M and 0.2 M NaCl,respectively.Among them,PPS2-A was neutral polysaccharide,accounting for 56.2±2.19%of PPS-2.PPS2-A was determined to be free of protein and nucleic acid by UV scanning and BCA detection,and was detected as a single peak by HPLC.The results showed that the uniformi of PPS2-A was high.3.Structure analysis of exopolysaccharide PPS2-A.The monosaccharide composition and molecular weight analysis showed that PPS2-A was glucan.with a molecular weight of 8526 Da.Methylation analysis showed that PPS2-A is dominated by α-D-(1.4)-Glcp,combined with one-and two-dimensional NMR maps,the first order structure of PPS2-Awas inferred as:PPS2-A has good solubility,does not change color when encountering iodine,and has a certain tolerance to α-amylase.AFM atomic force morphology showed that the images of PPS2-A were all round spots.Compared with the control group.λmax UV absorption of PPS2-A and Congo red complex showed no obvious redshift with the increase of NaOH concentration,and the maximum absorption peak of PPS2-Congo red complex was found at 350 nm,indicating that there was no triple helix structure and there might be more branches.4.Immunoactivity analysis of PPS2-A.PPS2-A could promote the proliferation of splenic lymphocytes in mice.At 100 μg/mL,the splenic lymphocyte proliferation rate in PPS2-A treatment group was 2.94 times higher than that in control group.PPS2-A not only promoted the proliferation and phagocytosis of mouse peritoneal macrophages,but also increased its NO secretion and acid phosphatase activity.Compared with control,the proliferation rate,phagocytosis capacity,NO secretion and acid phosphatase activity of peritoneal macrophages in 50.0 μg/mL PPS2-A treatment group were increased by 61.5%,87.7%,120.0%and 45.0%,respectively.RAW 264.7 is a mouse mononuclear macrophage cell line,and PPS2-A can promote cell proliferation,inhibit cell apoptosis,improve phagocytosis activity,and stimulate cytokine transcription and expression in mouse monocyte macrophage cell line RAW 264.7.The proliferation rate,apoptosis rate and phagocytosis index in 25.0μg/mL PPS2-A treatment group were 131.6%,45.9%and 187.7%of those in the blank control group,respectively.The secretion levels of NO,IL-1β,TNF-α and IL-6 of 25.0 μg/mL PPS2-A treatment group were 1.14,1.40,9.47,14.6 times of that of the negative control group,respectively.In conclusion,the exopolysaccharide production of Penicilliμm chrysogenum MS-02 decreased with increasing temperature.Its exopolysaccharide,PPS2-A,a glucan with α-1,4Glcp-linked main chain,can stimulate the activation of macrophages and enhance immune activity.This study provides theoretical basis and data support for the development of polar active polysaccharides to improve immune activity,and has certain significance for promoting the development and application of fungi extracellular polysaccharides from special environmental sources in Antarctica.