SGRBIC ACSD AS A FREfiERVATEVE FOR SWEET CUCUMBER PECKLES Thesis for We Degree of M: S. MICHIGAN STATE COLLEGE Jack Marsha” Sheneman 1954 THESIS Date O~169 This is to certify that the thesis entitled SORRIC ACID A3 A PiESIfiRVé‘tTIVE FOR. STIE‘ST CUCUl-IBSR. PICKLES presented hg JACK l-‘htfidflf‘tm Sthlflval‘I has been accepted towards fulfillment of the requirements for PUBLIC HEALTH m AUGUST 6, 19Sh SORBIC ACID AS A PRESERVATIVE FOR SHEET CUCUMBER PICKLES BY Jack Marshall Sheneman A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Bacteriology and Public Health 195M ACKNOWLEDGMENT The author wishes to express his sincere appreciation to Dr. Ralph N. Costilow, Assistant Professor of Bacteriology and Public Health, for his patient and willing guidance throughout this work. “3' ”‘4‘ "i (Jt) J 33L TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . . EXPERIMENTAL PROCEDURE . . . . . . . . . RESULTS . . . . . . . . . . . DISCUSSION . . . . . . . . . . . SUMMARY . . . . . . . . . . . CONCLUSIONS . . . . . . . . . . . REFEREE‘ICES o o o o o o o o o o e LIST OF TABLES TABLE _ Page 1. Growth and Acid Formation by Lactobacillus plantarum in Two Percent Sucrose Broth . . . . . 1h 2. Growth and Acid Formation by Lactobacillus plantarum in Ten Percent Sucrose Broth . . . . . lS 3. Growth and Acid Formation by Lactobacillus plantarum in Twenty Percent Sucrose Broth . . . 16 u. Growth and Acid Formation by Lactobacillus plantarum in Thirty Percent Sucrose Broth . . . 17 5. Growth and Acid Formation by Lactobacillus plantarum in Forty Percent Sucrose Broth . . . 18 6. Yeast Growth and Gas Production in Two Percent sucrose Broth . C O O C C . . . O O I O C O C O 21 7. Yeast Growth and Gas Production in Ten Percent Sucrose Broth . . . . . . . . . . . . . . . . . 22 8. Yeast Growth and Gas Production in Twenty Percent Sucrose Broth . . . . . . . . . . . . . 23 9. Yeast Growth and Gas Production in Thirty Percent Sucrose Broth . . . . . . . . . . . . . 2h 10. Yeast Growth and Gas Production in Forty Percent Sucrose Broth . . . . . . . . . . . . . 25 11. Effect of pH on the Inhibition of Spoilage Pickle Yeasts by 0.1 Percent Sorbic Acid . . . . . . . 29 12. Preservation and Spoilage of Experimental Packs of Inoculated Sweet Cucumber Pickles . . . . . . 31 LIST OF FIGURES FIGURE Page I. Inhibition of Lactobacillus plantarum in sucrose-acetic acid broths . . . . . . . . . . 2O II. Preservation prediction lines determined from growth studies of spoilage pickle yeasts . . . 27 III. Preservation and spoilage of experimental packs of inoculated sweet cucumber pickles . . . . . 32 ,INTRODUCTION Sorbic acid, a recently developed fungistatic agent, may have important applications as a preservative in the food industry. Its tentative acceptance as a harmless food preservative by the U. S. Food and Drug Administration has already led to its use in a mold preventive wrapping material by the cheese industry. Other possible applications include the control of both oxidative and fermentative yeasts during cucumber fermentations, and use as a superior substitute for sodium benzoate in such products as orange juice, fruits and fruit syrups, jams and jellies, pickles, margarine and candies. The application of this substance to the preservation of sweet pickles was suggested not only by its efficiency as a fungistat, but also by the possibility that a more desirable product may be developed as a result of using low concentra- tions of acid and sugar in combination with the preservative. The present investigation concerns the ability of sorbic acid in the presence of various concentrations of sugar and acid to prevent spoilage of sweet cucumber pickles without heat processing of the product. LITERATURE REVIEW Gooding (19h5) discovered that a group of alpha, beta- unsaturated fatty acids were effective as fungistatic agents. Of these, sorbic acid (2, h-hexadienoic acid) was found to be desirable for the prevention of spoilage in food products because of its lack of flavor and odor, and its low toxicity. Determinations of toxicity, reported by Deuel, Alfin- Slater, Neil, and Smyth (195k) indicated that sorbic acid was harmless as a component of food in concentrations greatly in excess of those required to insure preservation. Animal [tests showed sorbic acid to be far less toxic than sodium ben- zoate. The ratios of the LDSO of sorbic acid calculated as sodium sorbate to that of sodium benzoate, based on calcula- tions as free acids, were determined to be 1.72:1 and 1.90:1 in two separate experiments with rats. Thus, nearly twice as much sorbic acid as benzoic acid was required to produce similar lethal effects. The conclusion was reached that sor— bic acid is harmless to rats and to dogs when incorporated in the diet to the extent of 5 percent of the total weight. Further results of in vivo experiments by Deuel, Calbert, ‘Anisfeld, McKeehan, and Blunden (195h) indicated that sorbic acid was metabolized by the animal body in the same manner as the saturated six carbon fatty acid, caproic acid, a normally occurring component of foods. They concluded that under normal body conditions sorbic acid is completely oxi- dized to carbon dioxide and water, yielding its potential energy as calories. In comparison, Witter, Newcomb and Stotz (1950) found benzoic acid to inhibit fatty acid oxi- dation by liver enzymes. They noted that benzoic acid must be detoxicated in the liver with glycine yielding hippuric acid which is excreted in the urine. These data on the harmlessness of sorbic acid as a pre- servative have been tentatively accepted by the U. 8. Food and Drug Administration. This is indicated in a letter from that agency cited by Smith and Rollin (l95ha). Emard and Vaughn (1952) demonstrated that sorbic acid exerted a definite inhibitory action upon catalase positive microorganisms including yeasts, molds, bacteria and actino- mycetes. Both the growth medium and the pH influenced the degree of inhibition. Thus, a concentration of 0.12 per- cent was required to exert this activity in a liver infusion broth, whereas only 0.07 percent sorbic acid was needed in a 1 percent glucose medium. With 0.0? percent sorbic acid at a pH of 5.0 to 5.5, catalase negative organisms grew rapidly while catalase positive forms were inhibited in general. Above pH 5.5, sorbic acid had little effect on even the catalase positive types. These workers proposed the use of sorbic acid as a selective agent for use in pri- mary isolation of lactobacilli and clostridia. The inhibition of scum yeasts in cucumber fermentations by sorbic acid was reported by Phillips and Mundt (1950) and confirmed by Jones and Harper (1952). These investigators demonstrated that a concentration of 0.1 percent inhibited yeasts and molds in salt stock fermentations under conditions otherwise favorable to the growth of these organisms. How— ever, concentrations less than 0.1 percent did not completely inhibit growth of surface yeasts. They also indicated that lactic acid development in the presence of sorbic acid was slower than in control fermentations, but eventually a higher concentration of acid was developed. Because sorbic acid is strongly ionized, the initial pH of the brine was low. This increased as acid was absorbed by the fresh cucumbers, but dropped again as fermentation was initiated by lactic acid bacteria. No adverse flavoring of the finished pickle resulting from the presence of 0.1 percent sorbic acid in the brine fermentation was noted by Phillips and Mundt (1950). Flavor tests to determine minimum taste perception were performed with sorbic acid treated cheese by Smith and Rollin (l95hb). They reported that the lowest concentration resulting in a perceptible taste was 0.2 to 0.5 percent or about u to 10 times that concentration necessary for effective fungistatic activity. The mechanism of mold inhibition by sorbic acid was deter- mined by Melnick, Luckmann and Gooding (l95h). They found that the addition of alpha, beta- unsaturated fatty acids, including sorbic acid, to a test medium inhibited the dehy- drogenation enzyme system of the molds. Although such fatty acids are normal transitory metabolites resulting from the dehydrogenation processes in the oxidation of natural fatty acids, they are commonly present in very low concentrations. When this concentration was greatly increased, they became inhibitory. When a heavy inoculum of mold was used, it was found that oxidation of the sorbic acid by the mold occurred through the same mechanisms that operates in the animal metabolism; i.e., beta oxidation to the next lower fatty acid homologue of even number carbon atoms, and finally to carbon dioxide and water. The preservative effect of the various sugars and organic acids have been studied by a number of investigators. Levine and Fellers (19h0a) found that acetic acid inhibited the growth of bacteria in nutrient broth at pH h.9; the mold, Aspergillus niger, at pH h.l; and the yeast, Saccharomyces cereviseae at pH 3.9. They determined that the toxic effect of the acid appeared to be a function of the undissociated acid molecule, as well as of the hydrogen ion concentration. Acetic acid was shown to be much more toxic to these organisms than either lactic or hydrochloric acid. A later report by these workers (19h0b) indicated that 5 percent sodium chloride or 20 percent sucrose exerted but little effect upon the toxicity of acetic acid. Tarkow, Fellers and Levine (19u2) found that concentrations above 30 percent sugar has a sig- nificant inhibitive effect on microorganisms. Glucose was more inhibitory than sucrose because of the greater osmotic pressure exerted. Morse, Fellers and Levine (19h8) deter- mined that acetic acid was more effective against molds than yeasts in 30 to ho percent syrups, and exerted greater effect than either citric or lactic acids. Although the publications noted in the preceding para- graph purport to deal with inhibition of spoilage organisms, the assumption of the authors that the "bread yeast," Sac- charomyces cereviseae, is truly representative of spoilage yeasts is highly questionable. 0f five species of yeasts tested by Erickson and Fabian (19h2), Saccharomyces cereviseae was found to be one of the least resistant species to the in- hibitory activity of acetic acid and sucrose. In this inves- tigation of the effect of combinations of various sugars and organic acids on yeasts and bacteria, it was found that the acid was the more important factor in controlling the devel- opment of the organisms. The yeasts were more tolerant to acids than were bacteria, with acetic being the most inhibitory of the acids tested. Fabian and Wadsworth (1939), using an aciduric yeast isolated from catsup, noted that pH was not a reliable indi- cator of the preserving power of acids. They recommended an acidity of not less than 2 percent with a sucrose concentration of 15 to 22 Baume/(27 to ho percent by weight) to insure pre- servation of sweet pickle products. Fabian and Switzer (lth) later warned manufacturers of sweet pickles of the danger of spoilage in products having less than 2 percent acetic acid. A preservation prediction chart for yeast spoilage in sweet cucumber pickles was developed by Bell and Etchells (1952) from studies of sugar and acid tolerances of thirty- five yeast isolates from spoiled sweet pickles. Taxonomic studies revealed these to be closely related to the species Zygosaccharomyces globiformis Kroemer 2E Krumbholtz. Five representative strains were tested for growth and gas forma- tion in broth containing various concentrations of sucrose and glucose with acetic acid. The preservative, sodium ben— zoate, was also tested in the various combinations of sugar and acid. Preservation prediction lines were plotted from the determinations of acid and sugar concentrations necessary to inhibit yeast growth, and formulas to determine amounts of sugar and acid necessary to prevent spoilage were calculated. The validity of the prediction lines was confirmed by pre- paring small scale commercial lots of sweet pickles according to the formulas and determining the occurrence of spoilage. EXPERIMENTAL PROCEDURE The inhibition of sweet pickle spoilage organisms by sorbic acid was studied in two series of experiments. The first involved inoculation of pure cultures of spoilage yeasts and lactobacilli into broth media made up with various concen- trations of sugar, acetic acid and sorbic acid. The second series of tests involved the experimental preparation of sweet cucumber pickles at various concentrations of these agents to determine the efficiency of the sorbic acid as a preservative.under conditions resembling those encountered in commercial practice. The yeast cultures used in this study were three strains of spoilage pickle yeasts identified as SPY l5, SPY 21, and SPY 29. They were originally isolated by Bell and Etchells (1952) among 35 similar organisms from 15 samples of spoiled sweet pickles. Subsequent studies revealed these organisms to be closely related to the species Zygosaccharomyces globi- formis Kroemer gt Krumbholz. These cultures were obtained from the culture collection of the Northern Utilization Re- search Branch, Agriculture Research Service, United States Department of Agriculture, Peoria, Illinois. The lactobacilli cultures used were strains L-5 and L-lO of the species ngtg- bacillus plantarum. These were obtained from Dr. Ralph N. Costilow, Department of Bacteriology and Public Health, Michi- gan State College, who isolated them from commercial cucumber fermentations. The test media for the pure culture studies were prepared from basal broths containing the necessary nutrients for the growth of the organisms. The basal medium for culturing the yeasts was identical to that used by Bell and Etchells (1952) while the basal broth for the growth of the lactobacilli con- sisted of 1 percent Bacto tryptone and 0.5 percent Bacto yeast extract. Sucrose and sorbic acid were added to these broths on a percent by weight basis and glacial acetic acid on a percent by volume basis. The final concentrations of sucrose in the test media were 2, 10, 20, 30, and ho percent. Broths containing acetic acid concentrations of 0, 0.5, 1.0, 2.0 and 3.0 percent were prepared for each sugar concentration. Sorbic acid concen- trations of 0, 0.05, and 0.1 percent were used in conjunction with each sugar and acid combination. To dissolve the slightly soluble sorbic acid, the flasks of media were autoclaved for five minutes at 15 pounds pressure (1210 c). The pH of the broth containing no acetic acid was adjusted with a few drops of 0.1 N HCl to pH 6.5. The pH values of the remaining test media were not adjusted and the broths were not artificially buffered. Another series of flasks with 0.10 percent sorbic acid and 10 percent sucrose added to the basal yeast medium were 10 prepared. These broths were then adjusted with H01 to pH values of 3.0, 3.5, h.0, h.5 and 5.0.to determine the effect of pH on the inhibitory activity of sorbic acid. The various finished media were dispensed into 16 x 150 mm culture tubes so that each tube contained approximately 10 ml. Durham fermentation tubes were used for all tubes of yeast medium and all tubes were plugged with cotton.. The tubes of media were sterilized at 15 pounds pressure (1210 C) for 15 minutes after which the pH values of the different sets of tubes were determined electrometrically with a Beckman pH meter. The tubes were inoculated with one drop from a 2 ml pipette of 2h hour broth cultures of the organisms. Cul— tures used for inoculation were standardized by sub-culturing at 2h hour intervals at least 3 times prior to inoculation into the test broths. These cultures were prepared in the . same basal broths used for the test media with the addition of 10 percent sucrose for the yeast cultures and 2 percent sucrose for the cultures of lactobacilli. The inoculated test media and uninoculated controls were incubated at a temperature of 300 C for 21 days, during which they were observed frequently for growth determined by turbidity of the broth and presence of gas in the Durham tubes for the yeast cultures. Turbidity and increase in titratable ll acidity was determined in the broths inoculated with Lacto- bacillus plantarum. The acidity determination was accomplished by removing 1 ml of the broth at various intervals and titra- ting with 0.1 N NaOH using phenolphthalein as an indicator. To demonstrate the preservative effect of sorbic acid in the presence of prepared sweet cucumber pickles, 120 pint jars of such pickles were packed in various concentrations of sugar, acetic acid and sorbic acid. Two bushels of No. 1 size salt stock cucumbers (up to 1 1/8 inches in diameter) were obtained from the H. W. Madison Company salting station at Mason, Michigan. These were freshened in a crock by first covering with fresh water for about u hours. Then they were drained and recovered with water to which was added 3 ounces of alum and 0.2 ounce oil of tumeric per bushel of cucumbers. After this, they were heated in a crock with steam to a tem— perature of 1250 F (61.50 C) and allowed to soak 18 hours. Finally, they were again drained and covered with fresh water. The cucumbers were packed in pint jars and glacial ace- tic acid sufficient to give final desired concentrations of 0.5, 1.0, 2.0, 3.0 and h.0 percent was added. Sorbic acid was then added to the desired jars in an alcoholic solution prepared so that 5 m1 of the solution would provide a final concentration of 0.1 percent in the jar. For those jars which required a 0.05 percent concentration of sorbic acid, this solution was diluted in a one to one ratio with ethyl 12 alcohol, and 5 m1 of the resulting solution was added. To control the presence of the alcohol, 5 m1 of alcohol was added to those jars which had no sorbic acid. Sucrose was added in increments of 5 percent by weight every 12 hours until the total desired concentrations of 10, 20, 30 and no percent were attained. Thus, two jars each of 60 combina- tions of various concentrations of sucrose, sorbic acid and acetic acid were prepared. The average percent of sodium chloride present in a random sample of twenty of the jars, representing salt car- ried over after the freshening process, was determined by titration with silver nitrate using dichlorofluorescein as the indicator. Estimates of percent sucrose in the jars were made by the use of the hand refractometer from seven jars chosen at random from each of the four concentrations of sucrOse represented. After these determinations, the jars were sealed and incubated at room temperature for five weeks, during which time they were observed periodically for tur- bidity or other signs of spoilage. RESULTS The ability of Lactobacillus plantarum to grow, producing turbidity and acid in the presence of various combinations of acetic acid, sucrose and sorbic acid is indicated in Tables 1 through 5. The two strains of this organism tested, L-5 and L—lO exhibited very little difference in susceptibility to the inhibitory factors. Strain L-lO had a slightly greater tolerance for sucrose in the presence of 0.5 pereent acetic acid than the L-5 strain. Thus, the L-lO culture grew at 30 percent sucrose while L-5 was unable to grow in concentrations greater than 10 percent sucrose. Neither organism was able to tolerate more than 0.5 percent acetic acid in any sugar concentration. This could be expected since the pH of the media with 1.0 percent acetic acid were all below pH h.0. In the presence of 0.10 percent sorbic acid, both strains of Lactobacillus plantarum grew well in the absence of acetic acid, but neither was able to grow in a concentration of 0.5 percent. However, at a concentration of 0.05 percent sorbic acid, L-10 was able to grow in 0.5 percent acetic acid at low sugar concentrations. In general, the concentration of sucrose had but little effect upon the ability of these organisms to produce growth in the broth. However, their ability to produce acid was apparently greater in the higher concentrations of sucrose. 16 TABLE 3 GROWTH AND ACID FORMATION BY LACTOBACILLUS PLANTAHUM IN TWENTY PERCENT SUCROSE BROTH Lactobacillus plantarum strain Sorbic Acetic ‘ _ Acid 1 Acid 2 pH L'lo --JEJi- Increased Final Final a 7 Acidity3 Turbidity“ Turbidityu 0 0 6.50 .27 3+ 3+ 0.5 14,005 012 2+ '- 1.0 3.85 .03 .. .. 2.0 3.55 .06 - - 3.0 3050 015 - .- o.05 O 5.115 .2LL 3+ 3+ 0.5 LL00; 009 " "' 100 3.80 009 " - 200 3055 006 "' "' 3.0 3014-5 015 I- .- 0.10 0 h.90 .15 3+ 3+ 005 LLQOO 009 " '- 1.0 3.80 .09 - - 200 3055 015 ' ' 300 30“.; 012 "' '- Uninoculated control tubes 0 O 6050 009 - " 0005 0 5011.5 .09 - "' 0.10 0 h.90 .09 - - pr \phak’ Percent sorbic acid by weight Percent acetic acid by volume Percent increase in titratable acetic acid after seven days incubation. Sediment and turbidity of broth after seven days incubation: 3+ = good; 2+ = moderate; 1+ = scant, - = none. 19 This is indicated in Tables 1 through 5 by the higher titratable acidities determined from the broths containing the higher percentages of sucrose. Growth inhibition lines for the most resistant strain of Lactobacillus plantarum, L-10, are given in Figure I. It should be noted that in the presence of 0.10 percent sorbic acid and 0.5 percent acetic acid, inhibition of this organism was cer- tain. With no sorbic acid, an acetic acid concentration of 1.0 percent was required for inhibition in sucrose concentra- tions of 30 percent or less. In hO percent sucrose broth, only 0.5 percent acetic acid was required. Sorbic acid at 0.05 percent inhibited growth in presence of 0.5 percent acetic acid when the sucrose concentration was 20 percent or more. However, in sucrose concentrations of less than 20 percent, 0.05 percent sorbic acid had no appreciable effect; i.e., 1.0 percent acetic acid was required to inhibit growth. Growth and production of gas in the various broths by the three strains of spoilage pickle yeasts are indicated in Tables 6 through 10. From these data, five factors appeared to be operative in determining the ability of a given yeast inoculation to produce turbidity and gas in a given medium. These factors were the specific strain of yeast used; the concentrations of sucrose, sorbic acid, and acetic acid present in the medium; and the pH of the medium. The yeast strain most resistant to the inhibitory factors was SPY 21. This strain grew rapidly at 2 percent acetic acid in sucrose concentrations of 2 and 10 percent, and moderately WuQmemw QQV KQ Mum? KO 29k \ p33 20 are“... QmeQm, NQYURQi 1-. I I s to V ax ionxom vaufixxksg D [It A 4.. 3 “and? «a? 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I mo. m o. m I I I I I I I I I om.m o.m I I I I I I I I I 0:.m o.H I I I +H m m I I I oo.m m.o +m m H +N H +N 3 H ms.m o mo.o I I I I I I I I I oo.m o.m I I I I I I I I I om.m o.m +N m m +N 1 m +m w : o:.m o.H +m m H +m m H +m H ow.m m.o +m m H +m m H +m m H om.o o +m m o +m m H +m HH 0 on.n o o Amhdpv Amhmov Amh m3 3me 333 Amhwov . mm & assessese mu rezone apeeanssa mu rezone marseansss msesode Hedge 0 rated Hedda e pupae Hesse rude pupae used used om Mmm Hm Mmm mH wmm ma prood Wthom QHmppm ummmw maomm Hmomobm Bzmommm NBmHmB 2H ZOHBODQOmm mdw Dad mBZOmw Bm memmui Mudflats Mmofiiubm .NZMMQNQ W oi ow On or p _ b i _ _ H + o .\ g \\\I a I .IIIIQIIII I IIIIILQI .I IIIIIIII LI 92 ammom N 9.0 .o \\. a .17 04 -¢ III. ON IILTI O.” IJTI him. QCV QC m6< szumwn‘ The effect of pH on the inhibition of the spoilage pickle yeasts by 0.10 percent sorbic acid may be noted in Table 11. All three strains were able to grow and produced gas at pH 5.0, but at pH h.5 only SPY 29 was able to produce gas. The others grew without gas production at this pH. All strains were com- pletely inhibited by the sorbic acid at pH h.0. Since these organisms have'been shown to tolerate pH values considerably lower than this in the absence of sorbic acid (Tables 1 through 5) the effect of lowering the pH of the media to pH h.0 upon the inhibitory action of sorbic acid appears quite significant. From these studies it is apparent that the highest pH possible in which inhibition of the spoilage pickle yeasts by 0.1 percent sorbic acid is certain is pH h.0. To test the accuracy of the preservation prediction data 120 jars of sweet pickles were packed at various concen- trations of sucrose, acetic acid and sorbic acid. Because no visible signs of spoilage were detected during the first three weeks after packing, the jars were challenged with a 1 m1 inoculation of saline suspension of yeast cells and lac- tobacilli. The organisms introduced were prepared from 2h-hour broth cultures of SPY 21 and Lactobacillus plantarum strain L-5. After being centrifuged from the broth, the organisms were resuspended in isotonic saline, and pour plates were pre- pared to determine concentrations of organisms present. V-8 brom cresol green agar was used to determine the number of TABLE 11 EFFECT OF pH ON THE IKHIBITICN 0F SPOILAGE PICKLE YEASTS BY 0.1 PERCEKT SOREIC ACID SPY 15 SPY 21 SPY 29 Gas1 Final 2 Gas Final Gas Final (days) Turbidity (d ays) Turbidity (days) Turbidity 5.0 5 1+ M 1+ 5 1+ k.5 - 1+ - 1+ 12 1+ LL00 - - " " "' " 3.5 - - - - ' - - 3 00 "' "' "' " " "' 1 Expressed as days required for maximum evolution of gas. 2 Sediment and turbidity of broth after seven days incubatinn: 3+ = good; 2+ = moderate; 1+ = scant; "' = none. A 30 lactobacilli and acidified dextrose agar to determine yeast numbers. One ml of the suspension contained 9.5 x 106 lacto- bacilli and 1.5 x 106 yeast cells as determined by this method. The inoculated jars were allowed to incubate at room temperature for two weeks, during which they were observed for signs of spoilage -- turbidity and gas formation (Table 12). Spoilage occurred at concentrations of 0.5 . and 1.0 percent acetic acid with no sorbic acid present at all su- crose concentrations. Conversely, 0.05 and 0.1 percent sorbic acid in the presence of 0.5 percent acetic acid pre- vented spoilage in all concentrations of acid and sugar. Microscopic examination of those jars in which spoilage was inhibited revealed that non-viable yeast cells were present in only small numbers not exceeding the original inoculum. No viable yeast cells were detected in these jars. Examination of the physical appearance and taste of the preserved pickles revealed no undesirable color, texture or flavor as a result of the presence of the preservative. In Figure III, points have been plotted indicating con- centrations of sucrose, acetic acid and sorbic acid at which spoilage or preservation in the inoculated packs occurred. Superimposed upon these graphs are the preservation prediction lines of Figure II, which resulted from.growth studies of the spoilage pickle yeasts. It is evident from these graphs that the prediction lines for sucrose and acetic acid and for 0.1 percent sorbic acid are valid in that no spoilage occurred .UopeonaH maww ommHHomm 90 mm MQOprbammmam u m momeHomw u m .COHposooam new use huHoHQQSp hp occHanpop .mpwn popwHSOOCH Ho ommHHomm m .ampmSOBomawon wash an vocHepopoo omoaosm accused ommeHpmm N panoB hp cocoa omopodm uaooamm H m m Aoe.mv d how.mv d fled.mv m om.sm o: m m Amo.mv m Aom.mv m Rom.mv m oo.om om m m hoe.mv m Aom.mv d goo.mv m om.Hm om m d Amo.mv m Aom.mv m goo.mv m ms.mH 0H oa.o m d Ame.mv m Aow.mv m fled.mv e om.sm o: m m Amo.mv m Aom.mv m AQd.mv m oo.om om m m Ame.mv m Aom.mv m Aoo.mv m om.Hm om d d Ame.mv d AOd.mv d Aoo.:v m ms.ma OH mo.o m d Ame.mv m A©.m may m Ad.m may m om.sm o: m m Ame.mv m Am.m may m Ad.m may m oo.om om m m Hos.mv m Am.m may m Ao.m mew m om.Hm om m m Aos.mv m Am.m may m Ad.m may m ms.mH OH 0 0.: o.m o.m o.H m.o memotesw steed used pcooaem e oaoSm 0H£nom ©H0< OHpood unooamm oopwEHpmm psooamm unoomom mmdMOHm. rmmzbobo Hafizm QMBEDOOZH ho mMOHHm .HaHBZWAIHHmMmNH mo MojHomm Q24 ZOHBHSmmammm NH mqmde 4‘0 “I” 0 0 0 o 32 . S L/CVE’OISI; + A; E 71:" 4:19 ‘3‘0—- 0 o o / 2_0___ % 1‘7 / O L O -""' o o o , 0'5‘_ O O c . I 1 I J O I I I 7 4O 30 20 IO pE/PCE’W pERCENr sumose ACE/’76 AC/D + O "F- O 0 0 0 £1 05 Z S ORB/é“ A"? 112 30 -'- O O O o 2.0 __ o o A _0 ['0 —-IL-— 0 O o 0.5 __ G/" O O O l I I J O I I I | +0 30 20 /O PEREE/vr PERCENT ewe-05E ACET/C 246/0 4. 07.. o a o o - 1Q ZQ.Z_.SQZ I B 1151144512 3'0“” 0 O o o .2 .0_+_ O O o o to d— 0 o o o O I j j I 0 : 99555;? W! 176/? 40 V 30 20 [0 0 : SPOILHGE PEPE EN '/" PERCENT 51/6905 E ACE 77C 246/0 Fez/RE ZZZ PF? ESEPW/W/Vo SFO/MGE 0F worm. A 7E0 5 W557 (,7 row; 56/? P/CM E s, La L.) at points above these lines. The lines follow closely the zone of demarkation dividing spoiling concentrations of acetic acid and sucrose from non-spoiling concentrations. However, the absence of spoilage in any of those jars con- taining 0.05 percent sorbic acid indicate that under actual conditions of preservation, this concentration may be more effective than is indicated by the preservation prediction lines. A possible reason for the apparent increased ability of sorbic acid to inhibit spoilage in this concentration is lack of a favorable medium for optimum growth in the presence of the preservative. That is, the yeasts may find the sweet pickle syrup less desirable as a medium of growth in the presence of sorbic acid than was the broth culture medium. This could only be determined by further work with large numbers of experimental packs of sweet pickles. How- ever, it is always desirable to leave a wide margin of safety when it is possible to do this at no expense to the quality and cost of the product. DISCUSSION The preservation prediction line determined from this study for combinations of sucrose and acetic acid is, in general, similar to the one determined by Bell and Etchells (1952). However, these workers found that preservation by sucrose and acetic acid required slightly higher concentra- tions of these substances than was found necessary in the present investigation. For example, with 2 percent acetic acid at least ho percent sucrose was required to inhibit spoilage, while only 30 percent sucrose with the same acid concentration was found necessary in the present study. The differences apparent in these results may be due to the use by Bell and Etchells of a greater number of different strains of spoilage pickle yeasts including strains of higher toler- ance toward sugar and acid than those used in this investiga- tion. A preservation prediction line for sucrose and acetic acid concentrations in the presence of sodium benzoate was also plotted by Etchells and Bell (1952). They found that with 20 percent sucrose and 0.10 percent sodium.benzoate, 2 percent acetic acid was required for effective preservation. In contrast, with the same sugar concentration and 0.10 per- cent sorbic acid, only 0.5 percent acetic acid was found to be necessary in this study. Also, a concentration of 0.10 percent sodium benzoate appeared even less effective than 0.05 percent sorbic acid. Therefore, sorbic acid was shown to be a more efficient inhibitory agent than sodium benzoate, effecting preservation at lower concentrations of sugar and acetic'acid. The concentration of 0.10 percent sorbic acid required to inhibit spoilage of sweet pickles was found to be greater than that necessary to control mold growth in cheese. Smith and Rollin (195ha) reported that while concentrations of 0.005 to 0.01 percent retarded growth, 0.05 percent was fully ef- fective in completely inhibiting molds,in process cheese. The difference in effective concentrations of sorbic acid required in these two products is probably the result of the dissimilar characters of the two substrates as well as of the differences between the organisms involved. The studies of the effect of pH on the activity of sorbic acid indicated that the spoilage pickle yeasts grew well in the presence of 0.1 percent of the preservative at pH h.5, but were inhibited at pH h.0. This is a remarkable tolerance toward sorbic acid compared to that indicated by Emard and Vaughn (1951) for the yeast, Debaromyces membranaefaciens. This organism was completely inhibited at a pH of 5.h in a concentration of 0.07 percent sorbic acid. Also, Costilow and Ferguson (l95h) noted that 0.05 and 0.1 percent sorbic 36 acid completely inhibited 8 species of yeasts from cucumber fermentations at pH 5.0. It was noted in this study as well as those of Emard and Vaughn (1951), Phillips and Mundt (1950), and Jones and Harper (1952), that the lactic acid bacteria are not greatly affected by sorbic acid. The strain of Lactobacillus plantarum used was able to grow in the presence of 0.1 percent sorbic acid at a pH of h.1. At pH values lower than this, the hydrogen ion concentration is too high for the growth of these bacteria. Besides being efficient as a preservative, and odorless, colorless, tasteless and non—toxic in concentrations required for preservation, sorbic acid has yet another advantage in its use as a food preservative. Melnick, Luckmann, and Gooding (l95h) found that sorbic acid is metabolized by spoilage organ- isms if they are present in large numbers compared with the concentration of the preservative. Inhibitory activity would not be exerted under these conditions. Therefore, the pos- sibility of sorbic acid being overwhelmed by large concentra- tions of spoilage organisms makes impossible the use of this substance as a means of disguising inferior quality food products or masking poor sanitary practices in food processing. The organisms used in this study were selected because of their possible activity as causative agents of spoilage in sweet pickle products. It is evident that of the organisms used in this investigation, only the spoilage pickle yeasts may be considered active in this respect under the conditions NJ of sweet pickle preservation. Because the lactobacilli were far less tolerant toward the inhibitory factors than the yeasts, it was necessary to determine the preservation pre- diction lines from the growth studies of the yeasts. It is significant that some degree of variation of the different strains of yeasts in their tolerances toward the inhibitory factors was demonstrated. It should be emphasized that the preservation prediction lines proposed in this study were made from studies of 3 strains of a single species of spoilage yeast and are valid only until such time as other spoilage organisms may prove to be more resistant to the inhibitory factors. Therefore, the isolation and testing of spoilage organisms should be continued until such time that no spoilage occurs in prOperly prepared packs. SUMMARY From the results of growth studies involving spoilage pickle yeasts and Lactobacillus plantarum, preservation pre- diction lines werejplotted indicating the necessary concen- trations of sucrose and acetic acid, and of those agents in combination with sorbic acid required to prevent spoilage by these organisms. With sucrose and acetic acid alone, pre- servation was assured at 2 percent acetic acid in combination with sucrose concentrations of 20 percent or more. When the concentration of sugar was less than 20 percent, 3 percent acetic acid was required. When 0.1 percent sorbic acid was added, the presence of 0.5 percent acetic acid with even 2 percent sucrose was sufficient for preservation. Sorbic acid at 0.05 percent was effective in the presence of 2 per- cent acetic acid at 2 to 20 percent sucrose, while with 30 to ho percent sucrose, 1.0 and 0.5 percent acetic respectively were required. These preservation. predictions were verified by jars of preserved cucumber which were packed in various concen- trations of sugar, acetic acid and sorbic acid. All control jars containing less than 2 percent acetic acid without sorbic acid were spoiled. However, a concentration of 2 percent acetic acid resulted in preservation at all sugar concentratunm. No spoilage was evident in any of the Jars containing 0.05 percent sorbic acid with 0.5 percent acetic acid, regardless of the concentration of sucrose present. Thus, a concentra- tion of 0.05 percent sorbic acid resulted in the same preser- vative effect as 0.1 percent of the substance. Tests measuring the effect of pH on the inhibitory ac- tivity of 0.1 percent sorbic acid indicated that a pH value of H00 or less was necessary for the inhibition of pickle spoilage yeasts by this agent. This was also verified by the results of the cucumber preservation study which demonstrated no spoilage occurring at pH values less than h.0 l. 2. 3. CONCLUSIONS Sorbic acid effectively prevents spoilage of sweet cucum- ber pickles in a concentration of 0.1 percent in the presence of at least 0.5 percent acetic acid. The con- centration of sucrose has no effect upon the efficiency of the preservative under these conditions. The ability of 0.05 percent sorbic acid to insure preservation may depend upon the quantities of sucrose and acetic acid present. However, further investigation is indicated with this level of sorbic acid in experimental packs of sweet pickles. Sucrose and acetic acid in combination exhibit a comple- mentary relationship in effecting preservation; as the sucrose concentration increases the amount of acetic acid required for preservation decreases. However, at least 2 percent acetic acid is necessary for inhibition at any sucrose concentration.up to at least ho percent. The maximum pH value insuring preservation in the presence of 0.1 percent sorbic acid is pH h.0. Sorbic acid is a more efficient preservative than sodium benzoate. Spoilage is prevented in lower concentrations of sucrose and acetic acid, allowing sweet pickle products to be manufactured having low sugar (low Baumé$ and low kl acid concentrations. Thus it may be Inssible to produce more desirable products without heat processing by the use of sorbic acid. ADDENDUM After the preparation of this manuscript, continued observations of the experimental packs of inoculated sweet pickles were made. After three weeks of incubation at room temperature, spoilage was noted in additional jars. All packs containing 0.05 percent sorbic acid and 0.5 percent acetic acid showed definite signs of spoilage. The sucrose concentrations of these packs were 10, 20, 30, and he per- cent. No spoilage was evident in any of the packs contain- ing 1.0 percent acetic acid with 0.05 percent sorbic.acid. This was verified by microscopic examinations which re- vealed no viable yeasts present in these jars. These additional data further support the validity of the preservation prediction line for 0.05 percent sorbic acid, indicating that while spoilage may occur in acid- sucrose concentrations indicated below this line, no spoilage occurred for those concentrations above the prediction line. However, spoilage did occur in duplicate packs with he per- cent sucrose and 0.5 percent acetic acid, a point which lies on the preservation prediction line. Thus, the necessity of a wide margin of safety in calculating formulas for the pre- servation of sweet pickles is indicated. REFERENCES Bell, T. A. and Etchells, J. L. 1952. Sugar and Acid Toler- ance of Spoilage Yeasts from Sweet-Cucumber Pickles. Food Tech. 9 (immeamz. Costilow, R. N. and Ferguson, W. E. 19Sh. Personal communi- cation. Deuel, H. J. Jr., Alfin-Slater, R., Weil, C. 5., and Smyth, H. F. Jr. 195u. Sorbic Acid as a Fungistatic Agent for Foods. I. Harmlessness of Sorbic Acid as a Dietary Component. Food Research, 12:1-12. , Calbert, C. F., Anisfeld, L., McKeehan, H., and Blunden, H.I). 195k. Sorbic Acid as a Fungistatic Agent for Foods. II. Metabolism of Alpha, Beta-Unsatur- ated Fatty Acids with Emphasis on Sorbic Acid. Food Research, 12:13-19. Emard, L. 0., and Vaughn, R. H. 1951. Selectivity of Sorbic Acid Media for the Catalase Negative Lactic Acid Bacteria and Clostridia. J. Bact., é; (h):h87-h9u. Erickson, F. J. and Fabian, F. W. 19h2. 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