934.\A 
 Aitv 
 
 tte aad uly, 1923 Research Bulletin No. 80 
 
 HE VOLATILE ACIDS PRODUCED 
 BY STARTERS AND BY ORGAN- 
 ISMS ISOLATED FROM THEM 
 
 By B. W. HAMMER AND F. F. SHERWOOD 
 AGRICULTURAL EXPERIMENT STATION 
 IOWA STATE COLLEGE OF AGRICULTURE 
 AND MECHANIC ARTS 
 
 Cy H, Curis, Director 
 
 DAIRY SECTION 
 
 THE LIBRARY OF THE 
 SEP 24 1930 
 UNIVEKSITY OF ILLINOIS, 
 
 AMES, IOWA 
 
EXPERIMENT STATION OFFICERS AND STAFF 
 
 Raymond A. Pearson, M.S.A., LL.D, President 
 
 C. F. Curtiss, M.S.A., D.S., Director 
 
 W. H. Stevenson, A.B., B.S.A., Vice-Director 
 
 AGRICULTURAL ECONOMICS AND FARM MANAGEMENT 
 
 » Gee NourseseA, be eh onier 
 
 L. Holmes, A.B., Ph.D., Asst. Chief 
 OF Taylor oameasste 
 
 . la, Benner, A.M. VAsst. 
 
 John Hopkins, Jr., A.M., Asst. 
 
 aaas 
 
 AGRICULTURAL 
 
 Job a Davidson wb Se Mola Arhi me niet 
 W. A. Foster, B.S. in E., B.Arch., Asst. 
 Chief 
 
 Knute Bjorka, 'M.S., Asst. 
 
 C. W. Crickman, B.S., Asst. 
 
 W. H. Youngman, B.S., Asst. 
 
 E. J. Working, M.S., Asst. 
 Frank Robotka, B.S., Asst. 
 ENGINEERING 
 1D. Wo Oeil. AES), teak ANID 
 Agron., Asst. Chief 
 Verne W. Stambaugh, B.S. in A.E., Re- 
 search Fellow 
 
 Bisa 
 
 AGRONOMY 
 
 W. H. Stevenson, A.B., B.S.A., H.C.D., 
 Chief 
 B.S., M.S.A., Chief in 
 
 H. D. Hughes, 
 Farm Crops 
 
 Pe Ee Brown wb es Ace hes Ohreiein 
 Soil Chemistry and Bacteriology 
 
 L. C. Burnett, B.S.A., M.S., Chief in 
 Cereal Breeding 
 
 L. W. Forman, B.S.A., M.S., Chief in 
 Field Experiments 
 
 J. L. Robinson, B.S., M.S., Superintend- 
 ent of Cooperative Experiments 
 
 Eee OnTSOns ES. e ee hel ameAcsS us 
 Chief in Soil Chemistry 
 
 Paul Emerson, B.S., M.S., Ph.D., Asst. 
 Chief in Soil Bacteriology 
 
 F.. S. Wilkins, B.S., M-:S., Asst. Chief 
 
 in Farm Crops 
 
 H. Benton, B.S., M.S., Soil Surveyor 
 S. Gray, B.S., Soil Surveyor 
 
 L. Orrben, B.S., Soil Surveyor 
 
 M. O’Neal, Jr., B.S., Soil Surveyor 
 
 T. Auten, B.S., Soil Analyst 
 
 . G. Baker, B.S., Field Experiments 
 
 P. E. Nordaker, B.S., Field Experiments 
 
 J. L. Boatman, B.S., Field Experiments 
 
 q4Poon 
 
 ANIMAL HUSBANDRY 
 
 H. H. Kildee, B.S.A., M.S., Chief 
 
 J. M. Evvard, B.S.A., M.S., Chief in 
 Swine and Beef Cattle Production 
 
 Wray E. Hammond, B.S.A., M.S., Supt. 
 of Experiments in Animal Husbandry 
 
 CoC) Culbertsonys bios) in eAcwe ro mA ssts 
 Chief in Animal Husbandry 
 
 Q. W. Wallace, B.S., Assistant in Animal 
 Husbandry 
 
 A. R. Lamb, M.S., Chief in Nutrition 
 
 A. B. Caine, M.S., Chief in Horse In- 
 vestigations 
 
 P. S. Shearer, P.S., Chief in Animal 
 Breeding 
 Mee D2 Helser) BiSvAy MES: = Chichewa 
 
 Meat Investigation 
 
 G. E. Weaver, M.S., Chief in Dairy Hus- 
 bandry 
 
 James Waddell, B.S., M.S., Asst. Chief 
 
 Fordyce Ely, B.S., M.S., Asst. in Dairy 
 Husbandry 
 
 H. A. Bittenbender, B.S.A., Chief in 
 Poultry Husbandry 
 
 Re be Cochran 3B. S) eAsstan@hiet 
 
 BACTERIOLOGY 
 
 R. E. Buchanan, M.S., Ph.D., Chief, As- 
 sociate in Dairy and Soil Bacteriology 
 
 Buford H. Butcher, Graduate 
 
 Asst. 
 
 a ey 
 
 BOTANY AND PLANT PATHOLOGY 
 
 DE Pammels Bator MiSs) PhD Ohiek 
 
 Charlotte M. King, Asst. Chief 
 
 I. E. Melhus, B.S., Ph.D., Chief in Plant 
 Pathology 
 
 L. W. Durrell, B.S., M.S., Asst. Chief in 
 
 Plant Pathology 
 
 Jee Nee Martinge bs Sa emeASsts 
 Atlin Bakke Bes eM See be aeeAcs bs 
 Ray FEF. Crawford, B.S., Asst. 
 
 O.. H: Elmer, B.S., Asst. 
 
 Karl F. Petsch, B.S., Field Asst. 
 
 BY. PB. Sipe, B.S., Fellow { 
 
 CHEMISTRY p 
 
 W. G. Gaessler, B.S., M.S., Acting Chief 
 A. R. Lamb, B.S., M.S., Asst. 
 
 Edith Wilson, Asst. 
 J. A. Schullz, B.S., Asst. 
 
 DAIRYING 
 
 M. Mortenson, B.S.A., Chief 
 B. W. Hammer, Ph.D., Chief in Dairy 
 Bacteriology 
 
 F. F. Sherwood, M.S., Asst. Chief 
 Merle P. Baker, B.S., Asst. in Dairying 
 
 ENTOMOLOGY 
 
 Carl J. Drake, B.Sc., B.Ped., M.A., 
 Ph.D., Chief in Entomology 
 
 F, A. Fenton; B.A., M.S., Ph.D., Asst. 
 Chief in Entomology 
 
 Albert Hartzell, B.S., M.S., Asst. 
 
 Wallace Park, B.S., Asst. Chief in Api- 
 culture 
 
 Wallace Colman, A.B., M.S., Asst. 
 
 HORTICULTURE AND FORESTRY 
 
 ——_—________——_., Chief 
 T. J. Maney, B.S., Chief in Pomology 
 Harvey L. Lantz, B.S,, Asst. Chief 
 
 H. H. Plagge, B.S., Asst. in Pomology 
 A. T. Erwin, M.S., Chief in Truck Crops 
 
 W. C. Calvert, B.S., Asst. in Truck Crops 
 
 EH. <A. Piester, B:S:, Acting Chief, in 
 Landscape Architecture 
 
 G. B. MacDonald, B.S.F., M.F., Chief in 
 Forestry 
 
 RURAL SOCIOLOGY 
 
 G. H. Von *Tungeln; “Ph.B., MUA:" Chief 
 
 H. G. Loomer, B.A., Asst. 
 
 W. W. Weaver, B.S., Asst. 
 
 BULLETIN SECTION 
 
 F. W. Beckman, Ph.B., Bulletin Editor 
 
 Olive Sandford Alcox, Asst. Bulletin 
 Editor 
 
 PHOTOGRAPHIC SECTION 
 
 E. H. Richardson, Photographer 
 
i. 
 
 THE VOLATILE ACIDS PRODUCED BY 
 STARTERS AND BY THE ORGAN- 
 ISMS ISOLATED FROM THEM 
 
 By B. W. HAMMER AND F, F. SHERWooD 
 
 The work done in various laboratories during the last few 
 years has shown that starters, instead of being pure cultures of 
 Streptococcus lactis, as was at one time commonly supposed, 
 contain two or more organisms, at least one of which produces 
 considerable amounts of volatile acid. Good starters have regu- 
 larly been found to yield a high and rather definite amount of 
 volatile acidity but natural souring and sometimes poor starters 
 also give a high volatile acidity, so that such an acidity is no 
 guarantee as to the quality of a starter. This indicates that 
 while the amount of volatile acid produced is important with 
 a starter, the character of the volatile acids must also be con- 
 sidered and suggests the desirability of knowing what kinds of 
 acids are produced in starters and in pure cultures of the 
 starter organisms. The work herein reported represents an at- 
 tempt to secure information along this line and also on the kinds 
 of acids produced during prolonged natural souring such as 
 occurs in old cream. 
 
 METHODS USED 
 
 The solutions of volatile acids studied were secured by dis- 
 tilling the fermented milk or cream with steam after the addition 
 of a small amount of N/1 H.SO, to free any volatile acids that 
 might have been fixed by the milk constituents. With the 
 starters a liter of the fermented material was commonly distilled 
 in a five liter flask after the addition of 45 ¢.c. of N/1 H.SO,, 
 while with the pure cultures of organisms it was more common 
 to distil 250 ec. of the milk cultures in a two liter flask after 
 adding 15 ec. N/1 H,SO,; in both cases the usual amount of 
 distillate collected was 1 liter. In order to secure larger amounts 
 of volatile acid with the starters two distillations were commonly 
 made at the same time and the distillates mixed while with the 
 pure cultures distillates were also frequently combined. 
 
 Two methods were used in determining the kinds of volatile 
 acids present in the distillates, the estimation of the percent 
 Ba* in the barium salt and a modified Duclaux method. 
  *Before deciding on the use of the barium salt, the silver salt method (Barthel 
 Chr. 1910——Methods used in the Examination of Milk and Dairy Products, p. 219) 
 
 was tried but under the conditions used was not as satisfactory as the barium galt 
 method. 
 
 74 2°5R 
 
4 
 
 TABLE I—PERCENT Ba IN SALTS PREPARED FROM COMMERCIAL ACETIC AND 
 PROPIONIC ACIDS 
 
 % Ba in barium salt 
 
 A B Average Theoretical 
 
 Ba acetate: 
 
 4M ig 29 SOE Nar I Ce ae PR et 53.44 53.37 53.405 53.78 
 
 CTT ale ZY eee er ee Rees 53.70 53.59 SSC OMe t clloer se eee eS 
 
 ride Oot a ee 53.46 53.40 5S AS ee ase Sa eee 
 
 Triad <4 eee ee ee | 8 Toe 53.78 53.78 DB 18S olos2 eee 
 Ba propionate: 
 
 RW eR Meee ee ee Sy area ee 48.60 48 .29 48.445 48.46 
 
 Aisi ARE awe Bae ee re Oc oe 48.47 48.50 48-485) 2 | See ee eee 
 
 Brio. Rowe se 5 § eee a te ee er 48 .53 48.48 48: HOD Ms ae ee eee 
 
 ADS AEE Nig e: Seaplane on San “eles LS 48.44 48.41 A8;-42,5 gee seca ee eee 
 Barc bityrate. ee Ss 1 eee |e ee Se es Br eee | ee 44.10 
 
 The usual procedure in determining the percent Ba was to 
 titrate 100 ee. of the distillate with N/10 Ba (OH)., using phen- 
 olphthalein as an indicator, and then to add a little less than 
 the calculated amount of Ba(OH), to the remaining 900 cc. of 
 distillate, the 100 ec. portion to which indicator had been added 
 being discarded. The aqueous solution of the barium salt was 
 concentrated on the water bath to about 50 ec. and filtered. 
 After evaporating to dryness on the water bath the salt was 
 recrystallized, dried at 100° C. and the percent Ba determined 
 as follows: Two portions of about 14 gram each were weighed 
 out, dissolved in from 75 to 100 ce. of hot water, heated to boiling 
 and a slight excess of normal H,SO, slowly added while boiling 
 the solution. After digesting over night on the hot plate 
 (under a watch glass so that the free volatile acids would not 
 be lost smce a Duclaux determination was to be run on them), 
 the BaSO, was filtered off, ignited and weighed. From the 
 weight of BaSO, and the weight of the original salt the percent 
 Ba in the latter was calculated. Table I shows the results ob- 
 tained by this method with supposedly pure acetic and propionic 
 acids, from commercial sources. 
 
 The filtrates from the Ba determinations were used for the 
 Duclaux method which was carried out according to the modifi- 
 cation of Boekhout and Ott de Vries.t. The volume of the solu- 
 tion being distilled was kept constant at 110 ce. and distilled at 
 the rate of 100 cc. in about 45 minutes. The distillate was col- 
 
 1. (Dept. Landb.), Nijv. en Handel (Netherlands), Verslag en Meded. Dir. 
 Landb. 5, 12 (1916) Thru Exp. Sta. Record 387, p. 207. 
 
 49 
 a” 
 
5 
 
 lected in ten ce. fractions and each fraction titrated with 
 N/10 Ba (OH), solution using phenolphthalein as an indicator. 
 This method is based upon the fact that each volatile fatty acid 
 has a constant rate of vaporization when distilled under given 
 conditions. For example, if a definite amount of acid is sub- 
 jected to distillation, the amount that passes over in each suc- 
 cessive fraction bears a definite relationship to the total acidity 
 of the distillate. Constants were determined for acetic and pro- 
 pionie acids using about a 0.5 percent solution since this was 
 approximately equivalent to the strength of the unknown solu- 
 tions worked with; these are given in table II, where A repre- 
 sents the titration figure for each successive 10 ce. of distillate, 
 B the sum of these figures for a given amount of distillate, and 
 C the value given in B ealeulated as the percentage of the titra- 
 tion figure for the total 100 ce. of distillate. he values for 
 propionic acid are higher than the values for acetic acid indicat- 
 ing that propionic acid distills more rapidly than acetic acid. 
 The volatile acids worked with often gave constants which 
 fell in between those for acetic and propionic acids. When the 
 constants were much nearer those of acetic than those of pro- 
 plonie acid the mixture was considered as mainly acetic plus a 
 small amount of propionic acid and when the values obtained 
 agreed less closely with those for acetic acid the mixture was 
 considered to be acetic plus large amounts of propionic acid. 
 Table III illustrates the type of constants that were considered 
 to indicate a mixture of acetic acid plus a small amount of pro- 
 
 TABLE II—DUCLAUX VALUES FOR ACETIC AND PROPIONIC ACIDS 
 
 Acetie Acid 
 ee. of distillate 
 
 10 20 30 40) 50 60 70 80 90 100 
 
 Nee Gc hag. SSO Raia help ee Ane eee Deb e Ole eae meee OS 1.90) Soler ls SOW eke iol) mle O 
 eee ee ee ee 2.45) 4.75) 6.95) 9.05) 11.10) 13.00] 14.€5 16.65] 18.40) 20.10 
 Cl een cee ee UE BE 12.19) 26.63) 34.57) 45.02) 55.22) 64.68] 73.89) 82.838 et.56 100 
 Propionie Acid 
 ce. of distillate 
 10 20 30 40 50 60 70 80 90 100 
 Nee a Bey 2, ES Se Batol! asi! Siti Besa, Gui ee O DLE OF iat OA) ahateth, 
 NOS b, Geeta 2 4 ny pe enna Oe a ee 3.75| 7.10) 10.15) 18.00] 15.75) 18.35) 20.70) 22.85] 24.85) 26.65 
 Caen aay we Fate Pr) 14.07| 26.65} 38.08] 48.77 Pee 68.87| 77.67| 85.74} 93.26}100 
 
 *A—ce. N/10 Ba (OH):2 required for successive 10 ec. fractions. 
 **B—sum of values in A for a given amount of distillate. 
 ***C—values given in B ecale. as percent of the titration values for the 100 ce. of 
 listillate. 
 
 \ 
 
6 
 
 TABLE III—CONSTAN'T'S ILLUSTRATING ACETIC ACID PLUS A SMALL AMOUNT 
 OF PROPIONIC 
 
 ce. of distillate 
 
 ] 
 
 Source of material 10 20 30 40 50 60 70 80 90 100 
 Starter, oe See eee 12.39] 24.04) 35.22) 45.39] 55.46) 65.34] 74.67| 83.73] 91.94 100 
 SUATLOTA.2 cae 2 eee eee 12.45) 24.28) 35.27) 46.06] 55.14] 66.19} 75.95) 83.70) 91.94 100 
 Starter. eee eee ee eee 12.65) 24.53] 35.65] 45.99] 55.95) 65.54 14.78) 83.54] 91.98 100 
 
 plonie acid while table IV illustrates the constants considered to 
 indicate a mixture of acetic acid with large amounts of propionic 
 acid. While the Duclaux method as employed cannot be con- 
 sidered entirely satisfactory as a means of determining the 
 kinds of volatile acid present in a mixture, its use seemed de- 
 sirable in order to confirm the results secured with the method 
 of determining the percent Ba in the barium salt. 
 
 RESULTS SECURED 
 
 The types of volatile acids produced in starters ripened for 
 long periods are illustrated by table V. These results were ob- 
 tained during the preliminary work when the methods to be 
 employed were being studied; the long ripening periods were 
 used because considerable quantities of volatile acids were de- 
 sired and they were most easily secured from highly ripened 
 starters. The data presented show that with starters ripened 
 for considerable periods the barium values on the volatile acids 
 were over 03 percent in all cases, but not up to the values secured 
 with pure acetic acid; this suggests that the acid is very largely 
 acetic with a small amount of propionic. The Duclaux values 
 indicate the same thing. It is of interest to note how little 
 variation is evident in the barium values obtained on a number 
 of starters from a wide variety of original sources, when these 
 were ripened for considerable periods. 
 
 Table VI gives the results obtained on a number of starters 
 when two determinations of the types of volatile acids present 
 
 TABLE IV—CONSTANTS ILLUSTRATING ACETIC ACID PLUS A LARGE AMOUNT 
 OF PROPIONIC 
 
 ee. of distillate 
 
 Souree of material 10 20 50 40 50 60 70 80 90 100 
 Culture S. citrovorus___-__- 13.38] 25.97| 37.21) 47.67| 57.18) 66.07| 77.82] 88.83] 91.85 100 
 Starter ot ee eee 18.48) 25.37] 37.31) 48.51) 58.95) 68.66] 77.61) 85.82] 93.18 100 
 Starters ae eee 12.96) 25.92] 37.03) 48.22) 57.41| 68.67] 77.93) 85.26) 92.60 100 
 
7 
 
 TABLE V—BARIUM AND DUCLAUX RESULTS OBTAINED ON VOLATILE ACIDS 
 FROM HIGHLY RIPENED STARTERS 
 
 Acidity of % Ba in 
 1 lL. distillate barium salt 
 Starter Ineubation) from 1 J. — — Results of Duclaux 
 starter (steam| Det. | Det. |Aver- 
 distillation) AS 13}, age 
 
 DE SS ae ee eS eee 60.5 ec. N-/10} 53.02) 58.15/58.085|Acetie plus trace of propionic 
 Deeseeaeeens Bel, AIP COlaI) he) oe “ 53.08| 53.11/58.095)/Acetie plus trace of propionic 
 Hansen_____- 4 Cole AON aside 9 =¢ 53.10} 53.10/53.10 |Acetie plus trace of propionic 
 Whamplon=22 (Zed len On lGe. mes “ 53.27) 53.27158.27 | Acetic plus trace of propionic 
 Kricsson_-_-__- iol, Cal (l.|| Goa Ss G 53.23] 58.211538.22 |Acetic plus trace of propionic 
 Da bzeera ee. 26s DALY Bal ate (07 uC sé SB1850)| 53.30 |Acetie plus trace of propionic 
 Hansen___-__- A(t, Pal (Oral ey fe 58.02| 53.09|53.055|Acetic plus trace of propionic 
 
 were made at different times, the object being to see whether 
 the volatile acids were the same when the acidity of the starter 
 was low as when it was high. The data presented show that 
 with high acidities in the starter, the barium values on the vola- 
 tile acids were essentially the same as those given in table V, 
 being either above 53 percent or very close to this value in all 
 cases. In general with the lower acidities in the starters, lower 
 barium values were secured; in two instances with low acidity 
 starters the barium values were only shghtly over 51 perceni 
 and in two others only slightly over 52 percent. Since a iow 
 total acidity in the starter was accompanied by a low volatile 
 acid production, low barium values were obtained with low 
 volatile acidities. It is evident then that when a starter has 
 produced only a low total and volatile acidity, the volatile acids 
 have lower barium values than when the acidities are high and 
 accordingly include more volatile acid of a type higher than 
 acetic. The data presented indicate also that in general there 
 was less change in the type of volatile acid in the two determina- 
 tions when the first was on starter having. a total acidity near 
 0.8 percent than when it was on a starter having a lower total 
 acidity ; that is, the influence of 0.1 percent in the total acidity 
 of the starter on the type of volatile acid was less near the maxi- 
 mum acid production than it was at a lower acidity. The 
 Duclaux results reported in table VI confirm the conclusions 
 drawn from the barium values. 
 
 The results given in table VII which were obtained at a later 
 date and to a large extent on a different lot of starters than 
 those given in table VI are in the main confirmatory of those 
 presented in the latter table. The same high barium values were 
 secured on starters ripened to high acidities and the same gen- 
 eral relationship between low total (or volatile) acidities and 
 low barium values is evident. In the two instances where three 
 determinations were made on a lot of starter instead of only two 
 
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10 
 TABLE VIII—BARIUM AND DUCLAUX VALUES OBTAINED ON FIRST AND 
 SECOND LITER OF DISTILLATE FROM STARTER 
 
 First liter distillate 
 
 % Ba in barium salt 
 
 Starter — — Results of Duclaux 
 
 Det. A. | Det. B. | Average 
 
 DI0d a stadethes sseee 51.60 51.64 51.62 Acetic plus small amount propionie 
 20s dette see en 53.47 53.62 53.545 Acetie plus slight trace propionic 
 Ste WP S6 (ei rsp tere 53.20 53.27 53.230 Acetie plus slight trace propionic 
 
 Second liter distillate 
 
 % Ba in barium salt 
 
 Starter —— Results of Duclaux 
 
 Det. A. | Det. B. | Average 
 
 Dic lst dete =a 51.62 51.56 51.59 Acetic plus small amount. propionic 
 PLO sale rt 53.26 Dowell 53.285 Acetic plus trace propionic 
 SO OCG ae ee Hoek 53.32 53.265 Acetie plus trace propionic 
 
 — 
 
 the results simply tend to confirm those secured where two de- 
 terminations were used; in one instance the third determination 
 gave a slightly lower barium value than the second but the dif- 
 ference is too small to be of any significance while in the other 
 instance there was a rise in the barium value from the first to 
 the third determination. The Duclaux results reported in table 
 VII again confirm the conclusions drawn from the barium 
 values. 
 
 The data presented in tables VI and VII indicate that at a 
 given acidity starters may show quite different barium values. 
 The differences along this line seem to be as great in different 
 lots of starter inoculated from the same culture as in lots of 
 starter inoculated with different cultures. It seems reasonable 
 to conclude that the change from the volatile acids giving a low 
 barium value (acetic with considerable propionic) to those giv- 
 ing a somewhat higher barium value (acetic with only a small 
 amount of propionic) does not always occur at exactly the same 
 total acidity of the starter. 
 
 With the series of three determinations that were made on 
 one lot of starter 104 and reported in table VII barium and 
 Duclaux values were also secured on a second liter of distillate 
 from each determination. These are given in table VIII to- 
 evether with the barium and Duclaux values for the first liter 
 of distillate from each determination for comparison. The data 
 
11 
 
 show a close agreement between the values for the first and sec- 
 ond liters in all three comparisons and suggest that the first liter 
 of distillate is representative of the volatile acids produced in a 
 starter and can be used for studies on the kinds of volatile acids 
 formed. 
 
 The variation in the volatile acids produced at different 
 periods during the ripening of a starter indicates that the dif- 
 ferent groups of organisms may not produce the same volatile 
 acids, and suggests a consideration of the volatile acids formed 
 by S. lactis and by the associated organisms in pure cultures. 
 
 Material for the determination of the kinds of volatile acids 
 produced by SN. lactis was secured by combining distillates ob- 
 tained from cultures of S. lactis that were being checked to be 
 certain of their low volatile acid production. While a distillate 
 from a culture of one organism might have been preferable to 
 the material used, the work involved in securing such a distillate 
 with the low volatile acid producing S. lactis cultures seemed to 
 justify the other procedure. The results secured on the S. lactis 
 cultures are given in table 1X; from these it will be seen that the 
 pure cultures of S. lactis gave low barium values, comparable 
 to those secured on a starter rather early in the ripening period. 
 These barium values indicate that the volatile acid produced by 
 S. lactis is not largely acetic? as has been suggested in some in- 
 stances but is acetic with some higher acid presumably propionic 
 included with it in considerable amounts. The Duclaux values 
 confirm the conclusions drawn from the barium values. 
 
 TABLE IX—BARIUM AND DUCLAUX VALUES OBTAINED ON DISTILLATES 
 FROM CULTURES OF 8S. LACTIS 
 
 Number of| Average | % Ba in barium salt 
 distillates | volatile 
 
 mixed acidities* —.: Results of Duclaux 
 to supply |>f cultures : 
 
 acids S. lactis. | Det. A} Det. B] Av. 
 2 8.2 51.34 Bila 51.555| Acetic plus small amount of propionic 
 7 Oe 50.48 50.59 50.535| Acetic plus large amount of propionic 
 4 10.7 51.82 51.74 51.78 | Acetic plus small amount of propionic 
 2 it 0 50.23 50.49 50.36 | Acetic plus large amount of propionic 
 2, he 50. 64 50.93 50.785| Acetie plus large amount of propionie 
 4 7.5 50.34 50.49 50.415} Acetic plus large amount of propionic 
 4 8.1 51.86 | 51.90 51.88 | Acetic plus small amount of propioni¢c 
 6 (4 50.78 50.71 50.745) Acetic plus large amount of propionic 
 4 6.0 50.54 50.31 50.425] Acetic plus large amount of propionic 
 4 HAG 49.63 49.86 49.745| Acetic plus large amount. of propionic 
 4 Choiet 51.46 51.28 51.37 | Acetic plus small amount of propionic 
 9 8.7 50.12 50.24 50.18 | Acetic plus large amount of propionic 
 6 7-1 49.98 50.311 50.145] Acetie plus large amount. of propionie 
 
 *ee, of N:/10 alkali required to neutralize the first liter distillate obtained by 
 distilling 250 grams with steam after adding 15 ee. of approx. N/1 He2SOu. 
 
 2. See Evans, Alice C. A Study of the Streptococci Concerned in Cheese Ripen- 
 ing. Jr. Agr. Res. 13, Ap. 22, 1918, p. 235, for review of literature and original 
 data. 
 
iL 
 
 In determining the volatile acids produced by the organisms 
 associated with S. lactis in starters, various kinds of material 
 were employed. In two instances distillates secured from milk 
 cultures of S. paracitrovorus were used, while in the remainder 
 distillates from cultures of S. citrovorus or 8S. paracitrovorus 
 erown in milk to which citric acid had been added were em- 
 ployed. The addition of the citric acid increased very mate- 
 rially the amount of volatile acid produced and made it easier 
 to secure sufficient volatile acid for the preparation of the 
 barium salts in satisfactory amounts. The results secured with 
 the associated organisms are presented in table X. The barium 
 values with these organisms were higher than the values secured 
 with S. /actis and approximated the values obtained with starters 
 that had been allowed to develop considerable amounts of acid. 
 It is accordingly evident that the associated organisms produce 
 a volatile acidity that is largely acetic and thus quite different 
 than the volatile acidity produced by SN. lactis. The type of vola- 
 tile acids produced by the associated organisms accounts for 
 the type of volatile acids produced in starters that have de- 
 veloped considerable total acidity, which cannot be accounted 
 for by the action of S. lactis, and thus affords further proof of 
 the importance of the associated organisms in a starter. 
 
 In order to compare the volatile acids present in old cream 
 soured naturally with those present in starter, the volatile acids 
 were distilled from old cream with steam and the barium and 
 Duclaux values determined. The old off-flavored: cream was 
 secured by holding cream at room temperature for varying 
 lengths of time. The results obtained, which are given in table 
 XI, show that the barium values were very different than the 
 values secured on starters, as would be expected from the odor 
 of the lots of old cream. The barium values are low indicating 
 that acids higher than acetic must be present in considerable 
 amounts. Some of the barium values are so low that if a cer- 
 tain amount of acetic acid was present as was probably the case, 
 they could not be accounted for by the remainder of the volatile 
 acid being propionie so that some higher acid probably butyric 
 would necessarily be present. The Duclaux values on the dis- 
 tillates from old cream confirm the conclusions drawn from the 
 barium values. 
 
13 
 
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14 
 
 DISCUSSION OF RESULTS 
 
 The results presented show that in a highly ripened starter 
 the volatile acid is largely acetic, with undoubtedly a small 
 amount of propionic acid. These volatile acids must be assumed 
 to be of considerable importance from the standpoint of de- 
 termining the odor and flavor of a starter, altho by no means 
 the only factor. It is evident that the kind of volatile acid pres- 
 ent is not the same thruout the ripening period of a starter, the 
 acetic acid being less prominent early in the ripening and more 
 prominent later, and the propionic acid accordingly more 
 prominent early and less prominent later. The acetic acid pres- 
 ent in a starter seems to result mainly from the action of the 
 associated organisms, while the propionic acid comes largely 
 from the action of the S. lactis group. The time of the appear- 
 ance of these different types of volatile acids suggests that dur- 
 ing the early part of the ripening of a starter the S. lactis or- 
 anisms are the main ones developing and that later on the 
 associated organisms become active. This same idea is suggested 
 by the results previously reported by the Iowa station® showing 
 that the percentage of the total acidity made up of volatile acid 
 is greater late in the ripening period than early. 
 
 The data secured on the distillates from old sour cream show 
 that the kinds of volatile acids produced during prolonged 
 natural souring are quite different than those produced in the 
 ripening of starters, being higher in the series of fatty acids. 
 These acids are undoubtedly partly responsible for the very un- 
 desirable flavors and odors in old cream and in the butter made 
 from it. The results obtained in connection with the kinds of 
 volatile acids produced in cream held for a long period suggest 
 that certain organisms growing in such material may be of quite 
 different types than those ordinarily considered to be the most 
 prominent in causing changes in milk or cream. 
 
 3. Hammer, B. W., and Cordes, W. A. The Relation Between the Volatile and 
 
 Total Acidity in Starters and in Cultures of S. lacticus. Ja. Agr. Expt. Sta. Res. 
 Bul.. 66, July, 1921. 
 
15 
 
 CONCLUSIONS 
 
 1. Starters ripened for considerable periods contained vola- 
 tile acids made up largely of acetic with small amounts of pro- 
 plonie. 
 
 2. At low total (and accordingly volatile) acidities propt- 
 onic acid made up a greater percentage of the volatile acidity 
 than it did at higher total acidities. 
 
 3. The change from a volatile acidity in which propionic 
 acid was present in comparatively large amounts to one where 
 it made up only a small percentage of the total volatile acid 
 did not occur at any definite total acidity of a starter. 
 
 4. Streptococcus lactis produced a volatile acidity made up 
 of acetic with considerable amounts of propionic acid. 
 
 5. The associated organisms—S. citrovorus and S. para- 
 citrovorus—produced a volatile acidity that was very largely 
 acetic. 
 
 6. The volatile acids secured from old cream were quite dif- 
 ferent than those secured from starters in that acid higher than 
 acetic was present in large amounts. In some instances butyric 
 acid was apparently included. 
 
30112 11461