International Journal of Biological Macromolecules 50 (2012) 844–848Contents lists available at SciVerse ScienceDirectInternational Journal of BiologicalMacromoleculesj 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 /i j b i o m acAn immunostimulatory polysaccharide (SCP-IIa)from the fruit of Schisandra chinensis (Turcz.)BaillYong Chen a ,Jinbao Tang b ,∗,1,Xiaoke Wang a ,Fengxiang Sun a ,Shujuan Liang a ,∗,1a Department of Basic Medicine,Weifang Medical University,Weifang 261042,ChinabDepartment of Pharmacology and Biological Science,Weifang Medical University,Weifang 261042,Chinaa r t i c l ei n f oArticle history:Received 27October 2011Received in revised form 12November 2011Accepted 15November 2011Available online 23 November 2011Keywords:PolysaccharideSchisandra chinensisImmunomodulatory effect Cyclophosphamidea b s t r a c tA water-soluble polysaccharide named SCP-IIa was isolated from the water extract of the fruit of Schisandra chinensis (Turcz.)Baill by means of ethanol precipitation,deproteination,anion-exchange and gel-permeation chromatography.The molecular weight of SCP-IIa was ascertained via HPLC,and immuno-modulating effect was evaluated using the immunosuppressed model induced by cyclophos-phamide.SCP-IIa was a homogeneous form of polysaccharide,with an average molecular weight of approximately 7700Da.The detected parameters showed that SCP-IIa increased the thymus and spleen indices,as well as the pinocytic activity of the peritoneal macrophages in immunosuppressed mice.The splenocyte proliferation assay showed that SCP-IIa,in combination with Con A or LPS,positively affected splenocyte proliferation.Moreover,the polysaccharide promoted hemolysin formation.The results sug-gested that SCP-IIa was involved in immunomodulatory effects leading to the exploration for SCP-IIa as a potential immunostimulant.© 2011 Elsevier B.V. All rights reserved.1.IntroductionSchisandra chinensis (Turcz.)Baill is produced mainly in north-east China.Its fruit (referred to hereafter as S.chinensis )has been extensively used as traditional medicine and functional food in the Orient for thousands of years.S.chinensis has many traditional usages,such as collecting and arresting discharge,supplementing Qi,promoting the production of body fluids,nourishing the kidney,and calming the heart.It is a famous and precious Chinese tradi-tional drug,with frequency of 22times in the China pharmacopoeia [1].The interest of researchers in medicinal plants as natural sources of active compounds had remarkably increased during the past decades,and particular attention had been given to the polysac-charide components of various traditional Asian medicines [2–4].Recently polysaccharides were found to have many pharmacolog-ical actions,including liver protection,resistance to oxidation and aging,and anticancer properties [5–7].In recent years,the use of immunomodulators to enhance host defense responses had been found to be one of the most promising alternatives to classical antibiotic treatment [8].Polysac-charides isolated from various traditional medicinal plants had∗Corresponding authors.Tel.:+865368462266;fax:+865368462266.E-mail addresses:tangjb@ (J.Tang),liangshjuan@ (S.Liang).1Contributed equally to this work.been shown to profoundly affect the immune system both in vivo and in vitro through their ability to modulate immune function,including cytokine/chemokine production,reactive oxygen species (ROS)production,and cell proliferation [9].It has potential as an immunomodulator because it has no significant side effects,which is a major problem associated with immunomodulatory bacterial polysaccharides and synthetic compounds [8,10,11].However,there are few published data on the purification of S.chinensis polysaccharide (SCP)and its effects on immune responses.In the present study,we isolated and purified SCP-IIa from S.chi-nensis and investigated the immune status of SCP-IIa -treated mice.Since a temporarily weakened immune system is the indicator for therapy with herbal immunomodulators [12],we investigated the effects of SCP on macrophage function.Levels of serum cytokine and splenocyte proliferation in immunosuppressed mice treated with cyclophosphamide (Cy)were determined to identify whether SCP-IIa exerts its action through systemic effects and to investigate the extent of SCP-IIa’s ability to restore deviated immune parameters in immunosuppressed animals.2.Materials and methods2.1.MaterialsS.chinensis was purchased from Weifang Ben-Cao-Ge Chinese Matreria Medica Co.Ltd.and verified by Prof.Chongmei Xu (Depart-ment of Pharmaceutical and Biological Science,Weifang Medical University,Weifang,PR China).0141-8130/$–see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.ijbiomac.2011.11.015Y.Chen et al./International Journal of Biological Macromolecules50 (2012) 844–848845Sephacryl TM S-200High Resolution and DEAE-cellulose-52were purchased from Amersham Biosciences.Standard dextrans T-2000, T-110,T-70,T-40,T-10and Cy were obtained from Sigma(St. Louis,MO,USA).Concanavalin A(ConA)and lipopolysaccharide (LPS)were also from Sigma.All other reagents were of the highest available quality.2.2.General methodsUV–vis absorption spectra were recorded with a UNICO UV-2000spectrophotometer.Total protein concentration was esti-mated using the folin–phenol method proposed by Lowry et al.with bovine serum albumin as standard[13].Dialysis was carried out using dialysis tubing(Spectra/Por MWCO:6000–8000).2.3.Isolation and purification of the polysaccharideDried S.chinensis were crushed into particles(500g),defatted with95%EtOH,then extracted with5L of distilled water for2h at100◦C andfiltered.The residue was further extracted with3L of water bined aqueous extracts were concentrated in vacuo to500mL in a rotary evaporator under reduced pressure at 60◦C andfiltered.Thefiltrate was deproteinatedfive times using the Sevag method[14]and dialyzed against distilled water for2 days.The concentrated dark brown solution was precipitated by addition of4vol of95%EtOH and was kept overnight at4◦C.After centrifugation(6000rpm,20min),the precipitate was washed with anhydrous EtOH and then vacuum-dried at40◦C to obtain crude polysaccharide(SCP,27.4g).SCP(5.0g)dissolved in distilled water was applied to a DEAE-cellulose column(2.6cm×50cm),and eluted with water and sodium chloride(0–0.3mol/L).Fractions(4mL)were collected at aflow rate of0.8mL/min and monitored using the phenol-sulfuric acid method at490nm[15].Three fractions(i.e.SCP-I,SCP-II,and SCP-III)were obtained,with SCP-II constituting53%of the prod-ucts.SCP-II was further chromatographed on a Sephacryl S-200 HR column(1.6cm×60cm),eluted with0.1mol/L NaCl.Each2mL fraction was collected at aflow rate of1.0mL/min and monitored as described above.Collected fractions were dialyzed and lyophilized to obtain SCP-IIa,which was used in subsequent studies.2.4.Homogeneity and molecular weightThe homogeneity and molecular weight of SCP-IIa were deter-mined on a Waters HPLC system(1525HPLC pump)equipped with a Waters Ultrahydrogel250column(8.0mm×300mm)and a Waters2414differential refractometer.A sample solution(20␮L of0.1%)was injected in each run eluted with0.1mol/L NaCl as the mobile phase at theflow rate of0.5mL/min.The HPLC sys-tem was pre-calibrated with pullulan standards(Shodex Standard P-82,Waters).Commercially available standards(i.e.dextrans T-2000,T-110,T-70,T-40and T-10)were used as standard molecular markers.2.5.Measurement of immunomodulating activity2.5.1.Animal treatment and experimental designICR male BALB/c mice(8-weeks old;body weight20.0±2.0g) were obtained from the Animal Center of Shandong University of Traditional Chinese Medicine,Jinan,China.The animals were main-tained on a12-h-dark/12-h-light cycle at20◦C.They were allowed free access to standard laboratory pellet diet and water throughout the experiments.Mice were randomly divided intofive groups(10in each group) and were allowed to acclimatize for1week before the experiment. All animals received Cy intraperitoneally once every three days at a dose of80mg/kg body weight to establish the immunosuppressive animal model.The control group received normal saline.Different groups were treated with SCP-IIa at50,100and200mg/kg given orally,whereas control group and model group were given normal saline of the same volume via oral administration.Animals were sacrificed after10days of treatment.Twenty-four hours after the last drug administration,the ani-mals were weighed and killed by decapitation.The spleen and thymus were excised from the animal and weighed immediately. Thymus and spleen indices were calculated according to the follow-ing formula:thymus or spleen index(mg/g)=(weight of thymus or spleen/body weight).2.5.2.Peritoneal macrophages activity assay[16]Twenty-four hours after the last drug administration,mice were intraperitoneally injected with0.5mL of5%CRBC.Thirty min-utes later,animals were killed by decapitation and2mL of Hank’s balanced salt solution was used for peritoneal lavage.The cell suspension was smeared and incubated for30min at37◦C in a humidified5%CO2incubator.After incubation,non-phagocytosed CRBC and other cells were removed by washing.Macrophages werefixed with methanol and stained with Giemsa.Phagocyto-sis percent was measured by counting the number of macrophages phagocytosing CRBC per100macrophage cells.The phagocytosis index was measured by counting the number of phagocytosed CRBC per100macrophage cells.2.5.3.Assay of splenocyte proliferation[17]Cell proliferation was assessed using MTT-based colorimetric assay.Twenty-four hours after the last drug administration,the animals were killed by decapitation,and spleens were removed aseptically.Spleen cells of mice were obtained by gently plac-ing the organ in RPMI-1640medium under aseptic conditions, followed by centrifugation at2000rpm for10min at room temper-ature.Red blood cells were removed by hemolytic Gey’s solution. After two washes,cells were resuspended in RMPI-1640complete medium containing5%FBS,with cell concentration adjusted to 1×106cell/mL.The cell suspension was planted in a96-well cul-ture plate with or without ConA(5.0␮g/mL)or LPS(20.0␮g/mL). After incubation for72h at37◦C in a humidified5%CO2incubator, the number of proliferating cells was determined by MTT assay ata wavelength of540nm.2.5.4.Serum hemolysin formation test[18]On thefifth day of administration,each mouse was immunized by injection of0.2mL suspension of SRBC(109/mL).Twenty-four hours after the last drug administration,blood samples were col-lected.One hour later,these blood samples were centrifuged at 2000rpm/min for10min and20␮L of supernatant serum was diluted to500times with normal saline.About0.5mL of10%SRBC and1mL of fresh guinea pig serum(1:10dilution)was added to the reaction tubesfilled with1mL of diluted serum samples.After incubation for1h at37◦C,the reaction tubes were immediately moved to an ice bath and centrifuged again under the same con-ditions.About1mL of the supernatant was mixed with3mL of Drabkin’s solution.Ten minutes later,the absorbance at540nm was measured.2.5.5.Statistical analysisQuantitative data were expressed as mean±S.D.All statistical comparisons were carried out using one-way ANOVA test followed by Tukey’s test.P-values less than0.05were considered statis-tically significant,while P-values less than0.01were considered extremely significant.846Y.Chen et al./International Journal of Biological Macromolecules 50 (2012) 844–848Fig.1.Ion exchange chromatography (DEAE-cellulose)of the crude polysaccharide from S.chinensis .The polysaccharidie fraction was applied to the column equi-librated with water and eluted with a linear gradient of 0–0.3mol/L NaCl.The fractions were assayed by the phenol–H 2SO 4reaction to estimate the sugar content (A 490nm).3.Results3.1.Isolation and purification of SCP-IIaSCP was prepared from S.chinensis by hot-water extraction,EtOH precipitation,deproteination and dialysis.SCP yield was 5.27%of the dry weight.Total SCP extracted was fractioned by chromatography on DEAE-Cellulose-52column into three parts:SCP-I,SCP-II,and SCP-III with a ratio of 17:29:9(Fig.1).SCP-IIa was obtained from SCP-II by gel-filtration (Sephachryl S-200).Freeze-dried SCP-IIa appeared as pale yellow powder soluble in water.It had a negative response to the Lowry test and exhibited no absorption at 280and 260nm,indicating the absence of protein and nucleic acid.Total sugar content by the phenol–sulfuric acid method was 98.4%.GPC profile (Fig.2)showed a single and sym-metrically sharp peak,indicating that SCP-IIa was a homogeneous polysaccharide with an average molecular weight of ∼7700Da.3.2.Effects of SCP-IIa on thymus and spleen indices in miceTable 1demonstrated that the thymus and spleen indices of animals treated with Cy at 80mg/kg bodyweight decreased sig-nificantly compared with controls.This indicated that the mice immunosuppression model prepared by treatment with 80mg/kg bodyweight of Cy was built successfully.The thymus indices of animals treated with SCP-IIa at different concentrations combined with Cy did not show the significant difference except that of SCP-IIa (100mg/kg)combining Cy-treated group when compared with only Cy-treated model.The spleen indices of animals treated withTable 1Effects of SCP-IIa on the thymus and spleen indices of immunosuppressed mice.GroupThymus index (mg/g)Spleen index (mg/g)Control 2.79±0.38 5.14±0.76Cy1.38±0.29a 3.02±0.24a SCP-IIa (50)+Cy 1.41±0.31 3.78±0.36b SCP-IIa(100)+Cy 1.99±0.25b 4.22±0.18c SCP-IIa(200)+Cy1.56±0.174.45±0.22cCy and SCP-IIa +Cy denote mice groups treated with Cy and Cy plus SCP-IIa,respec-tively.The control group received the same volume of normal saline.The dose of Cy was 80mg/kg body weight for both the Cy and Cy plus SCP-IIa groups.Thymus or spleen index (mg/g)=(weight of thymus or spleen/body weight).The data are reported as mean ±S.D.(n =10).Significance was determined using the Student’s t -test.aP <0.01,compared with the matched control group.bP <pared with the Cy only-treated model group.cP <0.01,compared with the Cy only-treated model group.Table 2Phagocytotic function of macrophages and serum hemolysin formation in the immunosuppressed mice stimulated with SCP-IIa.GroupPhagocytose (%)Phagocytosis index Hemolytic plaque formationControl 41.12±2.310.79±0.120.318±0.016Cy25.80±3.30a 0.37±0.10a 0.102±0.010a SCP-IIa (50)+Cy 27.07±2.040.39±0.120.124±0.014b SCP-IIa(100)+Cy 31.13±2.19b 0.48±0.15b 0.119±0.026b SCP-IIa(200)+Cy35.08±2.43b0.47±0.08b0.247±0.019cCy and SCP-IIa +Cy represent the mice groups treated with Cy and Cy plus SCP-IIa,respectively.The control group received the same volume of normal saline.Dose of Cy was 80mg/kg body weight for both Cy and Cy plus SCP-IIa groups.Data are reported as mean ±S.D.(n =10).Significance was determined using the Student’s t -test.aP <0.01,compared with the matched control group.bP <pared with the Cy only-treated model group.cP <0.01,compared with the Cy only-treated model group.SCP-IIa at 50,100,or 200mg/kg combined with Cy at 80mg/kg bodyweight increased significantly in a dose-dependent manner compared with those treated with Cy only.The findings suggested that PAP overcame the immunosuppressed action induced by Cy.3.3.Effect of SCP-IIa on pinocytic activity of peritoneal macrophagesOne of the most distinct features of macrophage activation is an increase in pinocytic activity.Pinocytic activity of SCP-IIa-activated macrophages was indicated by uptake of SRBC.Results (Table 2)showed a dose-related enhancement of pinocytic activ-ity in macrophages treated with 50–200mg/kg doses of SCP-IIa.Phagocytosis percent and index in groups treated with moder-ate and high doses of SCP-II were significantly higher than the CY only group.SCP-IIa appeared to prime macrophages for enhanced pinocytic activity,which was comparable to that observed in con-trols.3.4.Effect of SCP-IIa on splenocyte proliferationThe Cy only-treatment significantly decreased the proliferation of the splenocytes compared with that of the normal control.In combination with Con A or LPS,SCP-IIa enhanced the proliferation of the splenocytes.The 50and 100mg/L SCP-IIa combined with Con A significantly promoted and strengthened the proliferation of the splenocytes.The 200mg/L SCP-IIa combined with Con A facilitated faster proliferation of splenocytes.The 100and 200mg/L SCP-IIa,combined with LPS provided obvious promotion and strengthening of the proliferation of the splenocytes.SCP-IIa showed a definite and clear synergistic effect on splenocyte proliferation after combining with Con A or LPS (Fig.3).3.5.Effect of SCP-IIa on serum hemolysin formationAs shown in Table 2,serum hemolysin formation in ani-mals treated with Cy only decreased significantly compared with controls.Serum hemolysin formation in animals treated with 200mg/kg SCP-IIa combined with Cy was significantly increased (p <0.01)compared with the Cy group.4.DiscussionS.chinensis is a famous and precious Chinese traditional drug.In recent years,Many studies have revealed that S.chinensis polysac-charides had widespread pharmacologic effects.We isolated and purified the polysaccharide SCP-IIa from S.chinensis using boiling water extraction,alcohol precipitation,ion-exchange chromatog-raphy,and column chromatography on silica gel.SCP-IIa was aY.Chen et al./International Journal of Biological Macromolecules 50 (2012) 844–848847Fig.2.Profile of SCP-IIa in HPLC.The sample was analyzed by Waters Ultrahydrogel 250column (8.0mm ×300mm)and eluted with 0.1mol/L NaCl at 0.5mL/min.homogeneous polysaccharide with an average molecular weight of ∼7700Da.Our findings provided basis for further research on biological activity mechanism of S.chinensis polysaccharides.The body’s immune function includes nonspecific and specific mechanisms.Nonspecific immunity refers to defenses against all pathogenic microbes having a certain degree of resistance.It has no special selectivity and is also known as natural or congenital immu-nity.Specific immunity protects the body from one kind of microbe or its products,and includes humoral and cellular mechanisms.In order to create a model of weakened immunity,we treated mice with Cy,which is a chemotherapeutic drug for cancer that has the side effect of injuring the DNA of normal cells.In our model,the effect of Cy was demonstrated by reduced spleen and thymus indices,since Cy treatment decreases the capacity of the immune system.The lower spleen and thymus indices in the Cy-treated mice in comparison with normal mice indicated an immunosuppressed state.The thymus and spleen are important immune organs.Their weights are important and intuitive indices for nonspecific immu-nity of the organism.Immunopotentiators could increase the weight of the thymus and spleen.The relative spleen and thy-mus weights in SCP-IIa test groups were notably higher than that of model groups.SCP-IIa apparently increased spleen indices in a dose-dependentmanner.Fig.3.SCP-IIa stimulation of the proliferation of splenic lymphocyte induced by mitogen.The data are reported as mean ±S.D.(n =10).Significance was determined using the Student’s t-test.a p <0.01,compared with the matched control group.b p <0.05,compared with the Cy only-treated model group c p <0.01,compared with the Cy only-treated model group.Macrophages are ancient and phylogenetically conserved cells in all multicellular organisms.Together with neutrophils,they represent the first line of host defense,second to the epithelial barrier.Macrophages can function as antigen-presenting cells and can interact with T lymphocytes to modulate the adaptive immune response [19].Furthermore,macrophages are involved in tissue remodeling during embryogenesis,wound repair,clearance of apoptotic cells,and hematopoiesis [20,21].Activated macrophages not only participate in both specific and non-specific immune reac-tions,but also is the “bridge cell”of these two immune reactions [22].The present study investigated the phagocytic activity of peri-toneal macrophages.The phagocytic rate in mice pretreated with SCP-IIa markedly rivaled the decrease in phagocytic rate induced by Cy.The group treated with 200mg/kg SCP-IIa showed the great-est increase in phagocytic function.These demonstrated that the administration of SCP-IIa significantly increased the phagocytic activity of peritoneal macrophages in a dose-dependent manner.Lymphocyte proliferation is a crucial event in the activation cascade of both cellular and humoral immune responses [23].Lym-phocytes induced by ConA in vitro may be used as a method to evaluate T lymphocyte activity,while those induced by LPS may be used to evaluate B lymphocyte activity [24].Cy-induced suppres-sion of Con A-induced T-lymphocyte proliferation and LPS-induced B-lymphocyte proliferation was identified in this study.SCP-IIa appeared to have greater sensitivity for T cells of splenocytes than B cells,which resulted in better T-cell-mediated immunostimulating effect.The formation of serum hemolysin with SRBC immuniza-tion reflects humoral immunologic function [25].Our results demonstrated that SCP-IIa significantly increased serum hemolysin formation in Cy-immunosuppressed mice.These suggested that SCP-IIa can enhance humoral immunity activities.5.ConclusionIn summary,SCP-IIa exhibited potent immunomodulating prop-erties,such as improving the weight of immune organs,enhancing the phagocytic activity of peritoneal macrophages,promoting hemolysin formation,and increasing lymphocyte transforma-tion.Our investigations suggested that SCP-IIa was involved in immunomodulatory effects in vivo.This study may provide a basis for the use of SCP-IIa as an efficacious adjuvant immunopoten-tiating agent,and it should be explored as a strong potential immunostimulant for the food and pharmaceutical purpose.Thus,it is clear that this polysaccharide extracted from S.chinensis has sig-nificant therapeutic potential and represent a rich source for future848Y.Chen et al./International Journal of Biological Macromolecules50 (2012) 844–848discovery and development of novel compounds of medical value which need to be taken more attention in further studies. AcknowledgmentsThis study was supported by the following funds:the Natural Scientific Foundation of Shandong Province(ZR2011HQ044); Science&Technology Development Program of Weifang (201101178);Traditional Chinese medicine Science&Technology Program of Health Bureau,Weifang(2009016).References[1]W.Y Wang,J.G.Chen,J.Beihua Univ.(Nat.Sci.)8(2007)128–133.[2]H.Kiyohara,T.Matsumoto,N.Takemoto,H.Kawamura,Y.Komatsu,H.Yamada,Planta Med.61(1995)429–434.[3]Y.W.Zhang,H.Kiyohara,T.Matsumoto,H.Yamada,Planta Med.63(1997)393–399.[4]T.Matsumoto,X.B.Sun,T.Hanawa,H.Kodaira,K.Ishii,H.Yamada,Phytother.Res.16(2002)91–93.[5]P.J.Gao,Y.F.Pu,X.L.Guo,D.Y.Ju,B.Ren,J.Norman Bethune Uni.Med.Sci.22(1996)23–24.[6]M.Miao,China J.Tradit.Chin.Med.Pharm.17(2002)187–188.[7]L.Huang,L.Chen,Z.L.Zhang,Zhong Cao Yao27(2004)202–203.[8]A.O.Tzianabos,Clin.Microbiol.Rev.13(2000)523–533.[9]A.I.Schepetkin,M.T.Quinn,Int.Immunopharmacol.6(2006)317–333.[10]Y.J.Jeon,S.B.Han,K.S.Ahn,H.M.Kim,Immunopharmacology43(1999)1–9.[11]K.Y.Lee,Y.J.Jeon,Int.Immunopharmacol.3(2003)1353–1362.[12]C.Bodinet,U.Lindequist,E.Teuscher,J.Freudenstein,Phytomedicine9(2002)606–613.[13]O.H.Lowry,N.J.Rosebrough,A.L.Farr,R.J.Randall,J.Biol.Chem.193(1951)265–275.[14]M.G.Sevag,ckman,J.J.Smolens,Biol.Chem.124(1938)425–436.[15]M.Dubois,K.A.Gilles,J.K.Hamilton,P.A.Rebers,F.Smith,Anal.Chem.28(1956)350–356.[16]X.Li,L.L.Jiao,X.Zhang,W.M.Tian,S.Chen,L.P.Zhang,Int.Immunopharmacol.8(2008)909–915.[17]X.F.Du,C.Z.Jiang,C.F.Wu,E.K.Won,S.Y.Choung,Arch.Pharm.Res.31(2008)1153–1159.[18]R.M.Yu,L.Y.Song,Y.Zhao,W.Bin,L.Wang,H.Zhang,Y.H.Wu,W.C.Ye,X.S.Yao,Fitoterapia75(2004)465–472.[19]R.W.Birk,A.Gratchev,N.Hakiy,O.Politz,K.Schledzewski,P.Guillot,C.E.Orfanos,S.Goerdt,Hautarzt52(2001)193–200.[20]M.W.Lingen,b.Med.125(2001)67–71.[21]A.H.Klimp,E.G.E.de Vries,G.L.Scherphof,T.Daemen,Crit.Rev.Oncol.Hematol.44(2002)143–161.[22]W.X.Chen,W.Y.Zhang,W.B.Shen,K.C.Wang,Cell.Immunol.262(2010)69–74.[23]C.Zhao,M.Li,Y.F.Lu,W.K.Wu,Carbohydr.Res.341(2006)485–491.[24]F.Cerqueira,A.Cordeiro-Da-Silva,C.Gaspar-Marques,F.Simoes,M.M.Pinto,M.S.Nascimento,Bioorg.Med.Chem.12(2004)217–223.[25]H.Y.Liu,J.Chang,K.Gao,H.Y.Wang,L.Wu,Y.H.Xue,China Dairy Ind.34(2006)37–39.。