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21 CFR PART 117美国联邦法规第21章117部分PART 117—CURRENT GOOD MANUFACTURING PRACTICE, HAZARD ANALYSIS, AND RISKBASED PREVENTIVE CONTROLS FOR HUMAN FOOD117---适用于人类食品的现行良好操作规范和危害分析及基于风险的预防性控制措施Subpart A—General ProvisionsA部分--- 总则117.1 Applicability and status.117.1 适用范围和法律地位117.3 Definitions.117.3 定义117.4 Qualifications of individuals who manufacture, process, pack, or hold food.117.4 从事生产、加工、包装或储存人员的资质认定117.5 Exemptions.117.5 豁免117.7 Applicability of subparts C, D, and G of this part to a facility solely engaged in the storage of unexposed packaged food.117.7 本法规C部分、D部分和G部分对仅从事密封包装食品储存企业的适用性117.8 Applicability of subpart B of this part to the off farm packing and holding of raw agricultural commodities117.8 本法规B部分对“在农场外进行初级农产品包装”和“初级农产品储存”的适用性117.9 Records required for this subpart.117.9 本部分要求的记录Subpart B—Current Good Manufacturing PracticeB部分—现行良好操作规范117.10 Personnel.117.10 人员117.20 Plant and grounds.117.20 工厂和场地117.35 Sanitary operations.117.35 卫生操作117.37 Sanitary facilities and controls.117.37 卫生设施和控制117.40 Equipment and utensils.117.40 设备和器具117.80 Processes and controls.117.80 过程和控制117.93 Warehousing and distribution.117.93 仓库和分发117.110 Defect action levels.117.110 缺陷行动水平Subpart C—Hazard Analysis and Risk Based Preventive ControlsC部分—危害分析和基于风险的预防性控制117.126 Food safety plan.117.126 食品安全计划117.130 Hazard analysis.117.130 危害分析117.135 Preventive controls.117.135 预防性控制117.136 Circumstances in which the owner, operator, or agent in charge of a manufacturing/processing facility is not required to implement a preventive control. 117.136 生产/加工企业的所有者、经营者或代理方不需实施预防性控制措施的情况117.137 Provision of assurances required under §117.136(a)(2), (3), and (4).117.137 按照§117.136(a)(2), (3), and (4).的要求提供保证117.139 Recall plan.117.139 召回计划117.140 Preventive control management components.117.140 预防性控制措施管理要素117.145 Monitoring.117.145 监控117.150 Corrective actions and corrections.117.150 纠正措施和纠正117.155 Verification.117.155 验证117.160 Validation.117.160 确认117.165 Verification of implementation and effectiveness.117.165 实施和有效性的验证117.170 Reanalysis.117.170 再分析117.180 Requirements applicable to a preventive controls qualified individual and a qualified auditor.117.180 适用于预防控制合格人员和合格审核员的要求117.190 Implementation records required for this subpart.117.190 本部分要求的实施的记录Subpart D—Modified RequirementsD部分—简化要求117.201 Modified requirements that apply to a qualified facility.117.201 适用于“符合条件的企业”的简化要求117.206 Modified requirements that apply to a facility solely engaged in the storage of unexposed packaged food.117.206 适用于仅从事密封包装食品储存的企业的简化要求Subpart E—Withdrawal of a Qualified Facility ExemptionE部分—符合条件企业的豁免撤销117.251 Circumstances that may lead FDA to withdraw a qualified facility exemption. 117.251 可能导致FDA撤销符合条件的企业豁免的情况117.254 Issuance of an order to withdraw a qualified facility exemption.117.254 符合条件企业豁免撤销命令的签发117.257 Contents of an order to withdraw a qualified facility exemption.117.257 符合条件企业豁免撤销命令的内容117.260 Compliance with, or appeal of, an order to withdraw a qualified facility exemption.117.260 对撤销符合条件企业豁免命令的遵守或申诉117.264 Procedure for submitting an appeal.117.264 申诉提交的程序117.267 Procedure for requesting an informal hearing.117.267 非正式听证会的申请程序117.270 Requirements applicable to an informal hearing.117.270 适用于非正式听证会的要求117.274 Presiding officer for an appeal and for an informal hearing.117.274 负责申诉和非正式听证会的的主管官员117.277 Timeframe for issuing a decision on an appeal.117.277 出具申诉决议的时限117.280 Revocation of an order to withdraw a qualified facility exemption.117.280 符合条件企业豁免撤销命令的取消117.284 Final agency action.117.284 最终行政措施117.287 Reinstatement of a qualified facility exemption that was withdrawn.117.287 被撤销符合条件企业豁免的恢复Subpart F—Requirements Applying to Records That Must Be Established and MaintainedF部分—针对必须建立并留存记录的要求117.301 Records subject to the requirements of this subpart.117.301 本章节所要求的记录117.305 General requirements applying to records.117.305 记录的一般要求117.310 Additional requirements applying to the food safety plan.117.310 针对于食品安全计划的附加要求117.315 Requirements for record retention.117.315 记录保存的要求117.320 Requirements for official review.117.320 官方复核的要求117.325 Public disclosure.117.325 公开117.330 Use of existing records.117.330 现有记录的使用117.335 Special requirements applicable to a written assurance.117.335 适用于书面保证的特殊要求Subpart G—Supply Chain ProgramG部分—供应链计划117.405 Requirement to establish and implement a supply chain program.117.405 建立和实施供应链计划的要求117.410 General requirements applicable to a supply chain program.117.410 供应链计划的通用要求117.415 Responsibilities of the receiving facility.117.415 接收企业的职责117.420 Using approved suppliers.117.420 使用获得批准的供应商117.425 Determining appropriate supplier verification activities (including determining the frequency of conducting the activity).117.425 确定适宜的供应商验证活动（包括确定实施的频率）117.430 Conducting supplier verification activities for raw materials and other ingredients.117.430 对原料供和其他辅料供应商的验证活动117.435 Onsite audit.117.435 现场审核117.475 Records documenting the supply chain program.117.475 供应链计划的文件记录AUTHORITY 授权: 21 U.S.C. 331, 342, 343, 350d note, 350g, 350g note, 371, 374; 42 U.S.C. 243, 264, 271.Subpart A —General ProvisionsA—总则§117.1 Applicability and status.§117.1 适用范围和法律地位(a) The criteria and definitions in this part apply in determining whether a food is:(a) 本部分中的标准和定义对判定以下情况时使用：(1) Adulterated within the meaning of:(1) 是否受到掺杂，此处“掺杂”是指：(i) Section 402(a) (3) of the Federal Food, Drug, and Cosmetic Act in that thefood has been manufactured under such conditions that it is unfit for food; or(i) 《联邦食品，药品和化妆品法案》中的402 (a) (3) 部分指出食品在不适合食品的条件下加工生产，或(ii) Section 402(a)(4) of the Federal Food, Drug, and Cosmetic Act in that the food has been prepared, packed, or held under insanitary conditions whereby it may have become contaminated with filth, or whereby it may have been renderedinjurious to health; and(ii) 《联邦食品，药品和化妆品法案》中的402 (a) (4)部分指出，食品在不卫生的条件下制备，包装或保留或者其他可能被污物污染或者已经被认为对健康是有害的；和(2) In violation of section 361 of the Public Health Service Act (42 U.S.C. 264).(2) 违反《公共健康服务法案》中361部分 (42 U.S.C. 264) 。
Gene expression profiling of Sinapis alba leaves under drought stress and rewatering growth conditions with Illumina deep sequencingCai-Hua Dong •Chen Li •Xiao-Hong Yan •Shun-Mou Huang •Jin-Yong Huang •Li-Jun Wang •Rui-Xing Guo •Guang-Yuan Lu •Xue-Kun Zhang •Xiao-Ping Fang •Wen-Hui WeiReceived:20June 2011/Accepted:17December 2011ÓSpringer Science+Business Media B.V.2011Abstract Sinapis alba has many desirable agronomic traits including tolerance to drought.In this investigation,we performed the genome-wide transcriptional profiling of S.alba leaves under drought stress and rewatering growth conditions in an attempt to identify candidate genes involved in drought tolerance,using the Illumina deep sequencing technology.The comparative analysis revealed numerous changes in gene expression level attributable to the drought stress,which resulted in the down-regulation of 309genes and the up-regulation of 248genes.Gene ontology analysis revealed that the differentially expressedgenes were mainly involved in cell division and catalytic and metabolic processes.Our results provide useful infor-mation for further analyses of the drought stress tolerance in Sinapis ,and will facilitate molecular breeding for Brassica crop plants.Keywords Sinapis alba ÁDrought stress ÁIllumina sequencing ÁGene expression ÁDrought tolerance genesIntroductionDrought is a meteorological occurrence in practice which displays zero rainfall for a long time,it firstly causes the depletion of moisture in soil,and finally works the decrease of water potential of plant tissues for water deficit [1].In the light of the agricultural point of view,its operating definition would be the insufficient of water availability from the soil during the life cycle of crop plants,which restricts a full exertion of genetic potential of the plants.At present,it is one of the grand restrictive factors in agri-cultural production by inhibiting crop plants reaching the theoretical maximum yield genetically determined.Drought stress is one of the most common stress factors decreasing crop output.Plants changes adaptively in cell morphology,gene expression,physiological and bio-chemical metabolisms to mitigate the damage caused by drought stress,and form a variety of drought stress adap-tation in aspects of growth habit and physiological and biochemical habits during long-term interaction with the environment and during evolution [2].As plants experience drought,many drought stress response genes are induced and a large number of specific proteins are produced to regulate physiological and biochemical and metabolic changes of plants cooperatively.Cai-Hua Dong,Chen Li and Xiao-Hong Yan contributed equally to this work.Electronic supplementary material The online version of this article (doi:10.1007/s11033-011-1395-9)contains supplementary material,which is available to authorized users.C.-H.Dong ÁX.-H.Yan ÁS.-M.Huang ÁL.-J.Wang ÁR.-X.Guo ÁG.-Y.Lu ÁX.-K.Zhang (&)ÁX.-P.Fang (&)ÁW.-H.Wei (&)Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences,Key Laboratory of Oil Crop Biology and Genetic Breeding of the Ministry of Agriculture,Wuhan 430062,China e-mail:whwei@ X.-P.Fange-mail:xpfang@ X.-K.Zhange-mail:zhangxk@C.LiCollege of Food Science and Technology,Agricultural University of Hebei,Baoding 071001,China J.-Y.HuangDepartment of Bioengineering,Zhengzhou University,Zhengzhou 450001,ChinaMol Biol RepDOI 10.1007/s11033-011-1395-9The mechanisms of drought tolerance in plants have already been studied at the gene level.Gene expression profiles of plant tissuesfluctuated under drought stress,this stress response made plants obtain drought tolerance.The genes related to drought tolerance can be grouped into two categories,thefirst one encode the special functional pro-teins directly involving drought tolerance,including pro-tective factors,osmotic adjustment proteins,ion tunnel proteins,ion transport proteins,oxidation-resistant pro-teins,etc.The second one encode those regulatory proteins, such as transcription factors,protein kinases,protein phos-phatases,calmodulin-binding proteins,etc.The expression levels of these genes are up-regulated or down-regulated during drought stress,which regulates the intra-and intercellular environments,and plants exhibit the trait tolerant to drought[3].With Arabidopsis[4],rice[5,6]and other plant genome sequencing completed,the research of genes of plants have entered the era of functional genomics,which studies not only the structure and function of genes,but also the temporal and spatial expression of plant genes and the regulation network.For a comprehensive understanding of the genetic basis of drought tolerance,exploring drought tolerance genes,cultivating resistant and water saving species,it is significant to discuss the sorts of genes induced by drought stress by methods of molecular biol-ogy,to construct the expression profiling of drought-related genes,to acknowledge the metabolism mechanism of plants under drought stress condition from the overall level.Arabidopsis thaliana is the model species used to study the mechanisms of drought tolerance and used to clone the genes that might code for the mechanisms leading to the tolerance to drought,several hundreds of drought-toler-ance-related genes have been identified from it[7–9]. There are four pathways for these genes to respond to drought stress,of which two pathways are dependent to abscisic acid(ABA),and two are not[8].Another good material for drought stress research is Thellungiella sal-suginea.Wong et al.[10]have studied gene expression of leaf tissue of T.salsuginea under drought stress using cDNA microarray,and revealed new abiotic stress response mechanisms in T.salsuginea.Sinapis alba(white mustard)is a crucifer classified into the genus Sinapis which includes about ten grass species.It is now widespread worldwide,although it probably originated in the Mediterranean region.It has many desirable agronomic traits including tolerance to drought[11,12].Until now,however,no research has been performed on the molecular mechanisms of drought tolerance in S.alba.It is necessary to analyze the gene expression profile of white mustard under drought stress. In recent years,various techniques,such as cDNA microarray(or cDNA chip),SSH and RT-PCR,were thought to be a powerful tool in the study of gene expression profiles induced by abiotic stress in plants [7,8,13–15].However,these techniques present some defects.They are laborious,rely on a prior knowledge of the sequence,or suffer from high noise or cross-hybrid-ization problem.With the Illumina sequencing(formerly Solexa sequencing)technique developed recently,this situation has changed,and it can execute quantitative and qualitative analyses of gene expression at low cost,even if the genome of a species have not been noted,and the Illumina sequencing data are highly replicable,with rel-atively little technical variation,it may suffice to sequence each mRNA sample only once[16,17].In this study,afine comparison of mRNA expression levels of S.alba leaves under rewatering growth conditions (SaW-A)and drought stress conditions(DL-B)was per-formed based on Illumina sequencing for thefirst time. These results provide novel information for studying the molecular mechanisms of drought tolerance in S.alba, since a number of candidate genes for drought tolerance were identified.Materials and methodsPlant materials and stress treatmentsPlants growth and stress treatment were performed as described by Wong et al.[10].Plants of S.alba were grown in controlled environments with a day/night temperature regime of22°C/10°C.An irradiance of250l mol photons m-2s-1over a21-h daylength was provided.When all the plants were4weeks old,some plants,named sample Saw-A,were subjected to drought stress treatment until they wilted visibly(3–4days),and then rewatered and allowed to recover.At the moment when sample Saw-A plants were rewatered,other plants,named sample DL-B,were sub-jected to drought treatment until they wilted visibly.0.1g leaves offive individual plants for each of Saw-A and DL-B were synchronously harvested8h after the lights came on in the growth chamber.Then the equal leaf samples fromfive individual plants were mixed together for RNA extraction of Saw-A and DL-B,respectively. Processing samples for sequencingTotal RNA was extracted using the TRIzol reagent (Invitrogen).After precipitation,RNA was purified with Qiagen’s RNeasy kit with on-column DNase digestion according to the manufacturer’s instructions.Purified RNA samples were dissolved in diethylpyrocarbonate-treated H2O,and the concentration determined spectroscopically. The quality of the RNA was assessed on1.0%denaturingMol Biol Repagarose gels in combination with the Bioanalyzer2100 (Agilent).Illumina sequencingIllumina sequencing was completed at Beijing Genomics Institute,with the use of an Illumina genome analyzer(San Diego,CA).Initially,we used poly(T)oligo-attached magnetic beads to isolate poly(A)mRNA from total RNA sample.First-and second-strand cDNA synthesis were performed while the RNA was bound to the beads.While on the beads,samples were digested with NlaIII to retain a cDNA fragment from the most30CATG to the poly(A)-tail.Subsequently,the GEX adapter1was ligated to the free50end of the RNA,and a digestion with MmeI was performed,which cuts17bp downstream of the CATG site.At this point,the fragments detach from the beads. After dephosphorylation and phenol extraction,the GEX adapter2was ligated to the30end of the tag.Finally,the short cDNA fragments were prepared for Solexa sequenc-ing on an Illumina genome analyzer(San Diego,CA), using the manufacturer’s protocol and reagents of the genomic DNA sequencing sample prep kit.The Illumina/Solexa approach involved sequencing of cDNA fragments,followed by counting of the number of times a particular fragment was observed.The terminators were labeled withfluorescent compounds of four different colors to distinguish among the different bases at the given sequence position.The template sequence of each cluster was deduced by reading the color at each successive nucleotide addition step.Image analysis and base calling were performed using the Illumina Pipeline,where high-throughput short-read sequence tags were obtained after purityfiltering.This was followed by sorting and counting the unique tags.Sequence annotation,comparison and functionalclassificationThe unigenes of Sinapis,Arabidopsis and Brassica were used as a reference sequence to align and identify the sequencing reads.To map the reads to the reference,the alignments and the candidate gene identification procedure were conducted using the mapping and assembly with qualities software package MAQ[18].Differentially expressed genes between two samples were analysed according to the digital gene expression detection methods reported by Audic and Claverie[19].To categorize transcripts by putative function,we have utilized the gene ontology(GO)classification scheme[20]. GO provides a dynamic controlled vocabulary and hierar-chy that unifies descriptions of biological,cellular and molecular functions across genomes.ResultsIllumina sequencing and gene annotationWe obtained4,123,307tags in sample Saw-A(accession: SRR353366)and4,340,054tags in sample DL-B(acces-sion:SRR352383)through Illumina sequencing(Fig.1a), the original data have been placed in public databases(http:// /sra/?term=SRA047029).204,279dis-tinct tags were obtained in sample Saw-A after eliminating low quality tags and single copy tags,and217,599distinct tags were obtained in sample DL-B(Fig.1b).Though some tag copy number is far more than100,this is not what we are interested in,because two samples may both have high expression genes.In the present study,our research focuses on those tags that have obvious differences between the two samples.Though different copy number of distinct tags displayed very similar distribution patterns on the whole,specific distinct tags are quite different in the two samples(Fig.2). In Fig.2,the regions on the left of the peak zones denote the distinct tag copy number of Saw-A are abovefivetimesMol Biol Repthe DL-B,these tags are down-regulation under drought stress.The regions on the right of the peak zones represent the distinct tag copy number of DL-B are above five times the Saw-A,these tags are up-regulation under drought stress.The peak zones differ in five times between two samples.Drought tolerance genes are probably found from these tags that have apparent change in expression.Through the comparison with the open reference sequences of Arabidopsis and Brassica ,all of the distinct tags were annotated.The expression levels of these annotated genes were quantitatively analyzed as their corresponding tag copy number,and they were classified into up-regulation,down-regulation and no significance change genes.Although this was a preliminary analysis of white mustard short-read data,we have gained valuable infor-mation,which lead to the identification of differentially expressed genes between Saw-A and DL-B samples.Figure 3shows the distribution of differentially expressed genes,the dexter and upper regions with dots reveal those genes with markedly expression difference,and the rest regions shows those genes with no obvious expression diversity.The upper region with dots displays the up-reg-ulated genes of sample DL-B after stress,248genes could be annotated.The dexter region with dots displays the down-regulated genes of sample SaW-A after stress,309genes could be annotated.More detailed information including data selection is provided in Supplementary Table S1.The unigenes of Arabidopsis and Brassica were used as a reference sequence,for Arabidopsis has good basis to study drought stress as model organism,and the application of Brassica will help to found drought-related genes for molecular breeding.GO classification and annotation of differentially expressed genesThese differentially expressed genes may involve different functions,and their function annotation is very helpful for us to roundly analyze the changes of the gene expression profiles under drought stress.Then GO classification of the differentially expressed genes was performed as their up-and down-regulation changes (Fig.4).A gene can be classified into different functional gene type,so the gene number shown in the chart is more than the total number of the differentially expressed gene.The GO classification results showed that there were not down-regulated genes in the classification involved in enzyme regulator and multi\-organism processes,and up-regulated genes were not found in the classification with functions of envelope and auxiliary transport protein.These results may reflect that up-and down-regulated genes participate in different metabolic pathways and are involved in different regulation mechanisms.The differentially expressed genes were mainly involved in the cell division and catalytic and metabolic processes.According to the annotation results,parts of the up-and down-regulated genes are related to the cDNAs and unigenes expressed under both biotic and abiotic stress,which shows that there may be some the same response mechanism for diverse stress.At the same time,there are a lot of unknown tags,some important functional genes will be found in them,especially those tags that have obvious change in expression level under stresscondition.Fig.2Distribution of ratio of distinct tag copy number between twolibrariesFig.3Down-regulation and up-regulation of gene expression in S.alba under drought stress conditionMol Biol RepDiscussionThe tolerance to biotic and abiotic stresses like low or high temperature,drought,salt and disease factors in plants is a defense response involving multiple genes.Drought stress causes a great change of gene expression profile,deep understanding of the cross-talk between the transcription factor of different pathways will help the improvement of the integrated characters of crops,it is important to study the changes of gene expression profiles from the overall level.The fine comparative analysis of mRNA expression levels of S.alba leaves under drought stress and rewatering growth conditions was performed by Illumina deep sequencing method in the present study.When the plants wilted,not only the expressions of the genes related to drought stress were changed,but also the expressions of partial genes related to plant growth and development were changed.When the wilting plants were re-watered,prob-ably the expressions of the genes related to plant growth and development still maintained the changed levels at the early stage.In addition,RNA-changes are not strictly correlated to protein levels,osmotic relations or membrane characteristics [21].So we could rightly screen the genes related to drought stress when the re-watering plants and wilting plants were used as the tested materials.Illumina sequencing,different from Sanger sequence method,can provide giant sequencing data with saving time and lower cost.It is also helpful for the study of molecular breeding,evolution and development,and stress response to envi-ronment in crop plants.In the present study,557annotated genes and a large number of no matched tags were found to be involved in drought stress response,some genes encode signaling components,transcription regulators or other proteins,these proteins are necessary for cell growth and develop-ment under drought stress [22].These results indicate that it is effective to analyze the gene expression profiles under drought stress by high-throughput sequencing technologies and many novel tags have been found,however,more reliable results in the present study could be obtained with biological repetition experiments.Lee et al.[23]has ana-lyzed 24,000unigenes using a B.rapa oligo microarray and many unigenes were found to be involved in the abiotic stresses,however,this technology relied on a prior knowledge of the sequences.It is now hypothesized that halophytes use salt-tolerance effectors and regulatory pathways very similar to those in glycophytes and that subtle differences in their regulation can account for large variations in salt sensitivity [24–26],other researchers have begun to test this hypothesis [27].Plants have many common response mechanisms under abiotic stress such as salt stress and drought stress.Molecular regulation mechanisms of salt stress and drought stress can be found through comparative analysis and genetic function analysis between halophytes and glyco-phytes,and new functions will be found in the genes that have been identified in glycophytes.Arabidopsis ,a relative of white mustard,was annotated completely in genomics,its genome was used as a reference to find some known and unknown functional genes related to drought stress in white mustard as possible as we can do.At the same time,transcriptome analysis using high-throughput short-read sequencing need not be restricted to the genome of model organisms [28,29].The gene expression profiles (Supplementary Table S1)showed that the annotated genes could be grouped into two categories,the first one encode protective proteins,such as oxidoreductase,the second one encode regulatory proteins,such as transcription factors.In the up-regulated genes,theFig.4Percentagerepresentation of GO mappings for drought-tolerance correlated clustersMol Biol RepFATTY ACID REDUCTASE1gene(AT5G22500.1)has the fatty-acyl-CoA reductase activity involved in salt stress,it is grouped into the protective protein.Another gene AT4G20890.1has GTPase activity,it is grouped into the regulatory protein.A lot of the annotated genes have not been found to be involved in drought tolerance,their function need to be identified in the future research.Brassica plants are also the relatives of white mustard, 329of the557genes related to drought stress were anno-tated as the reference sequences of Brassica,these329 genes include plenty of genes with unknown function.With the deep research on gene function we will know more about these genes in the role of drought tolerance,and at last those drought tolerance genes can be applied to the genetic improvement of Brassica crop plants with mass transforming.Acknowledgments This work was supported by the National Nat-ural Science Foundation of China(30671312),the Natural Science Foundation of Hubei Province(2008CDA083and2009CDB191),the Natural Science Foundation of Henan Province(114100510013),the Chenguang Program of Wuhan City(201050231022),the Interna-tional Science and Technology Cooperation Item(S2012GR0080), and the Science and Technical Innovation Project of Hubei Province. 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微透析技术在脑组织研究中的应用微透析技术是将推挽灌流和透析技术结合起来并逐渐完善的一种新型生物采样技术，可在麻醉或清醒的生物体上使用，特别适合于深部组织和重要器官的活体生化研究。
微透析技术最早应用于脑内是在1972年美国耶鲁大学报道的猴脑的微透析研究，之后微透析即开始应用于对脑内神经递质的改变，药物动力学监测以及一些疾病脑内神经生理改变的研究，至今已有30多年的历史。
本文就微透析技术在脑组织研究中的应用作一综述。
1 微透析技术的原理及探针回收率的测定微透析技术是以透析原理作为基础的在体取样技术，是在非平衡条件下即流出的透析液中待测化合物的浓度低于它在探针膜周围样品基质中的浓度，灌注埋在组织中的微透析探针，组织中的待测化合物沿浓度梯度扩散进入透析液，被连续不断地带出，从而达到从活体组织中取样的目的，通过测定流出液即透析液中待测物的浓度来研究组织中待测物的水平。
这是一种动态连续的取样方法。
简单地说，微透析技术的原理就相当于在组织中创造了一个“毛细血管”，使化合物在浓度差的作用下扩散而进入此“毛细血管”，然后随液体流动带出体外进行检测。
微透析技术最大的优点是可在基本上不干扰体内正常生理过程的情况下进行在体、实时和在线取样，透析过程探针周围的组织液成分不会发生改变，所以可以持续收集样品，而且被透析动物可以小到老鼠一般大小。
通过微透析技术可以单独取得细胞外液，因此可对大脑神经递质进行活体监测［1］，由于透析液中不含蛋白质和酶等生物大分子，能直接进样进行高效液相和毛细管电泳等生化分析，免除了复杂的进样前样品处理及由此而产生的样品损失和误差，也不会因在室温取样而酶解，提高了样品的稳定性，而且随着现代技术的进步可以对微透析试验进行全程的自动化控制。
微透析试验中一个重要的步骤是要测定微透析探针的回收率(透析液中待测化合物的浓度与其在样品基质中浓度之比)，由于微透析是在非平衡条件下取样，所以所测得的透析液中化合物的浓度只是探针周围样品基质中该化合物浓度的一部分。
直投式发酵剂（DVS）所发酵牛乳冷藏过程中生物化学变化规律的研究刘营，高学军*（东北农业大学乳品科学教育部重点实验室，黑龙江 哈尔滨 150030）E-mail：liu-ying3@摘 要：为测定本实验室自制直投式酸奶发酵剂的发酵性能，本实验通过对其发酵的牛乳在冷藏过程中的主要生物化学指标的测定，测定结果显示：该发酵乳在冷藏0～24d过程中，脂肪、蛋白质的含量无规律性变化；在0～15d过程中乳酸含量及酸度逐渐增加，乙醛百分含量、乳糖含量、pH值及己糖激酶活性逐渐降低。
结果证明：该直投式酸奶发酵剂所发酵牛乳的保质期为15d,冷藏0d的50ml酸乳中蛋白质含量为605、脂肪含量262 g/dl，该发酵剂发酵性能优良，已达到相应酸乳生产标准。
关键词：发酵乳，冷藏，生物化学变化规律酸乳是新鲜牛奶经乳酸菌发酵剂发酵制备的乳制品，酸奶质量的好坏主要取决于发酵剂的品质类型及活力[1]。
本实验利用自制的优质直投式酸奶发酵剂发酵牛乳，测定其发酵并储藏24天的相关生物化学指标的变化，确定了该直投式发酵剂所发酵牛乳冷藏过程中的生物化学变化规律，从而为进一步的应用提供实验依据。
1材料与方法1．1试验材料与试剂鲜牛乳（采自东北农业大学奶牛场），本实验室自行研制的优质直投式酸奶发酵剂，1． 2仪器设备GYB60-6S高压均质搅拌机(上海东华高压匀浆泵厂)，SPX-250C型恒温恒湿培养箱(上海博迅医疗实业有限公司医疗设备厂)，PB-10型酸度计（北京赛多利斯仪器系统有限公司），岛津GC-9A气相色谱仪（日本岛津公司）。
1． 3酸牛乳的制备将预发酵良好的鲜牛乳中添加2%全脂乳粉、6%蔗糖后在20Mpa的压力下均质，分装于50ml的三角瓶后95℃灭菌10min，按2％添加量将本实验室自制的直投式酸奶发酵剂投入后放于43℃恒温恒湿培养箱中发酵6h，发酵完成后放于4℃冰箱中冷藏，冷藏后的第20小时为0d，之后每3天取一次样品，至24d取样完毕。
河流污水处理的相关论述1前言随着工业化和城市化的发展，水环境污染、水资源紧缺日益严重，水污染控制、水环境保护已刻不容缓。
我国现在新建城市或城区采用雨污分流制，但老城市或老城区大多仍然是雨污合流的排水体制。
许多合流污水是直接排放到水体。
而将旧合流制改为分流制，受现状条件限制大许多。
老城区建成年代较长，地下管线基本成型，地面建筑拥挤，路面狭窄，旧合流制改分流制难度较大。
合流污水的一大特点是旱季和雨季的水质、水量变化大，雨季污水B O D浓度低，不利于生化处理。
国家提出2010的我国城市污水处理率要求达到40%,因此研究有效的合流污水处理方法对加快城市污水处理步伐具有重要的意义。
本文针对合流污水处理的有关情况，谈一些个人看法。
2污水处理工艺要求我国目前不少城市，新城区与老城区并存，合流制与分流制并存。
因此，新建或扩建的污水处理厂，在满足城市总体规划和排水规划需要的同时，还应能达到如下要求:1.具备接纳旧城区合流污水的能力，具有较强的适应冲击负荷的能力。
污水处理厂污水来源包括两部分，一是新城区分流污水，二是老城区合流污水。
与合流污水相比，分流污水水质、水量变化幅度小得多，对污水处理厂调节缓冲的要求小得多。
对于合流污3工艺流程选择和特点说明泥得以增长;2、在亚硝化菌和硝化菌作用下，4结语击负荷的要求，设置缓冲池均衡水质、储存水量比较适宜。
2.通过多个氧化沟构成若干个串、并联运行方式，在适应进水水质、水量、季节性变化方面能够发挥重要作用。
3.通过安排适当的进出水口位置、回流污泥入口位置，氧化沟可形式一个倒置A2/0工艺，在去除B O D 的同时，能取得较好的氮磷去除效果4.熟化塘的应用，为处理水安全排放水体，能够提供可靠的技术保证。
熟化塘投资省、运行费用低、管理维护方面、污水处理与利用相结合，在防治水污染、保护水环境及生态环境综合治理方面具有明显优势。
如果美化熟化塘表观，设置喷泉等设施，形成供人们休闲、游乐的人工景点，协调城市建设中土地资源的合理配置，那么熟化塘占地面积较大这一不足就不会成为突出的问题。
2019年度国家科技进步奖公示材料项目名称：肉品风味与凝胶品质控制关键技术研发及产业化应用提名者：教育部提名意见：肉品加工业是我国农产品加工及食品行业的支柱产业，但加工技术相对落后，缺乏自主研发的关键技术及装备，肉品风味和凝胶品质难以控制，产品质量不稳定，严重限制了产业发展。
该项目系统研究并揭示了我国传统腌腊肉品风味品质形成机理，阐明了低温肉制品肉蛋白乳化凝胶机制，研发出“低温低盐腌制-中温风干发酵-高温快速成熟”的风味品质控制技术和“高效乳化、注射-嫩化-滚揉一体化腌制和热诱导凝胶”的凝胶品质控制技术，突破了肉品风味和凝胶品质难以控制的技术难题，同时创制出火腿自动撒盐-滚揉腌制和智能化风干发酵成熟装备、高效乳化斩拌机、盐水高压雾化注射机、全自动变压滚揉设备和熏蒸煮多功能一体化装备等可替代进口的加工关键装备8台套，构建了肉品加工全程质量控制体系。
该项目获国家发明专利31项，发表SCI论文141篇，自主研发装备8台套，成果在雨润、华统等13家肉品加工领军企业得到产业化应用，开发出新产品75种，近三年实现累计销售额56.56亿元人民币，产生了显著的经济和社会效益，为中国肉品加工业的转型升级提供了技术支撑。
经同行专家评价，低温肉制品加工技术和干腌肉制品强化高温熟化技术达到国际领先。
成果获教育部科技进步一等奖2项。
推荐申报国家科技进步奖二等奖。
项目简介：肉品加工业是我国农产品加工及食品行业的支柱产业，但加工技术相对落后，缺乏自主研发的关键技术及装备，如代表我国传统肉品的腌腊肉制品以风味浓郁著称，但风味形成机理不明，缺乏风味品质控制技术，导致生产周期长、脂肪氧化严重、产品盐分过高；代表肉制品加工方向的低温肉制品以质地适口为优势，但凝胶质构形成机制不明，缺乏凝胶品质控制技术，导致产品质地差、出水出油严重。
该项目历时15年，系统研究了肉品风味与凝胶品质形成机理，研发出关键技术，创制了相关装备，建立了全程质量控制体系，突破了肉品风味与凝胶品质难以控制的技术瓶颈，显著提升了我国肉品加工科技创新能力和整体水平。
传统咸鱼风味快速形成技术杨锡洪1，吴海燕1，解万翠1 ，杨磊2，李思东2，陈建娣1（1.广东海洋大学食品科技学院，2.广东海洋大学理学院，广东湛江 524088） 摘要：为了解决传统腌鱼生产中出现的安全性问题，实现工业化生产，本文采用植物乳杆菌和木糖葡萄球菌混合发酵以提高金丝鱼干的风味，并应用正交试验法建立最佳发酵工艺条件。
结果表明：在35 ℃、植物乳杆菌和木糖葡萄球菌接种比例为1∶2和发酵10h的条件下，发酵后产品香气浓郁，鲜味足，回味好，质构得到改善；利用发酵法可改进传统水产品的加工工艺，缩短生产周期，提高产品质量，具有巨大的经济意义和显著的实用价值。
关键词：植物乳杆菌；木糖葡萄球菌；腌鱼；风味中图分类号：TS254.4；文献标识码：A；文章篇号:1673-9078(2009)11-1295-04Quickly Formation of Flavor in Traditional Cured FishY ANG Xi-hong1, WU Hai-yan1, XIE Wan-cui1, Y ANG Lei2, LI Si-dong2, CHEN Jian-di1(1.College of Food Science and Technology, 2.College of Science, Guangdong Ocean University, Zhanjiang 524088, China)Abstract: In order to resolve the safety issues in production of traditional cured fish, a new method for quickly formation of flavor in traditional cured fish production was developed via fermentation by Lactobacillus plantarum and Staphylococcus xylosus. The suitable fermentation conditions were determined by the orthogonal experiment as follows: temperature of 35 ℃, time of 10h and the ratio of Lactobacillus plantarum to Staphylococcus xylosus of 1/2. Under these fermentation conditions, the obtained product had rich flavor, good aftertaste and improved texture. This method could improve traditional aquatic processing, shorten the production cycle, and improve product quality, possessing significant economic significance and notable practical value.Key words: Lactobacillus plantarum；Staphylococcus xylosus；c ured fish；f lavor干/腌制品在广东省水产加工中占有重要地位，近些年来，由于传统加工腌鱼制品的食用安全性受到人们的质疑，如何改进传统的低效率、小规模、受自然气候条件限制的家庭作坊式加工工艺，受到越来越多人的关注和研究。
生物柴油循环过程描述英语作文Biodiesel Circular Process DescriptionBiodiesel, as an alternative and eco-friendly fuel, has gained significant attention in recent years. The production of biodiesel involves a systematic circular process that promotes sustainability and reduces environmental impact. This essay aims to describe the biodiesel circular process in detail.1. Feedstock Collection:The first step in the biodiesel production process is the collection of feedstock, primarily vegetable oils or animal fats. These raw materials are obtained from various sources such as soybean, rapeseed, palm oil, and waste cooking oil. The use of waste cooking oil contributes to the reduction of waste and enhances the sustainability of the process.2. Pretreatment:Before undergoing the transesterification process, the feedstock needs to be pretreated. This involves removing impurities such as water, free fatty acids, and solid particles. Pretreatment methods include degumming, neutralization, and drying. These steps ensure the quality of the raw material and improve the efficiency of the subsequent process.3. Transesterification:Transesterification is a chemical reaction that converts vegetable oils or animal fats into biodiesel. In this step, the feedstock is reacted with analcohol, typically methanol or ethanol, in the presence of a catalyst, such as sodium hydroxide or potassium hydroxide. The reaction results in the formation of biodiesel and glycerin as a byproduct.4. Separation and Purification:After transesterification, the mixture is separated into two phases: biodiesel and glycerin. The separation is achieved through gravity settling or centrifugal separation. Subsequently, the biodiesel is purified by washing with water to remove any remaining alcohol, catalyst, and soap. The purification step ensures the quality of the biodiesel, meeting the required specifications.5. Drying and Refining:The washed biodiesel is dried to remove any remaining water. This is crucial as water can lead to corrosion and reduce the quality of the fuel. Furthermore, the biodiesel may undergo refining processes such as filtration and deodorization to enhance its quality and stability.6. Quality Control:To ensure the biodiesel meets the desired standards, it undergoes rigorous quality control tests. These tests include measuring the fatty acid methyl ester (FAME) content, flash point, pour point, viscosity, and oxidation stability. Biodiesel that passes these tests is considered ready for use as a fuel.7. Utilization:The final step in the biodiesel circular process is its utilization. Biodiesel can be used as a blend with petroleum diesel or as a pure fuel in diesel engines. Its use as a transportation fuel reduces greenhouse gas emissions and helps combat climate change.8. Byproduct Utilization:The glycerin byproduct from the transesterification process can be used in various industries, such as pharmaceuticals, cosmetics, and animal feed. This further enhances the sustainability of the biodiesel process by utilizing all components of the feedstock.In conclusion, the biodiesel circular process involves the collection of feedstock, pretreatment, transesterification, separation and purification, drying and refining, quality control, and utilization. This process promotes the use of renewable resources, reduces waste, and offers a cleaner alternative to conventional fossil fuels.。
核磁共振横向弛豫时间与金属种类之间的关系宣艳;向义龙【摘要】采用低场核磁共振技术采集不同的硫酸盐溶液中的氢核的横向弛豫时间T2,比较了纯水和不同浓度硫酸盐溶液中的氢核的核磁共振信号,分析了不同金属阳离子对溶液中氢核的影响,并对金属离子盐溶液中的氢核的弛豫时间与金属种类和浓度之间的相关性进行研究.实验结果表明,金属离子钠、镁、铝、钾、铁、锰、铜和锌均能加快氢核的核磁共振弛豫衰减速度,具有促进水分子结合的作用.随着金属离子浓度增加,T2均变小.浓度相同的情况下,金属离子对液体中氢核的的影响由强到弱为:Mn2+＞Fe3+ ＞Cu2+＞Mg2+＞ Al3+＞Na+＞Zn2+＞K+.【期刊名称】《分析仪器》【年(卷),期】2018(000)004【总页数】5页(P149-153)【关键词】核磁共振;弛豫时间;盐溶液;相关性【作者】宣艳;向义龙【作者单位】南京林业大学现代分析测试中心,南京210037;南京林业大学现代分析测试中心,南京210037【正文语种】中文1 引言水是地球上生物赖以生存的最重要物质之一。
自然界中的水分子不是孤立的，而是通过氢键的作用使得若干个分子聚合在一起，形成水分子团。
自从Dyke等人[1]于1977年验证了二元水结构以来，水分子团 [2-6]的实验和理论研究已经成为一个重要的研究方向。
多数的硫酸盐易溶于水，溶液中的离子会使溶液中的水分子重新排列，而这样排列的水分子结构与纯水结构有较大的差异。
因此盐溶液中的氢核弛豫的研究对于水分子团的研究具有重要的理论和现实意义。
低场核磁共振技术[7]是基于自旋核在射频磁场中的性质对材料进行分析的的一种测试方法。
低场核磁共振因其成本低廉、无损、快速等优点在粮油食品[8-11]、地质勘探[12，13]、石油化工[14-18]等领域得到了广泛的应用。
本实验利用低场核磁共振技术研究了硫酸盐溶液中氢核，研究了盐中的金属离子的种类和溶液浓度对氢的弛豫时间的影响。
Biochemical changes during processing of traditional Jinhua hamG.H.Zhoua,*,G.M.ZhaobaKey Laboratory of Meat Processing and Quality Control EDU,College of Food Science and Technology,Nanjing Agricultural University,Nanjing 210095,PR ChinabFood Science and Technology,Henan Agricultural University,Zhengzhou 450002,PR ChinaReceived 7March 2007;received in revised form 28March 2007;accepted 29March 2007AbstractJinhua ham is the most famous traditional meat product of China and one of the most famed dry-cured hams in the world.Its pro-cessing consists of six stages:green ham preparation,salting,washing and sun-drying and shaping,ripening,and post-ripening.Intense proteolysis and lipolysis occur during processing period.As a result,the content of free amino acids in final ham products is 14–16times that of green ham,and 191volatile compounds have been identified during processing,which make a major contribution to the flavor of Jinhua ham.Ó2007Elsevier Ltd.All rights reserved.Keywords:Jinhua ham;Processing;Proteolysis;Lipolysis;Flavor development1.IntroductionChina has a long,glorious history and a splendid dietary culture.Many traditional meat products have been devel-oped in China,among which Jinhua ham is the most famous.Jinhua ham has an attractive color,unique flavor and bamboo leaf-like shape.Its rose-like muscle,golden yellow skin and pure white fat make Jinhua ham one of the most preferred items in Chinese cuisine.The processing technology of Jinhua ham was introduced to European countries by Marco Polo during the 13th to 14th century and had an important impact on the development of dry-cured ham processing technology outside China.Jinhua ham is formed and produced in Jinhua District,Zhejiang Province in China.The earliest legend regarding the method of processing Jinhua ham may be traced back to the Tang Dynasty (A.D.618–907);however,the name ‘‘Jinhua ham’’was formally bestowed by the first emperorof the South Song Dynasty about 800years ago (Wu,Sun,&Sun,1959).Typical Jinhua ham processing generally takes 8–10months,starting in winter and finishing in autumn of the following year.During a long ripening pro-cess,muscle protein and fat are hydrolyzed to some extent by internal enzymes,and many small peptides,free amino acids,free fatty acids and volatiles are produced,which eventually contribute to the unique flavor of Jinhua ham.This paper outlines the processing technology of tradi-tional Jinhua ham,and describes the flavor-and taste-related compounds that are produced and the enzymes that are involved during the course of processing.2.Processing of traditional Jinhua hamJinhua ham is traditionally processed under natural con-ditions where air temperature and relative humidity depend on climate and weather condition.Its unique quality is not only due to the elaborate processing technology,but also related to the unique local geographic terrain and climate.About 70%of the Jinhua District is mountainous with four distinct seasons.Air temperature fluctuates regularly with-out extremes,which is desirable for processing dry-cured0309-1740/$-see front matter Ó2007Elsevier Ltd.All rights reserved.doi:10.1016/j.meatsci.2007.03.028*Corresponding author.Tel.:+8602584395376;fax:+8602584432420.E-mail addresses:ghzhou@ (G.H.Zhou),gmzhao126@ (G.M.Zhao)./locate/meatsciMeat Science 77(2007)114–120MEAT SCIENCEmeat ually,air temperature in winter is between0and10°C,favorable for ham salting.In spring, air temperature goes up to20°C or higher,suitable for sun-drying.Summer in the Jinhua region is relatively hot. The temperature can reach40°C,ideal for ham ripening. Post-ripening begins in autumn when the temperature falls.The entire processing of Jinhua ham,beginning in winter andfinishing in the following autumn,takes8–10 months.Processing of Jinhua ham consists of six stages:green ham preparation,salting,washing,sun-drying and shap-ing,ripening,and post-ripening.In all,there are more than 90steps within the six stages.2.1.Green ham preparationTraditionally,only hind legs from the Jinhua‘Lian-gtouwu’pig or its cross offspring could be used for produc-ing Jinhua ham.Desirable legs should be fresh,with a thin skin and slim shank bone,well-developed muscle,and a thin layer of white fat.Broken bone should be particularly avoided.The exposed part of the bones as well as the fat, tendon and muscle membrane on the meat surface of selected legs are removed and the hams then trimmed into a shape like a bamboo leaf.The remaining blood should be squeezed out.A leg weight of between5.5and7.5kg after trimming is preferred(Zhao&Zhou,2003).2.2.SaltingSalting is a critical stage in Jinhua ham processing and inappropriate salting may cause spoilage.Ambient temper-ature and humidity have great effects on the salting process. With regard to temperature,it’s difficult for salt to pene-trate meat when it is below0°C,while fast growth of microbes will occur when temperature is above15°C.With regard to humidity,when ambient humidity is below70% RH,undesirable water loss will occur,which also causes insufficient salt penetration.When ambient humidity is above90%,salt willflow away in the form of brine,causing pastiness on the surface of the ham.Therefore,the desir-able ambient temperature and humidity for salting is5–10°C and75–85%RH,respectively(Gong,1987;Zhao& Zhou,2003;Zhao et al.,2004).Average duration of salting is about30d,varying from25d for small hams(<5kg)to 35d for large hams(more than8kg),during which time each ham is salted5–ually only dry salt is used during salting,however,nitrate may also be used during thefirst two times of salting when irregular weather condi-tion is encountered.2.3.Soaking and washingThe purpose of this procedure is to remove excess salt and wash offany dirty substances on the surface of the ually,hams are initially soaked in water for4–6h and then washed with bamboo brushes.Water is chan-ged after initial washing,and hams are again soaked in water for another16–18h.2.4.Sun-drying and shapingAchieving appropriate dehydration is the objective of sun-drying as insufficient dehydration may cause spoilage. For balance,a pair of hams of similar weight are tied with a rope and hung on a rack.Sufficient ventilation and expo-sure to sunshine are considered when positioning the hams. When hams are hung,hoofs are removed,water and dirt on skin is razed offwith blade and the brand is sealed on the skin.Then hams can be removed from the racks and shaped into a bamboo leaf-like shape.Sun-drying can be terminated when hams start to drop liquidified fat,which generally requires about7sunny days.2.5.RipeningRipening is the key process for generating hamflavor substances.During this period,muscle proteins and fat are hydrolyzed mainly by endogenous enzymes,which results increased amounts of peptides,free amino acids and free fatty acids.These products constitute the main part of hamflavor substances and may continue to react with one other or be hydrolyzed to produce volatiles that contribute to the unique aroma of Jinhua ham.Ham pairs are fastened to a centipede rack(named because of its centipede-like shape)with the meat surface toward the windows in the ripening room.Ham quality is susceptible to the microclimate in the ripening room.High temperature with low humidity stimulates weight loss and fat oxidation,while high humidity may result in ham spoil-age.On the other hand,low temperature,especially in the later phase of ripening,slows aroma formation in hams. The ripening room should be well ventilated.Room tem-perature should increase from15°C to37°C gradually and humidity is controlled to within55–75%during the 6–8months ripening.During ripening,skin and muscle shrink to some extent because of moisture loss and bones around joints protrude out of the surface of the meat.Therefore,hams are usually removed from the centipede rack and retrimmed in mid-April.This is normally the last shaping and it requires cut-ting offprotruding bones,superfluous skin and fat.After reshaping,hams are retied to the centipede rack for com-pletion of ually ripening process terminates in mid-August when temperatures begin to drop.2.6.Post-ripeningAfter ripening,the surface of the meat becomes very dry and is often covered with a thin layer of mould spores and dust.For this reason,hams arefirst brushed clean and a thin layer of vegetable oil applied to soften muscle and pre-vent excessive fat oxidation,then stacked with skin side up for post-ripening.The post-ripening stage is a processG.H.Zhou,G.M.Zhao/Meat Science77(2007)114–120115designed to stabilize and intensify hamflavor;it is carried out in a warehouse and usually takes two months.During post-ripening,the ham piles are turned over from time to prevent unexpected fermentation,which affects ham quality.2.7.Grading and storageIt’s common practice to grade Jinhua ham into different categories according to quality,depending primarily on aroma intensity.A grader appointed by the government assesses ham’s aroma by inserting a bamboo probe into a ham and smelling the probe when removing it.There are threefixed locations:‘up’,‘middle’and‘lower’position on a ham for this special purpose.Jinhua ham can be stored for years and peakflavor is reached when stored for around12months.3.Proteolysis and lipolysisProteins and lipids constitute the major chemical com-ponents of ham muscles and their proportions in muscle increase gradually with the loss of muscle moisture during processing.Intense proteolysis and lipolysis occur over the course of Jinhua ham processing.As a result,the content of free amino acids infinal Jinhua ham products is14–16 times that of green ham,and191volatile compounds have been identified,which make major contribution to the aroma andflavor of Jinhua ham.3.1.Effects of muscle enzymes in the processing of Jinhua ham3.1.1.ProteolysisMuscle proteins are hydrolyzed continuously during processing of Jinhua ham and small molecular productssuch as peptides and free amino acids(FAA)are generated (Zhao et al.,2005b).At the end of processing,the proteo-lytic index(non-protein nitrogen accounting for total nitro-gen,%)of Jinhua ham reaches14–20.During proteolysis, about10%of insoluble proteins are hydrolyzed to soluble proteins which are then actively hydrolyzed to small pep-tides and FAA.In the soluble fraction,the proportion of soluble proteins drops from71.63%before the beginning of the salting stage to27.57%at the end of post-ripening period,most of the increase of non-protein nitrogen (NPN)occurring at the same time is free amino acids and small peptides of molecular weight<1kDa,account-ing for more than95%of the total NPN at the end of pro-cessing(see Figs.1and2).This change is likely be the result of endogenous enzyme activity since low numbers of micro-organisms are found inside dry-cured ham(Toldra´&Etherington,1988;Zhen,He,Li,Zhou,&Zhang, 2004)and molds growing on the ham surface do not affect Jinhua hamflavor development(Lin et al.,1992).Thus, endogenous enzymes such as cathepsins,dipeptidyl pepti-dases and aminopeptidases have been the focus of further studies.3.1.2.CathepsinsIn vitro experiments show that cathepsin B and L retain 17.55%and22.92%of their initial activity at the end of the ripening stage and9.31%and13.66%at the end of the post-ripening stage,respectively.Their activities,however, are greatly affected by temperature,salt content and pH values that change continuously during Jinhua ham pro-cessing.The activity observed under defined in vitro condi-tions may not reflect that which occurs during ham processing because of the unfavorable environment in ham.Two quadratic regression equation models were built to express the effects of temperature,salt content and pH value on the activity of cathepsin B and L individually. According to the models,cathepsin B and L always remain active under production conditions of Jinhua ham,though <5%of the activity in an in vitro environment was observed during Jinhua ham processing(see Fig.3).Cathepsin B and116G.H.Zhou,G.M.Zhao/Meat Science77(2007)114–120L may play key roles in hydrolyzing insoluble proteins to soluble proteins,and soluble proteins to peptides as a results of in vitro conditions and the long duration of Jin-hua ham processing(Zhao et al.,2005c).3.1.3.Dipeptidyl peptidases(DPP)Experiments in an in vitro environment show that both dipeptidyl peptidase I(DPPI)and dipeptidyl peptidase IV (DPPIV)possess considerable activity during Jinhua ham processing.The activity of muscle DPPI decreased gradu-ally before the beginning of ripening and then increased gradually until the end of post-ripening while the activity of muscle DPPIV decreased continuously.At the end of processing,141.71%of initial DPPI activity and11.19% of initial DPPIV activity remained(Zhao et al.,2005a). The change in DPPIV activity contradicts research on European dry-cured hams(Sentandreu&Toldra´,2001c). This may be due to the differences of micro-organisms growing on the ham surface because some micro-organisms produce an isozyme of DPPI that may penetrate into inner muscle and play a role in DPPI activity(Zhao et al., 2005a).As in the case of cathepsins,the activities of DPPI and IV are also significantly influenced by temperature,salt content and pH value.In the mathematical models estab-lished to illustrate the effects of temperature,salt content and pH value on the activities of DPPI and IV,DPPI always retains very strong activity under practical Jinhua ham processing conditions,whereas the activity of DPPIV is always very weak despite its strong activity shown in a defined in vitro environment(see Fig.4).Therefore,muscle DPPI may be a key enzyme responsible for the generation of dipeptides in Jinhua ham processing(Zhao et al.,2005a).3.1.4.AminopeptidasesToldra´,Flores,and Sanz(1997)have shown that the free amino acids in dry-cured hams are primarily generated from muscle proteins and peptides by the actions of amino-peptidases.Experiments in vitro demonstrated that alanyl, arginyl and leucyl aminopeptidase(AAP,RAP and LAP)maintain strong activity during Jinhua ham processing, although only7.98%,5.70%and9.76%of the initial activ-ity of AAP,RAP and LAP are,respectively,retained at the end of ripening and3.05%,1.90%and6.02%,respectively, at the end of post-ripening.They are all affected by changes of temperature,salt content and pH value.The activities of AAP,RAP and LAP under practical Jinhua ham process-ing conditions are estimated at1.27–4.13%,1.14–2.21% and3.99–17.54%,respectively,according to the mathemat-ical models that include factors that affect the three amino-peptidases(see Fig.5).LAP and AAP exhibit strong activity throughout processing,while RAP shows consider-able activity only during thefirst three processing stages. The data shows that LAP and AAP are the most important aminopeptidases in Jinhua ham processing,while RAP may be more effective at the early stages of processing (Zhao et al.,2006;Zhao et al.,2005b).3.2.Lipolysis and oxidationLipolysis and lipid oxidation are a major source of ham flavor compounds.During Jinhua ham processing,intense degradation and oxidation occur in both muscle lipid andG.H.Zhou,G.M.Zhao/Meat Science77(2007)114–120117subcutaneous fat tissues(Huan,2005;She&Tong,2005; Yan,Li,&Jiang,2005).The lipolysis and,in part,the oxi-dation are attributed to activities of muscle lipases,includ-ing phospholipase and lipoxygenase(Huan,2005;Huan, Zhou,Zhao,Xu,&Peng,2005a).3.2.1.LipolysisThe results of research on Jinhua ham in the past few years show that,with the gradual loss of moisture,the pro-portion of lipid in ham increases from3.65%at the green stage to8.10%at the end of post-ripening(Huan,2005). Muscle lipid and subcutaneous fat experience different deg-radation processes.In muscle,the proportions of both tri-acylglycerol and free fatty acids increase during Jinhua ham processing,while phospholipids decrease dramatically with about66.67%of muscle phospholipids hydrolyzed during processing(see Fig.6).On the other hand,both the proportions of phospholipids and triacylglycerol in subcutaneous fat decrease during Jinhua ham processing, while only the proportion of free fatty acids increases dra-matically(see Fig.7).It seems that the phospholipid degra-dation in muscle and both phospholipid and triacylglycerol degradation in subcutaneous fat are the main reactions of ham lipolysis and it appears that phospholipids are a main source of precursors of hamflavor substances(Huan, 2005).3.2.2.Lipid oxidationLipid oxidation occurs throughout Jinhua ham process-ing with the most intense lipid oxidation occurring at the sun-drying stage.Free fatty acids are the main substrates of the lipid oxidation process.The proportions of saturated and monounsaturated fatty acids in both muscle and sub-cutaneous fat tissue increase during Jinhua ham process-ing.The proportion of monounsaturated fatty acids increases most dramatically,while the proportion of poly-unsaturated fatty acids decreases sharply.This indicates that intense oxidation of polyunsaturated fatty acids has occurred;especially of linoleic acid which is the fatty acid with the biggest decrease both in muscle and subcutaneous tissues during processing(Huan,2005).3.2.3.Lipases and phospholipasesAccording to the optimum pH of different lipases,at least three types of lipases can be categorized:acid lipases, neutral lipases and basic lipases.Considering that the pH value of ham is about6,basic lipases may not be effective and are not considered in our study.In vitro experiments show that lipases and phospholipases are not evenly dis-tributed in muscle and subcutaneous fat tissues.Neutral lipase demonstrates the highest activity in both muscle and subcutaneous tissues,while acid lipase and phospho-lipases the lowest.The activity of the three enzymes both in muscle and subcutaneous fat tissues decreases sharply during Jinhua ham processing(see Fig.8).No activity of muscle lipases or phospholipases can be detected after post-ripening.At the end of ripening,8.31%,4.18%and 7.56%of the initial activities of muscle acid lipases,neutral lipases and phospholipases,respectively,are retained,while only8.26%,2.94%and5.36%of the respective initial activ-ity remained at the end of post-ripening.In subcutaneous fat tissues,about16.16%,23.05%and14.77%of the initial activity of acid lipases,neutral lipases and phospholipases, respectively,occur at the end of ripening stage and no118G.H.Zhou,G.M.Zhao/Meat Science77(2007)114–120lipase or phospholipase activity can be detected in the fol-lowing stages(Huan,2005;Huan,Zhou,&Xu,2005b).Response surface experiments show that the activities of muscle lipases and phospholipases are affected by tempera-ture,salt concentration and the interaction effect of the two factors.Well-established mathematical models show that the muscle environment during Jinhua ham processing is quite favorable for muscle lipases and phospholipases,with very high activity during most of the processing(see Fig.9).This explains the concentration changes of free fatty acids in muscle during the processing of Jinhua ham (Huan,2005;Huan et al.,2005b).3.2.4.LipoxygenaseLipoxygenase is a major enzyme responsible for lipid oxidation.In vitro experiments show that subcutaneous fat does not possess lipoxygenase activity.On the other hand,muscle lipoxygenase displays very strong activity during Jinhua ham processing.This enzyme increases to its maximum activity at the end of the salting stage,then decreases during the following stages with about66%of its initial activity retained at the end of the post-ripening stage.Response surface experiments demonstrate that the activity of lipoxygenase is influenced by temperature,salt and nitrate concentration.Interaction effects also exist between temperature and salt content as well as tempera-ture and nitrate content.However,the established equation model indicates that the practical muscle lipoxygenase activity demonstrated during Jinhua ham processing explains only about20%of the oxidation effects.The result suggests that auto-oxidation,not enzyme catalyzed oxida-tion,is the major cause of muscle lipid oxidation during Jinhua ham processing(Huan,2005).4.Flavor compounds identification and their changes along processing stages4.1.Identification offlavor compoundsMore than330individual peaks are found in muscle samples of Jinhua ham during processing and191of them have been identified using headspace-solid phase microex-traction(SPME)and GC/MS techniques(Huan et al., 2005a;Zhao,2004).The compounds identified belong to alkanes and alkenes,aromatic hydrocarbons,alcohols, aldehydes,ketones,carboxylic acids,esters,oxygenous het-erocycle compounds,nitrogenous compounds,sulphur compounds,chloride compounds,amides,and terpenes (Huan,2005;Huan et al.,2005a;Zhang,Wang,Liu, Zhu,&Zhou,2006;Zhao,2004).At least22compounds make clear contribution to the characteristic aroma according to artificial sniffing(Tian,Wang,&Xu,2004). The compounds include eight aldehydes(3-methyl-butanal, 2-methyl-propanal,hexanal,(Z)-2-heptenal,2-methyl-propanal,octanal,phenyl acetaldehyde and nonanal),four sulphur compounds(dimetyl disulfide,methanethiol,3-methylthiol-propanal and dimethyl trisulfide),three ketones(acetone,2,3-pentanedione and2-heptanone), three heterocycle compounds(methylpyrazine,2,6-dimeth-ylpyrazine and2-pentyl-furan),one alcohol(1-octen-3-ol) and one ester(ethyl acetate)(Tian et al.,2004).4.2.Changes offlavor compounds during processingDuring Jinhua ham processing,all theflavor com-pounds increase in total peak area;however,their relative percentage changes.The percentages of alcohol and ketone areas drops sharply while aldehydes,acids,lactones,pyra-zines,sulphur compounds and oxygen heterocyclic com-pounds increase in area percentage during processing (Huan,2005;Huan et al.,2005a;Tian et al.,2004;Tian, Wang,&Xu,2005;Tian,Wang,&Xu,2006;Zhao, 2004)(see Fig.10).Flavor is extremely important for dry-cured ham.The development offlavor in dry-cured ham is a very complex process andflavor compounds mostly originate from enzy-matic action and/or chemical reactions such as lipid oxida-tion,Maillard reactions and Strecker degradations of muscle protein and fat.Proteolysis and lipolysis constitute the main biochemical reactions in the generation offlavor orflavor precursors(Toldra´,1998)and are mainly attrib-G.H.Zhou,G.M.Zhao/Meat Science77(2007)114–120119uted to the endogenous enzymatic systems in view of the low microbial counts found inside the hams,the conditions that are unfavorable for microbial growth(Toldra´&Ethe-rington,1988;Zhen et al.,2004)and low microbial enzyme activity levels(Molina&Toldra´,1992).Therefore,the quality andflavor characteristics of Jinhua ham depend on its raw meat properties and processing technology (Zhang et al.,2006)and the factors influencing enzyme activity during the course of processing.5.ConclusionTraditional Jinhua ham processing consists of six stages: green ham fabricating,salting,washing,sun-drying and shaping,ripening,and post-ripening.Intense proteolysis, lipolysis and oxidation reactions occur over the course of processing and,as a result,many characteristic volatilefla-vor compounds are produced.The content of free amino acids infinal Jinhua ham products is14–16times that of green ham,and191volatile compounds have been identi-fied during processing.Endogenous enzymes may play an important role in the development of the characteristicfla-vor of Jinhua ham.ReferencesGong,Y.L.(1987).Jinhua ham processing technology.Beijing:Popular Science Press(China).Huan,Y.J.(2005).Studying on the changes of lipid andflavor compounds during processing of Jinhua ham.Doctorial Paper of NanJing Agricultural University Nanjing,China.Huan,Y.J.,Zhou,G.H.,&Xu,X.L.(2005b).Study on time-related changes of phospholipase during processing of Jinhua ham using response surface method.Food and Fermentation Industries(China), 31(2),120–123.Huan,Y.J.,Zhou,G.H.,Zhao,G.M.,Xu,X.L.,&Peng,Z.Q.(2005a).Changes inflavor compounds of dry-cured Chinese Jinhua ham during processing.Meat Science,71,291–299.Lin,K.Z.,Yang,Y.H.,Zhu,S.W.,Wang,X.Y.,Zhang,S.H.,Bu,X.P., et al.(1992).Study on relationship of the quality and color,flavor development of Jinhua ham with mold growth.Meat Research (China)(2),10–16.Molina,I.,&Toldra´, F.(1992).Detection of proteolytic activity in microorganisms isolated from dry-cured ham.Journal of Food Science, 57,1308–1310.Sentandreu,M.A.,&Toldra´,F.(2001c).Dipeptidyl peptidase activities along the processing of Serrano dry-cured ham.European Food Research and Technology,213,83–87.She,X.J.,&Tong,Q.Y.(2005).Changes of fatty acid in intramuscular lipids during processing of Jinhua ham.Food and Fermentation Industries(China),31(1),139–142.Tian,H.X.,Wang,Z.,&Xu,S.Y.(2004).Characterization of odor-active compounds in Jinhua ham by GC-olfactometry.Food and Fermenta-tion Industries(China),30(12),117–123.Tian,H.X.,Wang,Z.,&Xu,S.Y.(2005).Research on volatileflavors of Jinhua ham.Journal of Wuxi University of Light Industry(China), 24(1),69–73,83.Tian,H.X.,Wang,Z.,&Xu,S.Y.(2006).Separation and identification of volatileflavors of Jinhua ham by gas chromatography–mass spectrometry coupled with head space solid phase microextraction.Chinese Journal of Chromatography,24(2),176–180.Toldra´,F.(1998).Proteolysis and lipolysis inflavour development of dry-cured meat products.Meat Science,49(Suppl.1),S101–S110.Toldra´,F.,&Etherington,D.J.(1988).Examination of cathepsins B,D,H and L activities in dry-cures hams.Meat Science,59,531–538. 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