ltano, Arao

Tsuji, Yasuhiko

Okayama University Scientific Achievement Repository

Microbiological Investigation on Tea. 1. Influence of Tea Extract on the Soi1 Microorganisms. By Arao ltano and Yasuhiko Tsuji. [FI自b.3， 1937.] 1n thi自泊V伺 tigation，もheinfluenoe of tea. extra.ct on the soil microorga.ni阻 mW帥 underto.ken旬剖certa.in幽 fa.rI.S P伺 sible副 tot，he a.ction of fa.llenも001崎刊自in the field on the回 ilmicroorga.nism自， a.nd朗a.prelimina.ry tωt the ext，rac旬 ofva.riou圃もea.lea.ve自wereprepa.red a.ndもheira.ction on the自tockcultur倒 wa.st倒防d泊出ela.bora.旬ry. The resulta of ~he prelimina.ηinve目もiga.tiona.re pr倒entedin this pa.per. The exもra.ctsof旬a.leav伺o.rediffere叫 bythe kind ofもea.嗣 wella.s th白temperature of wa.ter a.nd the til1le of extra.ction. However， a.s a.n exa.mple， a.report of EDDEU1) on the a.naly自i圃ofhot， wa.ter自主tra.ctof tea is noted嗣 fol1ow日:Compooitlon. %‘ Protein. 40 Thein. 1∞ Fat. 1∞ Tannin. 1∞ PO旬88inm. 76 Calcinm. 5 PhORphoruo. 10 ， Note: 侍 Thepercentag咽ofdiooolved 8uh尚ancei8 bBRed on the amonn色ofreo問ctiveconfltitl1ent originary present. A日thea.bove da.凶indi叫旬"tho自econstit.uentR such s.8 thein a.nd tannin which a.re considered句 bepeculia.r旬 tea.踊wella.s the fa.t a.re extra.cted completely回tha.t their自ub自叫uentinsuence on the microorga.ni自m圃.Reg町 dingthe action of ta.nnin andもhein，:MAR'IτN8) reporωd that although ta.nnin in 2-5 percent concenもra.tionha自nobactericidal a.ction on Es. coli and Eb. typhi， i旬 10ー 却 peroentconcentra.tion d倒 troyもhemwi出in24 hour自.492 A.ITANO and Y.百UJI:MEZZADROU4).7) report自dthat 0.5 percent solution deprωRed t.he growth of A自per-gillus niger while 0.25-1.0 percent自olutionRtirnulated the growth of yeasts. KISHlDA 3)report白dも，hat出自もeo.pr白vent自由egrowth of cholero. and typhoid bo.c-teria which may be出eo.ction of to.nnin o.nd thein pre目。nもinもheleo.ves. MATRU・1I0T05) investigo.ted th白germicido.lo.ction of di貸erentkind ofもeo.o.nd found tho.t 出eUhlong teo. ho.s stronger bo.ctericidal o.ction tho.nもheJo.po.ne自由もeo.but the bln.ck teo. in w白此erもho.nthe lo.t旬r，o.nd the ba.cもericido.la.ction of t倒 W朗 foundto be in pn.ro.lel wi出 thetannin content. From th白foregoingresul旬 itis cleo.r tho.t 出eもeo.po自由側同自 th白propertyto depre目白 thea.ction of microorgo.nism自o.lthoughもheinteIl8ity differs by the kind o.nd quo.ntit，y. As 0. prelimino.ry七割も toinv自白tigo.teもheo.ction of fn.len. ten. leave自onthe microorganism自inthe tea-farm自oiland al日oto investigateもhere自i自tanc白ofthe i自olat吋 organismago.iIl8t the tannin and itR similar substance， the action of teo. extract made by hot and cold wat白rupon the soil microorgani日m日wn.自 inveRti・ga句d，andも，here自ultso.re reported in thi自po.per.E玄perimental.The ten. extra.cts made by出ehoもo.ndcold water from Gyokuro， Sent.ya， blo.ckも価o.ndBo.ntya were added 加 the culture medium in 5， 10 snd 20 percent ratio， o.nd their insuence on B.白ubtiliR，Azo加b蹴 ter，yeast自o.ndA自pergillu自nigerW副総sted. At the sametime， tannin and出einwere determined quantito.tively. a.) Prφaraliol if hol waler exlracl: Ten p町匂 ofhot wo.ter were a.dded to one part ofもeo.leaves o.nd heo.teu for one hour in 0. boiler， filtered through cloth and cleared byもhecent討fuge.b.) P.ゆ araliolif cold z即断 exl百 1: The cold water extra.ct w倒 preparedin 也e同memsnner副 thehoもwsterextra.ct withou七boilingbut自hakenfor七wohours. c.) Pretaralioll if cullure media: (1.) The nutriell七agarmedium w朗 u日edfor B. RubtiliR; (2.) A自由主'sago.r for A.zotobacter chroococcum; (3.) LAURENT aga.r for Sacch. cerevi自io.eand (4.) CZAPEK'S ago.rめforAspergillu自niger.The LAUlIENT medium of the following composition wo.白U自ed:Ammonium sulfo.te Me.gn回 iumPotaRRium acid phospha加Mal旬自由Ago.r Wo.ter 4.71g. 0.10 " 0.75 " 卯.∞"15.∞" 1刈)().∞c.To the自eculture media， th白teaextra.cも wo.so.dded in 5， 10 a.nd 20 perce11もconcentration， keeping the toも0.1volume remo.in C011白ta.nt.The org岨 ism自usedwere those which have been kept in our sωck. Microbiological Jnve目色iga“onon Tea. I. 493 d.) Delermi・'na!irJnof lannin.. To 5 cc. of自olution，10∞. of Indigocarmin (0.5%)， 5 c. of H2S04 (20%) a.nd却oc. of wat.er were a.dded， a.nd titr叫edwith N/IO pota剖iurnpermangana.旬自olutionwhich hl.s been ca.refully standardized agmnst oxa.lic acid. At th自由a.metime，30 c. of water were added t.o 10 c. of th白 test自olutionto which 1 g.of ca.sein (the pure casein which has been subjected to ether extrao“on for several da.ys) w蹴 added，自hakenfor 15 minutes and cen-trifuged; a.nd anot.her 1 g. portion of the ca.sein w朗 added，自ha.kElnand cen-もruugedagain句 elimina色eta.nnin completely. Twenty cc. of the clear 801ution thus trea胎dwere taken and 10 c. of Indigoca.rmin (0.5%) and 5 c. of H2S04 (却%)were added， a.nd titrated with N/10 pot朗自iumperma.ngana.もesolution. One g. of oxa.lic acid correspond to 0.4648 g. gallotannin so tha.t the difference in qua.n均 ofpota.sium permanga.na.te used in two titra.t.ions gives the a.mount of ta.nnin pr倒ent. (This i日similarto RHIND a.nd SMITB method ci旬din TADOKORO'S Chemistry of Enzyme， 1929-Ja.pan倒白 edition.)e.) Delermina/i切 ofIhein.. 20 c. of the extract w制 dilu旬dwith wa.ter and 5 c. of satura.ted b朗iclea.d a.ceta.te w朗addedand make u p出etota.l volume旬250 c. a.nd filter; p朗自hydrogensulfide in句 thefiltl'ate to precipita.te lead which is随 pa.ratedby盆ltra.tion，a.nd 125 c. of the filtra.te is evn.poraもedめ dryness自O色，hathydrogen sulfide is eva.pora.ωd; the solid raidue i向 diωolvedin wa.ter a.nd 出einis extr郎総dwith chloroform five times; drive of the chloroform by evapかra.tion o.nd dry o.t 1∞。C.for 30 minutes or longer until the consta.nt weight is rea.ched. R伺 ul旬.1.) The results obぬinedwith B. subtilis are given in To.ble 1. (See Table 1 on nexもpage.)In the foregoing experiment日 itwo.自由usp伺 tedtho.t the tannin which w幽a.dded切出eculture medium might have been ta.ken up by the proもeincontent present in the medium o.nd na.turo.ly the insuence of ta.nnin might have been lost in the cou問eof experiment. To制 certainthis o.BFlumpt.ion， the exもra.ctof Sentya. W朗a.dded加 themedium in 20~杉 concentro.tion o.nd the o.mount of ta.nnin w制detern血eda.nd found a.s follows : 伽iginalta.nnin content in tea extra.ct Tannin found free in the medium Amount of tannin decroosed . • 0.1546 g. 0.1208 g. 0.0お8g.As the above da.ta indica旬， 0. 8ma.l portion of t.annin w嗣ta.kenup by the proもeinconstituent in the medium soもhat0.1 tannin initially a.dded does not insuence the orga.ni自m.Table 1 indicatea tho.t 20 percent hot water extra.ct prevented the growth completely while a白lightgrow出 tookpla.ce in 10 percent extra.ct o.nd g∞d growth wa.s obta.ined in 5 percent extract闘 wella自 in出econtrol except in 494: A.ITANO anrl Y. T日U.TI: Table 1. Influence of Tea Extracts on B. subtilis. Rate of growもhby days. Sample8. Extract Tannin Thein arlded. conもen色目. conもenもs.5 7 14 (%) (g・in1∞ cc.):(I~. in 1∞cc.) 。on甘01.柑 一 一 + 榊 榊5 0.0143 O.∞90 + 榊 榊. Gyokuro. 10 0.0286 0.0180 + + 榊(hoも‘)20 0.0573 0.0360 一 一 ー5 0.0084 0.0072 + 掛 掛Ibid. 10 0.0168 0.0144 + 榊 榊(oold・)20 0.0336 0.0288 + + 榊5 0.0306 0.01∞ + + 榊8entYB. 10 0.0613 0.02∞ + + + hot*) 20 0.1227 0.04∞ 一 ー ー5 0.0154 O.∞45 *+ 榊 柵Ibid. 10 0.0308 O.∞ω 側 柵 柵(cold・)20 0.0617 0.0180 一 ー 一Ibid. 一 一 一 + 榊 柵(control.嗣)5 0.0176 O.∞40 + 榊 e +t Ban色y(hao.S骨) 10 0.0352 O.∞80 + + 榊20 0.0704 0.0160 一 一 一5 O.∞48 O.∞62 制十 iI 柵Ibid. 10 O.α)96 0.0125 柵 柵 柵(cold・)20 0.0191 0.0250 + 榊 iI Ibid. (conもrol".) 一 一 一 + 掛 柵0.0155 0.0084 + 制 制Blaek旬a. 0.0311 0.0168 一 + + (hot *) 0.0623 0.0336 一 一 ー5 O.∞46 0.0044 + .榊 特+Ihid. 10 O.∞93 O.∞88 + +t 特+(oold・)20 0.0187 0.0176 + 件 + No旬日: 梼 Hoもandco1d designateもhehoもandcold water extrac制;柑 dillerentcon-trols were used 8ince the experimen旬瓜a凶edon di晶 renもd8Y8.The sumber of + illdica加s~h申 raWof grow向，Microbiological Inveat.ig叫onon Tea. 1. 496 8entya. 1n case of the co1d wa.ter extr蹴 t，good growth w朗 obta.inedin bo出5 a.nd 10 percent concentra.tion. 80 far闘 thekind ofもea.is concerned， Sentya. W舗 most effective in d自pre日目ingt，he grow出 while the others were ju自ta.bout 自ame. The b自ttergrowth was obta.ined wiもhthe co1d wa.ter extracもtha.nthat with もhehot water ext，mct， which日eems加 bedueもo出edi貸erentamount of ta.nnin and thein. The minimum do自eforもhe倒 epticn.ction of tannin w佃 0.0.573g. in 1∞∞. medium n.nd the hannfu1 infl.uence w闘 a1readynoted in 0.03 g. concen-もrationwhile， with出ein，0.016 g. in hot Bantya， was the minimum dOlle although 0.018 g. in Gyokuro and 0.025 g. in co1d Bn.ntya did not prevent the growtb. J udging from the foregoing r倒 ul旬， ta.nnin p1ays more importa.nt rら1eもba.nthein a目もhea.nti自eptic.2.) The r自由u1tsobta.ined with Azotobacter are pre自由ntedin Tab1e 2. Tab1e 2. Influence ot Tea Extr帥 tson Azoもobacter.Ra旬 ofgrow色hby day圃.Samples. added， I conten旬.Thein 3 I 4 I 7 conもen旬.(%) Kg. in 1∞c.) (g. in 1∞ec.) . Control. 一 一 一 件 一 t+ ー * 一O.∞39 O.∞21 件 + + + * + Gyoknro. O.∞79 0.0042 + ー + + + + (hoげ)0.0152 0.0265 一 一 ー ー ー 一O.∞12 O.∞12 + + * + * + Ibid. O.∞24 0.0024 + + + * + .。(cold ・j O.∞96 0.0102 一 ー ー ー 一 一S岨も(hao.H) 0.013t O.∞39 一 ー ー 一 ー ー0.0282 0.0220 ー ー ー ー ー 一lbid. O.∞62 0.0018 + 一 + ー t+ + (coll1 *) 0.0199 O.∞60 一 一 ー ー 一 ーBan色{vhao.H) O.∞71 O.∞47 + 一 + 一 * 件0.0185 0.0310 ー ー ー ー 一 一Ibid. O.∞19 0.0025 + + t+ 件 * + (cold *) O.∞95 O.∞70 ー 一 ー 一 一 ーBlack色帥. O.∞56 0.0034 + ー + 一 + + (hoも僑) 0.0167 0.0225 ー ー ー ー 一 一Ibid. O.α)()3 O.α)()9 + + * + t+ + (cold勺 O.∞65 0.0052 一 一 一 一 一 一Notes: 保 Hotanrl cold rl自由igna総色hehot and cold water extract. G.= growもh;P.= pigment pr四luction.Th白numberof + indica総自色hera民 ofgrDwth. 496 A. ITANO and Y. TSUJl : As Table 2 indi叫 tes，5 P自問enthot and ∞ld extra.巴tsof 0.1 kinds of tea prev印刷1t.he growth entirely although日omedi貸erenceRwere noted Ilimong the different kind of tea. One percent hot wa.ter extract of Gyokuro自howed幽 g∞dgrowth as the control but 2 percent depr自制edthe growt.h somewho.t while 2 percenもSentyo.prohibited th白 gl'owth，and very feeble growもhwas obtained in B阻 tya.o.nd blo.ck tea. It is notewol'山yt.lJat，もhepigm自ntwa自 produced泊0.1the cases a.nd itwa.自 inparo.1el with the a.mount， of ext.ract used， which 8eemsもoindicaぬ tha.tta.nnin a.nd t.hein a.re re叩onl¥iblefol' the production of pigment朗自uggestedby lTANo a.nd MAT自前JRi¥8)who repo此edtha.t the pigment productiQn is inlluenced by ultra.-violet ra.ys and自omeoth白rf舵 tors. In Cl自eof the cold wa.ter extract， 1 percent Gyokuro w阻 thebest and slightly wor自ein 2 percent. Witb other kind自oftea except Sentya， 2 percent extra.ct supported the growもhcqually we11朗出econtrol， and th白 pigmellもproducも，ionw闘 markedwhere the ext.ract W嗣 added.The cold water extra.ct wa.'1 more effective than tbe hot w叫erextract in producing betωr growth and stronger pigment proclucもionwhile Sentya. w嗣better a.nti日eptic七ba.n0もherkind白ofも帥.The minimum anもisepticdo白eof ta.nnin wa.s O.似>65g. but the grow出 ishindered by a.bout O.∞2 g. while O.∞52 g. is the minimum for thein. 3.) Inlluence on yeasts: The followillg re8ults were obta.ined 朗自hownin Table 3. (See Ta.ble 3 on nexもpage.)As noted above， the grow出 ofyea.sts w朗自もimula.tedin proportion to the concentration of bo出 hoもa.ndcold wa.ter exもra.ctwhich were differenもinca.8e自ofB. subtilis and Azotoba.cter. As to the hot wa.ter extraca the addition of 20 percent w朗 mo白も effect.ivewhile 10 and 5 percent were leBB e佳ectiveper order 0.1七hougheven 5 percent ohowed better growもhtba.n the control. The cold water extr郎旬 ga.veもhesimil町 r回 ul旬脳出ehot wa.もerextracta although no mal'ked di賃erencew剖 observed.Regarding出ekind of 旬、もbeb倒も extractwa.s obtained from Gyokuro and bla.ck tea was worse. The growもhwa.臼 stimula.t，edby the addition of ta.nnin， 0.1315，g. and thein， 0.015 g. which were the ma.:x;imum q¥la.nti旬introduced.4.) InftuenC'，(l on Aspergilluo niger: The following r自由ultswere obta.ined with A日pergillusniger 副自hownin Ta.ble 4. (See Ta.ble 4 on page 498.) As the a.bove da.ta indicate，もhebesもgrowthwas obt.oined in 5 p白rcenthot water exもracta.nd higher the percenta.ge， d白pr白ssedthe growlh more ma.rkedly. 011 the other hand no m町 keddi貸erenceW!l.8 ob日ervedamong the different con-cen tration of the cold wo.t自rextract although a 8ligbtly be枇，ergrowth than the control in 0.1出自 C嗣倒.Microbiological Investigation on Tea. I. 497 To.ble 3. Influence ot Tea Extracts on Yeasts. Rate of growth by day目.Extrac色 Tannin Thein 8amples. added. content日. contents. (%) (g. in 100 c.) (g. in 1∞∞.) 5 7 14 Con卸01. 一 一 一 + + + 5 0.0199 0.0129 fi * fi 10 0.0398 0.0258 fi * 州20 0.0796 0.0515 柵 柵十 附t5 0.0075 O.∞76 + * fi Ibid. 10 0.0149 (cold僻) 0.0149 州 柵十 州20 0.0299 0.0305 柵 附4 附15 0.0328 0.0102 + + + hMRω骨) 10 0.0657 0.0205 + * fi 20 0.1315 0.0410 fi 柵十 柵5 0.0157 0.0057 + + fi Ibic1. 10 0.0315 0.0115 + + fi (∞Id骨)20 0.0629 0.0230 柵 柵十 州0.0177 0.0129 fi * 柵Banw(hao. H) 0.0355 0.0257 柵 柵十 柵0.0710 0.0515 柵 柵十 附t5 0.0076 0.0076 + 件 榊Ibid. 10 0.0152 (∞Id骨) 0.0152 柵 * H附20 0.0305 0.0305 柵 柵十 柵5 0.0160 0.0092 + 件 + Black旬a. 10 0.0321 0.0185 件 件 柵(hot *) 20 0.0642 0.0370 州 榊 州5 O.∞42 O.∞31 件 件 + Ibid. 10 0.0084 (cold勺 O.∞62 + 榊 柵20 0.0168 0.0125 州 榊 州Note日:骨 Hotand cold designate the hot snd cold wster extract. The number of + indic8ιes the rs総 ofgrowth. 49ぬ A. ITANo and Y. TI日UJI:Table 4. Intluence of Tea. Extra.cts on Aspergillus副ger.Rate of growth by day目.Sample日. Extract Tannin Thein arlde(1. contenも8. 日onもen旬.(%) (g. in 1∞('c.) (胃.in 100cc.) 5 7 14 Conもrol. 一 一 + fi 柵5 0.0199 0.0104 掛 州 柵(hot *) 10 0.0399 0.0207 + 州 州20 0.0799 0.0415 + + fi 5 0.0061 0.0059 州 柵 柵Ibid. 10 0.0123 0.0117 掛 州 柵(cold *) 20 0.0245 0.0235 掛 州 柵5 0.0336 0.0098 掛 柵 柵島nsa(ho.H) 10 0.0671 0.0197 掛 柵 柵20 0.1342 0.0395 + fi 州5 0.0154 O.∞45 掛 柵 柵Ibid. 10 0.0308 0.0090 掛 柵 柵(colcl・)20 0.0617 0.0180 * 柵 柵5 0.0178 0.0116 * 柵 柵BmEb(yhao. も*) 10 0.0356 0.0232 掛 柵 柵20 0.0713 0.0465 + + m 5 0.0048 0.0062 制+ 州 制+Ibid. 10 0.0096 0.0125 柵 柵 柵(cold勺20 0.0191 0.0250 掛 州 制+5 0.0141 0.0086 制+ 柵 柵Black tea. 10 0.0282 0.0172 Ht 柵 柵(hoも勺20 0.0564 0.0345 + 制+ 制+5 0.0008 0.0024 + 柵 制+Ibid. 10 0.0016 0.0047 制+ 柵 制+(colrl *) 20 0.0032 O.α)95 制+ 州 制+Noもe8: 骨 HoもaJ0cold deRignauうもhehoもandcold water extract. The number of + indica旬日もhera旬。fgrowもh.Microbiological Investigation on Tea. I. 499 Th自coldwat自r自主tractwas more e宣白ctiv自thanthe hot water extract wh自na larger amount of the former is used which seems to indicate伽 insuenc白 oft岨 ninand thein. No difference awong出edi仇rentkind自 ofもeawas noもedin regard加 boもhkind日ofexもract.R.The maximum amount of旬nnin岨 dもheinwas 0.1342 g. and 0.046.'5 g. re・自pectively. More than 0.07 g. of tlLnnin WI.S harmful fcir the growth. 5.) ResistILn巴eof Soil Microorgo.nism日:Among four different kinds of microorga.nismsぬ自ted，Azotoba.cter w剖 thewea.k倒も andB. subもili白， AHpergillu目nigerwere slightly more r倒i自凶ntwhile ye嗣 tswere日もimulatffiin their growth by the addiもion01 extra.ct自. The minimum qua.ntity of tlLnnin which pr白ventsth白 growthwas 0.006 g. for Azotobacωr， 0.0573 g. for B. subtilis， 0.07 g. for Aspergillus niger bl1t for ye嗣 tseven 0.131.'5 g. stimula.もedtheir growth. Theぬaextract stimula.ted th自 growthof ye副知 butwas harmful for the others except when the quantity wa.s very small. The stimulation of yea.st growth by the t白a.extmct may be brought a.bout by thein as reported by MEZZA凶 OLl4)，a.nd a.ccording to the results obtained in this inve此iga.tion，they朗自mもoindica.te that theiu is the fa.ctor which influence the growth of目oilmicroorganisws in general. Summary. The influence of hot and cold water extra.cts of Gyokuro， Sentya.， Bantya and bla.ck tea. on the grow也 ofB. su btilis， Azo加b嗣 ter，Yea.s旬a.ndAspergillus niger wa.s inv佃 tigatedand出efollowing summ町ymay be mo.de. 1.) The rate of influence of extracts seem白tobe in parallel with the tlLnnin con色en旬.2.) As to the kind of ぬa，Sentya seems加 bethe strong倒 tin depr剖singthe growtb. 3.) Among the microorganism日used，Azotoba.cぬrwa.自由ewea.kest followed by B. subtili自andA日pergillusniger while yeas旬 W朗自timulated朗 theconcen-tration of extract increased so f町制u闘 din this in ve前iga.tion.Literature. ( 1) SAWAHURA， M.， Tea Manufactnring， 1909. (湾村~，製茶論.)( 2) FRED， E. B. and W AKSMAN， S.A.， J.Rboratory Mannsl of General Microbiol噌y.( 3) KISHIDA， Toijishin， 2756: 2829-2832， 1931. (岸田微，東宮事新.)( 4) MEZZADROI.， G. and AMATI， A.， 1m!. 8accar. ltal.， 25: 392-394， 1932. (From ab同，ract.)(5) 時A潤四OTO，K. and MATI!UDA， K.， Taiwan lkwai， 32: 514-522， 1933. (紙本一雄，松田主計， I陸海鹿倉・)民)() A. ITANO and Y. Tsu.TI: (6) MA町四， J&， J. D._ Rnd FOWLlI:R， C. D.， Ann. 8nrgery， 99: 993一部6，1934. (日唱mabstract.) (7) MSZZADBOLT， G. and AMATI， A.， A悦i，8Ccad. Lincei， 21: 46-50， 1935. (From I‘bstr8ct.) ( 8) ITANo， A. 8nd MATSUURA， A.， N均akn・kenkyfi，26: 349-369， 1936. (板野新夫，松浦寧.農事研究.)、

Microbiological Investigation on Tea. II. Influence of Tea Extract on the Soil Microorganisms.

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