| Millet is an ancient crop and used as a staple food grain in northern areas in China due to its strong vitality in drought prone areas and very poor soils where other crops fail to grow.It has the advantages of higher nutritional value,short growth period,strong adaptability,barren resistance and easy storage,too.With the progress of the society,the dietary and consumption idea of consumers have been greatly changed.The human being have paid more and more attention to increase the nutritional quality of plant foods.Germination can effectively increase the nutritional value and content of γ-aminobutyric acid(GABA)of millet,which is accord with the trend of " nutrition,hygiene and convenience" in China’s food industry.Thus,it is very necessary to increase the content of γ-aminobutyric acid in millet by germinating and then to analyze the composition changes of millet,the structural changes of millet protein and the changes of key enzymes in GABA-shunt during germination.The content of γ-aminobutyric acid in millet was improved by germination.And the composition changes of germinated millet were studied.At the same time,the difference of the structural characteristics of germinated millet protein were analyzed.The changes of key enzymes in GABA-shunt,the contents of key enzymes in GABA-shunt and γ-aminobutyric acid and the correlation analysis among the key enzymes during germination were studied,too.The main results were as follows:(1)Optimizing germination conditions to increase GABA content in milletBy single factor experiment and Plackett-Burman experiment,three significant factors affecting GABA were ensured: soaking time,Ca Cl2 concentration and germination temperature.The steepest ascent path experiment was used to approach the largest response region in GABA content and to measure the central point of the response surface experiment.Then response surface Box-Behnken experiment design was used to obtain the optimal levels of soaking time,Ca Cl2 concentration and germination temperature.However,under the optimal levels conditions,there was no significant difference between the measured GABA content and the measured GABA content at the design center of response surface experiment(P > 0.05).Therefore,considering the convenience of practical application,the optimal technological conditions for millet germination were as follows: soaked in 3.5 mmol/L Ca Cl2 solution at 35 ℃ for 13 h and germinated at 31 ℃ for 48 h.Under these conditions,GABA content in germinated millet was 2.90 times higher than that before optimization.Compared with millet,the contents of protein,fat,starch and ash in germinated millet all have decreased significantly(P < 0.05).The total fatty acid content of germinated millet increased from 3.15 g/100 g to 3.35 g/100 g,among them,palmitic acid,arachidic acid and linolenic acid did not change obviously(P > 0.05),linoleic acid content increased(P < 0.05).However,stearic acid and oleic acid content decreased significantly(P < 0.05).After millet is germinated,the contents of serine,threonine and cysteine did not change significantly(P > 0.05).However,the histidine content decreased.Apart from this,the contents of other amino acids all increased.At the same time,the content of hydrophobic amino acids and hydrophilic amino acids in germinated millet were all increased.They were respectively from 5.19 μmol/g and 8.40 μmol/g to 10.43 μmol/g and 8.92 μmol/g.They were respectively increased by 100.96% and 6.19%.Hydrophobic amino acids were significantly increased(P < 0.05).The total amount of amino acids in germinated millet increased from 13.59 μmol/g to 19.35 μmol/g.Scanning electron microscope was used to observe the microstructure of germination millet.It was found that the whole powder of millet was a bigger aggregate.After germination,the size of aggregates in millet reduced and most of them were irregular fragments.The size of starch particles also decreased with certain extent.At the same time,the number of pore on the surface of starch particles increased.The pore size increased and the surface was rougher.By analyzing the thermal characteristics of millet and germinated millet: compared with millet,the onset temperature(To),peak temperature(Tp)and conclusion temperature(Tc)of germinated millet increased from 77.09 ℃,77.09 ℃ and 81.01 ℃ to 71.11 ℃,78.10 ℃ and 82.04 ℃ respectively.At the same time,the gelatinization enthalpy(△H)also increased from 5.61 J/g to 6.18 J/g.(3)Structural changes of germinated millet proteinSDS-PAGE gel electrophoresis was used to analyze the influence of germination on the subunits of millet protein.The results showed that compared with millet protein,subunits of germinated millet protein had not changes.And the molecular weight was mainly distributed from 11 to 107 k Da,too.However,the content of each subunit changed.After germination,except the increase in the subunit content of molecular weight of 18.9 k Da,the other subunits decreased compared with before germination.By studying the particlediamite distribution of millet protein before and after germination,it was found that the range of the particlediamite distribution of millet protein before and after germination was 0.2-7 μm.And They all showed a bimodal distribution.After germination,the particlediamite distribution of millet protein between 0.5 to 3 μm increased from 54% before germination to 75.9%.However,the particlediamite distribution between 3 to 6.5 μm decreased from 46% before germination to 24.1%.At the same time,D50 of millet protein before germination was 1.48 μm.However,D50 of millet protein after germination decreased to 1.28 μm.By comparing the ultraviolet-visible absorption spectrum of millet protein before and after germination,it is found that compared with millet protein,the ultraviolet absorption intensity of germinated millet protein was increased and blue shift was 8.0 nm.FT-IR spectra was used to analyze the secondary structure of the protein.It was found that β-turn(2)Composition Changes of Germinated Millet occupied the main position in the secondary structure of millet protein.At the same time,compared with millet protein,α-helix structure in germinated millet protein increased from 14.37% to 18.49%,β-sheet and random coil structure decreased from 28.11% and 12.51% to 26.51% and 9.96%,respectively.The β-turn showed little change.By comparing the fluorescence spectrum of millet protein before and after germination,it was found that the fluorescence spectr um of millet protein had almost not changes before and after germination.However,compared with millet protein,the fluorescence intensity of germinated millet prote in decreased and red shift happened at the same time.The maximum emission wavelength changed from 354.6 to 358.6 nm and red shift was 4.0 nm.By comparing the hydrophobicity of millet protein before and after germination,it was found that the hydrophobicity of the protein after germination decreased obviously(P < 0.05)and changed from 294.90 before germination to 256.93.(4)The changes of key enzymes in GABA-shunt;correlation analysis of the key enzymes activity in GABA-shunt and contents of GABA and among key enzymes during germination.During germination,the activities of glutamic acid decarboxylase(GAD),γ-aminobutyric transaminase(GABA-T)and succinate semialdehyde dehydrogenase(SSADH)appeared a gradual increase with the extension of germination time.After 48 h of germination,the activity of GAD,GABA-T and SSADH increased to 3.97 U/L,2.37 U/L and 2.84 U/m L from 0.65 U/L,0.05 U/L and 1.02 U/m L of millet,respectively.The correlation analysis showed that GAD,GABA-T and SSADH were significantly positively correlated with each other(P < 0.05).Among them,GAD was significantly positively correlated with GABA-T and SSADH(P < 0.05).Correlation coefficients were respectively 0.934 and 0.928.GABA-T and SSADH were significantly positively correlated(P < 0.01).The correlation coefficient was 0.972.The content of GABA was significantly positively correlated with GAD(P < 0.01).And the correlation coefficient was 0.984.The content of GABA was significantly positively correlated with GABA-T(P < 0.05)and the correlation coefficient was 0.881.The related relationship between GABA content and SSADH was not significant(P > 0.05). |
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