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P.i P. NO. 400s

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TSMIG Z PONDLIMITED

INTER-OFFICE CORRESPONDENCEDATE: February 10, 1967TO Dr. Brouilla

FROM: Duane TackaberryRE: Minutes of Meeting on 99+ D .E . Corn Syrup andEnzymatic Inversion of Corn Syrups - 2/7/67

Persons attending :

D .2260COPIES TO

R. J. Reynolds : Claude Sawyer, Economic Eval . Section, Develop . DeptR i c h a r d M o r l e n , E n g i n e e r i n g I t ' ' 1 1 / D r . Wm. Squires, Biochemical " Research DeptDr. Paul Carey " " "

Penick & Ford Ltd : R. V. CroninDr. R. E . BrouillardW. C. BlackDr. C. C . KeslerE . T. HjermstadR: HarveyP . B . NelsonD . O. TackaberryC . E . Vesely

SUMMARYThis report covers the items discussed on February 7, 1967concerning the production of high D .E .syrups, enzymatic inversionof dextrose, enzyme production, and total sugar production .A description of the problems and processes outlined at thismeeting are in the discussion section .

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Minutes of meeting on99+ D .E. Corn Syrup Page 2 February 10, 1967DISCUSSION :A . Enzyme Production - Xylose Isomerase - Dr . Wm. Squires

The system used for the inversion (Xylose Isomerase) isknown and a Japanese and U .S . group both have patents on itsproduction. These patents do specify substrate levels that thework at Winston-Salem shows can be circumvented (2% Xylose levelmin . )The particular bacteria (Arthro bacter) used for the enzymeproduction came from a sample taken from our Blackfoot plant .

Based on the work thus far, it appears that genus coverage can beobtained by patent . Work defining the limits for the patent andadditional work to cover other areas to preserve this exclusiveposition is in progress. Dr. Squires pointed out that this systemhas been under study about six months and that normally up to twoyears is required to perfect such a system . However, work hasprogressed rapidly due to previous experience with a similar systemon nicotine .

The process entails growing the bacteria in a Xylosecontaining substrate . The Xylose level is 2%. Since the enzymeis produced within the walls of the bacteria, it may not be feasibleto produce the pure enzyme . The product and substrate could becentrifuged when growth is completed to supply the enzyme as apaste or acetone dry if necessary .

The enzyme paste appears to be stable under normal (40F . )refrigeration and the acetone dried powder stable at room temperaturThe presence of S02 has no detrimental effect .The enzyme will convert a dextrose solution in 24-36 hours

at 60C. to 45-50 mixture of glucose and fructose . They arecurrently using 685gm wet pack (80% H 2 0) enzyme per 100# dry solidsA Veltose syrup was inverted and also gave a 47-48% inversion ofthe dextrose portion . Little color formation was reported duringthe inversion. Dr. Squires emphasized that the reaction conditionsare not final and further work has to be done to optimize the proces

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Minutes of meeting on99+ D .E. Corn Syrup Page 3 February 10, 1967The use of coarse feed as a possible source of Xylose forthe enzyme growth was discussed . However, the inhibiting effectof glucose on enzyme production may limit its use without sometreatment to remove the starch present .However, a trial at 50-50 Xylose-glucose level gave asatisfactory substrate . Here again, definite limits have to beestablished .

B. Hydrolysis of Coarse Feed - Xylose ProductionMr. Sawyer commented on the patents in this area and theprocess as presently envisioned .A literature search has revealed several patents in thisarea . In addition, Dr. Bernasek had hydrolyzed coarse feed tsinga mixture of acids .The most promising process at present uses an excess ofS02 and cooks at 55j solids and 140-160F . at 25psig for one hourat pH 2 . in this process, the S02 would flash off after cooking

making it unnecessary to remove an acid salt . The process yields20-30% Xylose based on the corn hull weight .If other acids are used, an ion exchange step may benecessary .The requirements are :

2# Xylose/100# d .s . starch6# corn hulls,l100# starchIt is estimated that a fermentor with a 50,000 galloncapacity, associated equipment and an isolated building could costup to $1 million . This includes building enough versatility toprepare other enzyme requirements .

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Minutes of meeting on99+ D .E . Corn Syrup Page 4 February 10, 1967

Mr. Black commented on the fact that the Japanese havefound enzymatic inversion up to 2 to 3 times more costly thanalkaline inversion . They reported 80-90 hours reaction time andmore color formation with their particular enzyme system .

Production of 95+ D .E . Syrups - P . B. Nelson and Dr . Paul CareyThe use of high D .E . syrups in the 95+ D .E . range wasreported to be limited due to the attendant shipping, storage andhandling problems necessary to prevent crystallization . However,a high fermentable syrup or powder that could be more easily handledthan regular dextrose has excellent potential .Mr . Nelson has investigated three techniques to produce the95+ D .E . syrup. These are :

1) Starch to Water" In the method, a small amount of make-up water is heated to185-189F . The line starch slurry (40% D .S .) containing theliquefying enzyme ( .1/ Tenase) is then added slowly to minimize thepeak viscosity. Heating is continued at 185 until all starch is

added. The batch is held at 180-185F . for 1 to 1-1/2 hours. Theresulting D .E . is approximately 25 . The pH is adjusted and thebatch cooled to 140F . before adding the saccharifying enzyme ( .8%Miles Diazyme L-30) . The batch reaches 95-97 D .E . in 80-100 hours .The batch is filtered after saccharification . Filtrationrate is fair . This method was reported to give the faStest.filtration rate of the techniques used .

2) The second method uses the continuous cooker at 30-35% starchsolids with a heat stable enzyme at 200-205F . The paste ishold 1 hour 20 mins . to 1 hour 40 mins . at 185-189F ., beforetreating as in process #1 . The 95-97 D .E . product is moredifficult to filter than the syrup from #l .

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Minutes of meeting on99+ D .E. Corn Syrup Page 5 February 10, 19673) Another method using the cooker entails pressure cooking a 30%solids slurry at 300F . with excess steam. The liquefyingenzyme is added after cooking and the batch held at 185-189F .as before . The maximum D .E . obtained in this method is about95. The product is also very difficult to filter .4) Another method, as yet to be tried, would be to continuouslyliquefy and then pressure cook . Tt is planned to use thisprocedure when the equipment now on order is assembled . Thiswould be in conjunction with the project to prepare high solidsstarch pastes for coatings .

Dr. Carey's process entails stepsto minimize retrogradation .This procedure entails continuously cooking at 200-205Fo at 28fsolids using .1/ Tenase as the liquefying enzyme . The paste isallowed to cool slowly to 185F . in an insulated container withoutagitation. The starch paste is pourable in 15 minutes and has aD .E . of 15-17 in 1-1/2 hours . The paste pH is adjusted to 4 .0 withmild stirring (75-80rpm) and transferred and cooled to 140F . beforeadding .6/ Diazyme L-30 . The batch reaches 99 DoE . in 20-24 hours .Similar D .E . values have also been obtained using amyloglucosidaseproduced in Winston-Salem. No problems are encountered withfilteringo A residue of .6 to 1% is obtained, whereas, Mr . Nelsonencounters 1 to 2% with his procedure .Further work is planned by Dr. Carey using higher solidsand using line starch samples submitted by Cedar Rapids .

Work in Cedar Rapids is planned using Dr. Carey's technique .Total Sugar - W. C . Black

Mr. Black described the total sugar process Penick & Fordcurrently has under option .

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Minutes of meeting on99+ D .E. Corn Syrup Page 6 February 10, 1967

The process takes a 95-97 D .E . syrup but will operate(with some difficulty) starting with as low as a 90-92 D .E . syrup .The syrup is taken after carbon refining and ion exchange to ashock cooling crystallizer . This forms micro crystals with theremainder a solution of alpha dextrose and oglio-saccharides . Themasecuite is spray dried at 150 maximum to 15% moistur e . The '150F . maximum is to prevent the formation of anhydrous dextrose .The wet snow is allowed to cure for 4 to 5 hours to completecrystallization before drying to 91% solids . The resulting productis an instantly soluble free-flowing powder .The process can also be used to incorporate fruit juices,chocolate powders etc ., to obtain a variety of powdered drink

bases .During the general discussion that followed Mr. Black'spresentation, Dr . Carey indicated that some enzyme systems ofinterest to us had been developed and samples would be sent assoon as available . . The majority of the discussion centered aroundpossible additional reasons for Dr . Carey's higher D .E . syrups .It was suggested that, perhaps, an inhibiting effect of the solublesin our line starch could be a factor. Arrangements were made afterthe meeting to obtain line starch slurry for Dr . Carey to take backto Winston-Salem .At the conclusion of the meeting, arrangements were madefor a plant tour and for Mr. Sawyer to meet with Mr. Vesely toobtain pertinent cost information .

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