ABSTRACT THE EFFECT OF POTASSIUM SORBATE ON ORGANISMS COMMONLY ASSOCIATED WITH COTTAGE CHEESE SPOILAGE by Robert L. Bradley, Jr. Cottage cheese dressings containing several concentrations of potassium sorbate were inoculated with Pseudomonas fragi, Alcaligenes metalcaligenes, Geotrichum candidum, Penicillium frequentans, Rhodo- torula mucilaginosa or Torulopsis candida. The dressings were then mixed with the curd and stored at 50°F. Cheese samples inoculated with psychrophiles were analysed daily and those inoculated with yeasts or molds were analysed on alternate days. At each examination interval analyses were performed on each sample for total count, psychrophile count, yeast and mold count, pH, and percentage of potassium sorbate. The presence of potassium sorbate in the cheese in concentrations of 0.050, 0.075, and 0.100 per cent adversely affected the growth of the spoilage organisms. Potassium sorbate was both fungicidal and fungistatic. The fungicidal preperty was evidenced when the organism populations de- creased as the concentrations of potassium sorbate were increased. Fungis- tatic activity was shown by extended lag phases and retarded growth. Potassium sorbate appeared to be bactericidal and bacteriostatic when used against small concentrations of psychrophiles. Robert L. Bradley, Jr. _ 2 - In duplicate trials involving different populations of inocula, a longer lag phase was associated with a lower p0pulation. No off-flavor was noticed in creamed cottage cheese containing 0.100 per cent potassium sorbate. There was no evidence that any of the organisms metabolized potassium sorbate as a source of carbon. THE EFFECT OF POTASSIUM SORBATE ON ORGANISMS COMMONLY ASSOCIATED WITH COTTAGE CHEESE SPOILAGE By ROBERT LE BRADLEY, Jr. A THESIS Submitted to the College of Agriculture Michigan State university of Agriculture and Applied Science in partial fulfilhment of the requirements for the degree of MASTER.OF SCIENCE Department of Food Science 1960 To Jackie ii ACKNOWLEDGMENT My most sincere gratitude and appreciation are extended to Dr. L. G. Harmon for his professional counsel and guidance throughout the course of this study and deve10pment of this manuscript. Heartfelt thanks are also extended to Dr. C. M. Stine for his contributions of knowledge and advice. iii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . . . . . . . . . . . . . . . . 2 The Antimycotic Activity of Fatty Acids . . . . . . . . . . . . . 2 Physiological Properties of Sorbic Acid . . . . . . . . . . . . . 3 Physical and Chemical Characteristics of Sorbic Acid . . . . . . 4 Sensitivity of Organisms to Sorbic Acid . . . . . . . . . . . . . 7 EXPERIMENTAL PROCEDURE . . . . . . . . . . . . . . . . . . . . . . . . 9 Cottage Cheese Manufacture . . . . . . . . . . . . . . . . . . . 9 Propagation of Organisms . . . . . . . . . . . . . . . . . . . . 9 Preparation of Samples . . . . . . . . . . . . . . . . . . . . . 10 ANALYTICAL PROCEDURES. . . . . . . . . . . . . . . . . . . . . . . . . 10 Bacteriological Analyses . . . . . . . . . . . . . . . . . . . . 10 Determination of Sorbic Acid . . . . . . . . . . . . . . . . . . 12 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Analysis of Samples Inoculated with l) Pseudomonas fragi . . . . . . . . . . . . . . . . . . . . 15 2) Alcaligenes metalcaligenes . . . . . . . . . . . . . . . 22 3) Geotrichum candidum . . . . . . . . . . . . . . . . . . . 28 4) Penicillium frequentans . . . . . . . . . . . . . . . . . 33 5) Rhodotorula mucilaginosa . . . . . . . . . . . . . . . . 38 6) Torulopsis candida . . . . . . . . . . . . . . . . . . . 45 iv DISCUSSION . . . . . . SUMMARY AND CONCLUSIONS LITERATURE CITED . . . I. and II. III. and IV. and VI. VII. and VIII. IX. and X. XI. and XII. TABLES Organism Population, pH, and Per Cent Potassium Sorbate in Creamed Cottage Cheese Inoculated with Pseudomonas fragi and Incubated at 50° F. . . . . Organism Population, pH, and Per Cent Potassium Sorbate in Creamed Cottage Cheese Inoculated with Alcaligenes metalcaligenes and Incubated at 50° F. Organism.Population, pH, and Per Cent Potassium Sorbate in Creamed Cottage Cheese Inoculated with Geotrichum candidum and Incubated at 50° F. . . . Organism Population, pH, and Per Cent Potassium Sorbate in Creamed Cottage Cheese Inoculated with Penicillium frequentans and Incubated at 50° F. Organism Population, pH, and Per Cent Potassium Sorbate in Creamed Cottage Cheese Inoculated with Rhodotorula mucilaginosa and Incubated at 50° F. Organism Population, pH, and Per Cent Potassium Sorbate in Creamed Cottage Cheese Inoculated with Torulopsis candida and Incubated at 50° F. . . . vi 18 24 31 35 41 47 Page and and and and and and 20 26 32 37 43 48 10. 11. and 12. 13. and 14. FIGURES Page Weiland's dehydrogenation - hydration - dehydrogenation scheme . . . . . . . . . . . . . . . 6 Diagram illustrating the procedure for preparing samples of creamed cottage cheese in sterile 8 oz. jars, and indicating the analyses performed. . . . . 11 Effect of potassium sorbate on Pseudomonas fragi inoculated into creamed cottage cheese stored at 50° F. O O O O O O O O O O O O O O O O O O O O O O 17 Effect of potassium sorbate on Alcaligenes metalcaligenes inoculated into creamed cottage cheese Stored at 50° F. O O O O O O C O O O O O O O I O O 23 Effect of potassium sorbate on Geotrichum candidum inoculated into creamed cottage cheese stored at 50° F. O O O O O O O O O O I O O O O O O O O O I 0 30 Effect of potassium sorbate on Penicillium frequentans inoculated into creamed cottage cheese Stored at 50° F. O O O O O O O O O O O O O O O O O 34 Effect of potassium sorbate on Rhodotorula mucilaginosa inoculated into creamed cottage cheese Stored at 50° F. O O O O O O O O O O O O O O O O O 40 Effect of potassium sorbate on Torulopsis candida inoculated into creamed cottage cheese stored at 50° F. O O O O O I O I O O O O O O O O O O O O O O 46 vii INTRODUCTION Creamed cottage cheese, a protein concentrate containing about 475 to 500 calories per pound, is becoming an important food item with our diet conscious public. The per capita consumption has increased from 1.9 pounds in 1940 to 5.1 pounds in 1958. Continued improvement in the quality of cottage cheese is necessary to maintain increased consumption. Improper handling during manufacture, storage, and delivery together with adverse temperatures in grocery store and household refrigerators are detrimental to a long shelf-life. Research studies have proved that high quality acid foods will have a longer shelf-life when sorbic acid is added. Also, sorbic acid imparts no toxicity to the food. The scope of this study is to demonstrateathe efficacy of potassiun sorbate against surface spoilage organisms in creamed cottage cheese and to determine whether these organisms utilize sorbic acid during normal _storage periods. REVIEW OF LITERATURE Cottage cheese with a relatively low microorganism content may become unsalable in a short time unless it is constantly refrigerated at 40° F. or lower. Harmon gtflgl. (11) reported on the relationship of temperature to keeping quality of cottage cheese. By lowering the storage temperature from 50° to 42° F., they demonstrated that shelf- life could be increased by 12 to 100 per cent. Another section of their study revealed that temperatures of display cabinets in a cross section of retail markets ranged from 43° to 60° F. at the top of the cabinet and 38° to 55° F. at the bottom. Adverse storage temperatures have caused cheese manufacturers to consider the advantages of using sorbic acid and its potassium salt to increase the shelf-life of their product. The Federal Food and Drug Administration (28) has approved sorbic acid for use in cheese and cheese products in amounts up to 0.2 per cent sorbic acid by weight. The Antimycotic Acitivitylg£_Fatty Acids Sorbic acid (2,4-hexadienoic acid) is an , -unsaturated acid containing the same number of carbon atoms as caproic acid, but exhibit- ing greater fungicidal properties than caproic acid. Clark (3) was one of the first to report that the un-ionized portion of the fatty acid molecule was toxic to microorganisms. He demonstrated that acetic acid was much more toxic than the mineral acids. Kiesel (14) revealed many factors about the antimycotic character- istics of the fatty acids, most important of which are: a) the activity of the saturated fatty acids increases with the carbon chain length, and b) branched chain fatty acids are less active than those with straight chains and an equal number of carbons. Wyss 2E.2l- (29) also contributed to the knowledge of the inhibitory effect of the fatty acids and showed that: a) the activity of a fatty acid increases with decreasing pH, b) unsaturated fatty acids are slightly more active than saturated fatty acids, c) the point of unsaturation has little effect on the activity, d) multiple unsaturation gives about the same activity as mono-unsatura- tion and e) Optimum chain length is determined by the resistance of the organism and the solubility of the fatty acid. Cowles (6) showed that fatty acids are bactericidal at pH 4.7 and lower. Keeney (13) reported fungicidal prOperties for the 5, 6, 8, 10, and 11 carbon fatty acids, whereas, the shorter chain acids exhibited only fungistatic properties. Physiological Properties of Sorbic Acid One of the most efficient fungistats known today is sorbic acid and its potassium salt. The toxicity of these compounds as dietary con- stituents was investigated by Deuel gtflgl. (8). They proved that sorbic acid would not interfere with the digestion of foods and that egg-unsatur- ated fatty acids were readily metabolizable. In toxicity tests with rats, it was found that tissue changes did not occur when 0 to 8 per cent sorbic acid was included in the feed. However, a slight increase in liver weight was reported at the 8 per cent level. When an 8 per cent concentration of benzoic acid was fed to rats, there was a 50 per cent mortality and an increase in liver and kidney weight. These workers concluded that sorbic and benzoic acids fed to rats at a 4 per cent concentration showed no harmful effects in a 90 day trial. Demaree £5 £1. (7) investigated the effects of feeding sorbic acid at a 10 per cent level to two generations of rats. Their results showed that the parents and only the male off- spring had increased liver weights. The males in both generations showed an increase in depot fat. None of the test rats appeared to decrease in reproductive capacity. Physical and Chemical Characteristics of Sorbic Acid Melnick and Luckmann (16) proved that migration of sorbic acid occurred in six varieties of cheese at a rate prOportional to the moisture content of the cheese. One surface of a wrapped block of cheddar cheese was covered with a wrapper treated with 2.5 grams of sorbic acid per 1,000 square inches of surface. The concentration of sorbic acid at the treated surface decreased during the aging period. To determine the depth of migration the cheese was sliced into one-eighth inch strips, homogenated, and analysed for sorbic acid content. Some sorbic acid was actually lost from the wrapped cheese. The possibilities of sublimation through the wrapper and auto-oxidation were disProved. Melnick st 31. (17) found that mold enzymes, concentrated near the surface of the cheese, accomplish: metabolic degradation of the sorbic acid by reducing it to carbon dioxide and water; whereupon, the migrated sorbic acid attempts to re-establish the concentration equilibrium by re-migrating toward the lesser concentra- tion at the outer surface. The work of Mukherjee (20, 21, 22) elucidated the mechanism by which sorbic acid inhibits microorganisms. @>-oxidation of unsaturated fatty acids occurs according to the sequence in Figure l. The interme- diates formed are p-keto acids. In the degradation of butyric acid by the dehydrogenase enzymes of Aspergillus gige_r, hydration of the Q-unsaturage produced ©-hydroxybutyric acid. This compound was always present during the reaction, usually in small quantities. Continual oxidation prevented an accumulation of p-hydroxybutyric acid. In the later stages of the degradation, acetone accumulation and the absence of the unsaturated compound were noted. The presence of acetone indicated that the end- products were the same as in human metabolism. Accumulation of increased amounts of the(3-hydroxybutyric acid inhibited dehydrogenation which would produce the(3—unsaturate. Thisz-hydroxy acid inactivated the dehydro- genase enzymes of the mold. Since this inhibition is a function of the amount of theéfi-hydroxy acid present, the continual production of dehy- drogenase wouldieventually overcome the inhibitor after which the mold utilizes the inhibitor as a source of carbon. Sorbic acid, with the formula CH3-CH=CH-CH=CH-COOH is an 096» -un- saturated acid. Melnick fig. (17) noted that a O-hydroxy acid occurred two times during the degradation of sorbic acid, once after hydration, and again after it passed through a dehydration, oxidation, and hydration sequence. Samson 35 a1. (26) observed that the effectiveness of fatty acids as fungistats was a function of pH. The inhibitory effect increased with decreasing pH because the acid was permeable to the cell in the undissociated .omma.¢¢uma noummmmm uoom .cowuwnwsaH vac: mo Emwamcooz onu was moaoz hp omomno CH wao< aflnhom mo cowumvmuwov UHHocmamz .Hm um .n «xoflaamzm moaonum Goaumaowouphcow a cowumuomn u cowumcmwouvxnow m.wamHoH3 .H unawam omm + moo mooo-mmo-oo-mmo a; mooo-~mo-momo-mmu mom +_+ cmm + N8 + mooo-moumo-mmu No + mooo-Nmu-oo-monmo-mmo i moou-mmo-momo-moumo-mmu mom + moooéoumoéoumuhmo saga cannom muosooua pom oumuvhcocumo oHAMNHHocmumE mo monomoum ecu aw aowumwfixo Uwom comm aowumamwOHo%non vwom .98“ Panic aowumuohm coaumusummqsu a $0 Goaumaowouvmnon monotone was com oHom muumm nowumofixo owes comm cowumcowonp%coa owom 58:33-0 aowumuomm coauMHSummasu Q Jo aowumaowOHwhnoa Asao< uaouamov saga suumm anemones: omm + N8 mooo-~mm-oo-mmo a i mooo-~mo-momo-mmu at; mooo-=oumo-mmo mm - + omm + N8 + mooo mmo Nee mmo No +.+ mooo-~mo-ou-~mo-~mo-mmo .. I mooo-~mo-momu-~mo-~mu-mmo mo=++ mooo-moumo-~:o-~=o-mmu mm - + moou-Nmo-~mo-~mo-~mu-mmu 30¢ flush form only. According to O'Neill (22), any food product with a pH of 6.5 or lower and a potassium sorbate concentration of 0.1 per cent or less would have all the sorbate hydrolyzed to sorbic acid. Pfizer (24, 25) indicates the following solubilities for sorbic acid and potassium sor- bate: Sorbic Acid Potassium Sorbate Solubility in H20 at 20° C. at pH 4.4 0.22% * Solubility in H20 at 20° C. at pH 5.9 1.0270 * Solubility in H20 at 20° C. at pH 7.0 7.00% >40% *Hydrolyzed to the acid form at this pH. Cowles (6) reported that the germicidal activity of the salts of saturated fatty acids varies according to Traube's law which states that the lowering of surface tension is directly proportional to the length of the carbon chain. Hartman (12) stated that substances become absorbed at the cell interfaces by lowering the surface tension of the solvent. Sensitivity of Organisms E9 Sorbic Acid Bell EE.El- (1) reported on the influence of sorbic acid at several pH levels on many species of bacteria, yeasts, and molds commonly asso- ciated with cucumber fermentations. Thirty two Species representing 12 genera of yeasts grew in 0.1 per cent sorbic acid at pH 7.0 but not at pH 4.5. ‘Candida krusei was the most tolerant yeast of the 32 Species tested. Sixty six Species representing 32 genera of filamentous fungi would not grow in 0.1 per cent sorbic acid at pH 4.5. Six Species representing three genera of lactic bacteria were inhibited by 0.1 per cent sorbic acid at pH 3.5 but all grew at pH 5.0. These tests suggest that the efficiency - 7 - of sorbic acid as an inhibitor of certain microorganisms is based on the concentration of the undissociated molecule and not the total con- centration of the acid. Costilow ggnal. (4) reported that neither the lactic bacteria nor a large contamination of a yeast (Torulopsis holmii) metabolized sorbic acid in cucumber brines. Another study by Costilow 2; 31. (5) demonstrated that sorbic acid concentrations of 0.01 to 0.1 per cent in an 8 per cent sodium chloride brine were inhibitory to yeasts and molds. Perry and Lawrence (23) stated that sorbic acid would not suppress heavy contaminations but would effectively retard small contaminations of microorganisms. They also noted that samples containing up to 0.07 per cent sorbic acid by weight were similar in flavor to the controls. Geminder (10) reported that 0.075 per cent sorbic acid was effective in retarding the growth of yeasts, molds, and slime-forming bacteria on cottage cheese. Higher sorbic concentrations produced a bitter flavor in the finished product. EXPERIMENTAL PROCEDURE The purpose of this experiment was to measure the inhibitory effect of potassium sorbate on organisms commonly associated with cottage cheese spoilage and to determine if any of the organisms studied utilized po- tassium sorbate as a source of carbon. Cottage Cheese Manufacture Skimmilk pasteurized at 145° F. for 30 minutes was obtained from the Michigan State University Dairy Plant. Cottage cheese was made from this Skimmilk by the_short-set method. The creaming mixture containing 12 per cent fat was pasteurized by steaming in an autoclave for 30 minutes. Three per cent salt was added to the pasteurized creaming mixture. PrOpagation g; Organisms The following organisms commonly associated with surface spoilage in cottage cheese were used in this study: Pseudomonas fragi, Alcaligenes metalcaligenes, Geotrichum candidum, Penicillium frequentans, Rhodotorula mucilagiposa and Torulopsis candida. An active strain was developed by initiating daily transfers into nutrient broth three days before the organism was to be used. The cul- tures were incubated at 72° F. Four milliliters of the yeast and 4 ml. of the mold cultures were used to inoculate the respective creaming mixtures, whereas, only 2 m1. of the psychrophiles were used to give the desired contamination. The psy- chrophile pepulations inoculated into the cottage cheese dressings were determined on Violet Red Bile agar and.the yeasts and molds were enumer- ated on Potato Dextrose agar acidified to pH 3.5. Preparation‘2f_Samples A total of 12 lots of cheese were analysed with two lots being contaminated with each of the six spoilage organisms mentioned previously. Each trial (lot) was composed of five groups of cheese samples labelled as follows: group 1) non-inoculated control, group 2) inoculated control, group 3) inoculated + 0.050 per cent potassium sorbate, group 4) inocu- lated + 0.075 per cent potassium sorbate, and group 5) inoculated + 0.100 per cent potassium sorbate. The diagram (Figure 2) shows how the samples were prepared and indicates the analyses performed on each sample. Sixty seven grams of the dry curd and 33 grams of the creaming mixture were weighed into each sterilized sample jar. The samples were incubated at 50° F. until surface Spoilage was observed. ANALYTICAL PROCEDURES Bacteriological Analyses In the trials involving cheese samples inoculated with psychrophiles, one sample from each of the five groups was analysed daily for sorbic acid content, organism populations, and pH. In the trials with samples inocu- lated with yeasts or molds, one sample from each of the five groups was analysed every other day. Each sample was mixed for two minutes in a sterilized Waring blendor at slow Speed to assure thorough mixing. At high speed, curd particles adhered to the upper surfaces of the jar and were - 10 - .voauomuom momhaono map wnaudoavaa can anon .No w oHTuoum GT omooco owouuoo voawouo mo moaaaum magnumoua you ousvoooua waauouumSaHT Ewuwoan .N ouswdm _ A i H a [w T 1? T IF T F T i T rm T F T 1w T [F T L T F T .F T [T T F T .7 T 1F T [F T 4 T 1T T 1r T {T T E T [W L A T 1W T Ir T IF T H T 1r T iv T r? T Th T 17 T WT T 1r L [r T T T 1m T E T r? T i T 1r T 17 T 1 T i T T L T i T F i DmDU mmwmIO OEDO wmmmIO OEDU wmwwIU DO wmwwIU QmDU mmmeU 0oo N. m m u m N J .¢ ousmfim a _ T _\4 LUOJQ/JUI'IOQ 60"] .Moom us coho: omomno owduuob vofiwouo on”: pagoda Amman mmaoaovsomm so mosaics gammduom mo uoommm m>oo .e. 0 ms? m ____c .m muswfim N _ O _ 4 maootom. x .x. 09.0 + molasses o Sootom x .x. £06.. 83385 0 23.6m x .x. 03.0 + “caresses 4 2:63.35 0 b22305 u :02 oo m m .e. w n v m N _ o .0 ouswfim d 4 \ /A 4 A\ /v 1 /‘ 233m x .xoooiogaoseos n maootom x .x. 98.0 + stresses 0 23.6m x 8890822805 4 P363665 0 T322365 . :02 4 W) q. wmg / lunog 507 ‘9 (.0 .Moon on venoum mucosa owouuoo @8830 can.“ wouwgoosa mono H3332: moaowflmoja so Suwanee Bfimmmuom mo uoommm .m 0.33m m>oo n m m c n N _ o l 4 A l A 1 7 0 .D 9 l O n w / 9 J 0 w -23.. TABLE III ORGANISM.POPULATION, pH, AND PER.CENT POTASSIUM SORBATE* IN CREAMED COTTAGE CHEESE INOCULATED WITH ALCALIGENES METALCALIGENES (840,000 organisms per gram) AND INCUBATED AT 50°F. Description of Cheese Sample Inoculated + Potassium Sorbate Controls as Indicated Type of Non- Analysis inoculated Inoculated 0.050% 0.075% 0.100% 0 Day Total Count 3,300 2,100,000 480,000 360,000 220,000 Psychrophile <10 1,500,000 310,000 190,000 64,000 Yeast and Mold ‘ <10 <10 10 10 <10 pH 5.30 5.30 5.30 5.30 5.35 % K Sorbate 0.000 0.000 0.043 0.060 0.098 1 Day Total Count 11,000 2,800,000 840,000 390,000 270,000 PsychrOphile <10 2,100,000 520,000 290,000 180,000 Yeast and Mold <10 <10 10 <10 <10 pH 5.40 5.45 5.45 5.40 5.40 % K Sorbate 0.000 0.000 LA 0.074 0.106 2 Days Total Count 120,000 89,000,000 7,400,000 890,000 360,000 Psychrophile <10 71,000,000 4,200,000 530,000 260,000 Yeast and Mold <10 <10 <10 <10 10 pH 5.40 5.40 5.40 5.45 5.40 % K Sorbate 0.000 0.000 0.045 0.065 0.096 3 Days Total Count 160,000 220,000,000 11,000,000 1,400,000 540,000 PsychrOphile <10 130,000,000 ‘5,600,000 680,000 460,000 Yeast and Mold <10 <10 <10 <10 <10 pH 5.40 5.40 5.45 5.40 5.40 % K Sorbate 0.000 0.000 0.051 0.062 0.098 -24.. TABLE III (Continued) Description of Cheese Sample Inoculated + Potassium Sorbate Controls as Indicated Type of Non- Analysis inoculated Inoculated 0.050% 0.075% 0.100% 5 Days Total Count 9,200,000 ---- 23,000,000 7,100,000 2,200,000 Psychrophile 100 ---- 13,000,000 3,700,000 1,300,000 Yeast and Mold 30 ---- <10 <10 <10 pH 5.40 ---- 5.40 5.40 5.40 % K Sorbate 0.000 ---- 0.046 0.069 0.076 6 Days Total Count 40,000,000 ---- 76,000,000 19,000,000 10,000,000 Psychrophile 1,900 ---- 52,000,000 17,000,000 10,000,000 Yeast and Mold 620 ~--- 10 <10 <10 PH 5040 “"- 5.40 5040 5.40 % K Sorbate 0.000 ---- 0.034 0.075 0.086 7 Days Total Count 59,000,000 ---- ---- 100,000,000 74,000,000 Psychrophile 1,100 ---- ---- 38,000,000 15,000,000 Yeast and Mold 1,200 ---- ---- <10 <10 PH 5.35 "" "" 5.45 5.40 % K Sorbate 0.000 ---- ---- 0.065 0.085 8 Days Total Count 71,000,000 ---- ---- 130,000,000 110,000,000 PsychrOphile 2,700 ---- ---- 90,000,000 81,000,000 Yeast and Mold 1,300 ---- ---- <10 <10 pH 5.40 ---- ---- 5.40 5.40 ‘70 K sorbate 0.000 --"- "" 0.072 O. 086 0.000 corrected for irrelevant absorbancy *calculated as sirbic acid - 25 - TABLE IV ORGANISM POPULATION, pH, AND PER.CENT POTASSIUM SORBATE* IN CREAMED COTTAGE CHEESE INOCULATED WITH ALCALIGENES METALCALIGENES (700,000 organisms per gram) AND INCUBATED AT 50°F. Description of Cheese Sample Inoculated + Potassium Sorbate Controls as Indicated Type of Non- Analysis inoculated Inoculated 0.050% 0.075% 0.100% 0 Day Total Count 1,900 1,500,000 84,000 26,000 26,000 Psychrophile <10 590,000 13,000 15,000 10,000 Yeast and Mold <10 20 <10 10 30 pH 5.20 5.15 5.20 5.15 5.20 % K Sorbate 0.000 0.000 0.056 0.066 0.122 1 Day Total Count 7,400 3,500,000 73,000 31,000 28,000 Psychrophile <10 2,300,000 46,000 16,000 15,000 Yeast and Mold <10 <10 <10 <10 <10 pH 5.10 5.20 5.15 5.20 5.20 % K Sorbate 0.000 0.000 0.045 0.085 0.094 2 Days Total Count 7,600 52,000,000 1,700,000 560,000 33,000 Psychrophile 90 32,000,000 650,000 400,000 18,000 Yeast and Mold <10 <10 <10 <10 <10 pH 5.15 5.30 5.20 5.15 5.15 % K Sorbate 0.000 0.000 0.059 0.084 0.108 3 Days Total Count 77,000 120,000,000 19,000,000 790,000 130,000 Psychrophile 40 87,000,000 1,700,000 630,000 91,000 Yeast and Mold <10 <10 <10 <10 <10 pH 5.25 5.40 5.35 5.25 5.25 % K Sorbate 0.000 0.000 0.044 0.079 0.081 4 Days Total Count 460,000 ---- 87,000,000 1,300,000 610,000 PsychrOphile 110 ---- 51,000,000 640,000 290,000 Yeast and Mold 10 ---- <10 <10 <10 pH 5.30 ---- 5.30 5.25 5.30 % K Sorbate 0.000 ---- 0.046 0.074 0.083 TABLE IV (Continued) Description of Cheese Sample Inoculated + Potassium Sorbate Controls as Indicated Type of Non- Analysis inoculated Inoculated 0.050% 0.075% 0.100% 5 Days Total Count 6,100 ---- 210,000,000 9,900,000 2,900,000 Psychrophile 160 ---- 90,000,000 11,000,000 2,300,000 Yeast and Mold 2,200 ---- 40 20 <10 PH 5015 -""" 5020 5.15 5015 70 K Sorbate 0.000 "" 0.058 0.079 O. 104 6 Days Total Count 69,000 ---- ---- 61,000,000 43,000,000 Psychrophile 2,200 ---- ---- 58,000,000 33,000,000 Yeast and Mold 300 ---- ---- <10 <10 pH 5.15 ---- ---- 5.20 5.20 % K Sorbate 0.000 é;-- ---- 0.066 0.083 7 Days Total Count 27,000,000 ---- ---- ---- 120,000,000 Psychrophile 1,000 ---- ---- ---- 80,000,000 Yeast and Mold 700 --—- ---- ---- 10 pH 5.25 ---- ---- ---- 5.35 % K Sorbate 0.000 ---- ---- ---- 0.085 8 Days Total Count 7,600 ---- ---- ---- 41,000,000 Psychrophile 400 ---- ---- ---- 27,000,000 Yeast and Mold 1,800 ---- ---- ---- <10 pH 5.30 ---- ---- ---- 5.40 % K Sorbate 0.000 ---- ---- ---- 0.081 9 Days Total Count 19,000 ---- ---- ---- 62,000,000 Psychrophile 800 ---- ---- ---- 59,000,000 Yeast and Mold 4,400 ---- ---- ---- <10 pH 5.35 ---- ---- ---- 5.40 % K Sorbate 0.000 --—- ---- ---- 0.082 0.000 corrected for irrelevant absorbancy *calculated as sorbic acid _ 27 - The growth curve of the psychrophiles in the samples (Figures 5 and 6) containing 0.050 per cent potassium sorbate showed no lag. In the ino- culated samples containing 0.075 and 0.100 per cent potassium sorbate, the lags in the growth phase were apparent after tthe first day in the samples described in Figure 5 and during the first 1 or 2 days in the cor- responding samples in Figure 6. As a result of the addition of potassium sorbate to the cheese, the standard plate counts of the inoculated samples containing sorbate (Tables III and IV) were significantly less on the 0 day than the correSponding counts of the inoculated control sample. The pOpulations assumed a logarithmic growth rate when the inhibitory property of the sorbate ion was no longer effective. Since Alc. metalcaligenes grew at 89.6°F., the standard plate counts for the inoculated control samples were higher than the counts of the non- inoculated controls. Alc. metalcaligenes demonstrated no evidence of sorbate metabolism, and pH values of the inoculated samples showed no sig- nificant change as decomposition progressed. Analysis gf_Samp1es Inoculated with Geotrichum candidum.- The populations of Q, candidum (Figures 7 and 8) in the samples on the 0 day indicated that there was a greater destruction of the mold in the samples containing the higher percentages of potassium sorbate. A 2 to 4 day lag was evident in the growth curves of the mold in the inoculated samples con- taining 0.075 and 0.100 per cent potassium sorbate, whereas, the growth curve of the mold in the inoculated samples containing 0.050 per cent po- tassium sorbate showed relatively little lag. After the inhibitory effect of the sorbic acid was no longer apparent, the slope of the growth curves _ 28 - of the mold in the sample containing potassium sorbate was similar to the Slope of the growth curve of the mold in the inoculated control. In Table V, the inoculated samples that contained potassium sorbate in the following percentages: 0.050, 0.075, and 0.100 per cent, showed decreases in the population of the yeasts and molds on the 0 day of 68, 99, and 98 per cent, reSpectively. The correSponding samples (Table VI) Showed reductions in p0pulation on the 0 day of 54, 97, and 95 per cent, respectively. These decreases, calculated from the count of the inocu- lated control sample on the 0 day, exemplified the fungicidal property of the sorbic acid against 9. candidum. In most inoculated samples the total count decreased as the percent- ages of potassium sorbate increased; however, in one trial inoculated with fip candidum, the Standard plate counts (Table VI) on the 0 day showed greater pOpulations in the samples containing potassium sorbate than in the inoculated control samples. Large increases in the standard plate counts were observed after the second day of incubation. The numbers of psychrophiles were negligible throughout the sampling period. .9, candidum Showed no utilization of potassium sorbate as a source of carbon. During the storage period there were slight, but inconsistent fluctuations in pH with the terminal pH being slightly higher than the initial pH. - 29 - .MOOM UM VOHOUQ OmGQSO Owduuou fimaflOHO choom HQ fiOHOUm UmUQH—O one.“ won—manage.“ Etienne gsoauuoow amouuoo vofioouo one.“ woumgooa.“ E33250 no oumcnom Sammmuoa mo uoomwm .m 93me Encauuoow no muscuom 5.39369 mo uoommm .n Shaman m>oo m>oo m m c m N w m a. m N . O T _ a a i 1 a _ . i _ 4 \ .h _ 1|. D [U N A ‘ D m \_ m —OI .D .D 3 m 1 e m n W u / I, / \ n13 1 m 9 D J A w D O A w 1 0 269.6% x 0 $09 6+ b32309: Soocom x I N. I N . O o\o nuO 013263065 meootom x 4 $890 $22385 J m J m b22369: O b26309: .202 4 l m l m -30.. TABLE V ORGANISM POPULATION, pH, AND PER.CENT POTASSIUM SORBATE* IN CREAMED COTTAGE CHEESE INOCULATED WITH GEOTRICHUM CANDIDUM (560 organisms per gram) AND INCUBATED AT 50° F. Description of Cheese Sample Inoculated +-Potassium Sorbate Controls as Indicated Type of Non- Analysis inoculated Inoculated 0.050% 0.075% 0.100% 0 Day Total Count 1,900 5,600 3,000 2,700 2,600 Psychr0phile 30 <10 <10 20 10 Yeast and'Mold <10 3,100 1,000 40 60 pH 5.35 5.35 5.30 5.35 5.40 % K Sorbate 0.000 0.000 0.050 0.079 0.092 2 Days Total Count 4,900 13,000 7,200 5,700 4,300 Psychrophile <10 <10 <10 20 10 Yeast and Mold <10 17,000 2,400 50 90 pH 5.25 5.20 5.25 5.20 5.20 % K Sorbate 0.000 0.000 0.041 0.076 0.087 4 Days Total Count 260,000 250,000 430,000 530,000 540,000 Psychrophile <10 <10 10 60 30 Yeast and Mold 400 300,000 9,000 2,600 140 pH 5.25 5.45 5.50 5.45 5.45 % K Sorbate 0.000 0.000 0.047 0.084 0.103 6 Days Total Count 440,000 ---- 700,000 670,000 620,000 Psychr0phile 40 ---- 70 100 60 Yeast and Mold 1,700 ---- 730,000 120,000 350,000 pH 5.20 ---- 5.45 5.50 5.45 % K Sorbate 0.000 ---- 0.039 0.078 0.098 0.000 corrected for irrelevant absorbancy *calculated as sorbic acid - 31 _ TABLE VI ORGANISM POPULATION, pH, AND PER CENT POTASSIUM SORBATEklN CREAMED COTTAGE CHEESE INOCULATED WITH GEOTRICHUM CANDIDUM (150 organisms per gram) AND INCUBATED AT 50° F. ‘—_‘ Description of Cheese Sample Inoculated + Potassium Sorbate Controls as Indicated Type of Non- Analysis inoculated Inoculated 0.050% 0.075% 0.100% 0 Day Total Count 11,000 7,900 100,000 8,700 14,000 Psychrophile <10 <10 10 20 <10 Yeast and Mold <10 1,300 600 40 60 pH 5.40 5.40 5.40 5.40 5.40 % K Sorbate LA 0.000 0.046 0.077 0.098 2 Days Total Count 160,000 65,000 23,000 7,600 9,200 Psychrophile <10 <10 <10 <10 <10 Yeast and Mold <10 30,000 9,000 240 60 pH 5.40 5.45 5.40 5.40 5.35 % K Sorbate 0.000 0.000 0.043 0.067 0.092 4 Days Total Count 40,000,000 7,800,000 310,000 43,000 810,000 Psychrophile 1,900 20 <10 10 <10 Yeast and Mold 620 1,400,000 300,000 39,000 1,300 pH 5.40 5.35 5.40 5.35 5.40 % K Sorbate 0.000 0.000 0.035 0.082 0.095 Days Total Count 59,000,000 ---- ---- 850,000 1,200,000 Psychrophile 6,700 ---- ---- 90 40 Yeast and Mold 1,300 ---- ---- 710,000 60,000 pH 5.40 ---- ---- 5.40 5.45 % K Sorbate 0.000 ---- ---- 0.059 0.095 0.000 corrected # calculated as sorbic acid for irrelevant absorbancy - 32 - Analysis 9; Samples Inoculated with Penicillium frequentans.- The populations of P, frequentans in the samples on the 0 day showed no decrease when 0.050, 0.075, and 0.100 per cent potassium sorbate were added to the samples. In fact the three samples containing sor- bate (Figure 9) attained slightly higher populations than the inoculated controls, but these differences in population did not influence on the rate of spoilage of the samples. The lag phase of the growth curves of P, frequentans lengthened as the percentages of potassium sorbate in the samples of cheese increased from 0.050 to 0.100 per cent (Figures 9 and 10). In the samples illustrated in Figure 9 the growth curves of the mold in the samples containing 0.075 and 0.100 per cent potassium sorbate Showed a tendency towards a secondary lag between the sixth and eighth days, after which, logarithmic growth was resumed. Inconsistencies, such as the variations between the estimated population added to the samples of cheese and the actual count of the organism in the 0 day samples (Tables VII and VIII), are difficult to explain. After the second day, the populations increased less rapidly in the samples containing the higher percentages of potassium sorbate. This was the expected pattern of growth which was also exhibited by the other organisms used in this experiment. - 33 - moon um .hoom um caucus 030:0 owmuuoo 3530 one.“ wououm Smooco owmuuoo wofiwouo can.“ 63.6.3005 2523:qu .fiaTHHHoHaom T693909: maduaosuoum STSTOHSSN so oumcHOm Samson—om mo uoommm .3 0.33m so mosaics Samoan—on mo uoommm .a ouswfim msoo maoo m 0 ¢ N O N_ O_ m m v N .O 7 _ _ .4'4 _| _ _ _ _ a 4 \ oaoocom x 5 1 _ Sooeogmesaos o 1 _ maootom x O o\o ate .0 + P363065 1 N meootom x b .4 N .xoonoogoaosoos d 0 6223005 0 mm 7 an .l M 6263005182 0 O .D n n w .u. / / J J D 0 w w l 0 q m 1 h 1 N. 1 w 1 m -34.. TABLE VII ORGANISM POPULATION, pH, AND PER.CENT POTASSIUM SORBATE* IN CREAMED COTTAGE CHEESE INOCULATED WITH PENICILLIUM FREQUENTANS (19,000 organisms per gram) AND INCUBATED AT 50° F. Description of Cheese Sample Inoculated + Potassium Sorbate Controls as Indicated Type of Non- Analysis inoculated Inoculated 0.050% 0.075% 0.100% 0 Day Total Count 2,000 41,000 9,900 8,300 2,200 PsychrOphile 120 180 20 30 10 Yeast and Mold <10 270 780 3,000 1,700 pH 5.40 5.45 5.50 5.45 5.45 % K Sorbate 0.000 0.000 0 .050 0.081 0.101 2 Days Total Count 2,300 56,000 39,000 8,700 2,000 Psychrophile 100 130,000 16,000 2,700 11,000 Yeast and Mold 100 51,000 4,000 1,300 1,200 pH 5.30 5.30 5.30 5.25 5.30 % K Sorbate 0.000 0.000 0.052 0.070 0.097 4 Days Total Count 8,400 2,200,000 55,000 34,000 12,000 Psychrophile 20 LA 100 500 300 Yeast and Mold 2,700 2,000,000 49,000 35,000 6,800 pH 5.25 5.40 5.35 5.30 5.25 % K Sorbate 0.000 0.000 0.039- 0.075 0.101 Days Total Count 190,000 ---- 2,800,000 630,000 80,000 Psychrophile 120 ---- LA 1,000 300 Yeasts and Mold 6,800 ---- 3,000,000 570,000 120,000 pH 5.25 "" 5.35 5.30 5.30 % K Sorbate 0.000 ---- 0.047 0.071 0.088 - 35 _ TABLE VII (Continued) " m -_— Description of Cheese Sample Inoculated + Potassium Sorbate Controls as Indicated Type of Non- Analysis inoculated Inoculated 0.050% 0.075% 0.100% 8 Days Total Count 1,000,000 ---- 2,100,000 560,000 60,000 Psychrophile 710 ---- LA 3,000 1,000 Yeast and Mold 7,400 ---- 1,600,000 560,000 66,000 pH 5.25 ---- 5.35 5.35 5.35 % K Sorbate 0.000 ---- 0.037 0.068 0.102 10 Days Total Count 2,400,000 ---- ---- 4,000,000 2,700,000 PsychrOphile 2,300 ---- ---- 7,500 3,000 Yeast and Mold 17,000 ---- ---- 4,600,000 2,300,000 PH 5.35 "" ""-- 5.35 5.30 % K Sorbate 0.000 ---- ---- 0.068 0.095 12 Days Total Count 2,800,000 ---- ---- ---- 3,700,000 Psychrophile 3,000 ---- ---- ---- 10,000 Yeast and Mold 600,000 ---- ---- ---- 2,700,000 pH 5.30 ---- ---- ---- 5.30 % K Sorbate 0.000 ---- ---- ---- 0.090 0.000 corrected for irrelevant absorbancy * calculated as sorbic acid - 36 - TABLE VIII ORGANISM POPULATION, pH, AND PER.CENT POTASSIUM SORBATE* IN CREAMED COTTAGE CHEESE INOCULATED WITH PENICILLIUM FREQUENTANS (10,400 organisms per gram) AND INCUBATED AT 50° F. Description of Cheese Sample Inoculated + Potassium Sorbate Controls as Indicated Type of Non- Analysis inoculated Inoculated 0.050% 0.075% 0.100% 0 Day Total Count 1,900 5,500 4,900 4,800 4,100 Psychrophile <10 <10 .<10 <10 <10 Yeast and Mold <10 1,500 900 1,200 1,700 pH 5.20 5.15 5.20 5.20 5.20 % K Sorbate 0.000 0.000 0.039 0.070 0.117 2 Days Total Count 7,600 4,500 3,800 4,300 4,800 Psychrophile 90 <10 <10 <10 <10 Yeast and Mold <10 2,700 2,600 1,400 1,600 pH 5.15 5.15 5.20 5.20 5.15 % K Sorbate 0.000 0.000 0.037 0.080 0.102 4 Days Total Count 460,000 1,700,000 34,000 17,000 9,100 Psychrophile 110 20 <10 <10 <10 Yeast and Mold 10 940,000 21,000 7,000 6,200 pH 5.30 5.35 5.25 5.20 5.20 % K Sorbate 0.000 0.000 0.049 0.073 0.079 6 Days Total Count 69,000 ---- 3,400,000 610,000 29,000 Psychrophile 2,200 ---- 40 70 10 Yeast and Mbld 300 ---- 2,900,000 580,000 14,000 pH 5.15 -‘-'" 5.30 5.20 5.15 % K Sorbate 0.000 ---- 0.046 0.080 0.093 8 Days Total Count 7,600 ---- ---- 3,600,000 1,000,000 Psychrophile 400 ---- ---- 70 60 Yeast and Mold 1,800 ---- ---- 3,000,000 700,000 pH 5.30 "" -"'"" 5.35 5.35 0/0 K sorbate 0.000 "" -"""' 0.052 00080 0.000 corrected for irrelevant absorbancy * calculated as sorbic acid - 37 - Analysis gf_Samples Inoculated with Rhodotorula mucilaginosa.- 0n the 0 day, no decreases in population attributable to the presence of potassium sorbate were observed in the samples containing 0.050 and 0.075 per cent potassium sorbate and inoculated with R, mucilaginosa (Figure 11), but the sample containing 0.100 per cent potassium sorbate showed a 42 per cent decrease in count. However, in the duplicate trial (Figure 12), the p0pu1ations of the samples containing 01050, 0.075, and 0.100 per cent sorbate showed decreases in count of 82, 58, and 73 per cent, respectively. This experiment suggested that §:_mucilaginosa was very sensitive to the effects of potassium sorbate. Some fungicidal action was exhibited in addition to an extensive fungistatic activity which attenuated the growth of the p0pu1ations inoculated into the samples containing potassium sorbate. The growth curves of the yeast in the Samples containing 0.050 and 0.075 per cent sorbate (Figure 11) showed lag phses of 6 and 4 days, respectively. An increase in the populations occurred after the initial lag phase. However, the slope of the growth curves of the yeast in the inoculated samples containing sorbate showed less increase in elevation than the slope of the corresponding curve representing the inoculated con- trol sample. The growth curve of the yeast in the sample containing 0.100 per cent potassium sorbate indicated only a slight variation in population from the initial count, until the fourteenth day of storage when the only colonies that appeared on potato dextrose agar plates_were mold contamin- ants; therefore, a <10 population of yeast was plotted on Figure 11. The growth of the yeast in the inoculated samples containing 0.050, 0.075, and 0.100 per cent potassium sorbate (Figure 12) showed 4, 4, and - 3g - 8 day lags, respectively. During the phase of logarithmic growth that followed the lag phase, the growth curves of the yeast in the inoculated samples containing potassium sorbate approximated the growth curve of the yeast in the inoculated control sample. The lag phases shown in Figures 11 and 12 suggested that the potassium sorbate was functioning as a fungistat against the populations of R. mucilaginosa. The pH of the inoculated control (Tables IX and X) decreased sig- nificantly after the second day of storage. However, as the sorbate concentration increased from 0.050 to 0.100 per cent in the inoculated samples, a longer time elapsed before a decrease in pH occurred. The populations of the psychrophiles increased slowly in the samples inoculated with 3, mucilaginosa, and containing the concentrations of potassium sorbate used in this experiment. The total counts (Table IX) showed no decrease as a result of the inhibitory activity of the potassium sorbate until the second day of storage. Throughout the storage period the standard plate counts were substantially lower in the cheese samples containing potassium sorbate. The non-inoculated control samples (Tables IX and X) Showed mold growth on the surface after the twelfth and eighth days, reSpectively. The last yeast and mold count of the non-inoculated control sample (Table X, 8th day) could not be plotted accurately since insufficient dilutions made the plates impossible to count; therefore, a greater than 30,000 population was recorded. - 39 - .moon on caucus 000030 owmuuoo .moom um wououm 000050 owmuuoo passage and“ wouwasoosH emanawmfiwmmza. 0080090 ousT woumasoosfi «modqmmaaeafl. manuouowocm so ou0c000 asammwuoa mo uoommm .NH shaman masuouovocm :0 muscuom asammwuon m0 uoommm .HH madman mxoo m>0o m. mm. .v. N. Au. m. w .¢ N nu v; N. Au. m 10 .v N .u 0 1 _ 1 _ lootomx $090+oeoeooos n 4 200.5%! I N l N $000+ooaosoos o 0:00:0m.x 800000010385 a 4 1n W 1 m M 0203005 0 .D . .D o. . .0 00203005 1 :02 1.. .. .. \ . . 1.. ‘|‘ “I II V .oa \ ‘ - L. w n n u m. u . - a x a . . a nu . . .u w 4 1 w w 0 . 1.0 1 m 1.m - 4o - TABLE IX ORGANISM POPULATION, pH and PER.CENT POTASSIUM SORBATE* IN CREAMED COTTAGE CHEESE INOCULATED WITH RHODOTORULA MUCILAGINOSA (270, 000 organisms per gram) AND INCUBATED AT 50°F Description of Cheese Sample Inoculated + Potassium Sorbate Control as Indicated Type of Non- Analysis inoculated Inoculated 0.050% 0.075% 0.100% 0 Day Total Count 3,300 39,000 31,000 35,000 32,000 Psychrophile 5,100 3,000 5,800 3,200 3,100 Yeast and Mold <10 24,000 22,000 23,000 10,000 pH 5.25 5.20 5.30 5.20 5.30 % K Sorbate 0.000 0.000 0.050 0.073 0.096 2 Days Total Count 2,000 1,100,000 15,000 86,000 26,000 PsychrOphile 90 300 120 60 430 Yeast and Mold <10 490,000 19,000 16,000 12,000 pH 5.30 5.15 5.15 5.20 5.25 % K Sorbate 0.000 0.000 0.046 0.076 0.106 4 Days Total Count 2,800 6,000,000 15,000 20,000 21,000 Psychrophile 30 <10 160 230 650 Yeast and Mold <10 8,300,000 10,000 10,000 14,000 pH 5.25 4.80 5.15 5.20 5.20 % K Sorbate 0.000 0.000 0.047 0.068 0.101 6 Days Total Count 30,000 6,800,000 52,000 20,000 18,000 Psychrophile 90 360 740 300 190 Yeast and Mold 4,200 6,400,000 7,000 22,000 2,600 pH 5.25 4.75 5.05 5.15 5.15 % K Sorbate 0.000 0.000 0.046 0.073 0.105 - 41 _ TABLE IX (Continued) Description of Cheese Sample Inoculated + Potassium Sorbate Control as Indicated Type of Non- Analysis inoculated Inoculated 0.050% 0.075% 0.100% 8 Days Total Count 780,000 ---- 480,000 60,000 9,100 PsychrOphile <1 340 ---- 120 170 150 Yeast and Mold 13,000 ---- 340,000 73,000 5,400 pH 5.25 ---- 4.75 5.00 5.10 % K Sorbate 0.000 ---- 0.051 0.073 0.103 10 Days Total Count 1,700,000 ---- 320,000 320,000 61,000 Psychrophile 1,600 ---- 80 30 <10 Yeast and Mold 13,000 ---- 620,000 230,000 7,100 pH 5.25 """"'- 4o 65 4o 80 4.95 % K Sorbate 0.000 ---- 0.053 0.078 0.095 12 Days Total Count 2,100,000 ---- 730,000 770,000 42,000 Psychrophile 4,000 ---- 140 40 150 Yeast and Mold 93,000 ---- 80,000 38,000 9,000 pH 5. 25 "" 4o 65 4075 4. 85 % K Sorbate 0.000 ---- 0.050 0.080 0.093 14 Days Total Count ---- ---- 1,200,000 690,000 200,000 Psychrophile ---- ---- 280 100 50 Yeast and Mold ---- ---- 1,300,000 67,000 20 pH ---- ---- 4.65 4.75 4.90 70 K sorbate "" """"" 0.045 0.082 0.124 0.000 corrected for irrelevant absorbancy * calculated as sorbic acid -42- TABLE X ORGANISM POPULATION, pH, AND PER CENT POTASSIUM SORBATE* IN CREAMED COTTAGE CHEESE INOCULATED WITH RHODOTORULA MEICILAGINOSA (23,000 organisms per grog.) AND INCUBATED AT 50%. Description of Cheese Sample Inoculated‘+ Potassium Sorbate Control as Indicated Type of Non- Analysis inoculated Inoculated 0.050% 0.075% 0.100% 9 Day Total Count 3,300 110,000 26,000 20,000 22,000 Psychrophile <10 60 100 70 10 Yeast and Mold <10 110,000 9,000 19,000 8,000 pH 5.30 5.35 5.40 5.35 5.40 % K Sorbate 0.000 0.000 0.046 0.065 0.085 2 Days Total Count 120,000 2,700,000 23,000 12,000 8,700 PsychrOphile <10 <10 <10 <10 <10 Yeast and Mold 10 1,600,000 16,000 5,000 6,000 pH 5.40 5.35 5.35 5.35 5.35 % K Sorbate 0.000 0.000 0.039 0.079 0.085 4 Days Total Count 9,200,000 2,900,000 67,000 30,000 12,000 Psychr0phile <10 <10 <10 <10 <10 Yeast and Mold 30 1,900,000 16,000 9,000 6,000 pH 5.45 5.10 5.35 5.40 5.40 % K Sorbate 0.000 0.000 0.040 0.061 0.074 6 Days Total Count 59,000,000 5,000,000 530,000 86,000 19,000 PsychrOphile 1,100 <10 30 10 <10 Yeast and Mold 1,200 3,600,000 380,000 67,000 8,000 pH 5.35 4.70 4.90 5.00 5.10 % K Sorbate 0.000 0.000 0.044 0.081 0.099 8 Days Total Count 83,000,000 ---- 16,000,000 120,000 70,000 Psychrophile 10,000 ---- 120 290 60 Yeast and Mold >30,000 ---- 13,000,000 24,000 12,000 pH 5.40 "'-"- 4.80 4.95 5.05 0/o K sorbate 0.000 "" O. 052 0. 088 0.094 - 43 - TABLE X (Continued) Description of Cheese Sample Inoculated + Potassium Sorbate Control as Indicated Type of Non- Analysis inoculated Inoculated 0.050% 0.075% 0.100% 10 Days Total Count ---- ---- ---- 180,000 70,000 PsychrOphile ---- ---- ---- 630 190 Yeast and Mold ---- ---- ---- 170,000 93,000 pH ---- ---- ---- 4.80 4.95 % K Sorbate ---- ---- -—-- 0.083 0.088 12 Days Total Count ---- ---- ---- 260,000 140,000 Psychrophile ---- ---- ---- 900 400 Yeast and Mold ---- ---- ---- 350,000 270,000 pH ---- ---- ---- 4.70 4.75. % K Sorbate ---- ---- ---- 0.082 0.100 14 Days Total Count ---- ---- ---- 2,600,000 5,300,000 Psychr0phile ---- ---- ---- 1,100 700 Yeast and Mold ---- ---- ---- 2,200,000 3,700,000 pH ---- ---- ---- 4.65 4.75 % K Sorbate ---- ---- ---- 0.078 0.099 16 Days Total Count ---- ---- ---- ---- 430,000 Psychrophile ---- -—-- ---- ---- 1,100 Yeast and Mold ---- ---- ---- ---- 200,000 pH ---- ---- ---- ---- 4.60 % K Sorbate ---- ---- ---- ---- 0.102 18 Days Total Count ---- ---- ---- ---- 510,000 Psychrophile ---- ---- ---- ---- 1,500 Yeast and Mold ---- ---- ---- ---- 410,000 pH ---- ---- ---- ---- 4.60 % K Sorbate ---- ---- ---- ---- 0.108 0.000 corrected for * calculated as sorbic aCid irrelevant absorbancy -44- Analysis pi Samples Inoculated with Torulopsis candida.- The p0pu1ations of T, candida on the 0 day (Figures 13 and 14) were only slightly affected by the addition of potassium sorbate to the creamed cottage cheese. A progressive decrease in the population of the yeast was noted as the percentages of potassium sorbate were increased; however, the actual count of the yeast on the 0 day was many times higher than the estimated population added to the cheese. The growth curves of the yeast in the inoculated samples containing sorbate approximately paralleled the curve of the yeast in the inoculated control sample. This parallelism, which occurred from the beginning of storage, suggested that the sorbic acid had little fungistatic activity against this yeast. The counts of the yeasts and molds in the non-inoculated controls showed rapid increased after the second day of storage. Standard plate and psychrophile counts (Tables XI and X11) indicated a slight inhibition of the growth of the organisms particularly at the higher concentration of potassium sorbate in the inoculated samples. The pH values of the inoculated samples showed a slight increase as Spoilage progressed. An apparent decrease in the percentage of potassium sorbate . during Storage was attributed to discrepancies in the recovery of the sorbate from the creamed cottage cheese. - 45 - .moom .moom 00 00Houm 000050 00 wonoum 0000£0 0wmuuoo 00800H0 0w0uuoo 00800u0 oucw 0000asooaa 00T0000 000T 00u0H0000T 00H0000 0T000HSHOH 0.39.0508 no 0009000 Ewwgmuoa mo 000mmm .3 0.3me 00 0000.30 3.5000000 mo uo0mmm .mH 0.3me m o m>oo w m G m N _ O m m V m N _ O T _ a _ . _ A a _ _ _ _ T A 0200:00.x o\o 002 .0 + 002 03005 0 020900 2 o\o 0N 0.0 + 00203005 0200000 2 l v o\o 000 .0 + 002 03005 00203005 0 0203005 1:02 4 1* woxg/wnOQ 607 4 wmguunog 60'] -46- TABLE XI ORGANISM POPULATION, pH, AND PER CENT POTASSIUM SORBATE* IN CREAMED COTTAGE CHEESE INOCULATED WITH TORULOPSIS CANDIDA (130,000 Organisms Per gram) AND INCUBATED AT 50° F. Description of Cheese Sample Inoculated + Potassium Sorbate Control as Indicated Type of Non- Analysis inoculated Inoculated 0.050% 0.075% 0.100% 0 Day Total Count 12,000 940,000 430,000 250,000 150,000 Psychrophile 740 210 190 150 30 Yeast and Mold 10 2,200,000 480,000 310,000 240,000 pH 5.35 5.35 5.40 5.35 5.40 % K Sorbate 0.000 0.000 0.044 0.072 0.107 2 Days Total Count 3,100,000 11,000,000 4,400,000 1,700,000 1,400,000 Psychrophile 3,500 2,700 300 70 10 Yeast and Mold 40 14,000,000 4,900,000 1,200,000 1,100,000 pH 5.40 5.45 5.50 5.50 5.40 % K Sorbate 0.000 0.000 0.033 0.065 0.099 6 Days Total Count 150,000,000 250,000,000 130,000,000 94,000,000 21,000,000 Psychrophile 470,000,000 590,000,000 29,000,000 4,100,000 30,000 Yeast and Mold 4,900 100,000,000 170,000,000 130,000,000 34,000,000 pH 5.40 5.40 5.45 5.45 5.40 % K Sorbate 0.000 0.000 ' 0.027 0.053 0.086 0.000 corrected for irrelevant absorbancy * calculated as sorbic acid - 47 - TABLE XII ORGANISM POPULATION, pH, AND PER.CENT POTASSIUM SORBATE* IN CREAMED COTTAGE CHEESE INOCULATED WITH TORULOPSIS CANDIDA (3,200 organisms per gram) AND INCUBATED AT 50° F. Description of Cheese Sample Inoculated + Potassium Sorbate Control as Indicated Type of Non- Analysis inoculated Inoculated 0.050Z 0.075Z 0.100Z 0 Day Total Count 4,700 1,100,000 620,000 320,000 150,000 Psychrophile <10 <10 <10 <10 <10 Yeast and Mold <10 1,500,000 560,000 170,000 140,000 pH 5.35 5.25 5.20 5.25 5.20 Z K Sorbate 0.000 0.000 0.046 0.078 0.092 2 Days Total Count 6,900 39,000,000 5,800,000 4,800,000 3,000,000 Psychrophile 190 100 60 70 <10 Yeast and Mold 10 19,000,000 5,800,000 2,400,000 3,100,000 pH 5.25 5.20 5.20 5.25 5.20 Z K Sorbate 0.000 0.000 0.046 0.076 0.086 4 Days Total Count 1,400,000 54,000,000 29,000,000 34,000,000 17,000,000 Psychrophile 800 490 400 530 700 Yeast and Mold 3,900 95,000,000 67,000,000 58,000,000 13,000,000 pH 5.25 5.40 5.35 5.35 5.35 Z K Sorbate 0.000 0.000 0.034 0.064 0.094 6 Days Total Count 1,600,000 ---- ---- ---- 42,000,000 Psychrophile 2,600 ---- ---- ---- 1,500 Yeast and Mold 7,100 ---- ---- ---- 35,000,000 pH 5.35 ---- ---- ---- 5.35 Z K Sorbate 0.000 ---- ---- ---- 0.072 0.000 corrected for irrelevant absorbancy * calculated as sorbic acid - 43 - DISCUSSION Differences in the rate of Spoilage of the samples of creamed cottage cheese were observed in many instances in the duplicate trials. The ratio of two parts of curd to one part of creaming mixture was used in all trials. However, in some instances where the moisture content of the curd was low, the curd was not completely covered by the creaming mixture. '§,.gucilaginosa was sensitive to all concentrations of potassium sorbate used in this experiment and in the samples inoculated with this organism, surface Spoilage occurred much more rapidly when the surface of the curd was not covered by the creaming mixture. Surface spoilage, observed in the inoculated samples of cottage cheese, started on the particles of curd protruding from the creaming mixture. This suggested that the sorbic acid concentration was less on the exposed surface of the curd and greater in the creamed cottage cheese. Melnick and Luckmann (16) reported that the migration of sorbic acid was directly related to the moisture content of various varieties of cheese. Although cottage cheese was not studied by these authors, the high moisture content and the permeability of the particles of curd suggest that migration of sorbic acid should have permitted the concentration of sorbic acid to become equal- ized rapidly; but the Spoilage exhibited by 3, mucilaginosa indicated that equalization did not occur. _ 49 _ Several authors (16, 17, 27) have reported that molds metabolized sorbic acid as a source of carbon. Yeasts also have the necessary dehy- drogenase enzyme system; however, Costilow gtflgl. (4) demonstrated that a large inoculum of T, holmii would not decrease the concentration of the sorbate in pickle brines. In the work reported herein, there was no positive proof that any of the organisms used sorbic acid as a source of carbon. Apparently, spoilage of cottage cheese is accomplished by lower populations than required to cause measurable diminution of the sorbate. [No objectionable flavor was noted with the samples containing 0.100 per cent sorbate. When hydrolized, a 0.100 per cent solution pf potassium sorbate is only 0.075 per cent sorbic acid. These results were in agreement with the observations of Geminder (10) and Perry and Lawrence (23) who reported no perceptible typical "bitter" flavor in creamed cottage cheese containing 0.075 per cent sorbic acid or less. Since the creamed cottage cheese contained one part of the creaming mixture to two parts of curd, the individual creaming mixtures were pre- pared by adding three times the quantity of potassium sorbate desired in the finished product. 0n the 0 day, reductions in the populations attri- butable to the activity of the sorbate ion in the inoculated.samples containing sorbate were evident in all instances except the samples inocu- lated with 2, frequentans. Together with the three per cent salt, the tripled concentration of potassium sorbate could have caused some destruc; tion of the organisms during the interval after the organisms were added to the creaming mixture and before the mixture was blended with the cheese. - 5o - In the 0 day analysis of the samples containing no sorbate, the pOpulations of the inoculated controls were the same or higher than the calculated populations inoculated into the cottage cheese. In most deter- minations, the populations of organisms actually enumerated in the inoculated samples containing sorbate were lower than the calculated counts inoculated into the creamed cottage cheese. Exceptionsrwere noted in the samples inoculated with T, candida. These samples showed that some destruction had occurred within 4 hours after inoculation into the creamed cottage cheese and the degree of destruction was greater as the percentages of potassium sorbate were increased. Nevertheless, all the initial counts of T, candida in the samples of creamed cottage cheese were greater than the calculated population of the inoculum added to the cheese. The samples in both of the trials inoculated with g, frequentans and the samples in one trial inoculated with 3, mucilaginosa showed no decrease in the initial count when potassium sorbate was added to the cottage cheese. When inoculated into creamed cottage cheese, 3, mucilaginosa was the only organism used in this experiment in which the growth was retarded significantly, and some destruction continued from the 0 through the fourth day. Costilow gt 31. (4) reported that sorbic acid was a fungistat and slightly fungicidal. Keeney (13) stated that a 6 carbon fatty acid has fungicidal pr0perties. These findings with B, mucilaginosa confirmed the statements of these workers. There was some decrease of potassium sorbate in samples inoculated with E: freguentans and T: candida. The decrease was attributed to inherent - 51 - weaknesses in the analytical recovery of the sorbate rather than to metabolism of sorbate by the organisms. Variations experienced in analyzing the samples suggested that decreases in sorbate of less than 10 per cent should not be construed as an indication of utilization of sorbate by the organism. If this assumed standard is valid, there is no evidence that sorbate was metabolized by any of the organisms used in this work. It is possible that higher population concentrations of the organisms might have been able to effect some decrease in the sorbate, but under the conditions of this study in which the populations inoculated simulated extreme contamination, the approximate initial concentration of sorbic acid was maintained throughout the period the cheese was in storage. Yeasts and molds have a dehydrogenase system, but none has been reported for bacteria. Emard and Vaughn (9) demonstrated the inhibitory nature of sorbic acid on catalase positive organisms. ‘Such catalase activity for the Pseudomonas and Alcaligenes used in this experiment is not listed in Bergey's Manual (2). There is evidently a mechanism of in- ”hibition still undiscovered. The bacteriostatic nature of the sorbate ion was exhibited in the experiment with both of the psychrophiles. Some evidence of bactericidal tendencies is suggested by the decrease in the total count and the psychrophile count on the 0 day in the inocu- lated samples containing potassium sorbate. This property of sorbic acid was confirmed by Cowles (6) who stated that fatty acids had bactericidal properties at pH 4.7 and lower. However, the only samples of cottage cheese in which the pH decreased below 5.00 were those inoculated with B, mucilaginosa. - 52 - Also, a retarded growth of the psychroPhiles was observed in these samples at pH 4.7. Sorbic acid has a greater bactericidal power than the 6-carbon fatty acid because the two conjugated double bonds in the.structure of the sorbic acid add bactericidal activity. Therefore, since sorbic acid has a greater bactericidal power than a 6-carbon fatty acid, this greater power should exert an influence at a higher pH. Smith and Rollin (27) noted that the initial contamination of mold must be relatively light in order to obtain the high sorbic acid-to-mold dehydrogenase ratio necessary for inhibition. Melnick gtual. (18) stated \ similar findings with their work on bakery products. These findings ex- plained the reason for an increased initial lag phase when the p0pu1ation in the inocula decreased. All samples containing yeasts, molds, and psychrophiles except those samples inoculated with E} candida, exhibited this property. Perhaps a smaller population of‘T. candida would also demonstrate a lengthened lag phase in the growth curves, similar to those of 3. mucilaginosa. Bell g£_gl, (1) reported that six Species representing three genera of lactic bacteria were inhibited by 0.1 per cent sorbic acid at pH 3.5. pH 3.5 is a little low for most lactics to grow regardless of whether sorbic acid is present. No mention was made in their report as to whether growth tests were performed at pH 3.5 without sorbic acid added. Such a comparison would be necessary to determine the validity of the effect of sorbic acid on the lactics tested. - 53 _ SUMMARY AND CONCLUSIONS Samples of cottage cheese containing 0.050, 0.075, and 0.100 per cent potassium sorbate were contaminated with six organisms commonly associated with surface spoilage in cottage cheese. The organisms used were Pseudomonas fragi, Alcaligenes metalcaligenes, Geotrichun candidun, Penicillium frequentans, Torulopsis candida, and Rhodotorula mucilaginosa. Each sample of cheese was analyzed for total count, psychrophile pOpula- tion, yeast and mold p0pu1ation, pH, and percentage of potassium sorbate. Analyses were performed daily on samples contaminated with psychrophiles, and samples contaminated with yeasts or molds were examined on alternate days. None of the organisms tested metabolized potassium sorbate as a source of carbon before the sample was spoiled. In general, increases in p0pu1ation were inversely related to the percentage of potassium sorbate in the samples. Some destruction in the populations inoculated into the creamed cottage cheese was shown in the initial analysis, but usually this des- truction did not continue after the second day. In most instances, the amount of reduction in population was directly related to the percentage of potassium sorbate. Some of the organisms showed a definite lag in the growth curve which extended as the percentage of potassium sorbate in the sample increased. -54- Another factor influencing the lag was the pOpulation of the inoculum. A higher population in the initial samples resulted in a shorter lag phase. The observation that a low initial population would give a longer lag phase exemplifies the requirement for a ratio of high sorbic acid to low dehydrogenase enzyme to increase cottage cheese shelf-life. A bactericidal property was evidenced by the decrease in the ppp- ulations of the psychrophiles inoculated into the creamed cottage cheese. Potassium sorbate at a 0.100 per cent concentration imparted no objectionable flavor to the creamed cottage cheese. - 55 - (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) LITERATURE CITED Bell, T. A., Etchells, J. L., and Borg, A. F. Influence of Sorbic Acid on the Growth of Certain Species of Filamentous Fmgi. J. Bact., _7_7_: 573. 1959. Breed, R. S., Murray, E. G. D., and Smith, N. R. Bergey's Manual ‘~ 21: Determinative Bacteriology. 7th ed. The Williams and Wilkins Co., Baltimore, Md. 1957. 1094 pp., plus xviii. Clark, J. F. 0n the Toxic Effect of Deleterious Agents on the Germination and Development of Certain Filamentous Fungi. Bot. 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Harmlessness of Sorbic Acid as a Dietary Constituent. Food Res., )2: l. 1954. Emard, L. 0. and Vaughn, R. H. Selectivity of Sorbic Acid Media for the Catalase Negative Lactic Acid Bacteria and Clostridia. J. Bact., 925487. 1952. Geminder, J. Extending the Shelf-life of Creamed Cottage Cheese with Sorbic Acid. Milk Dealer, 4_8_: 44. 1959. Harmon, L. G., Trout, G. M., and Bonner, M. D. A Market Survey of Cottage Cheese. I. Some Characteristics Influencing Consumer Acceptance and Shelf-life. Mich. Agr. Expt. Sta. Quart. Bul., 185 146. 1955. -56- (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) Hartman, R. J. WW. 2nd ed. Houghton Mifflin Co., New York. 1947. 537 pp. plus xxxii. Keeney, E. L. New Preparations for the Treatment of Fngous Infections _Ig Vitro and £1 Vivo Experiments with Fatty Acid Salts, Penicillin, and Sodium Sulfathiavzole. J. Clin. Invest., _2_33 929. 1944. Kiesel, A. The Action of Different Acids and Acid Salts upon the Deve1Opment of ASpeiggillus niggr. 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Effects of Acetate and Other Short-Chained Fatty Acids on Yeast Metabolism. Arch. Biochem. and BiOphys., 24; 406. 1955. Smith, D. P. and Rollin, N. J. Sorbic Acid as a Fungistatic Agent for Foods. VIII. Need and Efficacy in Protecting Packaged Cheese. Food Tech., 8; 133. 1954. U. S. Dept. Health, Education, and Welfare. Food and Drug Adminis- tration. Cheeses and Cheese Products. Definitions and Standards. Part 19, Title 21. Reprinted Nov. 1959. wyss, 0., Ludwig, B. J. and Joiner, R. R. The Fungistatic and Fungicidal Action of Fatty Acids and Related Compounds. Arch. Biochem., 13 415. 1945. - 58 - 300M USE GEL“! ”fa. ‘10,!‘1a‘w 5‘? fl! §1_.'.Ll:%‘} 3.‘ n; it ‘ It; fl J .71.: 3‘ Ill IIIl ll||a 584