| Dictyophora indusiata（D.indusiata）is one of the famous edible fungi in our country.β-glucans contained in D.indusiata possess multiple health benefits,such as immune regulation,anti-oxidation,anti-inflammation,and anti-tumor effects,which show positive application prospects in nutrition and health food.The functional activity of edible fungi polysaccharides is closely related to its structure.However,the structure-activity relationship between the structural characteristics of D.indusiataβ-glucan and its biological activity is not completely revealed.Therefore,this study focused on the ultrasonic-assisted extraction,isolation and purification and potential structure-activity relationship of D.indusiataβ-glucan as well as its in vitro digestive and microbial fermentation characteristics,to provide theoretical and technical supports for the development of D.indusiataβ-glucan.The findings in this study are as follows:（1）Optimization of ultrasonic-assisted extraction of D.indusiataβ-glucanIn this study,the single factor experiments were adopted to evaluate the effects of ultrasonic time（2～20 min）,ultrasound amplitude（10%～70%）,and liquid-solid ratio（10～50 m L/g）on the yield of D.indusiataβ-glucan.Furthermore,the response surface experimental design was applied to optimize the extraction parameters.Under the conditions of ultrasound amplitud of 370 W,liquid-solid ratio of 22 m L/g,and ultrasonic extraction time of 15 min,the extraction rate of D.indusiataβ-glucan reached 1.54%±0.26%,which was significantly higher than that of traditional hot water extraction method（1.16%±0.17%）.（2）Isolation,structural characterization,and selective degradation of D.indusiataβ-glucanIn this study,a rapid and efficient separation of D.indusiataβ-glucan（DP）was achieved by fractional alcohol precipitation（30%,v/v）combined with membrane separation technology（100.0 k Da）.The structural features of D.indusiataβ-glucan were resolved in detail and found that D.indusiataβ-glucan was a uniform rigid rod conformation glucan with a molecular weight of about 1.077×106 Da,and the main chain was composed ofβ-（1→3）-glucan with a side chain ofβ-D-Glcp-(1→at its O-6 position.Besides,the ultrasonic-H2O2/Vc reaction system was used to selectively degrade DP.The results showed that three kinds of degradation products（DDP-1,DDP-2,and DDP-3）were obtained after degraded by ultrasonic-assisted H2O2/Vc reaction system（20.0 m M Vc,40.0 m M H2O2,ultrasound amplitud 60%）for 10,90,and 120 min,and the primary structures of the them were overall stable,and the monosaccharide composition and glycosidic bonds have not varied.However,the molecular weight and chain conformation of the degradation products have changed significantly.The molecular weights of the three degradation products were 5.955×105,3.531×105 and 1.870×105 Da.In addition,with the increase of degradation time,the chain conformation of degradation products changed from the rigid rod（DDP-1）to the random coil（DDP-2 and DDP-3）.（3）Study on the structure-activity relationship of the immune activity of D.indusiataβ-glucanIn this study,the results of in vitro immune activity experiments showed that D.indusiataβ-glucan and its degradation products had significant immune enhancement activity,which could significantly promote the release of nitric oxide（NO）,interleukin-6（IL-6）and tumor necrosis factor-α（TNF-α）,and to promote the polarization of RAW 264.7macrophages to the M1 direction.Both D.indusiataβ-glucan and its degradation products can bind to the toll-like receptor-4（TLR-4）on the surface of macrophages,activate the downstream nuclear factor-κB（NF-κB）signaling pathway,and promote the expression of inducible nitric oxide synthase（i NOS）,thereby exerting an immunositmulatory effect.Compared with D.indusiataβ-glucan at the same concentration,DDP-1,which maintained its stable higher solution chain conformation,had stronger immunostimulating activity:At320μg/m L,the highest secretion levels of NO,IL-6,and TNF–αcan reach 26.78μM,27.50pg/m L and 729.09 pg/m L,respectively.With the further reduced of molecular weight and the change of solution chain conformation（from the rigid chain to the random coil chain）,the immunostimulatory activity of DDP-2 and DDP-3 was obviously weaker than that of D.indusiataβ-glucan but not completely inactivated.（4）Study on the in vitro digestive and microbial fermentation characteristics of D.indusiataβ-glucan and its regulation effect on intestinal floraIn this study,the content of reducing sugar in D.indusiataβ-glucan digestion remained stable in different stages of simulated digestion environment in vitro,and the molecular weight,total polysaccharide content,and apparent viscosity of D.indusiataβ-glucan were also stable,indicating that D.indusiataβ-glucan can stably pass through the digestive system of the gastrointestinal tract.During the simulated colonic fermentation in vitro,the molecular weight（1.057×106 to 0.595×106 Da）and total polysaccharide content（95.01 mg/m L to51.12 mg/m L）of nondigestive D.indusiataβ-glucan（DPI）decreased significantly,and the reducing sugar in the fermentation broth of DPI significantly increased after 24 h of fermentation with producing free monosaccharides,indicating that DPI can be gradually degraded and consumed by human intestinal flora.After 48 h of fermentation,compared with the blank control,DPI can significantly increase the abundance of beneficial bacteria such as Bacteroides,Actinobacterium,Parabacteroides and Megamonas,and reduce the number of harmful pathogenic bacteria such as Escherichia-Shigella,Fusobacterium,Biliphila,Morganella,and Lachnochlorostridium.In addition,after 48 h of fermentation,DPI also promoted the production of short-chain fatty acids such as propionic acid（2.32mmol/L）and isobutyric acid（1.13 mmol/L）,thereby changing the acid-base environment of the intestinal tract,which is beneficial to the regulation of human intestinal health. |
PDF Full Text Request