The Effect of Medium Constituents on Penicillin Production fromNatural Materials1

BIJoy K. BHUYAN AND MARVIN J. JOHNSON

Departmnent of Biochemistry, University of Wisconsin, Madison, Wisconsin

Received for publication January 7, 1957

Since the discovery of the stimulating effect of cornsteep liquor on penicillin fermentation, various lactose-steep liquor media have been used in penicillin fermen-tations. The requirements for a good penicillin fermen-tation are fulfilled by the steep liquor-lactose media byvirtue of its composition (Johnson, 1952). The produc-tion of penicillin on a chemically defined medium (Jarvisand Johnson, 1947) is evidence for the absence of anyunknown essential factors in steep liquor. Therefore,natural materials other than steep liquor should besuitable for media for penicillin production, providedthat the constituents of such media are properlybalanced.

Several attempts have been made to replace steepliquor with various organic nutrients in surface culturemedium. Cook and Tulloch (1944) advocated the use ofpressed juice from fresh peas as a substitute for steepliquor. Wheat bran moistened with an equal weight ofwater was proposed by Rao (1944).

Foster et al. (1946) reported that cottonseed meal hadpotentialities as a possible substitute for steep liquor insubmerged penicillin fermentation. Perlman (1949)also showved that several protein materials may be usedas substitutes for steep liquor without marked reductionin penicillin yield.

In the work reported here, a number of organicmaterials were used as nitrogenous nutrients in penicil-lin production. In each case, an attempt was made toadjust the concentrations of the constituents of themedium so that the best environmental requirementsfor penicillin production would be obtained. Changesmade in the composition of the medium were on thebasis of information obtained from analyses madeduring the fermentations. These studies have shownthat with proper adjustment of the composition of themedium a wide variety of materials may be used ascarbon and nitrogen sources in the penicillin fermenta-tion.

METHODS AND MATERIALS

Fermentation techniques. The study was initiated withPenicillium chrysogenum strain W49-133 and was

1 Published with the approval of the Director of theWisconsin Agricultural Experiment Station. Supported inpart by grants from Wyeth Laboratories, Philadelphia,Pennsylvania, and Abbott Laboratories, North Chicago,Illinois.

262

later extended to include several strains descended fromit. Unless specifically mentioned the fermentations werecarried out with this strain.

Spores were grown on a spore plate medium whichcontained the following (in g per L): honey, 60; peptone,10; agar, 25. Ten ml of sterile water were dispensed intoeach plate to prepare the spore suspension. Five ml ofthe suspension were used to seed the synthetic inoculummedium which contained glucose, 4 g; calcium carbonate,1.3 g; ammonium sulfate, 1.3 g; and 10 ml of a saltmixture per 100 ml of medium. The salt mixture had thefollowing composition (in g per L): KH2PO4, 30; MgSO4*.7H20, 2.5; Fe(NH4)2(SO4)2. 6H20, 1.0; CuSO4. 5H20,0.05; ZnSO4*7H20, 0.2; Na2SO4, 5.0; MnSO4 H20,0.2; CaCl2 2H20, 0.5. After an incubation period ofapproximately 48 hr, 5 ml of the vegetative inoculumwere used to seed the fermentation medium.

Essential elements required by the mold were sup-plied in the fermentation medium either by the nutrientor when necessary by including 10 ml of salt mixture(of the above composition) per 100 ml of the medium.All fermentation media received 0.4 per cent calciumcarbonate and 0.1 per cent sodium sulfate.

After the medium was autoclaved the pH was ad-justed with sulfuric acid to about 5.5. All fermentationswere run in 500 ml Erlenmeyer flasks containing a totalvolume of 100 ml. The flasks were placed on a Gump2rotary shaker which described a 2-in. circle and operatedat 250 rpm. The temperature of incubation was 25 C.All fermentations were run in duplicate flasks, and thefigures reported here are the average of these 2 flasks.Samples for penicillin assay and chemical analyseswere taken under aseptic conditions and handled in themanner described by Gailey et al. (1946).

Potassium phenylacetate at a 0.1 per cent level wasadded as precursor to all fermentations every 24 hr afterthe pH of the fermentation had risen above 6.5.

Analytical procedures. Penicillin was assayed by theOxford cup method with Micrococcus pyogenes var.aureus strain H as the test organism and penicillin G asthe standard. The pH of each sample was detePminedimmediately after removal by means of a glass electrode.All sugars were determined by the method of Shafferand Somogyi (1933). Lactose was hydrolyzed with 1.5N hydrochloric acid in an autoclave at 120 C for 15 min.

2 B. F. Gump Company, Chicago, Illinois.

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NUTRIENTS FOR PENICILLIN PRODUCTION

The standard lactose curve was used with lactose-glucose combinations. The method for determination ofammonia nitrogen was that described by Gailey et al.(1946). Soluble Kjeldahl nitrogen was determined bythe method of Johnson (1941). Mycelial nitrogen was

determined by subtracting the soluble nitrogen presentat the time of samplingfrom the soluble nitrogen presentat the time of inoculation. It was not possible to deter-mine mycelial nitrogen in media containing insolublenitrogenous compounds. Mycelial nitrogen was notdetermined when the following substances were used as

the source of nitrogen: cottonseed meal, soybean meal,Curbay B-G,3 and distillers' solubles.

Materials. Corn steep liquor was obtained from theA. E. Staley Co. Difco tryptone, peptone, and yeastextract were used.The fish solubles used contained 4.5 g Kjeldahl

nitrogen per 100 g. The cottonseed meal used had a

Kjeldahl nitrogen content of 7.8 per cent.The solvent extracted soybean meal used had 8.5

per cent Kjeldahl nitrogen.The distillers' solubles used are available under the

trade name of Stimuflav.4 This contained approxi-mately 4 per cent Kjeldahl nitrogen.Curbay B-G is the dried residue obtained from the

molasses butanol fermentation. The residue had a highash and low nitrogen (2.3 per cent) content.

RESULTS AND DIscussIoN

Requirements for penicillin production. It has beenestablished (Johnson, 1952) that among the require-

3United States Industrial Chemicals Inc., New York, NewYork.

4Hiram Walker and Sons, Inc., Peoria, Illinois.

ments for good penicillin production are the following:growth of a good crop of mycelium; maintenance of themycelium with an adequate air supply; a pH value be-low 8 but not much below 7; conditions of partialcarbohydrate starvation; and presence of phenylaceticacid or other suitable precursor. For the growth phase,a pH below 7 is desirable, and readily available carbonsources must be present. The partial carbohydratestarvation required during the penicillin forming phaseis ordinarily brought about either by the use of a poorlyavailable energy source such as lactose, or by slowlyfeeding a readily available source such as glucose. In our

experiments lactose was used. The carbon and nitrogensource during the growth phase was either steep liquoror any one of a variety of natural materials.

Effect of salt mixture. When tryptone or peptone was

used as a nutrient it was found necessary to include saltmixture in the medium. The effect of including saltmixture in media containing tryptone is shown in partA of table 1. As may be seen from the table, the in-creased penicillin production obtained when salts were

used was not entirely due to increased growth, since theamount of penicillin produced per mg of mycelialnitrogen also increased.The chemical changes occurring with and without

salt mixture in media containing 1.5 per cent tryptoneand 3 per cent lactose are shown in figure 1. The aminoacids and peptides present in tryptone were utilized inpreference to lactose as the carbon source during thegrowth phase of the fermentation. This resulted in theliberation of ammonia which served as the source ofnitrogen during the next phase of the fermentation andalso caused a sharp rise in pH during the first 24 hr. Inthe absence of salt mixture the ammonia released was

TABLE 1. Effect of salt mixture on penicillin fermentation with Penicillium chrysogenum

Nitrogen Source Time ofSeries Strain Lactose Glucose Salt Mixture Penicillin pH Plateau* Max Yield

Compounds Concentration

u/mgl | % % a0u/ml mycelial hr

N

A 49-133 Tryptone 1.5 3.0 0.0 _ 240 535 7.73 941.5 2.0 1.0 _ 140 250 7.80 1181.5 2.0 1.0 + 600 800 7.50 941.5 3.0 0.0 + 800 1300 7.34 94

B 49-133 Steep liquor 2.5 3.0 0.0 - 735 7.46 722.5 3.0 0.0 + 810 7.13 72

Cottonseed 4.0 2.5 0.5 _ 1080 7.4 84meal 4.0 2.5 0.5 + 1130 7.4 114

C 51-20F3 Steep liquor 2.5 5.0 0.0 _ 1375 7.6 1142.5 5.0 0.0 + 1700 7.3 114

D 51-20F3 Soybean meal 2.0 4.0 0.0 _ 1140 6.9 962.0 4.0 0.0 + 923 6.9 120

All media contained 0.4 per cent calcium carbonate and 0.1 per cent sodium sulfate.* The pH plateau is defined as the average pH that exists in the medium during the penicillin production phase.

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B. K. BHUYAN AND M. J. JOHNSON

utilized at a slow rate. Due to the slow rate of ammoniautilization, the pH remained above the optimum forpenicillin synthesis. The rapid utilization of lactose andammonia in the presence of salt mixture served to main-tain the pH in a region suitable for penicillin synthesis.Therefore a higher penicillin yield was obtained on themedium with salt mixture than on the medium withoutsalt mixture. It was not determined which of the con-

stituents of the salt mixture were responsible for theobserved effects.

C)

z

z

I:'

3

2

1

800

600

400

200

24 48 72 96HOURS

FIG. 1. Effect of salt mixture on tryptone fermentation withPenicillium chrysogenum strain 49-133.

= Medium without salt mixture. The medium con-

tained 1.5 per cent tryptone and 3 per cent lactose.= Medium with salt mixture. The medium contained

salt mixture in addition to the above-mentioned constituents.Both media contained 0.4 per cent calcium carbonate and

0.1 per cent sodium sulfate.

An attempt was made to control the rise in pH of thetryptone-lactose medium by adding 1 per cent glucose.The glucose served as one of the carbon sources duringthe growth phase of the fermentation. Therefore only a

small amount of ammonia was released and the rise inpH was less than in the medium without glucose.Ammonia released after the first 24 hr caused a sharprise in pH. The slow utilization of ammonia in theabsence of salt mixture caused the pH to stay at a leveltoo high for optimum conditions for penicillin synthesis.The penicillin yields on the tryptone-lactose-glucosemedium were therefore low. When 1 per cent glucosewas added to the tryptone-lactose-salts medium therise in pH was delayed by 24 hr. Because of the shortperiod during which pH was maintained in the regionsuitable for penicillin synthesis, the penicillin yieldon this medium was lower than those obtained with thetryptone-lactose-salts medium.

Similar results were obtained when peptone was usedas the nitrogen source.

Salt mixture was usually not needed when complexmaterials such as steep liquor, cottonseed meal, or

distillers' solubles were used. The effect of salt mixtureon fermentations with P. chrysogenum strain 49-133when steep liquor or cottonseed meal was used is givenin part B of table 1. As may be seen from the table, saltmixture did not have any beneficial effect on the fer-mentation. However, an exception was the fermentationof steep liquor medium with P. chrysogenum strain51-20F3. The results of this fermentation are given inpart C of table 1. Since the effect of salt mixture on

fermentation of steep liquor medium was rather unex-

pected, the run was repeated. It was found that theresult was reproducible. The effect of salt mixture on

penicillin yield in a fermentation with P. chrysogenumstrain 51-20F3 when soybean meal was used is given inpart D of table 1. As may be seen from the table, saltmixture did not have any beneficial effects on this fer-mentation.

TABLE 2. Effect of glucose on penicillin fermentation with Penicillium chrysogenum

Nitrogen Source TSeries Strain Lactose Glucose Salt Mixture Penicillin pH Plateau* Max Yield

Compound Concentration

o % % u/ml hr

A 49-133 Yeast extract 2.0 2.5 0.5 + 816 7.8 932.0 3.0 0.0 + 120 8.36 48

B 49-133 Fish solubles 2.0 2.5 0.5 + 715 7.13 902.0 2.5 0.5 _ 680 7.22 902.0 3.0 0.0 _ 328 7.86 902.0 3.0 0.0 + 428 7.90 90

C 49-133 Steep liquor 2.5 4.0 1.0 _ 1345 7.3 1142.5 5.0 0.0 - 1375 7.6 114

All media contained 0.4 per cent calcium carbonate and 0.1 per cent sodium sulfate.* The pH plateau is defined as the average pH that exists in the medium during the penicillin production phase.

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NUTRIENTS FOR PENICILLIN PRODUCTION

Effect of glmedium, thewere utilizedsource duringThis resultedas the nitrogfermentationnecessary to c

replacement fyeast extractglucose in m(

FIG. 2. EffecPenicillium chr

- = Meper cent yeast e

and salt mixtui--- = Me

per cent yeast E

Both media0.1 per cent sod

ucose. When glucose was absent from the part A of table 2. The medium containing glucose gaveamino acids and peptides in the nutrient higher yields of penicillin than the medium withoutin preference to lactose as the carbon glucose. Chemical changes obtained, with and without

y the growth phase of the fermentation. glucose, in media containing yeast extract, lactose, andin the liberation of ammonia which served salt mixture are shown in figure 2. When glucose was

,en source during the next phase of the present in the medium the pH was maintained in a

and also caused a sharp rise in pH. It was region suitable for penicillin synthesis. However, in theontrol this rise in pH by adding glucose as a absence of glucose, the pH rose above 8 during thefor part of the lactose in media containing penicillin producing phase. Because the medium con-

,, or fish solubles. The effect of including taining glucose had a greater supply of rapidly utilizableedia containing yeast extract is shown in carbon available during the growth phase and because

the pH was maintained in a region more suitable forgrowth, more mycelial nitrogen was present in this

XV x _ 1 fermentation and a good penicillin yield resulted. The

'ROGEN (y/ML) MYCELIAL-N pH very poor yield without glucose is the result of the quitehigh pH obtained during the penicillin producing phase.

/ML) Glucose also was found to be a necessary constituent ofM YCELIAL-N / media containing fish solubles. The effect of including

/ /t__ Xglucose in different media containing fish solubles is

/EA

shown in part B of table 2. Because the pH of media/t /PE^CILLIN 1 9 containing glucose was maintained in a region more

M suitable for penicillin synthesis, the penicillin yields

-oHO ~o| on these media were higher than those obtained in

8 media without glucose.O pH Glucose was not beneficial when included at low

levels (0.5 to 1 per cent) in media containing the other* 7 natural materials studied. Obviously if glucose had been

used to completely replace the lactose in the medium,*_ PEMCILLIN then the growth phase would have continued until all

the glucose was exhausted. Such a medium would have,, 6 given a very low yield of penicillin. The effect of re-

20 HOURS 80 100 placing part of the lactose by glucose in a steep liquor

medium is shown in part C of table 2. The mediumt of glucose on yeast extract fermentation with**-ysogenum strain 49-133. containing glucose had a lower pH during the growthdium with glucose. The medium contained 2 phase of the fermentation than the medium without*xtract, 2.5 per cent lactose, 0.5 per cent glucose, glucose. However, the pH plateau during the penicillinre. producing phase was essentially the same in both media.dium without glucose. The medium contained 2 Effect of lactose concentration. The length of time that

extract, 3 per cent lactose, and salt mixture. . . .contained 0.4 per cent calcium carbonate and the fermentation contiues the penllin producig

lium sulfate. phase is determined by the concentration of lactose in

TABLE 3. Effect of lactose concentration on penicillin fermentation with Penicillium chrysogenum

Nitrogen Source pHSeries Strain N Source Lactose Penicillin pH | Time ofSeries ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~Max Yield

Compound Concentration 96 hr 120 hr | 144 hr

% % u/ml hr

A 49-133 Steep liquor 3.0 3.0 1480 8.3 963.0 4.0 1655 7.82 8.2 963.0 5.0 1610 7.82 7.98 120

B 51-20F3 Steep liquor 2.5 5.0 1375 7.7 7.8 8.2 1202.5 6.0 1410 7.6 7.7 7.8 144

C 51-20F3 Soybean meal 2.0 4.0 1140 7.18 8.0 962.0 5.0 1518 6.9 7.45 8.0 1202.0 6.0 1400 6.9 7.25 7.6 120

All media contained 0.4 per cent calcium carbonate and 0.1 per cent sodium sulfatc.

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B. K. BHUYAN AND M. J. JOHNSON

the medium. When all the lactose is exhausted, autolysissets in, thereby terminating the penicillin producingphase of the fermentation. Therefore the amount oflactose in a medium is one of the factors determiningthe penicillin yields on that medium.When lactose was used at varying concentrations in

the fermentation medium, it was found that 5 per centlactose was sufficient to meet the needs of the myceliumduring the penicillin production phase. Further increasein lactose concentration did not result in increasedyields. The effect of increasing lactose concentration, on

TABLE 4. Effect of the concentration of the nitrogenous nutrienton penicillin yield with Penicillium chrysogenum

Nitrogen Source pH TimeSeries Strain-- __ Lac- Peni- Pla- of Max

Concen- tose cillin teau* YieldCompound tration

% % u/ml hr

A 49-133 Tryptone 0.75 5.0 355 7.5 1201.5 5.0 825 7.4 1403.0 5.0 118 8.1 48

B 54-1255 Steep liquor 2.0 5.0 1008 7.64 1202.5 5.0 1536 7.45 1403.0 5.0 1830 7.54 1203.5 5.0 2440 7.60 1204.0 5.0 2680 7.60 1404.5 5.0 2160 7.52 120

All media contained 0.4 per cent calcium carbonate and 0.1per cent sodium sulfate.

* The pH plateau is defined as the average pH that existsin the medium during the penicillin production phase.

TABLE 5. Penicillin yields on various media with differentstrains of Penicillium chrysogenum

Nitrogen SourceSat TmStrain La-Glu- Mix- Penin of Max

Concen- tose cose ture cillin YieldCompound tration

% % % u/mi

49-133 Steep liquor 3.0 5.0 0.0 - 1660 96Tryptone 1.5 5.0 0.0 + 1170 96Peptone 1.5 5.0 0.0 + 930 96Yeast extract 2.0 4.0 1.0 - 940 96Fish solubles 2.0 4.0 1.0 - 1030 96Cottonseed meal 3.0 5.0 0.0 - 1930 114Soybean meal 2.0 5.0 0.0 - 1700 114Distillers' sol- 4.0 5.0 0.0 - 1330 96

ublesCurbay B-G 10.0 4.5 0.5 - 160 84

51-20F3 Steep liquor 2.5 5.0 0.0 + 1700 114Soybean meal 3.0 5.0 0.0 - 1540 137

F3-64 Steep liquor 3.0 5.0 0.0 - 1800 120Soybean meal 2.0 5.0 0.0 - 2150 120

54-1255 Steep liquor 3.5 5.0 0.0 - 2440 120Soybean meal 4.0 5.0 0.0 - 1870 120

All media contained 0.4 per cent calcium carbonate and 0.1per cent sodium sulfate.

penicillin yield, in a steep liquor fermentation with P.chrysogenum strain 49-133 is shown in part A of table 3.When the lactose concentration was increased from 3per cent to 4 per cent, the onset of autolysis was delayedby 24 hr and a higher penicillin yield was obtained.When the lactose concentration was increased from 4per cent to 5 per cent, no increase in yield was observedalthough autolysis did not set in until after 120 hr.Owen and Johnson (1955) also found that maximummycelial growth and penicillin yield in a shaken flaskfermentation with strain 49-133 was obtained in amedium containing 3 per cent steep liquor and 5 percent lactose. Further increase in steep liquor or lactoseconcentration did not result in increased yields. Theeffect of increasing the lactose concentration on penicil-lin yield in a steep liquor fermentation with strain51-20F3 is given in part B of table 3. When the lactoseconcentration was increased from 5 per cent to 6 percent, the onset of autolysis was delayed by 24 hr, but noincrease in yield was observed. The effect of increasinglactose concentration on penicillin yield in a soybeanmeal fermentation with P. chrysogenum strain 51-20F3is given in part C of table 3. When the lactose concen-tration was increased from 4 per cent to 5 per cent, theonset of autolysis was delayed by 24 hr and a higherpenicillin yield was obtained. When the lactose concen-tration was increased from 5 per cent to 6 per cent, nofurther increase in yield was observed although autolysisdid not set in until after 150 hr. On the basis of theseexperiments 5 per cent lactose was selected as theconcentration to be used in the medium.

Effect of the concentration of the nitrogenous nutrient.To determine the optimum concentration of a givennitrogenous nutrient, it was included in the medium at arange of concentrations with 5 per cent lactose. Theconcentration of the nutrient giving the best yield ofpenicillin was selected as being optimal. This method ofapproach is illustrated in parts A and B of table 4.

Penicillin production on various nutrients. The penicil-lin yields obtained on media developed for the differentnutrients studied are given in table 5. It is apparentfrom the table that steep liquor, cottonseed meal,soybean meal, and distillers' solubles are the best of theorganic nutrients studied.Due to the relatively low nitrogen content of Curbay

B-G, it had to be used at rather high concentrations.At these concentrations the inorganic salts in the nu-trient probably had a toxic effect which would explainthe very low yields of penicillin obtained with thisnutrient.

SUMMARYThe utilization of various organic materials as ni-

trogenous nutrients in penicillin production by severalstrains of Penicillium chrysogenum was studied. In eachcase an attempt was made to adjust the concentrations

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BACTERIAL BRINED OLIVE SPOILAGE

of the constituents of the medium so that the bestenvironmental conditions for penicillin production wereobtained. Peptone and tryptone were unable to supplythe essential elements required by the mold. Thereforea salt mixture was added to prepare a complete mediumwith these nutrients. Glucose was found to be necessarywhen media containing yeast extract or fish solubleswere used. Lactose at a 5 per cent level was found to besufficient to meet the needs of the mycelium during thepenicillin production phase. The optimum concentra-tion of the nitrogenous nutrient was determined in amedium containing 5 per cent lactose. It was observedthat steep liquor, cottonseed meal, soybean meal, anddistillers' solubles were the best of the organic nutrientsstudied.

REFERENCES

COOK, R. P. AND TULLOCH, W. J. 1944 The production ofpenicillin on media made from vegetable extracts, partic-ularly extracts of peas. J. Pathol. Bacteriol., 56, 555-559.

FOSTER, J. W., PERLMAN, D., MCDANIEL, L. E., WILKER, B.

L., AND HENDLIN, D. 1946 Microbiological aspects ofpenicillin. IX. Cottonseed meal as a substitute forcornsteep liquor in penicillin production. J. Bacteriol.,51, 695-698.

GAILEY, F. B., STEFANIAK, J. J., OLSON, B. H., AND JOHNSON,M. J. 1946 A comparison of penicillin producing strainsof Penicillium chrysogenum. J. Bacteriol., 52, 129-140.

JARVIS, F. G. AND JOHNSON, M. J. 1947 The role of the con-stituents of synthetic media for penicillin production. J.Am. Chem. Soc., 69, 3010-3017.

JOHNSON, M. J. 1941 Isolation and properties of pure yeastpolypeptidase. J. Biol. Chem., 137, 575-586.

JOHNSON, M. J. 1952 Recent advances in penicillin fermen-tation. Bull. World Health Organization, 6, 99-121.

OWEN, S. P. AND JOHNSON, M. J. 1955 The effect of tempera-ture changes on the production of penicillin by Penicilliumchrysogenum strain W49-133. Appl. Microbiol., 3, 375-379.

PERLMAN, D. 1949 Production of penicillin on naturalmedia. Bull. Torrey. Botan. Club, 76, 79-88.

RAO, S. S. 1944 Production of penicillin. Nature, 154, 83.SHAFFER, P. A. AND SOMOGYI, M. 1933 Copper-iodometric

reagents for sugar determination. J. Biol. Chem., 8,127-139.

Species of Propionibacterium Associated with ZapateraSpoilage of Olives

SPIROS PLASTOURGOS AND REESE H. VAUGHN

Department of Food Technology, University of California, Davis, California

Received for publication January 14, 1957

Characteristics of the undesirable malodorous fer-mentation of brined olives known as "zapatera" havebeen described in some detail by Ball (1938), Cruess(1924), Delmouzos, et al. (1953), Kawatomari andVaughn (1956), Vaughn (1946), and Vaughn et al.(1943). The first off-odors to appear have been called"cheesy" or "sagey" but, as the spoilage progresses,

they eventually develop into an unforgettable, foul,fecal stench. There is a continuous loss in acidity as thespoilage develops. As shown by Delmouzos et al. (1953),part of the odor results from the volatile acids developedin the spoiled brine samples. These include formic,propionic, butyric, valeric, caproic, and caprylic acids,together or in various combinations. In contrast, thenormal brines contain acetic, lactic and sometimessuccinic acids, but none of the more odoriferous volatileacids were found in the spoiled samples.

Recently, Kawatomari and Vaughn (1956) were ableto associate various species of Clostridium with zapateraspoilage. However, none of the cultures studied pro-

duced propionic acids under the conditions tested.Therefore, an additional study was initiated to deter-

mine whether species of Propionibacterium might alsobe associated with the spoilage. The characteristics ofspecies of propionic acid bacteria actually associatedwith zapatera spoilage of different kinds of brined olivesare described below.

EXPERIMENTAL METHODS

Sources of cultures. In addition to the 54 samplespreviously examined by Kawatomari and Vaughn(1956), 33 more suspected samples of zapatera oliveswere tested in this study. The 33 new samples wereobtained from Californian, Grecian, Algerian, andSpanish sources. The foreign samples were collectedthrough the courtesy of a number of importers. Addi-tional samples from California included brined olivesto be used for preparation of California canned ripeolives as well as Sicilian and Spanish type green fer-mented olives. The imported samples comprised brined,ripe (mature and colored) olives from Greece, Spanishtype green fermented olives from Algeria and Spanishgreen fermented olives.

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