Analysis of lysozyme in cheese on-linecombination of capillary electrophoresis mass spectrometry

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Biosensors and Bioelectronics 31 (2012) 84–89Contents lists available at SciVerse ScienceDirectBiosensors andBioelectronicsj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /b i osMolecularly imprinted polymer anchored on the surface of denatured bovine serum albumin modiﬁed CdTe quantum dots as ﬂuorescent artiﬁcial receptor for recognition of target proteinWei Zhang a ,Xi-Wen He a ,Yang Chen a ,Wen-You Li a ,∗,Yu-Kui Zhang a ,ba Department of Chemistry,Nankai University,94Weijin Road,Tianjin 300071,ChinabNational Chromatographic Research and Analysis Center,Dalian Institute of Chemical Physics,Chinese Academy of Sciences,Dalian 116011,Chinaa r t i c l ei n f oArticle history:Received 5July 2011Received in revised form 14September 2011Accepted 29September 2011Available online 6 October 2011Keywords:Molecular imprinting Quantum dotsDenatured bovine serum albumin Artiﬁcial receptorsSelective protein recognitiona b s t r a c tA new type of molecularly imprinted polymer (MIP)-based ﬂuorescent artiﬁcial receptor was developed by anchoring MIP on the surface of denatured bovine serum albumin (dBSA)modiﬁed CdTe quantum dots (QDs)using the surface molecular imprinting process.The approach combined the merits of molecular imprinting technology and the ﬂuorescent property of the CdTe QDs.The dBSA was used not only to mod-ify the surface defects of the CdTe QDs,but also as assistant monomer to create effective recognition sites.Three different proteins,namely lysozyme (Lyz),cytochrome c (Cyt)and methylated bovine serum albu-min (mBSA),were tested as the template molecules and then the receptors were synthesized by sol–gel reaction (imprinting process).The results of ﬂuorescence and binding experiments demonstrated the recognition performance of the receptors toward the corresponding template.Under optimum condi-tions,the linear range for Lyz was from 1.4×10−8to 8.5×10−6M,and the detection limit was 6.8nM.Moreover,the new artiﬁcial receptors were applied to separate and detect Lyz in real samples.This ﬂuorescent artiﬁcial receptor may serve as a starting point in the design of highly effective synthetic ﬂuorescent receptor for recognition of target protein.© 2011 Elsevier B.V. All rights reserved.1.IntroductionMolecular imprinting is a technique providing functional poly-mers able to recognize target molecules of interest.And the process of molecular imprinting involves the formation of polymers under the guidance of a molecular template to produce complementary binding sites with speciﬁc recognition ability.In contrast to the biological antibody,the substantial advantages of artiﬁcial coun-terparts are their mechanical and chemical stability,low cost of preparation and wide range of operating conditions.To date,MIP has been widely used in the ﬁelds of chromatographic separation,as antibody mimetics and artiﬁcial receptor,and catalysis (Matsui et al.,2007;Kempe and Mosbach,1995;Yang et al.,2004;Ouyang et al.,2007;Tong et al.,2001;Ye and Mosbach,2001;Bossi et al.,2001;Liu et al.,2011;Zeng et al.,2010).The majority of the MIP has been prepared using small molecules as template,much less attention has been paid to the protein recognition (Rusmini et al.,2007;Gale et al.,2009;Klepamik et al.,2011).This was mainly due to the properties of proteins that are large molecular size,ﬂexi-ble structure,and large number of functional groups available for∗Corresponding author.Tel.:+862223494962;fax:+862223502458.E-mail address:wyli@ (W.-Y.Li).recognition,which makes it impossible to use the same approach as imprinting small molecules for protein imprinting.Therefore,to develop highly selective and efﬁcient analytical methods for identi-fying and quantifying proteins by molecular imprinting technology is of great importance.Recently,QDs as sensing and recognizing element have attracted a prominent attention in the past few years when used as ﬂuorescent labels due to their properties,such as good photosta-bility,high luminescence efﬁciency and size-dependent emission wavelengths (Bruchez et al.,1998;Chan and Nie,1998).Those prop-erties render these materials suitable for various applications,such as chemical sensor for ion (Tang et al.,2005),small molecules (Tu et al.,2008),and biomacromolecules (Chen et al.,2009a,b;Han et al.,2009;Cao et al.,2009).The molecule imprinting technique is a promising way to improve the selectivity of the QDs through the formation of MIP on the surface of the QDs.Therefore,if we combine the high selectivity of molecular imprinting technology and excellent ﬂuorescent char-acteristics of QDs,a new afﬁnity material,with speciﬁc recognition cavity and responding to the binding event with signiﬁcant ﬂuores-cence intensity change,could be prepared.However,considerable interest has been focused on the development of small molecules imprinted polymer coated QDs,producing protein imprinted poly-mer based QDs was still a challenge (Haupt et al.,1998;Carlson0956-5663/$–see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.bios.2011.09.042W.Zhang et al./Biosensors and Bioelectronics31 (2012) 84–8985Fig.1.Preparative procedures for the fabrication of MIP-coated CdTe QDs asﬂuorescent artiﬁcial receptor.et al.,2006;Li et al.,2010;Lin et al.,2004,2009;Liu et al.,2010; Diltemiz et al.,2008;Wang et al.,2009;Zhang et al.,2011).The objective of the work is to develop a new kind ofﬂuores-cent afﬁnity material combining the merits of molecular imprinting technology andﬂuorescent property of the CdTe QDs for speciﬁc recognition of target protein.Herein,the dBSA was used not only to modify the surface of the CdTe QDs,but also as assistant monomer to create effective recognition sites(Kuo et al.,2008;Wang et al., 2006;Guo et al.,2006).Three proteins were chosen as the template molecules and then the receptors were synthesized by sol–gel pro-cess(imprinting process,see Fig.1).To the best of our knowledge, no reports to date have been published on the use of dBSA and QDs for protein imprinting.The performance of the receptors was esti-mated byﬂuorescence emission spectra,UV–vis absorption spectra and SDS-PAGE analysis.To illustrate the utility of MIP-basedﬂuo-rescent receptor,Lyz was chosen as the template protein.Lyz exists widely in body tissues and secretions.Its abnormal concentration in serum and urine is related to many diseases,such as renal diseases, leukemia and meningitis.Therefore,development of new methods for the analysis of Lyz in real samples is of considerable impor-tance.In this work,the proposedﬂuorescent artiﬁcial receptor was demonstrated as a simple and selective sensing system for selective separating and detecting of Lyz in real samples.2.Materials and methods2.1.Materials and chemicalsAll chemicals were of analytical grade reagents.Tellurium powder(Beilian Fine Chemical Factory,China),CdCl2·2.5H2O, sodium dodecyl sulfate(SDS)and NaBH4(Guangfu Fine Chem-ical Research Institute,China)were used to prepare CdTe QDs.3-Mercaptopropionic acid(MPA,Alfa Aesar Co.)was used as the capping agent.Tetraethoxysilane(TEOS)and3-aminopropyltriethoxysilane(APTES)were Wuhan University Silicone New Materials Co.,Ltd.(Wuhan,China).Bovine serum albumin(BSA,MW(molecular weight)=67kDa,p I(point iso-electric)=4.9),cytochrome c(MW=12.4kDa,p I=10.2),lysozyme (MW=14.4kDa,p I=11),and methylated bovine serum albumin (MW=68kDa,p I=8.5)were obtained from Sigma–Aldrich Co.(St. Louis,MO).2.2.Preparation of dBSA-coated CdTe QDsThe dBSA was prepared by treating BSA with NaBH4on the basis of a previous literature report(Kuo et al.,2008;Chen et al.,2009a,b). NaBH4was added to the BSA solution with stirring,with aim to reduce the disulﬁde bonds to sulfhydryl groups(–SH).After being stirred for1h at room temperature,the BSA solution was heated to 60–80◦C in water bath for20min to decompose the excess NaBH4. The water-soluble CdTe QDs were synthesized based on previous publication(Li et al.,2008;Zhang et al.,2011).The solution of CdTe QDs was mixed with the dBSA solution in proper molar ratios.The mixture was heated to60–80◦C in a water bath for15min.The solution was then incubated at room temperature for3days and the results were identiﬁed byﬂuorescence scan and SDS-PAGE.2.3.Synthesis ofﬂuorescent artiﬁcial receptorIn a typical synthesis of the MIP-based receptor,to a25-mL ﬂask,10mg of template protein,10mL of dBSA coated-CdTe QDs and60␮L of APTES(functional precursor)were added and stirred for30min.Then,0.10mL of TEOS(cross-linker)was added.Next, 0.10mL of the NH3·H2O(25%,w/v)was added and stirred for12h. The resultant MIP-coated QDs were centrifuged and washed with 0.5%SDS,which was repeated several times until no template was detected.Finally,the precipitate of MIP-coated QDs was redis-persed in buffer solution and then stored at4◦C prior to use.The NIP was prepared using the same procedure but without addition of the template molecule.2.4.Protein adsorption experimentsA mass of50mg of the particles was dispersed in certain vol-ume of protein solution and the mixtures were agitated in a shaken bed.At different time intervals,the mixtures were centrifuged and the concentration of protein in the supernatant was measured by a UV–vis spectrophotometer at a wavelength of280nm.The amount of adsorbed protein can be determined by the difference in concen-tration before and after the adsorption.The adsorption capacity(Q,expressed in units of mg/g)of the protein or analogue bound to the MIP-coated QDs is calculated by Q=(C0−C t)VWwhere C0and C t(mg/mL)are the initial concentration and the resid-ual concentration of the protein or analogue,respectively,V(mL) is the volume of the initial solution,and W(g)is the weight of the MIP-coated QDs.2.5.CharacterizationFluorescence measurements were performed on an F-4500 spectroﬂuorometer(Hitachi,Japan)equipped with a quartz cell (1cm×1cm),the slit widths of the excitation and emission were both5nm,and the excitation wavelength was set at470nm with a recording emission range of490–700nm.The photomultiplier tube voltage was set at950V.UV–vis spectra(200–800nm)were recorded on a UV-2450spectrophotometer(Shimadzu,Japan). Transmission electron microscopy(TEM)was obtained by a Tec-nai G220S-TWIN microscope(Philips,Holland).Fourier transform infrared(FT-IR)spectra(4000–400cm−1)in KBr were recorded using the AVATRA360FT-IR spectrophotometer(Nicolet,Waltham,86W.Zhang et al./Biosensors and Bioelectronics 31 (2012) 84–89Fig.2.Effects of the concentration of the monomer APTES (a)and the cross-linker TEOS (b)on the ﬂuorescence intensity of MIP-coated QDs and the ﬂuorescence change of MIP-coated QDs with template protein.USA).X-ray photoelectron spectroscopy (XPS)measurements were performed with an ESCALAB 250spectrometer (Thermo-VG Scien-tiﬁc)with ultra-high vacuum generators.2.6.Analysis of real samplesIn the sample analysis,chicken egg white was separated from a fresh egg and diluted to 50%(v/v)with Tris–HCl buffer (50mM,pH 7.0).After the diluted solution was immersed in an ice bath and centrifuged at 10,000rpm for 30min,the supernatant solu-tion was used as a Lyz source (Qin et al.,2009).The imprinted nanoparticles were applied to purify the Lyz from a 20-fold diluted real sample of egg white at room temperature.The imprinted nanoparticles were then treated with washing procedure to elute the speciﬁcally adsorbed protein.The eluate was desalted and concentrated 10-fold,using an ultraﬁltration membrane (molec-ular weight cutoff =3000),and 10␮L of each sample was used for SDS-PAGE analysis,using 12.0%polyacrylamide gel (Mini-protean,Bio-Rad,Hercules,CA).3.Results and discussion3.1.Preparation and characterization of MIP-and NIP-coated CdTe QDsThe MIP-based dBSA modiﬁed CdTe QDs were prepared via a surface molecular imprinting process similar to a previously reported approach (Wang et al.,2009;Zhang et al.,2011).The 3-aminopropyltriethoxysilane (APTES)was used as a functional monomer which had a noncovalent interaction with the tem-plate protein.The concentrations of the reactants were reduced to obtain a thin MIP layer and to minimize the homogeneous self-condensation of tetraethoxysilane (TEOS)and APTES.To determine the most favorable conditions for synthesizing the receptor,the amounts of the functional monomer APTES and cross-linker TEOS were investigated (Fig.2).It can be seen from Fig.2that the ﬂu-orescence intensity was gradually decreased with increasing the amount of APTES,which may be due to the native structure of the QDs was destroyed by the APTES through forming the silica shell on the surface of the QDs.Considering the effect of the APTES on the ﬂuorescence intensity of the MIP-coated QDs (curve 1of Fig.2a)and ﬂuorescence change of the receptor with template protein (curve 2of Fig.2a),40␮L of the monomer was chosen for the synthesis of the MIP-based receptor.According to the same consideration,60␮L of the cross-linker was selected for producing the receptor (Fig.2b).Moreover,a method to functionalize the QDs by coating with dBSA and the inﬂuence of the dBSA on the enhancement of ﬂu-orescence intensity for the QDs were investigated (Fig.S1).In orderto obtain dBSA,the BSA was denatured by NaBH 4.By this reaction,the disulﬁde bonds of BSA were opened,which made it possible for the stabilizing agent MPA on the surface of CdTe QDs to be substi-tuted by the thiol-group of dBSA through ligand exchange.Because of the high degree of surface defects of the CdTe QDs,dBSA was used to modify the surface via ligand exchange to increase the stability of CdTe QDs (Kuo et al.,2008).And utilizing the dBSA to modify the surface of CdTe QDs could not only improve the chemical stability and photoluminescence quantum yield of the QDs effectively,but also serve as assistant recognition peptide chains as an additional element of monomer to create effective recognition sites.After synthesis of the receptor,the ﬂuorescence uniformity of the receptor or the control particles (non-imprinted polymer (NIP)-coated QDs)was investigated (Fig.S2).It can be seen from Fig.S2that a linear ﬂuorescence intensity (MIP-and NIP-coated QDs)–concentration relationships were attained with a correlation of 0.996and 0.998,respectively.The results shown in Fig.S2indi-cated the stable emission of the MIP-and NIP-coated QDs.And the main reason for the stable emission was that the particles were protected by the silica shell layer (Liu et al.,2010).3.2.Characterization of the nanoparticles3.2.1.Characterization of IRFT-IR spectra of QDs,MIP-coated QDs and NIP-coated QDs are compared in Fig.S3.The strong and broad peak around 1063cm −1indicates the Si–O–Si asymmetric stretching.Other observed bands about 797cm −1showed the Si–O vibration.The presence of the bands around 2966cm −1was the C–H stretching band.The bands at 3426cm −1and 1542cm −1were the N–H stretch,suggesting the copolymer was successfully modiﬁed onto the surface of the QDs.All those bands showed that the MIP layer generated from sol–gel condensation was grafted on the surface of the QDs.In addition,the NIP-and MIP-QDs showed similar locations and appearance of the major bands,which was because the main compositions of MIP and NIP were similar.3.2.2.XPS characterization of the MIP-and NIP-coated QDsXPS is a state-of-the-art technique that is well-known as a quantiﬁcation tool for determining surface elemental compositions (Fig.S4).As shown in Fig.S4,the XPS survey showed intense sig-nals of Si 2p at 103eV,C 1s at 285eV,N 1s at 398eV and O 1s at 531eV,which indicated that the MIP layer generated from sol–gel condensation of APTES and TEOS was copolymerized and anchored onto the surface of the QDs.W.Zhang et al./Biosensors and Bioelectronics31 (2012) 84–8987Fig.3.Fluorescence emission spectra of(a)MIP-and(c)NIP-coated QDs with addition of the indicated concentration of protein Lyz solution.Stern–Volmer plots from(b) MIP-and(d)NIP-coated QDs with protein Lyz.3.2.3.TEM characterization of MIP-and NIP-coated QDsTo characterize the morphology of the nanoparticles,the TEM images of MIP-and NIP-coated QDs were investigated(Fig.S5).As shown in Fig.S5,it can be seen that the particles were nearly uni-form in size,and the mean diameter of the MIP-coated QDs was not distinctly different from that of the NIP-coated QDs.Therefore, the difference of recognition performance between the MIP-coated QDs and NIP-coated QDs in the subsequent study could not be attributed to the morphological difference of the MIP-coated QDs and NIP-coated QDs,but to the imprinting effect.3.3.Optosensing of template protein by MIP-coated QDs3.3.1.Effect of pHThe recognition ability of the receptor was investigated through the changes of theﬂuorescence signal.The effect of pH on the ﬂuorescence changes of receptor is given in Table S1.As can be seen from Table S1,the change ofﬂuorescence intensity for the MIP-coated QDs was larger than that of NIP-coated QDs.The best imprinting effect was observed at pH6.2with an imprinting fac-tor IF1of2.33(Table S1),so a pH of6.2was selected for further experiments.3.3.2.MIP-and NIP-coated QDs with template protein of different concentrationsCompared the response of MIP-coated QDs or control par-ticles with template protein,the speciﬁc recognition ability was evaluated(Fig.3).It can be seen from Fig.3that the ﬂuorescence intensity of the MIP-coated QDs was quenched grad-ually with the increasing concentration of template Lyz.Generally, theﬂuorescence quenching depends on the adsorptive afﬁnity of the particles with the template.In the case of the MIP-coated QDs,theﬂuorescence quenching was mainly achieved by the afﬁnity of the imprinted cavities with the template due to the speciﬁc inter-actions.The imprinting factor(IF),which is the ratio of the K SV,MIP and K SV,NIP(K SV,MIP and K SV,NIP was the linear slopes of Fig.3b and d,respectively),was used to evaluate the selectivity of the mate-rials.Under optimum conditions,the IF(K SV,MIP/K SV,NIP)was2.74, which indicated that the receptor can greatly enhance the quench-ing efﬁciency ofﬂuorescence,enlarging the spectral sensitivity of the MIP-coated QDs to the template protein.Fig.3b and d is plotted by the Stern–Volmer equation analysis for the MIP-and NIP-coated QDs with protein,respectively.F0F=1+K SV[Q]F0and F were theﬂuorescence intensity of QDs in the absence and presence of template,respectively,K SV was the Stern–Volmer constant,and[Q]was the quencher concentration.In order to show the generality of the imprinting method,the Cyt or mBSA was chosen as the template protein producing Cyt-or mBSA-receptors,respectively.And those results demonstrated the speciﬁc recognition ability of the receptors(Figs.S6and S7).3.4.Afﬁnity adsorption experimentThe binding performance of the receptor was investigated through afﬁnity adsorption experiment(Fig.4).Fig.4a shows the UV–vis spectra of the protein solution before and after the adsorption by MIP-or NIP-coated QDs,respectively.And the great difference between MIP-and NIP-coated demonstrates a good recognition ability of the receptor.Fig.4b compares the pro-tein adsorption amounts onto the MIP-or NIP-coated QDs,which directly exhibited the recognition ability of the receptor.88W.Zhang et al./Biosensors and Bioelectronics 31 (2012) 84–89Fig.4.The UV–vis spectra of the protein solution before (curve 1)and after the adsorption by MIP-(curve 3)or NIP (curve 2)-coated QDs,respectively (a)and the adsorption capacity of MIP-and NIP-coated QDs (b).Fig.S8shows the binding performance of the receptor with dif-ferent concentrations of the target protein.A constant amount of MIP-or NIP-coated QDs was incubated with increasing concen-trations of template and allowed to equilibrate for 12h at room temperature.After equilibration,the amount of protein remain-ing in the supernatant was measured by UV–vis analysis.In all of the concentrations studied,the MIP-coated QDs exhibited higher adsorption capacity to Lyz than the NIP-coated QDs.In the lower concentration of Lyz,the amount of Lyz was not enough to saturate the speciﬁc binding cavities.However,with the Lyz concentration increasing,almost all the speciﬁc imprinted sites were occupied by the Lyz and the adsorption capacity of the receptor was the highest.3.5.Cross-reactivity of the MIP-coated QDsThe speciﬁc recognition ability was further investigated through the cross-reactivity test of the receptors (Table 1).And the Cyt-and mBSA-receptors were synthesized in the same way with that of the Lyz receptor.To investigate the recognition ability of the Lyz-,Cyt-and mBSA-receptors,the aforementioned three types of imprinted particles were dedicated to the adsorption of the protein solution of the Lyz,Cyt,mBSA or BSA,respectively.And the results are summarized in Table 1.It can be seen from Table 1that each type of imprinted particles exhibited speciﬁc afﬁnity adsorption of the template protein over structurally related proteins,which clearly showed that the receptors displayed selectivity to the template protein and exhibited lower cross-reactivity toward structurally related species,and also demonstrated that the imprinting cavities were discriminating proteins on the basis of molecular shape rather than its size.3.6.Recycling of the MIP-coated QDsRecyclability,with this capability of being used again to bind the template,is one of the important strong points of MIP.In theTable 1Speciﬁcity of the bnding of homologous proteins to MIP-coated QDs.a ,bImprinted QDsProtein binding amounts (mg protein/g imprinted QDs)LyzCytmBSABSALyz-imprinted QDs 33.2±1.3 3.7±0.37.2±0.5 4.5±0.2Cyt-imprinted QDs 6.7±0.628.2±2.18.2±1.0 4.7±0.7mBSA-imprinted QDs6.4±0.54.5±0.637.4±2.64.3±0.4aExperiment was conducted by the addition of 50mg of MIP-coated QDs in 0.5mg/mL protein solution at room temperature.bThe results was obtained from three parallel experiments.test of MIP-coated QDs recyclability,the MIP-coated QDs were used to extract Lyz through six extraction/washing cycles.The wash-ing agent used in this experiment was 0.5M NaCl.The results of the recyclability studies are shown in Table S2.It can be seen that the receptor could be repeated many times with minimal binding capacity loss,which indicated that this receptor could be reused for many cycles rather than just as a one-time-use assay.3.7.Detection range and limitThe MIP-based dBSA modiﬁed CdTe QDs have distinctly linear ﬂuorescent quenching toward Lyz in the concentration range of 1.4×10−8–8.5×10−6M with a correlation coefﬁcient of 0.992.The detection limit,which was calculated as the concentration of Lyz that quenched three times the standard deviation of the blank sig-nal,was 6.8nM.And the precision for three replicate detections of 0.56␮M Lyz was 2.8%(RSD).Fig.5.SDS-PAGE analysis of the results for selective recognition of lysozyme from egg ne 1:molecular weight standards (markers are in kDa);lane 2:10␮L of 20-fold dilution of egg white before adsorption;lane 3:10␮L of 20-fold dilution of egg white after adsorption by MIP-coated QDs;and lane 4:10␮L of the eluate from the receptor.W.Zhang et al./Biosensors and Bioelectronics31 (2012) 84–89893.8.Application to real sample analysisTo illustrate the utility of the MIP-basedﬂuorescent receptor to discriminate between Lyz and other co-existence proteins,egg white as a real sample was used to separate the Lyz.The SDS-PAGE analysis(see Fig.5)showed that the eluate of the receptor exhib-ited a single band with a molecular mass of∼14.4kDa,in excellent agreement with that of the template Lyz.All other proteins in the egg white,such as ovalbumin and ovotransferrin,displayed no cross-adsorption by the receptor and did not interfere with the binding of Lyz,which suggested the potential practical applica-tion of the receptor.And this high selectivity of the receptor to the template was attributed to its imprinting effect to the tem-plate.Finally,the optimizedﬂuorescent artiﬁcial receptor has been applied to the analysis of Lyz in egg white samples(Sener et al., 2010;Jing et al.,2010).The average value of the three replicate,was 3.9␮M with a RSD of3.4%.A remarkable merit of using this type of MIP-basedﬂuorescent receptor for the separation and detection of target molecule in complex biological samples is that expensive antibody and tedious sample treatment procedures can be avoided.4.ConclusionsIn summary,we have demonstrated that the MIP which was anchored on the surface of the dBSA modiﬁed CdTe QDs can be used as selective materials for recognition of target protein.The dBSA was used not only to modify the surface defects of the CdTe QDs, but also as assistant monomer to create effective recognition sites. Theﬂuorescence of the artiﬁcial receptors was stronger quenched by the template versus that of control particles,which indicated the receptors could recognize the corresponding template.A series of binding experiments further demonstrated that the materials had good selectivity for template protein over analogues.The results of theﬂuorescent artiﬁcial receptor for egg white analysis further demonstrated its feasibility for target protein recognition.AcknowledgementsThis work was supported by the National Basic Research Program of China(973Program)(Nos.2011CB707703and 2007CB914100),the National Natural Science Foundation of China (Nos.21075069and20875049),and Tianjin Natural Science Foun-dation(No.11JCZDJC21700).Appendix A.Supplementary dataSupplementary data associated with this article can be found,in the online version,at doi:10.1016/j.bios.2011.09.042.ReferencesBossi,A.,Piletsky,S.A.,Piletska,E.V.,Righetti,P.G.,Turner,A.P.F.,2001.Anal.Chem.73,5281–5286.Bruchez Jr.,M.,Moronne,M.,Gin,P.,Weiss,S.,Alivisatos,A.P.,1998.Science281, 2013–2016.Chan,W.C.W.,Nie,S.,1998.Science281,2016–2018.Cao,M.,Cao, C.,Liu,M.G.,Wang,P.,Zhu, C.Q.,2009.Microchim.Acta165, 341–346.Carlson,C.A.,Lloyd,J.A.,Dean,S.L.,Walker,N.R.,Edmiston,P.L.,2006.Anal.Chem.78,3537–3542.Chen,C.,Peng,J.,Xia,H.S.,Yang,G.F.,Wu,Q.S.,Chen,L.D.,Zeng,L.B.,Zhang,Z.L.,Pang,D.W.,Li,Y.,2009a.Biomaterials30,2912–2918.Chen,W.,Xu,D.H.,Liu,L.Q.,Peng,C.F.,Zhu,Y.Y.,Ma,W.,Bian,A.,Li,Z.,Yuan, Y.,Jin,Z.Y.,Zhu,S.F.,Xu, C.L.,Wang,L.B.,2009b.Anal.Chem.81,9194–9198.Diltemiz,S.E.,Say,R.,Buyuktiryaki,S.,Hur,D.,Denizli,A.,Ersoz,A.,2008.Talanta75, 890–896.Gale,D.K.,Gutu,T.,Jiao,J.,Chang,C.H.,Rorrer,G.L.,2009.Adv.Funct.Mater.19, 926–933.Guo,M.J.,Zhao,Z.,Fan,Y.G.,Wang,C.H.,Shi,L.Q.,Xia,J.J.,Long,Y.,Mi,H.F.,2006.Biomaterials27,4381–4387.Han,B.Y.,Yuan,J.P.,Wang,E.K.,2009.Anal.Chem.81,5569–5573.Haupt,K.,Mayes,A.G.,Mosbach,K.,1998.Anal.Chem.70,3936–3939.Jing,T.,Du,H.R.,Dai,Q.,Xia,H.,Niu,J.W.,Hao,Q.L.,Mei,S.R.,Zhou,Y.K.,2010.Biosens.Bioelectron.26,301–306.Kempe,M.,Mosbach,K.,1995.J.Chromatogr.A691,317–323.Klepamik,K.,Voracova,I.,Liskova,M.,Prikryl,J.,Hezinova,V.,Foret,F.,2011.Elec-trophoresis32,1217–1223.Kuo,Y.C.,Wang,Q.,Ruengruglikit,C.,Yu,H.L.,Huang,Q.R.,2008.J.Phys.Chem.C 112,4818–4824.Li,J.,Mei,F.,Li,W.Y.,He,X.W.,Zhang,Y.K.,2008.Spectrochim.Acta A70,811–817.Li,H.B.,Li,Y.L.,Cheng,J.,2010.Chem.Mater.22,2451–2457.Lin,C.I.,Joseph,A.K.,Chang,C.K.,Lee,Y.D.,2004.Biosens.Bioelectron.20,127–131. 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解郁怡神茶饮对链脲佐菌素小鼠焦虑和抑郁行为的影响

10 Esterbauer H. Cytotoxicity and genotoxicity of lipid-oxidation products〔J〕．Am J Clin Nutr，1993； 57（ Suppl）： 779S-786S.
11 Blokhina O，Virolainen E，Fagerstedt KV. Antioxidants，oxidative damage and oxygen deprivation stress： a review〔J〕．Ann Bot，2003； 91（ 2）： 179-94. 〔2018-05-04 修回〕
1 材料与方法 1. 1 动物昆明（ KM）小鼠，SPF 级，雄性，体重 18 ～ 22 g，由河南省实验动物中心提供〔SCXK（豫）
基金项目：河南省高等学校青年骨干教师资助计划项目（ 2014GGJS072）；郑州市科技攻关计划项目（ 20150309）；河南中医学院大学生创新学习项目（ CXXM〔2015〕0112）
表 1 各组 EPM 和 LDT 实验中焦虑行为比较（ x±s，n = 10）
组别
OT%
OE% NT（次 /10 min）
对照组 STZ 组 JYYSCY 低剂量组 JYYSCY 中剂量组 JYYSCY 高剂量组
37. 9±7. 1 36. 3 ±4. 6 33. 9±4. 6 34. 4±3. 5 34. 7±5. 7 37. 9±3. 0 46. 9±7. 92） 4） 41. 1±4. 61） 4） 48. 8±8. 92） 4） 43. 0±5. 82） 4）
第一作者：王君明（ 1980-），男，博士，副教授，硕士生导师，主要从事中药药性药效机制及新药开发研究。

酰化修饰对牦牛乳酪蛋白结构及理化性质的影响

现代食品科技
Modern Food Science and Technology
2014, Vol.30, No.5
团队已研究了丁二酰化修饰对牦牛乳酪蛋白胶束结构 1.2.2 牦牛乳酪蛋白二级结构测定
及疏水性的影响，然而，关于不同酰化试剂对牦牛乳
牦牛乳酪蛋白红外光谱图采用傅里叶变换红外光
酪蛋白的修饰作用研究未见报道。
1.2.4 牦牛乳酪蛋白缓冲容量测定
酪蛋白缓冲容量测定参照 Raouche(2009)的方法
1.1 材料与仪器
并进行修改[12]。分别准确称取 2.000 g 各种酪蛋白样品于 250 mL 小烧杯中，加入适量蒸馏水，不断调节
牦牛乳酪蛋白（干酪素）由甘肃华羚生物科技有 pH 至 7.0 左右，40 ℃磁力搅拌直至酪蛋白制品溶解。
现代食品科技
Modern Food Science and Technology
2014, Vol.30, No.5
酰化修饰对牦牛乳酪蛋白结构及理化性质的影响
杨敏，史莹，梁琪，文鹏程，张卫兵（甘肃农业大学食品科学与工程学院，甘肃省功能乳品工程实验室,甘肃兰州 730070）
摘要：以丁二酸酐、丁二酸、乙酸酐为酰化试剂对牦牛乳酪蛋白进行酰化修饰，研究了酪蛋白空间结构、缓冲性能、钙镁磷含量、色度的变化。结果显示，丁二酸酐、乙酸酐、丁二酸酰化程度分别为 90.0%、87.9%、18.5%。酰化修饰后，牦牛乳酪蛋白二级结构以转角和 β-折叠的含量为主；丁二酸酐酰化促使酪蛋白形成少量规则的 α-螺旋结构，含量为 9.4%，其他样品中未检出。丁二酸酐酰化使其三级结构变化程度最大，丁二酸最小。酰化修饰后，牦牛乳酪蛋白最大荧光强度降低，除了乙酸酐修饰酪蛋白之外，其他样品最大发射波长红移；丁二酸修饰酪蛋白三级结构变化较小。酰化修饰后，酪蛋白缓冲性能增强。经酰化修饰后，牦牛乳酪蛋白中的钙、镁、磷含量均降低，但其色泽均变白。研究结果可为牦牛乳酪蛋白的改性及构效关系研究提供参考依据。

酒曲中产凝乳酶微生物菌株的分离筛选及鉴定

酒曲中产凝乳酶微生物菌株的分离筛选及鉴定腾军伟，赵笑，杨亚威，张健，赵爱梅，姜云芸，李柳，郑喆，杨贞耐*（北京食品营养与人类健康高精尖创新中心，北京市食品添加剂工程技术研究中心，北京工商大学，北京 100048）摘要：针对酒曲中的微生物进行分离纯化，得到11 株细菌和2 株真菌，并采用酪蛋白平板法和Arima 时间法筛选出了1 株产凝乳酶的细菌菌株编号为LB-51。

通过形态学观察、生理生化实验和16S rDNA 序列分析鉴定该菌株为解淀粉芽孢杆菌，将该产凝乳酶菌株命名为解淀粉芽孢杆菌GSBa-1。

该菌株在液体LB 培养基中发酵72 h 产凝乳酶的凝乳活力为（431.53±15.89）SU /mL ，蛋白水解活力为（5.05±0.59）U /mL ，所产凝乳酶凝乳活力高而蛋白水解活力低，凝乳酶粗酶单位酶活力为1.54×105 SU /g 。

解淀粉芽孢杆菌GSBa-1是分离筛选自酒曲中的一株高产凝乳酶细菌，因此其来源安全，可作为工业化候选菌株进一步研究开发。

关键词：酒曲；凝乳酶；分离鉴定；解淀粉芽孢杆菌Isolation and Identiﬁcation of Microbial Strains Producing Rennet from Jiuqu , a Traditional Chinese Fermentation StarterTENG Junwei, ZHAO Xiao, YANG Yawei, ZHANG Jian, ZHAO Aimei, JIANG Yunyun, LI Liu, ZHENG Zhe, YANG Zhennai *(Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Engineering and Technology Research Center ofFood Additives, Beijing Technology & Business University (BTBU), Beijing 100048, China)Abstract: Eleven bacterial strains and two fungal strains were isolated from Jiuqu , a traditional Chinese fermentation starter for rice wine. Out of these, one bacterial strain, designated LB-51, capable of producing rennet was screened by the casein plate method and the Arima method. The strain was identified as Bacillus amyloliquefaciens by its morphological characteristics, physiological and biochemical tests (API 50CHB system) and 16S rDNA sequence analysis and species specific gene analysis and named as B. amyloliquefaciens GSBa-1. The rennet and proteolytic activities were (431.53 ± 15.89) SU /mL and (5.05 ± 0.59) U /mL, respectively, after 72 h shaking culture (120 r /min) at 30 ℃ for 72 h in liquid LB medium. The enzyme exhibited high milk-clotting activity and low protein hydrolysis activity. The milk-clotting activity was 1.54 × 105 SU /g. B. amyloliquefaciens GSBa-1, an efficient producer and a safe source of rennet activity, is worth further research and development as a candidate strain for industrial production of rennet.Key words: Jiuqu ; rennet; isolation and identification; Bacillus amyloliquefaciens DOI:10.7506/spkx1002-6630-201716004中图分类号：TS252.1 文献标志码：A 文章编号：1002-6630（2017）16-0023-06引文格式：腾军伟, 赵笑, 杨亚威, 等. 酒曲中产凝乳酶微生物菌株的分离筛选及鉴定[J]. 食品科学, 2017, 38(16): 23-38. DOI:10.7506/spkx1002-6630-201716004. TENG Junwei, ZHAO Xiao, YANG Yawei, et al. Isolation and identiﬁcation of microbial strains producing rennet from Jiuqu , a traditional Chinese fermentation starter[J]. Food Science, 2017, 38(16): 23-38. (in Chinese with English abstract) DOI:10.7506/spkx1002-6630-201716004. 收稿日期：2016-11-03基金项目：国家自然科学基金面上项目（31371804）；北京市百千万人才工程资助项目（B 类）；国家自然科学基金青年科学基金项目（31601488）；2016年北京工商大学研究生科研能力提升计划项目；公益性行业（农业）科研专项（201303085）作者简介：腾军伟（1988—），男，硕士研究生，研究方向为食品生物技术。

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JIHEHULI主管北京市中医管理局主办中华中西医结合护理学会北京市中医护理能力提升工程办公室编辑《中西医结合护理》编辑部主编唐玲主任黄磊编辑尹佳杰吴银平编辑助理鄂海燕美术编辑王丽地址南京市龙蟠路155号联合立方广场3⁃203邮编210037电话************邮箱bjb@出版中西医结合护理杂志社社长叶振华电话************网刊http ：//邮箱tg@学术推广上海乐护文化传播有限公司电话021-********本刊刊出所有文章不代表编委会以及编辑部的观点ZHONGXIYI JIHE 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JIHE HULI2022年6月第8卷第6期案例分享糖尿病并发挪威疥疮1例护理体会及医院感染防控措施探讨………………………………………………………………………………………………黄秀娟，燕菊（144）1例血友病B患儿腺样体肥大的围手术期护理………………………………………………………………………………………………吕曼君，官小莉（149）1例氯化钙外渗致迟发性皮肤局部坏死的护理……………………………………………………………………………………陈明锐，高小红，陈美容（153）音乐引导性想象技术改善1例大隐静脉高位结扎加分段剥脱手术患者焦虑状态的护理………………………………………………………………………………………………张少娟，黄砚萍（157）综述国内外睡眠抱怨的影响因素研究现状……………………………………………………………………………………吴斯悦，梁蜜峻，徐林燕（161）结构化教育在脑卒中患者的应用进展……………………………………………………………………………………张礼秀，陈红，岑梅（166）快速康复外科理念在老年肺癌患者围手术期护理中应用的研究进展……………………………………………………………………………………赵欣，陈聪聪，张晶（171）老年糖尿病患者发生跌倒的原因分析及预防……………………………………………………耿雪姣，王华枝，贾英华，李蒙，曹雨，贾姗（175）Volume 8Number 6June 2022Inheritance and innovation ：Giving play to the unique advantages of Tradi⁃tional Chinese Medicine …………………………………XIE Wei （1）Effect of auricular acupoint sticking and pressing therapy on preventing postoperative nausea and vomiting in patients undergoing thoracolum⁃bar surgery under general anesthesia ……………………CHEN Ying ，XIE Wei ，YANG Mei ，REN Liwen ，ZHANG Yating ，YANG Ji ，REN Xiuya （2）A Meta analysis on effect of auricular point sticking and pressing therapy in the treatment of hyperemesis gravidarum …………………………………TIAN Tian ，XIE Wei ，DENG Yunyan ，LI zhongqing ，ZHANG yang ，ZHAO Su （8）Effects of auricular acupoint acupuncture therapy on postoperative pain in patients undergoing surgery for low anal fistula …YANG Jin ，XIE Wei ，XIAO Cheng ，ZHANG Fang ，HU Ying （13）Clinical application of the three -step auricular acupoint therapy for 23pa⁃tients with primary tinnitus …………………LIU Qing ，Xie Wei 3，YUAN Dan ，ZHAO Lingling ，REN Xiuya ，XIANG Yiming ，LUO Liyuan ，ZHOU Yihan （18）Application of a three -step auricular acupoint therapy in the treatment of 25elderly patients with tinnitus and insomnia …………………………HE Yanlin ，XIE Wei ，LENG Yu ，REN Xiuya ，Chen Xiaoqiong ，WANG Qin ，XIANG Yiming ，ZHOU Yihan （22）Treatment of 30cases of chronic insomnia by a three -step auricular acupoint therapy ……………ZHAO Lingling ，XIE Wei ，LIU Qing ，TAN Sha ，CHEN Xiaoqiong ，HE Yanlin ，YUAN Dan ，XIANG Yiming （27）Nursing of a patient with acne vulgaris treated with the three -step auricular acupoint therapy ………………ZHOU Yihan ，XIE Wei ，LIU Qing ，DONG Huaqian ，LIU Xiameng ，WANG Qin ，REN Xiuya ，LUO Liyuan （31）Nursing management of a case of primary hyperhidrosis treated by the three -step auricular acupoint therapy ………YUAN Dan ，XIE Wei ，LIU Qing ，TAN Sha ，DONG Huaqian ，LIU Xiameng ，ZHAO Lingling ，LUO Liyuan （34）A case report of the three -step auricular acupoint therapy for allergic rhinitis ………………………LUO Liyuan ，XIE Wei ，HE Yanlin ，YUAN Dan ，ZHAO Lingling ，XIANG Yiming ，ZHOU Yihan （38）Nursing experience of a patient with pain caused by periarthritis of theshoulder treated with the combined application of Traditional ChineseMedicine characteristic nursing techniques…………………LI Yuye ，PAN Lei ，SHEN Juan ，ZHANG Yuanyuan ，DOU Jinjie ，TANG Ling ，E Haiyan （41）Contents Responsible InstitutionBeijing Administration of Tradi⁃tional Chinese Medicine SponsorAssociationofIntegrativeNursingBeijing Traditional Chinese Med⁃icine Nursing Competence Im⁃provement Project OfficeEiditingEditorial Board of Chinese Jour⁃nal of Integrative Nursing Editor-in-ChiefTANG Ling DirectorHUANG Lei EditorsYIN Jiajie WU Yinping Editorial Assistant E Haiyan Art Editor WANG Li Address NO.155，LongpanRoad ，Nanjing ，China Post Code 210037Tel +86-25-85552880E⁃mail ：bjb@PublisherIntegrative Nursing Press Founder and CEO YE Zhenhua Tel +86-25-85630967Online Publishinghttp ：//E⁃mail ：tg@Academic PromotionShanghai Lehu Media Co.，Ltd Tel +86-21-31262772Volume8Number6June2022Nursing of an elderly patient with chronic constipation treated by auricular acupoint sticking and pressing therapy ………………………………………………………………………YE Yun，CAO Fang，TIAN Zheng（46）Nursing of a patient with nocturia treated by auricular acupoint sticking and pressing therapy………………DONG Huaqian，XIE Wei，CHEN Xiaoqiong，HE Yanling，REN Xiuya，YUAN Dan（50）A brief discussion on the theory and research status of auricular acupuncture in treating apoplexy sequelae…………………………DAI Li，XIE Wei，LI Haibo，CHEN Mengxian，HE Yanlin，ZHAO Lingling（54）Clinical application and research progress of auricular acupoint therapy in prevention and treatment of common gynecological diseases……………………………………………REN Xiuya，XIE Wei，HE Yanlin，CHEN Ying，XIANG Yiming，ZHAO Lingling，ZHOU Yihan，LUO Liyuan（60）A correlation analysis between the distributions of TCM constitutional types of patients with mammary cancerand anxiety and depression status………………………………ZI Yuxia，LI Li，YOU Dingyun，ZHANG Zhihua，MA Qiong，HEI Chunyan，CHEN Limin，LI Julin，LI Ya（66）Evidence summary for exercise therapy programs in patients after percutaneous coronary intervention ………………………………………………………………………………WEI Yajuan，WANG Shijie（73）Mediating effect of psychological capital between the psychological load and professional identity of nursing interns ………………………………………………………LU Sheng，WANG Zhenmeng，SHENG Yuehong（81）Effect observation of standardized nursing intervention after transjugular intrahepatic portosystemic shunt ……………………………………………………………………………WANG Chenlu，ZHANG Ting（88）Observation on therapeutic effect of acupoint application and massage technique in treating constipation after ce⁃rebral infarction…………………………GUO Haijiao，LIU Ganlu，MA Xiaoxu，SU Rong，CHEN Cheng，MA Dayong，HU Zhe，LIU Wenli，GENG Qingwen（93）Clinical observation on effect of external application of Rutong powder in the treatment of acute mastitis at stag⁃nation stage………………………………………………ZHENG Hongmei，HE Jing，CHEN Hong（100）Abdominal acupoint massage combined with auricular point sticking and pressing for a patient with postpartum constipation with Qi and blood deficiency and related nursing measures…………………………………………………WANG Xuejing，LU Ying，YANG MAN，TANG Ling（103）Nursing of a patient with acute mastitis during lactation treated by stone needle therapy combined with Tradition⁃al Chinese Medicine collapse stains…………………ZHENG Ruiwen，CHEN Hong，HE Jing，XU Jingjin，ZHANG Jing，TANG Ling（107）Auricular acupoint sticking-pressing and ear-apex bloodletting therapy for a young adult patient with acne of dampness-heat of spleen and stomach type and related nursing management……………YIN Hailan（111）Unblocking stuffy orifice and scraping technique for a child with allergic rhinitis of Qi deficiency of lung and spleen and related nursing measures……………………………………WANG Cai，CHANG Siming（115）Acupoint application in relief of abdominal pain and distension in a patient with pancreatic cancer and related nursing measures………………………………………………MA Xu，YE Yun，LI Xu，LI Qianqian（120）Evaluation on training about cardio-pulmonary resuscitation knowledge and first-aid skills for community-dwell⁃ings………………………………………………………………………………WANG Le，LUO Shan（123）Investigation on psychological stress of newly recruited nurses in a specialist hospital for hepatobiliary diseases and related countermeasures…………………………ZHANG Yujie，ZHU Hengmei，ZHAO Ningning，JIA Ling，FAN Hengwei（127）Volume8Number6June2022Application of Six Sigma methodology in the homogeneous nursing management for patients with acute pancreatitis …………………………………………………………LYU Jian，ZHOU Qun，WEI Yanli，XIE Lan（131）Application of mind mapping in quality management of nursing records in the Emergency Department ……………………………………………………………………………SHI Hailiu，CHEN Jiachun（135）Application of scenario simulation teaching method in the teaching of perioperative nursing of total knee arthroplasty …………………………………………………………CHEN Huiying，TIAN Li，ZHANG Tianqiang（139）Nursing of a diabetic patient with Norwegian scabies and related nosocomial infection prevention and control measures ………………………………………………………………………………HUANG Xiujuan，YAN Ju（144）Perioperative nursing of a child with hemophilia B and adenoid hypertrophy undergoing surgery ……………………………………………………………………………LYU Manjun，GUAN Xiaoli（149）Nursing management of a patient with delayed skin necrosis caused by extravasation of calcium chloride ……………………………………………………CHEN Mingrui，GAO Xiaohong，CHEN Meirong（153）Application of music-guided imagination on relief of anxiety in a patient undergoing High ligation and stripping of the greater saphenous vein…………………………………ZHANG Shaojuan，HUANG Yanping（157）Current status of research on influencing factors of sleep complaints at home and abroad………………………………………………………………WU Siyue，LIANG Mijun，XU Lingyan（161）Progress in application of structured education program for stroke patients………………………………………………………………ZHANG Xiuli，CHEN Hong，CEN Mei（166）Research progress of enhanced recovery after surgery in perioperative nursing of elderly patients undergoing sur⁃gery for lung cancer……………………………………ZHAO Xin，CHEN Congcong，ZHANG Jiang（171）Causes analysis and prevention strategies for falls in elderly patients with diabetes………………………GENG Xuejiao，WANG Huazhi，JIA Yinghua，LI Meng，CAO Yu，JIA Shan（175）。

奶油干酪生产关键工艺参数研究

奶油干酪生产关键工艺参数研究仰伟栋1，任发政2，张晓莹2，张玉秀1,*(1.中国矿业大学(北京)化学与环境工程学院，北京 100083；2.中国农业大学食品与营养工程学院，北京 100083)摘要：为研究奶油干酪的关键工艺参数，通过奶油干酪制作工艺过程中氯化钙添加量、凝乳酶添加量、凝乳温度、脂肪添加量这些工艺参数的设计，以产率、蛋白质含量、脂肪含量、水分含量、感官品质为评价指标进行实验，通过正交优化分析试验得到最佳的工艺参数组合为凝乳酶添加量0.002g/mL 、凝乳温度32℃、脂肪添加量12%、氯化钙添加量0.01g /mL 。

所得最佳工艺效果较好，可作为奶油干酪的实际生产工艺。

关键词：奶油干酪；工艺参数；评价指标Optimization of Key Technological Parameters for the Production of Cream CheeseYANG Wei-dong 1，REN Fa-zheng 2，ZHANG Xiao-ying 2，ZHANG Yu-xiu 1,*(1. School of Chemical and Environmental Engineering, China University of Mining and Technology, Beijing 100083, China ；2. College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China)Abstract ：In order to optimize the production technology of cream cheese, Single factor and orthogonal array design methods were used to investigate the effects of key technological parameters such as calcium chloride amount, rennet amount, coagulation temperature and fat amount on the productivity, protein content, fat content, water content and sensory quality of cream cheese.The optimum values of rennet amount, coagulation temperature, fat amount and calcium chloride amount were found to be: 0.002g/mL, 32 ℃, 12% and 0.01 g/mL, respectively.Key words ：cream cheese ；technological parameters ；evaluation index中图分类号：TS252.53 文献标识码：B 文章编号：1002-6630(2010)14-0309-04收稿日期：2009-10-15基金项目：国家转基因生物新品种培育重大专项(2009ZX08009-130B)；“十一五”国家科技支撑计划项目(2006BAD04A06)；中国矿业大学(北京)2009年大学生创新性实验计划项目(091209z)作者简介：仰伟栋(1985—)，男，硕士研究生，研究方向为奶油干酪加工工艺。

组织细胞坏死性淋巴结炎影像学表现及临床分析

组织细胞坏死性淋巴结炎影像学表现及临床分析Ana.ysoson ihec.o.oca.and iheomagongeeaiueesoehosioocyiocneceoioaong.ymphadenoios柯岩1，冯海凤'，郑义1，赵志伟1,贾岩龙21•湖北省咸宁市中心医院湖北科技学院附属第一医院放射科湖北咸宁437000；2.汕头大学医学院第二附属医院放射科广东汕头515041$摘要】目的探讨组织细胞坏死性淋巴结炎的临床特点、影像学表现、实验室检查及病理组织学等特点，旨在提高对该病的诊断水平。

方法选取我院经病理证实组织细胞坏死性淋巴结炎12例患者的一般临床资料、影像学资料(CT、超声)、实验室检查、病理组织特征及治疗方案进行观察并复习文献(结果12例患者(女性7例，男性5例)均出现浅表淋巴结肿大,直径约0.5~3,7cm,其中9例为颈部淋巴结肿大,1例右侧颌下淋巴结肿大,1例左侧腋窝淋巴结肿大，'例腹股沟区淋巴结肿大(12例患者中9例伴发热且其中'例伴发四肢皮疹。

影像学表现：8例行CT扫描直径约1cm以上肿大淋巴结中央见斑点状、线条状坏死灶，无钙化;4例行超声检查示肿大淋巴为实性结节回声，无坏死、钙化。

实验室检查：7例WBC计数减低,5例WBC计数正常,9例血沉加快,2例EB病毒衣壳抗体(EBV-WCAWgA)阳性、EB病毒早期抗体(EBV-PADgA)阳性。

病理组织学及免疫组化:淋巴结结构破坏，皮质及副皮质区见灶性组织细胞及免疫母细胞增生,有核分裂,见片状或灶状碎屑性坏死灶，无中性白细胞;免疫组化多为LCA+,CD3部分+,CD20部分+,CD68+(治疗措施:增强免疫力、使用糖皮质激素、伴病毒感染者加抗病毒药和对症支持治疗。

结论对不明原因的浅表淋巴结肿大并发热的青年患者(尤其女性)，当影像学(CT)发现肿大淋巴结中央出现少许点状坏死灶、无钙化,再结合临床症状及相关实验室检查，可做出疑似组织细胞坏死性淋巴结炎诊断,但最终结果有赖于病理组织学及免疫组化的综合诊断。

219316064_贻贝（Mytilus_edulis）酶解液酵母发酵法脱腥工艺探究及其风味变化分析

杨心怡，赵那娜，刘荔，等. 贻贝（Mytilus edulis ）酶解液酵母发酵法脱腥工艺探究及其风味变化分析[J]. 食品工业科技，2023，44（12）：319−327. doi: 10.13386/j.issn1002-0306.2022080175YANG Xinyi, ZHAO Nana, LIU Li, et al. Optimization of Deodorization and Analysis of Flavor Change of Blue Mussel (Mytilus edulis ) Hydrolysate by Yeast Fermentation[J]. Science and Technology of Food Industry, 2023, 44(12): 319−327. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2022080175· 分析检测 ·贻贝（Mytilus edulis ）酶解液酵母发酵法脱腥工艺探究及其风味变化分析杨心怡，赵那娜，刘　荔，马昱阳，曾名湧*（中国海洋大学食品科学与工程学院，青岛海洋食品保鲜技术工程研究中心，山东青岛 266003）摘　要：为了消除贝类酶解物的腥味和苦味，本文以贻贝酶解液为研究对象，使用安琪果酒酵母进行发酵并以感官评分和蛋白损失率为指标对酵母添加量、发酵温度和发酵时间进行研究，确定发酵参数。

利用感官科学结合氨基酸自动分析技术、质谱分析技术探究了发酵前后风味轮廓的差异。

结果表明，当果酒酵母添加量为0.2%，在35 ℃下发酵2.0 h 处理后，贻贝酶解液的鱼腥味、泥土味、哈喇味显著下降，发酵味和果香显著提升（P <0.05）。

甜味氨基酸含量由1.33 mg/g 显著上升至1.99 mg/g （P <0.05），鲜味氨基酸含量由1.60 mg/g 提高到1.93 mg/g ，苦味氨基酸含量由3.64 mg/g 下降至3.34 mg/g 。

直接稀释-UPLC-QTOF-MS_法快速测定辣椒粉中20_种合成染料

引用格式：贺光云, 韩梅, 邱世婷, 等. 直接稀释-UPLC-QTOF-MS 法快速测定辣椒粉中20种合成染料[J]. 中国测试，2023,49(11): 110-118. HE Guangyun, HAN Mei, QIU Shiting, et al. Rapid determination of twenty synthetic dyes in chilli powders by a dilute-and-shoot approach coupled with UPLC-QTOF-MS[J]. China Measurement & Test, 2023, 49(11): 110-118. DOI :10.11857/j.issn.1674-5124.2022050072直接稀释-UPLC-QTOF-MS 法快速测定辣椒粉中20种合成染料贺光云1,2，韩梅1,2，邱世婷1,2，吴亚姗3，李莹1,2，覃蜀迪1,2，夏斯琪1,2，陈思宇4，侯雪1,2，付才力4(1. 四川省农业科学院农业质量标准与检测技术研究所，四川成都 610066; 2. 农业农村部农产品质量安全风险评估实验室（成都），四川成都 610066; 3. 四川省农产品质量安全中心，四川成都 610041; 4. 新加坡国立大学苏州研究院，江苏苏州 215000)摘　要: 采用直接稀释前处理法，结合超高效液相色谱-四极杆串联飞行时间质谱（UPLC-QTOF-MS ），对辣椒粉中苏丹红等20种禁用合成染料进行快速分析。

该方法以水/乙腈/丙酮（v/v/v =2/3/3）混合体系为提取溶剂，提取液未经净化，用0.1%甲酸甲醇溶液稀释5倍后进行UPLC-QTOF-MS 分析，在8 min 内实现20种染料的良好分离及检测，定量限（LOQ ）为1~40 μg/kg ，回收率为54.72%~117.77%，RSD 在0.56%~16.27%范围，满足检测要求。

红酒提取物对南极磷虾贮藏过程中抗氧化效果的影响

红酒提取物对南极磷虾贮藏过程中抗氧化效果的影响迟海;李学英;杨宪时;杨峰;涂敏建【摘要】Influences of antioxidant on Antarctic krill during storage at 2℃ and 25℃ were investigated after pretreatment by different wine extracted solutions (0. 1 , 0. 5 , 1. 0 and 2. 0 g/L) in the research by testing the indexes of PPO activity, TBARS, color changes and sensory evaluation. The results exhibited that the optimal solutions of wine extracted for antioxidant effectiveness at 2℃ and 25℃ were 0. 1 g/L and 0.5 g/L, respectively. Under the situation, PPO activity, TBARS value and effects of blackening protection of Antarctic krill were better than any other solutions and control group, which could maintain the quality and extend shelf life for Antarctic krill effectively. Changes of A450 value and RGB value have significant correlation with storage time, therefore, those could be considered as the right indexes to reflect the degree of antioxidant.%采用不同浓度的红酒提取物溶液(0.1、0.5、1.0、2.0 g/L)对南极磷虾进行预处理,并以PPO活力、TBARS值、色泽变化和感官评价为指标,对贮藏在特定温度条件下(2℃和25℃)的南极磷虾抗氧化效果进行了研究.实验结果显示,2℃和25℃条件下红酒提取物最适质量浓度分别为0.1 g/L和0.5 g/L,在此条件下南极磷虾PPO活性、TBARS值及防黑变效果优于其他浓度及空白对照组,可以有效地保持南极磷虾的品质及延长货架期;南极磷虾波长450 nm的吸光度A450值变化和RGB值与贮藏时间呈良好的线性关系,可以考虑用作反映贮藏条件下南极磷虾抗氧化程度的指标.【期刊名称】《农业机械学报》【年(卷),期】2013(044)002【总页数】7页(P153-158,187)【关键词】红酒提取物;南极磷虾;色泽;贮藏;抗氧化效果【作者】迟海;李学英;杨宪时;杨峰;涂敏建【作者单位】中国水产科学研究院东海水产研究所,上海200090;中国水产科学研究院东海水产研究所,上海200090;中国水产科学研究院东海水产研究所,上海200090;上海理工大学医疗器械与食品学院,上海200093;大连海洋大学食品科学与工程学院,大连116023【正文语种】中文【中图分类】TS254.4引言作为当今世界资源量最大的单物种生物之一，南极磷虾(Euphausia superba)是南极食物链体系中最重要的生产者，其巨大的潜在资源以及在南极的特殊地位日益受到人们的关注［1］。

黑果枸杞花色苷的提取、纯化及降解动力学研究

连敏，高艺玮，年新，等. 黑果枸杞花色苷的提取、纯化及降解动力学研究[J]. 食品工业科技，2024，45（6）：24−31. doi:10.13386/j.issn1002-0306.2023080105LIAN Min, GAO Yiwei, NIAN Xin, et al. Study on Extraction, Purification and Degradation Kinetics of Anthocyanins from Lycium ruthenicum [J]. Science and Technology of Food Industry, 2024, 45(6): 24−31. (in Chinese with English abstract). doi:10.13386/j.issn1002-0306.2023080105· 特邀主编专栏—枸杞、红枣、沙棘等食药同源健康食品研究与开发（客座主编：方海田、田金虎、龚桂萍） ·黑果枸杞花色苷的提取、纯化及降解动力学研究连　敏，高艺玮，年　新，王梦泽*（宁夏大学食品科学与工程学院，宁夏银川 750021）摘　要：以花色苷提取量为主要考察指标，通过单因素和正交试验优化冻干黑果枸杞花色苷提取工艺，并在此条件下研究花色苷纯化工艺及其降解动力学，探讨不同温度、pH 下花色苷提取量的变化。

结果表明，提取最佳工艺条件为：料液比1:25（g :mL ）、乙醇浓度60%、pH4、提取时间2 h ，此条件下花色苷提取量达36.507±0.325 mg/g 。

研究显示AB-8大孔树脂纯化黑果枸杞花色苷效果最好，对花色苷吸附量和解吸量的影响效果最佳，其最佳条件为：上样液浓度200 mg/100 g ，解吸乙醇浓度80%，上样流速2 mL/min ，洗脱流速2 mL/min ，上样体积为5 BV ，纯化率为90.02%。

变波长高效液相色谱法测定糕点中的食品添加剂

分析检测变波长高效液相色谱法测定糕点中的食品添加剂李俊运1,2，李　丽1*（1.四川轻化工大学生物工程学院，四川宜宾 644005；2.宜宾市食品药品检验检测中心，四川宜宾 644600）摘　要：本研究旨在建立一种变波长高效液相色谱法同时测定糕点中5种食品添加剂的快速检测方法。

最终确定分离条件为博纳艾杰尔Venusil XBP Perservatives色谱柱（4.6 mm×250 mm，5 μm），流动相为甲醇和20 mmol·L-1磷酸二氢铵（pH值调至5.5）进行梯度洗脱，DAD紫外检测器（190～400 nm）进行变波长监测，外标法进行定量分析。

结果表明，5种食品添加剂的回收率为95.44%～96.96%，精密度为0.10%～0.20%。

关键词：食品添加剂；糕点；变波长；高效液相色谱法Determination of Food Additives in Pastry by Variable Wavelength High Performance Liquid ChromatographyLI Junyun1,2, LI Li1*(1.College of Bioengineering, Sichuan University of Science & Engineering, Yibin 644005, China; 2.Yibin Food andDrug Inspection and Testing Center, Yibin 644600, China)Abstract: This study aims to establish a rapid detection method for five food additives in pastries using variable wavelength high-performance liquid chromatography. The final separation condition was determined to be the Bonna-Agela’s Venusil XBP Preservatives column (4.6 mm×250 mm, 5 μm), The mobile phase consists of methanol and 20 mmol·L-1 ammonium dihydrogen phosphate (pH value adjusted to 5.5) for gradient elution. The DAD ultraviolet detector (190～400 nm) is used for wavelength monitoring, and the external standard method is used for quantitative analysis. The result showed that recovery rate range of five food additives is 95.44%～96.96%, the precision range is 0.10%～0.20%.Keywords: food additives; pastries; variable wavelength; high-performance liquid chromatography糕点是日常生活中常见的食品，深受各个年龄段消费者的喜爱，其质量和安全都至关重要。

简析凝乳酶凝乳机理及羔羊凝乳酶自制方法

Dairy Industry简析凝乳酶凝乳机理及羔羊凝乳酶自制方法周希梅，赵华，黄萌萌，李竞前，闫奎友*全国畜牧总站，北京 100125摘要：本文重点介绍了凝乳酶的种类和凝乳机理，以及自制羔羊凝乳酶及其活力测定的方法，对引导我国家庭牧场和小型羊乳企业突破凝乳酶自制核心技术，创新开展羊奶酪生产研发，填补国产羊奶酪市场空白与丰富羊乳制品种类，具有重要的指导意义。

关键词：凝乳酶；羔羊皱胃；活力测定；羊奶酪中图分类号：TS252.1 文献标识码：A 文章编号：1671-4393（2022）07-0070-060 引言凝乳酶（Chymosin）对奶酪生产至关重要。

早在公元3—4世纪，人类就偶然发现动物胃中某种成分可使乳凝固，这是人们认识凝乳酶的伊始[1]。

随着现代科学技术的发展，制作奶酪用的工业化商品凝乳酶已经取得了许多突破，但在国内家庭牧场和羊乳企业小规模生产制作奶酪上，凝乳酶的传统提取方法仍是解决其来源的最为简单易行的核心关键技术。

1 凝乳酶种类及作用机理1.1 种类根据凝乳酶来源的不同，可以分为动物凝乳酶、植物凝乳酶、微生物凝乳酶、遗传工程凝乳酶四大类。

1.1.1 动物凝乳酶在传统意义上，动物凝乳酶一般是指从多胃反刍动物（犊牛、羔羊等）的第四胃（皱胃）提取出来的，可使乳凝固的酶制品——皱胃酶（Rennet），有时也称“天然动物酶”。

皱胃酶的主要成分是凝乳酶、胃蛋白酶（Pepsin）等，均归类于天冬氨酸肽酶，除此之外，还含少量多肽、核苷、含氮碱基、氨基酸、甘油和脂肪酸等。

小牛皱胃中的凝乳酶占比高达90%。

经提取后，皱胃酶中的凝乳酶占比为50%～80%。

凝乳酶能特异性水解κ-酪蛋白产生酪蛋白巨肽，近而形成凝乳，而胃蛋白酶的水解特异性并不专一，可同时水解含苯丙氨酸（Phe）、亮氨酸（Leu）、苏氨酸作者简介：周希梅（1972-），女，山东茌平人，本科，研究方向为奶业技术服务推广；赵华（1988-），女，河北唐县人，畜牧师，研究方向为奶业技术服务推广；黄萌萌（1980-），女，北京人，高级畜牧师，研究方向为奶业技术服务推广；李竞前（1982-），男，甘肃天水人，高级畜牧师，研究方向为奶业技术服务推广。

牛乳酪蛋白源生物活性肽进展

乳及乳制品是目前公认最合适人类的动物蛋白食品，是人类膳食中蛋白质的重要来源。

大量研究表明，乳及乳制品不仅给人类提供丰富的营养，而且还是许多生物活性肽的重要来源。

自1979年，Brantl等首次从牛乳酪蛋白水解产物中分离得到阿片样肽β-酪啡肽-7以来，已经分离得到多种牛乳源蛋白水解的生物活性肽，包括：阿片样肽、阿片样拮抗肽、血管紧张素转化酶抑制肽、免疫调节肽、抗菌肽、抗血栓肽、矿物元素结合肽等。

本文综述了近几年来牛乳源的生物活性肽的研究进展。

1阿片肽（opioid peptide）和阿片拮抗肽（opioid antagonists）阿片肽是一类具有阿片肽活性的小分子生物活性肽，必须通过与之相对应的受体结合才能发挥功能。

阿片颉抗剂纳络酮（naloxone）对其有抑制作用。

研究表明，阿片肽几乎参与免疫应答的全部环节，并主要以内分泌和旁分泌的方式参与免疫调节。

表1列举了近年来已发现的来源于牛乳酪蛋白的阿片肽。

现已证实，乳源阿片肽具有内源性阿片肽的全部功能，如镇痛（静）、调节情绪、减缓呼吸、心跳变慢、增强耐受性、调节体温、调节胃肠道运动、影响营养素的吸收、调节内分泌及免疫作用等功能。

Tormpette等用β-酪啡肽-7对小鼠腹腔注射，发现能够500%的刺激肠道内肠黏膜的释放，从而起到调整肠胃功能的作用。

牛乳酪蛋白源生物活性肽研究进展卢姗姗1，张少辉1，2，*，付丽娜1，2，高艳玲1，2，钱炳俊1，2（1.上海交通大学农业与生物学院，上海200240；2.上海交通大学陆伯勋食品安全中心，上海200240）摘要：乳酪蛋白源生物活性肽以其良好的消化性、低敏性及生物活性而日益受到重视。

综述国内外在牛乳酪蛋白来源的阿片肽、阿片拮抗肽、ACE抑制肽、免疫调节肽和矿物元素结合肽等方面的研究，旨在为更好地利用牛乳酪蛋白源的生物活性肽提供参考。

关键词：牛乳酪蛋白；生物活性肽；免疫调解Review of Bioactive Peptide from Bovine CaseinLU Shan-shan1，ZHANG Shao-hui1，2，*，FU Li-na1，2，GAO Yan-lin g1，2，QIAN Bing-jun1，2（1.The School of Agriculture and Biology，Shanghai Jiao Tong University，Shanghai200240，China；2.SJTU－Bor Luh Food Safety Center，Shanghai Jiao Tong University，Shanghai200240，China）Abstract：The bioactive peptide from bovine casein attract ed more and more attention from people because of its digestibility，low hypersusceptibility and bioactivity．This passage reviewed the research of bioactive peptide in home and abroad，such as opioid peptide，opioid antagonists，ACE inhibiting peptide，immunopeptide and mineral binding peptide．This passage can serve as the reference and base for exploring and utilizing bioactive peptide from bovine casein．Key wor d s：bovine casein；bioactive peptide；immunodulatory作者简介：卢姗姗（1987—），女（汉），在读硕士研究生，研究方向：乳源功能性物质。

异甘草素对氧化偶氮甲烷和右旋葡聚糖苷钠诱导小鼠炎症相关结肠癌的预防作用及其机制

异甘草素对氧化偶氮甲烷和右旋葡聚糖苷钠诱导小鼠炎症相关结肠癌的预防作用及其机制冯言晓【摘要】Objective To investigate the effect of isoliquiritigenin (ISL) on the prevention of colitis associated colon cancer.Methods Fifty male BALB/c mice were randomly divided into 5 groups, each group was 10 mice: normal control group, model group, ISL low dose group, ISL middle dose group, ISL high dose group.Mice were observed for weight and other general condition, incidence of colon cancer and colon length changes;methylene blue staining was used to observe number of aberrant crypt foci (ACF);pathological morphology was used to observe morphological changes;immunohistochemistry method was used to detect macrophage mannose receptor (CD206), cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) expressions in colon tissue.Results The model group appeared emaciation, anorexia, hair removal, stool performance during the experiment.The three doses of ISL groups were significantly better than the model cont group.At the twelfth week, the weight of mice in model group was significantly lower than that in normal control group, while the mice in high dose group and middle dose group were significantly higher than that in model group, and the length of colon in model group was significantly lower than that in normal control pared with model group, the incidence of tumor was significantly lower than that in 3 ISL dose groups, and the number of ACFin model group was significantly lower than that in 3 ISL dose groups, and the number of iNOS, COX-2 and CD206 expressions in three dose groups were decreased significantly.Conclusion ISL has chemoprevention onAOM/DSS-induced mouse colon cancer.ISL can inhibit macrophages in the tumor microenvironment convert to M2 type and reduce COX-2 and iNOS expressions in colon tissue.%目的探讨异甘草素(isoliquiritigenin, ISL)对结肠炎相关结肠癌的预防作用及其机制.方法 50只雄性BALB/c 小鼠随机分为5组,每组10只: 正常对照组;模型对照组;低、中、高剂量组.每天观察小鼠一般情况,每周测体质量1次,12周后处死小鼠,观察结肠癌发生率及结肠长度的变化,美蓝染色法观察异常隐窝灶(aberrant crypt foci, ACF)数量,病理形态学观察小鼠结肠组织形态学改变,免疫组化法检测组织中巨噬细胞甘露糖受体(CD206)、环氧合酶(COX)-2 和一氧化氮合酶(iNOS)的表达水平.结果模型对照组小鼠在实验过程中均出现消瘦、厌食、脱毛、血便等表现.三个剂量给药组一般情况均明显优于模型对照组.在实验第12周,模型对照组小鼠体质量显著低于正常对照组,而中、高剂量给药组小鼠体质量显著高于模型对照组;模型对照组小鼠结肠长度显著低于正常对照组,而中、高剂量给药组小鼠结肠长度较模型对照组显著增加.三个剂量给药组与模型对照组比,肿瘤发生率呈浓度依赖性减少;三个剂量给药组ACF数量与模型对照组比较显著降低;与模型对照组相比,三个剂量给药组随着ISL浓度的增加,肠黏膜愈来愈完整,隐窝形态逐渐恢复,炎性细胞浸润愈来愈少;三个剂量给药组CD206、COX-2 和iNOS表达水平与模型对照组相比,呈剂量依赖性降低.结论 ISL对AOM/DSS 诱导的小鼠结肠炎相关结肠癌发生具有明显预防作用,其作用机制可能与其能抑制巨噬细胞向M2型转化及下调COX-2 和 iNOS表达有关.【期刊名称】《胃肠病学和肝病学杂志》【年(卷),期】2017(026)008【总页数】6页(P873-878)【关键词】异甘草素;结肠炎相关结肠癌;肿瘤相关巨噬细胞;CD206【作者】冯言晓【作者单位】浙江省台州医院急诊科,浙江台州 317000【正文语种】中文【中图分类】R574.62;R735.3+5近几年，我国结肠癌的发病率越来越高，原因是人们的生活水平不断升高，生活习惯发生变化，饮食结构也发生变化。

胰岛素对奶牛乳腺上皮细胞酪蛋白合成调节机理的研究

胰岛素对奶牛乳腺上皮细胞酪蛋白合成调节机理的研究田青;季昀;庞学燕;王洪荣【期刊名称】《动物营养学报》【年(卷),期】2013(025)003【摘要】This study was conducted to investigate the effects of different concentrations of insulin (INS) in medium on expression levels of αsl-casein ( CSN1S1) gene, mammalian target of rapamycin (mTOR) and Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway related genes. Hol-stein bovine mammary epithelial cells were treated with different concentrations (0, 2. 5, 25. 0, 250. 0 and 5 000.0 ng/mL) of INS, respectively. The expression levels of CSN1S1 gene, mTOR and JAK-STAT signaling pathway related genes were determined by real-time quantitative PCR. The results showed as follows: compared with 0 ng/mL group, INS could promote the expression levels of CSMS1, short-prolactin receptors (S-PRLR), protein kinase B (PKB) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) genes, and increase the expression levels of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5A (STAT5A) genes. Especially, the expression levels of these genes in 25 ng/mL group were the highest. The results indicate that INS can improve the expression level of CSN1S1 gene. The mechanism is as follows: firstly, INS can promote hormone receptor expression, and then promote genetic transcription; secondly , INS canpromote the positive regulation expression of mTOR and JAK-STAT signaling pathways related genes, and highly improve the expression of CSN1S1 gene, which provides a material foundation for increasing milk protein synthesis at the translation level.%本文旨在通过在培养液中添加不同浓度胰岛素(INS),研究其对奶牛乳腺上皮细胞中αs1-酪蛋白(CSNl Sl)基因、雷帕霉素靶蛋白(mTOR)信号通路及Janus激酶-信号传导和转录活化因子(JAK-STAT)信号通路相关基因表达的影响.试验选用中国荷斯坦奶牛的乳腺上皮细胞进行体外培养,在无血清无激素的培养基中分别添加0(对照)、2.5、25.0、250.0、5 000.0 ng/mL INS,利用实时荧光定量PCR方法检测CSNlSl基因、mTOR和JAK-STAT 信号通路中相关基因表达量.结果表明:与0 ng/mL组相比,添加不同浓度的INS后均能提高CSNlSl、短型催乳素受体(S-PRLR)及mTOR信号通路中上游蛋白激酶B(PKB)、mTOR和下游真核翻译起始因子4E结合蛋白1(4E-BP1)基因表达量,并且能够增加JAK-STAT信号通路中Janus激酶2(JAK2)和信号转导和转录激活因子5A(STAT5A)基因表达量,当INS浓度为25.0 ng/mL时各正向调节基因表达量最高.此结果提示,添加外源INS能提高奶牛乳腺上皮细胞CSNlSl基因表达量,其作用机理:一是添加INS后能够促进激素受体基因的表达,进而促进转录的启动；二是促进乳蛋白合成的mTOR和JAK-STAT信号通路中各正向调节基因的表达,进而使CSNlSl等乳蛋白合成基因能高水平表达,为进一步通过翻译水平增加乳蛋白的合成奠定了物质基础.【总页数】11页(P550-560)【作者】田青;季昀;庞学燕;王洪荣【作者单位】扬州大学动物科学与技术学院,扬州225009【正文语种】中文【中图分类】S823【相关文献】1.赖氨酸蛋氨酸配比模式对奶牛乳腺上皮细胞酪蛋白合成的影响 [J], 赵圣国;李喜艳;王加启2.不同氨基酸模式对奶牛乳腺上皮细胞酪蛋白合成影响的研究 [J], 张兴夫;高民;杜瑞平;敖长金;张航3.亮氨酸或组氨酸通过哺乳动物雷帕霉素靶蛋白信号通路影响奶牛乳腺上皮细胞中酪蛋白的合成 [J], 高海娜;胡菡;王加启;郑楠4.胰岛素对奶牛乳腺上皮细胞生长及k-酪蛋白和胰岛素受体基因表达的影响 [J], 田青;季昀;庞学燕;王洪荣5.葡萄糖对奶牛乳腺上皮细胞增殖、葡萄糖摄取和酪蛋白合成相关基因表达的影响[J], 李子南;李大彪;邢媛媛;金亚亚;母晓佳;曹越因版权原因，仅展示原文概要，查看原文内容请购买。

雅致放射毛霉添加量对牦牛霉菌奶酪品质的影响

雅致放射毛霉添加量对牦牛霉菌奶酪品质的影响李昂;陈炼红;李键;张岩【摘要】The influence of the addition of mucor on quality of yak mold cheese was researched to determine its optimal addition and fermentation time by the single factor experiment.In the case of the samecondition,Actinomucor elegans was added at the mass ratios of 0.2％,0.3％and 0.4％.The physicochem ical indexes such as sensory evaluation score,texture index,nutrient composition,pH value,decomposition index of protein and lipid were mensurated.The results indicated that the quality and sensory evaluation score of which added 0.3％ mucor were best.With the addition of starter rising,the content of moisture,protein,lipid and pH value showed a downward trend while ash was on the rise.Mature to the sixth day,the sensory index of yak mold cheese was the best.In the process of maturation,the content of soluble nitrogen at pH4.6 or 12％trichloroacetic acid soluble nitrogen,thiobarbituric acid value,acid value increased gradually.So the optimal addition amount of starter was determined to be 0.3％.The quality of yak mold cheese was best when fermented for 6 days.%研究毛霉添加量对牦牛霉菌奶酪品质的影响,确定最佳添加量及发酵时间.采用单因素实验设计,固定其他工艺参数,研究雅致放射毛霉的不同添加量(质量比:0.2％、0.3％、0.4％),测定成熟过程中奶酪的性能指标(感官评分、质构指标、营养成分、pH、蛋白质与脂肪分解指标).结果表明:毛霉添加量为0.3％,奶酪品质及感官指标最佳;随着毛霉添加量增加,水分、蛋白质、脂肪含量、pH均呈下降趋势,灰分呈上升趋势.成熟至第6d时,牦牛霉菌奶酪感官指标最佳;随成熟时间延长,pH4.6醋酸盐缓冲液可溶性氮含量、12％三氯乙酸可溶性氮含量、硫代巴比妥酸值、酸价不断升高.因此,发酵剂添加量0.3％,发酵6d时,牦牛霉菌奶酪品质最佳.【期刊名称】《食品工业科技》【年(卷),期】2017(038)024【总页数】6页(P120-125)【关键词】雅致放射毛霉;牦牛霉菌奶酪;发酵剂;品质【作者】李昂;陈炼红;李键;张岩【作者单位】西南民族大学生命科学与技术学院,四川成都610041;西南民族大学生命科学与技术学院,四川成都610041;西南民族大学生命科学与技术学院,四川成都610041;西南民族大学生命科学与技术学院,四川成都610041【正文语种】中文【中图分类】TS252.42奶酪是在乳中加入适量发酵剂、凝乳酶,使乳中酪蛋白凝固,经发酵成熟制成的一种营养价值极高的发酵乳制品[1]。

近红外光谱技术在奶酪品质评价中的应用_邹强(1)

第31卷,第10期光谱学与光谱分析Vol 131,No 110,pp2725-27292011年10月 Spectro sco py and Spectr al AnalysisO cto ber ,2011近红外光谱技术在奶酪品质评价中的应用邹强1,方慧1*,张维2,何勇111浙江大学生物系统工程与食品科学学院,浙江杭州 31005721浙江大学农业与生物技术学院,浙江杭州 310057摘要近红外光谱技术是一种快速、无损的分析方法,国外将该技术应用于奶酪品质的检测已有多年,国内在这方面的研究较少。

通过本文介绍了近红外光谱技术分析奶酪成分和在奶酪的加工生产、缩水收缩控制、成熟过程、货架期、组成成分和品牌分类鉴别等几个方面的应用,表明近红外光谱技术在奶酪品质分析中应用潜力巨大,促进近红外光谱技术的应用和我国奶酪行业的发展是一项紧迫的任务。

关键词奶酪;近红外光谱技术;应用中图分类号:T S25215 文献标识码:A DOI :1013964/j 1issn 11000-0593(2011)10-2725-05收稿日期:2011-01-10,修订日期:2011-04-23基金项目:浙江省科技厅项目(2009C12002),国家自然科学基金项目(60802038)和国家(863计划)项目(2006AA10Z234)资助作者简介:邹强,1987年生,浙江大学生物系统工程与食品科学学院硕士研究生 e -mail:513578155@1631com*通讯联系人 e -mail:h fang@zju 1edu 1cn引言奶酪是牛奶经浓缩,发酵而成的奶制品,它基本上排除了牛奶中大量的水分,保留了其中营养价值极高的精华部分,被誉为乳品中的/黄金0。

每公斤奶酪制品浓缩10公斤牛奶的蛋白质、钙和磷等人体所需的营养素,独特的发酵工艺,使其营养的吸收率达到了96%~98%。

虽然我国对奶酪的消耗较少,但随着人民生活水平的提高,对奶酪的消耗量也将逐年增加,同时对奶酪品质及时、快速、准确的分析也将对奶酪的生产起到极大的促进作用。

凝乳酶的研究进展_杭锋

DOI:10.7506/spkx1002-6630-201603047 中图分类号：TS252.1 引文格式：
文献标志码：A
文章编号：1002-6630（2016）03-0273-07
杭锋, 洪青, 王钦博, 等. 凝乳酶的研究进展[J]. 食品科学, 2016, 37(3): 273-279. DOI:10.7506/spkx1002-6630-201603047.
Abstract: Calf rennet is conventionally used as milk coagulant for the production of cheese. However, the supply of
calf rennet is not equivalent to the demand in cheese industry, which merely meets 20%–30% of the global demand for
块（图2）。Hsieh等[6]利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gels electrophoresis，SDS-PAGE）、二维凝胶电泳和质谱研究手段的蛋白质组学方法进一步阐明了凝乳酶的凝乳机理。通常κ-CN的水解度要达到80%～90%时才能发生凝乳，在凝乳第二步非酶反应过程，pH值的降低、温度的升高以及Ca2＋浓度的增加均可加速干酪凝乳过程[7]。
cheese production. Due to the scarcity and high price of calf rennet, it is necessary and urgent to ﬁnd potential substitutes.

表观遗传修饰调控阿尔茨海默病的研究进展_林苏扬

生物化学与生物物理进展 Progress in Biochemistry and Biophysics ISSN 1000-3282,CN 11-2161/Q
《生物化学与生物物理进展》网络首发论文
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收稿日期：网络首发日期：引用格式：
表观遗传修饰调控阿尔茨海默病的研究进展林苏扬，潘召韬，马宇涛，高君妍，单江晖，储超扬，谢凯，沈巍，王清娟，李丽萍 2021-08-25 2021-10-25 林苏扬，潘召韬，马宇涛，高君妍，单江晖，储超扬，谢凯，沈巍，王清娟，李丽萍．表观遗传修饰调控阿尔茨海默病的研究进展[J/OL]．生物化学与生物物理进展. https:///kcms/detail/11.2161.q.20211021.1428.004.html
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表观遗传修饰是指在不改变 DNA 序列的情况下，受环境因素影响导致基因表达发生可遗传改变. 表观遗传修饰主要包括 DNA 甲基化、组蛋白甲基化、组蛋白乙酰化、RNA 修饰和非编码 RNA 等，这些表观遗传修饰相互作用，可以调节基因表达，进而影响突触的可塑性、记忆的获得和巩固、神经通路的连接以及神经信号的传递等. 大量研究表明，表观遗传学在 AD 发生发展过程中发挥重要的作用[3]. 本文综述了近几

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Analytical MethodsAnalysis of lysozyme in cheese samples by on-line combination of capillary zone electrophoresis and massspectrometryMonika Kondekováa ,Víteˇzslav Maier b ,Pavlína Ginterováb ,Jozef Marák a ,⇑,Juraj Ševc ˇík c aDepartment of Analytical Chemistry,Faculty of Natural Sciences,Comenius University in Bratislava,MlynskáDolina CH-2,SK-84215Bratislava,Slovak Republicb Regional Centre of Advanced Technologies and Materials,Department of Analytical Chemistry,Faculty of Natural Science,Palacky´University Olomouc,17.listopadu 12,77146Olomouc,Czech Republic cDepartment of Analytical Chemistry,Faculty of Natural Science,Palacky´University Olomouc,17.listopadu 12,77146Olomouc,Czech Republic a r t i c l e i n f o Article history:Received 15April 2013Received in revised form 18December 2013Accepted 21December 2013Available online 3January 2014Keywords:Capillary zone electrophoresis Cheese LysozymeMass spectrometryMulti-component matricesa b s t r a c tSome methodological aspects of an on-line combination of capillary zone electrophoresis with massspectrometric detection (CZE–QqQ–MS)were studied in this work as well as the possibilities of using this combination for analysis of the high-molecular mass compounds present in multi-component matrices.All experiments using an on-line combination of capillary electrophoresis with mass spectrometric detec-tion were carried out in cationic mode in covalently-coated capillary.The optimised electrolyte system consisted of 100mmol/L formic acid.Prior to the CZE–QqQ–MS analysis,an extraction of lysozyme from cheese samples using 1mol/L of acetic acid was performed.The LOD was 3.6mg lysozyme per kg and the LOQ was 10.9mg lysozyme per kg.The concentration range of the lysozyme determined in four cheese samples analysed in this work was from 0.5to 3.3g of lysozyme per kg.The values of the relative standard deviations thus obtained were from 4.6%to 9.3%depending on the cheese sample.Ó2013Elsevier Ltd.All rights reserved.1.IntroductionHen egg-white lysozyme,also known as muramidase,is a hydrolase enzyme with an extensive bacteriostatic activity.The molecular mass of egg-white lysozyme,calculated from the sequence of 129amino acid residues,is 14,307Da.These residues are cross-linked by four disulphide bridges (Mine,Ma,&Lauriau,2004).The mechanism of the bacteriostatic activity of lysozyme is in the combination of the enzymatic hydrolysis of N -glycosidic linkages in the microbial cells and non-enzymatic damage of the cytoplasmic membrane by direct binding of the protein (Yeh,Dodds,Zuo,&Johnson,1997).This hydrolase enzyme exhibits lytic activity on the cell-wall of Gram-positive bacteria.Hence,lyso-zyme is used as a preservative agent in the cheesemaking process to prevent the late gas-blowing defect caused by Clostridium tyro-butyricum (Kvasnicˇka,2003;Schneider,Becker,&Pischetsrieder,2010;Schneider,Werkmeister,Becker,Pischetsrieder,2011).Lyso-zyme is also added as the preservative agent in the manufacturing process of soya milk,sushi and Chinese noodles.Fresh vegetables,ﬁsh,meat and seafood have been preserved by coating the surface of the food with lysozyme (Mine et al.,2004).In winemaking,the lysozyme is used to control spontaneous lactic acid-producing bac-teria (LAB),many of which cause wine spoilage (Guzzo,Cappello,Azzolini,Tosi,&Zapparoli,2011;Tirelli &De Noni,2007;Tolin et al.,2012).However,lysozyme can cause allergic reactions in susceptible individuals.For this reason,it is desirable to monitor (detect)its amount in food (NDA,2005).Lysozyme present in food can be determined by high-perfor-mance liquid chromatography (Guarino,Fuselli,La Mantia,&Longo,2011;Pellegrino &Tirelli,2000;Schneider et al.,2011;Tirelli &De Noni,2007).Schneider (Schneider et al.,2011)detailed the use of high-performance liquid chromatography combined with ﬂuorescence detection (HPLC–FLD)for the analysis of lyso-zyme in cheese samples,using a reversed-phase polymeric col-umn.An extraction of lysozyme from cheese samples using 1mol/L solution of sodium chloride was performed prior to the di-rect HPLC–FLD analysis.The limit of detection was calculated as 7.3mg of lysozyme per kg of cheese.Lysozyme can also be deter-mined by liquid chromatography coupled with tandem mass spec-trometry (LC–MS/MS)(Thammasirirak et al.,2010;Tolin et al.,2012),or mass spectrometry alone (Alomiraha,Allia,&Konishib,2000;Léonil,Gagnaire,Molleé,Pezennec,&Bouhallab,2000;Schneider et al.,2010).Schneider et al.(2010)described a method combining immuno-capture puriﬁcation and direct mass-spectro-metric analysis (MALDI–TOF–MS)for the detection of lysozyme in cheese samples.Cheese extracts were treated with magnetic particles coated with a monoclonal antibody directed against lyso-zyme.Lysozyme was ﬁnally detected by MALDI–TOF–MS.The limit of detection of lysozyme in cheese was approximately 5mg/kg.0308-8146/$-see front matter Ó2013Elsevier Ltd.All rights reserved./10.1016/j.foodchem.2013.12.078Corresponding author.Tel.:+421260296400;fax:+421260296706.E-mail address:marak@fns.uniba.sk (J.Marák).Another approach to the determination of the lysozyme was de-scribed by Kvasnicˇka(2003).He detailed the use of an on-line cou-pling of capillary isotachophoresis with capillary zone electrophoresis(CITP–CZE)for the determination of lysozyme in selected food products,using ammonium hydroxide with acetic acid as the leading electrolyte and e-aminocaproic acid with acetic acid as the terminating electrolyte.The limit of detection was 0.25mg/L.An extraction of lysozyme from selected food samples using1mol/L acetic acid was performed prior to the direct CITP–CZE separation.Capillary zone electrophoresis(CZE)represents a high-resolu-tion separation technique which requires very small volumes of the analysed samples(nL–l L)and a short separation time (10s–30min)and can be combined with mass spectrometry (MS)(Ahmed,2009).MS in comparison with other commonly used detection techniques in CZE provides information on the structure of the analyte,especially when MS/MS and MS n instru-mentation is used,and also gives information on the molecular mass of the studied analyte(Nielen et al.,2006).A suitable com-bination of CZE and MS affords the beneﬁts of each of the tech-niques,i.e.,high-efﬁciency separations of the analytes from the sample constituents and,at the same time,obtaining information on the molecular masses and/or structures of the analytes during the analysis(Cai&Henion,1995;Desiderio,Rossetti,Iavarone, Messana,&Castagnola,2010).The combination of CZE–MS has several advantages over the HPLC–MS combination,i.e.,(1)a much smaller sample is needed(nL versus l L);(2)higher separa-tion efﬁciencies can be obtained;(3)CZE works in a single-phase separation system unlike HPLC where at least two phases are used and,accordingly,the optimal separation conditions can be more readily and speedily found;(4)CZE separates the charged compounds and the neutral compounds present in the sample do not interfere in the detection whereas HPLC separates practi-cally all the compounds present in the sample and there is a high potential of interferences from the matrix;(5)the sample pre-treatment procedures used in CZE are much simpler than the complicated protocols used prior to HPLC;(6)minimum matrix effects have been described in the literature to date while the negative impact of the matrix effects is well-known in the HPLC–MS combination.A detailed summary of the use of CZE–MS in food analysis and its advantages can be found in the works of Ravelo-Pérez,Asensio-Ramos,Hernández-Borges,and Rodrí-guez-Delgado(2009)and Klepárnik(2013).This work sought the development of an analytical procedure suitable for the analysis of lysozyme in various cheese samples using the on-line combination of capillary zone electrophoresis with triple-stage mass-spectrometric detection(CZE–QqQ–MS) and a fast and simple method for the determination of lysozyme as an allergen.2.Materials and methods2.1.InstrumentsThe experiments were performed using an Agilent7100Capil-lary Electrophoresis System(Agilent Technologies,Waldbronn, Germany).Fused-silica capillaries were obtained from MicroSolv Technologies(Eatontown,NJ,USA).All CZE–QqQ–MS experiments were performed in a polyacrylamide-coated fused silicacapillary Fig.1.TIC record(a)and MS spectrum(b)obtained from CZE–QqQ–MS analysis of lysozyme standard.using the coating procedure published previously(Maier,Petr, Knob,Horáková,&Ševcˇík,2007).MS experiments were performed using an Agilent MSD mass spectrometer6460Series(Agilent Technologies)equipped witha Fig.2.SIM record and EIC chromatograms corresponding to m/z values of lysozyme obtained from CZE–QqQ–MS analysis of lysozyme standard.triple-stage quadrupole analyser.An Agilent coaxial sheath-liquid sprayer was used for CZE–MS coupling (Agilent Technologies).The sheath-liquid was introduced into the sprayer by a 1260Inﬁn-ity Isocratic Pump (Agilent Technologies).A constant voltage of +20kV was applied to the fused-silica capillary during the CZE run.The sample was injected using a 100mbar pressure for 5s.The CZE capillary was washed with a solution of background elec-trolyte (100mmol/L formic acid)for 180s prior to each CZE–QqQ–MS analysis.A constant voltage of +4kV was applied to the sprayer capillary in the mass spectrometry step,the temperature of the drying gas was 200°C,the gas ﬂow-rate was 10L/min,the nebul-iser pressure was 10psi and the ﬂow-rate of the sheath-liquid was 4l L/min.The sheath-liquid contained 50:49.5:0.5(v/v/v)of methanol,water and formic acid,respectively.2.2.ChemicalsThe lysozyme standard (hen egg-white)was obtained from Sig-ma–Aldrich (St.Louis,MO,USA).The solutions of electrolytes used for the CZE–QqQ–MS analyses were prepared from chemicals ob-tained from Sigma–Aldrich [formic acid,acetic acid,methanol (LC/MS quality)];water (LC/MS quality)was obtained from Merck (Darmstadt,Germany).Deionised water Simplicity (Millipore,Molsheim,France)was used for preparing the extracts.The elec-trolyte solutions were ﬁltered immediately after preparation through a syringe ﬁlter with a pore size of 0.22l m (Millipore).2.3.Cheese samples and their treatment prior to CE–MS analysis Cheese samples,i.e.,‘‘Edam’’(Produced for Kauﬂand,Germany),‘‘Parmesan’’(Gran Moravia,Czech Republic),‘‘Gouda’’(Produced for Kauﬂand,Germany),‘‘Mixture of natural cheeses’’(Zanetti,Italy),representing a multi-component matrix,were purchased from a local supermarket.To minimise the risk of interferents from the complex cheese matrix,extraction of lysozyme was performed.We applied a slightly modiﬁed procedure to that originally described by Kvas-nicˇka for the extraction of lysozyme from cheese samples (Kvas-nicˇka,2003).Two grams of each cheese sample were weighed into a 100mL volumetric ﬂask and 70mL of acetic acid (1mol/L in water)was added.The ﬂask was placed in a thermal bath at 40°C.The sample was homogenised in a T18basic Ultra Turrax homogeniser (IKA,Germany)at 25,000RPM for one hour.After that,deionised water was added to the ﬂask up to the mark and the mixture was mixed well.The extraction procedure was repeated 3times for each type of cheese sample to minimise differ-ences in the same samples.The extracted cheese samples wereﬁltered prior to the CZE–QqQ–MS analyses through syringe ﬁlters with a pore size of 0.45l m (Millipore).The syringe ﬁlters were dis-posed of after use.The recovery experiments performed in this work demonstrated that no loss of lysozyme occurred during the sample pre-treatment,hence there was no reason to handle and analyse the casein precipitates.An aqueous standard lysozyme solution of 1mg/mL was used for both calibration curve construction and sample-spiking.Two grams of lysozyme-free cheese were weighed into a 100mL volu-metric ﬂask.The lysozyme standard solution was added and di-luted with deionised water to achieve the concentration required for the calibration solutions as well as the recovery experiments.The cheese samples were extracted as previously described.The statistical evaluation of the data thus obtained was performed by statistical software QC.Expert 2.5(TriloByte Statistical Software,Pardubice,Czech Republic).All measurements were performed ﬁve times,unless stated otherwise.3.Results and discussion3.1.Optimisation of experimental parametersA typical problem in the capillary electrophoresis analysis of proteins is that the separation is often corrupted by strong adsorp-tion of the proteins onto the fused-silica capillary wall due to the electrostatic and hydrophobic interactions.To prevent this,back-ground electrolytes with either low or high pH values can be used (Haselberg,De Jong,&Somsen,2007).The isoelectric point of lyso-zyme is approximately 10.3which makes it different from other proteins and leads to its having a positive charge at pH of around 7(Wang et al.,2010).All CZE–QqQ–MS experiments were performed in a coated fused silica capillary (50l m i.d.)80cm in length to prevent electrostatic and hydrophobic interactions of the proteins with the capillary walls.A constant positive voltage of 20kV was applied to the fused silica capillary duringtheFig.3.SIM record obtained from CZE–QqQ–MS analysis of real cheese sample (Parmesan).Table 1Mean concentration levels of lysozyme found in cheese samples together with their standard deviations and relative standard deviations (RSD)(n =3).SampleLysozyme (g/kg)Standard deviation (g/kg)RSD (%)Parmesan 1.30.06 4.6Gouda 2.80.269.3Edam3.30.17 5.1Mixture of natural cheeses0.50.036.0TIC record(a),MS spectrum(b)and SIM record(c)obtained from CZE–QqQ–MS analysis of real cheese sample(Parmesan)with the addition of lysozymecoefﬁcient of determination of r2=0.9994conﬁrmed the linearity of the calibration line.In addition,the Mandel test conﬁrmed its linearity within the whole concentration range(p=0.01).The repeatability,accuracy,recovery,limit of detection(LOD) and limit of quantiﬁcation(LOQ)were determined in the spiked cheese samples(lysozyme-free cheese)for validation of the meth-od.For determination of the accuracy,the recovery rates were evaluated so that two grams of lysozyme-free cheese was spiked with the lysozyme stock solution to achieve the three concentra-tion levels,i.e.,90,450and900l g/L.After performing the extrac-tion procedure described in part2.3,the concentration of lysozyme was evaluated.The recoveries were94.7%,96.2%and102.5%for the concentration levels referred to above.The repeatability of the CZE–QqQ–MS method thus developed was determined by calculat-ing the relative standard deviation(RSD)in nine independent experiments.The RSD values ranged between3.4%and6.1%,deter-mined at three concentration levels.LOD and LOQ were calculated according to the literature(ICH Harmonized Tripartite Guideline: Validation of Analytical Procedures,Q2(R1),Step4version, November2005),as the ratio of standard deviation of intercept and the slope of the calibration line multiplied by factors of3.3 (LOD)and10(LOQ),respectively.The LOD was calculated as 3.6mg of lysozyme per1kg of cheese and the LOQ as10.9mg of lysozyme per1kg of cheese.The validation parameters obtained in this work indicate that the CZE–QqQ–MS method thus devel-oped is suitable for the quantiﬁcation of lysozyme in commercially available cheese samples.3.3.Analysis of lysozyme in cheese samples by CZE–QqQ–MS analysisThe CZE–QqQ–MS analysis of lysozyme standard(hen egg-white)was performed to determine the migration position of lyso-zyme and the TIC(Total Ion Current)record obtained and the MS spectrum of the lysozyme peak are shown in Fig.1.The m/z values obtained from MS spectra recorded during the CZE–QqQ–MS anal-ysis of the lysozyme standard were selected for its CZE–QqQ–MS analysis in SIM(Selective Ion Monitoring)mode(m/z 1432=[M+10H+]10+,m/z1591=[M+9H+]9+,m/z1789=[M+8 H+]8+).The precision of the m/z values measured was±0.5.After performing the CZE–QqQ–MS analysis in SIM mode(see Fig.2),it may be concluded that the peak shown in the TIC record in Fig.1 corresponded to the lysozyme standard.To minimise the inﬂuence of the interferents present in a multi-component matrix,an extrac-tion procedure was performed prior to the CZE–QqQ–MS analysis of lysozyme in cheese samples(the sample preparation procedure is described in part2.3).Fig.3shows the analysis of the acidic ex-tract of a selected cheese sample(parmesan).The SIM record(see Fig.3)was obtained by monitoring the selected m/z values associ-ated with the lysozyme standard.The SIM record shows the fully separated lysozyme from the sample matrix components which can be easily quantiﬁed.A standard addition method was used to determine the concentration of lysozyme in cheese samples and the results are summarised in Table1.The concentration range of the lysozyme determined in the cheese samples analysed in the present work was0.5–3.3g/kg and the relative standard devi-ations were from4.6%to9.3%depending on the cheese sample(see Table1).The lowest concentration of lysozyme was found in the cheese sample labelled‘‘Mixture of natural cheeses’’and the high-est concentration of lysozyme was found in the cheese sample la-belled‘‘Edam’’.The lysozyme concentration found in a Gouda cheese sample by Kerkaert et ing HPLC withﬂuorescence detection(Kerkaert,Mestdagh,&De Meulenaer,2010)was approx-imately0.2mg/kg.The difference in lysozyme concentration levels could be due to the fact that the Gouda cheese analysed in the present work was produced in Germany for Kauﬂand supermar-kets.On the other hand,the sample pre-treatment procedure used in their work was time-consuming(24h)while the procedure used in the present work lasted1h.A similar analytical technique,i.e., HPLC with UV-andﬂuorescence detection,was used for the deter-mination of lysozyme in cheese samples in studies by Guarino et al. (2011),Pellegrino and Tirelli(2000)and Schneider et al.(2011). The sample pre-treatment procedures performed by these authors used sodium chloride at a high concentration(1mol/L)which is not compatible either with capillary electrophoresis separation or with mass spectrometric detection.Moreover,the cheese samples analysed by both groups were different from the samples analysed in the present work,as the main focus was to analyse cheese sam-ples available on the local market.Schneider et al.(2010),also used sodium chloride at a high concentration for lysozyme extraction from the cheese samples.The high concentration of sodium in the extract was reduced prior to MALDI–TOF MS analysis by the C18ZipTips puriﬁcation procedure.It has been estimated that more than100tonnes of lysozyme are used annually in some foods and for therapeutic and pharma-cological applications(Mine et al.,2004).Hence,the quantity of the lysozyme in some food samples,including cheese samples,de-pends on the country where those samples are produced and also on the manufacturing procedure employed by each producer.The results of the CZE–QqQ–MS analysis of the selected cheese sample (‘‘Parmesan’’)with the addition of lysozyme standard are shown in Fig.4,where the relevant TIC record,MS spectrum and SIM records are displayed.4.ConclusionsIn this study,an on-line combination of capillary electrophore-sis with mass spectrometric detection was employed for the anal-ysis of lysozyme in four cheese samples.Optimisation of the separation conditions in the CZE stage,compatible with MS detec-tion,was performed to obtain the best signal at the MS stage.An extraction of lysozyme from cheese samples was used to eliminate potential disturbing interferents present in the cheese samples. Lysozyme at different concentration levels was found in four dif-ferent cheese samples i.e.,‘‘Edam’’,‘‘Parmesan’’,‘‘Gouda’’and‘‘Mix-ture of natural cheeses’’.The results presented in this work show that the CZE–QqQ–MS method thus developed is appropriate for the determination of lysozyme in cheese samples.AcknowledgementsTheﬁnancial support received from the following projects: Operational Programme Research and Development for Innova-tions–European Regional Development Fund(project no. CZ.1.05./2.1.00/03.0058of the Ministry of Education,Youth and Sports of the Czech Republic),Czech Science Foundation(P206/ 12/1150),the Slovak Research and Development Agency(project No.APVV-0583-11)and Slovak Grant Agency No.VEGA1/1305/ 12is gratefully acknowledged.ReferencesAhmed,F.E.(2009).The role of capillary electrophoresis–mass spectrometry to proteome analysis and biomarker discovery.Journal of Chromatography B,877, 1963–1981.Alomiraha,H.F.,Allia,I.,&Konishib,Y.(2000).Applications of mass spectrometry to food proteins and peptides.Journal of Chromatography A,893,1–21.Cai,J.,&Henion,J.(1995).Capillary electrophoresis–mass spectrometry.Journal of Chromatography A,703,667–692.Desiderio,C.,Rossetti,D.V.,Iavarone,F.,Messana,I.,&Castagnola,M.(2010).Capillary electrophoresis–mass spectrometry:Recent trends in clinical proteomics.Journal of Pharmaceutical and Biomedical Analysis,53,1161–1169. 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