Effects Of Temperature On Lipoxygenase-1 Of Barely Grain During Germination

Objective: The lipoxygenase(LOXs) in the seeds of malting barley results in a piece of bad taste of hardboard. It is regarded to be a stable strategy to improve beer flavor by reducing or removing LOXs content in malting barley. Although the deletion mutants have already been selected at present, the correspond production of this cultivar is limited and protected by patent. Apart from genotype, the content of LOXs in malting barley also relate to the temperature. Therefore, it could be useful to study the difference of LOXs activity during malting barley’s seeds germination in different temperature. Besides, the research also optimized the testing method, which clear the influence of various testing factors in the measurement of LOXs, also benefit to the application of UV spectrophotometry in LOXs measurement of malting barley.Method: Ganpi4 was used as research object, taking UV spectrophotometry as method to explore the effect of LOXs’ activity by some factors, which include the substrate concentration(0.05, 0.15, 0.25, 0.30, 0.50, 1.00 mmol L-1); the extraction buffer’s pH(4.0-9.0); the reaction buffer’s pH(4.6-9.6); extraction time(5min, 15 min, 30 min, 60 min, 120min); crude enzyme amount/substrate amount(1:1, 1:2, 1:3, 1:4, 1:5). Taking ZDM00279(LOXs deletion mutant), XinyinD9, Ganpi4 as objections to measure the LOXs activity and antioxidant enzyme activity as well as the content of malondialdehyde, hydrogen peroxide, ascorbic acid. The research used optimized LOXs testing method, and cultivated samples in light incubator to make samples germination in different temperatures(8℃, 15℃, 20℃, 33℃).Result: The research results are as follows:With the increasing of substrate concentration, the activity of LOX-1 firstly increased, then decreased; the peak appeared under the concentration of 0.30 mmol L-1,which significantly higher than the others. With the increasing of extraction buffer’s p H, LOX-1 activity shows bimodal curve, of which come to a significant peak(9.85U) in Ph = 5.0. The similar phenomenon happens to the change of reaction buffer’s pH, while the peak pH = 6.4. While the affection of crude enzyme extraction time appeared single peak curve, of which come to the significantly highest peak when it’s 30 mins. Under 50 ul crude enzyme, the relationship between LOX-1 activity and the amount of substrate addition shows similar trend with time, and it gets significant highest when comes to 200 ul.Under the condition of low temperature stress(8℃), the LOX-1 activity of Ganpi4 and XinyinD9 are lower than the correspond other temperature treatment during 12 h and 24h; it gets higher than the treatment of 15℃and 20℃treatments during the period of 36-60 h while lower than 33℃ treatment. With high temperature stress(33℃),during the process of two samples germination, the activity of LOX-1 are higher than the other treatments. Comparing the treatments of 15℃ and 20℃, we can see that LOX-1 activity in 15℃ is lower than 20℃ in the time points of 48 h and 60 h.With temperature stress treatments, 3 samples’ SOD activity appears higher than the corresponds 15℃and 20℃treatments before germination, while after the germination they comes to opposite. The similar trends appeared in the activity of CAT and the content of AsA. During whole germination progress, 3 samples’ SOD activity in 15℃ are higher compared to the correspond 20℃. When it comes to the late of germination, all samples’ POD activity are higher than the corresponds 15℃ and 20℃ treatments. With temperature stress, all 3 samples’ H2O2 and MDA content are higher than the correspond treatments of 15℃ and 20℃ during the whole germination period.Conclusion: It is a feasible method by improving the UV spectrophotometry measure parameters(the power sample of malting barley seeds; pH = 5.0; acetate buffer 1:20; extract 30 mins under 4℃; 0.25mmol·L-1 substrate concentration; use pH = 6.4 phosphate buffer as reaction buffer; crude enzyme/substrate = 1:4). The method can measure the LOX-1 activity of malting barley seeds in a better efficient way. And lower temperature in a proper way can decrease the activity of LOX-1 during malting barley seed’s germination. In addition, the malting barley variety which own high concentration of AsA show a lower activity of LOX-1 in seed.