APPUED MICROBIOLOGY, Sept. 1967, p. 1178-1184Copyright @ 1967 American Society for Microbiology

Vol. 15, No. 5Printed in U.S.A.

Occurrence of an Inhibitor of Lactic Acid Bacteria inGreen Olives1

H. P. FLEMING AND J. L. ETCHELLSU.S. Food Fermentation Laboratory, Southern Utilization Research and Development Division, and Department of

Food Science, North Carolina State University, Raleigh, North Carolina 27607

Received for publication 20 April 1967

Green olives were found to contain an inhibitor(s) of several species of lactic acidbacteria usually associated with the Spanish-type brined olive fermentation. Theinhibitor was demonstrated by the presence of inhibition zones surrounding tissuewhich had been cut from frozen olives and implanted in a seeded nutritive agarmedium. Relative potencies of aqueous extracts of frozen olives were determinedby a paper disc assay method. The Mission variety of olive contained the most in-hibitor, and the Manzanillo and Ascolano, about 50 and 40% as much as the Mis-sion variety, respectively. Sevillano and Barouni varieties contained comparativelylittle inhibitor. Effects of the inhibitor on growth rates of lactic acid bacteria weredetermined by adding various amounts of a concentrated aqueous extract of olivesto a nutritive broth medium contained in screw-capped tubes. Of the four species oflactic acid bacteria tested, Leuconostoc mesenteroides was the most sensitive, andLactobacillus plantarum was the least sensitive; Pediococcus cerevisiae and Lacto-bacillus brevis were intermediate in sensitivity. Extracts possessed a bactericidalproperty, as evidenced by their effect on L. mesenteroides. Sodium chloride, espe-cially at concentrations of about 5% and higher, greatly increased the effectivenessof the inhibitor. The inhibitor was ethyl alcohol-soluble and was stable when heatedat 100 C in aqueous solution. Potencies of extracts were reduced greatly by adjust-ment to pH 10, but no appreciable effect was noted by adjustment to pH 0.8.

The fermentation of Spanish-type green olivescharacteristically proceeds at a slower rate thandoes the fermentation of either cucumbers orcabbage. Occasionally, vats or casks of olivesfail to attain a normal fermentation, as evidencedby the failure of lactic acid bacteria to proliferate,utilize fermentable sugars, and produce preserva-tive levels of lactic acid. Consequently, microbialspoilage due to yeasts, clostridia, and other micro-organisms may ensue (13, 14). Attempts toestablish the cause of anomalous olive fermenta-tions are complicated by the treatments that theolives receive prior to brining for fermentation.First, they are treated with alkali to removebitterness, and then are washed to remove mostof the alkali. Thus, the natural bacterial popula-tion is greatly lowered as is the concentration ofgrowth factors and fermentable carbohydrates.Several factors, including those above, have beensuggested as being responsible for variability inthe fermentation of olives. The possibility that

1 Published with the approval of the Director ofResearch, North Carolina Agricultural ExperimentStation, as paper no. 2345 of the Journal Series.

olives may contain antimicrobial agents has beenconsidered (13), but little scientific evidence hasbeen reported regarding the effect that such com-pounds may have on the lactic acid fermentationof brined olives.

This laboratory has been successful with thefermentation of cucumbers and other vegetablesby pure cultures of lactic acid bacteria (4). Re-cently, efforts have been directed toward applyingthe technique to the fermentation of olives (3).Obvious differences in the fermentation ratesof cucumbers and olives (3) prompted us toexplore possible explanations for this observation.The present study was undertaken to determinewhether olives contain water-soluble substanceswhich are inhibitory to lactic acid bacteria.

MATERIALS AND METHODSStock cultures of bacteria used in this study were

carried as stabs in screw-capped tubes containing 10ml of Orange Serum Agar (Difco). These organismsincluded Lactobacillus plantarum (culture no. FBB-12,-15, -68, and L-442), L. brevis (culture no. FBB-50 and-70), Pediococcus cerevisiae (culture no. FBB-39 and-61), Leuconostoc mesenteroides (culture no. FBB-42,

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-43, -71, and -73), and an isolate from a commercialolive brine designated as WSO (tentatively, L. plan-tarum). Further identification of these cultures wasprovided earlier (3, 4). The cultures were activatedprior to study by inoculating 10 ml of cucumberjuice broth (CJB) in screw-capped tubes and trans-ferring daily in this medium for 4 days or more,allowing 16 to 18 hr for growth before refrigeratingand holding until the appropriate time for trans-ferring. All cultures were incubated at 30 C.Cucumber juice media were prepared from frozen

Model variety cucumbers [1 to 134 inches (2.5 to3.2 cm) in diameter]. The cucumbers were thawedand then macerated in a blender; the juice wassqueezed through cheese cloth. The juice was heatedto boiling, cooled, and centrifuged; the supernatantfluid was filtered through filter paper to remove anysuspended particles. The broth medium (CJB) wasprepared by dispensing 10 ml of the extract into 16-mmscrew-capped tubes and autoclaving for 10 min at121 C. This clear, light amber-colored medium wasexcellent for growth of the cultures under study.Cucumber juice agar (CJA) was prepared by dilutingthe filtered extract with an equal volume of distilledwater and adding 1.5% agar and 0.0075% bromo-cresol green. After heating to melt the agar, themedium was dispensed into bottles and autoclavedat 121 C for 15 min.Growth of the bacteria was followed by incubating

cultures in 10 ml of CJB and reading the opticaldensity at 650 m,u with a Bausch & Lomb Spectronic-20 colorimeter at specified time intervals. Appro-priate blanks were used to correct for color differencesin the broth, a particularly variable factor when oliveextracts were added.The olives used in this work were shipped, air

freight, by the Lindsay Ripe Olive Co., Lindsay,Calif. Upon receipt, the olives were sorted to removedamaged fruit and foreign material, washed, andstored at -15 C in plastic freezer bags. The varietiesused and the condition of the samples are given inTable 1.

In preparing extracts, olives were removed fromthe freezer, allowed to thaw partially at room tem-perature for a few minutes, and then pitted with ahand-operated pitting machine. The pitted oliveswere added to an equal weight of boiling watercontained in a foil-covered beaker, and the mixturewas heated for 30 min while being stirred. Afterbeing cooled to room temperature, the mixture wasblended; the slurry was adjusted to a standard volumeand then centrifuged for 20 min at 10,000 X g. Theaqueous phase was suction-filtered through Whatmanno. 7 paper. After removing 10% of this solution,designated herein as the aqueous extract, the re-maining portion was concentrated in vacuo at 70 Cto one-fifth volume. To this concentrate was addedfive volumes of 95% ethyl alchol, and the mixturewas held overnight at 5 C. The liquid was decantedfrom the gelatinous precipitate and was concentratedto about one-twentieth volume. Ten volumes of 95%ethyl alcohol was added to this second concentrate,and the mixture was again held overnight at 5 C. Theliquid was decanted from a dark-brown sticky residue.

TABLE 1. Characteristics of the experimental olives

Approxi-Variety Condition mate size

(count/kg)

Ascolano All green, a few bruises 101Barouni All green, no bruises 132Manzanillo All green, no bruises 172Mission Mostly light purple, 295

no bruisesSevillano All green, a few bruises 103

This liquid was concentrated to one-fifth volume andis designated herein as the ethyl alcohol-solublefraction.

Bacterial inhibition zones were determined forolive tissue and extracts. These assays were made indisposable petri dishes (100 by 15 mm; Falcon Plastic,Los Angeles, Calif.) which contained 20 ml of CJAseeded with one drop of a 16-hr culture of the testorganism. Tissue samples were placed in the mediumbefore it solidified and were positioned so as to exposeboth the pit and skin sides laterally in the dish.Samples of extracts were pipetted onto 13-mm diam-eter paper discs (Schleicher & Schuell no. 740-E) andwere allowed to dry partially before the discs wereplaced on the surface of the seeded agar. Assay plateswere placed in incubation at 30 C immediately afteraddition of the disc samples.

Total phenolic content of extracts was determinedby the Folin-Denis method (12). Assays were ex-pressed on the basis of catechol as the colorimetricstandard. Absorbance was determined at 725 m,uwith a Bausch & Lomb Spectronic-20 colorimeter.

RESULTS

Preliminary studies showed that aqueous ex-tracts of frozen Manzanillo olives were notfermented by the lactic acid bacteria customarilyused by this laboratory for the pure culturefermentation of cucumbers. Also, the fermenta-tion rate of cucumber extract was lowered by theaddition of olive extracts. Zones of inhibitionproduced by the disc method for extracts and alsoby implanting tissue sections in seeded CJAdemonstrated conclusively the presence of abacterial inhibitor in olives. It was found thattissue sections from olives that had been frozenexhibited a much larger zone of inhibition thandid tissue from the unfrozen fruit. Also, aqueousextracts of frozen olives appeared to be muchmore inhibitory. Thus, all extracts reported hereinwere made from olives that were frozen and heldat -15 C.

Inhibitor contents offive varieties of olives. L.mesenteroides FBB-42 was one of the most sensi-tive cultures to olive extracts and was chosen asthe test organism for determining relativepotencies of preparations. Olive tissue implants

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in seeded CJA plates resulted in measurableinhibition zones, although the zone boundariesfrequently were unsymmetrical. Inhibition zoneswere measured at the widest point on the pitside of the tissue, since these zones were usuallylarger than on the skin side. Average zone sizesof triplicate assays were 11.0, 4.7, 5.3, 10.7, and10.7 mm for the Ascolano, Barouni, Sevillano,Manzanillo, and Mission varieties, respectively.This same relationship was found when L.plantarum L-442, P. cerevisiae FBB-39, and L.brevis FBB-50 were the test organisms, exceptthat the Barouni and Sevillano varieties producedno zones of inhibition when any of the last threebacteria was used.Aqueous extracts of the five olive varieties were

prepared to relate the inhibitor contents on aquantitative basis. The paper disc assay methodwas used to determine the relative potencies ofthese extracts. Figure 1 illustrates a plate withtypical inhibition zones resulting from fourdifferent amounts of inhibitor. The ethyl alcohol-soluble fraction from an extract of Manzanilloolives served as the inhibitor standard with whicha standard curve was prepared (Fig. 2). Thissolution contained 20 mg of total phenols perml on the basis of catechol as the colimetricstandard. In preparing the standard curve, oneunit of inhibitor was defined as the zone diameter(millimeters) produced with a disc containing0.05 ml of the above solution. Appropriate dilu-tions were made so that 0.05 ml/disc provided

--.....=,......

FIG. 1. Inhibition zones of Leuconostoc mesen-

teroides FBB-42 in CJA plates produced by an ethylalcohol extract of Manzanillo olives. The zones dem-onstrate inhibitory responses over a fourfold range inamount of inhibitor applied to the discs.

1.0

u(I)

0CD

m

z

0

UI)i

0.8

0.6

0.4

0.2

0 . 1 I

21 23 25 27 29 31 33

ZONE DIAMETER, mm.

FIG. 2. Standard curve of inhibition zones ofLeuconostoc mesenteroides FBB-42 obtained by discassay of a Manzanillo olive ethyl alcohol extract. Oneunit of inhibition was definedas the zone, in millimeters,resulting from the application of 0.05 ml per disc ofthis standard solution, which contained 20 mg totalphenols per ml (catechol basis).

the units of inhibitor indicated. The test mediumconsisted of 20 ml of CJA seeded with one dropof a 16-hr culture of L. mesenteroides FBB-42.Solutions to be assayed were diluted to allowapplication of 0.1 to 0.8 unit of inhibitor per disc,as the semilogarithmic plot was linear in thisrange (Fig. 2). All assays, including the standardcurve, were performed in triplicate.Aqueous extracts of the Mission variety of

olives contained the largest amount of inhibitorof the five varieties analyzed (Table 2). Manzan-illo extracts contained about 54% and Ascolano41% of the potency of the extract from the Mis-sion olive variety. The Barouni and Sevillanoextracts gave no inhibition zones at the levelstested, but they may contain small amounts ofinhibitor since tissue sections gave small inhibi-tion zones when L. mesenteroides, the mostsensitive organism, was used to inoculate theassay medium. The inhibitory principle was quitesoluble in ethyl alcohol,as over 80% ofthe potencywas recovered in ethyl alcohol extracts of theconcentrated aqueous solutions. The precipitateformed upon addition of ethyl alcohol to theaqueous concentrates contained no detectableinhibitor.

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TABLE 2. Inhibitor and total phenolic content ofextracts from five varieties of olivesa

Inhibitor Inhibitor Total TotalOlive variety content recovery phenols recoveredOfivvarety per 100 g in ethyl per 100 g inethylre

of olives alcohol of olives 1anlcehol

units % mg %Ascolano ....... 196 83 380 74Barouni.. 218 64Sevillanoo. 184 65Manzanillo ..... 257 98 389 78Mission . . 478 84 510 77

a Inhibitor and total phenolic contents representthe quantities present in aqueous extracts, pre-pared as described in Materials and Methods,and are expressed on the basis of 100 g of pittedgreen olives.

Relative sensitivity of selected cultures of lacticacid bacteria. Thirteen cultures of lactic acidbacteria were tested for their sensitivity to theinhibitory principle in extracts of Manzanilloolives. An ethyl alcohol-soluble fraction ofdesired potency was diluted with 5 volumes ofdistilled water and then concentrated in a flashevaporator to remove the ethyl alcohol. Thissolution was centrifuged to remove a dark gummymaterial that usually formed. The solution wasthen filter-sterilized by filtration through a 0.22-,ufilter (Millipore Corp., Bedford, Mass.). Ap-propriate dilutions of the solution were madeand then added aseptically to 16-mm screw-capped culture tubes containing 10 ml of CJB.Table 3 summarizes the effects of inhibitor ongrowth of the test cultures. L. mesenteroideswas the most sensitive, and L. plantarum theleast sensitive; P. cerevisiae and L. brevis wereintermediate in sensitivity among the four speciesof bacteria tested. There were variations in sensi-tivity among different cultures of the same species.A study of the growth curves of the culturesindicated that the inhibitor had a greater effecton delaying the onset of growth than on retardingthe rate of growth subsequent to this lag period.This effect is typified in Fig. 3, with L. plantarumFBB-15 as the fermenting organism.

Synergistic effect of NaCI and inhibitor. Sincethe NaCl concentration in naturally fermentedgreen olives is quite high (13), it seemed desirableto test its effect in comhination with variouslevels of the inhibitory material. L. plantarumFBB-15 was selected to demonstrate the effect ofNaCl because of its comparatively high resistanceto the inhibitor. This organism grew at concen-trations of NaCl as high as 9% in the absence ofinhibitor, although the growth rate was loweredand the lag period was extended at higher concen-

trations (Fig. 4A). Results given earlier affirmthe relatively high resistance of L. plantarumFBB-15 to the inhibitor (Table 3 and Fig. 3).In combination, however, the effects of NaCl andinhibitor on growth were synergistic (Table 4).

TABLE 3. Effect of inhibitor level on growth ofcultures of lactic acid bacteriaa

Units of inhibitor/mlCulture

0 0.5 1.0 1.5 2.5

Lactobacillus plantarumFBB-12. + + + - _FBB-15....: + + + + +FBB-68.. + + + - -L442w... + + + + -

Pediococcus cerevisiaeFBB-39 .. ... + + + - _FBB-61 .. ... + + + - -

L. brevisFBB-50.. + + + +FBB-70.. + + + - _

Leuconostoc mesenteroidesFBB-42.. + + - _ _FBB-43.. + + - _ -FBB-71.. + + - - -FBB-73.+ + -_ -

a Visual turbidity of inoculated CJB tubescontaining the inhibitor was used as an indicationof growth. Growth within 5 days (+), no growth(-).

UNITS OF INHIBITOR /ML1.8 O- * 1.0

I1.6 8 E20.

=L1.4-E 1.2 -

0If) 1.0

a 0.8-0

0.4-

0.2-

0 2 67 821INCUBATION TIME, DAYS

FIG. 3. Growth curves of Lactobacillus plantarumFBB-I5 in CJB containing various concentrations ofinhibitor. The broth tubes, 16 X 150 mm screw-cappedtubes containing 10 ml of medium, were periodicallyshaken and the optical density was determined.

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I .8

1.6

I.4

1.2

1.0

0.8

0.6

z 0.4E 0.2

0

1.8

o 1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2

UNITS OF INHIBITOR/ML- O-0 6-0.1 -0.5,1.0,2.0PLUS 5% NaCI

(B)-H _

0 1 2 6 7 8 21

INCUBATION TIME, DAYSFIG. 4. Growth curves of Lactobacillus plantarum

FBB-15 in CJB containing NaCl with and withoutinhibitor. (A) Various concentrations of NaCI in theabsence of inhibitor; (B) 5% NaCl in the presence ofvarious concentrations of inhibitor. The broth tubes,16 X 150 mm screw-capped tubes containing 10 mlof medium, were periodically shaken and the opticaldensity was determined.

L. plantarum FBB-15 failed to grow with 5%NaCl when as little as 0.5 unit of inhibitor perml was present. The growth curves shown inFig. 4A represent typical effects of NaCl on thegrowth of L. plantarum, and the effect of variouslevels of inhibitor with 5% NaCl is shown inFig. 4B. Again, the lag in onset of growth seemedto be influenced more by the inhibitor than didthe growth rate when growth occurred (Fig. 4B).

Bactericidal effects. It was of interest to learnwhether the inhibitor possessed bactericidalproperties. L. mesenteroides FBB-42 was used asa test organism to answer this question. The ex-

periment was performed by incubating 6 x 107cells/ml at room temperature in a screw-cappedtube containing 10 ml of CJB and 0.5 unit ofinhibitor per ml. The cells were added to thesolution of CJB and inhibitor, and the tube wasthen shaken. Samples (1 ml) were removed atspecified times and pipetted into dilution bottlescontaining 99 ml of sterile 0.85% NaCi. Furtherdilutions were made and plated in CJA. There was

TABLE 4. Effect of levels of inhibitor and salt ongrowth of Lactobacillus plantarum FBB-15a

Units of inhibitor/mlNaCl

0 0.1 0.5 1.0 2.0

0 + + + + +I+ + + + +

3 + + + + +5 + + - - -7 + + - - -9 + - - - -

a Visual turbidity of inoculated CJB tubes con-taining the inhibitor was used as an indication ofgrowth. Growth within 5 days (+), no growth(-).

8

7

0

> 6

0 5wcroz 4

0-J

3

2~~~~~~~~

0 5 10 15 20INCUBATION TIME, MINUTES

FIG. 5. Survivor curve of Leuconostoc mesenteroidesFBB-42 in CJB containing 0.5 unit of inhibitor/ml.

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approximately a 99.999% reduction in viablecells within 20 min, as illustrated in the survivorcurve (Fig. 5). At an inhibitor concentration of5 units/ml, no viable cells were detected after 10min of incubation, whereas at 0.1 unit/ml noapparent reduction in viable cells resulted aftercells were in contact with the inhibitor for 1 hr.

Effect ofpH and temperature on stability of theinhibitor. The alkali treatment and subsequentwashings that Spanish-type green olives undergocould have a significant influence on the stabilityand content of inhibitory compounds which wereoriginally present in the olives. The followingstudy was designed to establish the effect ofpH onthe stability of the inhibitor. Six samples of 2.5ml, each containing about 20 units of inhibitor/ml, were pipetted into tubes (18 X 80 mm). Thesamples were divided into two sets of three eachand were purged with either air or nitrogen for1 min before the pH was adjusted. The sampleswere then adjusted with 6 N NaOH or 6 N HClto the pH indicated in Table 5. The gas was al-lowed to bubble through the solution for 10 minbefore the pH was readjusted to 4.7, which wasthe same as that of the control. All solutions werethen adjusted to the same NaCl concentration(5%), diluted to 10-ml volume, and filter-steril-ized through a 0.22-,.u filter (Millipore Corp.).The potencies of these solutions were assayed byadding portions to CJB tubes, inoculating withL. mesenteroides FBB-42, and observing growth

TABLE 5. Effect ofpHon the stability of the inhibitor

Growth within 5 dayspH with indicated units Recovery

Purging gas adjust- of inhibitor/mlb inhihitormenta by disc

0.8 0.4 0.2 0.1 assayc

Air 0.8 - _ - + 8510.8 - + + + 154.7d - - - + 98

Nitrogen 0.8 - - + + 8310.0 - + + + 254.7d - _ - + 100

a The pH was adjusted with 6 N NaOH or HCIto that indicated, held for 10 min while purgingwith air or nitrogen, and then readjusted to that ofthe control (pH 4.7).

b The inhibitor solutions were filter-sterilizedafter pH adjustments, and samples were added to10 ml of CJB in screw-capped tubes. The units ofinhibitor per milliliter are expressed on the basisof activity prior to adjustments.

c Recovery was based on activity relative to thenitrogen control.

d Control.

(Table 5). Both assay methods indicated about afourfold reduction in potency due to the alkalitreatment. The basic treatment was effective underan anaerobic as well as an aerobic atmosphere,and in both instances the bitterness was elimin-ated. Low pH had no observable effect on thepotency of the inhibitor, and the solution re-mained bitter.The inhibitor was stable at elevated tempera-

tures. No appreciable decrease in potency wasobserved by the disc assay method when aqueoussolutions of the inhibitor were heated at 100 C for30 min or after autoclaving at 121 C for 5 min.

DIscussIoNThe occurrence of antimicrobial agents ih

plants has been widely reported and has been theobject of surveys (2). Theories have been proposedwhich attribute various defense mechanisms ofplants to such substances (5, 6). Food productsare derived from some of these inhibitor-contain-ing plants. Although the occurrence of such sub-stances in foods that are preserved by manyprocesses may be only incidental, inhibitorycompounds may be very important in regulatingthe type of fermentation and the resulting productwhen foods are fermented.On the basis of the present findings, it seems

plausible that inhibitory compounds may exert asignificant influence on the survival and subse-quent activity of competing microbial groups thatpopulate olive brines, as was suggestedby Etchellset al. (3). The levels of inhibitor in extracts of thefive varieties of olives assayed were in approxi-mately the same order as their relative rates offermentation, which were reported by Vaughn(13). Vaughn noted that the Manzanillo andMission varieties ferment more slowly than theAscolano and Sevillano varieties. He stated thatthe first two varieties contain more of the bitterprinciple, oleuropein, and that this compound"may eventually be shown to suppress the ac-tivity of the lactic acid bacteria." However, asVaughn indicated, the Manzanillo and Missionvarieties are usually brined at higher levels ofsalt, which may account in part for their slowerrates of fermentation.

Inhibitory properties of olive tissue and ex-tracts were much more obvious when the oliveshad been frozen. It is not clear whether freezingcaused a release of the inhibitor due to physicalchanges in the olive or whether it resulted in asubsequent chemical alteration. In either case,the inhibitor or its precursor is subjected torather drastic conditions in the processing ofSpanish-type green olives. In the debitteringtreatment prior to brining, alkali is allowed topenetrate the olive about three-fourths or more

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of the way to the pit and then the alkali is re-moved by several washings (13). The inhibitorshould be reduced greatly in this treatment, asindicated ty its destruction when extracts wereadjusted to pH 10. However, the effects of anyresidual inhibitor might be enhanced significantlyby the presence of 4 to 7% NaCl, which is thenormal brine strength for green olives. It wasshown that the effectiveness of the inhibitor ismuch greater in the presence of NaCl.The inhibitory principle of olives remains to be

identified. A phenolic-containing compound is anobvious possibility. Varietal differences in water-soluble phenolic content were correlated withrelative amounts of the inhibitor in the fivevarieties of olives assayed. Many plants have beenreported to contain phenolic antimicrobialcompounds, either preformed in the tissue orformed as a physiological response to injury ordisease (5, 6). Among such compounds are mono-and dihydric phenols, phenolic glycosides,flavonoids, anthocyanins, aromatic amino acids,and coumarin derivatives. Anthocyanins typifythe occurrence of such compounds in food prod-ucts. The anthocyanins of certain grapes andwines have teen reported to be bactericidal (7).These pigments and related compounds fromseveral other plants possess varying degrees ofantibacterial activity (1, 10).

Oleuropein, the bitter principle of olives, hasbeen identified as a glucoside containing an o-dihydroxy phenol (8). This compound reportedlypossesses a hypotensive effect (8) and presumablyis related to the therapeutic properties long knownto be present in leaves and other parts of theolive plant (9). The color of black ripe olives hasbeen attributed at least partially to oxidationproducts of oleuropein (11). Oleuropein, orcompounds related to it, may possess antibacterialactivity. Although possibly coincidental, bothbitterness and inhibitory properties of oliveextracts were reduced greatly by adjustment topH 10.

LITERATuRE CITED1. BLANK, F., AND R. SUTER. 1948. The bacterio-

static action of anthocyanin and related com-pounds. Experientia 14:72-73.

2. CARLSON, H. J., H. G. DOUGLAS, AND J. ROBERT-SON. 1948. Antibacterial substances separatedfrom plants J. Bacteriol 55 :241-248.

3. ETCHELLS, J. L., A. F. BORG, I. D. KTrrEL, T. A.BELL, AND H. P. FLEMING. 1966. Pure culturefermentation of green olives. Appl. Microbiol.14:1027-1041.

4. ETCHELLS, J. L., R. N. CosTLOw, T. E. ANDERSONAND T. A. BELL. 1964. Pure culture fermentationof brined cucumbers. Appl. Microbiol. 12:523-535.

5. FARKAS, G. L., AND Z. KmAiLY. 1962. Role ofphenolic compounds in the physiology of plantdiseases and disease resistance. Phytopathol.Z. 44:105-150.

6. Kuc, J. 1966. Resistance of plants to infectiousagents. Ann. Rev. Microbiol. 20:337-370.

7. MASQUELIER, J. 1959. The bactericidal action ofcertain phenolics of grapes and wines, p.123-131. In J. W. Fairbairn [ed.] The pharma-cology of plant phenolics. Academic Press, Inc.,New York.

8. PANIZZI, L., M. L. SCARPATI, AND G. ORIENTE.1960. Costituzione della oleuropeina, glucosideamaro e ad azione ipotensiva dell'olivo. -NotaII. Gazz. Chim. Ital. 90:1449-1485.

9. POWER, F. B., AND F. TUTIN. 1908. The con-stituents of olive leaves. J. Chem. Soc. 93:891-904.

10. POWERS, J. J., D. SOMAATMADJA, D. E. PRATT,AND M. K. HAMDY. 1960. Anthocyanins. II.Action of anthocyanin pigments and relatedcompounds on the growth of certain micro-organisms. Food Technol. 14:626-632.

11. SIMPSON, K. L., C. 0. CHICHESTER, AND R. H.VAUGHN. 1961. Compounds responsible forthe color of black "ripe" olives. J. Food Sci.26:227-233.

12. SWAIN, T., AND W. E. HILLIS. 1959. The phenolicconstituents of Prunus Domestica. I. Thequantitative analysis of phenolic constituents.J. Sci. Food Agr. 10:63-68.

13. VAUGHN, R. H. 1954. Lactic acid fermentationof cucumbers, sauerkraut and olives, p. 417-478. In L. A. Underkofler and R. J. Hickey[ed.] Industrial fermentations, vol. 2. ChemicalPublishing Co., Inc., New York.

14. VAUGHN, R. H., H. C. DOUGLAS, AND J. R.GILILLAND. 1943. Production of Spanish-typegreen olives. Calif. Agr. Expt. Sta. Bull. 678.

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