Effects Of Solid State Fermentation With Cordyceps Militaris SN-18 On Physicochemical Characteristics And Functional Properties Of Chickpea

Chickpea,an annual herbage plant,is the second-largest consumer legume in the world.It is rich in protein,essential amino acids,unsaturated fatty acids,vitamins,minerals and biological compounds,and could be considered as the nutraceutical and functional food in health promotion.It occupies a very important position in the world's food production and consumption.The studies on chickpea in foreign contries have been for many years.However,to the best of our knowledge,there is little studies on chickpea and used it as food products in China.In addition,there is also little study about the processing and utilization of high-technology on chickpea,and the scale and intensification are not formed.In recent years,solid state fermentation(SSF)has been received great interest from researchers since several studies have demonstrated that this process may lead to higher yields and productivities or better product characteristics than the other methods.SSF is a low-cost,high-efficiency and environmentally-friendly food processing method.It could not only realize the large-scale utilization of chickpea resources,but also could fully improve its nutritional and health-stimulating effect,which has broad development and application prospects.Cordyceps militaris,a medicinal and edible mushroom generally recognized as safe(GRAS)filamentous fungus,which has been broadly used as a crude drug and a folk tonic food in East Asia countries.Like other filamentous fungi,C.militaris are rich in enzyme production,strong enzyme activity,and high catalytic efficiency during fermentation process.In addition,it is possible for C.militaris to bio-transform and synthesize compounds during fermentation with plant materials.However,there are few studies have been reported in this area.Besides,since cereal products such as bread are deficiency of essential amino acids(e.g.,lysine)and biologically active ingredients,considerable interest has been received in fortifying bread with high protein,high lysine and health-stimulating bioactive substances of materials such as legumes in recent years.However,the development and quality improvement of such grain breads is mainly focused on the supplementation of raw material multigrain ingredients and the bread improving agents such as fungal enzymes.There have been few reports on the processing of raw material components.Therefore,in this study,solid state fermentation(SSF)of chickpea with C.militaris SN-18 was performed.The production of enzymes during the C.militaris fermentation process was studied.Furthermore,the effect of SSF with C.militaris on nutritional value,physicochemical and functional properties,major antioxidant components,antioxidant activities of chickpeas was systematically investigated.Besides,use of C.militaris-fermented chickpea flour as a functional excipient to investigate its processing characteristics in bread was also performed,and developed a new type of chickpea bread with quality improved and health stimulating effect.This study consists of five sections and the main results are showed as follows:1.Study on enzymes production and changes of phenolics content during the solid state fermentation of chickpea with C.militarisC.miliaris could produce abundant fungus enzymes when fermented with chickpea,including a-amylase,(3-glucosidase,xylanase,protease,cellulase and esterase.The enzymes activity of the fungus grown chickpea substrate was low during early growth stages,and then started to increase significantly with the extension of the fermentation time.The content of total phenolics of chickpeas also increased significantly during the bioconversion process.After 8 days of fermentation,the total phenolics content increased from 659.17 ?g GAE/g d.w.(0 day)to 1928.21 ?g GAE/g d.w.(8 day).The results showed that the enhancement of phenolic compounds is closely related to the produced various enzymes during the C.militaris fermentation process.The correlation coefficient(R2 values)among total phenolics content and ?-amylase,P-glucosidase,xylanase,protease,carboxymethylcellulase,filter paperase and esterase were 0.9358,0.8228,0.7496,0.8157,0.8690,0.8591,and 0.4661,respectively.2.Study on the effect of solid state fermentation with C.militaris on the antioxidant activity and DNA damage protective effect of chickpeaSolid state fermentation with C.militaris significantly enhanced the DPPH radical scavenging activity,ABTS radical scavenging activity,reducing power,ferric reducing antioxidant power(FRAP)of chickpea.In addition,fermented chickpea demonstrated significantly(p<0.05)greater protection against oxidative DNA damage induced by Fenton's reagent than non-fermented samples.The results also indicated that the different polarity extraction solvents have a great influence on the extraction efficiency of antioxidant compounds such as phenolics and saponins in chickpea.Among various extracts examined,the 80%methanolic extracts of fermented chickpeas showed the highest DPPH radical scavenging activities,ABTS radical scavenging activities and reducing power,with EC50 values of 1.21,0.94,and 4.15 mg/mL,respectively.Besides,the antioxidant activity of 80%ethanolic extract of fermented chickpeas was significantly better than that of deionized water extract.HPLC analysis was performed for determination of the specific phenolics profile,which revealed shikimic acid,chlorogenic acid,rutin,daidzein,genistein and biochanin A were significantly increased during fermentation.Correlation analysis indicated total saponin and phenolic contents were closely related with antioxidant activities.3.Study on the C.militaris fermented chickpea against H2O2-induced oxidative damage in PC12 CellsA model of oxidative stress injury induced by H2O2 in PC 12 cells was established to investigate the protective effect CFC and UFC.Both C.militaris fermented chickpea(CFC)and unfermented chickpea(UFC)showed no toxic to PC 12 cells at concentrations of 50-800 ?g/mL by MTT assay.The MTT experiment results showed that CFC and UFC inhibited the oxidative damage of PC12 cells induced by H2O2 in different degrees,and increased the survival rate of cells in a dose-dependent manner.Inverted phase contrast microscopy showed that CFC/UFC treatment could significantly improve the morphology,growth status and survival rate of cells.Furthermore,the protective effect of CFC treatment groups on the oxidative damaged cells was significantly stronger than that of the respective UFC groups.At 800 ?g/mL,the cell viability of 80%methanolic,80%ethanolic and deionized water extracts of CFC(i.e.,M-CFC,E-CFC,W-CFC)treatment groups was 84.80%,77.24%and 71.99%,respectively,which were significantly higher than those of the respective solvent extracts of UFC at the same concentration.The M-CFC showed the highest cell viability.The results of biochemical analysis showed that different solvent extracts of CFC/UFC could significantly reduce the content of lactate dehydrogenase(LDH)and intracellular reactive oxygen species(ROS)level,and enhanced the intracellular glutathione(GSH)content,superoxide dismutase(SOD)and catalase(CAT)activity in PC 12 cells induced by H2O2,which also in a dose-dependent effect.It indicated that CFC/UFC extracts could play a protective role by improving the intracellular antioxidant system and eliminating intracellular ROS.Furthermore,the effect of various extracts of CFC groups treatment on enhancement of GSH levels,SOD and CAT activity,and the reduction of LDH and ROS levels were significantly stronger than that of the respective solvent extracts of UFC.The results of flow cytometry analysis also showed CFC groups could significantly inhibit the apoptosis rate of PC 12 cells when compared with UFC groups.After treatment with M-CFC,E-CFC and W-CFC,the apoptosis rate of PC12 cells was reduced from 54.67%(the model group)to 29.78%,32.80%and 36.21%respectively.The above results showed that SSF significantly enhanced the ability of chickpeas against H2O2-induced apoptosis in PC 12 cells,thereby protecting PC 12 cells from oxidative damage.Pearson's correlation analysis showed that the enhanced protection of oxidative damaged PC 12 cells of fermented chickpeas was mainly attributed to the increase of total phenolics content and total saponins content during C.militaris fermentation.4.Effect of solid state fermentation with C.militaris SN-18 on nutritional,physicochemical and functional properties of chickpea(Cicer arietinum L.)flourThe main objective of this study was conducted to investigate the effects of SSF on nutritional value and protein composition of chickpea flour,as well as the subsequent effect on proximate composition,physicochemical and functional properties.Results in the present study showed that SSF increased the contents of the crude protein,true protein and essential amino acids,and improved the in vitro protein digestibility of chickpea.SDS-PAGE demonstrated that proteins of fermented chickpeas were mainly composed of molecules of lower molecular mass compared to the non-fermented chickpeas.In the case of the fermented chickpea,the bands of large-sized proteins were disappeared(>60 kDa)and more small-sized proteins(<30 kDa,66.87%)were observed.It was obvious that the large-sized and mid-to-large sized proteins of chickpea were hydrolyzed into smaller molecular masses during C.militaris fermentation.Fermented chickpea flours showed higher water absorption index,and SSF improved water holding capacity,fat absorption capacity and emulsifying properties of chickpea flours.Additionally,some of new smaller molecular peptides with angiotensin I-converting enzyme(ACE)inhibitory activity were generated during SSF.Fermented chickpea extracts demonstrated significant ACE inhibitory activity,with IC50 value of 0.668 mg/mL.Thus,the study indicated that C.militaris fermentation improved the physicochemical and functional properties of chickpeas and it has great potential to design new nutritious food products and food formulations using C.militaris fermented chickpeas.5.Comparative study of the effects of fermented and non-fermented chickpea flour addition on quality and antioxidant properties of wheat breadThe main aim of this study was to compare the effects of C.militaris-fermented chickpea(CFC)and non-fermented chickpea(NFC)flour addition on quality(including proximate composition,specific volume,crust and crumb colour,molecular mobility,crumb texture,sensory evaluation)and antioxidant properties of wheat bread,and also compare with these properties on the wheat-only bread.The wheat flour was partially substituted by NFC and CFC at a level of 50 g/Kg flour basis,respectively.Addition of chickpea flour(both CFC and NFC)to wheat bread showed higher protein,fat content,yellowness index values as well as enhanced the phenolics content and antioxidant activity whereas lower lightness values were observed,and CFC-wheat bread showed higher phenolics and antioxidant activity than NFC-wheat bread.Furthermore,bread incorporated with CFC exhibited higher specific volume and lower crumb hardness than the wheat-only bread but addition of NFC resulted in a negative effect on specific volume and crumb firmness.LF-NMR study showed that 1H NMR mobility was different in the NFC-wheat and CFC-wheat breads as compared to the wheat-only bread,which may have affected the development of the gluten network resulting in different textural properties.Sensory evaluations revealed that breads substituted with CFC got the highest scores in terms of appearance,texture,colour and overall acceptance.Results suggested that bread fortified with CFC improved quality(specific volume,texture,colour,sensory)and antioxidant properties,and thus was consider of great potential in the production of high consumer-acceptable as well as quality and antioxidant properties improved bread.