Effect Of Water Migration And Microorganism On Quality Of Pine Nut Peptide Powder During Storage And The Exploration Of Action Mechanism

Pine nut protein is a good source of bioactive peptides.Developing the bioactive peptides from pine nuts effectively improves pine nut protein's value chain and industry chain.As a kind of antioxidant function raw material,pine nut peptide(PNP)powder has a broad application prospect.But it will appear hygroscopic,sticky,and produce an awful smell during storage,seriously affecting the preservation performance.One of the most obvious phenomena is the strong off-odor after the water migration of PNP powder during storage.The quality of the PNP powder during storage can be controlled if the potential degradation indicator volatile compounds can be identified.Therefore,the key factors affecting the quality of PNP powder were firstly determined in this paper.Then the deterioration mechanism of PNP powder caused by key influence factors was explored.The volatile indicator compounds were finally determined to guide the production and storage of terminal products.The research results of this paper are as follows:(1)The antioxidant activity of pine nut peptide was evaluated.The 3-10 k Da pine nut peptide(PNP)was prepared,isolated,and purified by Sephadex G-25 resin exclusion chromatography combined with high-performance liquid chromatography and tandem mass spectrometry(HPLC-MS/MS),and its structure was identified.DPPH radical scavenging activity,ABTS radical scavenging activity,and FRAP value were used as indexes to evaluate the antioxidant activity of peptide sequences from PNP.Combined with cytotoxicity test and cellular antioxidant activity analysis,the antioxidant activity of antioxidant peptide sequences was assessed at the cellular level.The results showed that the PNP had good antioxidant activity,and two novel antioxidant peptide sequences,KWFCT and AC-QWFCT,were isolated and identified.KWFCT showed higher DPPH radical scavenging activity(67.43%±1.33%)and FRAP value(67.86±1.03 m M Fe²⁺/mg)than Ac-QWFCT.In addition,Ac-QWFCT showed high ABTS radical inhibition activity with 74.90%±1.19%.KWFCT and Ac-QWFCT had no killing effect on Hep G2 cells at the concentration from 100?g/m L to 2 mg/m L.Both sequences have good antioxidant activity at the cellular level.The CAA value was 612.80?M QE/100 g and 916.30?M QE/100 g,respectively.(2)The dynamic changes and possible influencing factors of PNP powder degradation during storage were investigated.The PNP powder was stored under extreme conditions of 75%relative humidity(RH)and 25?for acceleration.DPPH radical scavenging activity,Fourier transform infrared spectroscopy(FT-IR),Cold field emission scanning electron microscope(SEM)and Solid-phase microextraction combined with Gas Chromatography-Mass Spectrometer(SPME-GC/MS)were used to investigate the dynamic changes of PNP powder during storage.The key factors affecting the quality of PNP powder were determined.The results showed that the DPPH radical scavenging activity of PNP powder decreased gradually during storage(from 76.91%±0.49%to 40.50%±0.84%).The surface structure of the PNP did not change,but the morphology and size of the particles did.The characteristic volatile compounds were produced during the storage of PNP powder,which indicated Maillard reaction,microbial fermentation,and oxidation reaction occurred.Combined with internal standard semi-quantitative quantitative analysis,cluster analysis,and principal component analysis(PCA),it was concluded that storage had a significant effect on the volatile compounds of PNP powder and the formation pathway of2-ethylhexyl ester and pyrazines was speculated.In addition,the study found that water migration occurred after 15 days'storage of PNP powder,and the microbial species are different.The results showed that water migration and microorganisms affected the storage of PNP powder,and water migration also affected the microbial community.(3)Investigating the substrate dynamic change of PNP powder during storage and determine the key factors affecting the degradation of PNP powder.Four comparative conditions were set to consider the effects of water migration and microorganisms on substrate structure during PNP powder storage,including normal accelerated water absorption(Normal),control water migration(CW),control microorganisms(CM)and control group.The dynamic changes of antioxidant activity of PNP powder were tracked by electron paramagnetic resonance spectroscopy(EPR).Combined with X-diffraction spectroscopy,circular chromatography(CD),fluorescence spectroscopy,UV-Vis spectroscopy,and amino acid analysis,the dynamic change of the substrate structure for PNP powder was analyzed to determine the critical influencing factors.The results showed that the DPPH radical scavenging activity showed a decreasing trend during the storage.The order of decreasing degree was Normal group>CM group>CW group>Control group from high to low.The number of·OH free radicals showed an increasing trend,and the order of increasing degree from high to low was Normal group>CW group>CM group>Control group.Under different storage conditions,new diffraction peaks appeared in the spectra,which indicated that water migration and microorganisms had a particular influence on the crystalline of the PNP powder.Microorganisms had no significant effect on the secondary structure,fluorescence intensity,and UV absorption spectrum of the PNP powder.But water migration could significantly increase the content of?-folding content(43.47±0.21%)and decrease the irregular curled structure content(56.53±0.20%).And the fluorescence intensity and UV absorption intensity also reduced with the increase of storage time.The results showed that the water migration caused the de-coloration reaction,and the conformation of PNP powder changed.Microorganisms had no significant effect on the contents of sulfur-containing amino acids and aromatic amino acids.But water migration could significantly increase the content of sulfur-containing amino acids and decrease the content of aromatic amino acids in PNP powder.Results have shown that the substrate structure of PNP powder will change during storage,and the influence of water migration is greater than the influence of microorganisms.(4)Investigating the water migration rule of PNP powder during storage and the effect of water migration on the fingerprint of volatile compounds.Low field nuclear magnetic resonance(LF-NMR)technique was used to compare the water migration patterns of PNP powder stored at 20%RH and 75%RH.In the headspace gas chromatography-ion migration spectroscopy(HS-GC-IMS)technique,the volatile compounds fingerprints of PNP powder during storage were constructed.Pearson correlation analysis and univariate analysis were used to determine the volatile indicator compounds that water migration affected the quality of PNP powder.The results showed that there was obvious water migration occurred at 75%RH storage conditions.With the increase of storage time,the content of bound water was gradually transformed into immobilized water,and then the immobilized water was transformed into free water.However,no water migration occurred at 20%RH storage conditions.According to the constructed volatile compound fingerprint of PNP powder,72 compounds were identified,among which 39 characteristic volatile compounds(including monomer and dimer)were produced by water migration.Correlation heat maps and matrices were obtained by Pearson correlation analysis,and 18 volatile compounds with high positive correlation with T2b and T23 were sorted out.Combined with FC analysis and t-test analysis,the results showed that 2,5-dimethylpyrazine-M was the best water migration indicator volatile compound.A total of 14 volatile compounds can be used as water migration indicator volatile compounds to improve the accuracy of the detection,including 2,5-dimethylpyrazine(M&D);furfural;3-methyl-3-buten-1-ol;1,2-propanediol;thiazole;2-cyclohexen-1-one;propanoic acid;2-heptanone-M;3-methylbutanol;2-methylbutanal;ethyl methyl ketone-D;benzaldehyde(M&D);3-methylbutanal-D and propyl acetate.(5)Exploring the self-equilibrium between PNP powder and environmental water molecules during storage and revealing adsorption and desorption mechanisms.The water adsorption isotherm was obtained by Dynamic Water Adsorption System(DVS).The adsorption-desorption model was established by calculating the adsorption characteristics and the kinetic parameters of adsorption-desorption.Then the self-equilibrium rule between PNP powder and environmental water molecules was studied.The surface morphology of the PNP powder was determined by atomic force microscopy(AFM).The content of the free sulfhydryl group and total sulfhydryl group was selected to evaluate the deterioration degree of PNP powder.The results showed that the adsorption curve was lower than the desorption curve,and the peptide bound 7.80%of the water in the way of chemical binding.The desorption curve of the PNP powder stored for 90 days was lower than the adsorption curve.In addition,the optimized adsorption model of PNP powder changed from the Henderson model to the Oswin model after 90 days'storage.In contrast,the optimized desorption model changed from the Halsey model to the Gab model.Vacuum freeze-drying can restore the adsorption and desorption models,but the fitting effect is reduced,which indicates that the PNP powder has irreversible structural changes during storage.The surface morphology changed during the storage process,water bonded to the PNP powder,and mercaptan content increased off-flavor compounds.(6)Investigating the dominant spoilage bacteria and the influence of microorganisms on the volatile fingerprint of PNP powder during storage and studying the impact of water migration on microorganisms.HS-GC-IMS technology was used to construct the fingerprint of the volatile compounds in PNP powder at the storage conditions with or without microorganisms,and the microbial indicator volatile compounds were determined.In addition,16S r DNA ampland sequencing technology,OTU cluster analysis,species annotation,and Alpha and Beta diversity analysis methods were used to explore the influence of dominant spoilage microorganisms and the water migration on the microbial community during the storage of PNP powder.The results showed that Exiguobacterium was the dominant bacteria during the PNP powder storage at the genus level,and its content was stable.In contrast,the content of Unidentified?chloroplast increased during storage.Firmicutes were the dominant bacteria during the storage of PNP powder at the phylum level,followed by Cyanobacteria and Proteobacteria.According to the volatile compounds fingerprint of PNP powder,30 kinds of compounds were identified,including 7 kinds of microbial characteristic volatile compounds.Pearson correlation analysis results show that the intensity of characteristic volatile compounds is positively correlated with the relative abundance of Unidentified?chloroplast.Therefore,Peak 17(Rt,247.845;Dt,0.967),Peak 18(Rt,218.205;Dt,1.336),Peak 19(Rt,218.010;Dt,1.23),Peak 20(Rt,187.59;Dt,1.266),furfural,2-methylbutyraldehyde(M&D),hexanal(M&D)can be used as microbial indicator volatile compounds.Based on OTU cluster analysis,species annotation,and Alpha and Beta diversity analysis,water migration had no significant effect on the species of microorganisms.Still,it could promote the dominant spoilage bacteria Cyanobacteria and Proteobacteria,thus promoting odor substances.