206
STU且）‡ES ON THE MANUFACTURE
OF SOYBEÅN P良OTEIN（ⅠⅠⅠ）
Extraction and Precipltation of Protein・SmalトScale
Manufacture of Soybean Protein・
By Sin’itjI6KAWAMURA
（Labor・a、tOヱy■Of】∋iologica1C‡1emistr・y） （Received November21，1952．）
This reportis the thirdand conclusivepartof the series，following the first part（81）
describingpreliminary experiments（Chapterl），and the second part（S2）concerning・the extractionofsoybeanOil（Chapter・2）and the purification of defatted meal（Chapter・3）1 This part consists of the followlngtwO Chapters：
Chapter4．Extraction and Precipitaltion of Protein．（Experiments No．17疇No．27；Tables
40・56）
Chapter5・Small・・Scale Manufacture of Soybean Protein．（、Experiment＄No．28−No．30； rrables57・64）
CHAPT畢R4．EXTRACTION AND PRECIPITATION OF二PROTEIN． Z；：trOducti’0プ∴ Probably thempstimportantprOCeSSOf soybean・prOteinmanufacture，
at1east from the viewpolnt Oflaboratory experimentS，is the extract；on process，in whic‡1
the defatted soybeanmealor flake with or without the pretreatmentis treated with a
P工Otein−e紳acting agent（orpeptizingagent）；thus the proteininsoybeansis made soluble （Oris dispersed．），and then this protein disperISionis separated from theinsoluble residue．
As the peptizing agent such one should be elected as willpeptize as much protein and a＄
1ittleimpurities as possible，Or・dinary extraclng■ag−entS are CauStic alkalies，alkaline＄alts， a、nd neutralsalts，、although water and acids may also be used．
CIRCLE（；）mentioned allthese extractants and discussedin detail”Tomention someICCentliteIatuIe，
alkalinesaltssuchasO．1％sodiumsulfite（35），0．3％sodiumsulfite（＄1），andO．06％sodium sul董ide（51）；lower guateInaIy ammOnium salts（4iラ）；SOit watelplus bacteIicide（13）；waIm WateI（50O）（51）；and acids subh as hYdrochlo＝ic（28），Phosphoric（28），Suifurous（28・：！3），andmi幻ure COJltain＝1g aCetic and phospholic acids（6）have been pIOpOSed
The pr・eSeht author studied the deta、iled conditions for uslng・sOdium sulfite（31，41）as the proteinqextra・Ctingagent（ExperimentS Nos・1ト21）and moreover carried out some ex− periments on sodiumhydroxide and sodilユm Carbonate（ExpeimentS Nos．22−24）．
The protein mustbe precipitated by adding・Suitable precipitating−ag・ent tOits ex岬 tract・Asthe precipitating agentS We Can name，aCCOrdingtOSATO（44）salts，alkaloid rea・ gentS，COlorlngSubstance，formaldehyde，electricmethod，heating・method，and fermentation method・But some of them may giveinferior product＄，and some may not’be pr・aCtical；
the only technicalmethodis the addition of dilute aCid to alkaline extract・Inorganic
acids（■s−ulfuric，Sulfurous，hydrochloric，phosphoric，and nitric）and organic acids（acetic a・nd oxalic）were triedin the author，s preliminaryexperiments．（31）cIRCLE（7）mentioned also
0rder to obtain maximum yield of soybean proteinisthe problem of theisoelectric point．
This poiut was not ultimately determined，although the experience agIreed that the addition
Of acid to pH4N5m耳de the precipitated proteineasy to settle andmade the upperlayer transparent．Exper・iments Nos・26and27concern theisoelectric point of soybean protein・
Sodi11m SuJfite as tlle ExtractingAg・ent（Experimeuts Nos．17−21）． 苫Ⅹperim￠ntSⅣ0．17．Pr￠liminaryExperim￠ntS七0工〉et￠rmiIle某Ⅹtraet￠dⅣitrogやn
and Whey Nitrogen、When the Naphtha−IlefattedれIealwas Treted with O．4％Sodium SⅥ1fite So且uti（〉m．
The raw Tnaterialfor Experiments Nos”17−24is of the sameorigin．It waSdefatted 壷ith na．phtha at the tnstitilte Of Physicaland ChemicalReseach，’Tokyo．It contained COnSiderable proportion of finer powder・”Fractionation by sievlngg・ave the followlng reSult：
coarser than50mesh，55．6L！％；50・■80mesh，13．30％；80・160mesh，ユ1ル00％；a．nd finer than
160mesh，20．06ゑ／．somelater experiments were carried out with fractionated flake by sieving．Teng・．Of the totalunsievedsample（totalN7．63％）was weighed Lin a beaker，
200ccL．Of O．，4一％sodium sulfite was added，and the mixtur・e waS filtered with cloth Lafter 1hrn The filtrate was diluted with O．4％sodium sulfite and made to 200cc．Twenty cc Out Of、this volume was decomposed to dete工mine extr・aCted nitrog・en‖ Then sulfuric acid
was added to80cc．of the protein extract to precipitat畠soybean protein，the precipitate was filtered off，and the filtrate，、i。e。Whey，WaS diluted to200cc．；Of which50cc．−pOrtion was put t0analysIS Of nitr・Ogren・ This ser・ies of experiments was performed alwaysin
duplicate．See Table4O．The pH af七er aCidification of the extract was allnearly4・2−4・4 （bytestpapers．）．ThevolumeofO．1Nsulfuricacidaddedper80cc．ofextract wastoム 1arg・e for No．1and No．3，and to Table40・Protein fIOm Naphtha−・De董atted Meal；
obtaintheaveragevalueofprecipitated
ExtIaCtedwithSodiumSulfite，andPrecIPiIatedwith Sulfuric Acid
nitrogen，these twowere excluded・This
ⅠVol．or O，1
tableshowsthatinthecase when the N。．lExtd，．N力持扇毎’孟dedlwh。yN
Pptd“Ntoち0（：C． Ext．
alkaline extract was completely sepa・Y rated from the residue60．6％ of tota1
nitrogen Of deiatted soybean meallwas
extraCted by a slng・1e treatment with￣
35．8 c（： 31 8 34．4 31．8
Avl46岬6）i
i391（51い2）0．4％sodium sulfite〔20times the weig・ht of the meal〉 forlhr。at the room temperature，and 51．2 ％ of total
The foImeIfiguIe8aIe％N foェraw material，and the
latter figuresin bIaCkets aIe％N divided by763，i、e
totalNin theIaW defatted meal
nitrogen waSpreCipitatedbyproperacidification（topH4l4）・ ■
Experimets No．18．ReJation between the Concentration of Extracting Ag’ent aIld t血e Qnan七ity of Extraeted Ni七rog・en・
AboutlOg．of thedefattedsoybean mealof 40・80mesh（totalN 7”753％）was weighedintOa beaker・Onehundredcc・Ofsodiumsulfitesolutionof various concentrations was added and the mixture was a1lowed foz・1hr．at the room temperature（about250）
aftermoderate stirring fotinitia12min・The protein extraCtWaS filter−ed by filter paper andふitrogenwaSdeterminedinthefiltrate（Table41）い工nthecaseofthetreatmentwith O％sodium sulfite，i．e．distilledt water（No。8）the filtrate wa．s turbid and much different
208
鉦om other cases． Be−
tween O．1andOu5％，the quantityofextractedni・ trOg■enincreaseswithin・
CreaSingCOnCentrations，
b11t the increase from
O・3t00．4％■is speciatly
；narked．For the concenN
trations of O．5，1，and 2％the quantity of ex− Sat6・The author a即eeS
Tadle41．ConcentIa、tion of Sodium Sulfite Solution vs”Quantity of Extracted Nitzogen
N
No・l慧芯：如3lsampl潤羞Iel耳血Nl諾ミ。l蒜慧謡惜梵慧誌）
tractednitr・Ogenincreases scarcely・This was observcd also by S・
with him to decide that the O．426solutionis of the most suitable concentration for t壇 yield and quality of the soybe？n prOtein・
Exerimen七sNo・・19・Rejation b9tⅥ・een the Extractiop Time and theQuantity
￠f】≡ⅩtraCted Ni毎ogeIl．
About ユO gI．Of the defatted soybeanmealof 40・80mesh（tOtalN7・34％）was
weig・hedina beaker，andlOOcc・Of O・4％■sodiumsll二fitesolution walS added”Two−min・
＄tirring wasperformedin each2Omin・．Of extraction at theroomtemp＄rature（about240）
′￣
（Table42）．Among3ca声eS， theincreaseinthe qtlEintityof exJ：raCtedト n；trogenlletWeen t壬1e qXtⅠaCtion of40min・and Of60min．wa、SlaTgeで・The Optimum extraction time was put to be60min・ Table42“ExtIaCtion Time〝・Squant中、Of ExtIacted NiItogen Ext？・N【慧s諾ムding ltolOOぢ・Sample （Extd・N）／ （Nin sa−叩1e） ％ 77．11 79，37 80．88 g 5 662 5．828 5．939 】00005 100001
g一・ 10．0015
描
ReJatiollbetlleen the Volume of Extracting Agent and
Exp￠rimeIltSⅣ0，20． tlle Quantity of Extracted N■itroge71・
AboutlO g．Of the defattedmealof40q80mesh（totalN 7・343％）was measured intb a suitablel〕eakeI．The O．，4％sodium sulfite solution of 5・50 times as much as the m由1was adde亘”Nitrogen was determined on a1iquots of theproteindisper’Sion，Obtained by fi．1tration afterlhr．Of extraction．Thedescriptjon，F，10times as much〝 means FFlOO
Table43，Volume o104％Sodium Sul董ite Solution vs
Quantity of ExtIaCted Nitrogen（1hr・）・
No No．of− times Vol．，CC Extd−N ea】cd．to mg．／100cc・・eXt（射 TotalextdN／ 10g sample Extd．N／Nin sample， ％、（B） Vol．min11S20cc Nin tbat vol，mg 〝／Nin
．
N′in that vdl、，mg・ 〝／Nin sample，％（D）
CC・Of pqptlZlngSOlution tolO g■・Of the meal．T［such expressionis．used throughout this paper・・The result of experimentsis shownin Table43and Fig・4”Tt was found that the
Fig′ 4・Volume of ExtIaCtant Solution veYSVS Quantity of Extacted Nitrogen quantity of extracted nitrog■enin the
Same VOlume of dispersion was gTadually
decreaslr塔aSthevolumeofsodiumsulfite SOlution、increased，i．．e．the concentr・ation
Of proteinin the disper−Sion decreaSed
（the curVe Ain Fig．4）and that the
quantity of extraCted nitrogen preSentin
thetotalvolumeof thedispersiongenera・11y decrea占ed（the curvel∋in Fig・．4．）．Butit isimpossible′tOSepar・ate the whole volume
Of the extract from the residue．Now
SuppOSe that there remain20cc．ofextract
with the residue（then，the sample used being・10g．，the residueis wet with67％ Ofthe extract．），′and−We get the curve C Of Fig・L4・Then thequantity of extracted nitr・OgenSeParatedincreases fromthecase
Of6times to that of25 times，Whileit
decreasesin the cases of30and50times．
％80 ・p意；［ 伽 BCD
．⋮粥A
o ○ 加 700 so 500 ヰ 100 ＄ l0 1S 20 受530 50Vol11me Of extIa（）tant SOlution tlmeS．
When the separ・ation of the extract from the residueis moreincomplete，and30cc．of the extractisI・etained with the residue（then th residue willbe wet with75％of the extract．）， we get the curve D of Fig■・4・The g・eneralaspectis similar to the curve C．
Discussi’0？10f ExperimentS No小20・・ttis concluded thatincrIeaSing・the volume of SOdium sulfite solulion to over25timesisnoteffectivein a slng・1e extraction巾Itis advisable to use sodium sulfite solution of6to10tines as much 乙S the meal，and to make the Separaltion of the extr’aCt from the residue asCOmplete ns possible．RecentliBECKEL，BE上rER， and SMIIH’1）studied the effect of wateI−flakcIatio on pilot−plant p70duction of soybean pzotein LIOふ the economic standpoint，andfound that，e‖g，When the pIICe Of proteinis20centsper poundthe proper wateI−
flakeIatio foImaXimum profit was appIOXimatelyll：1with slight dependence on the p＝Ce Of mealbut at
lO cents per pound theIatio was20：1withincIeaSed mealp11PeinfluenceThis couclusionis partly
understandable also judged from the cuIVeS C and D of Fig4UMEMOrO，SocABE，andINOKUI王61）used the
cxtracting agents（water，01％sodium hyd10Ⅹide，0IO016％sodium sul董ide）30 times the weight of the alcoholysis meal（2S−26）in oIder toobtain cleaISuPeInatantliquidamounting to more than80％of the volulne int；。d。C。d．HムweveI，they foundthatinthecaseoio‖3％sodium sulfite extIaCtant】Otimeslatherthah 30times the weight of the mealshouldbe used to obtain supelior pzoduct
Experiment＄ No．21．Effects of Repeated Extractions on tlle Quantity of
Extracted Nitrogerl．
Z；；trOductio；3tO瓦ⅩPeI・iments No。21．Itis the mostimportant point fT・Om the．prac− ticalviewpointtO deteェmine the number of repeated extractions（because a single extrac−
tion with sodium sulfite solutionis ncver complete）and the volu皿e Of sodium sulfite SOlution tQ be used”Hereitis necessary to know（1ノ）tbelimit of the vollユne Of sodium sulfite solution to be used when the volume（ntlmber of timeS）increases，and（2）the ratio
210
0f the quantity of extracted nitrogen Of eaCh sing1e extraCtion（e．g．withlO times as much）to the toは1quantity of extracted nitrogen Of，Say，five repeated extraCtions with
the sameVOlume of sodium sulfite solution．（1）From the preceding Experiments No．20， itis clear that，When a slng1e ex七r・aCtionis performedin order・tOincrease extracted nitrogen，makingthe volume applied toolarg・eis not advisat）1e，and that the separation Oftheextractfrom the residue has more pronounCed effect．Ifthe separationwere comblete，
the extracted nitrogen wOuld become morewith thelVOlume of sodium sulfite solution as
smallas5，6，Or8times，andit should be near・1y the same with the volume of sodium Sulfite solution oflO，15，20，and25times（see the curve B of Fig・4）・When the separa− tionis nearly practical，thee＄traCtednitrog・enincreaseswithincreaslng・vOlumeofsodium sulfite solution明）tO25time＄．ttis not at a11rationalto use more than 25 times．（2） The result of the experiment of5repeated extractions with＞0。4％ sodium sulfite．solut主on oflO times as much as the naphtha・eXtraCted defatted mealof50・80mesh（tota17．58％） by M・NONOYANfA（38）is asin Table44u When the′eXtraCtions are 貰・epeated，eaCh with the volumelO times，aS mueh as83％of totalextracted nitI・Ogenis extractedin the first
Table44・，ExtIaCtions Repeated5Times， EacllWitb the VolumelO Times as MucllaS Meal（NoNOYAMA（S8り
extraction，and we g■et Only neg■1ectable amountof nitrogen
extracted in the 4th and 5th
e又は・aCtion5．f’J・Om tbese and Other considerations the author
decidedto experiment on the3
repeatedextractions，first with
8times，SeCOndand third with6
n total，in order to save the
Totali61・31l83い2 100 O 1 5 03 1 66．3
times each，uSing20times asmuch extracting solutioni
extracting agent a・nd to accomplish sufficient extraction・
PγOCedure of FxperimentS Nou 21・Theprocedureisshownschematica11yasfollows：
・−Rl十300cc．0．4．％Na2SO8朝
一El＋H2SO4−トニ訂ein
50g・．unSievedmeal＋400cc・0・4％Na2＄08−
（tota1N7．634％．） teinN・R8十400cc・0・2％NaO王トl認・H2S叫認
−E8・H2SO4−E諾： 、‥￣−−ニ＝∴二∴。十
Each extraction was carried out forlhr．at the room temperature（July）．The4th extractionwith O”2％1sodium hydroxideis an additionalprocedure・The volume of 甲Ch
extract was measured．Some dilute sulfuric acid was added to a part of the filtered
extract，and the quantity of the aCid added was calculated as the numt）er・Of cc・Of ユ兄 H2SO4＝tOIL of extract・The pH after addition of acidwas made4巾2・4小4（BlC・G・） Nitrogen wasdeterminedihlO ccい Of each extract andof whey（filtatefr’OmPrOteincurd）
Result．ThevolumeoftheextractfilteredandthedegTeeOfacidificationare shown
inTable45．When the quantity of sulfuric acid added to the extract was calculated as
cc，Ofl％■acid per L．extract，it was about 300 for the first extr．act（El），and about 130−150 f6r the second and third extracts（E2and E8）．It was about180 for the4th extract withO．2％NaOH（E4＝）．The extraCtednitrogenand wheynitrogen・WeIe determined in10cc．of each extract（of Nos”1，3，5，and 7 0f Table45．）（Columnsland40f
Table45．Process of the Expeliments，E叩eCially thc DegIee Of Acidification・
was ca1culated as the diffeT・enCe between nitrog・eninlO Table46）・Nitrog・en preCipitated
cc．extr・aCt and nitrogenin whey fromlO cc．corresponding・extraCt・Extracted nitroien andprecipitated nitrog・en were Calculated for the volume bf eachliquid filtered，andtheTl agraincalculatedas％fo方・tOtalnitrogenin the or・ig・inalsample（Columns2and50f Table 46）ル ThepercentageOf eachvalue to the sum of thelst，2nd，and3rdextractionsi畠shown in ColtlmnS3and60f Ta，ble4：6．
TAble46．Data foI3Successive Extractions with On4％Sodium Sulfite（and the4th Extractidn with O、2％Sodium HydIOXide）
5 f 6 1 l 2 J 3 Pptd．N Extd．∴N Whey N mg」、／10cc・ mgい′10cc lグるt気tOtal】％竃1：貰Of ％to sⅥm Wl lO．46 W2 357 W＄ 1・74 W4 1・23 E1 4290 E2 13い09 E8 5‖30 E4 8‖23
Sodium Carbonate as the Extracting AgeIlt
Exp￠rimemtsⅣ0．22．Reはtion betw恍n the Concentra七iom．of Sodillm（プarbonate
aIld the Quantity of Extracted Nitrogell・
AboutlOg．Of naphtha・eXtraCted soybean mealof30−80 mesh（totalN7・871％）
was measured into a beaker and100cc．ofsodiumcarbonate soltltion of various concentra・ tionswasadded，andthemixture
was a，1lowed to sta．nd fo工・1bI・． at the room temperature（15− 170），and filtered with filter
Table4．7小 Concentration of Sodium CaIbonate vs Quantity of ExtIa℃ted Nitrogen（10 Times，1hrい）
paper．NitrogenwaSdeterminedinaliquots of this filtered extract・（Table47）
Sodiu．m昔王y血0Ⅹide as t血e Extraetimg・Agemt
ExperimentsNo．2吉．ReJation betⅥeen t血e Concentration ofSodium Hydroxide
SoJutiolland the Quantity of ETtraCted Nitrogen
About5g・小 Of naphtha−eXtraCtedmealof30−80mesh（totalN7・87％）was treated
withlOO cc．（20times）of sodium hydroxide solution of various concentration for・1hr
at the room temperature（MaICh）（Nos．l・4，Table48）・The sample for the other exper−
21．2
imentS（Nos．5・・9，Table48．）was tr・ichloroethylene−eXtraCted meallof 30・I80mesh（total N8・02％）；the peptization process was the same asin the former grOup Of exper・iments．
Table481Conr，entIation of Sodium HydIOXide vs
Quantity of Extracted NitIOgen・（20Times，1hr”）
Discussion of ExperimentS
No．23．When sodium hydroxide。一エ
WaS uSed as the extractingagent No
ConcnOf NaOH鱒ⅩtdlN／100g一SamP呵Extd．N／Nin samplefor soybean protein，the quantity
Of e史旺・aCted nitr・Ogen increased
O 005 0，05 01 0．2
gTeatly as the concentration of ＿二＿竺 5 O 05
0．1 02 0．3 0．4 4 85 6 17 6．08 ち78 5．28 50810U O7 52 5 ′0777′0 SOdiu叩hydroxidesolutionincreased up to O・2％，
decrease as the conqentr・ationin− 9］
CreaSe4aboveO・2％・ltisnotbelievedtodecre笹Se SubstantiallyinhigherICOnCentratipnsof SOdiumhydroxide（cf・Table49）・The errormayhavearisenfrom thefacttflat theextracts With sodiumhydroxide solutionw9re tO？Vi声COuS tO glVeSufficient volume of clear filtrate by filter paper・At any rate theincrease of extractingpOYIer Of sodium hydroxide solution OVer O・2％may be concluded to be very small・Itis naturally clear that the pH valueis
Of gr・eaterimportance than the concentration of sodiumhydroxidesolution used・According to A・K・SMITfr and SlJ・CIRCLE（4：う）the relation between thepHofthepr・Otein extract
with sodium hydroxide and the perce叫age of extracted nitrog■en was as follows：
pH ofextla￠t 80 910 10・0 1】0 1210
％N extd 90．8 り2．8 94．5 95．4 96、4
Compar主son of Sodium S山fite，Soditlm Carbonatb，and Sddium Hydroxide as
the Extrae七ing Agen七
Experiments No．24一 To5glOf naphtha−defatted soybean flake of 30・・80 mesh
（totalN7．871先）waSaddedlOO cc… Of thesolutionof the extf・aCting・agrent∴Themixture
wasallowedtdstandforlhr．at・heroomtemperature（18−20◇）and filteredwith filter
paper・Nitrogen WaSdeterminedin100cc・Ofthe、filtrate・SeeTable
extracteく旦nitrogen was thellighest with Tablc49・ComparisonoiSodium Sulfite，Sodium
Carbonate，and Sodium HydIOXide（2P Times．）
sodium】1ydroxide andthelowest with sodium carbonヨte．，In the caSe Of sodium hydroxide the conce叫ration need not be higher than Ou2％LT（cf．the preceding Experiments No． 23）．Theincreasing ratio of the extracted nitrogen from O′2 to Oo4％■is ghreater for sodium sulfite than for sodium carbonate
Additionalcomparative observations weremade about（■a∴）the viscosityof the extract and（b）the quantityof sulfuric acid tO preCipitateptotein‖（a）Viscosity Of the protein−
aceou§ extract：Te．n cc．of somewhat turIbid extract filtered with cloth was takenin an Table50．Viscoslty Of the ExtIaCt．
Ostwaldviscosimeter（’Tabl与50）・＝
These figures of relative viscosity
Time of dropping，′See． Rel Viscosity Observed values F Av．Clearly show the diEficulty of ma・ DibtdHけ0 ′0′0ワ∵0ノ 3 ′h﹀′0っ・0ノ 2413 ′0′070ノ 5′D l l ′0′0 0ノ 2‘U ﹁▲
雨pulation（e”g・，filtIation）ofthe Extu withOl2％Na℡SO8 〝 〝 〝 NaゴCO男
ヶ 〝 NaOIす
213
hydroxideand，tO alesser degree，Withsodium carbonate・（b）The quantity ofsulfuricabid necessa・ry tO preCipitate soybean protein from each extract：h the Table5l，tO′a CC．Of each extract was added b cc．of O・1Nsulfuric acid to pH4・6．The value cis the volume in cc．of O．1N sulfuric acid forlOO cc．of the
extract accordingtO the equation，a：b ＝100： Table51一Nece＄Sary Amount of Acid
c．T．t became evidnet from these observations， iacc・一bcc・
Ext．with ′′ ′′ OUOU 岬岬 a N ％％％％ 4224 0000
that（1）the concentration of sodium hydroxide and the quantity of sulfur・ic acid necessary were 〝 〃 indirectproportion；（2）the extract with sodium
Carbonate required the same quantity ofsulfuric acidas the extractwith sodium hydrokide
Of the same concentration；and（3）the extr・aCt With sodium sulfiteI・equired only・18％ of
sulfuric acid compared to that With sodium hydroxide of the sELme COnCentration．
Mixture of Sodium Su5fite and Sodium Hydroxide as the E反tractingAgent
ExperimentsNoo 25・The experience showed that sodium sulfite gave soybean
protein of the highest plasticity，While sodium hydroxide g・arVe the highest yield of the
pr・Oteinl・Thus the mixed solution of these two extractants was examined．S．Sat6（44）used the mixture of O・20％sodium sulfite and O・24＝｛％sodium hydroxideけ（1）At fir・St the author
COmpar・ed the extracting■ pOwer・Of the mixture of the equalvolumes of O．2％ sodium
hydroxideandO・4％’sodiumsulfite，0・2％hydroxide and O・3％sulfite，and O．2％hydroxide
and O・2％rsulfite・The result（Table52）was alittleirr・egulaT，but was convincingtO
make further comparisons uslngthe mixtures with O・2％■ sodium sulfite ratherthan those
with O．4％sulfite・The sample was trichloroethylene・defatted meal（totalN 8．023％）．
ThemiⅩtureOf5g■‖OfmealandlO〕cc・Of Table52∴MixtuIeOfO．2％Sodium HydIOXidepl
extracting・SOlution was filtered＼afterlhr； 04，Ol3，OIOl｝2％Sodium Sulfite・（20Tihes） Withfilterpaperandnitrog■enwaSdetermined N。。i■NaOH
Ext完N，愕島1ご／㌫in
NaクSO8 in・20 cc．of the fi血・ate．（−2．）T’he simila，T
estimation was;nade for the mixtules off various ratios of two extract孔ntS（Table53）．
I．tis advisable to use the mixture of No．6 （−hydroxide 20％ pluS Sulfite80％）or No，7 （’hydroxide40％■pulssulfite60％）ifanymiⅩ− ture be desirIable，because the other mixtur・eS （No．’9、and8）would g・ive th昌proteinof inferior plasticity without anナ substantial increasein the yields
〝 fO・2ク右〝
Table53′ Mixtu工e Of Sodium HydIOXide and Sodium Sllliite（20Times）
S鱒mmaryQf’Ex少erime；ltS凡）S17q25小 Amongthe a1kaline extracting agentS eX一
年mined，i・e・，SOdiumsuliite，SOdium carbonate，andsodium hydroxide，（1）0・4％ sodium
Sulfite solution was the bestinthe quality of the product，in the easiness of manipulation
（the viscosity ofthe extract was thelowest（Table50）），andin the smallest quantity of the acid needed（Table51）；（2）0．．2％sodium hydroxide solution was the most desirable When the maximum yieldlほSthe only object（Experiments No．23）；and〔3）the mixture
214
0f・0・2％sodiumhydroxibdeand O．2％P sodiumsulfite（2：80主4：6．）wouldbeadvisable’in SOmeCaSe（’ExperimentsNo．25）uTheoptimumconditionsofextracting・prOteinfr・Om themeal Were determined“（1）The concentration of sodium・Sulfite solution should be O．4％■（Exper・ iments No・18，Table4ユ）；（2）the extraction sholコ1d be for60min‖atthe100mlempera・ture
（ExperimentS No．19，Table42）；（3）the volume of the extracting solution should be5・8 times as much as the meal，in E！Sing1e extra・Ction（Experiments No：20，Table’43）；（4） itis ne9eSSary tOmake the sep二i・且tion of the extract 土工01n the residue as complete as possible（■Experiments No・20）；（5）the volume of the extracting・SOlution more than25 timesisnot
（0．f8，6，and6times）were performed，52％of the totalnitrogen was extracted，73．5％ Of which was extrac七ed at the first ex七raCtion（■Table46）u These findingS COnfir：med the
results of S巾 SATe．．（44）Itis claimedina recent paten｝t（18a）that the ad−dition of sodium acetateto the alkali used foI dissolving the protein causes the gummy and muciIaginous impuIities to agglomerate and
Settle to the bottom of the vat，allowing the cleaISOlution of the pIOtein to be decanted o仔i0Ilater
p工eCユPitatio氾WitllaCid
Precipitation of Protein．
Zntroduction．ttis wellknown that the solution or，mOre COrreCtly，dispersion of the proteinis minimalatitsisoelectr・ic point．Thus the alka1ine extractshould be acidified
to叫eisoelectric point to get the maximalyield of the protein precipita・ted．Butit should be noted here（1）that the＄Oybean pr・Oteinis not a sing・1e compound ofa definite physical
pr・Operty，andノ（2）、that the soybeanitself manifests so−Called biologica1fluctu．ation・ Theざ■寧fore we cannot settle a certaiTlisoelectric pointfor the soybean protein，but ra・ther
We maymention spme SetS Ofisoelectric rang・e fQr the soybean proteins o至 such and such
Varieties under such and such conditions．Many exper・iments andlong experience tellus that theisoelec七r・icrange of thesoybeanproteinlies betweenpH4and5，OrmOrenarrOWly betveen pH4・2alnd4．6．Some experiments were tried with a potentiometer，butin many CaSeithe pH test papers（B”C・G・Or bromophenolblue）of T6y6Filter Paper Co．′，Ltd・， WaS relied upon．
Experimen七sⅣ0・28一′ T血￠iso壷etriepointofsoybeanpro七einbyt血ema軍imum precipitation method．（h collaboration with HidetosiIwAMOTO（2Sa））
The saTq）1e mealwas obtainedbyextr・aCting residualoilwithnaphtha（b・p，60−90O） fromhalfqdefattedflake（by cold pressing− w坤the pressure of 20001b・／sq・in・）・The
SOybeanis the1948crop from Hokkaido．The defatted mealcontained water8．55，Crude fatl．80，Crude protein51．57，fiber4、30，and ash5．23％■巾
Protein disper’Sion of the fir・St eXtraCtion was obtained by filteringthe mixture of
defatted mealand O。4％sodium sulfite．Addition oflO cc．of this dispersion tolO cc．of the MACILVANEbuffer solution（pH3”0−6・0）ra・ised the pH‘，e．g・．，from 3・・O to3・・4．Even
when only O。5cc．dispersion was added tolO cc．of the buffer soluti6n，there occurred the rねise of pH valuesこ Thu＄this method was not suitable
TolO cc・Of proteindispersionl。0−12．O cc．of o．1Nsulfuric acid was added，and the pH by test palperS and the☆eig・ht of the preciptated protein weremeasured（Table54，
Table54．Weight of Precipitated PIOtein fIOmlO cc．of PIOt占in Dispersion．
method，the nitrogen remalnlngln Whey was
measured．TotalnitrogeninlO cc小 Of this protein dispersion was 45・95mg・In Table 55，the ratio of precipitated nitrogenis cal・ culated from（45．95・Whey N．）／45・95・ Table55．PIeCipitated NitIOgen Weight of precipitatt，，mg・
No几諾霊IpH
1 蓼 2l3lAveIage
000︵U O O O O OハV nV O l小9仙34■れ′07000ハ︵U 1 1 1 ．−】 58．4 00U428′04ウ︼0∠U40 ′0−ゝ良5444443︵aへa 123■45′070U OノO12 1 1 1 ■ − ■ ■ ■ − ■ ■ −− ■ − 1 1 1 1 1 1 1 1 1 1 1 1 177．．7 214い6 239．．9 258．3 269い4 277い1 26．6．∧4 258．7 242い9 230：5 210．0 〓一一〓 〓一一∵∵こ∴二∴∵二 ∵∵∵∵
Acid。：チded，l温帯Sured
5 70ノ500 030ノ035 7／05431 4／08503 122 222222 ︶︶︶︶︶︶︶︶︶︶︶︶︶︶︶ヽ−′︶︶︶︶︶︶︶︶ 603603605928162816282528 221︵UハリO／8nO7ノb′05544332？−1100q′ ． 5．鼠5554444444444▲■4444444。3 ︵︵︵︵︵︵︵︵l︵︵︵︵︵︵︵︵︵︵︵︵︵︵︵ 22200000nOの0ノb′0′○′D44■A−42222ハリOO555説55444444444▲・44444444
4310987︰b43 3333▲22222222111111122n∠22
4′0ハ07845485q′500／′O l′073310／100 0ノつJ q′875329′O1040003q′′05873′00000000000000000000000000 024′O nO O24‘U OUO2468024′08024′O
l ル ● ● ◆ ● ハ ◆ ● ● − ● “ I い ぃ い い ● 岬 ● ひ ■ 444445555S‘U‘UよU6∠U777778888 12345′0700q′O12345／078q′O1234 111111111■J22 2ウ］ 2 ＿ 00000000ハU O O O O O O O O O O O O O O O ′02004−0∠U2840′D20040′D20040′0284 〇11233各45661778990〇1223134 1 1 1 1 1 1 1 1 ∠U′0555．5554144ム14443．a33132222 208′044208′0／044208′04208′042 12へ∂41∂′b7の0︵‖ソO12345‘リア80／O lウ］へ∂．皿︻ l l l l l l l l l1 2 2222 t t − ■ ■ ■ ● ■ ■ 一 − I −一 222222222222222222222222 820ノ82 5 78′D7′05
（Note：PH（calcd一）is the value calculated by the gIapllic method‖）
Exp￠riments No．27．T血eIsoele￠tri￠ 208．劉 210．31 203．5
Poin七ofSoybeanProtein by t血e Miniml互m Solubi且i七y：Me七血od・（tn CollaboI−ation with
由idetosilwAMOTO（28a））
Thesampleforthisseriesofexper・imentSWaSpreparedfromdefattedsoybeanmealof
the preceding Exper・iments by dispersing
Table56。WeightofInsoluble MatteIandwithO．4％sodium sulfite，preCipitatingwith dilute sulfuric acid at pH 4．4，filtering，
DissoIved NitIOgen・
Dissolved N mg・／g・ Wt．ofinsol．mattel
washing with water（pH6．6．），and air・No・1pH
2 1 3 1Av一．聖跡 dr・ying．This preparation contained water
lO小06，N12“68，and ash 2・42％’・
tnto12test tubes contalnlng10cc・ of the buffer solution of S6RENSEN（’pH3．6 q5．8）0．1g・．eaCh of protein preparation was added． A壬terlhr．the mixture was filtered and to the filtrate（2cc．）0．5cc． Of］0％ sodium tungState SOlution was added．The turbidity of the mixtur’e waS
compared．The minimum turbidity occurred
0′0245′04
4444447 ハリ0／0ノ0／0ノq′q′
︵︵︵︵︵︵
aへ∂︵∂33444 024′n﹀ハ0024亡UnO O24′0
1234−b6700q′O1234 1 1 1 1 1
（Note：thevaluein parentheses excludedin averagingい） at pH4。6，Which must be theisoelectric point・
216
To20cc．of the blユffer solution oE MACILVANE（pH3・0・r5・・6）lor O”5g小Of protein
preparation was added．After5hrs… Of gentle shaking■the welght ofinsoIuble matter．．pwas measured（3measurements）・Besides，the dissolved nitrOgenⅥ苧1S determin箪du The data （Table56）indicate theisoelectr・icpo三nt of soybean acid・preC；pitatedprote：n to be pH4・6・
Discussion．”TheliteIature Values ofisoelctIic point of 占oyhcan protein aIe COllectedin the accompanylng tableThisis arIanged chronologlCallyScientific studies conceIn Chiefly the globuliTl，glyclnin， wムichhasthei＄OelectIicpointpIi47・5．4t；itisinteIeSting tonotethat the olde；t value（1926）（9）isthe
lowest（4l7），Whilelatestvalue（1950）封is the highest（54）；OtheIValues are pIi49（4；），50（18），5・2（29）， and5l32（37）‖ TheisoelectIic point pH50 was accepted by HoRVA＝ト甘，though he TePOItedit to be pH
5￠pIeViou＄1ylThevalue5O was accepted also by WA＝S‘53）h NAKAZIMA37）IePOIted that denatuIed glyclnin Show￠申loweIisoelectIic point（pH486）than nondenatured（5l32）1According to BRrGGS and MANN（4）at lea？t7electIOphoretically distinct p10teins weIe PreSentin aqueous extIaCt Of deiatted soybean meal，COn− taining95％of totalnitIOgen；an electIOPhoIetica11y homogelleOuS prOtCinIePIeSenting60％ of the g】obulin fIaCtion，PreCIPitated by cooling an aqueous extIaCt Oithe meal，ShowedisoelectIic polnt at PH514TADO− RoRO′49）reported，that glyclnin had higheIisoelectIic point，thanlegumelin”AccoIding to Suminokura（4き）
The IsoelectIic Point of Soybean PIolein
pH 蔓 Autl10ⅠS CsoNEA（1926）し？ノ JoNES＆CsoN濫A（1932）（29） 2 ′ケ HARIMAN＆CHENG（1936J（lS） Ⅵ7ArrS（ユ937）（52） SuMINOXURA（1939）りり ′′ †′ ′′ ノケ CIRCIE＆SMI柑（】941）（8） ノケ ′′ 0 ノケ IsII＆KAWAMURA（1949）（27〉 BRIGGS＆MANN（1950）（4） Solubility Solubility Solubility ノ′ Mass transpoIt，Solubility， CataphoIeSis Solubility
Viscosity，Conductivity l′
〝 ′′ ′′ Mic工OelecいOphoIeSis，Sol11bility ′′ ′′ Sol11bilit〉 〝 Solllbility 4り7 5い2 5 4．86 5．0 4い】 4け5◆5．0 4．2＿4り4 4．9 45＿4り7 34−38 4．1 4い6 43 43 4り34 44 5．4 qlycinin qlycinin Glycinin（nondenatured・） Glycinin（denaturpd） Gly￠inin Total，nOndenatu軍edTotal，Of soybeans stoIed2months
〝 8 months G1y戸車nln Gl11telin Phaseolin Total Indusいial A￠idてpptd・ Albumin alfa Albumin beta Tムtal Glycinin jMicIOelectlOphoIeSis
theIeチre2albumin＄in the soybean，and slbumin alla（corIeSpOndingtolegumelino10sBORNE41））had isムelectIic point pH430，and几1bumin beta（coIreSPOnding to soylegumelin oiMuRAMAIU36J）had that at pH
4。34。These2values azeloweIthan the value for glycinin（pH4l9）
Theisoelectric point oithe totalprotein oL nondenatuIed deLatted soybean mcalis pH4」1according to wAIIS：52）and CIRCLE and SMlrHSl小 HowevelWe Obtained the value4、4confiImlng OuIfozmeIreSult（2；）・ Although ouImethods are not so delicate，aS those oiAmezican authors，We Wish to note that de董atted soybean mealstoIed董0Ⅰ8months hadisoelectric point pH各2−4・4，COmPaIed to pH4l5−5・O Lor the meal stoIed foIOnly2months（47）一 Difference＄in thelnateIials and method should also be considered
Americanindu＄tIialprotein hadisoelcctIic point pH4、6accordingto CTRCIE and SM＝欄S）”The acid− precipitat9d proteinhad the point pH4l3according to the same authors・Ou＝eSultis pH4”6，JoNESand CsoNKA′2O）noted，that thcisoelectlic point of glyclnin was higheIthan that of nonfIaCtionated preparation The morc the purity of preparationisIaised，the higheltheisoelectIic point becomeslAIso we see・that theisoelectIic point has the tendency to be higher，When the prepaIationIeCeivesless del−atur、ation
C担APTER5小SM二阜‡乱一SCAIJ瓦MANUFACTUREOFSOYBEANPRQTEIN
When the2uthorノbelon三・ed t．othe Mansy丘Daidu K6gy6K．K．（ManchuriaSoybean I．ndustrialCoい，Ltd．），Kawasaki，Small−SCalemanufacture of soybeanproteinⅥaSpraCticed ヰuiingmorethanoneyear（1937・・・38）accordingtOhismethodincollaborati6nwithLMAEDA
and K．00MI．The pr・Oductsl∼ere uSedin affiliatedlaborator・ies aS the raw materia1fof soybeang・1ue（30），SOybean plastics，（拘 sizing・materia1fo‡COated paper，（21）and artificial Ⅵ001．（5G−57）The manufacturing pr・OCeSSⅥ・aS put tO apilotplant，Which was opera・ted t〉y K
0NO（30）and A．TAKANI（5｛｝）for some years（1938−1944）．At p土eSentindustria1utilization of SOybean proteinis notCOnSid由・edinJapan，butitis much studied and practicedin・the United States of America（E）1a）
Abridg・ed Record of SmaJB−ScaJe〕Ⅶanufac餌re of Soybean Protein．See Table 571r The raw mater・iE！1defatted soybean flakeused daily amoumed to a half to severa1
Table57h Sma11・Scale Manufactule Of Soybean Protein． 、、
−
−、
No．of
ChaIgeS Av）rield Method
315kg 365 46 64 64 55 S，（S−Ac） S S，（H−S） S，（S−Ac） S 408わ085 22222 リ︼ 4タあ 4 6 9 ．1 8 S−S，（S n−S， 軋甘 S 説 H−S，（S 5 5 53224
′“U32 7′hU 1 1 1 1 5dノ5とU51 0ハU 700−／ 2 5′O1 51 222222 3 7′八U2 3 2 14ウ︼13 5 SSC S
S，（C−Cl） Cl，（S−Ac） S，S−CI Cl，（S−S） Cl 52 213 1 121 1 568 i144．70
Note：The method of manufactuIeislepIeSented by palrS Of sYmbol＄，OL wもich the foImeIi8 forthe extIaCtig agent（＄：SOdium sulfite，H：Sodium hydroxide，C：SOdium caIbonate，M：miⅩtu−e Of sulfite and hYdroxide），and thelatter＆iteI the hyphe】】iき joIthe precipitating agent（S：SulfuIic acid，Sl： Sulfu10uS aCid，Ac：aCetic acid，and Cl：hydrochlolic acid）
（mosf often4，andup to6）kilogranlS”l’）uringthesemanuねcturess？me eXperime甲SWere made，aS described below．
l。T血e Ra．w Materia且
Eプゆerime；ltal．Throughout the 罰nanufacture defatted soybean flake（pressed，but not pulveri2；ed）used as the rawmateria1was that obt息ined t）y eXtraCtionⅥith naphtha Qr
trichloroethylene atlower temperature．There were three kinds of themuSed：（1）soybean flake extracted with naphtha at theInstitute for・Physicaland Che皿icalResearch，Tokyo， （2）soybean壬1ake extracted with trichloroethylene about550（the same as the flake used
in Experiments Noり8（3L）），a．nd（3．）soybean flake extracted with trichloroethylene at room temperature（see Experiments No・28）巾
軌叩心・imIle七sⅣ028．Ex七raction of O封fromりT品se七ほ”at Room Tempera毎Te． （1）To5kg・Of the raw materialFjT6setu′′（Part1ydefatted soybean fl乙ke）（31）10L・Of triu（trichloroethyl？ne）Ⅵ，a＄added，and・themiⅩture WaS allowed tO Standat the room tempel・ature（about200．）for24or・48 hrs．，the miscella was separated by pressing，and theoiトextr乙Ctionresidlユe Wa苧WaShed tWice withlOIJ”Of tri・The washed residue wa苧
pressed and dried by free evaporation．The yield and the analysis of the defatted flake
are shownin rrElble58… This result shows that the cold extraction of oilwith tri．is well attained王n24−守S hrs・When the washingis thorough・（2）Some extractions of oilwith tri・ Were perfolユ王ed r！Ot On riT6setu′′，but on rolled soybeans，but on thelatter the extraction
2：18
was carr・ied dut about 550．To 5．、5・7JO kg．Of r011ed soybeans lO1．12 Ⅰ，．Of tri．was added．
The yield of the defatted flake
WaS about 75％−．The soybean pIbtein p工0′due由fr・Om tIli占餌ke ふasノ ＄imila‡・七O tbat froTn tbe flake obtained from／／T6setuPP．（■3）
Tab158．Cold Extraction of Oilhom ＝TeI＄etllI， with TrichloIoethylene T∂set潤S崇？e芸慧！ Weight，uSed oIObtained，kg・ Ratio，％ Analysis：MoistuIe％ Fat，％ Fat，％on moistuIe−fIee、basis
In thelatter half of the manufactur・ing，tO8kg小 Of
〝T6setu′′12L・Of”triclene”（tri・manufacturedby MituiK6zanK．K．（MituiMining・Co．，
Ltd・））was added，andthe coldextraction was continuedfor2・4days．The analysis of
SOmeraw血aterialsisgIivenin Table59
TableS9．AnalysISOf theRaw MateIialsConditions of extn．
t
‡Ⅰ・駄tr批tionofProtein o。
EWerimental・Asthealkalineex−hr・
A
tracting agentOfprotein，SOdiumsulfite（0．4 MoistuIe，％
24148 13‖82 1．33 Fat， ％ TotalN，％ Asll， ％
％solution）wasma血1yused・OtheI・agents
usedノa‡e O．2％ sodium hydroxide，0．4％
SOdium carbonate，Or mixtur・e Of O．2％sodium sulfite plus O．2％sodium hydroxide・（1）In the former period（up to No．151）：0．4％■ sodium Sulfite solution（’or other extrIaCting
ag■en七）of8q5times asmuCh as the weight of the raw material，defatted soybean flake， was used．The3「4extractions each for aboutlhr．were per・formed repeatedly・The mixture WaS first士iltered roughly with wire gauZe Of40mesh，and then extract was filter．ed with a filter press or by other means．（’2）In thelater period（from No・152）：0・4％一 sodium
stllfitesolution（Or Otherextracting agent．）oflO，8，and then5times as much as the weight of the defatted flake was used successively，eaCh extraction being・COntinuedforl hr・The extract was separatedin other vessels，allowed for al〕Out3hrsn，and freed fIOm
insoluble matter，and then decanted．Thb clear supernatantliquid was filtered through CqttOn Or Silk cloth・The filtr・ate waS again sett1ed and freed frominsoluble sediment・
5hMlemeniaryDisc久SSiOn．Theauthor carIidouttheextIaCtionalways atthe room tempezatu工e巾 Theleis a pdtent（19）conceming the extraction temperature：dilute alkaline solutionis added to soybean flake，
the mixtuIC after sufficint swe11ingis fIeeZed，and then warmed‖ UMEMOIO，SoGABE，andINOXUIZ（51）added
SOme aCid to themiⅩture Of defatted flake and alk虫1ito facilitate the filtration（25a）”AccoIdingto Davidson（11） boIaX，alakliphosphates，and sulfites a王e uSed as extIaCtantSin such a manner that the aqueous extract of
the proteinisfiltered thIOugh a filteIbed of the wateI−SWOllen cellulaImateTialof the residue
‡‡Ⅰ巾 Pr￠eipi七atiom．of Pro七ein
ExPeYimental・As the precipitating・agent Of protein，Sulfuric acid was mainly
used。But also sulfurous，aCetic，and hydrochloric acids were used．Dilute acid was added Slowly to the clear extract untilthe pH of the mixturel〕eCame 4．2q4．7一．with sulfuric
acid theisoelectric rangeliedinpH4u2−4・7’（B．C．G．），While with hydrochloric acid the
Opti血alprecipitation seemed to be attaind at pH4．0・4．5（．B．C巾G．）．tt was observed that
the precipitate withhydrochloric acid was coarser than withsulfuric acid・Theprecipitate
Experiments No．29．The Use−Of StlJftRrOuS Acid as the Precipitating Agent．
The precipitaingagent uSed was（1）commerCialsulfurous acid solution，（2）sulfur
dioxide gaS prepared（i．）by droppingdown concentrated sulfuric acid to the concentrated
SOlution of sodium sulfite：Na2SO8＋H2SO4 ＝ Na2SO4＝＋SO2−L鞘2（二），Or（ii）by
heatingrslowly sulfur and concentratedsulfuric acid：2H2SO4＝＋S墓3SO2＋2H20
（ih主hes色2methods，（i）gaVe Sulfur diokide申SmOre eaSili）and（3）sulfuroもs acid
よblutioh，☆hichwas pr・eParedbydissolving sulf最・’dioxide thus obtainedinto wat6r．Th6
qtiahtity of atid 鹿cessary to preCipithte prbteih Table6b・SulfulicandSulfurous Acids a岳
’h
is瓦sinT包ble60．Fr6mthetable，iti畠。1earthat：冒sF
。ne muSt addsulfur。uS aCiddf about3．4 times a岳 NaBSO缶（715times）
血uch as the quantity of sulfuric acid・Tn this case Acidl concn． L vol．iQilantity the p‡寸Of the mixture was about4．4・4”5．
芸……3芸は警5g．′1。。。。．
≡芸5……二1写二……喜二ⅠⅤ．WasllingOf Protein．
EWerimental。The protein aS preCipitated with acid naturally contains soluble impur・ities，eSpeCially theinorganic saltS prOduced by neutrali2，ation・For exampleif the extracting−ag−entis sodium sulfite and the precipitatingagentis sulfuric acid，the preclpl・
tatedproteincurdshouldcontainsodium Sulfate・Therefore，itis verylmPOrtant tOWaShthe PreCipitatedprotein curd thorougIhly with water．・The protein curd was ordinarily washed with pure water3timeS by decantation。The washed protein was filteredwith filterpaper． When hydrochloric acid was added，the pretipitated protein（atpH4．0・4．5．）was a11bwed to settle for15・17hrs”，and the super・natantliquid wasremoved off・Thecoagulatedcurd was＜filtered by naturalfiltr・ation with suitable silk cloth and stirTed with pure water of about3times as much oveI・the filter cloth・The washing wasrepeated3times．After the washing the pH became 5．O−5．2こ Miyazaki（e5）also noted that the protein precipitated
reaChed an equilibrium after washingWith water3times．
VIJryillg Of Protein・
E畑eYime；2tal．Tn thelatter period the soybean protein precipitated and washed was directly delivered tootherplabor・atOry（as raw malterialfor artificialwool）in theform Of paste，butin other casesit was driedin a vacuum drier below 600．
Exp餅imentsN仇 30．封ehydration（〉f七血eProt￠inもy恥eeヱing・．
Precipitatedprotein with75q85％moisture wasputin the coldest place（av・q5O
C．）in an electric refrigerator・．Whenit was allowed to sta，nd for severalhours at this
temperature，it completely freezed・Freezedprotein becamelight yellowishbrown，andwas
easily dehydratedowlng・tO fine cracksu When this was dried，it was nearly transparent
and became harder．When soybean proteinis fr・ee2；ed，itis not only easily dehydrated，
but the product containS deccreased ash，Owlng tO the solution ofinorganic compounds
htothe separatedwater・tnOneeXample，theash content（on air−dry basis）decreチSed
from2．5％to O．83％．See the pa・tent（5B）．
Swlem9nlaryDiscussionAccording to a patent（3，1P）proteirlPIeCipitateis dehydTated by siow
220
is七hawnIAccoIding to anotheIPatent（11）soybean proteinis dIied by passing heated air vertica11y and coun−
t七ICuIIently through a foraminous belt ol−Which the cuIds ale tIaVeling・
VI．YieJd of tlle Product
EWerime；ltal・．The yieldof tIle SOybean protein must be expressed，aS theratキ0 0f protein obt左ined to the totalprotein containedin the raw material．．But here tb．e
percentageoftheweightofprQteinobtained to that of theraw materialusedis calculated
as the yield．The average yieldsfor eachmonth areshownin Table．57．The totalaverage yieldof protein to the raw materialwas25．5％■．Nowtheyields wer・e Calculated according totheprocedur．eofmanufactur？（Table61）・Insomela・terCaSeS，the pr’Oduct was not
Tahle61‖ Yield of the Soyhean PIOtein According to
the MetIlOd of ManufactuIe
Ex略 2ptg‖
agent − agent Range I Average
1−3，10−16，20−3】，a5・−38， 40−51、53二8l，83−88，92−93 4，8−9，1、7−19，32−34，82，89， 90．．91 94＿96 5・−6，52，97 39；98 10ト133 139＿149 153−179 Na2SO8−H2SO4＝ Na2SOg・H9SO4 Na2SO汚−H2SO3 Na2SO8・−AcOH NaOH−H2SOI Na2SOろ−H9SO1 Na2CO乃−H2SO4 Nぬ2SO男一恥SO4 Na2SO只−HCl 1（i7＿214
dried，but weighedaspaste。tnCalculatingthe yield，One・fourth theweightof the paste
WaS taken as the weight of airqdr・ied pTptein・Putting■ the moisture of air・qdried protein
tobel0％，thiscorresponds tomoisturecontent，Of thepaste tobe77．5％．In evaluating
the resultofTable61，the following pointS Should be noted：1。）The protein contents of the raw materia1s were not the same・Generally the trichlor・Oethyene′qeXtraCted soybean
flake containedmore protein，than the naphtha・eXtraCted・Thus the yield of protein was higherin the case of former・2．）The procedure of the pr・Otein man！ユfacutre was also a factor for the yield. Zt was found that the quantity of soybean flake used has some effect
Onthe yield・For theappar・a・tuS uSed（2cxtraCtion vats of 551itefS，12cylinders for
PreCipitating− and washing prOtein of 31iters，and2washingvatS Of 501iters），the
quantity of the flake used for one batch should be4kg．at best．When more flake was used，the yield appEirently decreassed（Table Table62・Yield oiSoybean PIOtein AccoIding to the
Quantity of the Raw MateIialUsed 62）．3．）The yield of protein variedin some
From flakes extd．with
degTee aCCOrding tO the protein−eXtraCting・Quantity ofllake
used foI One batch lTrichloroethylenel Naphtha
agent，but varied scarcely according・tO
theprotein−pr・eClpltatingagent，Withwhich
kg
44 22 31，28り3％ 35，2∈‡5 】6．7 24−26，248％ 18−26，24．8 18．3rather the quality of the product varied．4．）
Asformerlydescribedtheyield for the productaS paSte WaS Calculated supposlng・the
ふatercontentofthepaste tobe77．5％∴But′thepaste or・dinarilycontainedless moisture
than775％（see Table64）・5）Theaverag・eyieldfor thelastpartofmanufacture（Nos．
167−214）was21％・Thislow yieldwas obtai壷d because the filtration ofthe protein
VIIu T血e Qnali七y of t毎Prod11（豆s
Exberime71tal．Thep童・Od血ts manuiactured byordinarymethods・Were rather good in color，adheSive power，and other respects as soybean protein・For example the result Of examinationS Oftheproducts as substitut1ng■materialfor milk caseininthe manufacture
Of coated paperis shownin
Table 63・Sodium hy4roxide as protein・eXtraCting agent No
Table63．The Quality of tbe Soybean Proteinin PapeICoatingl（21）
玩完盲萌「下蒜iein−押鴨‖l蒜 jsolubi叫coloI蔓viscosity
agent
gaVe the highestryield，but
could not be t三Sed forCOated paper・．The mixture of sodium llydT0Ⅹide and sulfite wa．sfaiT・．
′rhe anal†SeS Of the
H2SO4 I12SO, AcOH H2SO4 〝 ＋ ＋ ＋ 十
（三詣。ult）（禁盈）（
寸心igh 一−lowSOybean protein（Table 64）show that on moisturel・free basis ether extractis ordinarily les占thanO・2％，aShcontentabout2斉，andnitrogencontent14・03q15・19鬼（average14・6 ％）（except for the first andlast samples）。
Table64．Analyses oL Some Products一（NoNOYAMA（S8））
On rIeSh basis ヲさ water＿r工ee basis
▼．，．．．．．▼▼＿▼ No ￣ 士b 154 155 210 211 39 Extractant−PIeCipltant
25j
Fat l Asll Moist1Tot．．N上 Fat l Ash Tot。NIN√×6Na9SO8−H2SO4 ノソ ′′ Na2SO8−HCl（paste） 「′ （r′） 〝 （〝） Na2CO8−HCl OU1484′0− 0／∩フ 7 70の0 112120 15 S9 14．．72 14 03 14 59 1礼28 15／19 13．82 97．44 92．．00 87小69 91．14 89 23 94 97 86．38
DiscussionlAmong the acids a＄PrOtein・・PIeCIPltating agents，aCetic acid was uniquein glVlng the PlOductofthehighestviscosit≠・Thiswasohs占ⅠVedals占hy P・DJArSCHENXO」（12）AccoIding
Of soybean pIOtein（dispersedin On2％NaOH）pIeCipitated by hydIOChlolic，SulfuIic，and acetic acids sho示ed viscosity（the Ostwald method）of310，3】1，and410seconds，1eSPeCtively．Sulfurous acid has bleaching
effect；SulLite as the extIaCting agent also has the simi1ar effect．It wasleCentlv obseIVedin the case of Peanut and cottonseed pIOteins、（15）
か套SC〟SSわ花＝げ0紘甜P玩旭読喝」血肌肋冊
NeweIliteIatuIenOt mentioned（0ISufficientlyexplained）by CIRCLE7）noIby BtJRNErIさ∼）is bfiefly IeVi占wed”TheliteratuIeIelatlng tO SOme StePS Of manuiactuIe WaS discussed abovelHcre the method；of modif、ylng SOybean protein and tota11y diflerent methods of pIePaIing soybean pIOtei’n are the subjects’・
Soyb如n protein canhe modilied during alkaline extractionlThus urea wasincoIPOlatedinto the alkaline plOtein dispeISion；the uIea」may be added to the soybean mealp＝Or tO the extraction oiprdtein・0I to the alkaline solution used董0IeXtraCtion oItO the alkaline pIOtein dispeISiont（W）0Ithe aqueous sluIIy Of PIOteinis tIeated with O・1−05gn−mOl′Of a cupIIC OImeICuIic compound・aS CuPIIC nitrate，CuPrlC Sulfate，
cupric chloride，CuPIic oxide，CuPIiC hydIOXide，merCuIic acetate，0ImerCuIic sulfate，and with O・3 to4％ ola gl−mOlOf an alkalimetalsulfite，aS SOdium sr111ite，PerlOO g”Of recoveIable plOtein‖（42）As the
thiId method，hyd工OXide oISulfate of aluminum，ChIOmium，0ICuPPeIis added to the alkaline protein disperSion to obtain soybean pIOtein LoIPlywood adhesive‖（1；2
Protein cuId may be modifiedlAqueous solution oical￠ium chloride oz）nagneSium chloride、、aS added to protein curd，and ca】cium hydIOXidelormed hy adding alkalito′themixtuIe WaS remOVed together with nonpzoteinicimpurities・（5］）01tO the emulsion pIePared bymixing soybean protein cu王d with water， peptizing，芦Oftening，antiseptlC，Or bleaching agent was added，and the mixtuIe WaS SPray・driedl（勅Acidl・ precIPitated pIOtein wa＄mOdified by addition of aluminum sulfate（・11a）
222
0f soybean proteinin dilute acid a calcium salt solublein this solution，added alkalito the mixtuIe，and− filteIed thc calcium hydIOXid＄pIeCIPltate With nonplOteinic 如PuIitiesBLOCK and HowARD2）puIified
soybean globulinやy dissoIvingin alkaliand then pIeCIPitating with sulfuIdioxide・AIeCent Pa．tent（…、（｝） descIibed a method ofIeiiningindustIialsoybean pIOtein hy dispersing the pIOteinin alkalisol11tion，
pIeCipltating the calciumions，SePaIating the c
pIOtein
Two entirely diHeIent methods o董prepaIing soybean plOteinloIadhesive puzpose aェe descrilbed F6r adhesive puIPOSe nOt Only thもsolybean plOteinisolate，butalso the deiattedsoybean flouI‘isusedwidely
Now soybean wheyfIaCtionis not desiIable foIadhesive、INAGAKI，KATAOXA，and YAMADA（22）did not sepaIate insolubleIeSidue afteISteePlng defatted soybean mealin dilute acid or alkali，but added alkalioIaCid to
pェecIPitate protein at pH410・50；wheyiraction was removed andりprotein plu＄insolubleIleSidue〃was dIied and pulvelizedlDAVIDSON（10）IeCOVered protein and hemicellulose simultaneously hom soybean meal；the soybean mealis digested with Oh4％sodium hydroxide solution（41％hased on the meal）and the−dispeISion PaSSed through a200・−meSh screen to remove cellulose；theIeSultingliquidis acidified to pH 4′5 and the
PIOtein and hemice11ulose，Simultaneously pleCIPltated，aIe WaShed and dIiedlThe pIOduct was supeIior to
；oybean
SU】ⅦMARY
The extrゑction of soybean protein fromdefatted flakewith alkaline agentS，Chiefly Sbium sulfite solution，WaS Studiedin detailto develop the most suitable pr・OCedue．・Maxi− mum yield of soybean protein was attained at pH 4．4・Theisoelectric point of soybean pr■Oteinsis discussed
Small・SCale m包nufacture of soybean protein was carried out for about one year；
213chargeS Of O．5・6lO kg・・defatted flake daily gaVe mOrethan145kg．soybean casein． ExpeIiences regardin三eaCh step of manufa6tur・e are described，andliterature r・eView of
manufacturlng pr・OCeSSeSisincluded・
AC鼠NOW‡J取Ⅰ）GM二ENTS
Th畠 most part of the studies was performedin thelaboratory of the ManSyG Daidu Ki）gy6 K．K・under the guid乳nCe Of thelate Dr・Bunsuke SUZURI，the then Professor of University of Toky’0，and Dr・MasumiKANAIof that Company，tO Whom t
expr・eSS my Sincere thankslMy thanks are due to Professor TaiiiKUROKAMIand Profes・ SOr KenziKATAKURA of Kagawa Agr■iculturalColleg■e，Who gave me the convenience to
COntinue and publish the studies．
Ⅰ．1TERATURE CITEI）
TheliteratureCOnsultedisgiven firstIC・Al＝ChemicalAbsiracis；N・K・S ＝N妙On Kaga貞u
So7an（Complete Abstracts ofJapanese ChemicalLiteratuIeS）
（1）13ECEKl，AlCり，EELTER，P‖ A”，and SMIIfr，AKl，CA40，51684＝（194｝6）；1946；hld・Eng Cゐの明38，、731−4（1946）；（7），297
（2）BLOC】こ，RJ，，and HowARD，H・｝ W・（to BoIden Corp）C，A”43，5136d（1949）；UlS、Patl2，468，−
730（May3，1949）；d・（7），32Ⅰ
（3）BoYER，R」Al，CRUPPI，T、and ATXINSON，WT（to Ford MotoICo），（5），1012；USPat一2，377，−
853（194S）；cfい（16）
（5）BuRNEII，R．S．SoybeanpIOt占inindustrialpIOducts．Sqybean and Sqybean PlOducts，editedbyK
SMARKLEY，2，1003−53（1951）．
（6）CALDWELLりJ．R小（to Ea＄tman Kodak Co介），C・A．i6，629a（1952）；US∴ Pathr2，592，120（ApIi18， 1952）
（7）CIRCIE，S”JIPIOteins and otheInitrogen constituentsSoybeans and Soyうean Producis，edited by K小 S・・MARKIEY，1，275−370（1950）．
（8）CIRCLE，SJ．，and SMIIH，A．X．，（7）333，325；］甥ys．Chem・45，916−30（1941） （9）CsoNKA、，FA，MuRPIIY“J…C‖，and：ToNES，D‖B．，（7），332；JAm．ChBm Soc48，763・8（1926） （10）DAVIDSON，G，CA、43，3663b（1949）；US Pat2，4164，075（Mar18，1949）
（11）I）AVIDSON，G，C・A‖ 44，356d（1950）；U小 SPatl2，479，040（Augt16，1949）
（12）I）JAISC耶NKO，P。（D，YACHENXO，P”）A4bsloboino−ZhiYOVOe DelolO，Noh 8，39（1934）Cited by HEt胤UM，属お紹5f血β〝gβγ払 エβ言明32，369・73（1935）
（13）EBERL，J，J，andlTRELFA，RTい（to Hercules PowdeICo），C A。44，356e（1950）；U・S小 Pat・
2，479，481（Aug・16，1949）
（14）ERICXSON，E、（tol‡er（；ules Powder＜Cor），C”A．44，4604e（1950）；U一SlPatI2，502，134（MaI小 28， 1950）
（J15）FoNTAINE，T．D．，DErWILER，九，S．Bh，andIRVING，TI，GlW・，1nd Eng・Cheml，37，1232・・6（1945）・ （16）FoId MotoICo”Ltd．，C．A．409702（1946）；Brit．Patl559，848（Mar・8，1944）；Cf一（3）・
（17）HAGIWARA，T．，MrYAZA町，Yい，and MIMURO，T．・（to壬Ionen SeiyuKu K），N K Sp 25，9582 （1951）；Japan．Patい Appl，3272（June22，1951）；Japan・Path189，875（Sept巾 25，1951）・
（18）HARIMAN，R．丁い，a】1d CHENG，L T．．，・（7），382；J．物ざ，Cゐβ∽・40，’453−9（1936）・ （18a）馳NMNG，S．臥，C”A…45，4067．f（1951）；U・SlPatl、2，540，669（FebLt6”1951）・
（19）HoRr，S。，ⅠIIKAWA，S．，and MoIOZAKI，S．（to Mansy仏N6san KagakuKu K・），N K・S 20，184（1946） （Pub．1949）；Japan．Pat．168，520叩帆16，1944）・
（20）HoRVArH，A．A．，（7），332；ヱ乃d．E乃g… Cカβ∽・，∧屯紺S＆g・14，500（1936） （21）ⅠIMURA，MitunoIi（Nippon Kak6siK・：K・），pIivate communications
（22）INAGAKI，E“，KATAOEA，K．，and YAMADA，T．（to MitubisiKaseiK6gy6K・K），N K・S・20，303 （1946）（Pub−1950）；Tapan．Pat．168，274叩0vL・8・・1944）・
（23）INOKUTち K，〟㌧麒ぶ．16，537S（1942）∴哨卸商亘通徹卿薇月師伽れ軌伽肋（Reptl・Japan・Acad・ Sci）lE，39ト6（1941）
（24）INOEUTI，R．，互ogγク励gい ZαS5套（．丁．Soc．ChemいInd．，Tapan）47，156−7，157−9（1944）；〟麒・且
18，3617（1944）；C．A．43，5209ゐ（1949）・
（25）INOKUrI，K，荘rrAGAWA，K．，and YAHIRO SI，KogyoKag．Zassi47，235−6，236−9（1944）；NK S
18，537＄（1944）；C，A，43，5209♂（1949）
（25a）INOKUrl，軋，TAKAGI，Yい，andUMEMOTO，Z．，N K S。16，4i8S（1942）L；7apanPat”150，911（Juue
l，1942）．
（26）INOKUII，Kけ，UMEMOTO，Z小，and SIMADA，Y．，茸og一γ∂励g・ZαSS去49，31，31−2（1946）；〟小麒・ぶ・21 478（1947）（Pubり1952）；C．A．43，5209β（1949）
（27）IsII，Kenzi，and KAWAMURA，Sin，itir6（Nihon Daigaku）n ExpeIimentsin1949；tO he published （28）IwAMAE，甘（to MitubisiKaseiK6gy6KK）N K S 20，184（1946）（Puh1949）；Japan・Pat・
167，066（Septい16，1944）
（28a）IwAMOIO，Hidetosi．PIiizoelektra punkto de sojfabo−PrOteinoh Thesis for gIdauating from Coll・Agrh，
Nihon Univ．（1951），PreSented to SKAWANMURA
（29）JoNES，D．B。，and CsoNXA，Fl，Al，（7）332，291；JBioLChemh 96，ⅩXiv−XXX（1932）・ （30）JuuAN，P‖ L・，CIRCLE，S．J．，and MACDoNALD，RT（to Gliddcn Co）”CA・46，5225h（1952）：
U，SPat，2588，392（Ma王り11，1952）．
（31）KAWAMURA，Sin，itir6，BullCollAgr．，N之hon t7nlL・l・2，2−16（1951）；Cい At・46，11741d（1952）
（32）KAWAMURA小 Sin’itiI6，ibidtlL7−36；C．A．ibid
（33）Mc KINNEY，L。L，，and SoLIARS，WF小IndEngChem．4l，10S8−60（1949）；C．A・43，S133a
224
MAEDA，lk00（Mansyd Dqidu K6gy6R・K・），unPublished；
MIYAZAEI，Y，C．A44，5413メ（1950）；．Jぷ裾い 7も一灯路ねC￠偽（わざβJ〝d／（ゆ￠押3，152−4（1947） MuR岬A叩，ノ㌧s．，J7袖γOC鹿沼‥ぶ￠C．4Ⅰ，3Il−53（1920）
NAXAZIMA，K−，〟∬．且 7，226（1933）；ノ．飽c〟J秒Agγ小 ガ0ゐ如gdoJ研♪，乙玩査び．31，165・・356
＼ノ ︶ ヽ．ノ ︶ ︶ ︶ ︶ ■4 5 ′0 7 ノ 8 0ノ 0 3 3 3 3 3 3 ■槌 ︵ ︵ ︵ ︵ ︵ ︵ ︵ 2 3 0ノ l NoNOYAMA，Mitukane（MansytiDaidu K6gy6K・K），PlivateマOmmu‘nication声・ ONO，Ⅹaoru（MansytiDaidu K6gy6K．K），unPublished
OppER，A．、Ⅴ”L．，and TER HoRSr，WP：（to Virgini？・・Cazolina Chem・Gorp・），C．A．43，333d （1949）；US．Pat・2，450，810（伽t一ノ 5，1948）・
（41）OsBORNETn B，and CAMPBEu，G小 FhL，J・Am・ChemlSoc”2O，419・28（1898）
（41a）RowE，S（to Bnckeye Cotton OilCo∴），CいA・45，10433e（1951）；肌 S．．Pat・2，5419，526（ApI 17，1951）
（42）RowE，S．．，Cい A・46，5870≠（195召）；U・・Sト Pat・2，589，8￠7（．MaIり18，1952）． （43）SMlrH，A，R・・，and CIRCLE，Sり丁，Ind・Eng Chem・30，1414−18（1938）；（7），302，Fig・S2・ （4・4）SAr∂，S，Kogyo Kag」Zassi（J・SocChemImdJapan）23，1・25，109−3S，219・・36・321−42（1920）・ （45）SHIBE，JI，W・J，CA44，10216d（1950）；U”S」Pat」2，516，531（7uly25，1950）
（46）、SIBAZAK一・T，and OGIMURA，Ml（t0・DaiNippon Ce11uloid K・K・），N K S・21，542（1947）（Pub 1952）；ねpah・− Pat174，340（Decl26，・1946）
（47）SuMINOKURA，濫，入物0タ亨∧わgdz励g∴償加滋（J・AgIい仇em Soc・Japan）15A，ユ33・42（ユ939）；−C
A．45，2037∂（1951）
（48）SuMINOKUXA∴K・，路辺・1g，213−14（1943）；Cり Al・45、2037／−（1951）
（49）TADOEORO，Tり amd Yo5IMURA，瓦∴，ノ〟属＼ぷ2，249（ユ928）；一ん」物挽物㌧頗笹．ガ祓肋泌クカ噸 ÷助套む．21），355−62（1928）
（50）TAKANl，Akisige（Mansy止Daidu K6gy6］軋K・），unPublished
（5】）UMEMOIO，Z・，SoGABE，Yい，andINOKUrI，Kり，Kogyo KaglZassi（JJSoc．Chem・Indl−Japan）47， 883−5（19く沌）；Cい A・43，4789ゐ（1949）
（51a）tJnited States Depa工tment Of AgricultuIe，ノMaIketing Potential董0IOilseedPIOteinMaterialsinIndustIial Uses‖ 7もcゐ〝．月お払 No1043，120 pp・（Se・pteIゆeI，1951）
（52）WAITS，B．M，リ（71）L 333，290；J〝dβ〃g、C血多沼．29；10b9−11（1937）． （53）WATでS，B．Mい，and tJIRICIi，D．，（7），333，290；J紹d・月形g，C血z∽、31，1282・3（1939） （54）YAMADA，T‖，and MuKAWA，T‖（MitubisiKaseiK6gy6K．．Kい），凡瓦‥g小20，184（1946）（Pub．1949）； Iapan−Patけ166，599（Aug18，】944） （55）YAMADA，T．，and MuKAWA，T・，NKSlbid；Japan．Pat・167，185（Sept．22，・1944・）． （56）YAMAGA，Masuzるuβ〝JJyα購αgαfαAgγ・CoJよ2，46ノ・53（1950）；〟こ 風5・24，374q（1950）
（57）YAMAGA，Masug61XJⅣOS‡IA，、Ry茂zi，and X工S柑E，Ya＄uO（Mansya Daidu X∂卯∂Ⅹ．瓦小），unpublisムed （58）YAMAGA，Masuz6，and MAEDA，Ikuo（to Nihon YusiK・・K）。N・K・S14，985L5（1941）；JapanPat
