”H H W IM‘ “Mil I I h l U I! ‘y ‘ I ‘ l x I ll ‘ '3. ,H‘, :l I THE MAILLARD REACTION 3N MKRORSOLQGiCAL ASSAY Thesis for flu Degree of M. S. MiCHiGAN STATE COLLEGE Jean Eberrard Mammal-1 i949 This is to cwrtifg that the llwsis mnillml 'The Maillard Reaction in Microbiclogical Assay' presented In] Jean Maatman has been arvelncd luwzmls fulfillmunl nl thv requirements for M.S. MW n. Chemistg /) Q fjlfiij/YL’ \I'linl‘ Iv’qulf SHI‘ [late May 11’ 191‘9 0169 _——-——— ——-.- THE WED REACTION IN MICROBIOLOGICAL ASSAY JEAN SHERARD mm A THEIB Submitted to the School of Graduate Studlee of Michigan Stete College of Agriculture end Applied Science in pertlel fulfillment of the requlrunente for the degree of mm OF SCIENCE Department of Chemistry 1949 ACKNOWLEDGMENT The author wishes to express sincere epprecietion to Profeeeor C. A. Hoppert, end eepecielly to Profeeeor K. B. McCall, for their ueietenoe end guidenoe in the work for this thesis. *ttttttt *tttt. **#t it t TABLE OF CONTENTS Page Intrwuction...OOOOOOOOOOOOIOOOOOOOOOOOOOOOOOOOIOCC 1 EIporimon'tlJ. mathOdB‘eeeeeeeeeee'eeeeeeeeeeeeeeeoeee 3 Dita. and Result...eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 8 Conclufliomeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 31 BibliogrQPhYeeeeeeeeeeeeeeeoeeeeeeeeeeeeeeeeeeeeee 34 INTRODUCTI ON In 1912 Maillard (1) showed that solutions of anino acids became brown when they were heated with reducing sugars. This is a reaction of great importance to the food industry and to may other industries mich are concerned with materials of biological origin. The Iicro- biological technique my be well suited to the study of the millard reaction since the preparation of bacterial media invokes heating amino acids or simple proteins with a reducing sugar. usually glucose. Hill and Patton (2) have shown that a better growth response by Streptococcus i'aecalis occurred when graded levels of tryptcphane were added to a tryptophlne deficient medium if sucrose ms used as the for- lsentable carbohydrate and heated with the medium than when glucose us so .ployed. a better growth response also occurred if glucose was autoclaved seprately and added aseptically to the medium after both had cooled. In a later publication, Patton and Hill (3) dasonstrated that the poorer growth response obtained when glucose was heated with the nediun was due to the partial destruction of anino acids and vita- nias of the B—complex group. The amount of destruction of mino acids in the process of heating with a reducing sugar us determined by sub- sequent nicrobiological assay of the test solution. A sucrose nediun was used to prevent further browning. Significant destruction of l.- tryptophane, D-L aethionine, and L-lysine occurred. In addition, when L-tryptophane and D-glucose were heated in a solution buffered at pH 10 and in an unbuffered solution, greater browning, but less apparent loss of Iptrypotophane occurred in the buffered solution. The latryptophane us determined by microbiological assay. This suggests tint in the reaction there occurs a partial destruction of tryptophane simultaneous- ly with the production of a substance or substances which stimlate bac- terial growth throughout the total period of growth. Orla-Jensen (4) demonstrated that activators necessary for the optimum growth of certain lactic-acid bacteria were produced by heating glucose with nitrogenous substances common to bacterial media Optisnm growth was also produced when pyruvaldehyde. xylcse, arebinose, or furfural was heated‘with the nitro- genous components of the'nediun. Acetaldelwde was shoe: to be without effect. Hill. Patton, and Foreman (5) demonstrated.by subsequent microbio- logical essay that when casein was heated with glucose and hydrolysed ensynetioelly, only L-lysine, L—erginine, and Ldtryptophane'were appre- ciably inactivated. In addition, McInroy, Hirer, and Thiessen (6) eutoclaved casein with glucose and found that it did not support the growth of weanling rats when.employed es the sole source of protein in the ration. Thus, the'work of these two groups illustrates the effect of the‘Maillard re- action on intact protein. The results of these experiments do not sees: to give a complete picture of the Maillerd reaction.es it occurs in bacterial media. The work presented here was undertaken in an attempt to determine the amino acids involved in the reaction, some of the products formed, and their effect on growth as a measure of the suitability of snythetic media in nicrobiological assay. EEPERIIMTAL 1313013 1 The organisms used in this study were Streptococcus faecelis R( 8043 ) , Leuconost oc Meroides P-60 (8042 ) , Iactobacillus erabinosus 17-5 (8014). The cultures were minteined as agar stabs in a modified Hunter (7) medium composed of 2% agar, 1% beef extract, 2% glucose, 1% peptone, 0.6% NaZHPO4OZHZO, pH 6.8 without adjustment. The medium was dispensed into tubes , plugged with cotton, and autoclaved for 16 minutes at 1200 C. Duplicate or triplicate transfers were nde every two or three weeks in this medium. One culture was always retained un- used for subsequent transfer. The same medium, devoid of agar, was need he growing the inoculum. Usually, several transfers were nde in the inoculun before use. In most cases, excellent growth was observed within ten hours. Before use the inoculum medium containing the desired organism eas centrifuged in e wind-shielded horizontal centrifuge for five or more minutes. The organisms were resuspended in 0.8% sterile saline and re- centrifuged twice. The cells were then suspended in ten milliliters of sterile saline. One dr0p of this esspensicn from a sterile ten milli- liter pipeite was placed in each of the essay tubes. Care was taken so that the inoculun did not touch the sides of the tube. The essay medium was that of Haderson'and Snell (8). The composi- tion is given in Table I. l The organism were secured from the American Type Culture Collection. The numbers given in parenthe- ses are those of the Amaican Type Culture Collection. -3- nets: COMPOSITION OF ASSAY MEDIUM Amt/100 in. Constituent ‘ of medium a glucose 2.0 gm. D-l. alanine 100 mgm. sodium citrate 2.0 D-L aspartic acid 100 sodium acetate 0.1 ' L-glutamic acid 100 amenium chloride 0.3 L-arginine 1vdro- dipotassium phoiphate 0.5 ‘ chloride 20 - adenine sulfate 1.0 mgm. L-lysine dihydro- guanine hydrochloride 1.0 chloride monotwdrate 20 uracil 2 1.0 L-histidine 10 xenthine 1.0 D-L' isolenoine 20 ngnesiun sulfate L-leucine 10 heptahydrate 80.0 . D-L methionine 20 ferrous sulfate D-L phenylalanine 20 heptahydrate 4.0 L-proline 10 sodium chloride 4.0 D-L threonine 20 moment?” sulfate L-tyrosine 10 hepta drate 16.0 D-L valine 20 thiemin .100 L-tryptophane 4 10 riboflavin . 100 L-cystine 10 pyridoxel .020 D-L serine 20 calcium pantothenate .100 glycine 10 niacin .100 water to nuke 100 m1. biotin‘ .001 folic acid‘ \ .001 1 Adenine sulfate, guanine hydrochloride, and uracil were dissolved in dilute hydrochloric acid so that one milliliter contained the ammgt needed for one hundred milliliters of medium and stored at 2-10 Ce 2 Xenthine nus dissolved in dilute ammonium hydroxide in a concentra- tion such that one milliliter contained the amount needed for one hundred milliliters of media and stored at 2-10° c. 5 A mixture of the solid vitamins as made so that when 11.0 mgm was dissolved in dilute hydrochloric adid and made to twenty-five milliliters, one milliliter contained the amount needed for one hundred milliliters of medium. The solution was stored at 2-10° C. and kept for no longer than one week. 4' The biotin was received in five milliliter ampulu which contained twmty-fivelu‘gmaflfbiotin. This was diluted to twenty-five milli- liters and stored at 2-10° c. -4- 6 Twenty milligrams of folic acid was dissolved in twonty milli- liters of ethanol and stored at 0°C. A one to one hundred di- lution of this was rude just before use. One-tenth of a milli- liter contained the amount needed for one hundred milliliters of medium. '6 The amino acids were kept in a solution of a concentration such that twenty-five milliliters contained the amount necessary for ‘ one hundred milliliters of medium. The cystine and tyrosine were added first and dissolved in dilute hydrochloric acid and diluted before adding the other amino acids. Since most of the work presented here was done with graded levels of trypto- phane, the tryptophene was dissolved and stored separately. The solutions were stored at 2-10" 0. The medium was usually diluted to 0.8 its final volume and pipetted automatically by the Cannon Automatic Dispenser (9). This dispenser operates on the principle of measuring a quantity of liquid by an inter- val cf time since the volume of fluid tich will flow through a given orifice will depend upon the lugth of time which it is allowed to flow if the temperature, viscosity, and pressure of the liquid reamin con- stant. The deviceprovides means by which the size of orifice. the pres- sure, and the duration of flow can be controlled to give any desired vol- ume. Four milliliters of medium were pipetted into each tube. Other additions were made from pipettes. . Aluminum racks containing forty-eight tubes were used for incuba- tion. During the early part of the work, stainless steel covers lined with gaused-covered cotton wool were placed on the rack of tubes before autoclaving. Solutions from which additions were to be ends to file medium after autoclaving were autoclaeed at the same time as the medium. The conditions of autoclaving are stated with each experiment. The pi- pettes were autoclaved in an aluminum pipette can for fifteen or more minutes at 120°C. When additions were made to the medium after autoolaving only one or two rows were uncovered at a time. The addi- tions and inoculation were mde rapidly to prevent contamination by spore-formers. A room as free from drafts as possible was washed down with a 131000 solution of mercuric chloride and was used when additions to the medium and inoculation were smde. During the later part of the work individual aluminum caps were used for the tubes. These proved to be very satisfactory. When sterile solutions of volatile compounds were to be added after autoclaving to the basal medium, they were sterilized by passage through a Jenkim fil- ter. The Jenkins filter was used in connection with a filter flask, the letter containing a tube into which the tip of the filter could be placed. Pressure tubing and a calcium chloride tube filled with cotton wool were attached to the flask. The assembled apparatus was autoclaved for one and one-half hours or longer at 120°C. After filtration the tube was plugged with sterile cotton wool and taken from the flask. During the early investigation the essay medium was incubated in an air incubator at 35-38°C. but it was found that the [recision of repetitive assays was not as good as one should expect. Large water baths in tich more exact temperature control and a shorter therml adjustment could be made were built and subsequently used for incubation chambers. The time at which growth response us determined is stated with each experiment. Growth response at specified times during the incubation period was determined by measuring the turbidity at 660 In in a Hellige photoelectric cclorimeter using water as a blank. . The optical density of uninooulated media was usually 0.008 at this wave length. At the end of -6- 70-74 hours growth response was determined either bywneesuring turbidity, or in most cases by titration of the acid produced. An adaptation of the Cannon dispenser us used for titration. Electrometric titrations were nmde to pH 7.0 using celomel and quinhydrone half cells. The time of flow of 0.1 N sodium hydroxide was determined by a counter iiich was evaluated daily as to its volume equivalent. -7- DBIL.£NDIRESULES The preliminary work consisted ofdetermining the effect of the extent of autoclaving on the ability of various media containing glucose or an equimcler quantity of sucrose, added before or added after auto- claving, to support the growth of Srep. faecalie and E. arebinosus. The results are given in Table II. Six tubes were used for each condition under investigation. Table II EFFECT OF'VARYING.£UTOCLKVING PRESSURE.KND*GARBOHYDRATB'USED ON THE GROWTH RESPONSE G? STRF. FAECALIS AN D 2. WWW Carbohydrate with respect to l in lbs. W.E.m ST. . autoclaving glucose before 10 95 l 178 2 glucose after 10 88 1 190 3 sucrose before 10 96 l 187 2 sucrose after 10 83 l 211 13 glucose before 12 112 3 206 6 glucose after 12 100 2 218 3 sucrose before 12 100 2 216 4 sucrose after 1 101' l 215 4 glucose before 15 134 3 224 3 glucose after 15 125 l 230 4 sucrose before 15 124 2 237 2 sucrose after 15 126 l 235 2 *Determined at 72 hours as counts of 0.1006 E'NeCH; 224 counts 3 10 ml. {-1 washed saline suspension of a 16 hour culture was used for inoculum. *The time of autoclaving was 10 minutes. Table II shows that growth response increased as the eutecleving pres- sure increased with both organisms used. However, Strep. faecalie end I? erabinosus differed in.their reaction to the fermenteble carbohydrate which was used. In general, the best growth response of Strep. faecalie no obtained when glucose was added to the medium before autoclaving. The best growth response of E. arabinosus was obtained when sucrose was ' used as the fermentable carbohydrate or when glucose us added after autoclaving. A possible explanation for this would be the simltaneous destruction of essential mtrients and production of activators. It would seu that m. faecalie might respond to a smaller amount of the activator substances cr substances than would 3' arabincsue. When the mediun was taken from the autoclave as soon as the pres- sure had returned to atmospheric pressure, that is, after approximtely fifteen ninutes the results were of the sane order as those given in Table II. If the medium was allowed to rennin in the autoclave without disturbance until cool the growth respeue cf _8_t_r_e_p. faecalie was quite similar to that given in Table II. The growth response of I‘.‘ arabinosus me somewhat different. The results obtained under such conditions are given in Table III. The time of autoclaving was ten mimtes as before. TABLE III mm mm on BACTERIAL mm or vmoos horocmrmc 9mm renown) BY 1 PROLONGED comm PERIOD me o a t on essure Carbohydrate in respect to in lbs. #- Growth Response autoclaving? j§irep. talnllls §.D§}L. unshinnsns 3.] glucose before 10 106 1 297 6 glucose after 10 92 4 265 6 sucrose before 10 101 2 274 1 sucrose after 10 92 3 251 8 glucose before 12 111 1 292 4 glucose after 12; 85 3 266 8 sucrose before 12 96 2 266 1 sucrose after 12% 91‘, 2 251 5 glucose before 15 119 ' 6 248 4 glucose after 15 120 2 212 6 sucrose before 15 117 3 211 1 sucrose after 15 109 2 224 9 It Determined as counts of 0.1027 R No.03; 224 counts 3 10 m1. . 1- A washed saline suspension of a 16 hour culture was used for inoculum. -9- The growth response of Strep. faecalie us greatest when glucose was autoclaved with the medium at the lower pressure. At 15 pounds preemre no appreciable difference is noted. Here 1;. _a_rabincs3_e_ also showed the greatest growth response ten glucose was autoclaved with the medium. The longer heating period my have produced cough of an activator or activators to produce this effect. Considerable time us spent attempting to set up a standard curve for lysine using sucrose in the medium. For any experiments, no appre- ciable production of acid us noted. Sucrose and glucose respectively were added in triplicate before and after autoclaving and the results were noted in Table IV. TABLE IV EFFECT CF THE MAILLARD REACTIGION E2132; MEETENTEROIDES .. 5rovithTesponse 0W 6T:— , 24 hrs. 48 hrs. 72 hrs. 1. sucrose added to nediun before autocla'ging at 5 * g + ninth” at 120 c. e007 " e000? e008 " e002 0 " em 2. sucrose added to medium + * * after autoclaving .010 - .0007 .014 - .002 0 - .00 3. glucose added to medium ‘ ‘ before autoclaving .019 t .007 .332 - .053 75.7 - 2.92 4. glucose added to medium after .aut colaving .010 I? i .046 69.7 t 3.54 .000 .320 At 24 and 48 hours the growth was deterflned in term of density ‘t 660 me At 72 hours growth was determined as counts of 0.0998 N NaOH where 139 counts 3 10 1:1. It is evident that the Maillard reaction produced something which as necessary for the growth of Leuc. Meson. on what had pre- viously been considered a complete medium. ( . . 7‘) ‘ ,a W“ 2']. '1'qu e F fiehq'1LIb A J ' ‘ a 4 T Y 1' Y A A A Y V v 1 2 3 4 5 6 7 8 9 10 micrograms 4 L-trfiotophane - Determined as counts of 0.09j8N. NsOH; 197 counts: 30 ml. Inoculation was made with a washed saline suspension of a 16 hr. culture. As discussed in the introduction, L-tryptophane is believed to be partially destroyed when heated with glucose. An attempt was nde to determine the extent of tryptophane destruction when only L-tryptophane and D-gluccee were heated together in various concentrations and for various periods of time. Accordingly 40 mg of L-tryptophane and 2.0 g. of D-glucose in 50 milliliters of nter were adjusted to pH 7.0 and autoclaved for one hour at 120° C. Fran this solution were nde dilutions of 1:100; 1:150; 1:200, representing initially 8, 6, and 4 nicrogram (m g.) of L—tryptcphans per milliliter. It Ins felt at this time that the concomitant addition of D-glucose (approximtely 400 to 200 mu g.) would have insignificant effects upon the growth response. Subsequent microbiological assay in quadruplicate of these solutions was carried out using the usual Hender- son and Snell aedium with sucrose as the 'fernentable carbohydrate to prevent further browning. The nediua (4 Isl/tube) and the diluted solu- tions were autoclaved for fifteen minutes at 120° 0. The results are presented in Table V and Figure 1. TABLE V ASSAY 0F TRYPI'OPHANE - GLUCOSE MIXTURE Mu. g. oFtryptophane ‘erai Response at 72 ‘ Mu. g. of tryptcphane from test solution hrs. of j-Siigep. faecalie D.D. 16 28 33 38 44 51 4 46 6 67 67 QQGOONH OHNGOHHHN ' 8 It fieterxiined as counts of 5.0998 F iafi; I97 counts - 10 if. 1- A washed saline suspension cf a 16 hour culture was used as inoculun. -1 1- These data indicate that the test organism did not respond to the sample in the same “nor as it did to the pure standard and are evi-_ dence against the validity of the assay. Thompson and Kirby (10) have reported a method for the assay of lysine in a urine which contained material toxic to the test organism. For this method a standard curve was employed to determine whether or not the values obtained in the assay represented a linear dose-response curve. The technique consisted of adding a and 12 'm. g. of L-iysiue respectively to 1, 2, and 3 milliliters of urine in the assay medium. The growth response 60 the urine, and the growth response to each addi- ' ticn was found. By dividing the growth response of the urine by the average growth response to each addition and multiplying by the increase of each addition of lysine over the previous the concentration of lysine in the urine was determined. It was suggested that this night be a general method for assaying substances in the presence of tonic nter- ials. In orda' to test this approach in the presence of possible stim- latory substances 1, 2, and 3 m. g. (initial concentration) of L- tryptophane from the sterile L-tryptophane-D-glucoee solutions were added aseptically to tubes containing respectively, 0, 1, and 2 m. g. of I—tryptophane in the otherwise complete Henderson and Snell medium. The standard response curve is given in Figure 2. The results of the assay appear in Table VI. mflr‘I ‘.f ‘r.| n~~r ' so I .C .‘ mishbatw unweln H i O "r V) t ) .9 r“ Iv-d (7') l 2 3 4 , 5 6 ,7 8 9 10 ll 12 ‘3 micrograms of L-tryptophane it Determined as counts of 0.0998N Neon; 212 counts a 10 ml. ' Inoculation was made with a washed saline suspension of a 16 hour ‘ t CU.- TABLE VI DETERMINATION OF TRYPTOPEANE CONTM OF TRYPTOPHANE-GIBCCBE SOLUTION BY THE METHOD OF THOMPSON AND KIRBY nu g. L-tryptophane oErcwth Response in g. btryptophane of test solution of i Strapi fagcalig S. . 0 O 32 53 66 56 61 79 79 74 89 uuuwuwoooo OINHOINHOINH NHNNNHHHHOU tDeternined after 72 hours incubation as counts of 0.0998 N NaOH 212 counts 3 10 n1. {-A washed saline suspension of a 16 hour culture was used as inoculua. Calculations were undo according to the method of Thompson and Kirby. Values above 75 counts were not used since this was considered the linit of the linear dose-response portion of the standard curve. The calcula- tions follow: Response due to 1 flag. of L-tryptcphane of test solution 3 32-0 : 32 Response due to 1 nu. g. of L-tryptcphane 3 56-32 a 24 Response due to 1 an. 5. of L-tryptophane a 79-56 g 23 Average response the to l m. g. of L-tryptophane : 24 + 23 g 23.5 T Tryptophane content of test solution -_-, 1 x 32 e 1.36 mg. Responsg due to 2 as: g. of L-tryptcphane of test solution 3 53-0 3 53 Response due to l m g. of L—tryptophane 3 61-53 a 8 Response due to l as: g. of L-tryptophane I 74-61 g 13 Average response che to 1 an g. of L-tryptophane ? Tryptofiiane content of test solution 3 l x 53 g 5.05 n1 5. 10.5 These calculations illustrate that in most cases the growth re- . spouse is not in proportion to the probable tryptophane content of the reaction mixture. 'Another nethod which can be used for. calculating the dose response in an assay is the five point method of Elmens (11). A common point, often an extrapolated sero point is used in the calculation. Calcula- tions are made in this manner: To simplify calculations T-G is used to mean the L-tryptophane D-glucose test solution. The number used as a prefix indicates the tryptophane content in micrograms which was originally present. For the standards (s) Response to: l T-G l T-G plus 1 in: g. of 1 T-G plus 2 an g. of L-tryptophane L-trypt ophane 32 66 79 For the unknown (u) 1 no 2 r-0 3 r-c 32 53 as Ls=-c*§.2-2§s1- 32t79-2(56):-l Ihsfoeiu -2§u1- 32466-2(53)=-8 b. g (1.2 - To) +. (6 Lu - L.) . 3:3: + 6(-8)-(-1) . 22.8 2 70 2 70 bu : (in - To) + (6 L, - L“) 3 (66-32) t 6(-1)-(-s) g 17.0 2 7O 2 70 1t GmOwTh mLSPUhEh or ;. Am;¥lld 05 AT 48 dUUdS 133 'i § 53 is microrrams of i-trvntophane \Ldb- H O H H H [U FISLHLE 14 a GPO‘n‘Ti‘I RhSECher OF L. Art-\EINOSJS AT 72 dJURS 3 6 8 7 B s 712 F4 R)” [‘0 H» O H 1...: micrograms of L-tryptophane it Determined as turbidity at 660m; mu at 48 hours, and as counts of 0.0998N Neon; 128 counts: 10 m1. ' ThnA‘I1D34ny - un-k-A hfl14“- HIV u‘ne—e A“ r R:bu=17.0=0.74nug. b? 2276' This value is undoubtedly a truer indication of the amount of tryptophane actually present. L. and Lu measure the departure of the slopes from linearity bll and bu are respectively the slopes of the lines of the stand- ard and unknown. 7 a mean of values for a particular response. R 3 amount of the substance which is. present. This experiment was repeated using L. arabinoeus as the test organ- isn. The medium was that of Henderson and Snell deficient in tryptophane. The medium, a tryptophane solution, and the T-G solutions were auto- claved for five minutes at 120° C. Additions of tryptophane and T-G were nade aseptically after autoclaving. The results appear in Table VII, and the standard dose response curves are given in Figures 3 and 4. TABLE VII TRYPTOPHANE ASSAY 01“ TEST SOLUTION USING 1:. 11211311705131: IIt‘irow‘th RUspons .7? n1 g. tryptophane T-G ' +1.. arabinoeus A A at_24 hour? at 48 hours at 72 hours 0 0 0:075 "IO. 57‘ O 1 7 .155 .198 53 0 2 .194 .251 56 0 3 .217 .277 56 1 l .200 .247 56 l 2 .244 .303 64 1 3 .247 .324 70 2 l .226 .289 65 2 2 .272 .349 73 2 3 .300 .376 78 II'lieasured as turbidity at 660 n m, using nter as a blank for 24 hour and 48 hour period and as counts of 0.0998 N NaOH; 128 counts 3 10 ml. for 72 hour .period. {A washed saline suspension of a 16 hour culture was used for inoculum. Calculations were made according to the method of Thompson and Kirby (10). For 24 hour period of growth: Response due to 1 T-G : 0.155 - 0.075 g 0.080 Response due to 1 m g. of tryptophane - 0.200-0.155 : 0.045 Response due to 1 m g. of tryptophane _' 0.226-0.200 g 0.026 Average response due to 1 up g. of tryptophane : 0.045 .3025 e 0.0355 2 . Tryptcphane content of l T-G g 0.080 g 2.25 m g. 0.5355 Response due to 2 T-G : 0.194-0.075 : 0.119 Response due to 1 m g. of tryptophane : 0.244-0.194 . 0.050 Response due to 1 on g. of tryptophane - 0.272-0.244 : 0.020 Average response to l m g. of tryptophane : 0.050 4 0.028 a 0.039 - 2 Tryptophane content of 2 1—0 g 0.115 . 5.05 an g. #059 ' Response due to 3 7-0 .-_- 0.217-0.075 : 0.142 Response due to 1 mu g. of tryptophane - 0.247-0.217 .. 0.030 0e3m.00247 a 0.053 Response due to 1 .1 g. of tryptophane Average response to 1 m g. of tryptophane - 0.03040.053 - 0.0415 ._._..§.___. Tryptophane content of 3 T-G : 0.142 = 3.43 m g. 575115 For 48 hour period of growth: Response due to 1 m g. of tryptophane - 0.247-0.198 0.049 Response due to l m g. of tryptophane - 0.289-O.247 - 0.045 -16- Arerage response to 1 mu g. of tryptophane = 0.049 4 0.045 g 0.047 ‘2 Tryptophane content of l T-G : 0.041 g 0.87 mu g. 0.047 Response due to 2 T-G = 0.251-0.157 g 0.094 Response due to 1 nu g. of tryptophane : 0.305-0.251 : 0.052 Response due to l mu g. of tryptophane : 0.349-0.303 : 0.046 Average response to 1 mu g. of tryptophane : 0.0522: 0:94§_= 0.049 2 Tryptophane content of 2 T-C : 0.094 g 1.92 an g. Response due to 3 T-G : 0.277-0.157 : 0.120 00324-0sz77 8 0004? Response due to l mu g. of tryptophane Response due to 1 mu g. of tryptophane _ 0.376-0.324_= 0.052 Average response to l mu g. of tryptophane g 0.047 4 0.052 3 0.0495 2 . Tryptophane content of 3 T-G g 0.120 = 2.41 mu g. 5.0495 For the 72 hour period of growth: Response due to l T-G : 53-30 g 23' II 6‘ Response due to 1 mu g. of tryptophane : 56-53 I 0 Response due to l mu g. of tryptophane 55-55 _ Average response due to 1 an g. of tryptophane .- 3 4 9 = 6.0 T Tryptophane content of l T-G = 23 g 3.83 m g. Response due to 2 r-o . 55-50 = 25 Response due to 1 nu g. of tryptophene 54-55 . a Response due to l mu g. of tryptophane : 73-64 g 9 Average response to 1 mu g. of tryptophane : 8 4 9 g 8.5 2 Tryptophane content of 2 T-G 8'6 26 = 3.06 m g. -30 .-_ 26 Response due to 3 T-G": 56 Response due to 1 m g. of tryptOphane - 70-56 g 14 Response due to l m g. of tryptophane 78-70 g 8 Average response due to 1 mu g. of tryptophane : 1448 = 11.0 Tryptophane contmt-of 3 T-G : 26 g 2.36 mu g. 1170 These results show that a very marked stimlation occurred at 24 hours. By the end of the 45 hour growth period the stimlation had disappeared to a comiderable extent. The results at 72 hours some higher than expected. This resulted because the dose response to each added I: g. of tryptophane was snnll. ' In the next experiment reaction mixtures of 40 mg. L-tryptophane and 2.0 g. of D-glucose and 400 mg L-tryptophane and 2.0 g. of D-glucose in 50 milliliters of water were adjusted to a pH 7.0 and autoclaved for four hours at 120° C. In addition : 400 mg. of L-tryptophane and 2.0 g. of D-glucose in 50 milliliters of nter unadjusted to pH 7.0 and auto- claved for one hour at 120° C. Dilutions of the solutions were aside so that one milliliter represented 1, 2, or 3 an. g. of tryptophane origin- ally present. The solutione were assayed for tryptophane content. In this case all additions to the medium were rude before autoclaving at 120° C. for five minutes. The results appear in Table VIII. TABLE VIII COMPARISON OF VARIOUS TB? SOLUTIONS tGroitE‘Reeponse er Etrep. Tascalisf T-G Test Solution ' 24 hours 48 hours 7'2 hours 1 40 mg. L-tryptophane 0.070 0.087 37 2 2.0 g. D-ghucose 0.091 0.123 58 3 heated for 4 hrs. 0.111 0.150 74 l .400 mg. L-tryptophane 0.063 0.093 38 2 2.0 g. Dbglucose 0.093 0.128 64 3 heated for 1 hour 0.126 0.160 81 l 400 mg. L-tryptophane 0.065 0.091 36 2 2.0 g. Dbglucose 0.096 0.12! 58 3 heated for 4 hours 0.128 0.157 78 ItDe'termined as turbidity at 24 and 48 hours at 660 m m. using water as a blank and at 72 hours as counts of 0.0980 N NaQH; 262 counts 3 10 m1. - $1 washed saline suspension of a 16 hour culture was used as inoculunu The best growth response was observed with the solution which con- tained 400 mg. of tryptophane and 2.0 g. of D-ghucose and had been heated together for one hour despite the fact that the dilution.was ten.timss greater. Therefore. this ratio and time of heating was chosen for fur- ther investigation. The next,experiment‘was undertaken to determine theeffect of naking the»additions of the tryptophane and the test solution (T-G) before or after autoclaving. The results appear in Table Ix and the standard growth response curves appear in Figures 5 and 6. -19- FIGURE 5 _ a GnOWTH RESPONSE OF STREP. FAECALIS AT 18 nOUdS 00300} L 3 _ _.,__ 7 o .200{ .100? 1 2 3 4 5 6 7 8 9 10 micrograms of L-tryptoohane FIGURE 6 GROWTH RESPONSE OF STRiP. FAiCALIS AT 72 HOURS' 180 “ ,.-«—-fiF==:W- e¢/“' 190 ' }/’ ,4? 89 “ .;é7’ [/e 60 i //' '0 no i» / 20 4b ./ 3 A 5 6 7 8 9 10 micrograms of L-tryptoohane .____) tryptophane autoclaved Ilth media * ‘T'fi tryptophane added after autoclaving Determined as turbidity at 650 m. mu at 18 hours, and as counts of 0.09 NaOHiat 72 hours; 233 counts ; 10 ml. Inoculation was made with a washed saline suspension of a 16 nour cultu TABLE II EFECT OF MAKING ADDITIONS BEFORE OR AFTER AUTOCLAVING In. g. e?‘ Farowth‘RSsponse of sire . faecalie} tEEEEophane T-G at 18 hours 3.D. at 52 Hours 3.D. one e are Intoclaving 0 0 0.018 0.001 0 0 0 1 0.083 .003 42 3 0 2 .149 .001 70 l 0 3 .189 .002 92 l l l .133 .002 74 l 1 2 .197 .002 93 2 1 3 .237 .004 103 1 2 1 .176 .005 94 1 2 2 .247 .003 108 1 2 3 .258 .002 114 3 Additions.After Autoclaving 0 0 0.017 .001 3 0 0 1 0.078 .001 43 1 0 2 .129 .001 70 0 0 3 .203 .007 93 l l 1 .138 .001 74 O 1 2 .177 .001 91 1 1 3 .226 .001 107 2 2 l .186 .003 92 1 2 2 .229 .007 104 1 2 3 .238 .015 113 l *Determined at 18 hours as turbidity at 660 m m using water as a blank; at 72 hours as counts of 0.0992 N NaOH; 233 Counts g 10 m1. 4A washed saline suspension of a 16 hour culture'was used as inoculum. The'amount of L-tryptophane present appears much greater than the .actual content of the test solutions (T-G) both when additions were made before or after autoclaving. The amount of L-tryptophane present was calculated by the method of Thompson and Kirby (10) as follows: At 18 hours when additions were made before autoclaving: Response due to 1 T-G g 0.083-0.018 : 0.065 Response due to ltmu g. of L-tryptophane a 0.133-0.083 a 0.050 Response due to l mu g. of Intryptophane a 0.176-0.133 a 0.043 -20- Average response to 1 m g. of L-tryptOphane = 0.050 4 0.043 = 0.0465 2 Tryptophane content of l T-G = 0.065 - 1.40 nu g. .0465 Response due to 2 T-G . 0.149-0.018 = 0.131 Response due to 1 m g. of Iptryptophane -.- 0.197-0.149 _ 0.048 Response due to 1 nu g. of L-tryptophane = 0.247-0.197 g 0.050 Average response to l m g. of L-tryptophane = 0.048 4 0.050 : 0.049 2 . Tryptophane content of 2 T-G : 0.131 g 4.06 m g. Response due to 3 T-G :- 0.189-0.017 : 0.171 Response due to 1 m. g. of L-tryptophane : 0.237-0.189 g 0.048 Response due to 1 an g. of L-tryptophane g 0.258-0.237 g 0.021 Average response to l m g. of L-tryptophane : 0.048 + 0.021 3 0.0345 + Tryptophane content of 3 T-C : 0.171 g' 4.95 m g. At 18 hours when additions were nde after autoclaving: Response due to 1 T-G g 0.078-0.017 . 0.061 Response due to l m g. of L-tryptophane . 0.138-0.078 . 0.060 Response due to l m g. of L-tryptophane : 0.186-0.l38 : 0.052 Average response to 1 nu g. of L-tryptophsne : 0.060+0.052 : 0.056 2 . Tryptophane content of 1 T-G : 0.061 g 1.09 m g. Response due to 2 T-G : 0.129 - 0.017 g 0.112 Response due to l m g. of lie-tryptophane _ 0.177-0.129 : 0.058 Response the to 1 an g. of L—tryptophane _, 0.229-0.177 :- 0.052 -21- .kverage response to 1 mu g. of L-tryptophane = 0.058 - 0.052 3 0.055 2 Tryptophane content of 2 T-G g 0.112 g 2.04 mu g. 5.055 Response due to 3 T-G = 0.203-0.017 g 0.186 Response due to l mu g. of L-tryptophane : 0.226-0.203 : 0.023 Response due to 1 mu g. of L-tryptophane 0.238-0.226 g 0.012 Average response to 1 nu. g. of L-tryptophane 2 Tryptophane content of 3 T-G g 0.186 g 10.33 mu g. 0.018 At 72 hours when additions were made before autoclaving: Response due to 1 T-G : 42-0 2 42 Response due to 1 mu g. of L-tryptophane a 74-42 g 32 Response due to 1 mu g. of L-tryptophane = 94-74 g 20 Average response to l mu g. of L-tryptophane : 32 4 20 g 26.0 Tryptophane content of l T-G grggv g 1.62 nu g. ROBPOBIO due to 2 T-G = 70-0 3 70 Response due to l mu g. of L-tryptophane - 93-70 = 23 Response due to l mu g. of L-tryptophane 3 108-93 : 15 Average response to 1 mu g. of L-tryptOphane . 23 + 15 3 19.0 Tryptophane content of 2 T-G : g; g 3.68 mu g. The growth response to 3 T-G plus tryptophane additions is above the linear portion of the curve, so valid calculations for 3 T-G cannot be mdQe -22- At 72 hournvwhen additions were made after autoclaving: Response due to l T-G = 43-3. 3 40 Response due to 1 mu g. of L-tryptophane 74-43 g 31 Response due to 1 mu g. of L-tryptophane : 92-74 g 18 Average response to 1 mu g. of L-tryptcphane g 31 4 18 g 24.5 Tryptophane content of 1 T-G g 42_ g 1.57 mu g. 24.5 Response due to 2 T-G = 70-3 = 67 Response due to 1 mu g. of L-tryptophane 3 91-70 3 19 Response due to 1 nu g. of L-tryptophane a 104-91 . 13 Average response to 1 nu g. of L-tryptophane g 19 4 13 g 16 .__!__.. Tryptophane content of 2 T-G ; gg’ g 4.18 nu g. Here again valid calculations can not be'made on the series of tubes containing 3 T-G plus tryptophane additions since the growth response is above the linear portion of the standard curve. The apparent tryptophane content of the L-tryptcphane-D-glucose (T-G) test solution was considerably greater than the amount original- ly present. This is true when additions of L-tryptophane and the‘test solution are made either before or after autoclnving. The calculated tryptophane values are slightly less when these additions are made after autoclaving than when the additions are made before autoclaving. In general, the growth response to the addition of the second nicrc- gram of L-tryptophane to tubes which contain the test solution is not as great as the growth response to the first added nicrogram of L-tryptc- phane. The presence of a stimulatory factor or factors even in the greatest dilution (1:8,000) nukes an accurate assay of the test solu- tion impossible with the synthetic medium of Henderson and Snell. Calculations were also nnde according to the method of Mom (11). At the end of 24 hours: For the standards (I) : lT-G lT-Gpluslsug lT-Gplue2-1g. of L-tryptophane cf L-tryptophane Addition made before auto- ‘ slaving 0.078 0.138 0.186 Addition nude after auto- 0.133 0.176 Ola-fins 0.083 For the unknown (u) 1 T-G 2 T-G 3 13-0 Additions made before auto- claving: 0.078 0.129 0.205 Additions snde after auto- claving: 0.083 0.149 0.189 Calculations based on additions undo after autoclaving: I... g 0.078 4 0.186 - 2(0.138) g 0.012 I“ . 0.078 + 0e203 " 2(Oe129) . 00043 b. n 0.186-0.078 + 65.043;-S-0.0122 = 0.0555 b}! = W- * 6(a‘0e01gl- 0.043 : 0e0621 2 7O R : 0e0621 : 1.12 ‘1 g. Calculations for additions made before autoclaving: -24- h, n 'o.17§-o.oez + 6 -0.026 a Locofl = 0.0252 bu g 0.189-0.088 + 6(-0.027 - -0.026) a 0.0507 2 v R = bu . 0.050? I 2.01 nu 6e 3 e Calculations for 72 hour datas For the standards. (s): lT-G 1T-Gpluslmg.of 1T-Gp1us2mg.of L-trypt optnne L-tryp‘tophane Additions made before autoclaving: 42 7‘4. 94 Additions made after autoclaving: 43 ' 74 92 For the unknown (u).- 1 M} 2 r-c 3 T-G Additions nde before autoclaving: 42 70 92 Additions snde after autoclaving: 43 70 93 For additions made after autoclaving: I...43+92-2(74)=-13 In.“+93-2(70)g-4 53:92-43 46-4 - -13 324.34 """""2 .‘j—l‘tj‘Jv bu . 93 - 4s + s -13 - .4) g 23.94 T RIM-23.94 =O.98mge 5‘; 24.34 -25- O r -« , O - e.- a . .e. . .. e.— -- , . n — l For additions made before autoclaving: 1.. e 42 + 94 - 2(74) : -12 Ige42+92-2(70).-6 b, ,-_- 94-42 - 6 -6 - -12 = 25.76 ‘2'" o =92-42 -6-12 --6 =24.06 bu .2. _<__,>m_<_2 R = I? .-_- 24.06 g 0.93 m g. 8 £5.76 Again this method gives a lower value for the apparent tryptophane content. However, this value also seem to be such above the actual tryptophane content, especially when calculations are nude from data collected during the early growth period. The conclusion was reached that a stimulatory factor inch was concerned with the metabolism of tryptophane was produced by the Maillard reaction. Orla-Jensen (4) stated that in the process of heating reducing sugars with nitrogenous substances, degradation products of the sugars were formed. Among these products were pyruvaldehyde. diacetyl, furfur- aldehyde, and acetaldehyde. In the next experiment pyruvaldelwde was heated with L-tryptophane to determine what the effect of pyruvaldehyde would be on a subsequent microbiological assay of the test solution for tryptophane. Since pyruvaldehyde is a volatile substance it as easy to remove the pyruvaldehyde so that the pyruvaldehyde itself would have no effect on the assay. I Fifty filligrams cf L-tryptophane were refluxed with ten milliliters of 0.005 M pyruvaldehyde for one hour. About five milliliters of the solution were distilled off; pymvaldehyde was detected in the distillate. .26- FIIUHE 7 a * STANDARD GRUWIH RLSRONSE CURVE FOR FTnEF. FAEOALIS 48 hrs 0.280 ’ .240 .200 _ :8 hr: .160 .120 0080 .040 +7 4 t 4 + 4 if ‘4 4 +9 4 l # ' 3+ 5 6 7 8 9 10 11 12 1 2 3 9. micrograms of L-tryptopnane i Determined as turbidity at 660 m. mu, using water as a blanm. it Iroculation was made with a washed saline suspension of a 16 nour cu The reaction predict was assayed for tryptophane in quadruplicate, using the usual Henderson and Snell medium. The results appear in Table X, and the standard curve is given in Figure 7. mate 2 ASSAY or ramommrs WHICH m0 semi 3mm mm mumps mu. g. off an. g. of tryptophane *Growth Response tryptophane according to original of f Stre faecalie content it'I8_HEE?3I1§35§‘=1fi?inr1fiifii——BID7 0 0 0.017 0.001 0.025 0.002 0 1 .057 .001 0.066 0.004 0 2 .083 .005 0.113 .004 0 3 .114 .003 .150 .001 1 1 .096 .004 .121 .003 1 2 .127 .004 - .170 .003 1 3 .141 .003 .197 .004 2 l .139 .002 .166 , .004 2 2 .143 .002 .196. .004 2 3 .152 .001 .220 .001 l“Determined as turbidity at 660 m 1111., using nter as a blank. {-A washed saline suspension of a 16 hour culture was used as inoculum. The apparent tryptophane content of the test solution as calculated from the first five series of tubes as the results are determined from thevstandard curve is 0.73 mu g. The values for the other tubes fall above the linear dose-response portion of the standard curve. This is probably a good index of the amount of tryptophane which remained inrthe test solution. Evidently no stinulatcry factorwas produced by the re- action of pynrvaldehyde and L-tryptophane, and a significant portion of tryptophane was destroyed by the reaction. The effect of various carbohydrate degradation products on the growth of §E£gp. faecalie was determined.’ The first of these tested.was the pyruvate ion. (he milliliter of a sodium pyruvate solution (0.5 mg.) 00400 .550 0300 .200 0.300 .200 0100 0 Fieuas 8 EFFhCT 0? sooluw PXRUVATE IN COMPLLTE Msolum ’ ' F‘" ‘ f" "‘L.‘ TC' L. "A r 'Q '~ 01V lnL G.TJ\A.1 OF 3.4L . .1...- : .‘LAJ‘Q.‘¢L.1U* l—no additions 2-pyruvate added before autoclavin 3-pyruvate added after autoclaving 4-pyruvate aided at intervals ‘ o i. E l 4 4 4 + ‘fi 10 2O 33 4D 50 hours 4» FIGUnE 9 iv GLUCOSE ADDSU AFIER AUTOCLAVING L L A j ' Y I Y :7 T 10 20 30 40 50 hours *Determined as turbidity at6€0 L. nu, using water as a blank. Inoculation was made with a washed saline suspension of a 12 hr. cuN 0.400 .300 @4300 .100 VYmUVA.e IA has thPlutH KL thIdM 71 " 1 L4\}" 3. LL“: LCI JP mutant». ‘7 V P ‘~ / \ (171 k h ‘ ' ON l‘."if.‘.a 'u' {‘Jer. U: VI .‘.LI e IO 20 30 40 50 hours FIGUHE ll GLUCOSE ADDED AFTnn AJIOCLAVIKG litions its added before autoclavi ate adiod after autoclavh; rte added at intervals l) 29 3) 49 50 hours *Determined as turbidity at 660 m. nu, using water as a blank. Inoculation was made bith a wasn i saline suspension of 3 l2 hr. cultur‘i ns added in guadruplicate to the cmnplete Henderson and snell medium before autoclaving and to another series aseptically after autoclav- ing.‘ To a third series the sodium pyruvate was added in amounts of 0.1 mg. at 0, 2, 4, 8, and 12 hours after inoculation. This same pro- cedure was followed using the Honda'son and Snell medium with glucose added aseptically after autoclaving. The results are shown in Figures 8 and 9. A similar study was mde in a low tryptophane (5 m g. of L- tryptophane per tube) medium using exactly the same proceedure. The results are shcm in Figures 10 and 11. Other substances which were tested included acetaldelnrde, pyruval- dehyde, acetol, and diacetyl. Solutions of each of these compounds were made so that one milliliter contained 0.5 mg. Since these are volatile compounds, their solutions were sterilised by passage through a Jenkins filter. One milliliter of the sterile solutions was added aseptically to the medium in quadruplicate after cooling. The results of the growth response of _8_t_i_'_ep. faecalie to added acetaldetvde in comparison to a series to which no additions had been nde is found in Table II. The results of the growth response of m. faecalie when acetol, di- acetyl, and pyruvaldehyde were added to the medium of Henderson and Snell is given in Figure 1.2. Acetol produced moderate stimlation of growth, diacetyl produced marked stimlation, while pyruvaldehyde appar- ently inhibited growth in the early hours after inoculation. Goto (12) showed that acetol and diacetyl were formed in the decomposition of sugars by weal: alkali. The other amino acids present in the Henderson and Snell medium were individually autoclaved for one hour with 2.0 g. of D-glucose. ~28- ‘r”. EIJJmn 12 [x‘ V“: 91“ fr (4 ’— ‘ 3r 1. . - \ .4 ’.' ,‘ ,!_,..'. I“ . . . "(PIC' U: :,\Ji‘.l;. ;.'\['L-3"JT1£JL\AAL u‘LdiLhJstA ‘UAO f .sKJLJUUAh.) ‘ “w ~ 4' -- ~ |v (Jr‘s A's—LIL Jlflluhld .r -).‘,‘...1- .-q ‘ A. \ a. — ,4 lr—«H -e D-EQLDA‘A-’L Us -..A..A.ao - 0.406 0 3004 no additions of 0 20C)“ .'pyruvaldenyde H (a U o«v i A T 2.0 ()1 {L U 60 hours “Determined as turbidity at 663 m. flJ, using water as a clans. Inoculation was made with a washed saline suspension of a 6 hr. Cult FIGURH;13 EFFECT OF AMIHO ACID-GLUCOSE PRODUCTS 05 THE GROWTH OF STEERQECALISs 9 , ‘11!" _ ':§-' .400 .. <,e 230 ' - ‘1.