Study On GAD Enzyme Catalytic Ability Of Bee Pollen And Biological Activity,Metabonomics Of Bee Pollen With High Hydrostatic Pressure Treatment

Bee pollen is considered to be "the best food in the world",rich in various functional nutrients needed by human body.Moreover,bee pollen contains a large number of enzymes from living pollen cells and some enzymes from bee saliva,which has the potential to be used as natural biocatalysts.However,the current research mainly focuses on the analysis of active components of bee pollen and the evaluation of its biological function,and the research on bee pollen as an enzyme source for the biosynthesis of functional molecules has not been reported so far.In addition,the therapeutic effect of bee pollen on prostate diseases has long been recorded in ancient medicine and confirmed by modern clinical medicine,but the material basis and mechanism of bee pollen in the treatment of prostate diseases are still unclear.Secondly,high hydrostatic pressure(HHP),as a non-thermal sterilization technology,is widely used in the sterilization of spoilage bacteria and pathogenic bacteria in food,and has the functions of protein denaturation and intracellular pH value change to inactivate or activate endogenous enzymes in food or improve enzymatic reaction.However,there are few studies on the effects of HHP treatment on glutamic acid decarboxylase(GAD)and other metabolites in bee pollen,and there are few studies on the antioxidant and anticancer active components of bee pollen after HHP treatment.Therefore,in this study,the synthesis efficiency of gamma aminobutyric acid(GABA)using bee pollen as a natural catalyst was investigated,and the HHP treatment conditions were optimized.Secondly,the mechanism of HHP on GABA synthesis in bee pollen was preliminarily confirmed by analyzing the changes of GAD enzyme activity,amino acids,soluble egg content and small peptide content after HHP treatment.On the basis of the above studies,we further studied the antioxidant activity of bee pollen extract treated with HHP and the mechanism of inhibiting the activity of prostate cancer PC-3 cells.At the same time,we analyzed the effect of HHP treatment on metabolites in bee pollen and the correlation between the changes of active components and anticancer activity by non-targeted metabonomics.The main results were as follows:1.The ability of enzymatic synthesis of GABA in bee pollen was preliminarily identified and quantitatively analyzed by paper chromatography,high performance liquid chromatography(HPLC)and multiple reaction monitoring(MRM)of secondary mass spectrometry.It was found that the enrichment ability of unbroken pollen was significantly higher than that of broken pollen.After high temperature sterilization,unbroken and broken pollen not only failed to synthesize GABA,but also affected the content of GABA It is indicated that the process of transforming MSG into GABA is enzymatic reaction.The expression of GAD enzyme in fresh lotus powder was detected by real-time quantitative PCR(RT-PCR).The results showed that the expression of GAD enzyme in lotus powder was 2.5 times higher than that in stamen.The activity of GAD enzyme in fresh pollen was similar to that in bee pollen,which reasonably explained the high efficiency of lotus powder synthesis The formation of GABA is attributed to the increase of GAD enzyme expression,but not to the external factors(a small amount of bee saliva mixed in bee pollen and some microorganisms that may be carried).Finally,the GABA synthesis reaction and determination of bee pollen from different plants or different areas showed that they all had strong ability to synthesize GABA,especially the GABA content of bee pollen from Jiangxi Province transformed into MSG was as high as 101.8 mg/g,so we chose the bee pollen without wall breaking as the next research material.2.Water extraction and alcohol extraction were used to extract GABA from bee pollen with different water content,and the content was determined by HPLC.The results showed that the effect of water content on GABA content was negligible,but the content of GABA extracted by water extraction was higher than that extracted by alcohol extraction.Under the condition of bee pollen solid-state reaction,the GABA yield reached 49.31 mg/g,which was 20%higher than that of the blank control group(41.07 mg/100 g),but the residual MSG content was 16.2 mg/g,which indicated that the solid-state reaction was more suitable for the transformation of low concentration MSG.The higher the concentration of MSQ the higher the GABA content,but the residual amount of MSG also increased gradually,and the transformation rate was lower.The results showed that when all the reaction conditions were optimized(the reaction time was 4 h,the reaction temperature was 40℃,the pH was 5.8,and the reaction environment was 80 mM phosphate buffer),the content of GABA was as high as 388.94 mm,and the conversion of MSG was 65.9%.Furthermore,HHP technology was used to induce GABA synthetase reaction.It was found that HHP technology could significantly increase the yield of GABA,which was 17%higher than that of the ordinary single factor optimization.Response surface methodology(RSM)analysis of HHP treatment conditions showed that the measured value of GABA reached 508.79 mm(209.8 mg/g)at 40℃ and 200 MPa for 10 min,and the conversion of MSG was 86.25%.3.The mechanism of HHP treatment on GABA synthesis and the effects of HHP treatment on protein composition,small peptide content and functional properties of bee pollen were studied by automatic amino acid analysis,GAD activity determination and SDS-PAGE electrophoresis.After HHP treatment,the covering material on the surface of pollen grains gradually deformed or removed with the increase of pressure,and the structure of pollen outer wall changed significantly.Most of the pollen appeared broken wall phenomenon,and the outer wall protruded a lot of irregular contents,the surface of pollen grains was smooth,the area gradually increased,and the phenomenon of cross-linking of pollen grains appeared.The results showed that HHP treatment increased the release of content substances in bee pollen,and thus increased the fluorescence intensity of bee pollen.The highest activity of GAD in LBP was 2679.24 U/g,which was 28.75%higher than that in control group.The results of determination of GABA and free amino acids showed that when the pressure was 200 MPa,the contents of total amino acids and total essential amino acids in LBP increased by 15.69%and 30.23%respectively,the highest content of GABA increased by 38.52%,and the contents of soluble protein and small peptide below 10 HDa increased by 22.84%and 6.4%respectively.SDS-PAGE showed that HHP treatment could change the structure of some macromolecular proteins,but did not cause protein degradation.Taking the total phenol content as an example,the effects of HHP treatment on the content of active substances in bee pollen were compared.80%ethanol,80%methanol and water were used for extraction respectively.It was found that the total phenol content of methanol extract was higher than that of ethanol and water extract.The change of total phenol content after HHP treatment increased with the increase of pressure the total phenol contents of 80%ethanol,80%methanol and water extracts were 235.76,360.77 and 192.13 mg/100 mg,respectively.4.To study the changes of ACE inhibitory activity,antioxidant activity of different bee pollen extracts before and after HHP treatment,as well as the inhibitory effect on prostate cancer PC-3 cells.First of all,the ACE inhibitory activity of the samples treated with 50 MPa HHP was higher than that of the samples without pressure treatment(P<0.05),and the ACE inhibitory rate was 89.72%,which was 2.54%higher than that of the control group.HHP treatment of bee pollen significantly improved its antioxidant activities,such as DPPH radical scavenging capacity,ABTS+scavenging capacity,reducing potential,FRAP,AgNPAC,and showed a dose-dependent effect.The results were consistent with the expected results.The antioxidant capacity of 80%methanol extract was higher than that of ethanol and water extract.The antioxidant capacity of bee pollen extract was expressed by the content of VC or GA.The DPPH radical scavenging capacity of bee pollen extract was the strongest.The DPPH scavenging capacity of different solvent extracts of samples treated with different HHP ranged from 290.3 to 4049.29 μg VC/g D w.The scavenging ability of ABTS`+ was 81.49～666.02 μg VC/g d w,the range of reducing potential was 146.77～425.41 μg VC/g d w,the range of FRAP ability was 1.61～140.15 p GVC/g DW,and the range of nano silver reducing ability was 223～581 üg GA/g d w.The antioxidant capacity of LBP aqueous extract without HHP treatment was the lowest,and that of LBP methyl alcohol extract under 200 MPa pressure was the highest.Pearson’s bivariate method was used to analyze the relationship between the antioxidant activity and total phenol content of bee pollen treated with HHP.The results showed that the antioxidant activity of bee pollen was closely related to the total phenol content,and the correlation coefficients were above 0.726.HHP treatment could significantly increase the antioxidant activity of bee pollen.The results showed that the extracts of different solvents after HHP treated bee pollen had higher toxicity to PC-3 cells of human prostate cancer,and the results showed concentration and time dependence.The MTT results of PC-3 cells treated with different extracts for 24,48 and 72 hours showed that the inhibition rate of bee pollen extract on PC-3 cells increased with the increase of treatment time,and IC50 value decreased.Compared with positive control(Cis-platin),the extracts of bee pollen showed similar cytotoxic effect after the treatment time was prolonged to 72 hours.The results showed that the mortality and damage rate of PC-3 cells were increased significantly under the condition of IC50 concentration.The apoptosis rate of PC-3 cells was significantly higher than that of normal group under the condition of IC50 concentration.The apoptosis rate of all samples was over 20%,and that of methanol extract was even more than 50%.Meanwhile,the apoptosis rate of HHP treated bee pollen was significantly increased.The results of cell cycle distribution showed that after treatment with PC-3 cells with different solvent extracts,cell cycle was disordered,water extract blocked G1 region,methanol and ethanol extract blocked G2/M cells,positive control block G2/M region,HHP treatment significantly increased the effect of bee pollen extract on cell cycle.After different solvent extracts treated PC-3 cells,GSH content was reduced,which caused apoptosis.5.Finally,the metabolomics of the pollen of the lotus wasp before and after HHP treatment was studied.The results of the experiment were evaluated and analyzed by the method of liquid chromatography mass spectrometry.The results showed that the metabolic pattern of the samples and the sample groups were all fitted without any fitting.The metabolites of 200 MPa treatment group and control group were compared by principal component analysis.The results showed that the metabolites of 200MPa treatment group were significantly different from those of control group.3874 different metabolites were screened according to p value(P<0.05),1739 of them were up regulated,and 448 different metabolites were further screened by inserting VIP value.317 up regulated differential metabolites and 131 down regulated differential metabolites,including flavonoids,fatty acids,amino acids,glycerophosphin,sterols,bile acids and other compounds.The results of metabolic pathway enrichment analysis showed that 200 After MPa treatment,37 metabolic pathways were enriched in bee pollen,59 of which were involved in the enrichment of these metabolic pathways.Among them,the most significant metabolic pathways were glycerin phospholipid,α-linolenic acid,galactose,cysteine and methionine,flavonoid,etc.the map and Bubble Diagram showed that after HHP treatment,the difference metabolites involved in the enrichment of these pathways were 59 The effect of bee pollen on the enrichment of metabolic pathway is very significant.