Rancidez Hidrolítica e Lipases em Alimentos: Nutrição e Segurança Alimentar

Disciplina SR Bioquímica de Alimentos Identificação da tarefaTarefa 1 Envio de arquivo Pontuação 50 pontos Tarefa 1 Esta atividade se dividirá em duas partes Observe os comandos de cada uma e responda o que se solicita 1 As lipases são responsáveis por um tipo de deterioração dos alimentos conhecido como rancidez hidrolítica Você deve pesquisar em revistas científicas da área de Alimentos um artigo sobre o tema Rancidez Hidrolítica e em seguida descrever o assunto do ponto de vista nutricional e da segurança alimentar O artigo utilizado para a tarefa deverá ser anexado ao memorial com a indicação Artigo sobre rancidez Esperase um texto entre 1000 e 1500 palavras 2Vocês deverão escrever um texto de duas páginas sobre o tema gorduras fazem maldiscutindo a relação com a saúde e os diferentes tipos de gorduras Bom trabalho Frontiers in Plant Science 01 frontiersinorg Lossoffunction of triacylglycerol lipases are associated with low flour rancidity in pearl millet Pennisetum glaucum L R Br Rasika Rajendra Aher 12 Palakolanu Sudhakar Reddy 1 Rupam Kumar Bhunia 3 Kayla S Flyckt 4 Aishwarya R Shankhapal 1 Rabishankar Ojha 3 John D Everard 4 Laura L Wayne 4 Brian M Ruddy 4 Benjamin Deonovic 4 Shashi K Gupta 1 Kiran K Sharma 1 and Pooja BhatnagarMathur 1 1 International Crops Research Institute for SemiArid Tropics ICRISAT Patancheru Telangana India 2 Department of Biotechnology Osmania University Hyderabad Telangana India 3 National AgriFood Biotechnology Institute NABI Mohali Punjab India 4 Corteva Agriscience Johnston IA United States Pearl millet is an important cereal crop of semiarid regions since it is highly nutritious and climate resilient However pearl millet is underutilized commercially due to the rapid onset of hydrolytic rancidity of seed lipids postmilling We investigated the underlying biochemical and molecular mechanisms of rancidity development in the flour from contrasting inbred lines under accelerated aging conditions The breakdown of storage lipids triacylglycerols TAG was accompanied by free fatty acid accumulation over the time course for all lines The high rancidity lines had the highest amount of FFA by day 21 suggesting that TAG lipases may be the cause of rancidity Additionally the high rancidity lines manifested substantial amounts of volatile aldehyde compounds which are characteristic products of lipid oxidation Lipases with expression in seed postmilling were sequenced from low and high rancidity lines Polymorphisms were identified in two TAG lipase genes PgTAGLip1 and PgTAGLip2 from the low rancidity line Expression in a yeast model system confirmed these mutants were nonfunctional We provide a direct mechanism to alleviate rancidity in pearl millet flour by identifying mutations in key TAG lipase genes that are associated with low rancidity These genetic variations can be exploited through molecular breeding or precision genome technologies to develop elite pearl millet cultivars with improved flour shelf life KEYWORDS pearl millet Pennisetum glaucum L R Br millet milled flour lipase triacylglycerol rancidity shelf life TYPE Original Research PUBLISHED 04 October 2022 DOI 103389fpls2022962667 OPEN ACCESS EDITED BY Palak Chaturvedi University of Vienna Austria REVIEWED BY Sunil S Gangurde University of Georgia United States Abhishek Rathore International Maize and Wheat Improvement Center CIMMYT Mexico Geetha Govind University of Agricultural Sciences Bangalore India CORRESPONDENCE Pooja BhatnagarMathur pbmathurcgiarorg PRESENT ADDRESSES Pooja BhatnagarMathur The International Maize and Wheat Improvement Center CIMMYT Texcoco Mexico Kiran K Sharma The Energy and Resources Institute TERI Gram Gurugram Haryana India These authors have contributed equally to this work SPECIALTY SECTION This article was submitted to Plant Biotechnology a section of the journal Frontiers in Plant Science RECEIVED 16 June 2022 ACCEPTED 11 August 2022 PUBLISHED 04 October 2022 CITATION Aher RR Reddy PS Bhunia RK Flyckt KS Shankhapal AR Ojha R Everard JD Wayne LL Ruddy BM Deonovic B Gupta SK Sharma KK and BhatnagarMathur P 2022 Lossof function of triacylglycerol lipases are associated with low flour rancidity in pearl millet Pennisetum glaucum L R Br Front Plant Sci 13962667 doi 103389fpls2022962667 COPYRIGHT 2022 Aher Reddy Bhunia Flyckt Shankhapal Ojha Everard Wayne Ruddy Deonovic Gupta Sharma and Bhatnagar Mathur This is an openaccess article distributed under the terms of the Creative Commons Attribution License CC BY The use distribution or reproduction in other forums is permitted provided the original authors and the copyright owners are credited and that the original publication in this journal is cited in accordance with accepted academic practice No use distribution or reproduction is permitted which does not comply with these terms Aher et al 103389fpls2022962667 Frontiers in Plant Science 02 frontiersinorg Introduction Pearl millet Pennisetum glaucum L R Br is the principal staple food for millions of people in arid and semiarid regions of Asia and Africa such as India and Nigeria It is primarily grown in the driest regions due to its ability to tolerate and thrive under both continuous and erratic drought This C4 plant requires low irrigation and resource management inputs when compared with other staples such as rice and wheat effectively reduces atmospheric CO2 has high water use efficiency and keeps drylands productive ensuring food and nutritional security for smallholder farming communities Nambiar et al 2011 A powerhouse of nutrients like protein minerals vitamins phytochemicals and antioxidants pearl millet is glutenfree and contains high levels of polyphenols and other biologically advantageous compounds thereby designating it as a Smart food Tako et al 2015 Its beneficial health properties such as lowering of fat absorption in the intestines and spikefree sugar release low glycemic index helps to reduce blood pressure heart disease and diabetes Varsha and Narayanan 2017 Despite its nutritional profile and advantage over other cereals pearl millet has remained unpopular due to the short storage life of the milled flour Goswami et al 2020 The rapid development of off flavors and taste within 57 days of milling poses a major hindrance for wider consumer acceptance creates postharvest waste and causes drudgery to consumers particularly women who are traditionally involved in food preparation Over the last decade pearl millet consumption has declined in urban areas and in commercial outlets Rani et al 2018 leading to lower incentives for its cultivation by smallholder farmers To address rancidity efforts have been made to develop postharvest processing and premilling techniques to inactivate the biological components that lead to accelerated rancidity However these mechanical and physicochemical techniques have been shown to negatively influence the nutritional quality of both the micro and macronutrients Goyal et al 2015 Therefore to revive the importance of this nutricereal in dryland agriculture further understanding of the biological processes that lead to the development of rancidity in pearl millet is urgently needed Pearl millet grain has a larger germ layer than other cereals apart from maize Taylor 2004 and has a higher lipid content 57 characterized by high levels of unsaturated fatty acids Sharma et al 2015 During milling of the whole grain the bran and germ layers rupture and release endogenous lipases that commence hydrolysis of stored lipids and release of free fatty acids FFAs Lipases are more thermostable than lipoxygenases and show higher enzyme activity in low moisture conditions Hence inhibiting the initial formation of FFAs is critical for controlling seed lipid oxidation While lipases are reported to play a primary role in the susceptibility of pearl millet to postmilling rancidity Kumar et al 2021 the underlying biochemical and molecular mechanisms have not yet been studied in detail and to our knowledge the specific lipases responsible have not been identified Here we demonstrate that flour from pearl millet characterized as having low rancidity has lower expression of TAG lipases and is more resistant to TAG degradation and lipid oxidation than flour milled from high rancidity germplasm Two TAG lipase genes PgTAGLip1 and PgTAGLip2 were identified in the low rancidity pearl millet line and contained polymorphisms that rendered the enzymes nonfunctional These key TAG lipases may be used to develop seeds with greater post milling shelf life without affecting the beneficial nutritional profile of pearl millet Materials and methods Plant material and growth Initial screening experiments were performed on a representative panel of 12 inbreds and germplasm association lines PMiGAP developed by the millet breeding program at ICRISAT Supplementary Table S1 These lines were grown at ICRISATPatancheru India and harvested during the rainy season of 2018 and 2019 and analyzed in 2019 and 2021 respectively Grain milling and storage conditions Seed was stored in vacuum packed pouches in a cold room prior to analysis 30 g of seeds from each sample were ground to fine powder in a custombuilt burrmill grinder or in a Cyclotec Foss grinding mill The ground flours were spread into evenly distributed layers in lidless food grade trays 4 oz118 ml under accelerated storage conditions in incubator chambers at a temperature of 35 01C and 75 3 relative humidity Subsamples of the flour were collected for the biochemical determination of acid value AV triacylglycerol TAG breakdown FFA generation and volatiles Highlights Nonfunctional triacyglycerol TAG lipases from low rancidity pearl millet lines had less degradation of TAG and lipid oxidation postmilling Mutations in these genes can provide targets for developing pearl millet germplasm with extended flour shelf life Aher et al 103389fpls2022962667 Frontiers in Plant Science 03 frontiersinorg Acid value Total crude fat was extracted in a Soxhlet apparatus from flour using the standard method AOAC 1990 with a few modifications Briefly 5 g of flour was taken in a cellulose thimble which was suspended in a preweighed extraction beaker containing 100 ml petroleum ether boiling point 6080C and kept on a hot plate at 180C until the sample started to boil For the complete extraction of fat the process was carried out for at least 90 min Following removal of the solvent the extracted oil was titrated against 01 N KOH using phenolphthalein as indicator and the end point recorded The acid value was calculated using the formula Acid value mg KOH g Titrant volume Normality of KOH 56 1 Weight of sample where 561 is the molecular weight of KOH Hakoda et al 2006 Quantification of lipids by GCFID and HPLCELSD Total lipid extractions were based on the method of Bligh and Dyer 1959 Briefly lipids were extracted using 3 ml of 12 vv chloroformmethanol by shaking at room temperature for 15 min Phase separation was induced by the addition of 1 ml chloroform and 18 ml water The lower lipid layer was collected and samples were reextracted twice with 2 ml chloroform The lipid extracts were subsequently filtered and dried under a stream of nitrogen gas In the 2021 experiment the extraction method was modified to include an initial step to quench lipase activity by treatment with isopropanol heated to 75C for 15 min 001 BHT was added to all reagents Neutral lipids were quantified by HPLC equipped with an ELSD Evaporative Light Scattering Detector using a cyanopropyl column Luna 5 μm CN 100 Å 250 46 mm Phenomenex with hexane as mobile phase A and methyl tertiarybutyl ether MTBEisopropanol 955 vv plus 02 acetic acid as mobile phase B with a gradient of 0 to 100 B and reequilibration of the column to 0 B Standard curves of triC182 TAG and 182 FFA were run with each sample set to quantify total TAG and FFA as a percent of dry weight DW For quantitation of total fatty acids by GCFID 01 mg of 170 TAG was added to each sample as an internal standard Acid catalysis was performed by adding 1 ml of 5 sulfuric acid in methanol followed by heating at 90C for 1 h Phase separation was induced by the addition of 1 ml of 1 M NaCl and 1 ml heptane The upper heptane layer was transferred to a sample vial for analysis FAME separation was performed on a GC system Agilent 7890A with an OmegaWax 320 column Supelco followed by FID analysis The GC oven temperature was set at a starting temperature of 190C then increased to 240C 5C min with a total run time of 10 min Analysis of volatiles by solid phase microextractionGCMS Samples for each pearl millet line were subject to Solid Phase MicroExtraction Gas Chromatography with Electron Impact Ionization Mass Spectrometry SPMEGCEIMS after 21 day of accelerated storage from the 2019 experiment Triplicate flour samples 05000 00001 g of each line were weighed into 20 ml amber headspace vials that were brought to room temperature before incubation at 35C for 1 h Analysis was performed on an Agilent 7890 GC system equipped with an Agilent 5977B detector and a Gerstel MultiPurpose Sampler MPS Robotic Gerstel with automated SPME sampling capability MS data files were preprocessed and analyzed with Genedata Expressionist software version 1451 Asiago et al 2012 Details on chromatography hardware parameters software for processing and statistics is provided in Supplementary Methods Appendix A RNA isolation cDNA synthesis and quantitative real timePCR analysis Pearl millet tissue samples milled grain were collected at 0 h 6 h 12 h and 24 h of accelerated storage and used for total RNA extraction using RNeasy Plant Mini kit Qiagen and analyzed quantitatively and qualitatively using a NanoVue plus spectrophotometer GE HealthCare One and a half μg of RNA was used for cDNA synthesis using Superscript III Invitrogen and samples diluted 110 times were used as the template Quantitative RTPCR was carried out in 96well optical reaction plates with a total reaction volume of 10 μl containing 05 μm of each primer 08 μl cDNA 10 μl Sensi Master Mix 2X and dH2O added up to 32 μl The quantitative real timePCR qRT PCR primers were designed using Primer3 software v040 with GC content of 4060 a Tm of 6062C and primer length 2025 nucleotides Supplementary Table S3 for an expected product size of 90180 bp The qRTPCR reactions were carried out using a standard thermal profile 95C for 10s 40 cycles of 15 s at 95C 15 s at 61C with fluorescent signal recording and 15 s at 72C After the 40th cycle amplicon dissociation curves were measured by heating at 58 to 95C with fluorescence measured within 20 min All qRTPCR data were obtained from three biological replicates with three technical replicates Normalized expression was calculated with qBase software Schmidt and Delaney 2010 with reference genes eukaryotic initiation factor4α PgEIF4α and malate dehydrogenase PgMDH Reddy et al 2015 List of all primers used for molecular studies is given in Supplementary Table S2 Gene expression analysis was carried out using vegetative tissues leaf shoot and roots seed developmental stages embryo milky seed immature seed and harvested grain flour under 1 wwwgenedatacom Aher et al 103389fpls2022962667 Frontiers in Plant Science 04 frontiersinorg accelerated storage for 24 h Lipid reserve mobilization studies were conducted on filter paper germinated seedlings sampled at regular intervals during 07 days after imbibition DAI Sequence analysis of the pearl millet TAG lipase genes Rice lipase protein sequences were used as the query and searched in the latest version of the pearl millet genome Varshney et al 2017 Following removal of all duplicate and redundant sequences the remaining sequences were analyzed for prediction of domains and motifs using the ExPASy PROSITE tool2 Sigrist et al 2012 Multiple sequence alignment was carried out with fulllength protein sequences of PgTAGLips with ClustalW MacVector V171 Evolutionary relationship within lipases of pearl millet were investigated using the neighborjoining algorithm of MacVector Software and the subcellular localization of proteins was predicted using the WoLF PSORT tool3 Molecular modeling and docking Molecular models of PgTAGLips were generated using homology modeling server SWISS MODEL Waterhouse et al 2018 by utilizing the known structure of the template protein Ramachandran plots were generated using PROCHECK Laskowski et al 1993 for validation of structures Three dimensional structures of triglycerides and ester ligands substrate were retrieved using the PubChem database Autodock 42 Morris et al 2009 was used for docking the triglycerides substrates to the lipase gene structures from the simulation A grid box was prepared with dimensions 30 30 30 and centered on the ATP binding site with grid spacing of 0478 Å Ten docking runs were performed for each substrate with a population size of 50 and 1500000 energy evaluation All other algorithm parameters used the default setting The docked poses for each ligand were clustered with an RMSD tolerance of 15 Å Predicting the possible proteinligand interactions and the final pose of the substrate was selected based on its docking score Gene cloning Selected PgTAGLips were PCR amplified using cDNA as a template and cloned in pCR8GWTOPO TA Cloning vector Invitrogen All sequence confirmations were carried out with at least four to five colonies from each genotype The vector backbone was trimmed and sequences analyzed and aligned across genotypes using ClustalW to identify sequence variabilities 2 httpprositeexpasyorg 3 httppsorthgcjpformhtml Yeast transformation The cDNAs of PgTAGLip genes from selected genotypes were cloned in pYES2 vector under the control of galactoseinducible promoter and URA3 as a yeast selection marker The yeast triple lipase mutant tgl3tgl4tgl5 ΔTGL Klein et al 2016 was used for TAG lipase heterologous characterization All pYES2 PgTAGLip variants were transformed into ΔTGL according to the Ojha et al 2021 Positive yeast transformants were selected on minimal synthetic defined SD base Takara media supplemented with complete supplement mixture without uracil Ura Himedia India and further identified by colony PCR using conditions described above Selected single yeast colonies were transferred into dropout base growth medium Himedia India with 2 galactose and 1 raffinose and incubated at 30C for 24 h for lipid and FACS analysis BODIPY493503 staining and yeast lipid degradation analysis Yeast ΔTGL cells transformed with different PgTAGLip variants were stained with BODIPY493503 dye 44 Difluoro 13578 Pentamethyl4Bora3a4aDiazasIndacene Invitrogen and used to quantify lipid degradation as an indication of lipase gene function BODIPY dye was used to label the yeast TAGs according to Bhunia et al 2021a In brief 2 μl of 1 mgml BODIPY stock solution prepared in DMSO was added to 1 ml yeast culture carrying different lipase variants and the samples were kept at 28C for 20 min Following the incubation period yeast cells were centrifuged at 10000 rpm for 10 min and the cell pellets washed thrice with PBS buffer and suspended in 1 ml of PBS buffer Yeast cells were further diluted 110 in PBS buffer Overnight cultures of yeast cells expressing TAG lipase were diluted into fresh medium containing cerulenin 10 μgml and terbinafine 30 μgml to block the de novo fatty acid biosynthesis and collected at 2 h 4 h and 6 h intervals Finally BODIPY dye was used to label the yeast cells for flow cytometry analysis Bhunia et al 2021a Labeled yeast cells collected at different intervals were diluted 110 in PBS buffer and used to measure the fluorescence intensity in a flow cytometer The excitation and emission wavelengths were set at 493 nm and 503 nm respectively Flow cytometry analysis Flow cytometry analysis was performed using highspeed flow cytometer BD FACSAria Fusion Becton Dickinson to measure the uptake of yeast lipids according to Bhunia et al 2021a Every flow cytometry event was measured using side scatter SSC and LB fluorescence using FITC 530 15 nm excited at 488 nm filters The mean fluorescence intensity values were analyzed using BD FACSDiva 801 The flow cytometer settings of all channels remained the same for all yeast cell sorting procedures Aher et al 103389fpls2022962667 Frontiers in Plant Science 05 frontiersinorg Statistical analysis Determination of all chemicalbiochemical parameters were carried out in triplicates and analyzed using Genstat Several Loglinear hierarchical mixed models for TAG and FFA were fitted using the brms software package Bürkner 2017 in the R programming language The model with the lowest leaveoneout information criteria Vehtari et al 2017 was chosen as the final model for inference For more information see supplementary materials Results Lipid degradation and offflavor volatiles are increased in high rancidity pearl millet lines To examine the rancidification of pearl millet flour acid values AV were determined in the oil extracted from flours of selected pearl millet lines subjected to accelerated storage conditions of high temperature and high humidity Crude fat contents of the flours ranged between 42 and 72 wt at the start of the experiment day 0 The AV of freshly ground flours at day 0 varied between 515 to 136 mg KOHg among the genotypes Under accelerated storage flour conditions AV increased significantly in all lines Figure 1A although the rates varied substantially between genotypes For instance while several lines eg I6 and P19 had AV approaching 100 mg KOHg after 14 day of accelerated aging others eg I2 I3 and I8 showed significantly lower AV 6733 mg KOHg Based on these results a subset of inbred lines with early onset of rancidity indicators were selected and reevaluated in a 21 day experiment Figure 1B The I3 inbred line had lower AV while the I5 and I7 lines had higher AV indicative of greater FFA hydrolysis A sensory panel also characterized the I3 line as less rancid eg no odor dry flour and the I5 and I7 lines as more rancid eg pungent bad smell after 21 day of accelerated aging Supplementary Table S3 The lipids from I3 I5 and I7 flours were analyzed to further characterize lipid hydrolysis All three inbred lines had similar fatty acid profiles dominated by unsaturated linoleic and oleic acids 75 which are prone particularly linoleic acid to primary and secondary oxidation Figure 2A Additionally pairwise comparisons using two sample MannWhitney tests across all fatty acids profiled showed no significant differences suggesting that fatty acid composition is likely not the cause of differences in the propensity of the lines to develop rancidity While there are some differences in total fatty acid values Figure 2B there were no significant differences between the lines KruskalWallis test and pairwise MannWhitney tests in both experiment sets 2019 and 2021 and no significant differences in total fatty acid measurements were observed between the two experiments Two independent 21day acceleratedaging experiments were performed to determine differences in lipid catabolism for the I3 I5 and I7 lines The TAG and FFA levels were measured via HPLC from subsamples taken at each time point For all lines TAG levels showed a decreasing trend over time and a corresponding increasing trend in FFAs Figure 2C Pairwise posterior median differences on the log scale averaged across the datasets were computed to assess the significance of the differences between these three lines Supplementary Table S4 The TAG values of the I7 line were significantly lower than the other two lines at all time points The TAG values of the I3 and I5 line were not significantly different until day 14 All pairwise FFA levels were found to be significantly different except for the I5 and I7 lines at day 14 The I3 line maintained the lowest levels of FFA throughout the time course Interestingly the I7 line exhibited elevated FFA amounts at the onset of the time course across both datasets it is unknown if I7 seed has elevated FFA prior to milling Another 2021 dataset using TLCGCMS showed similar trends with increasing FFA especially in I7 and decreasing TAG levels over the time course Supplementary Figure S1 however this experiment was not quantitative and there was most likely sample loss These results suggest that flour from the I7 line is more A B FIGURE 1 A Comparison of acid value AV of flour of 12 pearl millet inbred and germplasm association lines PMiGAP during 14day accelerated storage conditions The genotypes include ICMB 843 I1 ICMB88004 I2 ICMB95222 I3 ICMB81 I4 ICMB 89111 I5 ICMP842 I6 ICMB863 I7 ICMB98222 I8 IP5931 P13 IP13840 P14 IP 22419 P19 IP6099 P20 B Change in AV in the flour of selected contrasting lines on day 10 and 21 The data are represented as the mean standard error of three biological replicates Variables showing Fvalue less than 005 were significant Also the means displaying nonmatching lowercase letters were significantly different at p 005 by the Duncan test Aher et al 103389fpls2022962667 Frontiers in Plant Science 06 frontiersinorg prone to TAG degradation during storage at high temperature and high humidity To confirm that the hydrolysis of TAG and the release of FFAs in flours is directly associated with the development of rancidity the volatile chemicals released from the flours after accelerated storage were analyzed using SPMEGC EIMS Principal component analysis of all autoscaled components indicated that each line was distinct Figure 3A however line I3 appeared to be markedly different from the lines I5 and I7 due to the presence of aliphatic aldehydes and other known markers of lipid oxidation Figure 3 Supplementary Table S5 These data clearly indicate differential oxidation of fatty acids in the tested lines From over 3000 detected features in this analysis four of the top 15 most statistically significant metabolites were aldehydes Figure 3 which are known secondary oxidative products of unsaturated lipids The I5 and I7 lines had significantly higher levels of hexanal octanal nonanal and benzeneacetaldehyde compared to the line I3 in the headspace above flours exposed to 21 days of accelerated aging Figures 3BC Since the fatty acid profiles for the three lines were similar Figure 2A the lines I5 and I7 seem to have greater susceptibility to rancidity than the I3 line due to higher amounts of FFAs and the oxidation of these FFAs A B C FIGURE 2 Lipid catabolism in milled flour under acceleratedaging conditions A Fatty acid profiles obtained by GCFID B Total fatty acid quantitation by GCFID average over three replicates Each brace corresponds to a MannWhitney test with the resulting value of p indicated above the brace C TAG and FFA levels measured by HPLCELSD in two datasets 2019 and 2021 Loglinear hierarchical mixed models were utilized to assess the differences in TAG and FFA levels of these three inbred lines Points represent observed data lines represent posterior mean of the predictive distribution and the ribbon corresponds to the 95 credible interval of the posterior mean predictive distribution Aher et al 103389fpls2022962667 Frontiers in Plant Science 07 frontiersinorg Polymorphisms in TAG lipases identified in the low rancid line are nonfunctional The lipid and oxidative analyses suggest that these lines may have differences in TAG lipases Our analysis identified a total of 44 lipase genes in pearl millet accessions in the International Pearl Millet Genome Sequence Consortium IPMGSC data These lipases were divided into three major subfamilies based on sequence alignment and evolutionary relationships Subfamily I and III characterized under PLDPLCesterase comprised 12 and 18 genes respectively Figure 4A Subfamily II contained 14 genes with a conserved LID domain and a catalytic triad which are the characteristic features of TAG lipases Subfamily II lipase gene and protein characteristics are summarized in Supplementary Table S6 Based on the expression analyses of various plant tissues of the popular variety ICMB 9333 and 24 h accelerated storage of milled flour of the contrasting I3 I5 and I7 inbreds three lipases were selected for further analyses These lipases had higher similarity to the previously reported rice and Arabidopsis lipases LOC Os11g43510 and AT5G18640 respectively Tiwari et al 2016 The lipases PglGLEAN10023115 PglGLEAN10024115 and PglGLEAN10011213 were renamed PgTAGLip1 PgTAGLip2 and PgTAGLip3 respectively PgTAGLip2 had high expression in immature embryos of ICMB 9333 compared to other tissues such as milky seed immature seed leaf stem and roots Figure 4B While PgTAGLip1 expressed in most of the tissues except leaf with the highest expression at the milky seed stage PgTAGLip3 showed higher expression in milky seeds and leaves Figure 4B During 24 h of accelerated storage of flours from the contrasting inbred lines there were significant increases in the expression of all three genes in the flour from lines I5 and I7 compared to the flour from line I3 Figure 4C Interestingly the expression levels of PgTAGLip1 and PgTAGLip2 were considerably lower in I3 flour compared to the flours of I5 and I7 15fold during the first 24 h Figure 4C suggesting that these lipase genes may be responsible for the differences in TAG degradation between these lines Figure 2 Due to the differences in quantitative expression the genetic sequences were analyzed for structural polymorphisms across genotypes Sanger sequencing confirmed that the fulllength coding regions of all three lipases carried a LID domain an active site containing a serine GXSXG motif and a catalytic triad Supplementary Figure S2 However the PgTAGLip1 gene sequences revealed polymorphisms among the lines I3 I5 and I7 Figure 5A To further confirm the correlation of these allelic A B C D FIGURE 3 Volatile analysis of pearl millet I3 I5 and I7 lines using Solid Phase MicroExtraction Electron Impact Ionization Gas Chromatography Mass Spectrometry SPMEEIGCMS A First and second principal components for processed autoscaled datasets for the I3 I5 and I7 lines blue triangles aquamarine inverted triangles and pink stars respectively A 95 confidence range is indicated by a dashed line B Most abundant fragment mz total peak intensity bar charts for lowest value of p aldehydes C5C10 for the I3 I5 and I7 lines CD Volcano plots generated from 2 groups sample comparison tests for I3 vs I5 C and I3 vs I7 D Most statistically significant aldehydes as measured by value of p are shown labeled See Supplemental Table S5 for the detailed statistical analysis Aher et al 103389fpls2022962667 Frontiers in Plant Science 08 frontiersinorg variations with the observed and reported flour shelf life additional genotypes hybrids and inbred lines based on a subset of the rancidity matrix were profiled for structural variations in PgTAGLip1 The PgTAGLip1 gene in the I7 and high rancidity line 86 M88 formed a functional protein encoded by a 1110 bp coding sequence while the line I5 had a 807 bp coding region Interestingly the I3 line showed two transcript variants of PgTAGLip1 having fragment sizes of 528 bp and 276 bp Supplementary Table S6 these polymorphisms were named LR1 and LR2 respectively The LR1 transcript contained a stop codon within the LID domain that was expected to result in a lossoffunction of this gene while the LR2 variant formed a much smaller peptide lacking the LID domainactive site and was also presumed to be nonfunctional Figure 5A In addition to the I3 line other low rancidity lines Super Boss and RHB 177 also revealed mutations resulting in stop codons upstream of the LID domain at 420 bp and 285 bp respectively Figure 5A Supplementary Table S6 It was intriguing to note that in contrast to the PgTAGLip2 transcript 1068 bp in line I7 single base variations and deletions up to 6 bp were observed in both lines I3 and I5 causing premature stop codons Figure 5B Supplementary Table S6 No sequence variations among the three tested genotypes were observed in PgTAGLip3 These results provide further evidence that the maintenance of TAG lower accumulation of FFA and lower amounts of aldehyde volatiles in the line I3 are associated with allelic variations within the PgTAGLip1 and PgTAGLip2 genes To further support our annotations molecular docking was used to predict the affinityspecificity of these pearl millet TAG lipases to several triacylglyceride ligands Supplementary Table S7 Rhizomucor miehei lipase RML PDB id 3TGL was used as a template to generate the threedimensional models and shared a 3255 peptide identity with the PgTAGLips The modeled structure when superimposed with the template PDB id 3TGL showed an overall root mean square deviation RMSD of 166 suggesting close structural similarities among the modeled and template structures Geometrical aspects of the modeled structures revealed that 70 of the modeled secondary structure was favorable and based on the predicted Ramachandran plots only 09 of residues fell in the disallowed region thereby confirming the high quality of the modeled structures Proteinligand docking A B C FIGURE 4 Phylogenetic relationships and expression analysis of the pearl millet lipases A The phylogenetic tree constructed using MacVector software by the NJ method for PgTAGLips Asterisks denotes the genes used for further analysis PglGLEAN10023115 was renamed PgTAGLip1 Pgl GLEAN10024115 renamed PgTAGLip2 and PglGLEAN10011213 renamed PgTAGLip3 B Expression analysis of three selected PgTAGLips in the various developmental stages of pearl millet variety ICMB 9333 Data points represent the expression values obtained after normalization against the reference genes EUKARYOTIC INITIATION FACTOR4Α PgEIF4α and MALATE DEHYDROGENASE PgMDH Each data point represents the mean of three biological replications with standard error SE representing the mean of three technical replicates C Expression of the selected PgTAGLips in contrasting genotypes I3 I5 and I7 under accelerated storage of milled flour The xaxis represents the identities of flour samples stored at 0 h 1 h 5 h 24 h The yaxis indicates the normalized gene expression values against the reference genes PgEIF4α and PgMDH Each data point represents the mean of three biological replications with standard error SE representing the mean of three technical replicates Aher et al 103389fpls2022962667 Frontiers in Plant Science 09 frontiersinorg revealed that PgTAGLip1 PgTAGLip2 and PgTAGLip3 had very high affinitiesspecificities for triglycerides containing unsaturated fatty acid residues suggesting that these lipases have a predisposition for degrading TAG containing linoleic and oleic fatty acids in pearl millet flour Figure 5C Supplementary Table S7 To determine if the PgTAGLip allelic variants were functional they were assessed for complementation in a lipasedeficient yeast mutant The yeast triple lipase mutant Δtgl3Δtgl4Δtgl5 ΔTGL lacks all three endogenous lipases PgTAGLip1 PgTAGLip2 and PgTAGLip3 genes were cloned from the lines I3 I5 and I7 and transformed into ΔTGL To indicate the germplasm source and genotypic identity of allelic variants mutants superscripts were added to the gene names eg PgTAGLip1I7 PgTAGLip1I3LR1 etc The total amount of TAG from the transformed yeast cells was analyzed by GCMS Yeast cells expressing fulllength nontruncated PgTAGLip1 PgTAGLip2 and PgTAGLip3 accumulated less TAG 1651 1872 and 1465 μmolg respectively when compared to the ΔTGL control 2693 μmolg Figure 6A The truncated allelic variants PgTAGLip1I3LR1 PgTAGLip1I3LR2 PgTAGLip2I3 and PgTAGLip2I5 accumulated comparable TAG content to ΔTGL Figure 6A confirming that these truncations are nonfunctional To corroborate the lower amounts of TAG from the truncated PgTAGLip variants mean fluorescence intensity of yeast TAGs stained with BODIPY493503 were analyzed by flow cytometry There was a significant decrease in fluorescence intensity in the populations of yeast cells P3 expressing fulllength lipase genes compared to the control ΔTGL expressing empty vector The yeast cells carrying nontruncated versions of PgTAGLip1 PgTAGLip3 and PgTAGLip2 cloned from the I7 line had lower intensities 6425 5810 5415 respectively reflecting TAG breakdown In contrast the cells expressing truncated variants PgTAGLip1I3LR1 PgTAGLip1I3LR2 PgTAGLip2I3 and PgTAGLip2I5 showed higher fluorescence intensities viz 8265 8475 8255 and 898 respectively Supplementary Figure S3A Hence these results demonstrate that all the truncated allelic variants PgTAG Lip1I3LR1 PgTAG Lip1I3LR2 PgTAG Lip2I3 and PgTAG Lip2I5 had reduced functionality TAG lipases with lossoffunction mutations do not mobilize TAG and have no impact on seedling establishment The reduction in TAG content in yeast strongly suggested that all three nontruncated PgTAGLip exhibit lipolytic function To demonstrate that deleterious mutations in the variants affect their function timedependent turnover of TAG was estimated during the yeast growth up to 6 h and measured using flow cytometry Results indicated that the ΔTGL mutant and truncated variants PgTAGLip1I3LR1 PgTAGLip1I3LR2 PgTAGLip2I3 and A B C FIGURE 5 Structural variations of selected TAG lipases A Comparative amino acid sequence alignment of PglGLEAN10023115 PgTAGLip1 in high I7 86 M88 I5 and low rancidity genotypes I3 Super Boss RHB177 The LID domain is in bold and the Lip serine active motif GxSxG is in bold and underlined B Comparative amino acid sequence alignment of PgTAGLip2 in high rancid I7 and I5 and low rancid I3 lines The LIP domain is bold and Lip serine active motif GxSxG is bold and underlined C Predicted protein models and docking affinities of three PgTAGLips PgTAGLip1 PglTAGLip2 and PgTAGLip3 with triolein and trilinolein The predicted substrate binding is shown in a ball and stick model with dashed pink lines denoting the hydrogen bonds The putative LID domain GxSxG motif is in green the catalytic triad is in yellow with ball and stick the putative glycosylation sites are in red and the ligand ball and stick is in pink Aher et al 103389fpls2022962667 Frontiers in Plant Science 10 frontiersinorg PgTAGLip2I5 did not mobilize TAG products In contrast the nontruncated versions cloned from the line I7 were significantly active in mobilizing TAG products during the 2 h to 6 h time course Figure 6B To determine if the observed differences in lipid degradation between the lines I3 and I7 had any effect on seedling establishment plumule elongation and lipid mobilization were measured during germination No differences were observed in postgermination seedling establishment or growth Figure 6C indicating that while the mutations in both candidate lipases may be useful for minimizing TAG hydrolysis these enzymes are not essential for germination Additionally there were minimal differences in TAG mobilization between the lines I3 and I7 during germination Figure 6D most likely other TAG lipases are involved in mobilizing TAG for germination Taken together these results suggest that mutations in PgTAGLip1 and PgTAGLip2 contribute to lower amounts of FFA accumulating in seeds postmilling Therefore allelic variations in these genes may provide an opportunity to decrease the amount of FFA and thus their oxidation thereby alleviating the propensity of rancidity development in pearl millet flour Discussion Considered as the mother of all problems in pearl millet flour rancidity is the most important intervention required for ensuring a sustainable demand for this nutricereal While stabilization techniques have been effective in minimizing rancidity to some extent only largescale processing would justify their economic feasibility which may not be practical in rural agricultural communities The improved stability of shelf life following stringent treatments suggests that biological enzymatic driven processes drive the generation of offflavor volatiles in pearl millet flours Thus genetic improvements are a promising alternative to physical stabilization techniques for extending the storage life of flour Biochemical analyses suggest rancidity of pearl millet flour is due to lipid catabolism A set of 12 pearl millet genotypes based on an earlier study Majumdar et al 2016 were investigated for identifying genotypes with contrasting susceptibility to rancidity The change in AV an indicator of esterified lipid hydrolysis and the presence of FFA over 10 days from seed grinding was used to categorize the pearl millet lines Contrasting inbred lines were categorized as low AV I3 high AV I5 and intermediate AV I7 that were further confirmed by sensory panel analysis Clear differences between the selected lines were observed in the amounts of TAG and FFA present in the seeds at the time A B C D FIGURE 6 Functional validation in yeast expression system and seedling growth of the contrasting pearl millet seeds lines during germination A GCMS TAG analysis of yeast cells expressing TAG lipase variants Each data point represents the mean of three biological replications with standard error SE B in vivo mobilization of TAG reserve in yeast triple lipase mutant expressing TAG lipase variants from 2 h to 6 h Each data point represents the mean of two biological replications with standard error SE C Seedling establishment of I3 and I7 lines D TAG mobilization during germination and postgerminative growth Each data point represents the mean of three biological replications with standard error SE Aher et al 103389fpls2022962667 Frontiers in Plant Science 11 frontiersinorg of grinding through the acceleratedaging treatment which were in broad agreement with the initial AV studies Further analysis after 21 days of accelerated storage showed that aldehydes and other markers of fatty acids primary and secondary oxidation 1octen3ol and 2pentyl furan were the dominant discriminators in comparisons between the headspace volatiles of the flours from lines I3 vs I5 and I3 vs I7 Not only are these volatile chemicals the key markers of oil oxidation Franklin and Mitchell 2019 but they are also associated with the development of rancidity in foods rich in polyunsaturated fats including seeds and flours Gorji et al 2019 Other nonlipid related offflavor metabolites such as 2acetyl1pyrroline and apigenin have been identified in high moisture 30 pearl millet flours within 15 h of grinding and wetting Reddy et al 1986 Seitz et al 1993 where both have been associated with a mousy acidic odor detected in millet flours Mass spectral searches of the volatile chemistry profiles of the 21day aged flours analyzed in this study using the NIST AMDIS application failed to detect these two compounds thereby indicating that they were not principal factors in this material and that products of lipid oxidation were closely associated with flour rancidity These results indicate that differential hydrolysis of esterified lipids is linked to the release of FFA and is closely associated with the development of rancidity under acceleratedaging conditions Although PgTAGLip1 and PgTAGLip2 in the I5 line are prematurely truncated the flour from the I5 line accumulated more FFAs This discrepancy could possibly be due to functional redundancy among TAG lipases We presume that the amphipathic polyunsaturated FFAs are more prone to oxidative degradation once released from lipid bodies and membranes resulting in the generation of short chain aldehydes and other secondary oxidation products characteristic of oil rancidity Bhunia et al 2021b Considering that the fatty acid profiles for the three lines were similar these results indicate that the lines I5 and I7 had higher propensity for rancidity than the line I3 due to higher amounts of FFAs and the subsequent oxidation products Furthermore this direct association between TAG degradation and offflavor volatiles suggest that there may be transcript or genomic differences in the TAG lipases between these pearl millet lines Nonfunctional mutations in PgTAGLip1 and PgTAGLip2 are associated with low rancidity Three PgTAG lipases with close homology to those reported from O sativa and A thaliana Tiwari et al 2016 were found to have differences in transcript levels and sequence between the three tested pearl millet lines Expression analyses showed that PgTAGLip1 PgTAGLip2 and PgTAGLip3 were expressed in stored flour with line I3 having the lowest expression of PgTAGLip1 and PgTAGLip2 suggesting a catalytic role in the degradation of triacylglycerols TAGs and FFA generation even after milling Further insights connecting these TAG lipases to the differential susceptibility to rancidity in the I3 I5 and I7 lines were revealed through sequence analysis where mutations were observed in the coding sequences of PgTAGLip1 and PgTAGLip2 of lines I3 and I5 Structural polymorphisms SNPs and deletions in PgTAGLip1 and PgTAGLip2 led to premature stop codons that terminated the PgTAGLip gene products before or close to the catalytic center TAG lipases contain a catalytic serineaspartatehistidine triad that is required for activity Wada and Murata 2009 Tiwari et al 2016 A LID domain covering this active site is present in lipases and is critical for the interaction with TAG substrates ElKouhen et al 2005 Our study revealed that in the I7 high rancidity line PgTAGLip1 had an intact LID domain and catalytic triad while in the I5 line PgTAGLip1 lacked the histidine residue Supplementary Table S8 In contrast the low rancidity I3 line altogether lacked these active site motifs in this gene Interestingly while premature truncations were apparent in the PgTAGLip2 gene in lines I3 and I5 the catalytic center was intact in all three lines While our study did not confirm the effect of truncations on the enzymatic activity the protein docking of various ligands triglycerides with both PgTAGLip1 and PgTAGLip2 predicted very high affinityspecificity for triolein and trilinolein substrates These substrates are most likely some of the endogenous substrates since oleic and linoleic fatty acids are major components of pearl millet flour Zaplin et al 2013 In addition to the PgTAGLip1 polymorphisms uncovered in line I3 detection of similar mutations in other low rancidity lines Supplementary Table S8 like Super Boss and RHB177 further imply that mutations in this TAG lipase are the primary cause of rancidity in pearl millet These findings are in line with previous reports where mutations in the serine residue in the GXSXG motif of Pseudomonas aeruginosa patatinlike phospholipase and yeast TAG Lipase 4 with an alanine residue almost demolishes lipase activity Kurat et al 2006 Similarly complete deletion of the GXSXG motif in mouse phospholipase A2 PLA2 almost eliminates its enzymatic activity Wada and Murata 2009 A yeast lipasedeficient system can be used to test the functionality of heterologous lipase genes Bansal et al 2021 Since the yeast storage lipids are structurally and functionally similar to seed storage lipids it serves as an ideal in vivo system to ascertain the role of candidate lipases in TAG hydrolysis Jacquier et al 2013 A comparison of the truncated lipases from the line I3 PgTAGLip1I3 and PgTAGLip2I3 with the nontruncated lipases from the line I7 PgTAGLip1I7 and PgTAGLip2I7 revealed that the lipase variants in line I3 were less functional in mobilizing yeast storage lipids These results demonstrate that rendering PgTAGLip1 andor PgTAGLip2 nonfunctional through mutations in the active site motifs can provide a mechanism for maintaining TAG levels and decrease offflavor volatiles in the flour Aher et al 103389fpls2022962667 Frontiers in Plant Science 12 frontiersinorg TAG lipase polymorphisms can be used to develop pearl millet varieties with low susceptibility to rancidity For the application of mutating TAG lipases to alleviate the rancidity of pearl millet flour and increase shelf life physiological and agronomic effects must be minimal Lipases are important in the catabolism of lipid reserves especially during germination and postgermination seedling establishment Eastmond 2006 Our results show minimal changes to seedling establishment and TAG mobilization in the I3 line containing polymorphisms in PgTAGLip1 and PgTAGLip2 thereby suggesting that these genes are not essential for germination or postgermination growth While both of these TAG lipases may be useful for TAG hydrolysis since the variants do not mobilize TAG there are most likely functional redundancies among PgTAGLip enzymes Conclusion and future prospects Specific polymorphisms in pearl millet lipases have been identified that can be used for breeding high yielding hybrid pearl millet varieties with prolonged flour shelf life Our study also demonstrates strategies for future chemical mutagenesis or CRISPRbased approaches to create lossoffunction mutations in PgTAGLip1 or PgTAGLip2 thereby mitigating rancidity in elite milled pearl millet germplasm Increasing the storage life of flour from this nutritious grain offers tremendous opportunities for primary and secondary processing creating markets and enhanced profits for smallholder farmers in South Asia and SubSaharan Africa Data availability statement The datasets presented in this study can be found in online repositories The names of the repositoryrepositories and accession numbers can be found in the articleSupplementary material Author contributions RA investigation and validation PR methodology original draft and analysis RB methodology investigation and review and editing KF investigation and review and editing AS investigation and validation RO investigation JE conceptualization methodology investigation and review and editing LW investigation analysis and review and editing BR data curation and visualization BD data curation SG seeds and review KS conceptualization analysis original draft review and editing and project administration PBM conceptualization funding acquisition methodology formal analysis original draft supervision and project administration All authors contributed to the article and approved the submitted version Funding RA and RB acknowledge fellowship support from the Department of Science and Technology in the form of INSPIRE fellowship DSTINSPIRE032018000417 and INSPIRE Faculty award DSTINSPIRE042017000484 respectively PBM acknowledges the financial support from the CGIAR Research Program on Grain Legumes and Dryland Cereals CRPGLDC supported by CGIAR Fund Donors Acknowledgments Authors thank Central Instrumentation Facility CIFNABI for providing key instruments used during the study Thanks to Joe Shambaugh for assistance with workflow development on the Genedata Expressionist software platform Authors also thank Günther Daum Graz University of Technology for kindly providing the yeast lipasemutant strain Conflict of interest KF JE LW BR and BD were employed by the company Corteva Agriscience The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest The reviewer AR declared a past coauthorship with the author SG to the handling editor Publishers note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations or those of the publisher the editors and the reviewers Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher Supplementary material The Supplementary material for this article can be found online at httpswwwfrontiersinorgarticles103389fpls2022962667 fullsupplementarymaterial Aher et al 103389fpls2022962667 Frontiers in Plant Science 13 frontiersinorg References AOAC 1990 Official Methods of Analysis 15th Edn Washington DC Association of Official Analytical Chemists Asiago V M Hazebroek J and Harp T 2012 Effects of genetics and environment on the Metabolome of commercial maize hybrids a multisite study J Agric Food Chem 60 1149811508 doi 101021jf303873a Bansal S Sardar S Sinha K Bhunia R K Katoch M Sonah H et al 2021 Identification and molecular characterization of rice branspecific lipases Plant Cell Rep 40 12151228 doi 101007s00299021027144 Bhunia R K Sinha K Chawla K Randhawa V and Sharma T R 2021a Functional characterization of two type1 diacylglycerol acyltransferase DGAT1 genes from rice Oryza sativa embryo restoring the triacylglycerol accumulation in yeast Plant Mol Biol 105 247262 doi 101007s1110302001085w Bhunia R K Sinha K Kaur R Kaur S and Chawla K 2021b A holistic view of the genetic factors involved in triggering hydrolytic and oxidative rancidity of rice bran lipids Food Rev Int 126 doi 1010808755912920211915328 Bligh E G and Dyer W J 1959 A rapid method of total lipid extraction and purification Can J Biochem Physiol 37 911917 doi 101139y59099 Bürkner P 2017 Brms An R package for Bayesian multilevel models using stan J Statist Soft 80 128 doi 1018637jssv080i01 Eastmond P J 2006 SUGARDEPENDENT1 encodes a patatin domain triacylglycerol lipase that initiates storage oil breakdown in germinating Arabidopsis seeds Plant Cell 18 665675 doi 101105tpc105040543 ElKouhen K Blangy S Ortiz E Gardies A M Ferté N and Arondel V 2005 Identification and characterization of a triacylglycerol lipase in Arabidopsis homologous to mammalian acid lipases FEBS Lett 579 60676073 doi 101016j febslet200509072 Franklin L M and Mitchell A E 2019 Review of the sensory and chemical characteristics of almond Prunus dulcis flavor J Agric Food Chem 67 27432753 doi 101021acsjafc8b06606 Gorji S G Calingacion M Smyth H E and Fitzgerald M 2019 Effect of natural antioxidants on lipid oxidation in mayonnaise compared with BHA the industry standard Metabolomics 15 114 doi 101007s1130601915684 Goswami S Asrani P Ali T A Kumar R D Vinutha T Veda K et al 2020 Rancidity matrix development of biochemical indicators for analyzing the keeping quality of pearl millet flour Food Anal Methods 13 21472164 doi 101007 s12161020018312 Goyal P Chugh LK and Bajaj S 2015 Improving shelf life of pearl millet flour through conventional plant breeding approach Proceedings of the India International Science FestivalYoung Scientists Meet Department of Science and Technology Government of India Paper Code Innov 61 Hakoda A Sakaida K and Suzuki T 2006 Determination of the acid value of instant noodlesInterlaboratory study J AOAC Int 89 13411346 doi 101093 jaoac8951341 Jacquier N Mishra S Choudhary V and Schneiter R 2013 Expression of oleosin and perilipins in yeast promotes formation of lipid droplets from the endoplasmic reticulum J Cell Sci 126 51985209 doi 101242jcs131896 Klein M Islam Z U Knudsen P B Carrillo M Swinnen S Workman M et al 2016 The expression of glycerol facilitators from various yeast species improves growth on glycerol of Saccharomyces cerevisiae Metabol Eng Commun 3 252257 doi 101016jmeteno201609001 Kumar R R Bhargava D V Pandit K Goswami S Shankar S M Singh S P et al 2021 Lipasethe fascinating dynamics of enzyme in seed storage and germinationa real challenge to pearl millet Food Chem 361130031 doi 101016j foodchem2021130031 Kurat C F Natter K Petschnigg J Wolinski H Scheuringer K Scholz H et al 2006 Obese yeast triglyceride lipolysis is functionally conserved from mammals to yeast J Biol Chem 281 491500 doi 101074jbcM508414200 Laskowski R A MacArthur M W Moss D S and Thornton J M 1993 PROCHECK a program to check the stereochemical quality of protein structures J Appl Crystallogr 26 283291 doi 101107S0021889892009944 Majumdar K Sanyal S K Dutta S K Satyanarayana T and Singh V K 2016 Nutrient mining addressing the challenges to soil resources and food security in Biofortification of Food crops eds U Singh C Praharaj S Singh and N Singh New Delhi Springer 177198 Morris G M Huey R Lindstrom W Sanner M F Belew R K Goodsell D S et al 2009 AutoDock4 and AutoDockTools4 automated docking with selective receptor flexibility J Comput Chem 30 27852791 doi 101002 jcc21256 Nambiar V S Dhaduk J J Sareen N Shahu T and Desai R 2011 Potential functional implications of pearl millet Pennisetum glaucum in health and disease J Appl Pharm Sci 1 6267 Ojha R Kaur S Sinha K Chawla K Kaur S Jadhav H et al 2021 Characterization of oleosin genes from forage sorghum in Arabidopsis and yeast reveals their role in storage lipid stability Planta 25497 doi 101007 s00425021037448 Rani S Singh R Sehrawat R Kaur B P and Upadhyay A 2018 Pearl millet processing a review Nutr Food Sci 48 3044 doi 101108 NFS0420170070 Reddy V P Faubion J M and Hoseney R C 1986 Odor generation in ground stored pearl millet Cereal Chem 63 403406 Reddy P S Reddy D S Sharma K K BhatnagarMathur P and Vadez V 2015 Cloning and validation of reference genes for normalization of gene expression studies in pearl millet Pennisetum glaucum L R Br by quantitative realtime PCR Plant Gene 1 3542 doi 101016jplgene201502001 Schmidt G W and Delaney S K 2010 Stable internal reference genes for normalization of realtime RTPCR in tobacco Nicotiana tabacum during development and abiotic stress Mol Gen Genet 283 233241 doi 101007 s0043801005111 Seitz L M Wright R L Waniska R D and Rooney L W 1993 Contribution of 2acetyl1pyrroline to odors from wetted ground pearl millet J Agric Food Chem 41 955958 doi 101021jf00030a023 Sharma M Yadav D N Singh A K and Tomar S K 2015 Rheological and functional properties of heat moisture treated pearl millet starch J Food Sci Technol 52 65026510 doi 101007s1319701517351 Sigrist C Castro E Cerutti L Cuche B Hulo N Bridge A et al 2012 New and continuing developments at PROSITE Nucleic Acids Res 41 D344D347 doi 101093nargks1067 Tako E Reed S M Budiman J Hart J J and Glahn R P 2015 Higher iron pearl millet Pennisetum glaucum L provides more absorbable iron that is limited by increased polyphenolic content Nutr J 14 19 doi 101186147528911411 Taylor J R N 2004 Millets in Encyclopedia in Grain Science eds C Wrigley H Corke and C E Walker London Elsevier 253261 Tiwari S SL K Kumar V Singh B Rao A R Mithra SV A et al 2016 Mapping QTLs for salt tolerance in rice Oryza sativa L by bulked segregant analysis of recombinant inbred lines using 50K SNP chip PLoS One 11e0153610 doi 101371journalpone0153610 Varsha R and Narayanan A 2017 Storage stability of bio fortified pearl millet flour Int J Agric Innov Res 5 23191473 Varshney R K Shi C Thudi M Mariac C Wallace J Qi P et al 2017 Pearl millet genome sequence provides a resource to improve agronomic traits in arid environments Nat Biotechnol 35 969976 doi 101038nbt3943 Vehtari A Gelman A and Gabry J 2017 Practical Bayesian model evaluation using leaveoneout crossvalidation and WAIC Stat Comput 27 14131432 doi 101007s1122201696964 Wada H and Murata N 2009 Lipids in Photosynthesis Essential and Regulatory Functions 30 Germany Springer Science Business Media Waterhouse A Bertoni M Bienert S Studer G Tauriello G Gumienny R et al 2018 SWISSMODEL homology modelling of protein structures and complexes Nucleic Acids Res 46 W296W303 doi 101093nargky427 Zaplin E S Liu Q Li Z Butardo V M Blanchard C L and Rahman S 2013 Production of high oleic rice grains by suppressing the expression of the OsFAD21 gene Funct Plant Biol 40 9961004 doi 101071FP 12301 Disciplina SR Bioquímica de Alimentos Identificação da tarefaTarefa 1 Envio de arquivo Pontuação 50 pontos Tarefa 1 Esta atividade se dividirá em duas partes Observe os comandos de cada uma e responda o que se solicita 1 As lipases são responsáveis por um tipo de deterioração dos alimentos conhecido como rancidez hidrolítica Você deve pesquisar em revistas científicas da área de Alimentos um artigo sobre o tema Rancidez Hidrolítica e em seguida descrever o assunto do ponto de vista nutricional e da segurança alimentar O artigo utilizado para a tarefa deverá ser anexado ao memorial com a indicação Artigo sobre rancidez Esperase um texto entre 1000 e 1500 palavras O artigo escolhido trata dos mecanismos bioquímicos e moleculares no desenvolvimento de ranço em farinha de milheto O milheto é tratado como um alimento básico principalmente em regiões da Ásia e da África Como a cultura é resistente a secas é uma boa opção para cultivar em áreas áridas Além disso o cultivo do milheto não demanda muitos insumos em irrigação e fertilizantes sendo uma boa opção para o cultivo de pequenos agricultores O milheto garante a segurança alimentar e nutricional de pequenas comunidades devido ao seu fácil cultivo e alto valor nutricional além de trazer benefícios a saúde como a redução de pressão arterial e de açúcar no sangue Contudo uma grande dificuldade ocorre na implementação do milheto na alimentação do dia a dia Isso ocorre devido ao processo de rancidez hidrolítica o processo garante ao milheto processado em farinha aromas e sabores estranhos apenas cinco a sete dias após a moagem A rancidez hidrolítica corresponde a deterioração de alimentos quando os lipídios dos alimentos sofrem quebra em suas ligações Em geral os triglicerídios em contato com a água leva a formação de ácidos graxos e outros compostos que alteram as características organolépticas do alimento como sabor textura e odor No caso do milheto a formação de ranço na farinha fez com que o alimento fosse cada vez mais impopular Novas tecnologias de póscolheita e moagem visaram melhorar e diminuir a formação de ranço Contudo tais técnicas impactaram negativamente no aspecto nutricional do vegetal Com isso em vista o artigo foca em descobrir os processos biológicos que levam a rancidez hidrolítica na farinha de milheto Como o milho o grão do milheto apresenta uma camada germinativa maior que a maioria dos vegetais tendo um alto teor de lipídios principalmente altos níveis de ácidos graxos insaturados Tais ácidos graxos são liberados durante o processo de moagem do grão quando a camada germinativa é rompida e ocorre a liberação de lipases As lipases são enzimas endógenas que uma vez liberadas iniciam a quebra dos lipídios do milheto em ácidos graxos livres Os ácidos livres então levam a oxidação do grão e alteração do sabor e valor nutricional Sendo assim as lipases são essenciais quando se fala da formação de ranço no processo de moagem Para entender melhor o papel das lipases na rancidez do milheto foram utilizadas 12 linhagens de milheto as quais foram cultivadas na Índia durante a estação de chuvas em 2018 e 2019 As sementes após colhidas foram armazenadas em bolsas embaladas a vácuo em câmara fria Esse armazenamento cuidadoso é importante pois protege o vegetal e garante um maior nível de segurança alimentar Isso acontece porque a presença de água e ácidos graxos livres que se formam em alimentos que sofreram rancidez hidrolítica pode levar ao crescimento de bactérias e fungos tornando o grão impróprio para o consumo Além disso como a rancidez causa a mudança em odores é possível que uma contaminação por microrganismos patogênicos passe despercebida por causa dessas alterações O armazenamento em câmara fria em temperaturas mais baixas também protege o grão contra crescimento de bactérias e fungos Após armazenado os grãos passaram por moagem e foram colocados em bandejas de qualidade alimentar umidecidas e armazenadas em câmaras incubadoras durante 14 e 21 dias Amostras foram coletadas para a determinação do índice de acidez quebra de triacilgriceróis e geração de ácido graxos livres e voláteis Tais fatores forma analisados tendo em vista que a rancidez hidrolítica altera o aspecto nutricional levando a perda de vitaminas e proteínas além da geração de compostos prejudiciais à saúde como aldeídos e peróxidos Para compreender os processos biológicos envolvendo as lipases presentes no milheto foi feita isolamento de RNA e análise de PCR em tempo real e as sequências dos genes de lipase foram analisadas e utilizadas para a transformação de leveduras Assim a quantificação da degradação lipídica foi analisada verificando a capacidade de degradação dos genes na levedura E para medir a captação de lipídios de leveduras foi utilizado uma citometria de fluxo O teor de gordura bruta na farinha variou entre 4272 em 14 dias de armazenamento pósmoagem Em condições de armazenamento em temperaturas mais altas como 35ºC todas as linhagens analisadas apresentaram um maior nível de acidez ao final de 14 dias Como resultado o artigo utilizou dos valores de acidez para determinar linhagens com início precoce a rancidez que foram então analisadas durante 21 dias Desse grupo ao final de 21 dias uma linhagem I3 não apresentou odores desagradáveis sendo uma farinha seca Enquanto que duas linhagens I5 e I7 desenvolveram um mau cheiro característico do processo de rancidez Essas três linhagens I3 I5 e I7 apresentaram perfis lipídicos semelhantes com grandes quantidades de ácidos linoleicos e ácidos oleicos insaturados O que demonstra que a composição lipídica do milheto não interferiu na capacidade de ranço precoce Analisando o catabolismo lipídico a linhagem I7 é mais propensa a degradação do triacilglicerol ao ser armazenada em alta temperatura e alta umidade Verificando o ranço nessas amostras os produtos químicos voláteis formados foram diferentes principalmente na linhagem I3 Apesar dos perfis lipídicos semelhantes as linhagens I5 e I7 apresentaram uma maior quantidade aldeídos Desse modo tais linhagens apresentam maior susceptibilidade ao ranço devido a maior quantidade de ácidos graxos livres e sua oxidação Além disso em condições de maior teor de umidade 30 foi possível perceber outros voláteis presentes tais como apigenina e pirrolina que conferiu um odor desagradável mais forte as amostras Com os resultados dessas análises apresentadas os autores sugeriram que nas linhagens com pouca susceptibilidade a formação de ranço os genes das lipases devem ser diferentes Escolheuse três genes de lipases PgTAGLip1 Pg TAGLip2 e PgTAGLip3 nas linhagens I3I5 e I7 E ao analisar esses genes percebeuse que na linhagem I3 houve um nível de expressão 15 vezes menor em comparação com as outras linhagens O que sugere que uma menor expressão de genes da lipase garante uma menor susceptibilidade da formação de rancidez hidrolítica em menor tempo Houve ainda a identificação de polimorfismos na estrutura da enzima principalmente no sítio de ligação de ácidos graxos A linha I3 não apresentava um sítio catalítico o que está relacionado a baixa atividade da enzima e a baixa formação de ranço Contudo apesar desse polimorfismo a atividade da enzima se faz presente principalmente quando necessário no desenvolvimento da planta de modo que o mesmo pode ser utilizado como alvo para melhorar a qualidade da vida útil da farinha do milheto De modo geral o artigo verificou que as lipases são as responsáveis pela formação de ranço na farinha do milheto Condições de altas temperaturas e umidade no armazenamento da farinha diminuem a vida útil da mesma pois aceleram o tempo para o estabelecimento da rancidez hidrolítica Assim um bom armazenamento correto e uma linhagem de alto rendimento com baixa atividade de lipases como a linhagem I3 pode impactar na comercialização da farinha e por fim no lucro para os pequenos agricultores 2 Vocês deverão escrever um texto de duas páginas sobre o tema gorduras fazem maldiscutindo a relação com a saúde e os diferentes tipos de gorduras Muito se questiona sobre as gorduras na alimentação Muitos falam que gorduras fazem mal a saúde contudo as mesmas também são necessárias na alimentação Tal contraste se faz presente nos questionamentos dos cientistas e estudantes e para melhor compreensão é preciso entender que existem diferentes tipos de gorduras que por sua vez apresenta diferentes impactos na saúde Os principais tipos de gordura que fazem parte da alimentação são as gorduras saturadas gorduras insaturadas e gorduras trans As gorduras saturadas são encontradas em alimentos de origem animal As insaturadas são divididas em dois grupos monoinsaturadas e poliinsaturadas e estão presentes em alimentos como azeites sementes e peixes Já as gorduras trans estão presentes em alimentos processados e ultraprocessados que são parcialmente hidrogenados Carnes e leites integrais fazem parte da alimentação da maioria das pessoas Sendo alimentos ricos em gorduras saturadas muito se debate se esses alimentos fazem mal Isso porque o consumo de gorduras saturadas muitas vezes está associada a um aumento no colesterol e na incidência de doenças cardiovasculares Contudo isso não quer dizer que tomar um copo de leite integral todo dia vai levar a um infarto Quando se fala de gorduras saturadas a qualidade do alimento bem como a quantidade consumida será o verdadeiro responsável pelos benefícios ou malefícios do consumo desse tipo de gordura Conhecidas como gorduras saudáveis as gorduras insaturadas são muitas vezes associadas a benefícios a saúde principalmente na redução de doenças cardiovasculares Como a fonte mais conhecida desse tipo de gordura o azeite está associado a redução do colesterol e a prevenção de problemas cardíacos O ômega 3 também é bom para a saúde do coração Como uma gordura poliinsaturada além desses benefícios o mesmo é capaz de reduzir os níveis de pressão arterial e de processos inflamatórios Por isso o consumo de peixes como o salmão rico em ômega3 é muito indicado na alimentação Responsável pela má fama das gorduras as gorduras trans são conhecidas por seu malefício a saúde aumentado o risco de problemas cardíacos diabetes e distúrbios metabólicos As gorduras trans estão disponíveis em alimentos processados que muitas das vezes passam por hidrogenação parcial para alteração de sabor e textura Importante lembrar que as gorduras trans também se fazem presente em produtos de origem animal como as gorduras saturadas Contudo a fonte principal desse tipo de gorduras na alimentação atual são os alimentos processados Na saúde as gorduras trans são capazes de aumentar o colesterol LDL contribuindo para a formação de placas de gorduras no interior das artérias Além disso um alto consumo de gorduras trans aumenta o risco de AVC acidente vascular cerebral Também já foi possível correlacionar o consumo abusivo de gorduras trans com um aumento no risco de resistência à insulina levando ao desenvolvimento de diabetes tipo 2 Uma dieta desbalanceada nesse tipo de gordura também pode levar ao aumento de peso e até mesmo evoluir para obesidade Apesar das gorduras em si não serem caloricamente densas alimentos ricos em gorduras trans muitas vezes não possuem um valor nutricional considerável levando ao aumento de peso O alto consumo de gorduras trans também pode desencadear processos inflamatórios no corpo levando ao desenvolvimento de doenças crônicas como artrite Sendo assim gorduras são boas ou más Depende Naturalmente os baixos níveis de gordura trans em alimentos como carne não fazem mal Assim como o consumo de alimentos ricos em gorduras saturadas como produtos lácteos integrais e carnes Contudo um abuso no consumo desses tipos de gorduras traz malefícios a saúde Considerando que a fonte principal de gorduras trans são alimentos processados que além de ricos em gorduras não saudáveis muitas das vezes não carregam valor nutricional nenhum uma redução do consumo desse tipo de alimentos e das gorduras trans no geral é benéfico a saúde Já as gorduras insaturadas são conhecidas por trazem benefícios a saúde por isso o consumo de alimentos ricos nesse tipo de gorduras não precisa ser tão regrado De modo geral uma alimentação balanceada será aquela com baixo consumo de gorduras trans pouco consumo de fontes de gorduras saturadas e ricas em alimentos com gorduras insaturadas Isso acontece porque a relação entre a saúde e os tipos de gordura é complexa devido aos diversos tipos de gordura que atuam de modo diferente no nosso corpo