Identification Phenolics Antioxidants In Wheat Grain By Metabolomics And The Regulation Effect Of Nitrogen Fertilizer Application

Wheat is one of the most important crops in the world,and apart fromproviding carbohydrate,it also contains many unique nutrients,such as mineral element,phenolics compounds.In order to identify the metabolites of wheat grain at immature and mature stage,targeted and non-targeted metabolomics analysis were used to analyze the wheat grain with different grain color(black,purple,blue and white).At the same time,the effect of heat processing on the antioxidants content and antioxidant activity were also analysed.Meanwhile,clarifying the distribution of phenolic compounds in layered grinding wheat flour and its response to nitrogen fertilizer application would provide useful information for wheat quality improvement and high-quality cultivation.Two wheat cultivars,purple wheat(Jizi439)and white wheat(Xinhuamai818),were planted with two nitrogen application rate(HN,N 210 kg hm–2;LN,N 105 kg hm–2)in Zhengzhou and Yuanyang experimental sites during growing period in 2019 and 2020.The mature grains were ground into five milling fractionations(LY1,LY2 LY3,LY4,and LY5)from the bran layer to the endospermic layer by layered grinding,and the total phenolic content(TPC),the total flavonoids content(TFC),and the anthocyanin content(AC),and their antioxidant activity were determined.The main results are as follows:(1)The metabolites of wheat grain with different grain colors at immature and mature stageMetabonomic analysis showed that the flavonoid biosynthesis pathway was closely related to the formation of grain color.A total of 866 metabolites was identified in the tested wheat grain,of which 105 flavonoids and 39 phenolic acids showing significantly different expression abundance.Most of the expression abundance of flavonoids compounds in black wheat and purple wheat were higher than that in blue wheat.Quantitative targeted metabolic analysis showed that purple wheat grain(JZM4)got the highest content of rutin,paeoniflorin-3-O-glucoside,geranium-3-O-glucoside,peonidin chloride and diosmetin,whereas blue wheat(LXM1)got the highest content of taxifolin,luteolin and cynaroside.Only trace amounts of the above components were detected in white wheat(XHM818),but the content of trans-hydroxycinnamic acid in XHM818 and colored grains were comparable.After steaming treatment,the up-regulated flavonoid components in JZM4 mainly included rutin,pelargonidin 3-O-glucoside,peonidin-3-O-glucoside chloride,and cynaroside,whereas the down-regulated were peonidin chloride,diosmetin,luteolin and trans-hydroxycinnamic acid.Among them,the content of p-coumaric acid decreased from 1752.19 μg kg–1 in fresh grains to 462.08μg kg–1 in steamed grain,while Paeoniflorin chloride decreased from 60.91 μg kg–1 to 50.24 μg kg–1.However,rutin showed an upward trend,from 140.33 μg kg–1 to 167.94μg kg–1.(2)The phenolics contnent and antioxidant activity of different wheat grainTotal phenolics,total flavonoids,and antioxidant activity increased with the deepening of grain color,which showing as fallow: black wheat>purple wheat>blue wheat>white wheat.The total free phenolic content of fresh grain got the lowest value(0.63~0.87 mg g-1)under heat treatment,whereas the total bound phenolics content got the lowest(1.53~1.83 mg g-1)value in mature grains.The highest TEAC values of free phenolics and bound phenolics(9.05μmol g-1 and 9.46 μmol g-1)were obtained in fresh black wheat grain(ND3753 and ZHM1),while the lowest value were showed in mature white wheat(XH818)(3.81 μmol g-1 and 7.13μmol g-1).In addition,the highest FRAP value and DPPH value of bound phenolics were also obtained in fresh grains,while lowest value were showed in mature grains.The results indicate that fresh wheat grain(late milk ripening-dough stage)would be used to make nutritious foods.(3)Distribution of phenolic compounds and antioxidant activities in layered grinding wheat flourThe study of antioxidants in wheat flour at different grinding levels showed that the contents of total phenolics,flavonoids and anthocyanins in the extracts of free phenols and bound phenols increased from the inner layer to the outer layer.The LY1 layer was the lowest(1.05~2.03 mg g-1),(0.24~1.19 mg g-1),(0.64~0.97 μg g-1),and the LY5 layer highest(1.76~3.98 mg g-1),(0.74~3.36 mg g-1),(1.01~7.54μg g-1).The antioxidant content of the whole wheat flour was between LY2 and LY3.The purple wheat(Jizi439)had a higher antioxidant content and antioxidant activity than white wheat(Xinhuamai818),but the difference between the two cultivars displayed a decreasing trend from the bran layer flour fractionation to endosperm layer flour fractionation.(4)Effect of nitrogen fertilizer application rate on antioxidants content and antioxidant activityThe TPC,TFC,and AC of LY1 to LY3 flour fractionation increased with the increase of nitrogen fertilizer application except for TPC of LY1 in Yuanyang.However,the contents of total phenolics and total flavonoids of LY4 to LY5 flour fractionation had a weak response to nitrogen fertilizer application.The content of ferulic acid accounted for more than 93% of the TPC in wheat grains and had a higher value under low nitrogen condition.In conclusion,purple wheat had higher antioxidant substances such as phenolics than white wheat,and the difference between purple wheat and white wheat decreased with the deepening of grinding degree.The content and activity of antioxidants in the outer layer flour fractionation were significantly responsive to nitrogen regulation,and the content increased with the increase of nitrogen.