Nutritional Functions Of Whole-grain Hulless Barley Treated By Various Thermal Processing And Its Alleviative Effect On Type 2 Diabetic Rats

High-fat and high-sugar diet has gradually become an important part of human diet in recent couple of decades.Long-time excessive intake of energy is positively correlated with the incidence of chronic non-communicable diseases.For example,the global prevalence of type 2 diabetes mellitus（T2DM）is increasing year by year.Growing body of evidence has revealed that increasing the intake of whole grains has been considered to be an important and beneficial strategy to reduce the prevalence of T2DM,which is the most cost-effective precaution in the preventing of T2DM.Hulless barley（HB）,as an important cultivar of cereals,is rich in functional ingredients,such as dietary fibre and polyphenols,has various functions including anti-oxidation,anti-inflammatory,and regulating intestinal flora.However,the low acceptance of HB and the low bioavailability of dietary fiber and polyphenols in HB limit its application in the field of food processing.Therefore,increasing the accessibility of dietary fiber and polyphenols in HB by employing appropriate thermal processing has an important reference for improving the nutritional value of HB and the processing of HB-related food.The whole-grains HB was pre-treated by four thermal processing methods including flacking and roasting（FR）,stir-frying（SF）,steam-flash explosion（SFE）,and popping expansion（PE）,followed by analysis the basic ingredients in the present study.Moreover,the effects of various thermal processing andβ-glucan on the digestive characteristics,antioxidant activity,and regulation for the intestinal microbes of HB were investigated in vitro.Further,the structural feature,physicochemical properties,and prebiotics ofβ-glucans extracted from HB processed with different thermal processing were characterized by using high performance size exclusion chromatography coupled with multi-angle laser light scattering and refractive index detection,X-ray crystal diffractometer,laser scanning confocal microscope,atomic force microscope,16S r DNA sequencing technology,etc.Afterwards,an ultra-high pressure liquid chromatography-Xevo triple quadrupole mass spectrometry was used to measure the content and composition of polyphenols in digestive tract of T2DM rats,followed by determining the content and antioxidant activity of polyphenols in each HB sample before and after digestion in vitro.Additionally,the index associated with glucose and lipid metabolism,the level of oxidative stress and inflammatory factors,the short-chain fatty acids（SCFAs）and microbial composition in ceacal contents,intestinal functions,and the expression levels of NF-k B proteins in intestinal tissue and PI3K/AKT signaling pathway-related proteins in the liver and skeletal muscle of T2DM rats were investigated by using Western blot technology and other methods.The underlying mechanism regarding the effect ofβ-glucan in HB and thermal processing on starch digestion was preliminarily clarified.The effects of HB pre-treated by various thermal processing on the amelioration of T2DM rats and its possible mechanism were studied based on theβ-glucans and polyphenols.The main findings were as follows:（1）Theβ-glucans located on the cell wall of HB and formed the intact cellular structure and/or the network structure,which inhibited the starch digestion.The order of starch and protein digestibility of HB in different processing groups was PE>SFE>FR>SF>Control.The integrate structure was completely destroyed by SFE and PE,whereas considerably retained in HB processed by FR and SF.Moreover,the ability of scavenging·O₂^-,FRAP reduction,and total antioxidant capacity（T-AOC）of each HB sample increased significantly after gastrointestinal digestion in vitro.The antioxidant capacity among different HB samples decreased in the order of SFE>PE>SF>FR>Control.Meanwhile,the antioxidant ability（except the inhibition ability for·OH）of each HB sample elevated significantly once theβ-glucan was hydrolyzed.Additionally,the ratio of Firmicutes to Bacteroidota at phylum level and the relative abundance of Megasphaera,Bifidobacterium,Lactobacillus,unclassified-o-Veillonellales-Selenomonadales and unclassified-p-Firmicutes at genus level in human feces increased significantly after fermentation with HB samples in vitro.Importantly,the PE-HB and SF-HB exhibited higherβ-glucan utilization and total SCFAs content,especially the content of acetic acid and butyric acid.（2）PE,SF and FR significantly increased the yield（>0.7%,w/w）,molecular weight（M_w～5243.6 k Da,1245.6 k Da and 210.2 k Da）and distribution（R_g～125.5 nm,59.7 nm and 126.4 nm,respectively）ofβ-glucan from HB,whereas SFE decreased its yield,purity,and molecular weight（M_w～89.9 k Da）.Furthermore,the primary structure ofβ-glucan from each processed HB was not changed remarkably.The water-holding capacity ofβ-glucan was positively correlated with its molecular weight,while the solubility and gelling properties were not only affected by its molecular weight,but also by its molecular conformation.Importantly,the relative abundance of Bacteroid at phylum level and the relative abundance of Megacoccus,Lactobacillus,and Bifidobacterium at genus level of the microbes from human feces was significantly reduced and increased,respectively,after fermentation with eachβ-glucan sample,which occurred dramatically in the Control,PE,and SFE groups.Moreover,the utilization rate ofβ-glucan,the concentration of acetic acid,butyric acid,and isobutyric acid were higher in the PE and SFE groups.（3）The bound polyphenols content in raw HB were significantly higher than that of free polyphenols;the content of free polyphenols and free flavonoids from HB and its antioxidant ability increased significantly after pre-treatment with each thermal processing,especially the SFE and PE.Notably,the content of free polyphenols and antioxidant capacity of polyphenols extracted from each HB samples was significantly increased after hydrolyzing theβ-glucan and gastrointestinal digestion in vitro.At the same digestive phase,the free polyphenols content and antioxidant capacity of each extracts from HB increased in the order of SFE>PE>SF>FR>Control.Moreover,the polyphenols content and antioxidant capacity in the Control,FR,and SF groups increased gradually with the time in colonic phase,whereas increased from 0 to 12 h and decreased from 12 to 48 h in the SFE and PE groups.Analysis by using LC-MS suggested that the thermal processing,especially the SFE and PE,increased significantly the content of ferulic acid,phloretin,kaempferol,etc,while significantly reduced the content of quercitrin,epicatechin and procyanidin B2.Expectedly,the content of each phenolic acid and flavonoid increased considerably after hydrolyzing withβ-glucanase.（4）The weight-loss,the level of fasting blood glucose and insulin,Hb A1C,lipid and Hb A1c in blood,and the gluconeogenesis level were effectively decreased;the impaired glucose tolerance and ketoacidemia were also obviously alleviated;the level of oxidant stress,inflammatory factors,and the expression of NF-k B protein in intestine down-regulated remarkably,and the intestinal barrier function enhanced significantly in T2DM rats intervened with SFE-HB and PE-HB.Importantly,the absorption of chlorogenic acid and ferulic acid enhanced in the digestive tract of the Non BG group rats,while SFE delayed the absorption of quercetin,epicatechin,phloretin,and rutin,and accelerated the absorption of gallic acid,p-hydroxybenzoic acid,and proanthocyanidin B2;the PE delayed the absorption of quercetin and phloretin,and accelerated the absorption of p-hydroxybenzoic acid,p-coumaric acid and procyanidin B2.It was found that the relative abundance of Bifidobacterium,Lactobacillus,Faecalibacterium,Allobaculum,Kurthia and Phascolarctobacteriu,and the relative abundance of Nk4A214 and Romboutsia was higher and lower,respectively,in each HB group than that of T2DM model group.Especially,the relative abundance of Blautia in SF and SFE group,Dubosiella in PE group,Akkermansia and Lactobacillus in Non BG group was remarkably increased.Moreover,the content of butyric acid,isobutyric acid and valeric acid in Non BG,SFE and PE groups increased dramatically compared to the model group.Western blot results indicated that the expression level of AMPK protein in the skeletal muscle of the rats intervened with all HB increased significantly,and the expression levels of IRS-1/PI3K/AKT protein in the liver and skeletal muscle of T2DM rats intervened with SFE-HB and PE-HB up-regulated significantly,while the expression levels of GSK-3βprotein down-regulated dramatically,compared to the model group.Therefore,the profound effect of SFE and PE on the anti-diabetic function of HB mainly through increasing the bioavailability and bioavailablity ofβ-glucan and polyphenols,and thus reducing the level of oxidative stress and inflammation,regulating the composition and abundance of gut microbes,regulating the expression level of PI3K/AKT signaling pathway related proteins,resulting in the amelioration of T2DM rats.