M I l \ “IMWHM ’—— '4 ___'——— .____'—— ’4. ____——- '_— ___——— —.___—- ?’ ____——— _—__——— .___—_— ———— f.— —__— '_—— —_—— .___—_— ___—— ____—— ___4 '— ___—’ .— -|_._s Ima CD\l—¥ LEUCQNQSTGC MESENTERWDES AS A CASE ('2? RGP'ENESS IN Czi‘stfifl Ffizkfiié'éfi ‘I’ixesis far i'i'm liken-am 3?“ (vi. 3. MiCHEGM S-TATfi COLE-13? fwbefl Eéowaii Hanéei‘sm W542 This is to certify that the thesis entitled mm W as a Can” of Boplneas in Canned Peaches. presented bl] Robert Howell Henderson has been accepted towards fulfillment ._———..—.v- ——-—— -—-—q—-. I- u- of the requirements for E Master mega mlannnmogy a: Public Health; h - Major professor DateWs—_ 0-169 ) g»..- .. '1 ovum; M :2, _.~ .- .- W walls 3“ 4 T?“- gagflfinu w: p s - u 7 .| ‘ . 61“] .’” {g L‘Iflfl’e‘ #5 4 WW ' . . ’ - ' '90 6') ’~ , ms. 935 . I l . 4 , fl ._ . «49$» ,. - l»:- £33 ' .' LJ’. .' Y . v .. x .1 if) ‘1 "I: I l 'I I. ,. l I“ t‘. :w Lil“ , "'1 .“f f”. l‘f I. “I I '3 ‘w T , hi" I‘ 1“}; \r ‘I' A030, "A. ' (IV. ‘ l H. J r- f" " 'I a’ ‘7 ' ' v _. r“ «M .. viii: .-". ‘ .21! II." 7 W‘ l. ['1]. leg??? Kg? .15. . " a; If . .'I .. \ ' " ’ -s: fix" x, I”. ‘ _.-3'~ I 1“ L :1 \s‘r’ g” c 4“. .rl {v.5}. ”Ii ‘, 1" 23k”: I ,1."!‘[\1‘, 'I ' ‘0”) '~‘ in! 43;." ‘ I ‘v .I'.‘ 'I " (’l.'Tv .L.’ ‘l’h :1 "‘I'" ‘5 '4“. 11 I 2" us: {x “1:34.“ . . t_ ‘- TX . I. ”A KI/‘iy‘li'd of? ' 1* \‘;":Y?’_.~“. 5‘" . " ’1‘; 7.. 7&3". . "Mafifi " I "I ' ‘1‘ “1’ T‘K 31,}. F x .. “1.4g 1 . . ' In: ‘ LEUCONOSTOC MESENTEROIDES AS A CAUSE OF ROPINESS IN CANNED PEACHES ROBERT HOWELL EENDERSON A THESIS Submitted to the School of Graduate studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Bacteriology and Public Health 1950 HES! .T u] L'\ ACKNOWLEDGEMENT The author wishes to make known his appreciation of the assistance and guidance of Doctor F.W. Fabian. Research Professor in Bacteriology and Public Health. during the course of investigation and preparation of this manuscript. .44 6"“? "f" TABLE OF CONTENTS INTRODUCTION . REVIEW OF THE LITERATURE PRESENT WORK . . SUMMARY . . . . LITERATURE CITED 0 10 13 16 Introduction ROpy fermentation is often encountered in the different food industries. It occurs most often in the dairy and sugar industries. The cause of ropiness. as determined by investigators, has been bacterial in origin. In the fall of l949, the Michigan Agricultural Laboratory. Lansing, Michigan. submitted samples of canned peaches in which the syrup had become ropy. Bacteriological analysis was made of the samples and representative colonies were picked from the plates which were purified and identified according to the methods outlined in “The Manual of Methods for Pure Culture Study of Bacteria“. Literature Review Ehrenberg (1840) was probably the first to observe specific organisms in rcpy milk. Since that time over forty different microorganisms have been shown to cause ropiness in milk and milk products. Buchanan and Hammer (1915) in a scholarly and comprehensive paper presented a key to thirty-three organisms implicated in ropy milk outbreaks. Hammer and Cordes (1920) reported Micrgcoccus cremoriggviscosi as the causative organism in a rcpy milk outbreak. Stark and Foter (1931) grouped repy milk organisms into the following groups: (1) A gram negative rod, which produced gas. and curdled milk. (2) A gram negative rod. which produced gas. but did not curdle milk and produced ropiness only in the presence of a fermentable sugar. (3) Gram positive cocci, which fermented none of the sugars used. caused the milk to have an alkaline reaction. and at 37°C gave rise to organisms which appeared to be rods. (A) Gram positive cocci which digested milk at room temperature. and at A5‘DC produced an acid curd which reduced litmus milk. (5) A gram positive staphylococcus which fermented glucose. sucrose. and lactose, with no gas formation, and produced ropiness only when a fermentable sugar was present. (6) A gram positive staphylococcus which fermented only glucose. (7) A gram positive coccus. with no other noted characteristics. Sarles and Hammer (1933) found that rcpiness in dairy products may also be caused by; Escherichia neapolitang, Aerobacter aerggenes, and Aerobacteg cloacae. ' Davis (1935) reported that Pseudomonas and Qgrnyebacteria may cause ropiness in milk. Three strains of Alcaligenes viscosug produced pronounced ropiness in milk at 18°C. 25°C, and 30°C, but failed to produce ropiness in milk which became acid due to action of lactic acid bacteria present; according to Beck and Chase (1938). Rodenkirchen (1939) described a transitional form between a micrococcus and a sarcina (apparently Sarcina auratica) which caused ropiness in cream. Ropiness is often associated with the production of acid. Streptococcus lactis, Lactobagillus gaggi, and Lactobacillus bulgaricus. show ropiness which is evident before much acid is produced. Microcoocus or Achromobacter were isolated from rcpy chocolate milk by Tarnutzer (1939). These organisms would not produce a rcpy condition in regular milk. Apparently they required carbohydrates other than lactose. An outbreak of ropiness in commercial pasteurized milk was studied by Prouty <19u3) and a thermcduric micrococcus closely related to Eigrococcus fgeudenreichii was isolated. This organism survived a pasteurization temperature of 143°F for 20 to 35 minutes. In Great Britain a similar organism was isolated from new hay by M'Kenzie. Morgan. and Parker (1943). Ropiness in sauerkraut according to Pederson (1931) is ordinarily caused by certain strains of Lactobacillus cucumeris or Lactobagillus plantggum. These organisms grew rapidly and became enveloped in a slimy material which caused the organisms to adhere to each other. The kraut may become normal after further curing. Ropy cider was studied by Kayser (1911) who was able to isolate four different organisms. However he felt that the rcpiness might be due to factors other than the presence of bacteria. Edson (1910) isolated a new species Bacillus aceris as a causative organism of ropy sap. Later Edson. Jones, and Carpenter (1912) reported strains of Pgeudomonaglflgurescens which caused ropiness in maple sap. They also found a new species named Bacillus papillelus causing ropiness in sap. Fabian and Buskirk (1935) isolated A- aerogenes as a causative organism of ropiness in maple syrup. Fabian and Nienhuis (1934) working with ropy brine in cucumber fermentation isolated an organism which closely coincided with the characteristics of the genus Achromobacter. Another group of organisms isolated from ropy brine were thought to be involution forms of Leuconostoc. A case of ropiness in tea was investigated by Greene. Judd. and Marx (19M0). They found that the rcpiness was caused by §,aer9genes in the water supply. Schroder (1930) described ropiness in sausage which he found was due to cocci which grew best on. blood and serum agar but was nonpathogenic to men and white mice. The first to isolate the organism causing rcpiness in bread was probably Laurent (1885). in Belgium. He named the causal organism Bacillus panificans. In Vienna Kratschmer and liemilowicz (1889) showed that Bagillus mesentericus vulgatgg was the organism that caused rcpy bread. The two organisms are probably the same. In the United States Russell (1898) reported g, mesenteriggg vulgatus as the causal organism. Workers generally agreed that B, mesentericus vulgatus is the organism responsible for ropy bread. Ropy meat curing solutions are caused by encapsulated bacteria. Heideman (1923) isolated five organisms capable of causing rope in curing solutions. Another organism causing ropiness in curing solutions was named Micrococcus lgpglyticus by Horowitz-Vlasova. and Livshitz (1931). It closely resembled Eicrococcus glpps and Micrococcus flavusliguefgpiens. The deveIOpment of rcpiness in sugar solutions and in fruits possessing a large amount of sugar in the Juice is not uncommon. Ropiness as we have seen may be caused by a large number of different organisms; one of which is the genus Leucgnostoc. The genus Leuconostgg is made up of but a few species. The great majority of which live on plant and vegetable materials. Leuconostoo mesentergides has been isolated from sugar beets. sugar cane. milk and milk products. by Hucker and Pederson (1930). Vaughan. Douglas. and Gililland (19h3) reported on its role in fermenting olives. They found that L, mesenteroideg was the most prevalent of the lactic acid organisms present during the first stage of fermentation of Spanish Type olives. During the second or intermediate stage it (L, mesenteroides) gained the ascendency over all types of organisms; however it disappeared in the early part of the third stage when L, plantarum predominated. L, mesenterpides has been reported by Pederson (1930) to be largely responsible for the early production of acid in the fermentation of sauerkraut. Hohl (1942) found it present in the early stages of lettuce fermentation. but it disappeared after the sixth day. Hartsell (19#%) was able to isolate it from spray dried egg powder. L, mesenteroides has also been reported by Spiegelberg (1940) to be the cause of swells in canned fruit. ‘L. mesenteroides may be cultivated on nutrient best or cane sugar juices in which. at favorable temperatures. it will decompose large quantities of sugar with the resulting production of a slime or rcpy condition. In sugar factories this condition is known as "frog-Spawn"; which inhibits crystallization of the sugar from the liquor. Abundant growth of the slime may block the pipes and fittings of the sugar factories. Kircher (1839) and Felts (1874) both observed microorganisms which were probably Lgpgpnggtgg in slimy beet Juice. The ”frog-spawn' phenomenon led Scheibler (1869) to study slimy beet juice in an effort to determine its cause. He believed that the slime was formed from the protoplasm of the best cell by enzymic action. Jubert (157k) did not agree with Scheibler‘s work and showed that the power of slime formation was destroyed by heating to 90°C (194°F) or treating with phenol. thus indicating the presence of a microorganism. Scheibler in further studies isolated a gum-like substance which he called dextran. Cienkowski (1878). was the first to study the slime formation from a microbiological approach. isolated a species he called Ascococcus mesenteroides as the cause of "frog-spawn”. Van Teighem (1878) determined the size of Cienkowski's organism and observed the endospore formation. The organism reminded him of the blue-green algae genus Ngstoc so he named the bacterium genus Leuconostoo or white Ngsto . Liesenberg and Zopf (1892) were the first to isolate strains of L. mggenteroides in pure culture form. and succeeded in purifying them by virtue of their resistance to heat. After Liesenberg and Zopf a number of workers isolated and studied strains of Leuconostoo. There was much duplication of effort and different names were given to the same species. It was not until Hucker and Pederson (1930) in an excellent paper on the cultural characteristics and taxonomic relationships of the Leuconostoo was order established. They isolated Leuconostoo from slimy sugar solutions, fermenting vegetables, and from milk and milk products. They considered the Leuconostgc as a morphological intermediate between the Streptococci and Lactobacilli. Under certain conditions the cells were elongated, while in slowly growing cultures the spherical form predominated. In ordinary yeast extract broth they grew as cocci and sometimes formed chains. In acid sauerkraut Juice or in acid fruit Juices they often grew as rods 0.5 to 1.0 microns in length. Hucker and Pederson (1930) also found that the different species within the genus could be differentiated on the basis of sucrose fermentation. Those species which did not ferment sucrose were usually found in milk and milk products. Those species fermenting sucrose were further divided into two groups on the basis of pentose fermentation. a. Those fermenting sucrose and pentose (xylose and/or arabinose). b. Those that fermented sucrose but not pentosss. The by—products of the carbohydrate fermentation were found to be; levo-laotic acid, acetic acid. 002, and ethyl alcohol. L: mesenteroides belongs to the first group - the sucrose and pentose fermenters. It is the most common and the most active member of the Leuconostoo genus. It produces dextran from sucrose. and forms acid from glucose. fructose. galactose. mannose. xylose. arabinose. sucrose. generally forms acid from lactose. raffincse. salicin. mannitol. and rarely from dextrin. starch. inulin. sorbitol. rhamnose. or glycerol. The temperature range is from 5C>C to #50 C, The Optimum temperature being 21°C to 25°C . Alford and McClesky (1942) found that 25°C was the optimum for slime production. At 37°C the organism did not produce slime. The gum (slime. ropiness) or dextran formed by L, ggsenterpides was studied by Levi. Hawkins. and Hibbert (l9u2). They showed that the dextran elaborated by the organism was made up of three glucosides: (a) Tetramethyl glucoside (b) Trimethyl glucoside (c) Dimethyl glucoside The ratio of tetra. to tri. to dimethyl glucoside was 1.3.1. Three of the linkages between the building units were 1-6 linkages. The remaining two were either 1-H or 1—6 glucosidic linkages. Duodoroff (l9k5) also demonstrated that the hexcse 10 units of the dextran were Joined through a 1-6 linkage. but the type of linkage was undetermined. Jeanes. Wilham. and Micro (1948) stated that the dextran was a polysaccharide having predominantly alpha 1-6 glucosidic linkages. Isolation and Identification of Leuconostoo mesenteroides Streaks were made on tomato Juice agar and on yeast extract-glucose agar from the samples of rcpy peaches obtained from the Michigan Agricultural Laboratory. The yeast extract-glucose agar gave the best growth so it was this medium which was used for the isolation of the organism. Yeast extract- glucose agar plates were streaked and single colonies were picked and streaked on another series of yeast extract-glucose agar plates. This was repeated six times. To further insure the purification of the organism single colonies were picked from the sixth plate and inoculated into 15 m1 vials of yeast extract-glucose broth. The vials were well shaken and allowed to stand for twenty minutes. Streaks were then made on yeast extract-glucose agar plates. The dilution of a single colony through 15 ml of sterile yeast extract glucose broth and the resulting streaking gave greater assurance of the isolation of a pure strain. 11 When certain that a pure strain had been obtained slants of yeast extract-glucose agar were prepared and inoculated with eleven different colonies of the pure strain. The Manual of Methods for the Pure Culture Study of Bacteria recommends that a minimum of six isolates of the organism to be identified be run through the different tests for identification. A gram stain of the organism was made from a twenty-four hour yeast extract-glucose broth culture. The stain showed gram positive cocci or very short rods in pairs or short chains. Colonies twenty-four hours after streaking on yeast extract-glucose agar were uniformly round. pinpoint. translucent. raised. domeshaped. granular. and entire. A can of peaches was obtained and macerated in a sterile Waring blendor. The macerated peaches and their Juices were placed in small flasks and autoclaved. After autoclaving the flasks were inoculated with the isolates. Ropiness developed in all flasks in forty-eight hours. The ropy strings when stretched reached about one half inch or more before breaking. A gram stain revealed the same gram positive 00001 in pairs or chains. Morphological characteristicgrpf the isolates: (Size and shape) - Gram positive cocci 12 0.95 to 1.3 microns in diameter. occurring in pairs or in short chains. Non-motile Aerobic ggltural characteristics: Gelatin stab — Slight surface growth. no liquefaction. Agar slant - Small. round. pinpoint. transparent. raised. dome-shaped. Yeast extract—glucose agar (24 hours) - Round. pinpoint. translucent. raised. dome-shaped. granular. and entire. Yeast extract-glucose broth - turbid. PhysioIOgical characteristics Litmus milk - no reduction Indol test — negative Nitrates not reduced to nitrites Voges-Proskauer test - acetyl-methylcarbinol not formed. Acid no gas in - xylose. arabinose. dextrin. maltose. galactose. lactose. raffincse. glucose. and sucrose. 13 Very slight acid and no gas in - mannose. glycerol. and starch. No acid nor gas in - mannose. inulin. and mannitol. Habitat: Isolated from rcpy canned peaches. Checking the characteristics against Bergey's Manual of Determinative Bacteriology. the organism to which the above characteristics most nearly corresponded was 2, mesenteroideg. The only difference noted was that no acid was produced from mannose. 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