THE IDfiNTfl-‘Y Am} HEAT Tmmw Gfi WWW 5M5 CW SPOELAQ OF PASTEUREZE SWEET H6055 ME: for the Dow of M. S. MiG-{mm STATE UNNERSITY “we"! K. Robbins i957 ""“!.l' . ‘“‘—----IIIIIIIIIIIIIIII _ Has IIIIWUIHHIIIlllllllllllillllllllllllllllilllllillllllllll 31293 10401 3283 ‘ RETURNING MATERIALS: IV1£31_] P1ace in book drop to remove this checkout from 1::::;§2Ei_ your record. flfl§§.wil1 be charged if book is returned after the date stamped be1ow. l m. _ 1W8 THE IIENTITY AND HEAT TOLERANCE CF MICROCRGANISHS CAUSING SPCILAGE CF PASTEURIZRB SHEET PICKLES By EDMARD K. ROBBINS AN A ETRACT Submitted to the College of Science and Arts Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1957 Approved by Edward K. Robbins l The purpose of this study was to identify and determine the heat resistance of microorganisms causing spoilage in pasteurized sweet pickles. Twenty-one cultures of acid-forming bacteria were isolated and identified as Lactobacillus plantarum (3 strains), Lactobacillus brevis (2 strains), and Lactobacillus fermenti (16 strains). All cultures isolated were screened to determine their approximate heat resistance; and the one strain of each species found to be most resistant selected for thermal death time studies. The thermal death time of E; fermenti (F160=2.S) in phosphate buffer at pH 7.0 showed that this organism was the most heat resistant of the three species. L:.£EEX$§ (F160=l.l9) and E: plantarum (F160=l.08) were not greatly different in heat tolerance. Therefore, E; fermenti was used for all further studies. Concentrations of sucrose above 30 per cent resulted in a decrease in population of the test organism,even at room temperature. Sucrose solution of 15 and 25 per cent had little or no significant effect at room temperature but exerted a great protective action at pasteurizing tem- peratures. g; fermenti in 15 and 25 per cent sucrose solutions had F160 values of h.9 and 6.6 respectively. The thermal death time of E; fermenti was greatly reduced when acetic acid was added to the substrate in which the cells were heated. with 0.5 per cent acid and 25 per cent sucrose, the F160 was further reduced to 0.365. The addition of 3 per cent salt (NaCl) to a solution containing 1 per cent acetic acid and 25 per cent sucrose did not greatly affect the thermal resistance of L: fermenti. Edward K. Robbins 2 These results indicate that the pasteurization of sweet pickles at 160°F for 20 minutes or l6S°F for 15 minutes (center of the jar tempera- ture) would be sufficient to eliminate spoilage due to the organisms isolated in this study. TEE IDENTITY LED HEAT TOLERANCE 01" MICROORGANISMS GAUSING SPOILAGE 03‘ PASTEURI- SWEET PICKLES 3: mm 1. 30331153 A THESIS Submitted to the College of Science and Arts Michigan State University of Agriculture and. Applied Science in partial fulfillment of the requirements for the degree 0! MASTER OF SCIENCE Department of Microbiology and Public Health 1957 WWW!!! To Dr. Ralph ll. Costilov of the Department of Microbiology and Public Health. Michigan State University. for his formiation of the plans for this thesis. and for his many kindnesses in its preparation. advice. and assistance: to Dr. Harold L. Badoff of the Department of Microbiology and Public Edith. Michigan State University. for his help in preparing the final figures. I wish to express 1w sincere ap- preciation. Also. to the Carbon and Carbide Chemical Corporation which painted the sssistantship which made this work possible. I wish to express w thanks. 11 TABLE OF CONTENTS NEW . ...................................... . IBERODUCTION ................................ mm or LIEW 000.000.00.000 OOOOOOOOOOOOOOOOOOOO Organisms of High Acid Foods . ..... . .......... .. Pasteurisation of Fresh Cucumber Pickles .. ...... . Influence of Sugar. Acid and Salt Concentra- tion upon Heat Resistance of Bacteria . ........ . WWW . ............. ................ . Isolation of Speilege Organisms ....... .. Identification of Bpoilage Organisms ..... ....... . Determination of Heat Resistance ................. Emu 00000000000000.0000 0000000000000000000000000000 Spoilsge Organisms from Pasteurised l'resh Sweet Pickles . ....... .................... ...... Determination of Thermal Tolerance .. ............. DISCUSSIW .0... OOOOOOOOOOOOO .00.. OOOOOOOOOOOOOOOOOOOO 0 mm 0.00.00000000000. ....... O 000000000 0000000000000 Bmtmmm .0000 000000000000000000 00.0000 000000000000 mu 1: sumvoa comm non mums In momma sums (pH 7.0) ..... . man 11: comma comma or _I._. mm man was In moss som'fon' 3 (pH 7.0) mm III: SURVIVOR GWEN arms MING IN 25 PER CENT BUOROSE SMONS VITH 0.5 PER CENT AGING ACID . ...... . ...... . ..... .. A APPENDIX IV: SURVIVOR GOMAHEBMINGIHZBPER CENT BUOROSE SOLUTIONS WITE1.0PER MT mnc MID oeeeeeee eeeeeeeeeee eeeeeeo APPENDIX 7: SURVIVOR COUMS ma HEATING IN 25 m CENT SUCROSE SOLUTION PLUS 1 PER CENT ACETIO ACID AND 3 PER cm 39.0]. ........... iii 10 lit in 16 18 18 31+ 38 1+0 1&3 ’45 In Table 1. LIST OF TABLES Page pH. Acid. Sugar and Salt Concentrations of Brines from Sweet Pickles .. .............. ...... l9 Populations of Bacteria and Yeasts Observed in Experimental Jars of Sweet Sliced Pickles ... 20 Morphological Characteristics of Organisms Stud-10d 00000000000 000000000000 00000000000000000000 21 Physiological Characteristics of Organisms Stud-10d 0000000000000000000000 00000000000000000000 0 a Bosults of Screening Tests for Thermal Tolerance of Bacteria from Spoiled Pickles .. . . . . .. an The 1:. l' 60’ and s Values for Three Species of Lactobacilli Heated in Phosphate Buffer (1.37.0) .......... ........ .28 The 1:. 1160’ and 8 Values for L fermenti Heated in 15 and 25 Per Cent Sucrose solutions 000000000000000000000000.0000... 0000000 00 30 he 1:. 1' . and 3 Values for L. f’ementi floated in 25 Peil' t Sucrose Solution- Plus 0.5 and 1.0 Per cut mtic kid 00000000000000000 00000 0000 32 iv LIST 0! FIGURES figure P860 1. Thermal death times curves of L. fermenti. _I_._. brevis and L. plantarum in phosphate buff-TFPH7. 0.0000000000000000000000000000000 000000 0 27 2. Effect of 15 and 25 per cent sucrose concen- trations on thermal death times of L. _f______ermenti (PH7.0)....000....0.00......0...................... 29 3. Effect of the addition of acetic acid on the thermal death times of L. fermenti in 25 per cont mm” 0.00000000000000000000.0000000000000000 31 it. Effect of the addition of 3 per cent sodium chloride on the thermal death time in 25 per cent mms. 00000000000000000.00000000000000000000. 33 INTRODUCTIOH The manufacture of fresh cucumber pickles has increased from an insignificant volume in 1930 to an amount equal to about 30 per cent of the total pickle production today. The preservation of fer- mented pickles involves ally the addition of sufficient levels of acid and salt. acid and sugar. or lower levels of these agents combined with sodium benzoate. Barely are these products pasteurized. However. the manufacture of fresh cucumber pickles requires pasteurization. This has created many new problems for the industry. Many sizes of whole cucumbers and many sizes and shapes of out cucumbers are packa‘Od. mess are packed with many different formula- tions of acid. sugar. salt. and spices. A. large variety of glass con- tainers are used for sales appeal. All of these variables may affect the pasteurization process required for preservation. There are recommendations in the literature for pasteurizing cucum- bers. However. some of these recommndations are not in such a form as to penit their splication to continuous pasteurization processes. Also. in son instances. they have failed to tales into consideration the many variables between different types of pickles. fie purpose of this study was to isolate and identify microorganisms causing spoilage a fresh pasteurized pickles. and to determine their heat tolerance in various combinatims of sugar. acid and salt. his data should provide a basis for determining processes for various types of pitles to insure preservation from microbial spoilage. .1- REVIEW 01' LIERLTU'EE Organisms g_:_f_ High Acid Foods High acid foods (pH 3.7 or below) are relatively free from spoilage caused by spore-forming bacteria (26). hiring pasteuriza- tion. the vegetative cells are destroyed leaving spores in the product. he low pH inhibits geruination of these spores resulting in dormancy. Such foods as sauerkraut. cucumber pickles. berries. grapefruit. citrus fruit Juices. and rhubarb are included in this group. Etchells and sci-ems. (11) and ntchells and Ohmsr (12) have denom strated that there are two main groups of organisms responsible for the spoilage of high acid foods. Probably the most economically important spoilage organisms are the gran positive lactic acid bacteria of the genus Lactobacillus. he gas forming Lactobacillus 3.211.! is sell known as the causative agent in the spoilage of tomato ketchup. worcester- shire sauce and other similar products (3). Etchells and miner (12). studying the bacteriology of the manufac- ture of fresh cucumber pickles. found that resistant spore-formers re- mained after pastarization. However. during storage these spores did not germinate. If lactic acid bacteria or yeast survive the pasteuriza- tion. spoilage of the product usually occurs. Anderson gt g_l_. (2) isolated several cultures of organisms from spoiled fresh kosher style pickles. Each culture was inoculated into I. sound Jal‘ of fresh pack pickles and tubes of brine (0.814 acetic acid. 25$ laOl). After incubation. only three organisms produced typical -2. -3- spoilage characteristics. One of these was a yeast and the other two were lactobacilli. No further identification was reported. The family Lactobacteriaceae was designated by Orla-Jensen (19) to a physiological group of gram positive rods and cocci that ferment carbohydrates to a variety of end products; 113:, lactic acid, acetic acid, alcohol, and carbon dioxide. These lactic acid bacteria obtain their energy by fermentation of sugar without the utilization of free oxygen. This necessitates that large amounts of sugar be fermented to obtain sufficient energy for growth (22). The Species of the genus Lactobacillus are separated into two categories; (a) homofermentative and (b) heterofermentative (28). Homofermentative species produce 85 to 95 per cent lactic acid on the basis of sugar fermented and only traces of other end products. The most common organism in this group associated with food fermentation is-Lactobacillus plantarun, Orin-Jensen. Probable synonyms of this species are. Bacillus pabuli acidi II Weiss; Bacillus cucumeris fermen- tati Henneberg; Bacillus wartmanni Henneberg; Bacillus listeri Hennebergg Bacillus maeockeri Henneberg; Bacillus leichmani II Henneberg; Bacillus beijerincki Henneberg; Lactobacillus pentosus Fred, Peterson, and Anderson; Lactobacillus arabinosus Fred, Peterson and Anderson (5, 20). Results of a study of 400 cultures of L. plantarum demonstrated that the majority of strains formed acid from glucose, fructose, mannose, galactose, arabinose, sucrose, maltose, lactose, raffinose, ’61 '1’. ll ‘ Ji— md salicin (5s 20). The lactic acid produced is usually optically inactive with only small amounts of acetic acid and carbon dioxide being formed. 1'.- plantarum is microaerophilic having an eptimm temperature of 30 to 35°C and a maximum of 1lO°C:. In a recent report. Gostilow and Humphreys (6) have demonstrated that some strains of .13.. plantarum can be separated by their ability to reduce nitrates to nitrites by using indole-nitrate medium (BBL). Heterofermentative lactics are characterised by the conversion of up to 50 per cent glucose to lactic acid. 20 to 25 per cent to carbon dioxide. and 20 to 25 per cent to ethyl alcohol and acetic acid (28). Heterofermentative lactics are separated into four species on the basis of temperature range of growth and ability or inability to ferment sugars (5. 21). he most common species of this group Lactobapilclus Lrpvis Orla- J'ensen. has an optimum temperature range of 30 to 35°C and will grow well from 18 to h0°0 with an occasional strain; growing at 10° and h5°0. his organism ferments arabinose. glucose. fructose and galac- tose. but usually shows negative or partial fermentation of lactose. sucrose. mannose. and raffinose (5. 21). Probable synonyms for this organism are: Mbacillus pantoaceticu; l'red. Peterson. and Davenport: Lactobacillus fermentatus Bergey gt 3.1.: Lactobacillus lzggpgrsici Nickle: Léejobacillg acidMl—aeromes Bergey _e_t_ 44.: and Lactobacillus IE1}; Borne: as. all... The second species of the heterofementative group. Lactobacillus . -5- fermenti Beijemnnk. usually does not ferment arabinose or xylose. but does ferment sucrose. lactose. and raffinose (5. 21). Its temperature range for Optimum growth is between 35° and ’45” with a maximum of w to 50°C; the minimum growth temperature is approxi- mately 20°C. Probable synonyms for this species are: Lactobacillus M Bergey 93 _a;l._.. Lactobacillujg m Pederson. and Lactobacillus intermedium Bergey gt, 9;. he third species. Lactobacillug mg; Bergey _e__t_ £519. ferments arabinose and has a wider range of growth temperatures than the previous two species. Its Optimm temperature range is 32° to 37°C. maxim l$866. and minimum 10°C. Probable synonyms for this species are Bacillus buchneri Henneberg and Bacillus wehmeri Henneberg. Inactobacillu! pastorianus Bergey gt _a_l_.. the fourth hetero-fermenta- tive species. was described by Pederson (21) as long rods which grow at low temperatures. hese organisms are less active in the fermenta- tion of sugars than previously described species. Probable synonyms for this species are Baccharobacillus pastorianns var. berolinenesis Henneberg. Bacillus lindneri Benneberg. and Bacillus fasciformis Schomfeld and Rommel. Pasteurization of hash cucumber Pickles Gomercial pasteurization studies of fresh cucumber pickles were first reported by Etchells in 1938 (10). Rate of heat penetration was measured by placing thermocoqiles in the center of all-oz. containers of -6- fresh cucumber pickles and water. he increase in container tempera- tures during specific time intervals was determined and process curves plotted using time and temperature relationships. Results of heat studies showed that 60 minutes was required to reach a temperature of 160°r in containers of fresh cucumber pickles in a water bath at 180°}. hirty minutes was required for water in the same containers to reach 16o°r. An internal temperature of 160°]! for 20 minutes we. recommended as a process. his was based upon bacteriological studies. but data to this effect were absent. Btchells and Goresline (11) reported on the bacteriological examination of pasteurized and unpasteurized pickles. Ewe main groups of organisms were found responsible for the spoilage of the unpasteu1~ ized products; _v_i_z_.. acid-forming bacteria and yeasts. Pasteurised products contained spore-forming organisms. but these did not increase during three months' storage. rtehens and miner (12). in 191a. published the results of a four year bacteriological study of pasteurized pickles using pro- cesses of 160°]? for 20 minutes or 165°r for 15 mimtes. i total of 1.758 Jars were examined during the 1% year period. Results indicated that all spoilage organisms were destroyed by these procedures. Btchells and Jones (13) classified e11 pasteurized pickle products ' into three classes: (I) unfermented. (II) partially fermented. and (111) mu: tormented. Controlled pasteurization of 73.9% (165°!) for 15 minutes was used on two representatives of each group to -7- determine if spoilage organisms would.be destroyed. The products were checked for’the effect of’this heat treatment‘upon crispness. firmness and flavor. The specific items studied were as follows: (a)'unfermented sliced fresh cucumbers. (b) unfermented fresh dills. (c) partially fermented overnight dills. (d) partially tormented genu- ine dills. (e) fully fermented genuine dills (3-5 grains acetic acid). and (f) fully fermented.genuine dills (6-8 grains acetic acid). The acidpforming'bacteria and.yeasts which were capable of causing spoilage were destroyed. Observations indicated that quality in crispness. firmness. and flavor were retained after eight months' storage. Reten- tion of crispness. firmness. and flavor was reportedly due to using a,minimnm pasteurization time and.temperature. Extensive work on the thermal resistance of acidpforming bacteria and yeast was carried out by Etchells and.Jcnes (13). Contrglled pasteurisation of 71.1°O (160°!) for 20 minutes or 73.9°O (165°!) for 15 minutes was necessary in order to destroy the test organisms-4 Lagg- bacillus and two yeasts. The investigation involved the use of already pasteurized.sliced pickles containing liquors of these concentrations: (a) full strength‘brine from original Jars (1.5% acetic acid. 16.7o Baume). (b) l/2-strength brine (1.26% acetic acid. lhoBanms). and (c) IIN-strength brine (1.07% eoetie acid. invents). The per cent acidity and sugar stated above existed in lots (b) and (s) only after heating for 80 minutes and equilization.between diluted liquor and pickles had occurred. .8- J’ars of sliced pickles from each lot were inoculated with large populations of acid-forming bacteria and yeasts. Samples from each lot were pestourisod at 120. 130. 11m. 150. and 160°r allowing a holding time of 15 minutes after the center of the Jar reached the desired temperature. Center of the Jar temperature was determined by boring a hole in the center of the cap and inserting a thermometer. The thermometer was placed so as the tip was located in the slowest heating part of the Jar. Results indicated that there was a definite relationship between survival counts and acid content of the liquors at low temperatures. The size of the inoculum also played a part in thermal destruction. Pasteurisation studies of trosh whole pickles (8) indicates that the rate of heat penetration varies with the uniformity of the product and the nature of the pack. If the product is loosely pacloed. free circulation of brine by convection currents occurs while a tight pack impedes circulation. Within certain limits. the preportion of brine to pickles also affects the rate of heat penetration (8). in increase in brine to pickle ratios tends to increase rate of heating. ’ rtohons and Jones (13) using a pasteurizing temperature of 73% (165°r) for 15 minutes. pointed out that care should be taken to avoid overheating fresh pickles since this would result in loss of firmness and possible development of coolned flavors. In a recent study (8). it was found that hosting fresh whole pickles at 82°C (180°?) for to sin- utes had very little effect upon firmess. cooked flavors were not -9- detected even after pasteurising up to three times. Pickles tend to develop an off-flavor during storage if not preperly pasteurised. This appears to be related to the presence of peroxidase and related ensymes (2). therefore it is necessary to base the pasteurization of pickle products upon the heat tolerance of peroxi- dase. as well as bacterial tolerances. Nebesky 2.3 _e_1_. (13) reported that peroxidase from commercial packs of fresh whole kosher style dill pickles varied greatly in its thermal resistance. Thermal stability studies on this ensy'ne at a temperature of 88°C (190°?) indicated that ho to 15 minutes was required to destroy the ensyne of one lot and a second lot required 150 to 200 minutes. This difference was reportedly due to variation in peroxidase concentration within different lots of the fresh cucumbers. hrther studies (2) indicated that after one years' storage at room temperature. residual peroxidase could be denom- strated in quart Jars of fresh whole kosher style dill pickles which had been processed 35 minutes at 85°C} (185%). However. after processing 110 minutes. no evidence of peroxidase activity could be demonstrated. Anderson 9_t_ 5L. (2) studying the thermal resistance of microorganisms and peroxidase. found that two lactobacilli and a yeast were capable of causing spoilage of kosher dills. No further identification was mde. Only the yeast was enable of surviving one ninute at(130°3' in brine containing 0.5 per cent acetic acid and 5 per cent salt. hsidnal bacterial counts remained after pasteurising at 82% (130°!) for no minutes. Ihese organisms were largely peptonising spore-formers and -10- did not increase upon storage. These data were in agreement with the findings of Etchells and Jones (13). To rethice peroxidase activity completely. more severe heat treatment was found necessary than for microbiological control. However. it was demonstrated that sufficient heat to reduce peroxi- dase to a low level was adequate to prevent off-flavors (2). The' total heating on pasteurisation treatment was found to provide an '160' value of 36 ninutee. An '160 of 27 minutes was obtained by calculation from the data presented by Etchells and Jones (13) for fresh sliced cucurbers in 25-os. Jars (2). his value was based upon their controlled pasteurization of 71°C (160°?) center of the Jar temperature for 20 mimites. Influence g; M. Acid and Salt Concentration M Heat Resistance 91 Bacteria he effect of sugar concentration on microorganisms has been studied by several workers. When sugar is present in sufficient con- oentrations. protection from heating in most cases is sigificant. Pederson. Levine. and Buchanan (2h) determined that yeast were destroyed nore rapidly in distilled water than in syrup when heated at 100°C (212%). The thermal death tine was 6 minutes in crap at 2n°3aune and 28 minutes in 36°Baune syrup. :Brann _e_t 9;. (h) determined the effect of sugar concentration "160 u 'fotal lethality of process expressed in minutes at 160°l'. -11.. won the thermal resistance of bacterial spores associated with spoilage of canned foods. The investigation demonstrated that as sugar concentrations increased so did heat tolerance of Bacillus stearotheflilus. In some cases. heat tolerance was reduced at concentrations of 15 to 50 per cent as typified by the putrefactive anaerobe (PA-3679) and cioetridiun botulinun. Tarkow _e_t_ _e_._l_. (27) demonstrated that sucrose solutions above 30 per cent had a significant inhibitive effect on microorganisms. However. results indicated that tl-O to 50 per cent glucose solutions were nore ef- fective as inhibitors than the same concentrations of sucrose. The ap- parent difference in inhibitor effect is probably the result of differ- ent osonotic pressures. Glucose has an osonotic pressure 1.7 times that of sucrose. Tarkow g_t_ 9}... (27) found that the heat resistance of Baccharogce! cerevisiae was reduced in solutions of 30. 1&0 and 50 per cent sucrose. However. these data indicate that the spores of argillus p.351; appeared to be affected very little by heat treat- seats in either glucose or sucrose solutions. Etchells and Jones (13) reported that upon inoculation of acid- forming bacteria and yeasts into Jars of fresh sliced pickles (1.5% acetic acid. 16.7°Bauae' sugar). a great reduction in total pepula- tion occurred after 1 and 2 hours' incubation at room temperature. This effect was presumably due to acid content of the brine. The effect of acids in inhibiting the growth of microorganisms may be due to the hydrogen ion concentration or the toxicity of the .12.. undissociated molecule. The toxicity of mineral acids is related to hydrogen ion concentration. but organic acids affect toxicity by the undissociated molecule (3). Fabian and Vadsworth (1)4) indicated that acetic acid was a better preserving agent than lactic acid for pickle products and that pH was not a reliable indication of preserving ability. Levine and rollers (16) reported that acetic acid is more effective as an inhibiting agent than lactic acid for bacteria. yeasts. and mold species. Using a synthetic medium and adjusting pH with acetic acid. it was found that bacteria were inhibited at pH lL9. Saccharogces cereviseae at pH 3.9. and Ajpgrgillus gigs; at pH lI».l. The titratable acidities were 0.014 per cent. 0.59 per cent. and 0.27 per cent acetic respectively. It should be noted that the acidities mentioned relate only to .‘inhib’i'tion 1 of a few species in laboratory media and that im commercial application higher concentrations of acetic acid (1.5 to 2 per cent) are needed to preserve a product. Levine and Tellers (17) reported that the addition of 5 per cent salt and 20 per cent sugar did not affect the amount of acid required to prevent growth and that the toxicity of acetic acid was not due to pH alone. However. an increase in hydrogen ion concentration re- sulted in a decrease in thermal resistance. Erickson and rabien (7) observed that the preserving and germici- dal powers of acetic. citric. and lactic acids followed in the order listed when based upon pH. When determined upon per cent of acid present. the order was lactic. acetic. and citric. Yeasts were found -13- more tolerant to acid than bacteria. For yeasts. toxicity to acids was in the order of acetic. lactic. and citric regardless of pH or concentration of acids present. EXPERIMENTAL METHODS Isolation g_f__ M Organisms a number of 16-01:. Jars of fresh sweet sliced pickles were heated in an 180"!. water bath for various intervals of time (10. 20. 30. 11-0. 50. 60. 80. 120 minutes). All Jars were stored at room temperature for 21 days. Jars in which spoilage had occurred were characterised by turbidity of the brine or swells. Often. both turbidity and swells occurred simultaneously. as acid content of the brines was determined by titrating against a standard base. A Beckma liner-operated pH meter with a glass electrode was used to determine pH. Sugar concentrations of the brines were determined by direct readings from a hand refractometer. Salt concentrations were determined by titrating with a standard solu- tion of silver nitrate. Jars which showed turbidity or swells were Opened and samples of the brines plated using a standard plating technique (1). 1.8 agar (15) was used for the cultivation and enumeration of acid-forming bacteria. Dextrose agar plus 0.5 per cent yeast extract acidified with 1: ml of 5.0 per cent tartaric acid per 100 ml of medium was used for the enumeration of yeast. Twenty-one cultures of acid-forming bacteria were isolated from plates of the highest semis dilutions. cultures were maintained in stabs using trypticase sugar agar (BBL) plus 0.5 per cent yeast extract. 4)!- -15. Identification _o_f_ £2233! Orga_n__isms from the 21 organins isolated. three separate groups were estab— lished. These groups were based on morphological characteristics. gas production. optima temperature of growth. oxygen requirements. litms milk reactions. catalase production. nitrate reduction. and ability or inability to fermnt certain cugars. these three groups were desigated i. B. and 0. Gas production was determined by using a method described by Pederson and dlbury (23). is a safeguard. tubes of pasteurised brine were inoculated and sealed with 3 per cent agar to test further for produc— ticm of gas. Optimum temperature of growth and oxygen requirements were determined simultaneously using the same tubes. Tubes containing 9 ml of trypticase sugar agar were melted in flowing steam and allowed to cool. hese tubes were placed in a water bath at 110% and inoculated using 0.1 ml of a abhor: broth culture of each organism. After inoculation. the tubes were shaken and placed in incubators at 10. 20. 30. 35 and 15°C. Optimum tauperature of growth was determined by estimating the quantity of growth present in each set of tubes. Oxygen requirements were deter- mined by the level at which most rapid growth occurred in the tubes. litrate reduction (6) was determined by using indole-nitrate medium (331.). All carbohydrate fsrmentations were determined by using oystine tryptioase agar (BBL) as a basal aedium. l'inal identifications were according to their morphological and ~16- physiolcgical preperties described in Berggz's Manual of Determinative Bacteriolog (5) and by Pedersom (20. 21). Determination 91 Beat Resistance Pasteurisatiom is based upon the time and temperature necessary to destroy all organisms capable of causing spoilage. Therefore. it is necessary that time and temperature determinations be made upon the most heat resistant spoilage organisms. In order to separate the 21 cultures of acid-forming bacteria into groups based upon thermal toler- mice. a screening technique was used. for this purpose. 0.1 ml of a 211- hour broth culture of each organism was inoculated into sterile tubes of trypticase sugar broth. Rubber stoppers were inserted on top of the cotton plugs and the tubes placed in a water bath at various temperatures (130. no. 150. 160%). Individual tubes were removed at specific time inter- vals and cooled in ice water. he heated cultures were incubated for be hours. Relative thermal tolerance of an organism at any temperature was determined by noting the time at which negative tubes (no growth) smeared. The most heat tolerant culture from each group was chosen for further study. has multiple tube method described by rsty and Williams (9) was first employed for determination of thermal death times. Sealed tubes contain- ing a 1.0 m1 suspension of cells in phosphate buffer (pH 7.0) were placed in an oil bath at various temperatures (130. lit-0. 150. 160°”. rive tubes were removed from the bath for each of eight time intervals and .17.. placed in ice water to cool. Contents of the sealed tubes were trans- ferred to tubes of trypticase sugar broth and incubated for to hours. hr each heating period. positive and negative tubes were plotted on semi-log paper to obtain the thermal death time curve. The second method of determining the thermal resistance of the cultures was reported by speck (25) studying the heat resistance of Micrococcus freudenreichii in milk and ice cream mix. Tubes contain- ing 9 ml of phosphate buffer (pH 7.0) were sealed with a rubber dia- phragm stopper and placed in a water bath with the water extending 2 in. above the level of the liquid in the tube. Esmerature of the water bath was varied to include 135. no. and 1h5°r. use temperature of the water bath was regulated so that a variance of only plus or minus 0.5°l‘ occurred. Tubes were inoculated with 1.0 ml of a cell suspension in. phosphate buffer. Gells' suspensions were obtained by centrifuging 2’4- hour broth cultures of each organism and resuspending cells in phosphate buffer (pH 7.0). Inoculation was performed by using a 5 ml mpodermic syringe and a 17 681180: 3 in. needle. hie needle length allowed the inoculum to be placed in the tubes without splashing. After heating. all tubes were removed and placed in ice water until cool. A standard plating tech- nique was used to determine survivor counts using trypticase sugar agar (331.) as an enumeration medium. Survivor counts were plotted on semi-log paper resulting in straight line thermal death rate curves for each temperature . RE SULTS Spoilage Organisms from Pasteurized Fresh Sweet Pickles Twenty-one cultures of acid-forming bacteria were isolated from Jars of spoiled fresh sweet cucumber pickles. The acid-forming bacteria were taken from plates of the highest dilution. Table 1 gives the pH, acid, sugar, and salt concentrations obtained from the spoiled and un- spoiled pickle brines. It should be noted that the pH values were lower and the per cent acid correspondingly higher in the spoiled brines than in the unspoiled brines. Differences in sugar, salt, and acid concen- trations among the jars of unspoiled pickles were probably due to differ- ences in tightness of pack. Table 2 gives counts obtained from the spoiled and unapoiled brines using a standard plating technique. Acid-forming bacteria were isolated from the Spoiled jars which had been processed for 20 minutes. Therefore, only the most heat tolerant organisms were obtained. NOD! acid-forming bacteria were not isolated for further study since the re- sults indicated that they were incapable of growing in this product. All cultures of acid-forming bacteria isolated were found to be gram positive, non-spore forming, non-motile, rods, which did not produce catalase, were microaerophilic with respect to oxygen, and had an optimum temperature of 30° to 35°C (Table 5). This placed them all as members of the genus Lactobacillus. mm“ ps. icIn. BUGLE AND sen conmmmrs or muss mom me Home Jar Process Fastic so. time" :93 noid $ Sucrose i you 10" 10 3.35 1.66 18.0 1.11 15" 10 3.10 1.58 17.5 1.07 20"- 20 3.».2 1.52 15.0 .92 21” 20 3J2 1.117 16.0 .99 35 30 3.85 .81 15.0 .89 mi 1&0 3.73 .97 17.5 1.10 51 50 3.70 1.07 18.5 1.22 62 60 3.7!: .91 16.5 1.06 85 80 3.57 1.23 21.5 1.h0 125 120 3.75 .9h 111.0 1.07 '- uinutes held in 1so°r water bath " Jars in which spoilage had occurred -20- TABLE 2 POPULATIONS OF BACTERIA AND YEASTS OBSERVED IN EXPERIMENTAL JARS OF SWEET SLICED PICKLBS Acid-forming Non-acidefcrm- Jar Process bacteria ing bacteria Yeasts No . times No ./m1 No ./ml No ./ml 10... 10 1.6x107 1.0x109 0 less 10 2.2x107 8.4x107 0 20st 20 5.0x109 1.01107 0 21st 20 3.1x109 1.0x107 0 35 so 4- 1o 410 0 44 40 4 10 L 10 o 51 50 A 10 ‘- 10 0 62 6O 4 10 4 10 o 85 80 4 10 4 10 o 125 120 a 10 L 10 0 e Minutes held in ISOPF water bath at Jars in which Spoilage had occurred m3 MORPHOLOGICAL CHARACTERISTICS 01' ORGANI $48 STUD!!!) Ween GROUP No. of Cell Temperature Require- Cultures Morpholgg; relation mentp__ A 3 Rods: Occurring singly Optimm temp- Micro- and in short chains. erature 30°C. acre-- with rounded ends. Minimum philic Show elongation as 10-15°C. c acid concentration in- Maximum 1&0 C. creases. lon-Motile. Gram positive. EB 16 Beds: Variable. usual— Optimum temp- Micro- 1y short. in pairs and erature 35°C. aero- short chains. Non- Minimum philic motile. Gram positive. 20°C. Karim 15°C. 0 2 Rods: Occurring sing- Optimum temp- Micro- 1y. short cinins and erature 30°C. aero- occasionally in long Minimum 18°C. philic filaments with round- Maximum 110%. ed ends. Hon-Motile. Gram positive. -22.. The Group A. cultures were found to be homofermentative and identified as Inactobacillujg plantarum. The 16 cultures in Group 3 and the 2 in Group 0 all produced gas. However. they could be separ- ated on the basis of carbolndrate fermentations and reduction of ni- trates (Table n). Thus in Group 3 were similar to Iactobacillus fermenti in all respects except for nitrate reduction. However. this may not necessarily exclude them from the species since it has been recently demonstrated that some lactobacilli will reduce nitrates when tested in indole-nitrate medium. Therefore. these cultures were tenta- tively identified as such. Group C cultures were identified as Lactobacillus 112911-30 It was of interest to note that all strains of L. 1133}; failed to produce gas in glucose broth. Determination g_f_ merlal i'olerance In the initial phase of this study. a screening technique was used to detemine the heat tolerance of the acid-forming bacteria isolated. his method provided a basis for further heat studies and indicated which organisms would survive higher temperatures at specific time inter- vals. Table 5 gives the results obtained from the initial screening. One strain (15-2) of Lagtobacillus fermenti was seemingly more heat tolerant than any of the other strains studied. However. the remaining strains of the same species did not show the same characteristic. Little difference was noted between strains of Inactobacillus brevis. Lactobacillus -23.. mt PHYSIOLOGICAL CHARAG'ERISTICS 01' OM13” STUDIED Properties r MEL—‘1: Fermentation reactions: J Arabinose - .. ... Dextrose + + 4. Fructose ‘ + + ... Galactose + + + Lactose + + - Maltese + + + Raffinose + + - heroes 4- ... .. 171080 - 3;" + 9;; production: Cucumber brine - + + Glucose broth - + - M 9.1.1.! Acid coagulation No change No change gitrate reduction. - _ , + - Catslase 3gp; - - '- *HaJority of strains fermented alose. but a few did not. 'ioww :mtt mum 10: run was some Owns so °srncq;gn perequou; pus sunsodre scureredmeg-euxc qcee oz peaoecqus area seqna 3 . “neg-u; '1 133 xfififiififififififfififii NNNNNNNNNNNNNNN NNNNNHNNNNNNNNN HNNNNHNPNNNPNHN OOOOOOOOOOOOOOH OOOOOOOOOOOOOOO NNNNNNNNNNNNNNN HNNNNNNNNNNNNHN HOHNNNNPNHNHNNN OOOOOOOOOOOOOON OOOOOOOOOOOOOOO PNNNNNNNNNNNNHN ONOOONNNNNNNNPN OOOOOOOOOOHOOON OOOOOOOOOOOOOOO OOOOOOOOOOOOOOO OOOOOOOOOOOOOON OOOOOOOOOOOOOOH OOOOOOOOOOOOOOH 000000000000000 000000000000000 wa- srmguq '1 MN NH MN 00 CO NM MN 00 00 00 OH 00 OO 00 OO unprequefd 1'18 11-91 3‘01 eat-OI NNHN NNNN ONNN 0000 GOOD NNNN NNNN 0000 0000 0000 NNNN 0000 0°00 0000 0000 000° 0000 0000 0000 0000 __-:__ 3'! sepceds Con “MM I W seruurn I '[OI I 0 sequntn I £0011 .squemqeezg seen suntan; zeayv aapztsog seqpm JD on I or 004mm: 0 "mum .1 o t 01 931101! 0331106 mm 71mm! 8 IO EDWIN? mm 301 smsm sumac ‘0 8mm 9mm -25. plantarun. and Inactobacillus fermenti with exception of strain (15-2). Due to the small number of tubes used. “skipping.“ and ap- parent similarity of all strains to heat tolerance. this method could not be considered quantitative. In order to make a comparative study of these organisms. one culture of each species was selected for study. Selection was based upon ability of the organism to withstand higher temperatures. 'i'herefore. strain 15-2 of _I_a. fermenti and 15-3 of _I-_. 232.11.! were selected due to their slight tolerance over other strains. No differences were observed between strains of a. plantarum. but one strain was selected for comparative purposes. The multiple tube method described by Esty and William (9) was abandoned in the initial phase of this study. Considerable error oc- curred due to 'skipping' and contamination from air-borne spore-forming bacteria. The time and equipment necessary were considered impractical for further work. _ he heating of the cell suspensions was conducted according to the method of Speck (5) and survivor counts determined at various tines. ‘i'hese counts were plotted on semi-log paper to obtain thermal death rate curves. The thermal death rate constant (1:) was calculated for each of these curves as follows: $2171 I: a thermal death rate cmstant t1 .- time at first reading t2 = time at second reading -26- H1 :- population at t1 N2 .- population at t2 Ihe lower the 1: values for any specific temperature. the slower the thermal destruction of cells. thermal death time curves were constructed by determining a point on the thermal death rate curves for each temperature and menstrum at which 99.999 per cent destruction: of cells had occurred. Ehese points were plotted on semi-log paper (log time vs. temperature) to give a straight line themal death time curve. the time required at 160°: to destroy 99.999 per cent of the cells present in a suspension was designated as the 1160 value. he 1160 value was determined by extending the thermal death time curve until it crossed 160°! on the temperature scale and reading the number of minutes required to destroy 99.999 per cent of the cells at this point. “a“ values were expressed as the maber of degrees l'ahrenheit on the temperature scale over which the line passes in traversing one logarithmic cycle on the time scale. '2' values indicate the slope of the thermal death time curve. he results of the studies on the three species of lactic acid bacteria in phosphate buffer (pH 7.0) are given in fable 6 and figure 1. L. fermsnti was decidedly the nest heat tolerant of the organisms studied. Results indicate that k values for the three species were similar at 135%. but at inc. 1u5°r ;. fermenti had meh snaller values than the other two. Ehis resulted in a high s value for this species and an 1160 LOG TIME - MINUTES -27.. o L. fermenti x L. brevis e L. plontarum l 1 I330 I40 I50 IGO T EMPERATURE-°F Figure]. Thermal death times curves of L. fermenti, £3 brevis and £’ plantar-um in phosphate 'c'qu‘er pH 7.0. ~28- of 2.5 minutes as compared to 1.19 for _I_-. plantarum and 1.08 for 3. brevis. Therefore. 1:. fermenti was selected for all further studies. warms m k. 1160' AND 8 VAIIJBB J'OR rm SPECIES OF LAD'IOBAOIILI M II PHOSPHATE BOMB (pH 7.0) V 1: values cramm- We: #11er '1 F533- r160- . Le 5371! cam ' .‘I-31l-ajo656 1:19 mes L. fermenti .260 A00; ‘1; .m 2.5' f: 27.5 L. 212tm 03m 0571;;l41‘)1008 1.&A~I ago * Based upon 99.999 per cent destruction The next factor to be investigated was the influence of sucrose concentrations on the heat tolerance of the lactic acid bacteria. The first step was to check the effect of various sucrose concentrations on cell suspensions without heat. Cells were suspended in 15. 25. 30. ’40. and 50 per cent sucrose solutions. held at room temperature 1 hour. and survivor counts determined. Ihe following per cent reduction in cell populations were observed: 15$ sucrose. 1$ reduction: 25% sucrose. 2% reduction: 30$ sucrose. 80% reduction: M sucrose. 95% reduction: and 50$ sucrose. 99$ reduction. therefore. only the 15 and 25 per cent sucrose concentrations were used for further studies. figure 2 shows the effect of sucrose concentrations on the thermal -29.. O PHOSPHATE BUFFER o :5 °/. 'sucaose 2 - x 25 °/. SUCROSE LOG TIME-MINUTES EXTRAPOLATED T0 FISO/ i I _I I30 I40 I50 I60 TEMPERATURE - F ° Figurea Effect of 15 and 25 per cent sucrose concentrations on thermal death times of a. fermsnti (pH 7.0) -30- death time curves of E. fermenti. The resistance of the organism to heat was increased with increasing sugar levels. However, the slope of the curve (a value) was not significantly affected. The F160 values increased from 2.5 in phosphate buffer to 4.9 in 15 per cent sucrose and 6.6 in 25 per cent sucrose (Table 7). TABLE 7 THE k, F 0: AND z VALUES FOR L. FERmENTI HEATED IN 13 AND 26 PER CENT sucfibsE‘sdtUTTONs Per Cent 0 k values Sucrose 135 F 140°F 145°F 150°F F160. z 15 .198 .235 .740 -- 4.9 33 .0 26 -- .266 .53 .684 6.6 50.5 e Based upon 99.999 per cent destructibn Table 8 and Figure 3 give the effect of the addition of acetic acid on the thermal death times of E. fermenti in 25 per cent sucrose concentrations. The results demonstrate that the presence of acetic acid greatly reduces the thermal tolerance of this organism. However, there were no significant differences demonstrated between the 0.5 and 1.0 per cent levels of acid at the temperatures tested. This apparent similarity of results at the two levels of acid may have been the result of allowing a time lag before heating the samples containing 0.5 per cent acid; therefore, permitting the combined action of acid and sugar to produce a detrimental effect upon the organism. .31.. e 25 °/e SUCROSE 3 __ 0 25 % SUCROSE + 0.5 °/o ACETIC ACID ' 3 x 25 “lo SUCROSE + |°/e ACETIC ACID .— . :3 E 2 ' 2 I m .2. I- I” <9 0 .J o_ -l 1 I J I30 I40 l50 l60 TEMPERATURE - F° Iigure3 Effect of the addition of acetic acid on the thermal death times of la. fermenti in 25 per cent sucrose -32- was our. 1160mm gums rang. mm mm 25 m cm moss somror rms 0.5 mm 1.0 m mm mm mm Per cent 1! Values ‘ acetic ggid 13521:: jiEr 1E5“r"' 1'160 s 0.5 .707 1.50 2.00 .55 25.0 1.0 .638 1.33 2.50 .365 16.5 * Based upon 99.999 per cent destruction The effect of sodium chloride on the therml death time curve was determined using a combination of 3.0 per cent laCl. 1.0 per cent acetic acid. and 25 per cent sucrose. he some cell suspensions were used to obtain the curves represented imrigdre h. The addition of sodiua chloride to the heating menstrua had little effect upon thermal tolerance. In all practical purposes. both curves were identical. Survivor counts in all aenstra may be found in the Appendix. LOG TIME - MINUTES e 25% SUCROSE+ I°/e ACETIC ACID 0 25 % SUCROSE+ I°/oACET IC ACID +3°lo NoCI 2 - I .— 0 .. -I 1 1 I I I30 I40 I50 I60 TEMPERATURE- F° Iigureh Effect of the addition of 3 per cent sodium chloride on the thermal death time in 25 per cent sucrose DI 8013881“ he isolation ad identification of three species of lacto- bacilli causing moilage in fresh sweet pickles was accomplished in this study. he organisms were placed in the genus Etobacillus according to their morphological and physiological characteristics as described by Pederson (20. 21) and Dergez's M_a_n_1_l_el__ of Determinative bacterioiog (5). is indicated by the data. a slight variation from the standard description of the genus was encountered. Sixteen strains of Lactobacillus fermenti were found capable of reducing nitrates to nitrites when grown on indolecnitrate medium (331.). Costilow and Humphreys (6) demonstrated that certain strains of Lactobacillus plantarus reduced nitrate when groin on this medium. herefore. not all members of the genus I-aotobacillus are nitrate negative. ltchells and Goresline (11) and ltchells and Queer (12) have desen- strated that two Iain groups of organisms are responsible for the spoil- age of pasteurised pickle products: 1.1.2. . acid-toning bacteria ... yeasts. Anderson 33 g. (2) also isolated acid-forming bacteria and yeasts from Jars of spoiled Kosher style pickles. lo identification of these organisms was offered. In part. the data acquired in this investi- gation agreed with the above mthors. However. the results of the present work indicated that the lactic acid bacteria would survive higher pasteuri- sation levels than the yeasts. Anderson gt g. (2) indicated that the thermal resistance of the lactobacilli isolated from spoiled Kosher style pickles was. less than .3);— -35- the test organism—a yeast. Beat tolerance was determined in brine containing 0.5 per cent acetic acid and 5.5 per cent salt: pH of the brine was 2.9. hermal death times indicated that the lactobacilli would not tolerate l ninnte at 130% in this nenstmnn. he yeast had an 1160 value of only 0.5 in such a brine. his is about the same re- sistance noted here for E. fermenti in 0.5 per cent acetic acid and 25 per cent sucrose. 33. fermenti was found to be the most heat tolerant of the three species of lactobacilli tested. he '16 of this species was about two 0 times that of the other two organisms and its s value was Inch greater. Therefore. one light expect it to be acre frequently encountered in the spoilage of such products than I:- bgoli; or 3.5.- plautaruu. but this is not evident in the literature. Various conbinations of sugar. acid, and salt were used to determine their effects upon the thermal death time of L. fenenti. As indicated by a number of workers (2,1h.16.l1). acetic acid produces a marked ef- fect upon the heat tolerance and growth of sicroorganisls. It was sono- what surprising that as the per cent acid was increased fron 0.5 to 1.0 per cent. the thermal tolerance of L. m did not decrease accord- ingly at low temperatures. However. this was demonstrated in the presence of 25 per cent sources which night affect the results. As the tempera- ture was increased to lh5°r, the effect of the increased acid level be- came apparent. E Several concentrations of sucrose were used to detemine their effect -36.. upon the growth and thermal death times of 3'.- fermenti. Two concentra- tions (15 and 25 per cent) were found to afford protection during heat- ing without inhibiting the organism to any great extent. , However. _ concentrations of 30. 110. and 50 per cent sucrose produced an inhibitory effect. These data were in agreementwith Tarbow _e_t_ 2;. (27) who re- ported that high concentrations of sucrose inhibited microorganisms. he inhibitory action is believed to be One to osmotic pressure and af- finity of sucrose for water. Braun gt 9;. (1L) demonstrated a protective effect of high concentrations of sucrose upon food spoilage organisms during processing. but this was with bacterial spores which are extremely tolerant to osmotic influences. he addition of 3 per cent sodium chloride to a 25 per cent sucrose solution containing 1.0 per cent acetic acid did not greatly affect the thenal death time curves of _I_.. fermenti. Levine gt 9;. (l7) and Anderson 93 _a_l_. (2) determined the effect of salt concentrations in solu- tions containing acetic acid and sucrose upon microorganisms. hose data indicated that salt concentrations of 5 and 15 per cent produced little or no effect @on the organisms studied. Lower concentrations of salt were not evaluated. he data presented in this study indicates that the controlled pasteurization procedures recomended by Etchells (10). Etchells and Jones (13). or lssolen gt; 5;. (8) would be sufficient to eliminate any spoilage which might result due to the three species of lactic acid bacteria isolated and tested. However. variations in the nature of the .37.. pack mu produce conditions in which organisms might survive. In many experimental packs. it has been demonstrated that organisms may be pro- tested this to pickles pressing tightly together or against the sides of the Jar. In such cases. heat penetration is retarded and. the lethal effect of the acid is lost. Also. there is always the possibility that the cucumber tissue may exert some protective effect aiding the bacteria. his is certainly a possibility which deserves investigation. WAR! Twenty-one cultures of acid-forming bacteria were isolated from spoiled experimental packs of fresh sweet pickles. These spoiled Jars of pickles had been pasteurised in a 180°? water bath for 20 minutes. (h identification. 16 cultures were found to be Lactobacillus femonti: 3. Lactobacillus plantarum: and 2. Laotobacil- inn arena. ill cultures isolated were screened to determine their approximate heat resistance: and the one strain of each species found to be most resistant selected for further studies. herml death time studies in- phosphate buffer at pH 7.0 showed that ;. fermenti "160 e 2.5) was the most heat resistant of the three species. .Iio m (1160 a 1.19) and .1;- plantarum (1160 s- 1.08) were not greatly different in heat tolerance. Therefore. _Ig. fermenti was used for all further studies. Sucrose solutions of 30 to 50 per cent were found to have a lethal effect upon the organism. even at room temperature. However. solutions of 15 and 25 per cent had no siglificant effect at room temperature and exerted a great protective action at pasteurising temperatures. he 1160 values for _In_. fermenti in 15 and 25 per cent sucrose solutions were k9 and 6.6 respectively. he addition of acetic acid to the substrate in which the cells were heated reduced the thermal death tin of the organism greatly. Vith 0.5 per cent acid and 25 per cent sucrose. the 1' was only 0.55; 160 and with 1.0 per cent acid in this solution. the 3160 was further re- .33.. .39.. dnced to 0.365. he inclusion of 3 per cent salt (NaCl) in the solution contain- ing 25 per cent sucrose and 1.0 per cent acid had no measurable effect on the thermal death time of .13. fermenti. Iron these data. it is evident that any of the processes recom- mended in the literature today (s. 10. 13) should be sufficient to pro- tect sweet pickles from microbiological spoilage. In fact. they should provide adequate safety factors. However. since such spoilage still occurs in these products. further studies of the effects of other factors on the heat tolerance of spoilage organisms are indicated. 1. 9. 10. ll. 440— LIIEBATUHE CITED American Public Health Association. Standard Methods for The Examination 9; Daigz Products. 9th ed.. 19348. New York. H. I. Anderson. I. 3.. Elder. In. 1.. Hsselen. V. B. Jr.. Hebesb. E. A" and Lahbee. H. Pasteurised fresh whole pickles. II. Thermal resistance of microorganisms and peroxidase. Food. Technol.. 5,. 361; (1951) . Baumgartner. J. S. Canned Hoods. 3rd ed. J. do A. Churchill Ltd" London. England (1959). Braun. 0. G.. Hays. G. I... and Benjamin. H. i. he effect of sugar concentrations upon the thermal resistance of spore-formers asso- ciated in spoilage of canned foods. l'ood _I__n_(1. 3,2. h? (1941). Breed. H. S.. Murray. H. 0.. and Etchens. A. Parker. Berggz's Manual _q_f_ Determinative Bacteriolog. 6th ed. williams and “MI 00.. Baltimore. Md. 19 e Costilow. H. H.. and Humphreys. E. V. Nitrate reduction by certain strains of Inactobacillus plantarum. Science. 2.3. 168 (1955). Erickson. 1'. J.. and l‘ab'ian. r. V. The preserving powers of acetic and lactic acid. l‘ood Research. 1. 69 (1912). EIuIOn’ W. 3.. Jr” “damn. 1e lee masts Ila re, and. Pfl‘ug. Is Jo Pasteurised fresh whole pickles. I. Pasteurisation studies. l'ood Technol.. 5. 279 (1951). Esty. J. H.. and Williams. 0. 0. Heat studies I. A new method for the determination of heat resistance of bacterial spores. ,1. Infect. ai__s_.. 516 (1921;). Htchells. J. I. Hate of heat penetration during pasteurization of cucumber pickles. l'ruit Products 1.. id. 63 (1938). Htchells. J. I... and Goresline. H. 1!. Methods of examination of fresh cucumber pickle. l‘ruit Products _J.. 19. 331 (19%). 12. Htchells. J. I... and Ohmar. H. B. A bacteriological study of the manufacture of fresh cucumber pickle. Il'ruit Products 1.. _29_. 13. Etchells. J. I... and Jones. I. D. liortality of microorganisms during pasteurization of cucumber pickle. Food Research. g. 33 (19143). 11$. 15. 16. 17. 18. 19. 20. 21. 22. 23 . 25. 26. 4&1- l'abian. 1. '.. and Wadsworth. O. K. Experimental work on lactic acid in preserving pickles and pickle products. II. Preserving value of acetic acid and lactic acids in the presence of sucrose. rood Research. a. 511 (1939). Fabian. 1'. 1L. Palde. R. 6.. and Mei'i'ick. J. E. A new Y-S medium for determining lactobacilli. Food Research. _J_._§. 280 (1952). Levine. A. 8.. and Tellers. G. R. Action of acetic acid on food spoilage micro-organisms. g. Bact.. 19. 1199 (19110). Levine. A. 8.. and rollers. G. R. Inhibiting effect of acetic @on micro-organisms in the presence of sodium chloride and sucrose. 11,. Mn 29. 255 (1990). HOBO”. 3e Ass Elulene V. Be :1... “13183.. As no. and 1911.1... 6. R. Thermal destruction and stability of peroxidase in acid foods. I‘ood Research. Ii. 119 (1950). Orla-Jenson. 8. he main lines of natural bacterial systems. :1. 29.05." Q. 263 (1921). Pederson. G. 8. A study of the genus Lactobacillus gantarum. 3.1-. Bacte! fl”- (1936). Pederson. O. 8. The gas roducing species of the genus Lactobacg- 12!.- 1. Bact.. 12119383 Pederson. O. S. The fermentation of glucose by certain gram posi- tivfi non-spore-forming anaerobic bacteria. _J_. Beet" fl. 1L75 19 5 . Pederson. O. S. and Albury. H. II. Effect of temperature upon the bacteriological and chemical changes in fermenting cucumbers. Mlfimxao ER. 532. 2229.- EA- .2555. (19119)- Peterson. 3. 3.. Levine. 14.. and Buchanan. J. E. The effect of sucrose upon yeasts. Iowa State Coll. Jour. 9_f_ §_c_i_.. 2 (1927). Speck. H. L. The resistance of Micrococcus freudenreichii. in laboratory high temperature short-time pasteurization of milk and ice cream mix. ,1. Dairy _S_c_i_.. &. 975 (1937). renner. r. v. nicrchicicg _o_f_ Foods. 2110. ed.. 191m. Harvard Press. .142- 27. Iarkow. L.. rollers. G. 3.. and Levine. A. 8. Relative inhibition of micro-organisms by glucose and sucrose sirups. 9:. Bact.._‘$_1_h 367 (1992). 28. Iittsler. 3. R.. Pederson. O. 8.. Snell. E. E... Hendlin. 1).. and Niven. G. 3'. Symposium on the lactic acid bacteria. Bact. Reviews. 1_§, (1952). APPEHDIX 4.3- .I" as! 8." .l'slss ...—03 e F was e ” mfifi 8n rename ...... ...... u mean; can mum“ gage madam; 1...... ...... .. page m3 ..th ...m ...... madam; nonhuman ...... ... ~03; 9: node; mafia; mods; ...... .. have.” oi a mode.” moan? magnum ...... .. node...“ .92 «museum. ..W. ...m 1!... 1!... madam...” 303.." .. moaned .9: 8 mega i1354 mode; .. moire 9: a H6 node.» 1.35.,“ mound. moan; .. madame an «83%.. .m on rim. h m o a 1% sensual-.5» minnow .aaua asaqsuao moss» $13»an 25.73» nacho Mandrake H.453 Hag 8;. may Em Bahama a amass 3E BB8 SE ~hh- APPENDIX II SURVIVOR COUNTS 03‘ In. MENTI Am HEATING In smnosn mun“ ons (pH 7.0) g 3.3.11.5 Tamrature of matgi(°r) 15 0 5.02106 7.02106 1.0:107 ...- 5 9.01:105 5.0::105 9.0::10“ ...-- 8 2.0:105 5.0110” 1..?.~.r.ioh ...- 1o 8.0::10" 15:10” 3.0::103 ...-- 20 1.0::103 1100 60 .....- 25 o ...— s.o:105 6.02105 14.2m106 1 ......— --—-- ---- 1.0!:106 5 .... .-- n.01155 3.02163 10 ...- 2.02105 2.0210“ ...- 20 ...—— 6.0:103 60 ~— APPENDIX III SURVIVOR ems AMER HEATING IN 25 PER GENT SUOBOSE SOLUTIONS WITH 0.5 PER GENT ACETIC AGID Heating Tegperature of heating Pr) 1‘1“ his! 1 5 Inc ins gig. No m1 No ml 11 . m1 0 v 5.0mm: 1.0mm; 3.0.:105 3 5.0::10 mono 1.01107 1 A 1.01.108 6.0::106 1.0arioh 3 1.0110 -.- 1.0ano5 h 2 A --- 2.o:1 1.011 B ...-'- 7‘M13.t 3eM§ 3 A 15:10” 2.01103 20 3 --- 3.0::103 so 2 5 A 7.orio 6 ...... B 5. o3 20 ...... s a ...... ...... ...... B 100 ...... ...... 10 A ...-.. .....- ..-- B 60 ...... ...... v.— _— "' Data obtained from repeating experiment ~146- APPEFDIX IV SURVIVOR COUNTS arm HEATING IN 25 PER GENT SUCROSE SOLUTIONS WITH 1.0 PER GENT ACETIC ACID Temperature of heating (OP) Heating time; rriai g? 1143 T51 " 31g. lo. ml loglml No. ml 0 A" 1.0210; 6.0:102 11.51105 13 8.0x10 6.0:10 5.0x105 1 A 1.02105 1.01105 amino“ 3 ....- -.-- 2.01105 2 A 1.02105 n.0x105 20 n a ....- s.0xio5 7.0110 3 A 1.5x10h 1.01102 --- 5 A 20 ...... ...... B 1.0::106 5.0mm” 50 8 A “- # ..- n 2.0::105 2.52103 ---- 10 A .... ...... .... 13 1.01:105 1150 20 A .....- ...- -..... 3 1.02103 -—- ...- " Data obtained from repeating experiment 4+7- APPENDIXV SURVIVOR COUNTS am EATING IE 25 PER CENT SUCROSE SOIUTION PLUS 1 PER GEM ACETIC ACID AND 3 PER GENT Nacl Temperature of heating (°l') Heating 33: 1.35.1 a3; xiii-1 0 8.01106 6.02105 3.01105 1 ...... ...... 2.01105 2 ---—- 8.0::105 7.0m ’5‘ 3 -- 3.0r105 n.0rio3 5 1.0::106 5.0:101‘ 50 s 2.0.:10'5 2.51103 --- 10 1.0x105 1.50 .... 20 1.03103 ...... ...... Thesis Robbins, Edward K. The identity and heat tolerance of microorganisms __ __ 9933136 Spoilaae of Thesis Robbins, Edward K. The identity and heat tolerance of microorgan- isms causing spoilage of pasteurized sweet pickles *211(333 "111111111 111 11111 11:11} 11111111“ 3129310401