Purification, biochemical characterization and dye decolorization capacity

Purification（净化）这个词在不同的语境中有不同的含义，主要包括以下几种：
1. 化学与生物科学中的净化：指通过物理、化学或生物等方法去除混合物中的杂质以获得纯度较高的物质的过程。

例如，在实验室中，可以使用蒸馏、萃取、结晶、层析、过滤等方式对化合物进行提纯。

2. 水处理领域的净化：指的是将受污染的水体通过各种工艺如沉淀、过滤、吸附、消毒、反渗透等手段，使其达到生活饮用、工业生产或其他用途所需的水质标准。

3. 环境科学中的净化：包括空气净化和土壤净化，旨在减少或消除环境中存在的有害污染物，恢复环境质量。

4. 宗教与精神层面的净化：在某些宗教和哲学体系中，净化是指涤除心灵或灵魂的罪恶、杂念、不净之物，使人回归纯洁或神圣状态的过程。

5. 日常生活中：也指清洁、杀菌、消毒等过程，如净化空气、净化食材等。

综上所述，purification一词的核心概念是通过某种方式去除不想要的成分或影响，从而提高某物的质量、纯净度或价值。

双抗制备流程The process of preparing dual antibodies involves several key steps that are essential for the successful production of these important biochemical reagents. 双抗制备流程涉及到几个必不可少的关键步骤，对于成功生产这些重要的生化试剂至关重要。

First and foremost, the process begins with obtaining the appropriate cell lines that will be used to produce the antibodies. 首先，这个流程始于获取适用于生产抗体的细胞系。

Without the right cell lines, it is impossible to proceed with the production of antibodies. 没有合适的细胞系，就无法继续进行抗体的生产。

This step involves careful selection and characterization of the cell lines to ensure that they are capable of producing the desired antibodies. 这一步需要仔细选择和表征细胞系，确保它们能够产生所需的抗体。

Once the appropriate cell lines have been obtained, the next step in the process is to culture and expand the cells under controlled conditions. 一旦获得了合适的细胞系，流程的下一步是在受控条件下培养和扩展细胞。

伯杰氏细菌鉴定手册引用伯杰氏细菌鉴定手册引用参考内容：1. Bergey's Manual of Systematic Bacteriology- Whitman, W.B., et al. (eds.). Bergey's Manual of Systematic Bacteriology. 2nd edition. Springer, 2012.- This manual provides a comprehensive and authoritative resource for the identification and classification of bacteria. It includes information on morphological, physiological, and genetic characteristics of bacteria, along with detailed descriptions of individual taxa.2. Manual of Clinical Microbiology- Jorgensen, J.H., et al. (eds.). Manual of Clinical Microbiology. 11th edition. ASM Press, 2015.- The Manual of Clinical Microbiology is a widely used reference in clinical microbiology laboratories. It covers a broad range of topics related to bacterial identification, including specimen collection and processing, laboratory techniques, and interpretation of test results.3. Laboratory Identification of Bacteria- Elaichouni, A. (ed.). Laboratory Identification of Bacteria. CRC Press, 2014.- This book provides a practical guide to laboratory identification of bacteria, focusing on different techniques and tests used to identify and characterize bacteria. It covers topics such as microscopy, culturing, biochemical tests, and molecular techniques.4. Identification of Bacteria by Gas Chromatography-MassSpectrometry Analysis- Seng, P., et al. Identification of Bacteria by Gas Chromatography-Mass Spectrometry Analysis of Urine Samples. Clinical Microbiology and Infection, 2013, 19(4), 335-341.- This research article describes the use of gas chromatography-mass spectrometry (GC-MS) analysis for the identification of bacteria in urine samples. GC-MS analysis can provide accurate and rapid identification of bacteria based on their specific chemical profiles.5. MALDI-TOF Mass Spectrometry for Bacterial Identification- Patel, R. MALDI-TOF Mass Spectrometry for Bacterial Identification. Expert Review of Molecular Diagnostics, 2013,13(2), 167-180.- This review article discusses the applications of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for bacterial identification. It provides an overview of the technique, its advantages, and limitations, along with examples of its use in clinical microbiology.6. Molecular Methods for Bacterial Identification- Petti, C.A. Detection and Identification of Microorganisms by Gene Amplification and Sequencing. Clinical Infectious Diseases, 2007, 44(8), 1108-1114.- This review article discusses the use of molecular methods, such as gene amplification and sequencing, for the detection and identification of microorganisms. It provides an overview of different molecular techniques and their applications in clinical microbiology.Note: The above references are for informational purposes only and do not constitute an endorsement of any specific product, manufacturer, or organization. It is always important to consult multiple sources and use professional judgement in scientific research and practice.。

Purification and characterization of antioxidative peptides from round scad (Decapterus maruadsi )muscle proteinhydrolysateHaiping Jiang a ,c ,Tianzhe Tong b ,Jianhua Sun a ,Yuanjin Xu a ,Zhongxing Zhao a ,Dankui Liao a ,c ,⇑aSchool of Chemistry and Chemical Engineering,Guangxi University,Nanning 530004,Guangxi,ChinabSchool of Chemistry and Materials Science,University of Science and Technology of China,Anhui 230026,China cGuangxi Key Lab of Petrochemical Resource Processing and Process Intensification Technology,Nanning 530004,Guangxi,Chinaa r t i c l e i n f o Article history:Received 18July 2013Received in revised form 22November 2013Accepted 18December 2013Available online 28December 2013Keywords:Round scad (Decapterus maruadsi )Antioxidative peptide PurificationCharacterizationEnzymatic hydrolysisa b s t r a c tMuscle protein from round scad (Decapterus maruadsi )was hydrolyzed with five commercial proteases,namely,Alcalase,neutral protease,papain,pepsin,and trypsin.Round scad hydrolysate (RSH)prepared with Alcalase demonstrated high antioxidative activity.After ultrafiltration,RSH-III fraction (M W <5kDa)exhibited the strongest activity.Then,RSH-III was purified by gel filtration chromatography (Sephadex G-15)and separated into four fractions (A,B,C,and D),of which fraction B showed the highest antioxi-dative activity and was further purified using reverse-phase high-performance liquid chromatography twice.The purified peptides were identified as His-Asp-His-Pro-Val-Cys (706.8Da)and His-Glu-Lys-Val-Cys (614.7Da)by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spec-trometry.Subsequently,the identified peptides were synthesized,and their antioxidative activities were verified.Results indicated that the two novel peptides isolated from round scad muscle protein can be developed into antioxidative ingredients in functional foods.Ó2013Elsevier Ltd.All rights reserved.1.IntroductionAntioxidants play an important role both in human health by protecting the body against various degenerative diseases and in food preservation by retarding oxidation-induced deterioration.Free radical-mediated oxidation and antioxidants have been widely discussed.Many synthetic antioxidants of phenolic com-pounds,such as butylated hydroxyanisole,butylated hydroxytolu-ene,and tert-butylhydroquinone,are widely used in the food industry.However,their usage is strictly regulated because of weak carcinogenic effects in some animals at high levels (Shahidi and Zhong,2010).Therefore,identifying natural and safe antioxi-dants from various natural sources as alternatives to synthetic ones is a topic of great interest among researchers.In general,bioactive peptides usually contain 3–20amino acid residues per molecule,and their activities are based on the inherent composition and sequence of amino acids (Pihlanto-Leppälä,2001).Peptides within the sequence of their parent proteins are usually inactive,but can be liberated during enzymatic digestion or fermen-tation.Applying enzymes to modify protein is an efficient way torecover potential bioactive peptides (Ismail &Sarmadi,2010).Dif-ferent kinds of animal and plant proteins released by enzymatic hydrolysis have been reported to exhibit antioxidative activity,including porcine myofibrilla protein (Saiga,Tanabe,&Nishimura,2003),rice protein (Qiu,Liu,&Beta,2009),and barley glutelin (Xia,Bamdad,Gänzle,&Chen,2012).Moreover,various bioactive peptides with antioxidative activity have been derived from various marine organisms,such as marine rotifer (Byun,Lee,Park,Jeon,&Kim,2009),sardinelle by-products (Bougatef et al.,2010),and oys-ter (Qian,Jung,Byun,&Kim,2008).Round scad (Decapterus maruadsi ),a kind of marine fish,be-longs to the family of mackerel.In Southern China,the peak of pro-duction for round scad is during the hot season,this kind of fish is limited in use because of their dark colour,susceptibility to oxida-tion,and off-putting flavour (Thiansilakul,Benjakul,&Shahidi,2007a ).Therefore,this species is usually regarded as a byproduct in fishery,discarded or processed into fish meal or fertilizer.Pro-cessing low-valued pelagic fish into high market-value products,such as surimi production (Benjakul,Artharn,&Prodpran,2008),and into protein hydrolysates with bioactive activity (Thiansilakul,Benjakul,&Shahidi,2007b )has seen a surge.However,no informa-tion regarding the purification and characterization of antioxida-tive peptides from round scad protein hydrolysate has been reported.In the present study,the main purpose is to isolate and characterize antioxidative peptides derived from round scad mus-cle protein (RSP).Furthermore,to verify their potential use,the0308-8146/$-see front matter Ó2013Elsevier Ltd.All rights reserved./10.1016/j.foodchem.2013.12.074⇑Corresponding author at:School of Chemistry and Chemical Engineering,Guangxi University,Nanning 530004,Guangxi,China.Tel.:+867713272702;fax:+867713233718.E-mail address:liaodk@ (D.Liao).purified peptides are synthesized,and their antioxidative activities are confirmed.2.Materials and methods2.1.MaterialsFresh round scad were collected from a localfish market in Guangxi,China and kept frozen atÀ76.0°C.Round scadfish loin portions were rinsed with distilled water,minced,and then stirred into meat.Protein content of round scad was determined using Kjeldahl,whereas lipid content was measured by Soxhlet extrac-tion method.Alcalase,neutral protease,papain,and pepsin were provided by Pangbo Biological Engineering Co.,Ltd.(Nanning,China).Trypsin was purchased from Sinopharm Medicine Holding Co.,Ltd.(Shang-hai,China).2,2-Diphenyl-1-picrylhydrazyl(DPPH)and vitamin C were purchased from Sigma-Aldrich Chemical Co.(St.Louis,MO, USA).Reduced L-Glutathione(GSH)was purchased from Beijing Biodee Biotechnology Co.,Ltd.(Beijing,China).Sephadex G-15 was purchased from Pharmacia Fine Chemicals Co.,Ltd.(Uppsala, Sweden).All other chemicals and reagents used were of the highest grade available commercially.2.2.Preparation of enzymatic hydrolysatesHydrolysis experiments were conducted in a500mL glass reac-tor.RSP was dissolved in ultrapure water with ratio of2:100(w/v) and placed into boiling water for10min to ensure deactivation of endogenous enzymes.Then,the proteins were digested byfive kinds of enzymes at an enzyme/substrate ratio of1:100(w/w). Temperature and pH were adjusted to the optimum values for each enzyme:Alcalase,50°C and pH9.5;neutral protease,50°C and pH 7.0;papain,55°C and pH7.0;pepsin,37.5°C and pH2.0;and tryp-sin,37.5°C and pH7.8.During the reaction,hydrolysis pH was maintained at the prescribed value by continuously adding 0.05M NaOH for5h under continuous stirring.Reactions were ter-minated by heating the solution in a boiling water bath for15min to ensure enzyme deactivation,and then pH was adjusted to7.0. After the hydrolysate was centrifuged at11,671g for10min,the supernatant was collected and then lyophilized for further use. The hydrolysis process was reproducible for preparation of round scad hydrolysate.2.3.Measurement of antioxidative activity2.3.1.DPPH radical-scavenging activity assayThe DPPHÅscavenging activity of protein hydrolysate was mea-sured as previously described with slight modifications(Alma, Mavi,Yildirim,Digrak,&Hirata,2003).In the experiment,0.5mL of hydrolysate was added to0.5mL of DPPHÅsolution(0.1mM in methanol).The mixture was shaken thoroughly and incubated at 30°C for30min in darkness.Absorbance was determined at 515nm using UV-2550Spectrophotometer(Shimadzu,Kyoto,Ja-pan).The scavenging effect was expressed as the formula Scavenging activity of DPPHð%Þ¼½1ÀðA sÀA1Þ=A0 Â100;ð1Þwhere A s is the absorbance of the sample,A0is the absorbance of the control of the DPPH-methanol solution,and A1is the absorbance of the sample added to methanol.Free radical-scavenging activity was quantified by a regression analysis of scavenging activity(%)versus peptide concentration and defined as an EC50value,that is,the pep-tide concentration required to produce50%of scavenging activity under described conditions.2.3.2.Reducing power assayThe reducing power of RSH to reduce Fe(III)was measured using the modified method of Oyaizu(1986).In brief,0.5mL of hydrolysate and0.2mL of phosphate buffer(0.2M,pH6.6)were mixed with50l L of1%K3Fe(CN)6.The mixture was incubated at 50°C for20min and mixed with0.1mL of10%TCA,followed by centrifugation at875g for10min.Then,0.2mL of the supernatant was drawn and mixed with0.2mL of water,followed by20l L of 0.1%FeCl3.After10min of incubation at room temperature,the absorbance of the resulting Prussian blue solution at700nm was measured.A higher absorbance value indicated greater reducing power.2.3.3.Superoxide radical OÅÀ2scavenging activity assaySuperoxide radicalðOÅÀ2Þscavenging activity was measured according to a previous method,with modifications(Li,Jiang, Zhang,Mu,&Liu,2008).The reaction mixture included5.7mL of 50mM Tris-HCl-(1mM)EDTA(ethylene diamine tetraacetic acid) buffer with pH8.2(incubated at25°C for20min),0.2mL of pro-tein hydrolysate,and0.1mL of5mM pyrogallol solution,added in sequence.Reaction was initiated by the addition of pyrogallol. The rate of pyrogallol antoxidation was its optical density,which was measured at320nm using a spectrophotometer and recorded every30s for4min.The capability for scavenging superoxide an-ion radicals was calculated asScavenging activity of OÅÀ2ð%Þ¼½ðD A0=minÀD A s=minÞ=ðD A0=minÞ Â100;ð2Þwhere D A0/min is the change in absorbance per minute of the con-trol solution containing pyrogallol and buffer,and D A s/min is the change in absorbance per minute of the sample.2.4.Purification of the antioxidative peptides2.4.1.UltrafiltrationThe lyophilized RSH powder was dissolved in ultrapure water and fractionated by ultrafiltration using Labscale System(Labscale TFF System,Millipore Co.,Billerica,MA,USA)with the molecular weight cut-off(MWCO)membranes of10and5kDa(Millipore Co.,Billerica,MA,USA),respectively.Hydrolysates were initially processed in a10kDa membrane to generate a retentate(termed RSH-I,M W>10kDa),then the10kDa permeate was further frac-tionated into a5kDa retentate(termed RSH-II,5<M W<10kDa) and permeate(termed RSH-III,M W<5kDa).The fractions RSH-I, RSH-II,and RSH-III were collected and lyophilized for antioxidative activity assay and further purification.2.4.2.Gelfiltration chromatographyThe fraction with the highest antioxidative activity among RSH-I,RSH-II,and RSH-III was further purified by loading onto a Sephadex G-15gelfiltration column(2.0Â40cm),which was previously equilibrated with ultrapure water,then eluted with ultrapure water at aflow rate of1.0mL/min.Each fraction of the eluted solution was monitored at280nm,collected,and pooled for lyophilization.Their antioxidative activities were investigated. The fraction with the strongest antioxidative activity was subjected to the next purification step.2.4.3.High-performance liquid chromatography(HPLC)The fraction with the most effective antioxidative activity from the gelfiltration chromatography purification was further purified using reversed-phase(RP)HPLC(Agilent1100)on a Hedera ODS C2 column(10Â250mm,10l m,Hanbon Science and Technology Co.,Ltd.,Jiangsu,China).Elution was carried out with a linear gradient of acetonitrile(0–60%in50min)containing0.1%H.Jiang et al./Food Chemistry154(2014)158–163159trifluoroacetic acid (TFA)at a flow rate of 1.5mL/min.Elution peaks were detected at 220nm.Then,the active fraction of eluted peak was concentrated and freeze-dried.The peak that represented the highest antioxidative activity was subjected to a second round of RP-HPLC purification using a Hypersil ODS-C18column (4.0Â250mm,5l m,Agilent Technologies Co.,Ltd.,Santa Clara,CA,USA)with a linear gradient of acetonitrile (0–20%in 40min)containing 0.1%TFA at a flow rate of 0.5mL/min.The purified pep-tides were then used for the analysis of amino acid sequence.2.5.Characterization of purified peptidesAccurate molecular mass and amino acid sequence of the puri-fied peptides were determined by using 4800plus MALDI-TOF/TOF™Analyzer (Applied Biosystems,Beverly,MA,USA),based on Liu,Wang,Duan,Guo,and Tang (2010).The purified peptides were first lyophilized and dissolved in 50%acetonitrile containing 0.1%TFA.Then,1l L of the sample solution was mixed with 1l L of ma-trix solution (a -cyano-4-hydroxycinnamic acid solution,prepared with 50%acetonitrile containing 0.1%TFA),and allowed to dry on a conventional steel matrix-assisted laser desorption ionization target.The sample was desorbed and ionized at 337nm,and oper-ated in the positive ion delayed extraction reflector mode.Spectra were recorded over the mass/charge (m /z )range of 500–1500.Mass spectrometry/mass spectrometry experiments were achieved by collision-induced dissociation.Peptide sequencing was per-formed via manual calculation.2.6.Antioxidative activities of synthetic peptidesThe purified peptides were also synthesized by GL Biochem Co.,Ltd.(Shanghai,China).Antioxidative activities of purified peptides were measured through assays for DPPH Åscavenging activity,reducing power,and O Å2Àscavenging activity.2.7.Statistical analysisAll assays for the antioxidative activities were conducted in triplicates.Data were presented as mean ±standard deviation (SD).Statistical analysis was done in MS Excel (Microsoft Windows 2003)using Student’s t -test,significant difference in means be-tween the samples was determined at the 5%confidence level (p <0.05).3.Results and discussionAs protein-rich marine fish,round scad muscle has high proteincontent (15.5±0.43%)and low lipid content (1.8±0.08%).Šlizˇyte,Daukšas,Falch,Storrø1,and Rustad (2005)suggested that a higher amount of lipids indicates a lower percentage of solubilized pro-tein in raw material.Low fat and high protein contents indicate that an appropriate amount of raw protein in round scad muscle can be processed into more functional and nutritional proteins.Fig.1shows the simplified flow chart for the preparation of antioxidative peptide from round scad.To produce antioxidative peptides,five commercial enzymes were used to hydrolyze RSP for the preparation of RSH.3.1.Preparation of RSH and its antioxidative activity3.1.1.DPPH Åscavenging assayDPPH Åis a relatively stable radical of organic nitrogen that can be characterized by a typical deep purple color,with absorbance in the range of 515–528nm when dissolved in ethanol or methanol,and it reacts with hydrogen donors (Sánchez-Moreno,2002).When DPPH radicals encounter a proton-donating substrate,such as an antioxidant,the radicals would be scavenged.The DPPH Åscaveng-ing activities of various hydrolysates are shown in Table 1.The hydrolysates all possessed the capability to quench the DPPH rad-ical and showed no significant difference (p >0.05).3.1.2.Reducing power of hydrolysateDetermining the reducing power is via electron-transfer assay,whereby oxidants are reduced by hydrolysates in redox-linked col-orimetric reaction.As shown in Table 1,the five hydrolysates allTable 1Antioxidative Activities of Round Scad Protein Hydrolysates with Different Enzymes for 5h.Hydrolysate DPPH Åscavenging activity a (%)O À2Åscavenging activity b (%)Reducing power a(A 700)Alcalase 39.36±1.3423.06±1.500.309±0.012Neutral 32.33±1.0825.25±1.580.234±0.015Trypsin 39.37±1.2818.85±1.670.330±0.019Papain 40.21±0.8520.78±1.440.260±0.018Pepsin32.63±1.137.57±1.25c0.229±0.019Each data is expressed as mean ±standard deviations from three replications (n =3).aScavenging effects were tested at a concentration of 1mg/mL.bScavenging effects were tested at a concentration of 10mg/mL.cValues in the same column are significantly different at p <0.05.160H.Jiang et al./Food Chemistry 154(2014)158–163possessed reducing power,and no significant difference (p >0.05)was observed.3.1.3.O ÅÀ2scavenging activity assay Superoxide radical is a biologically unstable and toxic species.However,ðO ÅÀ2Þcannot directly initiate lipid oxidation,and is nor-mally formed first as potential precursor of other kinds of free rad-icals and oxidizing agents,such as hydrogen peroxide and hydroxyl radicals.Superoxide radical and its derivatives damage cells,which leads to cell or tissue injury (Macdonald,Galley,&Webster,2003).Therefore,scavenging of superoxide radicals is important.The assay for ðO ÅÀ2Þscavenging activity is dependent on thereducing activity of hydrolysate by a ðO ÅÀ2Þ-dependent reaction,which releases chromophoric products.As shown in Table 1,neu-tral protease-treated hydrolysate was found to be the most active superoxide radical-scavenger,followed by Alcalase-treated hydro-lysate and papain proteolytic hydrolysate.The pepsin-treated hydrolysate showed a significantly low (p <0.05)radical scaveng-ing efficiency.However,the DPPH Åscavenging abilities of trypsin hydrolysate (39.37±1.28%)and papain hydrolysate (40.21±0.85%)were nearly the same as that of Alcalase hydrolysate (39.36±1.34%).The trypsin hydrolysate (0.330±0.019)exhibited a stronger reduc-ing power than Alcalase hydrolysate (0.309±0.012).In comparing the results comprehensively,Alcalase-treated RSH presented the most effective antioxidative activity.Moreover,Alcalase has been reported to be capable of producing bioactive peptides from natu-ral protein hydrolysates (Wijesekara,Qian,Ryu,Ngo,&Kim,2011).Machie (1974)also indicated that proteases from plants and microorganisms are highly suitable in the preparation of fish hydrolysates.Therefore,RSH generated by Alcalase was selected for further study.3.2.Purification of antioxidative peptidesThe Alcalase proteolytic hydrolysate was fractionated using dif-ferent MWCO membranes.The antioxidative activities of fractions RSH-I,RSH-II,and RSH-III are shown in Table 2.The three fractions showed a significant difference (p <0.05)in DPPH Åscavenging avtivity and reducing power.RSH-III (M W <5kDa)showed the most effective antioxidative activities among the three in vitro antioxidative evaluation methods.This finding reflects that the low molecular mass of antioxidative peptides may be enriched by ultrafiltration,which is consistent with previous reports.According to Sun,He,and Xie (2004),bioactive peptides with a molecular mass less than 6000Da possess antioxidative properties.As shown in Fig.2(a),fraction RSH-III was fractionated into four separate fractions (A,B,C,and D)by gel filtration chromatography (Sephadex G-15column,2.0Â40cm).All fractions exhibited anti-oxidative activities but at significantly different levels (p <0.05).The molecular weight distribution of the four major fractions (A,B,C,and D)were measured to be located at 1.7–8.4kDa,0.3–1.2kDa,0.3–0.8kDa,and 0.15–0.3kDa,respectively (Shodex Protein KW-802.5column).As such,antioxidative peptides with various molecular weights were distributed in the hydrolysates.Among the fractions,fraction B demonstrated the most effective scavenging activity of 59.77±1.24%for DPPH radical,followed by fraction A.Table 2Antioxidative Activities of Fractions RSH-I,RSH-II,and RSH-III,obtained from Alcalase hydrolysation process.FractionMolecular weight (kDa)DPPH Åscavenging activity a (%)O À2Åscavenging activity b (%)Reducingpower a (A 700)RSH-I >1047.23±1.12c 13.00±1.460.177±0.012c RSH-II 5–1036.27±1.06c 15.21±1.490.271±0.019c RSH-III<550.54±0.94c28.11±1.60c0.343±0.018cEach data is expressed as mean ±standard deviations from three replications (n =3).aScavenging effects were tested at a concentration of 1mg/mL.bScavenging effects were tested at a concentration of 10mg/mL.cValues in the same column are significantly different at p <0.05.S Retention time (min)A b s . a t 280 n m (A )A B C D1020304050600204060050100B7B6B5B4B3Retention time (min)B1B2H.Jiang et al./Food Chemistry 154(2014)158–163161Furthermore,fraction B was lyophilized and separated by RP-HPLC on a semi-preparation column into seven major fractions (B1–B7),as shown in Fig.2(b).A significant difference (p <0.05)was observed between fractions B1,B2,and B3and other fractions B4,B5,B6,and B7.Fraction B1possessed the highest DPPH radical scavenging capability with a value of (78.70±1.04%)and was fur-ther applied onto an ODS-C18analytical column (4.0Â250mm,5l m,Agilent),then divided into three peaks (B11,B12,and B13),as illustrated in Fig.2(c).Peaks B11and B12showed the antioxida-tive activities of 98.69±0.76%and 41.70±0.93%,respectively.Fractions of B11and B12were collected and rechromatographed on the same column using a linear gradient of acetonitrile (0–10%in 40min)in 0.1%TFA at a flow rate of 0.5mL/min,respec-tively,and then used for identification by mass spectrometry.3.3.Characterization of antioxidative peptides by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS)The amino acid sequences of fractions B11and B12were identified as His-Asp-His-Pro-Val-Cys (HDHPVC)and His-Glu-Lys-Val-Cys (HEKVC)by MALDI-TOF/TOF MS (Fig.3),respectively.The theoretical molecular mass of HDHPVC was determined as 706.8Da,which was close to the observed mass of 707.11(M+H +)of fraction B11,The deviation between the theoretical mass of HEKVC (614.7Da)and observed mass of fraction B12(615.22Da [M+H +])was deemed acceptable.Regarding the relationship between the properties and antioxi-dative activity of amino acids,peptides of HDHPVC and HEKVC contained mainly hydrophilic amino acids of Asp,Glu,Cys,His,as validated by results in Fig.2(b)and (c),whereby polar fractions (B1and B11)were eluted first.The highest activity for fractions B1and B11indicated that antioxidative activity is relevant to hydro-philic amino acids.Sacchetti,Di Mattia,Pittia,and Martino (2008)also reported that the antioxidative activity of a hydrophilicfraction is much higher than that of a lipophilic fraction extracted from chicken meat.3.4.Antioxidant activities of synthetic peptidesPeptides of HDHPVC and HEKVC were synthesized (98%purity),and their antioxidative activities were tested,as shown in Table 3and Fig.4.Both HDHPVC and HEKVC exhibited effective antioxida-tive capacity compared with vitamin C and GSH.The scavenging activities of HDHPVC and HEKVC were near to those of fractions B11and B12,respectively.As shown in Fig.4,compared with HDHPVC,HEKVC and GSH,the lowest reducing power (p <0.05)was obtained for vitamin C,which donated a single electron to Fe 3+.HDHPVC and HEKVC showed higher reducing capability than GSH when the concentra-tion was less than 0.5mM.By contrast,the reducing capacity of GSH was higher when the concentration ranged from 0.5to 0.7mM.This discrepancy may be attributed to the charged amino acids of His,Asp,Glu and Lys contained in HDHPVC,HEKVC andGSH.HDHPVC and HEKVC displayed weaker ðO ÅÀ2Þscavenging activities compared with GSH and vitamin C (Table 3).The reason for the most effective inhibition of DPPH radical and auto-oxida-tion of ðO ÅÀ2Þby vitamin C was its content of phenolic hydroxyl group that reacted with free radicals.Meanwhile,among GSH,HDHPVC,and HEKVC,structural difference was responsible forthe more effective ðO ÅÀ2Þscavenging capability obtained by GSH.In addition,for its His,Pro,Lys,Asp,Val and Cys residues,HDHPVC displayed similar DPPH Åscavenging capability with GSH.Mean-while,the DPPH Åscavenging capability of HEKVC was half of that of GSH.The antioxidative activities of peptides was enhanced by the participation of hydrophobic amino acids and one or more res-idues of His,Pro,Met,Cys,Tyr,Trp,Phe,and Met,based on the study of Ren et al.(2008).Histidine is reportedly important to the antioxidative activities of peptides for its imidazole characteristics that indicate proton-donation capability (Li,Chen,Wang,Ji,&Wu,2007).Histidine res-idue,which was located at the N -terminus,was a component of both HDHPVC and HEKVC,but it was included in the centre of the sequence for HDHPVC,which accounted for the stronger DPPH Åscavenging activity and reducing power of the former compared with the HEKVC.This result indicates that the antioxidativeTable 3Antioxidative Activities of DPPH ÅScavenging and O À2ÅScavenging (EC 50mM).SampleEC 50valuesa(mM)DPPH Åscavenging activitiesO À2Åscavenging activities HEKVC 0.0677±0.0012b 0.3744±0.0021b HDHPVC 0.0310±0.00110.3817±0.0017b GSH0.0300±0.00100.2317±0.0027b vitamin C0.0102±0.0012b0.0895±0.0011baResults are expressed as mean ±standard deviations from three replications (n =3).bValues in the same column are significantly different at p <0.05.162H.Jiang et al./Food Chemistry 154(2014)158–163activity may not only be related to histidine residue,but also to its amount and position in the peptide sequence.Murase,Nagao,and Terao(1993)found that compounds can suppress the oxidation of phosphatidylcholine and methyl linoleate when a histidine residue is located in the N-(long-chain-acyl)terminus.Saito et al.(2003) suggested that a Histidine residue in the center of the peptide se-quence plays an important role in antioxidative activity.Moreover,HDHPVC and HEKVC were composed of Lys,Asp,Pro, and Val residues,which are believed to be relevant to antioxidative activity.According to Kim et al.(2001),Pro residue contributes to the scavenging of free radicals.Research on the antioxidative activ-ity of porcine myofibrilla protein hydrolysate,demonstrated that Asp and Lys residues could deactivate the prooxidant activity of metal ions(Saiga et al.,2003).By using DPPHÅscavenging assay, Di Bernardini et al.(2011)suggested that Val residue-containing peptides display antioxidative activity.Furthermore,Cys residue,which may contribute to antioxida-tive activity,was found and located at the C-terminus of HDHPVC and HEKVC.Li,Li,He,and Qian(2010)suggested that Cys plays a significant role in antioxidative activity when it is located next to the C-terminus as it has a high tendency for hydrogen bonding. Moreover,the reaction of cystine with reactive oxygen species can be facilitated by cystine disulfide bonding and sulfuric acid (Garrison,1987).GSH is recognized as a potential antioxidant base on its sulphydryl group and Cys residue.Cys residue was present in HDHPVC,HEKVC,and GSH,but these peptides exhibited different antioxidative capacities,which may be attributed to the presence of other amino acids,such as His,Lys,Pro,Asp,and Val residues, as well as structural difference.The two antioxidative peptides showed some common features. The presence of specific amino acids and their sequences could ac-count for the antioxidative activity.Although the structural activity relationship of antioxidative peptides has not yet been fully estab-lished,HDHPVC and HEKVC may be incorporated into functional foods as novel antioxidative ingredients.4.ConclusionsIn this study,two novel antioxidative peptides,namely, HDHPVC and HEKVC were successfully purified and identified from RSH for thefirst time.After being synthesized,the two peptides exhibited effective antioxidative capacity in vitro compared with vitamin C and GSH.Hence,these peptides may be used to aid in developing potential applications of low-valued marinefish pro-teins as functional food ingredients after being tested in vivo.A more detailed study of the antioxidative mechanism and in vivo activity of the two peptides is underway.AcknowledgmentsThis work was supported by the programs of Science and Tech-nology Development of Guangxi Province(No.10123008-20), Guangxi Natural Science Foundation of Key Project(2012 GXNSFDA53004),and Key Project of Guangxi Experiment Centre of Science and Technology(No.LGZX201208).ReferencesAlma,M.H.,Mavi,A.,Yildirim,A.,Digrak,M.,&Hirata,T.(2003).Screening chemical composition and in vitro antioxidant and antimicrobial activities of the essential oils from Origanum syriacum L.growing in Turkey.Biological and Pharmaceutical Bulletin,26,1725–1729.Benjakul,S.,Artharn,A.,&Prodpran,T.(2008).Properties of protein-basedfilm from round scad(Decapterus maruadsi)muscle as influenced byfish quality.LWT-Food Science and Technology,41(5),753–763.Bougatef,A.,Nedjar-Arroume,N.,Manni,L.,Ravallec,R.,Barkia,A.,Guillochon,D.,& Nasri,M.(2010).Purification and identification of novel antioxidant peptidesfrom enzymatic hydrolysates of sardinelle(Sardinellaaurita)by-products proteins.Food Chemistry,118(3),559–565.Byun,H.G.,Lee,J.K.,Park,H.G.,Jeon,J.K.,&Kim,S.K.(2009).Antioxidant peptides isolated from the marine rotifer,Brachionus rotundiformis.Process Biochemistry, 44,842–846.Di Bernardini,R.,Rai,D.K.,Bolton,D.,Kerry,J.,O’Neill,E.,Mullen,A.M.,Harnedy,P., &Hayes,M.(2011).Isolation,purification and characterization of antioxidant peptidic fractions from a bovine liver sarcoplasmic protein thermolysin hydrolyzate.Peptides,32,388–400.Garrison,W.M.(1987).Reaction mechanisms in radiolysis of peptides, polypeptides,and proteins.Chemical Reviews,87,381–398.Ismail,A.,&Sarmadi,B.H.(2010).Antioxidative peptides from food proteins:A review.Peptides,31,1949–1956.Kim,S.K.,Kim,Y.T.,Byun,H.G.,Nam,K.S.,Joo,D.S.,&Shahidi,F.(2001).Isolation and characterization of antioxidative peptides from gelatin hydrolysate of Alaska pollack skin.Journal of Agricultural and Food Chemistry,49,1984–1989. Li,B.,Chen,F.,Wang,X.,Ji,B.,&Wu,Y.(2007).Isolation and identification of antioxidative peptides from porcine collagen hydrolysate by consecutive chromatography and electrospray ionization–mass spectrometry.Food Chemistry,102(4),1135–1143.Li,Y.H.,Jiang,B.,Zhang,T.,Mu,W.M.,&Liu,J.(2008).Antioxidant and free radical-scavenging activities of chickpea protein hydrolysate(CPH).Food Chemistry, 106(2),444–450.Li,Y.W.,Li,B.,He,J.,&Qian,P.(2010).Structure-activity relationship study of antioxidative peptides by QSAR modeling:the amino acid next to C-terminus affects the activity.Journal of Peptide Science,17,454–462.Liu,R.,Wang,M.,Duan,J.,Guo,J.,&Tang,Y.(2010).Purification and identification of three novel antioxidant peptides from Cornu Bubali(water buffalo horn).Peptides,31,786–793.Macdonald,J.,Galley,H.F.,&Webster,N.R.(2003).Oxidative stress and gene expression in sepsis.British Journal of Anaesthesia,90(2),221–232.Machie,I.M.(1974).Proteolytic enzymes in recovery of proteins fromfish waste.Process Biochemistry,9,12–14.Murase,H.,Nagao,A.,&Terao,J.(1993).Antioxidant and emulsifying activity of N-(long-chain-acyl)histidine and N-(long-chain-acyl)carnosine.Journal of Agricultural and Food Chemistry,41,1601–1604.Oyaizu,M.(1986).Studies on products of browning reactions antioxidative activities of products of browning reaction prepared from glucosamine.Japanese Journal of Nutrition,44,307–315.Pihlanto-Leppälä,A.(2001).Bioactive peptides derived from bovine whey proteins: Opioid and ACE-inhibitory peptides.Trends in Food Science&Technology,11, 347–356.Qian,Z.J.,Jung,W.K.,Byun,H.G.,&Kim,S.K.(2008).Protective effect of an antioxidative peptide purified from gastrointestinal digests of oyster, Crassostrea gigas against free radical induced DNA damage.Bioresource Technology,99,3365–3371.Qiu,Y.,Liu,Q.,&Beta,T.(2009).Antioxidant activity of commercial wild rice and identification onflavonoid compounds in active fractions.Journal of Agricultural and Food Chemistry,57,7543–7551.Ren,J.,Zhao,M.,Shi,J.,Wang,J.,Jiang,Y.,Cui,C.,Kakuda,Y.,&Xue,S.J.(2008).Purification and identification of antioxidant peptides from grass carp muscle hydrolysates by consecutive chromatography and electrospray ionization–mass spectrometry.Food Chemistry,108,727–736.Sacchetti,G.,Di Mattia,C.,Pittia,P.,&Martino,G.(2008).Application of a radical scavenging activity test to measure the total antioxidant activity of poultry meat.Meat Science,80,1081–1085.Saiga, A.,Tanabe,S.,&Nishimura,T.(2003).Antioxidant activity of peptides obtained from porcine myofibrillar proteins by protease treatment.Journal of Agricultural and Food Chemistry,51,3661–3667.Saito,K.,Jin, D.H.,Ogawa,T.,Muramoto,K.,Hatakeyama, E.,Yasuhara,T.,& Nokihara,K.(2003).Antioxidative properties of tripeptide libraries prepared by the combinatorial chemistry.Journal of Agricultural and Food Chemistry,51, 3668–3674.Sánchez-Moreno,C.(2002).Review:Methods used to evaluate the free radical scavenging activity in foods and biological systems.Food Science and Technology International,8,121–137.Shahidi,F.,&Zhong,Y.(2010).Novel antioxidants in food quality preservation and health promotion.European Journal of Lipid Technology,112,930–940.Šlizˇyte,R.,Daukšas, E.,Falch, E.,Storrø1,I.,&Rustad,T.(2005).Yield and composition of different fractions obtained after enzymatic hydrolysis of cod (Gadus morhua)by-products.Process Biochemistry,40,1415–1424.Sun,J.,He,H.,&Xie, B.J.(2004).Novel antioxidant peptides from fermented mushroom Ganoderma lucidum.Journal of Agricultural and Food Chemistry,52, 6646–6652.Thiansilakul,Y.,Benjakul,S.,&Shahidi, F.(2007a).Compositions,functional properties and antioxidative activity of protein hydrolysates prepared from round scad(Decapterus maruadsi).Food Chemistry,103,1385–1394. Thiansilakul,Y.,Benjakul,S.,&Shahidi,F.(2007b).Antioxidative activity of protein hydrolysate from round scad muscle using Alcalase and Flavourzyme.Journal of Food Biochemistry,31,266–287.Wijesekara,I.,Qian,Z.J.,Ryu,B.,Ngo,D.H.,&Kim,S.K.(2011).Purification and identification of antihypertensive peptides from seaweed pipefish(Syngnathus schlegeli)muscle protein hydrolysate.Food Research International,44,703–707. Xia,Y.,Bamdad,F.,Gänzle,M.,&Chen,L.(2012).Fractionation and characterization of antioxidant peptides derived from barley glutelin by enzymatic hydrolysis.Food Chemistry,134(3),1509–1518.H.Jiang et al./Food Chemistry154(2014)158–163163。

皮肤组织的热力学行为表征：Ⅰ. 拉压行为（作者:___________单位: ___________邮编: ___________）【摘要】目的重点研究在不同温度下猪皮肤组织的拉压行为，探讨温度和真皮层胶原质热变性对皮肤组织力学性质的影响。

方法实验采用新鲜猪皮，在特殊设计的温控制拉伸系统和压缩系统条件下观察猪皮肤在不同温度下的拉压行为。

结果在拉伸实验中，皮肤的刚度随着温度的升高而降低，而在压缩实验中则相反。

结论温度对皮肤组织的拉伸和压缩行为都有影响：拉伸性质的变化主要是由于随着温度的升高皮肤胶原蛋白的热变性所引起，而压缩性质的变化则有可能是由于在热变性的影响下水合的变化所引起。

【关键词】皮肤组织变性热力学行为拉伸行为压缩行为ABSTRACT: Objective This paper aims to characterize the tensile and compressive behaviors of skin tissue under different temperatures and to study the effect of temperature and corresponding dermal collagen denaturation on the mechanical properties of skin tissue. Methods The uniaxial tensile and compressive tests of fresh pig ear skin underdifferent temperatures have been performed by using two specifically designed hydrothermal experimental systems. Results In tensile tests, the skin stiffness decreases with increased temperature, while a contrary trend is observed in compressive tests. Conclusion The results show that temperature has a great influence on both tensile and compressive properties of skin tissue, but the mechanisms are different. The variation of skin tensile properties is caused by the thermal denaturation of skin collagen with increased temperature, while the variation of skin compressive behavior of skin tissue may be due to the hydration change with thermal denaturation.KEY WORDS: skin tissue; thermal denaturation; thermomechanical behavior; tensile behavior; compressive behavior皮肤覆盖于人体表面，约占人体重量的16%，容纳了人体约1/3的循环血液和约1/4的水份，具有多种重要的生理功能。

Purification and Characterization of a NovelGalloyltransferase Involved in Catechin Galloylation in the Tea Plant (Camellia sinensis )*Received for publication,July 20,2012,and in revised form,October 31,2012Published,JBC Papers in Press,November 6,2012,DOI 10.1074/jbc.M112.403071Yajun Liu ‡1,Liping Gao ‡1,Li Liu §,Qin Yang ‡,Zhongwei Lu ‡,Zhiyin Nie ‡,Yunsheng Wang ‡,and Tao Xia §2From the ‡School of Life Science and §Key Laboratory of Tea Biochemistry and Biotechnology,Ministry of Education in China,Anhui Agricultural University,130West Changjiang Rd,Hefei,Anhui 230036,ChinaCatechins (flavan-3-ols),the most important secondary met-abolites in the tea plant,have positive effects on human health and are crucial in defense against pathogens of the tea plant.The aim of this study was to elucidate the biosynthetic pathway of galloylated catechins in the tea plant.The results suggested that galloylated catechins were biosynthesized via 1-O -glucose ester-dependent two-step reactions by acyltransferases,which involved two enzymes,UDP-glucose:galloyl-1-O -␤-D -glucosyl-transferase (UGGT)and a newly discovered enzyme,epicat-echin:1-O -galloyl-␤-D -glucose O -galloyltransferase (ECGT).In the first reaction,the galloylated acyl donor ␤-glucogallin was biosynthesized by UGGT from gallic acid and uridine diphos-phate glucose.In the second reaction,galloylated catechins were produced by ECGT catalysis from ␤-glucogallin and 2,3-cis -fla-van-3-ol.2,3-cis -Flavan-3-ol and 1-O -galloyl-␤-D -glucose were appropriate substrates of ECGT rather than 2,3-trans -flavan-3-ol and 1,2,3,4,6-pentagalloylglucose.Purification by more than 1641-fold to apparent homogeneity yielded ECGT with an estimated molecular mass of 241to 121kDa by gel filtration.Enzyme activity and SDS-PAGE analysis indicated that the native ECGT might be a dimer,trimer,or tetramer of 60-and/or 58-kDa monomers,and these monomers represent a het-erodimer consisting of pairs of 36-or 34-of and 28-kDa subunits.MALDI-TOF-TOF MS showed that the protein SCPL1199was identified.Epigallocatechin and epicatechin exhibited higher substrate affinities than ␤-glucogallin.ECGT had an optimum temperature of 30°C and maximal reaction rates between pH 4.0and 6.0.The enzyme reaction was inhib-ited dramatically by phenylmethylsulfonyl fluoride,HgCl 2,and sodium deoxycholate.Flavonoids,a major class of secondary metabolites in plants,have a number of important physiological roles as endogenous auxin transport regulators (1–3),root development (4,5),seed germination (6),allelopathy (7),plant-bacterium interaction (8,9),UV-B protection (10),and plant defense against pathogens and environmental stress (11).Flavonoids can be grouped into several subgroups including chalcone,flavone,flavonol,flavandiol,anthocyanin,proantho-cyanidin (oligomer or polymer of flavan-3-ols and flavan-3,4-diol units)and other specialized forms (12).Flavan-3-ols (cat-echins),which comprise ϳ70–80%of tea polyphenols,are rich in young leaves and shoots of the tea plant (Camellia sinensis (L.)O.Kuntze).Catechins,with a basic 2-phenylchromone structure,are characterized by the di-or tri-hydroxyl group substitution of the B ring,the 2,3-position isomer of the C ring,and presence of a galloyl group at the 3-postion of the C ring (Fig.1).On the basis of the classical definition proposed of galloyl group structural features,catechins are divided into gal-loylated and nongalloylated compounds.Galloylated catechins,including (Ϫ)-epigallocatechin gallate (EGCG)3and (Ϫ)-epi-catechin gallate (ECG),esterified often with gallic acid (GA)in the 3-hydroxyl group of the flavan-3-ol units are major catechin compounds that account for up to 76%of catechins in the tea plant (13,14).Catechins,especially EGCG,possess antioxidant activity,antimutagenic,anticarcinogenic,antidiabetic,antibacterial,and anti-inflammatory potential,antihypertensive and anticar-diovascular disease effects,solar UV protection,body weight control effects,and therapeutic properties for Parkinson dis-ease (15).The health-promoting effects of galloylated catechins are stronger than those of nongalloylated catechins (16,17).Flavonoid biosynthesis has been a major focus of investiga-tion in recent decades (12).As the building blocks of most pro-*Thiswork was supported by the Natural Science Foundation of China (30972401,31170647,and 31170282),the Natural Science Foundation of Anhui Province (11040606M73),the Collegiate Natural Science Founda-tion of Anhui Province (KJ2012A110),the Program for Changjiang Scholars and Innovative Research Team in University (IRT1101),and the Major Pro-ject of Chinese National Programmes for Fundamental Research and Development (2012CB722903).1Both authors contributed equally to this work.2To whom correspondence should be addressed.Tel.:86-551-5786003;Fax:86-551-5785729;E-mail:xiatao62@.3The abbreviations used are:EGCG,(Ϫ)-epigallocatechin gallate;ECG,(Ϫ)-epicatechin gallate;ECGT epicatechin:1-O -galloyl-␤-D -glucose O -gal-loyltransferase;GA,gallic acid;C,(ϩ)-catechin;EC,(Ϫ)-epicatechin;GC,(ϩ)-gallocatechin;EGC,(Ϫ)-epigallocatechin;␤G,␤-glucogallin,1-O -gal-loyl-␤-D -glucose;ConA,concanavalin A;GCG,(ϩ)-gallocatechin-3-gallate;SCPL,serine carboxypeptidase-like;UDPG,UDP glucose.THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL.287,NO.53,pp.44406–44417,December 28,2012©2012by The American Society for Biochemistry and Molecular Biology,Inc.Published in the U.S.A.F1Fn3anthocyanidins,the 2,3-trans -flavan-3-ol (ϩ)-catechin and 2,3-cis -flavan-3-ol (Ϫ)-epicatechin biosynthetic pathways have been investigated intensively at the biochemical and genetic levels (18,19).The biosynthetic pathway of nongalloylated cat-echins,which include (ϩ)-catechin (C),(Ϫ)-epicatechin (EC),(ϩ)-gallocatechin (GC),and (Ϫ)-epigallocatechin (EGC)is well documented.Some key genes and enzymes in the pathway include dihydroflavonol 4-reductase (EC 1.1.1.219),leucoan-thocyanidin reductase (EC 1.3.1.77),anthocyanidin synthase (EC 1.3.1.77),and anthocyanidin reductase (EC 1.3.1.77)(20–23).Despite recent progress in improving our understanding of flavan-3-ol synthesis,the mechanism involved in galloylation of catechins remains a mystery (19).In the 1980s,the biosynthesis of galloylated catechins and GA in the tea plant was investi-gated using radioactive tracer techniques.It was found that GA was presumably esterified with epigallocatechin and epicat-echin to form catechin gallates in young tea shoots,and the amount of GA might be a key limiting factor for the formation of EGCG and ECG (24).Understanding the galloylation of flavan-3-ols has been hin-dered by an absence of spontaneous genetic mutants for cate-chin biosynthesis.Niemetz and Gross (25)have done much research on the biogenetic pathways of hydrolyzable tannins.Their research confirmed that ␤-glucogallin (1-O -galloyl-␤-D -glucose (␤G))exerts a dual role functioning not only as an acyl acceptor but also as an efficient acyl donor.This work indicates ␤G plays the same role in biosynthesis of galloylated catechin (Fig.2).Galloylation of glucose with GA to yield ␤G,the first specific metabolite in the route to hydrolyzable tannins,was catalyzed by enzyme extracts from oak leaves with UDP-glu-cose serving as an activated substrate (26).This result indicated that,in galloylated catechin biosynthesis,␤G rather than GA might be a precursor of galloylated flavan-3-ols.In this study we sought to identify the enzymatic reactions and purify the key enzyme involved in galloylated catechin biosynthesis.Enzyme assays in vitro were designed to inves-tigate the biosynthesis of galloylated catechins,and the enzymes involved in galloylated catechin biosynthesis werepurified and identified.In addition,the enzyme properties were investigated.EXPERIMENTAL PROCEDURESPlant Materials —Leaves of the tea plant (C.sinensis (L.)O.Kuntze)were plucked from the experimental tea garden of Anhui Agricultural University during early summer.All of the samples were immediately frozen in liquid nitrogen and stored at Ϫ80°before analysis.Enzyme Extraction and Enzyme Assays —Enzyme extraction was performed in accordance with the method of Zhang et al.(27).All enzymes assays were conducted in phosphate buffer.In the multienzyme incorporative reaction system,theUGGT/FIGURE 1.Basic units of typicalcatechins.FIGURE 2.Reaction diagram of the galloylated catechin biosynthetic pathway.In the first reaction (I ),the galloylated acyl donor ␤G was biosyn-thesized by UGGT from the substrates GA and UDPG.In the second reaction (II),galloylated catechins ECG or EGCG were produced by ECGT from the substrates ␤G and nongalloylated catechins EC or EGC.Galloyltransferase Involved in Catechin GalloylationAQ:AAQ:BF2ECGT (UDP glucose:galloyl-1-O -␤-D -glucosyltransferase/epi-catechin:1-O -galloyl-␤-D -glucose O -galloyltransferase)assay solution was incubated at 30°C for 1.5h in a total volume of 1.5ml containing 50m M phosphate buffer (pH 6.0),0.4m M EGC or EC,1.4m M GA,2.3m M UDP glucose (UDPG),4m M ascorbic acid,and crude enzyme extract (0.55mg of total protein).The UGGT reaction solution was incubated at 30°C for 1.5h in a total volume of 1.5ml containing 50m M phosphate buffer (pH 6.0),2.3m M UDPG,1.4m M GA,4m M ascorbic acid,and crude enzyme extract (0.55mg of total protein).The ECGT assay solution was incubated at 30°C for 1h in a total volume of 1.5ml containing 50m M phosphate buffer (pH 6.0),0.4m M EGC or EC,0.96m M ␤G (Advanced Technology and Industrial Company,Hong Kong,China),4m M ascorbic acid,and crude enzyme extract (0.55mg of total protein).The above enzyme reactions were terminated by the addition of ethyl acetate.Each of the reaction products was extracted three times with 3ml of ethyl acetate.The ethyl acetate extract was evaporated and redissolved in 500␮l of methanol and then used directly for analyses of the enzymatic reaction products.For gel filtration-purified enzyme activity analysis,the ECGT assay solution was incubated at 30°C for 10min in a total vol-ume of 100␮l containing 50m M phosphate buffer (pH 6.0),0.4m M EGC or EC,0.96m M ␤G,and 5.6␮g ofenzyme.Enzymatic reactions were terminated by the addition of 10␮l of 5M HCl to the assay solution,and then the solution was used directly for analysis of the enzymatic reaction products.The protein concentration was determined by the Bradford method (28)using bovine serum albumin as a standard.In the control treatment,crude enzyme extract was heated to 100°C to inactivate enzyme activities.Analysis of UGGT and ECGT Enzyme Reaction Products —Extraction of enzyme reaction products was performed accord-ing to the method of Liu et al.(29).The solution (20␮l)of reaction products was spotted on a silica GF254TLC sheet (5ϫ20cm;HeFei BoMei Biotechnology Co.,Hefei,China)that was developed in chloroform:methanol:formic acid (28:10:1,v/v)and then sprayed with 1%vanillin-HCl (w/v)reagent.The spots of reaction products in the methanol extract were identified by R f values,and their visual color compared with those of cate-chin standards.The reaction products extract was analyzed by HPLC on a Phenomenex Synergi 4u Fusion-RP80column (5␮m,250ϫ4.6mm)with detection at 280nm.Ultraviolet spectra were recorded with a Waters 2487UV array detector (Waters Corp.,Milford,MA).For HPLC analysis,the solvent system consisted of 1%(v/v)acetic acid (A)and 100%acetonitrile (B).After injec-tion (5␮l),a linear gradient at a flow rate of 1.0ml/min was set as follows:B from 10to 13%(v/v)in 20min was initiated,then B from 13to 30%(v/v)between 20and 40min;B from 30to 10%(v/v)between 40and 41min.Peaks were identified by compar-ison of the retention times with those of standards.Analysis of enzyme reaction products by LC-MS followed the method of Miketova et al.(30).Enzymatic products were ana-lyzed by HPLC,and the area of the product peaks were collected and identified by LC-MS.Liquid chromatography electrospray-ionization-MS analyses were performed on a Thermo Finnigan LCQ Advantage instrument using the following conditions:negative ion detection mode,centroiding mode,multiplier at 1600keV,1000atomic mass units/s,source at 4.5kV,sheath gas at 70p.s.i.,auxiliary gas at 25p.s.i.,capillary temperature at 220°C,and UV detection at 220nm.Preparation and Identification of ␤-Glucogallin from Tea Plant —For ␤G analysis,1g of fresh leaves was crushed in liquid nitrogen and extracted with 5ml of methanol by sonication at room temperature for 10min followed by centrifugation at 4000ϫg for 15min,and the residues were re-extracted twice as above.The supernatants were pooled and evaporated and redissolved in 5ml of water.The pooled supernatants were then extracted three times with chloroform.The supernatant water phase was purified further with a SEP-PAK C 18cartridge,and after filtration the supernatant was separated by HPLC,and the ␤G peak was collected and freeze-dried.The powder was used for chemical identification by HPLC,MS,and 1H and 13C NMR spectroscopy.For HPLC analysis,the solvent system consisted of 1%acetic acid in water (A)and acetonitrile (B).After injection (5␮l),a linear gradient at a flow rate of 1.0ml/min was established as follows:B from 0to 10%(v/v)in 10min was initiated,then B from 10to 30%between 10and 30min.Peaks were identified by comparing the retention times with those of standards.The LC-MS analysis of ␤G employed the same method as that used for analysis of enzyme reaction products described above.1H and 13C NMR spectra were recorded in methanol-d 4on a Bruker Avance 400MHz spectrometer using TMS as an internal standard.Chemical shifts were expressed in ppm (␦).ECGT Isolation and Purification —ECGT purification con-sisted of acetone powder preparation,ammonium sulfate pre-cipitation,hydrophobic interaction chromatography,conca-navalin A (ConA)chromatography,and gel filtration.The first two steps were performed at 4°C,and the last three steps were conducted at room temperature.Gel filtration was performed to estimate the relative molecular mass of the enzyme.Step 1;Ammonium Sulfate Precipitation —The acetone pow-der was prepared by homogenization of 50g of tea leaves in cold acetone (Ϫ20°)with a Waring blender.The finely ground pre-cipitate was collected by vacuum filtration.The precipitate was washed several times with acetone until the washings were col-orless.This precipitate powder was used as a crude material for ECGT preparation.Precipitate powder (20g)was homogenized with 400ml of extraction buffer (50m M phosphate buffer (pH 7.0),4m M ␤-mercaptoethanol,1%(w/v)polyvinylpolypyrroli-done (Sigma))and filtered through cheesecloth.The homoge-nate was centrifuged at 15,000ϫg for 15min at 4°C,and the supernatant was fractionated with 20–40%ammonium sulfate.Step 2;Hydrophobic Interaction Chromatography —The pre-cipitate was dissolved in 20m M phosphate buffer (pH 7.0)con-taining 1M ammonium sulfate and loaded onto a butyl-Sephar-ose column (20cm ϫ2.5-cm inner diameter,Bio-Rad).For hydrophobic interaction chromatography,the solvent system consisted of 20m M phosphate buffer (pH 7.0)including 1M ammonium sulfate (A)and phosphate buffer (pH 7.0)(B).After application of the enzyme solution,the column was washed with five volumes of buffer A and subsequently eluted with a stepped gradient of 38,95,and 100%B at a flow rate 2.5ml/min.Step3;Affinity Chromatography—The active fractions were subjected to ConA-Sepharose4B chromatography(column10 cmϫ1.6cm inner diameter;GE Healthcare).For ConA chro-matography,the solvent system consisted of20m M Tris-HCl (pH7.0)containing0.5M NaCl(A)and20m M Tris-HCl(pH 7.0)containing0.5M␣-D-methylglucoside(B).After applying the enzyme solution,the column was washed with5volumes of buffer A and subsequently eluted with100%B at a flow rate of1 ml/min.Step4;Gel Filtration Chromatography—The active fractions were subjected to gel filtration on a Superdex200column(50 cmϫ1.6cm inner diameter;GE Healthcare)and eluted with20 m M phosphate buffer(pH7.0)containing0.15M NaCl at a flow rate of0.8ml/min.Step5:SDS-PAGE Assay—SDS-PAGE was performed in accordance with the method of Laemmli(31),after which the proteins were visualized with Coomassie Brilliant Blue using the methods of Oakley(32).Protein Identification by MALDI-TOF-TOF MS—Protein spots were cut from gels,destained for20min in50m MNH4HCO3solution containing30%acetonitrile,and washed inMilli-Q water until the gels were destained.The spots wereincubated in0.2M NH4HCO3for20min and then lyophilized.Each spot was digested overnight in12.5ng/ml trypsin in0.1MNH4HCO3.After trypsin digestion,the peptide mixtures wereextracted with8␮l of extraction solution(50%acetonitrile, 0.5%TFA)at37°for1h.Finally the extracts were dried underthe protection of N2.Samples were reconstituted in3␮l of50%acetonitrile containing0.1%TFA before MS analysis.A1-␮l drop of this peptide solution was applied to an Anchorchip target plate.After drying at room temperature,a0.1-␮l droplet of CHCA matrix was applied to the plate at the same position. Samples were analyzed with ultrafleXtreme(Bruker).All acquired spectra of samples were processed using flexControl-software(Bruker)in the default mode.Parent mass peaks with a mass range of500–3500Da were detected with a minimum S/N filter of10for precursor ion selection.The five most abun-dant MS peaks were selected for MS/MS analysis.The com-bined MS and MS/MS data from the MALDI-TOF-TOF anal-ysis were submitted to Mascot2.3.02for a search against the NCBI C.sinensis protein database(;827 sequences),C.sinensis Genome Database“cam.pep”to con-struct a protein data bank(40,551sequences,data not shown), and C.sinensis Genome Database“tie.pep”to construct a pro-tein data bank(49,413sequences,data not shown).The identi-fication was accepted based on results from three biological replicates.Properties of the ECGT Enzyme—For characterization of ECGT enzyme properties,gel filtration-purified enzyme was used.For determination of the optimum pH for the ECGT, citrate-phosphate buffer(pH4.0–5.5),phosphate buffer(pH 6.0–7.0),and Tris-HCl buffer(pH7.5–8.0)were used.The optimum temperature range for ECGT activity was tested from 0°to70°C at pH6.0.Other assay conditions were identical to those used in the routine assay.To test the effect of inhibitors on ECGT activity,the enzyme was incubated with the inhibitors for5min at30°C before the enzyme assay.Enzymatic activity was measured in the presenceof PMSF,ZnCl2,EDTA,and␤-mercaptoethanol at a final con-centration of0–50m M,and sodium deoxycholate and HgCl2 were used at a final concentration of0–5m M.Other assay con-ditions were identical to those used in the routine assay.For investigation of the effects of temperature and pH on enzyme stability,ECGT activity was tested after enzyme stor-age atϪ20,0,4,10,20,30,40,or50°for48h and after storage atpH4.0to9.0at4°for48h.In addition,the temporal stability of ECGT was determined after storage at4°C for0,2,7,20,or40 days.RESULTSEvidence for Biosynthetic Enzymes of Galloylated Catechins—Niemetz and Gross(25)confirmed that␤G acts not only as anacyl donor but as an acceptor in the biosynthesis of hydrolyz-able tannins.To determine whether catechin galloylation was similar to that of hydrolyzable tannin biosynthesis,a two-step enzyme assay incorporating the substrates GA,EC,or EGC and cosubstrate UDPG was designed,and the enzymatic products were analyzed via TLC and HPLC.The assay showed that UDPG was indispensable in the two-step enzymatic reaction,and a significant amount of␤G was detected in the enzymatic products by HPLC analysis(Fig.3).This result suggested a UDPG-dependent glucosyltransferase existed in the tea plant,and␤G was the enzymatic product.In addition,the galloylated catechins EGCG and ECG(Fig.3),but not(ϩ)-gallocatechin-3-gallate(GCG),were detected by the two-step enzyme assayvia TLC and HPLC(data not shown),which indicated that thecis-catechins EGC and EC were appropriate substrates of a gal-loyltransferase instead of the trans-catechin GC or C.Thesedata further confirmed that EGCG and ECG in the tea plant are biosynthesized via enzymatic galloylation of EGC and EC with␤G,whereas GCG in green tea beverages is derived from isomerization of EGCG during green tea production(33).To test the above assumptions further,two separate enzyme-reaction assays were performed.The first enzyme assay was designed to detect UDPG-glucosyltransferase activity with the substrates GA and UDPG,and the second assay was to detect galloyltransferase activity with the substrates␤G(or1,2,3,4,6-pentagalloylglucose)and EGC(or EC and GC).The enzymatic products were identified by TLC,HPLC,and LC-MS.In the UDPG-glucosyltransferase assay,the product␤G could not be analyzed effectively by TLC for lack of an appro-priate staining reagent.However,HPLC(Fig.4A)and LC-MS confirmed that the product was␤G(Fig.5A)and indicated the enzyme UGGT existed in the tea plant.In the galloyltransferase assay,TLC analysis of the enzyme reaction products showedtwo magenta spots with Rfvalues of0.43and0.28correspond-ing to the ECG and EGCG standards that were displayed in TLC sheets by staining with1%(w/v)vanillin-HCl reagent(Fig.6).This conclusion was confirmed by LC-MS.The parent ionswith m/z457and441corresponding to EGCG and ECG stand-ards,respectively,were observed(Fig.5,B and C).EGC,EC,and␤G were appropriate substrates of the galloyltransferase instead of GC(Fig.6)and1,2,3,4,6-pentagalloylglucose(PGG; Fig.4D).The deduced galloylated catechin biosynthetic path-way is depicted in Fig.2.Galloyltransferase Involved in Catechin GalloylationF3F4F5AQ:CF6Identification of ␤-Glucogallin in Tea Plant —To gain further evidence for the existence of ECGT and UGGT in the tea plant,␤G was extracted and identified from the leaves.An improved method for extraction and quantification of ␤G was estab-lished.A SEP-PAK C18cartridge was used in sample prepara-tion,and the purity of ␤G in the solvent was enhanced mark-edly.To prevent interference from noisy peaks,the linear gradient of the solvent system was optimized for ␤G analysis based on the method used for HPLC analysis of catechins.A single peak with retention time and spectral information con-sistent with those of the ␤G standard appeared at 11.75min in the chromatogram (Fig.7).The ␤G peak was collected largely via HPLC and identified by MS and NMR (Fig.8).The parent ion of the compound was detected at m /z 331,and majorfrag-FIGURE 3.HPLC analysis of UGGT/ECGT enzyme assay extracts.A and C ,the UGGT/ECGT assay solution was incubated at 30°C for 1.5h in a total volume of 1.5ml containing 50m M phosphate buffer (pH 6.0),0.4m M nongalloylated catechins (EGC or EC ),1.4m M GA,2.3m M UDPG,4m M ascorbic acid,1.5m M salicylic acid,and crude enzyme extract (0.55mg total of protein).The products ␤G and galloylated catechins EGCG or ECG were detected clearly in this two-step reaction enzyme assay.Peaks were identified by comparing the retention times with standards.B and D ,control treatments of the UGGT/ECGT assay extracts with the crude enzyme extract were heated to 100°C to inactivate enzyme activities.There were no enzymatic products in control treatments.AU ,absorbanceunits.FIGURE 4.HPLC analysis of UGGT and ECGT enzyme assay extracts.A ,the UGGT assay solution was incubated at 30°C for 1.5h in a total volume of 1.5ml containing 50m M phosphate buffer (pH 6.0),2.3m M UDPG,1.4m M GA,4m M ascorbic acid,1.5m M salicylic acid,and crude enzyme extract (0.55mg of total protein).The product ␤G was detected clearly in this assay.B and C ,the ECGT assay solution was incubated at 30°C for 1h in a total volume of 1.5ml containing 50m M phosphate buffer (pH 6.0),0.4m M nongalloylated catechins (EGC or EC ),0.96m M ␤G,4m M ascorbic acid,and crude enzyme extract (0.55mg of total protein).The galloylated catechins EGCG or ECG were detected clearly in this assay.D ,the ECGT assay solution was conducted with substrates 0.96m M 1,2,3,4,6-pentagalloylglucose (PGG ),0.4m M nongalloylated catechins (EGC ),and conditions otherwise identical to those of the ECGT assay.No galloylated catechins were produced effectively from the substrates 1,2,3,4,6-pentagalloylglucose and EGC.The product peaks of ␤G in A ,ECG in B ,and EGCG in C were collected and identified by LC-MS (see Fig.5).AU ,absorbance units.Galloyltransferase Involved in Catechin GalloylationF7F8ments were detected at m /z 271and 169,which were consistent with those of the ␤G standard.1H and 13C NMR spectra were recorded in methanol-d 4on a Bruker Avance 400MHz spec-trometer with TMS as an internal standard.Chemical shifts were expressed in ppm (␦).1H NMR ␦:7.086(2H,s,H-2Јand H-6Ј),5.615(1H,d,J ϭ8Hz,H-1),3.813(1H,dd,J ϭ12.0,1.6Hz,H-6a),3.658(1H,dd,J ϭ12.0,4.8Hz,H-6b),3.35to 3.45(4H,m,H-2-H-5).13C NMR ␦:167.06(C ϭO),146.53(C-5Ј),140.34(C-4Ј),120.78(C1Ј),110.54(C-2Ј,C-6Ј),95.97(C-1),78.84(C-5),78.23(C-3),74.15(C-2),71.11(C-4),62.36(C-6).These data were consistent with those of the standard com-pounds,and thus the compound was identified as ␤G.Purification of ECGT —Activity of ECGT was monitored throughout the purification.An ϳ1.64-fold purification was obtained by ammonium sulfate precipitation.The main active parts existed in the 20–60%ammonium sulfate fraction.About 10-fold specific activity was increased by hydrophobic interac-tion chromatography separation (Table 1).Monitoring of ECGT activity showed that ECGT enzyme was eluted from the column with a low ion eluent (Fig.9A ),which suggested ECGT was a highly hydrophobic protein.ConA-affinity chromatogra-phy was the most effective purification step for ECGT (Fig.9B ).Separation by ConA-affinity chromatography yielded an ϳ46-fold increase in purification (Table 1)and indicated that ECGT was a glycoprotein.An ϳ2-fold increase in specific activity was achieved by sep-aration on a Superdex 200column (Table 1).Obvious enzyme activities were detected approximately from 52min (fraction 2Ј)to 74min (fraction 6Ј,Fig.9C )with estimated molecular masses of 241to 121kDa based on a standard curve.To inves-tigate the subunit molecular masses of this enzyme,SDS-PAGE was routinely used for identification.The enzyme activity of lane 2was about double that of lane 6,whereas there were a large number of superfluous bands in lane 6from 40to 80kDa,so we speculated that three bands of estimated molecular masses of 36,34,and 28kDa were the subunits of this enzyme (Fig.9D ).One noteworthy phenomenon was that the SDS-PAGE Coomassie Brilliant Blue-stained bands changed with the degree of protein denaturation.SDS-PAGE analysis of lane 2showed that only two bands of 60and 58kDa remained after the enzyme was denatured in loading buffer containing 1%SDS,and three bands of 36,34,and 28kDa were present when the enzyme was denatured in loading buffer containing 5%SDS (Fig.10A).FIGURE 5.Mass spectra of products in the UGGT,ECGT,reaction assays.A ,shown are mass spectra of peaks corresponding to ␤G in the UGGT enzyme reaction assay (see Fig.4A ).The ions of full MS correspond to the ␤G standard.B and C ,MS assay of products peaks in the ECGT enzyme reaction assay (see Fig.4,B and C )are shown.The ions of 441and 457correspond to galloylated catechins ECG and EGCG standards,respectively.FIGURE 6.TLC assay of the ECGT reaction ne 1,ECG was produced with substrates ␤G and ne 3,EGCG was produced with substrates ␤G and ne 5,no product was produced with substrates ␤G and nes 2,4,and 6are catechin standards.Boxed band a ,ECG;boxed band b ,EGCG.FIGURE 7.␤-Glucogallin assay in tea leaves by HPLC.For HPLC analysis,the solvent system consisted of 1%acetic acid in water (A )and 100%acetonitrile (B ).After injection (5␮l),a linear gradient at a flow rate of 1.0ml min Ϫ1was established as follows:B from 0to 10%(v/v)in 10min was initiated,then B from 10to 30%(v/v)between 10min to 30min.Peaks were identified by comparing the retention times with the standard.mAU ,absorbance units.Galloyltransferase Involved in Catechin GalloylationAQ:QAQ:D,T1F9F10。

生物学基本名词详细解释（中英文对照）/english/speciality/5091.html组织相容性Histocompatibility字面上讲是指不同组织共存的能力；严格地讲是指所有移植蛋白的一致性，这是阻止移植和器官排斥的需要。

组织相容性的分子基础是修饰几乎所有人类细胞表面的一套移植蛋白。

这些蛋白是由位于6号染色体上的一段称为主要组织相溶性复合体基因，MHC编码的。

这些蛋白高度多态。

例如，它们在不同的人中显示差异。

尽管很多人会有一些相同的MHC分子，极少数人有完全相同的MHC分子。

微小的差别导致这些蛋白质被移植受体的免疫系统识别为外来的而进行破坏。

对成功的移植来说这些蛋白质应该在供体和受体之间相匹配。

双胞胎相配的几率最高，接下来是兄弟姐妹。

在一般人群中只有10万分之一的比例是MHC匹配的，可以允许移植。

Literally, the ability of different tissues to “get along”; strictly, identity in all of the transplantation proteins, which is a requirement for the prevention of graft or organ rejection. The molecular basis of histocompatibility is a set of transplantation proteins that decorate the surface of nearly all human cells. These proteins are encoded by genes that are grouped on a part of chromosome 6 called the major histocompatibility complex, or MHC. These proteins are highly “polymorphic” i.e., they show variation in different individuals. Although many in dividuals may share some identical MHC molecules, a very low number share all the MHC molecules. The consequence of these minor differences is that these proteins are recognized by the transplant recipient’s immune system as being foreign, and so are targe ted for destruction (since the immune system’s job is to eradicate any foreign proteins or cells that invade the body). For successful transplantation these proteins ideally should be matched between donor and recipient. Twins have the highest rate of matc h, followed by siblings. In the general population only 1 in 100,000 individuals is sufficiently “MHC matched” to another person to allow transplantation.X射线结晶学X-ray Crystallography阐述蛋白质、DNA或其它生物分子的原子水平的三维结构的技术。

Isolation,Identification,and Characterization of Small Bioactive Peptides From Lactobacillus GG Conditional Media That ExertBoth Anti-Gram-negative and Gram-positiveBactericidal ActivityÃRuiliang Lu,Ãy Serena Fasano,z Nandakumar Madayiputhiya,§Nicholas P.Morin,§James Nataro,and ÃAlessio FasanoÃMucosalBiology Research Center,{University of Maryland College Park,{Proteomic Core Facility,and §Center for Vaccine Development,University of Maryland School of Medicine,Baltimore,MDABSTRACTObjectives:Diarrheal diseases remain a major human plague that still claim millions of lives every year.Probiotics,including Lactobacillus GG (LGG),are known to have a beneficial effect on diarrheal diseases,but their mechanism of action has not yet been completely established.Therefore,the main objective of this work was to identify and characterize moieties elaborated by LGG that exert antibacterial activity.Materials and Methods:Lactobacillus GG conditional media was subjected to liquid chromatography/mass spectrometry.The identified peptides were synthesized by Symphony peptide synthesizer and purified by HPLC using Dynamax reverse-phase C ing A600measurement and tested for their antibacterial activity.Results:We identified 7small peptides from LGG cultured media,2of which are NPSRQERR and PDENK,retained theantibacterial activity detected with LGG conditional media.The antibacterial activity was exerted against both Gram-negative (Escherichia coli EAEC 042and Salmonella typhi )and,with less potency,Gram-positive (Staphylococcus aureus )bacteria.Conclusions:Lactobacillus GG elaborates small peptides showing various degrees of antibacterial activity.NPSRQERR showed the most potent antibacterial effect that was detected both in Gram-negative and Gram-positive microorganisms.These synthetic peptides may represent novel tools for the treatment of bacterial infectious diseases.JPGN 49:25–32,2009.Key Words:Antibacterial peptides —Diarrhea —Lactobacillus GG —Probiotics.#2009by European Society for Pediatric Gastroenterology,Hepatology,and Nutrition and North American Society for Pediatric Gastroenterology,Hepatology,and NutritionDiarrheal diseases claim more then 2million lives per year,80%of them being children younger than age 2years (1).Disease and death caused by diarrhea are a global problem;however,it occurs predominantly in developing countries where sanitation remains an unresolved issue and education is limited (2).Although antibiotics are theoretically available for the treatment of these devastat-ing diseases,their use is contraindicated because of the risk of selecting bacterial strains that display antibiotic resist-ance (3).Vaccines remain unaffordable and are not alwayseffective or available (1).Therefore,there is an urgent need for a safe and affordable treatment to solve this serious public health problem.Treatment of diarrheal diseases remains a major chal-lenge due to the number of disease-causing pathogens,including pathogenic Escherichia coli (4,5).The primary cause of death from diarrheal diseases is dehy-dration,which causes extreme thirst,shock,and organ failure.Studies suggested that diarrheal diseases can also have a negative long-term impact on both physical and psychological growth due to reduction in appetite,altered feeding practices,and decreased absorption of nutrients (6).Probiotics are nonpathogenic bacteria that are claimed to have several beneficial effects related to their capa-bility to prevent the growth of several pathogenic micro-organisms,including E coli (7).According to the cur-rently adopted definition by FAO/WHO,probiotics are Received August 6,2008;accepted October 22,2008.Address correspondence and reprint requests to Ruiliang Lu,MD,University of Maryland School of Medicine,Mucosal Biology Research Center,Health Science Facility II,20Penn St,Baltimore,MD 21201,USA (e-mail:rlu@).Drs Lu and Fasano hold stock in Alba Therapeutics.The other authors report no conflicts of interest.Journal of Pediatric Gastroenterology and Nutrition49:25–32#2009by European Society for Pediatric Gastroenterology,Hepatology,and Nutrition and North American Society for Pediatric Gastroenterology,Hepatology,and Nutrition25‘‘live microorganisms which when administered in ade-quate amounts confer a health benefit on the host’’(8). There are many forms of probiotics currently commer-cially available as both pills and liquid medicine(9). Probiotics are known to have a beneficial effect on diarrheal diseases;however,their mechanism of action has not yet been completely established(10).It is well known that probiotics help in maintaining a healthy intestinal microbiota(11).There are several theories proposed to explain the antibacterial effects of probiotics,including their capa-bility to compete for nutrients,the establishment of a microenvironment in which pathogenic microorganisms are not able to survive,or the elaboration of toxins lethal for pathogenic bacteria(10).To explore these possibilities, we used a combination of microbiological,biochemical, and genetic approaches that led to the identification for the first time of Lactobacillus GG(LGG)-derived small pep-tides that retain the antibacterial properties of LGG against both Gram-negative and Gram-positive bacteria.MATERIALS AND METHODSMaterialsLuria Broth Base was purchased from GibcoBRL(Carlsbad, CA);MRS Broth was purchased from Becton Dickinson Com-pany(Franklin Lakes,NJ);MRS agar was obtained from Fluka (Buches,Switzerland);and LB Agar plates were purchased from TEKnova(Hollister,CA).Macro-prep DEAE Support anion exchange resin and Criterion precast gel(4%–20%)were obtained from Bio-Rad(Hercules,CA).Spectrophotometry was performed using a spectrophotometer Beckman Coulter DU530 (Fullerton,CA);cultures were prepared using a Forma Orbital Shakers from Thermo(Waltham,MA).StrainsLactobacillus GG,enteroaggregative E coli strain EAEC 042,Salmonella typhi,and Staphilococcus aureus strains were obtained from the collection of the Center for Vaccine Devel-opment,University of Maryland School of Medicine.Preparation of LGG Conditional Media Lactobacillus GG was cultured in5mL MRS broth,at378C, with shaking at225rpm overnight.The following day,0.1mL cultured MRS broth was diluted to10À10,10À11,10À12,spread on the MRS agar plates,cultured at378C for24hours,and then colonies were counted.The4.9mL of the cultured mixture was centrifuged at5000g for45minutes,and conditional media (CM)collected,filtered,and used for the studies described below.Ion Exchange ChromatographyThree milliliters of LGG CM was added to an anion ex-change column(d¼1.5cm,L¼2.0cm flow rate0.1mL/min).Before loading,washing the column using12mL of Tris-HCl (pH8.0);after loading,the column was washed with12mL Tris-HCl(pH8.0)again and then eluted by ImmunoPure Ig G elution buffer(pH2.8,Pierce,Rockford,IL).The fractions collected for activity assay and sodium dodecyl sulfate-poly-acrylamide gel electrophoresis(SDS-PAGE).Sodium Dodecyl Sulfate-Polyacrylamide GelElectrophoresisEach fraction eluted from anion exchange column was mixed with protein sample buffer(1:1),heated at958C for5minutes, and then applied to Criterion precast gel(4%–20%),using Tris-Glycine-SDS buffer(Bio-Rad)as running buffer at constant 170V for1hour.The gel was stained by2.5%Coomassie blue and destained by10%methonol,7.5%acetic acid solution.LC/MS Analysis and Identification of PeptidesLiquid chromatography/mass spectrometry(LC/MS) analysis of peptides derived from proteins present in the con-ditional media(CM)was performed on Thermofinnigan LCQ mass spectrometer(Thermofinnigan,San Jose,CA),which was connected to a nanoelectrospray ionizer.Initially the super-natant was prefiltered and concentrated using10,000MW cut of membranes(Microcon;Millipore,Billerica,MA).The Sur-veyor chromatographic system with auto sampler(Thermofin-nigan)was used for peptide separation.The LC system was connected to10.5cm fused silica reverse-phase C18column (Pico Frit Column;New Objective,Woburn,MA).The peptides were separated during90-minute linear gradient of5%to90% acetonitrile/water mixture,containing0.1%formic acid at a flow rate of300nL/min.The spectra were accumulated and the acquired MS scans were searched against the Lactobacillus database(IPI)using the SEQUEST search algorithm.Several peptides with different MW distribution were detected and synthesized to check for their antibacterial activity.Peptide synthesis,purification,and identification were car-ried by the Biopolymer Laboratory at the University of Mary-land School of Medicine.Briefly,the peptides were synthesized on a Symphony peptide synthesizer(PTI Instruments,Boston, MA),using the Fmoc coupling strategy.Peptide purification was performed on a Beckman Gold system consisting of two 110B pumps and a167detector(215nm)using a Dynamax reverse-phase C18column(8m L,25.6Â250mm)(Varian,Wal-nut Creek,CA).Peptide characterization was performed by reverse-phase HPLC and MALDI-TOF.Antibacterial Activity AssaysE coli Growth Time CourseTen microliters of culture from E coli strain EAEC042 (2.16Â1014CFU/mL)were added in1-mL LB broth and incubated in378C,shaking at225rpm,measuring A600every 30minutes.Measurement of Antibacterial Activity by CultureSpectrophotometry at A600The assay was performed as previously described(12),with minor modifications.Briefly,10m L E coli EAEC04226LU ET AL. J Pediatr Gastroenterol Nutr,Vol.49,No.1,July2009(7.7Â1014CFU/mL)was added to1.0-mL LB Broth,mixed, and100m L of each LGG-derived synthetic peptide solution dissolved in MRS(for peptide final concentration,see Fig.6B) was added to the mixture.MRS alone(100m L)and LGG CM 100m L(LGG concentration:19.7Â1012CFU/mL)were used as negative and positive controls,respectively.The mixture was cultured for3hours with shaking at225rpm,378C,measuring A600at the end.The relative inhibition activity was calculated according to the following formula:Sample A600Control A600Â100and expressed as percentage:Experiments With Peptide NPSRQERR for Staphylococcus orSalmonella(CVD908)GrowthIncreased concentrations of peptide NPSRQERR were dis-solved in100m L MRS and added to150-m L Staphylococcus culture(44Â106CFU/mL)or150-m L Salmonella culture (38Â106CFU/mL)in LB broth.The same Staphylococcus or Salmonella culture conditions without the peptide were used as control.The mixture was cultured at378C,225rpm for 3hours.At the end,100-m L culture mixture was spread onto LB agar plates,cultured overnight at378C,and colonies counted the next day.The relative inhibition activity calculation was performed according to the following formula:1ÀSample coloniesÂ100and expressed as percentage:Statistical AnalysisTwo-tailed Student t tests were used to test differences between2groups.Data were paired wherever appropriate. Values of P<0.05were regarded as significant.RESULTSEffect of LGG on E coli GrowthTo determine the effect of LGG on pathogenic bac-terial survival,increasing amounts of LGG cultures were added to McConkey petri dishes plated with10À8 dilution of an overnight culture of E coli EAEC042 ctobacillus GG caused a dose-dependent negative effect on E coli growth(Fig.1),suggesting LGG and/or factor(s)secreted by LGG present in the CM exert an antibacterial effect on E coli.To establish whether the LGG antibacterial effect was related to its direct action on E coli or to the secretion of an antibacterial factor(s),LGG CM was used to repeat the experiments described in Figure1.Figure2shows that LGG CM media is responsible for the antibacterial effect observed with LGG.Similar results were obtained when the antibacterial activity was monitored byspectropho-FIG.1.Effect of increasing amounts of LGG culture on EAEC042 ctobacillus GG cultures caused a dose-dependent inhibition of EAEC042growth.Open bars:EAEC042alone;gray bars:EAEC042þ100m L of LGG culture;solid bars:EAEC042þ1000m L of LGG culture.ÃP<0.001compared with EAECalone. FIG.2.Effect of LGG CM on EAEC042survival.Either50or 100m L of EAEC042cultures(starting concentration 2.2Â1014CFU/mL)were mixed with either100m L LGG con-ditional media(starting concentration19.7Â1012CFU/mL)(open bars)or100m L of nonpathogenic E coli conditional media(starting concentration19.7Â1012CFU/mL)(closed bars)used as a nega-tive control.Cultures were then plated on petri dishes,incubated overnight,and colony ctobacillus GG conditional media caused significant decrease in colony counts compared with nonpathogenic E coli CM,irrespective of the initial EAEC042 inoculum.ÃP<0.006;ÃÃP<0.0008compared with nonpatho-genic E coli conditional media.CHARACTERIZATION OF LACTOBACILLUS GG SMALL BIOACTIVE PEPTIDES27J Pediatr Gastroenterol Nutr,Vol.49,No.1,July2009tometry,with an average inhibition rate of95.03% (n¼8).Heat StabilityTo establish whether the factor secreted by LGG was thermostable,CM was heated at958C and added to EAEC042bacterial cultures.When grown at a10À4 dilution,EAEC042growth was quantitated to be 800.5Æ96.9CFU/ctobacillus GG CM inhibited the growth of EAEC042either when the culture was heated(24Æ2.8CFU/mL,P<0.00005)or not heated (22.5Æ4.3CFU/mL,P<0.00005)(Fig.3).Similar results were obtained at higher EAEC042culture dilutions(Fig.3).These results proved that the antibac-terial moiety present in LGG CM is heat resistant.Ion Exchange Chromatography and SDS-PAGEAnalysis ResultsFive fractions were collected from ion exchange chromatography.On the overnight plates culture assay,only fraction3showed antibacterial activity(Fig.4). However,no protein bands could be detected by SDS-PAGE analysis.These results suggested that the concen-tration of the active peptide(s)was low,the molecular weight of the protein was too small,or the molecule(s) was not a protein.To address this issue,LGG CM was concentrated by dialysis against phosphatebuffered FIG.3.Effect of heat treatment on LGG conditional media anti-bacterial activity.Heat treatment of LGG conditional media did not affect its antibacterial effect,irrespective of the dilution of E coli 042cultures plated.ÃP<0.00005compared withcontrol. FIG.4.Effect of LGG conditional media(CM)fractions obtained by anion exchange chromatography on E coli042growth.Ion exchange chromatography was carried out on a column with d¼1.5cm,L¼2.0cm,flow rate0.1mL/min,washing buffer Tris-HCl(pH8.0),elution buffer ImmunoPure IgG(pH2.8).Five fractions were collected and tested on EAEC042overnight cultures used at increasing dilutions. EAEC042culture alone and EAEC042cultures plus LGG CM were used as negative and positive controls,respectively.Of the5fractions collected,only fraction3showed antibacterial activity(ÃP<5Â10À7),irrespective of the original EAEC042inoculum concentration. 28LU ET AL.J Pediatr Gastroenterol Nutr,Vol.49,No.1,July2009saline by using 1000Da molecular weight cutoff bags.The dialyzed LGG lost its antibacterial activities and,therefore,attention was paid to searching for molecular peptides smaller than 1000Da.LC/MS Analysis ResultsThe LC/MS spectra of the LGG CM were analyzed and the mass spectrometry sequences of the <1000Da pep-tides detected in the media were compared with the Lactobacillus database (IPI)using SEQUEST search algorithm.Many peptides with different molecular weight distributions were detected during the process of LC/MS (Fig.5).Of the several fragments of $1000Da molecular weight,the following 7peptides resulted being part of the LGG genome:NPSRQERR,PDENK,YTRGLPM,VHTAPK,LSQKSVK,MLNERVK,and GKLSNK.These peptides were synthesized to 95%to 99%purity and tested for potential antibacterial activity.Activities Assay ResultsThe antibacterial activity of these 7peptides was compared with the linear growth of EAEC 042over time as determined by spectrophotometry A600(Fig.6A)and analyzed at the 180-minute time point.Figure 6B shows the inhibitory effects of the 7peptides on the growth of E coli in liquid culture.The comparative antibacterial activity of the 7peptides was NPSRQERR >PDENK >VHTAPK >MLNERVK >YTRGLPM >GKLSNK >LSQKSVK.Only NPSRQERR showed an activity (81.4%E coli growth inhibition)comparable with LGG CM (95%growth inhibition).PDENK had a mod-erate activity (68.7%growth inhibition),whereas VHTAPK had a mild activity (30%growth inhibition).The remaining 4peptides had little or no activity.To establish whether the antibacterial activity of pep-tide NPSRQERR was specific for E coli ,we repeated our biological assay using both S typhi andStaphylococcusFIG.5.Identification of antibacterial peptides from LGG CM by LC/MS/MS ctobacillus GG CM was filtered and concentrated,and peptides present in CM were separated by Surveyor chromatographic system using 90-minute linear gradient of 5%–90%acetonitrile/water mixture,containing 0.1%formic acid at a flow rate of 300nL/min.The spectra were accumulated and the acquired MS scans searched against the Lactobacillus database (IPI)using SEQUEST search algorithm.The figure shows the typical chromatogram marked with the 7identified peptides (MS1)by LC/MS and SEQUEST Lactobacillus database search.CHARACTERIZATION OF LACTOBACILLUS GG SMALL BIOACTIVE PEPTIDES29J Pediatr Gastroenterol Nutr,Vol.49,No.1,July 2009aureus as bacterial targets.Although the effect of peptide NPSRQERR on S typhi was similar to that observed in E coli EAEC 042(Fig.7),its effect on S aureus was only mild but dose dependent (Fig.7)DISCUSSIONProbiotics were defined by Fuller in 1989as ‘‘live microbial feed supplements that beneficially affect the host animal by improving its intestinal microbial bal-ance’’(13).Fuller’s definition emphasizes the require-ment of viability for probiotics and introduces the aspect of a beneficial effect on the host.Probiotics,which means ‘‘for life,’’have been used for centuries as natural com-ponents in health-promoting foods.The original obser-vation of the positive role played by certain bacteria was first introduced by Russian scientist and Nobel laureate Eli Metchnikoff,who in the beginning of the 20th century suggested that it would be possible to modify the gut flora and to replace harmful microbes by useful microbes (14).Experiments into the benefits of probiotic therapies suggest a range of potentially beneficial medicinal uses for probiotics.However,for many of the potential benefits,research is limited and only preliminary results are available.Among others,probiotics are claimed to protect against pathogens by means of competitive inhi-bition (ie,by competing for growth)or by improving immune functions (15,16).As concerns infection of the gastrointestinal tract,it has been reported that probiotics present in food or supplements are effective intheFIG.6.(A)EAEC 042growth time course.Ten microliters of E coli (2.16Â1014CFU/mL)were added in 1mL LB broth and incubated in 378C,shaking at 225rpm,measuring A600every 30minutes.EAEC 042shows a liner growth between 90and 210minutes.(B)LGG CM and its 7synthetic peptides relative inhibition activities on E coli growth.Ten microliters of EAEC 042(7.7Â1012CFU/mL)were added to 1mL LB broth mixed,and 100m L of each peptide solution dissolved in MRS was added to the mixture.MRS alone (100m L)and LGG CM (100m L)(initial concentration 19.7Â1012CFU/mL)were used as negative and positive controls,respectively .The mixture was cultured for 3hours and the A600measured at the end of incubation.Figure shows mean percentage inhibition ÆSD.N ¼3for all synthetic peptides tested.N ¼7for LCC CM.30LU ET AL.J Pediatr Gastroenterol Nutr,Vol.49,No.1,July 2009treatment and prevention of acute diarrhea;decreasing the severity and duration of rotavirus infections in chil-dren as well as travelers’diarrhea in adults (15,16).Despite major research efforts aimed at finding an effective treatment,diarrheal disease remains a human plague claiming millions of lives every year (1).EAEC 042is among the leading enteric pathogens in pediatrics,causing prolonged diarrheal diseases in children (4).The discovery and characterization of a small peptide elabo-rated by LGG opens unexplored horizons for an effective treatment of diarrheal diseases affordable for third world countries.Our stepwise approach was initially focused on the effect of LGG on E coli growth and on the realization that the antibacterial activity was related to a moiety secreted by LGG in the CM.These results confirmed previous reports of an antimicrobial substance elaborated from lactobacilli (17).However,the nature of this inhibitory substance has never before been characterized.For the first time,we report in this article the identification and characterization of this moiety as a molecule(s)that is heat resistant and small in size.Anion exchange chroma-tography showed that this factor(s)is peptide in nature with an approximate molecular weight of less than1000Da.MS/MS analysis identified 7peptides elabo-rated by genes present in the LGG genome,3of them showing variable antibacterial activity (see Fig.6B).Interestingly,probiotics also elaborate peptides that regulate intestinal cell survival and growth (18,19),suggesting that their beneficial action on infective gas-troenteritis can be the combined effect of both direct antibacterial activity and intestinal mucosal protection.The use of small peptides as antidiarrheal drugs is novel and would offer several advantages over current treatments (20).Being small peptides,it would be easy and cheap to produce them in large amounts and would be virtually devoid of the side effects experienced with current antidiarrheal remedies (drug resistance for anti-biotics,immune reaction for vaccines)(21).Due to the fact that these peptides are elaborated by probiotics found in common food (such as yogurt),is it conceivable to hypothesize that their use would be safe for the treatment of children and adults experiencing diarrhea.However,proper clinical trials are necessary to confirm the safety of these peptides from their clinical use.Of the 7peptides isolated and characterized,the peptide NPSRQERR showed the highest antibacterial properties,both on Gram negative and Gram positive.We note that each of the peptides is predicted to carry a net positive charge at neutral pH by virtue of the preponderance of basic amino acids.Such cationic peptides may act similarly to cationic antibacterial peptides produced by mammalian species,although this hypothesis remains to be tested.Notably,the small LGG-derived peptides are also thermostable,a characteristic that would be advan-tageous for the transport and usage of the drug in devel-oping countries.The synthesis of this small peptide would not require sophisticated equipment.Therefore,developing countries could produce their own LGG-derived peptide at relatively low,affordable costs.Never-theless,peptide formulation for proper protection against degradation and delivery to specific gastrointestinal regions are pitfalls that need to be addressed before possible clinical applications to decrease the burden of diarrheal diseases in developing countries.The object of this article may far reach beyond the development of novel treatment of diarrheal diseases.There are a growing number of reports suggesting that human-associated microbes influence human health and vice versa.The estimated 10to 100trillion microorgan-isms that inhabit the human intestine actually outnumber the body’s own cells by a factor of 10.The human microbiome is the genetic sum of this community of microorganisms living in symbiosis with their host.Therefore,it is conceivable to hypothesize that the metabolic activities of the bacterial population in the colon can be manipulated to promote health.Unfortu-nately,this hypothesis has not been rigorously challenged because of several shortcomings.First of all,the com-plexity of the colonic biota (flora)is vast and alsolargelyFIG.7.Peptide NPSRQERR antibacterial activities on both Sal-monella typhi and Staphylococcus aureus .Increased concen-trations of peptide NPSRQERR were dissolved in 100m L MRS and added to150m L S aureus culture (44Â106CFU/mL)or 150m L S typhi culture (38Â106CFU/mL)in LB broth.S aureus and S typhi cultures (using the same initial inocula)alone were used as controls.The mixture was cultured at 378C,225rpm for 3hours.At the end,100m L culture mixture was spread onto LB agar plate,cultured overnight at 378C,and colonies counted the next day .The relative inhibition activity was calculated using the formula shown in the material and method section.The results show that NPSRQERR shows a dose-dependent antibacterial effect that was more potent on S typhi (in which 100%inhibition was reached at the peptide concentration of 8mmol/L)than on S aureus (in which 100%was reached at a peptide concentration of 64mmol/L).CHARACTERIZATION OF LACTOBACILLUS GG SMALL BIOACTIVE PEPTIDES 31J Pediatr Gastroenterol Nutr,Vol.49,No.1,July 2009undefined.Consequently,many studies have lacked the proper scientific stringency necessary for meaningful readouts on the impact of probiotics on gut microflora. Another important issue is to establish the role of microbial biota in the pathogenesis of several gastroin-testinal(eg,inflammatory bowel diseases)and extrain-testinal(eg,autoimmune diseases)diseases.A third and related issue is defining conditions that may be amelio-rated by probiotic therapies.Therefore,it is possible to conceptualize the use of probiotics or probiotic-derived peptides for the treatment of these other conditions in which manipulation of the intestinal microbiome can represent a novel and yet unexplored alternative approach.REFERENCES1.Nataro JP.Vaccines against diarrheal diseases.Semin Pediatr InfectDis2004;15:272–9.2.O’Ryan M,Prado V,Pickering LK.A millennium update onpediatric diarrheal illness in the developing world.Semin Pediatr Infect Dis2005;16:125–36.3.Marano NN,Rossiter S,Stamey K,et al.The National Antimicro-bial Resistance Monitoring System(NARMS)for enteric bacteria, 1996–1999:surveillance for action.J Am Vet Med Assoc2000;217:1829–30.4.Harrington SM,Dudley EG,Nataro JP.Pathogenesis of enteroag-gregative Escherichia coli infection.FEMS Microbiol Lett2006;254:12–8.5.The US Centers for Disease Control and Prevention(CDC).Out-breaks of Escherichia coli serotype O157:H7infections in con-taminated spinach.September29issue of the Morbidity and Mortality Weekly Report.6.Mata L.Diarrheal disease as a cause of malnutrition.Am J TropMed Hyg1992;47:16–27.7.Guandalini S.Probiotics for children:use in diarrhea.J ClinGastroenterol2006;40:244–8.8.FAO/WHO.Health and Nutritional Properties of Probiotics in Foodincluding Powder Milk with Live Lactic Acid Bacteria.Report of a Joint FAO/WHO Expert Consultation on Evaluation of Health and Nutritional Properties of Probiotics in Food Including Powder Milk with Live Lactic Acid Bacteria;2001.9.Santosa S,Farnworth E,Jones PJ.Probiotics and their potentialhealth claims.Nutr Rev2006;64:265–74.10.Ljungh A,Wadstrom ctic acid bacteria as probiotics.CurrIssues Intest Microbiol2006;7:73–89.11.Broekaert IJ,Walker WA.Probiotics asflourishing benefactors forthe human body.Gastroenterol Nurs2006;29:26–34.12.Kreander A,Vuorela P,Tammela P.A rapid screening method fordetecting active compounds against erythromicin-resistant bacterial strains of Finnish origin.Folia Microbiol2005;50:487–93.13.Fuller R.Probiotics in man and animals.J Appl Bacteriol1989;66:365–78.14.Metchnikoff,E.Essais optimistes.Paris.The prolongation of life.Optimistic studies.Translated and edited by P.Chalmers Mitchell.London:Heinemann;1907.15.Reid G,Jass J,Sebulsky MT,et al.Potential uses of probiotics inclinical practice.Clin Microbiol Rev2003;16:658–72.16.Ouwehand AC,Salminen S,Isolauri E.Probiotics:an overview ofbeneficial effects.Antonie Van Leeuwenhoek2002;82:279–89. 17.Silva M,Jacobus NW,Deneke C,et al.Antimicrobial substancefrom a human Lactobacillus strain.Antimicrob Agents Chemother 1987;31:1231–3.18.Yan F,Polk DB.Probiotic bacterium prevents cytokine-inducedapoptosis in intestinal epithelial cells.J Biol Chem2002;277:50959–65.19.Yan F,Cao H,Cover TL,et al.Soluble proteins produced byprobiotic bacteria regulate intestinal epithelial cell survival and growth.Gastroenterology2007;132:562–75.20.Yeaman MR,Yount NY.Mechanisms of antimicrobial peptideaction and resistance.Pharmacol Rev2003;55:27–55.21.Dennehy PH.Active immunization in the United States:develop-ments over the past decade.Clin Microbiol Rev2001;14:872–908.32LU ET AL. J Pediatr Gastroenterol Nutr,Vol.49,No.1,July2009。

Industry Perspective on the Validation of Column-Based Separation Processes for the Purification of Proteins蛋白质柱层析别离纯化验证现状I.Foreword前言The purpose of this document is to outline some of the significant issues related to the validation of columnbased separation processes used in the purification of proteins produced by recombinant DNA (rDNA), hybridoma technology, or peptide synthesis. While validation of these processes has been identified as a first priority by the PDA Biotechnology Task Force, the issues raised in certain sections of this document may have broader applications, including processes for non-protein pharmaceutical products purified by HPLC, as well as protein pharmaceuticals which are not produced by rDNA, hybridoma, or peptide synthesis technologies.这个文件的目的是概述一些关于重组DNA〔rDNA〕，杂交瘤技术，或多肽合成过程中有重大意义的柱别离纯化验证。