I I I I II I I I | IIIIIIII I I III __THS DETERIORATION OF WHITE SUGAR I. CHARACTERISTICS OF YEASTS FOUND IN DETERIORATED SUGAR II. SOME STUDIES ON DETERIORATED SUGAR Thesis for the Degree of M. S. Harlow H. Hall 1936 .THESI; 1 . 'G ' , ' M. "“ M x - G . . f ‘ ‘f '\ ‘ V ~ . \. ‘\ s \ I _ w: Pig-$137». ;.f ‘.. 7 PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE I I ‘ _fi “—c. .gm-“n—- -- “W 1-", '3 "n 2 ' " '. TW,"""“”‘"”**‘%M%WrWN¥ 5 ' a - 6’01 c-JCIRC/DateDuepss-p. 15 DETERIORATION 0F WHITE SUGAR I. CHARACTERISTICS OF YEASTS FOUND IN DETERIORATED SUGAR II. SOME STUDIES ON DETERIORATED SUGAR A THESIS Submitted to the Faculty of the Michigan State College in partial fulfillment of the requirements for the degree of Master of Science by .) Harlow H; gall June 1936 III-its}: I. Characteristics of Yeaete Found in Deteriorated Sugar 11. Some Studies on Deteriorated Sugar fl V\ V" ‘ .« 1 ‘ §4_£-.;‘)fl::a " a we.“ fiw?) ACKNOWLEDGMENT It is with deep appreciation that I acknowledge the assistance and constructive criticism offered by Dr. F. W. Fabian while studying the yeasts and in the preparation of this thesis and, to Dr. J. W. Grist and Prof. C. D. Ball for helpful cooperation while conducting this study. Finally, I wish to acknowledge my appreci— ation to those of the Food Research Division and the Carbohydrate Research Division of the Bureau of Chemistry and Soils, United States Department of Agriculture, who have made this work possible. TABLE or CONTENTS Title Acknowledgment Part I. Introduction Historical Present Work Characteristics of Yeasts Schizosaccharomyces octosporgue Biejerinck. Zygosaccharomyces lgponicus SaitO. gaccharomyce§,sp. (unidentified). Saccharomyces sp. (unidentified). gyggsaccharomyces mellis Fabian and Quinet. Summary Part II. Deterioration by Yeasts Moisture Absorption by Sugar Cause of Sugar Deterioration by Microorganisms Summary and Conclusions References Characteristics of Yeasts Found in Deteriorated Sugar. INTRODUCTION During the course of an extended study in which a large number of white sugar samples were analyzed several samples of domestic plantation cane sugar were obtained each year that posessed an alcoholic odor and were in an unusually moist condition. The samples were as a rule dis~ colored and were further characterised by a tendency to creepiness. A microscOpical examination of crystals dissolved in a waternglycerine mixture showed the presence of yeast cells. Chemical examination of the samples showed excessive moisture, invert sugar and a low sucrose content. Considerable monetary losses are suffered annually by the sugar industry through the development and action of microorganisms in stored sugar. In an attempt to charac~ terize and study more fully the spoilage agents several samples of domestic plantation cane sugar representing various stages of deterioration were selected for a study to determine the reason for this condition and, to study the ideal condition for its occurgnce. HISTORICAL Numerous studies have been made on the microbial deterioration of raw sugar and other forms of sugar products. Few reports are available, however, in which the exact H1" identification of the causative agents of spoilage are given. This is especially true in deterioration studies in which yeasts were the causative agents. The first scientific paper to deal with the phenomenon of sugar deterioration was that of Payen (1851) the French chemist. Payen describes a type of deterioration in which loaves of solid sugar became pitted, the cavities being stained a reddish, and at times greyish color which rendered the product unsalable. This type of spoilage was observed in France as early as 18%} and was described by Montague, a contemporary mycologist, as being due to the action of two closely related fungi, Clyciphila erythrospora_and Glyciphila eleaospora. Browne and Church (1928) likewise observed the action of yeast upon the surface of crystals of soft and raw sugars in which the sugar had become pitted. Two cultures of yeast and a culture of a yeast—like mold were isolated from a pitted sample of raw sugar. In commenting on the observam tions of Payen, they cite the probable importance of atmos~ pheric humidity in sugar deterioration, a point not mentioned by Payen. They state “the attraction of moisture from the air by the impurities of the sugar favoring the germinatian of the spores and the activity of the decomposing enzymes. The invert sugar, produced by the latter, would in turn attract additional quantities of atmospheric moisture, owing to its exceedingly strong hygrosc0pic preperties, and the loaf of sugar, although perfectly dry at first, would thus n2» become a favorable medium for the growth of molds and other microorganisms.“ Browne (1918) isolated three nonnsporulating forms of yeast while studying the microflora of Cuban raw sugar. They were identified as a species of Torula and two species of Monilia. Torulagcommunis Browne was found to be the most predominant type in the sugar and was characterised by its ability to attack invert sugar rather than sucrose. Monilia g$g£§.Browne and honilia Egggg_Browne were characterised by their ability to attack and invert sucrose. Browne conn siders these Monilia to be the most destructive organisms in sugar on account of their ability to readily adapt themn selves to different environmental conditions. ‘ Owen (1926) in making claims for a patent for a method for the preservation of raw sugar against fungi deteri— oration cites the need for the removal of the hygroscOpio invert sugars, especially levulcse, from the molasses film. To accomplish this, he recommends the inoculation of the sugar mass with a culture of a nonusporulating yeast (torulae). Cultures that readily adapt themselves to the conditions of high osmotic pressure and that do not utilize the sucrose but which do attack the invert sugar are used. As a result of the utilization by the torulae of the monosaccharides the molasses film becomes saturated with 002 which prevents the growth of fungi. The claim is also made that since the torulae utilize mainly levulcse, a levorotatory sugar, the polarization of the sugar increases. Also by the elimn ”3.. ination of the levulose the color of the raw mass is improved. The ability of yeasts to adapt themselves to their environment has been shown by Schutt (1925) and Church, Pains and Hamilton (1927) in their studies on sugar tolerant yeasts that cause bursting in chocolateucoated cream~center candies. They observed the presence of yeast cells on the faces of the sugar crystals from the cream centers. These yeasts were found to be capable of active growth in the cream centers and caused bursting through the formation of gas. Present Work Representative samples of deteriorated white sugar, posessing an alcoholic odor, high moisture and invert sugar content and a condition of creepiness were selected for a microbiological and chemical examination. Water solutions of the samples were diluted serially and plated on clarified honey agar (Hall and Lothrop, 1933) and on malt extractu sucrose agar (Browne and Church, 1928). Since the yeasts were growing under conditions of apparently high osmotic pressure in films of liquid on crystals, it was felt that these media would be suitable for their isolation. At the end of seven days of incubation at 30°C. the number of viable cells in each sample was determined (Table 1.) and several colonies were fished from each medigyand trans— ferred to slants of the medium from which the isolation was made. Twenty—two cultures were obtained which were H A]. studied for their physiological, cultural and morphological characteristics. The chemical analysis of the sugar samples included moisture, invert sugar and sucrose content as well as the pH. The results of these determinations are given in table 1. Table 1. Results of Microbiological and Chemical Examination of Deteriorated Sugar. Sample Plat Count Co osition of 3 ar No H0557 F'Llart-g'xu ROI s't's- I nver uc rose ‘ agar sucr. agar pH ure sugar gm. gm. per cent per cent Hper cent 3M60 #00 100 6.h 0.39 2.20 93.03 3&61 2200 2600 5.5 0.31 1.73 93.86 3u62 19,000 30,000 5.7 1.27 3.49 91.52 3163 ...,.... 1...... 6.9 ..._...._ ...... —--— 333° """""" w'“ 607 0.7“ 1007 96013 These data are too few from.which to correlate the condition of the sugar with the number of viable yeast cells. However, sample N0. 3462 which contained the greatest number of yeast cells also contained the highest moisture and invert sugar content as well as the lowest pH value and the lowest sucrose content. infected with yeasts capable of destroying sucrose.’ This condition might be expected to prevail in sugars As a result of its decomposition there would be an increase in the invert sugar content which would result in increased .9 5 a attraction for moisture. The growth of yeasts is usually accompanied by an increased hydrogen ion concentration. Yeasts present in sugar which do not ferment sucrose but which attack only the invert sugars would be limited in their action by the amount of these sugars remaining in the molasses film surrounding the crystal. In this case the yeast activity would cease when the available sugars had been utilized. Characteristics of Yeasts The yeasts isolated and studied may be divided into five groups, based upon their physiological, (table 2.) cultural and morphological characteristics. Since all cultures included in this study produce spores, they would belong to the family, Saccharomycetaceae. Based upon their methods of spore forming characteristics they have been divided into three genera, Schizosaccharomyces, Zygosacch— aromyces, and Saccharomyces. In attempting to classify and identify the several species the classification of N. M. StellinguDeeker (1931) has been used. For species not listed there Guilliermond (1920) and the more recent pubs lications in scientific Journals were consulted. Y Schizosaccharomyces octosporous Beijericnk. IT' I Group I, contains cultures numbers 3330—1 and 3h60~l. They are the most difficult of any of the cultures in the groups to cultivate on artificial media. They ferment glucose, mannose, fructose and maltose but not arabinose, «r6ru xylose, galactose, sucrose, lactose, raffinose or rhamnose. An incomplete ring is formed on malt extract broth. The medium is clear and the sediment is compact. The cultures are characterised by a scant growth on plain and 10 per cent dextrose agar, while on honey agar and malt extract—sucrose agar the growth is moderate al~ though slow. At first the growth is smooth but later be— comes verrucose. The color of the growth is tan, the luster is dull; it is raised and convex, and is entire. Gelatin stabs are liquified and the form of the liquefaction is cratengorm. The growth on carrot slants is smooth, glisten~ ing, white, raised and butyrous. The medium is not changed and there is no chromogenesis. The growth of giant colonies on dextrose agar and on malt extract agar is very slow. After #0 days the diameter of the colonies is less than two centimeters. The color is dull white to tan. The primary colony is slightly roughened and has a warty appear~ ance. The secondary growth, which extends beyond the pri» mary growth, is white, smooth and has an entire edge. Two types of cell forms are usually present in actively growing cultures. (fig. 1.) They are (1) round to slightly oval of uniform size which do not show internal structure and, (2) large cells that are frequently rectangu» lar and which always appear to be granular. Reproduction by the vegetative cells is by trans» verse division. A cross wall appears in the middle of the cell, which increases in size until the cell is separated, giving rise to two daughter cells. Clusters of cells are frequently formed by the failure of the daughter cells to separate from the parent cells after division. Clusters also have their origin by the failure of new cells derived from spores to separate when released from the ascus. The average size of the vegetative cells is M.5 microns. Fig. l. A. Vegetative cells grown on malt extract agar. (a) Cells reproducing by transverse division. B. Sporulating cells grown on 10 per cent dextrose agar. (a) An ascus containing eight spores. (b) An ascus from which eight spores have been liberated; two of the spores undergoing conjugation. (c) Ascus from which spores have been liberated. Sporulation is preceded by isogamic conjugation which takes place between two cells. The cells unite by a conjugating canal through which the contents of the two cells mix. (Guilliermond 1901). A large oval zygospore, frequently rectangular, results from the fusion. I1- , An even number of spores are always formed there being either four or eight in each ascus. The walls of the ascos— as... pores are stained blue with Lugol's iodine. Just prior to sporulation the starchy materials of the asci wall disappear and are probably utilized during sporulation. The members of this group correspond with those of Schizosaccharomyces octosporgus Beijerinck, as given by Stelling~Dekker. This species was first isolated by Beijerinck (1896) from figs and raisins obtained from warm climates. zygosaccharomyges jgponicu§_8aito. Group II, consists of cultures numbers 3460~2 and 3, 3h61~1,2,3,fl,5,6 and 7 and 3462~2,4 and 6. They are char~ acterised by the fermentation of glucose, mannose, fructose and maltose. They do not ferment arabinose, xylose, galactose, sucrose, lactose, raffinose or rhamnose. They form a heavy ring but no scum 0n malt extract broth. The medium is clear and the sediment is compact. The cultural characteristics of this group are a moderate growth on dextrose, honey and malt extractnsucrose agar. The growth is glistening, grayish, convex, echinulate and butyrous. 0n carrot slants the growth is moderate to abundant, raised, convex and verrucose. The luster is usually glistening and the color is tan. In gelatin stabs the growth is filiform, with no liquefaction. The growth.of giant colonies on dextrose agar and malt extract agar is moderate. After six weeks the colonies are approximately four centimeters in diameter. The primary growth is smooth, glistening, and raised. After 15 days of incubation the colony becomes rough and later is folded and wrinkled having a veily appearance. I I We * bait? I . I or“- lief: _,_ I /j I Fig. 2. Vegetative cells grown on malt extract agar. (a) Cells showing budding. B. Sporulating cells grown on malt extract agar. (a Ascus with ascospores resulting from isogamic conjugation. b Parthenogenes1s. Asexual reproduction is by budding. The daughter cells tend to adhere to the mother cells forming clusters. (fig. 2.). This is observed both in liquid carbohydrate media and on solid culture media. The vegetative cells were elliptical and range from 3.0~H.0 x 5.2~8.0 microns. The formation of ascospores follows isogamic conjugation. Spores are readily produced on carrot, honey and malt extract agar. The asci present a dumb~be11 appearance which results from the uniai of two vegetative cells by means of a conjugating canal. The spores appear in the enlarged ends of the asci, usually two in each end. Frequently one may be seen in one end and two in the Opposite end of the asci. Parthenogenesis is frequently observed. The size of the epores range from 2.5 to 4.0 microns; the average size is 3.5 microns. The characteristics of this group of yeasts agree essentially with those of gyggsaccharomyce§_japonicus Saito. This yeast was first isolated by Saito (1906) from the products of fermenting Soya. It has since been isolated from fermenting honey by Fabian and Quinet (1928) and from fern menting maple sirup by Fabian and Hall (1933). Saccharomyces sp. (unidentified). Group III, consists of cultures numbers 3463~l,2,3 and 4. They are characterised physiologically by the fer— mentation of glucose, mannose, fructose, galactose, sucrose and raffinose with the production of alcohol and carbon di~ oxide gas. They do not ferment arabinose, xylose, maltose, lactose or rhamnose. They produce a ring on malt extract broth and an abundant compact sediment. The cultural characteristics of this group are a moderate growth on dextrose, honey and malt extract~sucrose agar. The growth is glistening, filiform, entire, convex and butyrous. The growth on carrot slants is moderate, filiform and smooth. The growth in gelatin stabs is fili~ form but there is no liquefaction. The growth of giant colonies on dextrose agar and malt extract agar is abundant. In young colonies the growth is white, glistening, and smooth, later becoming greyish and dull. The colony is raised and the edge is irregular. The vegetative cells of this group are nearly round. Asexual reproduction is by budding; there is no tendency to form clusters. (fig. 3.). The size of the vegetative cells on honey agar is 3.5 to 4.7 microns, the average size is “.1 microns. The asci occur singly with from one to three spores. The most common number of spores is two in each asci. The spores measure from 1.7 to 3.0 microns, the average size is 2.6 microns. The ascospores are formed directly in the asci without evidence of previous sexual n phenomenona is the vegetative cells. ' 4 I0 0(5) e O C?) 1). Q ' ad (9) I I Fr Fig. 3. A. Vegetative cells grown on malt extract agar. (a) Cell showing budding. B. Sporulating cells from carrot medium. (a) Ascus with three ascospores. (b) Ascus showing the deve10pment of two ascospores. This yeast agrees very closely with Saccharomyces mangini Guilliermond (1914). A point of difference is noted, however, in the physiological characteristics of the culture isolated by Guilliermond and the cultures isolated ".12 H from sugar. Guilliermond's culture of g, mangini ferments lactose, whereas, those isolated from sugar failed to ferment this sugar. This is considered to be a major point of difference. It is possible that the cultures isolated from sugar are a variety of g, mangini. A known culture of g. mangini was not immediately available for comparative purposes, however, it is felt that the cultures isolated from sugar agree closely in other respects so that addition— al work is necessary to check the species differentiation more fully. Saccharomyces sp. (unidentified). Group IV, consists of culture No. 3462—7 only. It is characterised by the fermentation of glucose, fructose, mannose, and sucrose with the production of alcohol and carbon dioxide gas. Arabinoee, xylose, maltose, lactose, raffinose or rhamnose are not fermented. A heavy compact ring is formed on malt extract broth. The broth remains turbid for several weeks after inoculation with the culture. The growth of this culture on dextrose agar is abundant, glistening, white, smooth, entire and butyrous. The growth on malt extract—sucrose agar is moderate, glistening, and smooth. 0n carrot slants the growth is abundant, glistening, smooth and noticeably raised above the surface of the medium. The growth in gelatin stabs is filiform but there is no liquefaction. The growth of giant colonies on dextrose agar and malt extract agar is abundant, white, glistening and smooth, later becoming dull. The cells of this culture are elliptical. (fig. 4.). Asexual reproduction is by budding and there is no tendency of the cells to attach themselves together to form clusters. The size of the vegetative cells on honey and dextrose agar range from 3.5—4.2 x 4.5—5.5 microns, the average size is 4.0 x 5.2 microns. The asci occur singly with from one to two spores. The ascospores are formed without evidence of sexual relationship in the vegetative cells. With the formation of the spores the asci becomes more nearly spherical than when in the vegetative state. Spores were obtained with difficulty and were produced only after transferring the growth from carrot slants to blocks of gypsum. They then occurred after three days incubation at room temperature. I 9}“ ”Tier I 9 f ' P2309 at I I casffo C7 ; (E I a ' /I 5 a L Fig. 4. A. Vegetative cells grown on malt extract agar. (a) Budding cell. B. Sporulating cells from gypsum blocks. (a) Ascus with two ascospores. The most common number of spores is one in each ascus, The cells of this culture are elliptical. (fig. 4.). Asexual reproduction is by budding and there is no tendency of the cells to attach themselves together to form clusters. The size of the vegetative cells on honey and dextrose agar range from 3.5—4.2 x 4.5~5.5 microns, the average size is 4.0 x 5.2 microns. The asci occur singly with from one to two spores. The ascospores are formed without evidence of sexual relationship in the vegetative cells. With the formation of the spores the asci becomes more nearly spherical than when in the vegetative state. Spores were obtained with difficulty and were produced only after transferring the growth from carrot slants to blocks of gypsum. They then occurred after three days incubation at room temperature. J 9 Q00 5 90‘9” i fld’b W96 ‘ ex: 0 C7 : (7 i a E A I 13 Fig. 4. A. Vegetative cells grown on malt extract agar. (a) Budding cell. B. Sporulating cells from gypsum blocks. (a) Ascus with two ascospores. The most common number of spores is one in each ascus. H 14 H two rarely being present. The spores are from 3.5 to 4.5 microns in diameter. They are smooth and are centrally located in the ascus. A search of the literature for a species of yeast corresponding in all respects to the culture contained in this group has been in vain. It was thought at first that it corresponded to Saccharomyces acerisrsacchari Fabian and Hall. (1933). There are no apparent differences in the cultural or physiOIOgical characteristics, however, agree- ment in the morphological characteristics could not be ob~ tained. The cells of g, ggeris—sacchari are round, almost without exception, whereas, the cells of culture 3462~7 are elliptical. Another point of difference is the ease with which spores are formed in cultures of g, aceris~sacchari. They are readily formed on carrot media, while spores are formed with difficulty in the cultures isolated from sugar. Finally, it has been observed that g, aceris~sacchari vigorously ferments a medium of cider producing a distinct, pleasant aroma. Fermentation of the same medium by the culture under study was slow and did not produce the aroma. It is felt that this culture may represent an un~ identified species, however, before it can be named a con— tinued search of the literature must be made. Also, come parisons with other known cultures will be necessary before naming this species. a 15 w Zygosaccharomyces mellis Fabian and Quinet. Group V, is comprised of cultures numbers 3462Hl, 3 and 5. They ferment glucose, mannose and fructose but not arabinose, xylose, galactose, maltose, sucrose, lactose or rhamnose. A well deve10ped ring is formed on malt extract broth. The medium is clear and the sediment is compact. The cultures of this group are characterised by a moderate growth on dextrose agar, malt extract—sucrose agar, malt extract agar and on carrot medium. The growth on dextrose agar is smooth, greyish and butyrous; in the case of culture No. 1 it is glistening while that of cultures Nos. 3 and 5 it is dull. The growth of the cultures on malt extract~sucrose agar is smooth, tan, entire and convex and on carrot medium it is white, smooth, spreading and raised. The growth of giant colonies on dextrose agar and malt extract agar is characterised as being moderate; the colony attained a diameter of approximately three centi~ meters in six to eight weeks after inoculation. The growth was smooth at first later becoming folded and wrinkled. The vegetative cells of the cultures of this group are round to oval, with the occasional production of cylindrical cells. The size of the vegetative cells ranges from 2.5»4.5 x 4.5—6.5 microns. The average size of the vegetative cells is 4.0 x 5.0 microns (fig. 5.). Spore pro~ duction results from isogamic conjugation between two vegetative cells. The most common arrangement of spores is one in each end of the ascospore. However, this may vary from two spores in one end to one in the Opposite end. Parthenogenesis is not uncommon in these cultures and there are usually two spores in each ascus. The diameter of the spores is from 2.9 to 4.0 microns. 1 a . l 9006’ a? 3% 3” fian are as as" f l ‘ L_ ‘A [3 Fig. 5. A. Vegetative cells grown on malt extract agar. (a) Budding cell. B. Sporulating cells from malt extract agar. (a) An ascus with three spores resulting from isogamic conjugation. (b) Cells showing isogamic conjugation. Since the characteristics of the members of this group correspond essentially with those of Zygosaccharomyces mellis Fabian and Quinet, they are considered as members of this species. Fabian and Quinet (1928) first isolated this yeast from fermenting honey. It has since been isolated by Lochhead and Heron (1929) of Canada and Marvin et al (1931) of the United States and Sacchetti (1932) of Italy from samples of fermenting honey. This same species ..17.. was also isolated by Fabian and Hall (1933) from samples of fermented maple sirup. SUMMARY 1. The condition of wetting and creepiness in white sugars indicated that some agent might be responsible for this condition. The odor of alcohol in the deteriorated samples suggested that the spoilage agents were possibly yeasts. 2. Large numbers of yeast cells were demonstrated when crystals of the sugar were dissolved in liquid and examined microscOpically. Quantitative determinations of yeast cells showed them to be present in numbers from 100 to 30,000 per gram of sugar when grown in a medium of malt extractwsucrose agar. 3. Five groups of yeasts were obtained when samples of deteriorated sugar were plated on several media. They were identified according to their physiological, cultural and morphological characteristics as follows: a Group I. Schizosaocharomycg§_octospordus Beijerinck. Group II. Zygosaccharomyces lgponicus Saito. Group III. Saccharomyces sp. (unidentified). Group IV. Saccharomyc s sp. (unidentified). Group V. Z gosaccharomyces mellis Fabian and nineto "18—. [Physiological_Characteristics of Yeasts Found in Deteriorated Sugar. Table 2. 25°C. film I Scum formation malt ext. broth ring, Gelatin liu sxasm g axes&?n 8193M g anaemia xeemlr I I'll I [I I 11.! 1I.I III! I ‘ I ssorons OSOUIJJ'GH ssoumsqg osoaosq ssoitsn Fermentation in‘a 1 a solution of guefication ssoaostsa ssouusn as along ascents esotfix ssourqsrv Culture No. a 19.H NMTNmeoN-TNMmmNTmM r4 (U \o \o {I} 2- d- z. m N'\ r¢\ ”War I" 7" .17: Some Studies 0n Deteriorated Sugar Deterioration by Yeasts. Preliminary attempts to reproduce deterioration in samples of domestic plantation cane sugar and refined granulated cane and best sugar, were unsuccessful when. inoculated with portions of deteriorated sugar containing a mixed yeast culture and incubated in cpen containers at room temperatures. Slight visible evidence of deterior» ation by discoloration was frequently observed soon after inoculation in areas-immediately surrounding the inoculum but as desiccation of the inoculum continued the deter~ iorative action ceased. It seemed then, that a method of supplying and maintaining the moisture content of the inoculum or, the sample under study must be employed. The method used was to place 70 gram samples of granulated white sugar, representing different methods of manufacture, in sterile covers of petri dishes, and inn oculating them with two grams of deteriorated sugar conn taining yeast. The samples were incubated in desiccators containing water—sulfuric acid mixtures to produce and maintain relative humidities of 50~60 and 70 per cent. These values were chosen since Wilson (1921) reports that 50 per cent relative humidity is the average for indoor conditions, while 65 per cent relative humidity is the average for outdoor conditions. The inoculated samples n20o-o were incubated at room temperature for a period of nine months. At the end of this period the samples were removed for analysis. The physical condition of the samples inn cubated in an atmosphere of 50 and 60 per cent relative humidity was unchanged while those incubated in an atmos~ pherc of 70 per cent relative humidity were wet, discolored and creepy. They were analyzed for sucrose, invert sugar and moisture by the methods of analysis of the Association of Official and Agricultural Chemists (1930). Sucrose was determined by the double polarisation method, using the enzyme invertase. Invert sugars were determined by the Munson~Walker volumetric method and moisture was determined by drying on sand at 70°c, The results of the analysis of the samples are given in table 3 as follows: Table 3. Composition of Original and Deteriorated Sugar Original Deteriorated samples sugar ‘1 2’ j; ‘4 "Per cent er cent Per cent Per centher centher cent Sucrose 99.7 98.71 64.49 78.19 72.21 71.84 Sucrose (dry 99.30 78.49 83.54 83.11 83.37 basis) Invert 0.20 0.30 11.81 4.22 11.01 9.98 sugar fifigzit o.us 14.37 4.50 12.67 11.53 (dry basi ) ' Moisture f 0.04 0.60 21.72 6.85 15.10 16.05 The loss of sucrose, calculated on the dry weight basis, in the incubated samples was from 0.40 per cent in sample No. 1. to 21.21 per cent in sample No. 2. The loss of sucrose was accompanied_by an increase in the invert sugar content of from 0.28 per cent to 14.17 per cent. There was an increase in the moisture content of from 0.56 per cent to 21.68 per cent in these samples. The loss of sucrose in sample No. 3. was 16.16 per cent while the in— crease in the invert sugar content was 4.20 per cent. The loss in total sugar due to deterioration was 11.66 per cent. Losses in total sugars were noted in the remaining samples. In an attempt to determine the reason for the failure of sample No. 1. to deteriorate an examination of the original sample was made for yeast growth promoting sub— stances. The method of Hall, James and Stuart (1933) was used in which sterilized 10 per cent solutions of the sugars were inoculated with a definite number of yeast cells from a 24 hour culture. The numbers of cells deveIOping during a 72 hour incubation period was used as an index of the amount of stimulative substances present in the sugar. There was no develOpment of cells in sample No. 1. while varying numbers developed in the remaining samples. The degree of deterioration of the remaining samples could not be correlated, however, with the relative amounts of stimulants shown by the develOpment of yeast cells. Moisture Absorption by Sugar. Previous results demonstrated the necessity of moisture in order for the deteriorative agents of sugar to react. The phemonena of the adjustment of deterior— ative agents to the sugar medium seems analogous to that prOposed by Fabian and Quinet (1928) regarding yeast fermentation in honey. The basis of their theory was that sufficient moisture is absorbed, under conditions of suitable humidity and temperature, by the hygrosoOpic sub~ stances in honey to result in the dilution of the medium at its surface to a concentration which would.permit the yeasts to grow. When the yeasts became adjusted to the conditions of high sugar concentration they were then able to grow throughout the medium. In order to study the relationship of atmospheric moisture and temperature to the absorption of moisture in inoculated sugar samples a series of samples w§;; prepared for incubation under controlled conditions of these elements. A quantity of white granulated sugar was prepared by comm positing several lots of various quality sugars. The com~ posite had a single polarization of 99.7 and its moisture content was 0.04 per cent. The composite was divided into five portions for inoculation with representative cultures of the groups studied in Part I. It was also desired_to determine if there was a relationship between the absorption of moisture in samples inoculated.with sucrose fermenting yeasts and non-sucrose fermenting yeasts. The prepared samples of sugar were inoculated by mixing into the sugar portions of sporulated cultures of the several yeasts. Five 70 gram quantities of each inoculated sample #35 then weighed into sterile covers of petri dishes for incubation under the following conditions of humidity and temperature: Condition I. Humidity 60 per cent; temperature 2200, Condition II. Humidity 60 per cent; temperature 30°C, Condition III. Humidity 70 per cent; temperature 22°o, Condition IV. Humidity 70 per cent; temperature 30°C. The samples of sugar were inoculated.with yeast cultures Nos. 3460~2, 3461—1, 3462—7, and.3463—1 representing Groups II, III and IV. One uninoculated quantity of sugar was incubated under each of the four conditions and served as a control. The samples were incubated for a period of 100 days and were then examined for their moisture content and physical appearance. The results are given in table 4. Two of the samples of sugar incubated under both conditions of temperature (2200. and 30°C.) and in an atmosphere of 60 per cent relative humidity showed physical changes. The sample that was inoculated with yeast culture No. 3460~2, a nonnsucrose fermenting yeast, and incubated at 22°C. was wet in several isolated spots at thejuncture of the sugar and the container. The sample had absorbed 0.43 per cent moisture as compared to 0.12 per cent for the un— inoculated control. The sample that was inoculated with a n 24 a Table 4. Relationship of Humidity and Temperature to Moisture Absorption by Inoculated Sugar. After 100 days. Humidity_:Temperaturel,__Culture , Moisture 2hysical . No. absorbed characteristics per cent 60 22°c. control 0.12 unchanged 3460~2 0.43 wet spots 346l~l 0.12 unchanged 3462—7 0.12 unchanged 3463~1 0.25 unchanged 60 3000. control 0.16 unchanged 3460~2 0.20 unchanged 3461—1 0.22 unchanged 3462~7 0.23 unchanged 3463-1 0.47 wet spots 70 22°C. control 0.12 unchanged 3460~2 0.16 unchanged 3461~l 0.16 unchanged 3462~7 0.Z2 damp areas 3463~l 0. l damp areas 70 30°C. control 4.34 wet, creepy 3460~2 4.64 wet, creepy 346l~1 9.81 wet, caked, moldy 3462~7 8.68 wet, moldy 3463~l 4.28 wet, creepy_ sucrose fermenting yeast, culture No. 3463—1, absorbed 0.25 per cent moisture but did not show evidence of deterioration. The sample that was inoculated with the same culture but incubated at 30°C. under the same condition of humidity showed wet spots at the juncture of the sugar and the container. This sample absorbed 0.47 per cent moisture. Budding yeast cells were found in the wet spots indicating that they had begun to adapt themselves to the conditions of their environment. The samples that were inoculated with the sucrose fermenting cultures Nos. 3462-7 and 3463~l and incubated at 22°c, in an atmosphere of 70 per cent relative humidity were damp at the Junction of the sugar and the container. They had absorbed 0.32 and 0.41 per cent moisture, respective— ly, as compared to 0.12 per cent for the uninoculated control. It was not possible to demonstrate budding yeast cells in these samples, however, some non—sporulated cells were ob« served that had lost their characteristic granular appear— ance, which is indicative of dormacy in these cultures. Considerable wetting of the samples occurred when incubated at 30°C. in an atmosphere of 70 per cent relative humidity. The condition of these samples is in contrast to those incubated at 22°C. under the same condition of humidity. The samples that were inoculated with cultures Nos. 3461—1 and 3462~7 evidenced the greatest physical change. The samples were unusually wet, slightly discolored and showed evidence of pitting. Analysis showed that they had absorbed 9.81 and 8.68 per cent moisture, respectively, as compared to 4.34 per cent in the uninoculated control. A microsCOpical examination of the samples showed them to be heavily infected with molds. Mold mycelia predominated over the yeast cells in the samples of this series and it was concluded that molds were the deteriorative agents. It is apparent from these results that excessive deterioration of white sugar, resulting from microbialaction, 0:. v The samples that were inoculated with the sucrose fermenting cultures Nos. 3462-7 and 3463~l and incubated at 2200, in an atmosphere of 70 per cent relative humidity were damp at the Junction of the sugar and the container. They had absorbed 0.32 and 0.41 per cent moisture, respective— ly, as compared to 0.12 per cent for the uninoculated control. It was not possible to demonstrate budding yeast cells in these samples, however, some non—sporulated cells were ob« served that had lost their characteristic granular appear— ance, which is indicative of dormacy in these cultures. Considerable wetting of the samples occurred when incubated at 30°C. in an atmosphere of 70 per cent relative humidity. The condition of these samples is in contrast to those incubated at 22°C. under the same condition of humidity. The samples that were inoculated with cultures Nos. 346l~1 and 3462~7 evidenced the greatest physical change. The samples were unusually wet, slightly discolored and showed evidence of pitting. Analysis showed that they had absorbed 9.81 and 8.68 per cent moisture, respectively, as compared to 4.34 per cent in the uninoculated control. A microscopical examination of the samples showed them to be heavily infected with molds. Mold mycelia predominated over the yeast cells in the samples of this series and it was concluded that molds were the deteriorative agents. It is apparent from these results that excessive deterioration of white sugar, resulting from microbialaction, a 26 n occurs under conditions approaching the upper limits of normal humidity and at temperatures approximating 30°C. It was further observed that moisture must be attracted to some point in the mass of sugar, either by the presence of hygrosCOpic substances on the crystal or by some object in contact with the sugar. Since wetting of the samples incubated under conditions A, B and C occurred without apparent regard for the fermentation characteristics of the yeast it is felt that accumulations of hygroscopic substances were responsible for this condition rather than the type of yeast present. The results also suggest that several months are required for the adjustment of the yeast to the environment prior to deteriorativs action. Numerous studies have been made to determine the causes for the dampening of sugar in storage. Svatek (1932) observed that crystals of completely dried sugar~ became damp when placed under conditions of changing temper~ ature. He observed that during the temperature changes water condensed upon the crystals and formed a film of sirup. Vondrak (1932) suggests that damp sugar, especially raw sugar, may be caused by contact of bags of sugar with damp floors, exposure to adverse weather conditions during transportation and to the condensation of moisture from damp and warm sugar on the colder surface of the bag in contact with cold air. Sanders (1933) observed that the condensation of moisture on the surface of the bags was ~27... the most prevailing cause of damp sugar. Cause of Sugar Deterioration by Microorganisms. Sufficient evidence has been accumulated in these err periments to offer a plausable explanation for the deterin oration of sugar during storage. The results of these ex~ periments indicate that the moisture collects at the juncture of the sugar with the glass in sufficient quantities to support the growth of certain sugar tolerant yeasts. The accumulation of water is facilitated by several factors. The principle ones being relative humidity, temperature and the presence of hygrosc0pic substances on the sugar. Sufficient water is held at the Juncture by capalliarity to permit certain yeasts to grow. Their growth may be facilitated by the presence of growth stimulating substances. Once they become adjusted to the environment, they break down the sugar very rapidly. These experimental observations removes the suggestions of Vcndrak from the realm of specu~ laticn to one of fact and confirm Sandera's Observations on the prevailing cause of damp sugar. They likewise fit in very nicely with Svatek‘s observations on the relationship between changing temperatures and the dampening of sugar. It would, therefore, appear that any factor such as humidity, temperature, the presence of hygros00pic sub~ stances as levulcse and contact that increases the moisture content correspondingly increases the possibility of sugar deterioration by microorganisms. If molds are present, «28—. they may cause the deterioration as Owen, cheloff and others have shown. If yeasts are present, they may cause the deterioration as these experiments show or, if both molds and yeasts are present, it may be a combination as the results show. Summary. 1. Seventy gram samples of white sugar from widely different sources when inoculated.with two gram samples of sugar containing yeasts and incubated nine months at room temperature at relative humidities of 50, 60 and 70 per cent respectively showed deterioration only at a relative humidity of 70 per cent. 2. The loss of sucrose ranged from 0.4 to as high as 21.21 per cent while the increase in moisture content in the same samples ranged from 0.56 to 21.68 per cent. 3. There seemed to be no definite correlation between yeast growth promoting substances and the deterioration of the samples. 4. A second group of samples inoculated with pure cultures of yeasts isolated from deteriorated sugar and incubated at 22° and 30°C. and at 60 and 70 per cent relative humidity respectively showed that the combination of the high humidity and higher temperature was the most conduéive to deterioration. 5. It was Observed in this series of experiments "29.. that the growth of the yeasts and the deterioration of the sugar occurred at the Juncture of the sugar with the petri dish where the moisture collected and was held. This observation is important since it would explain why sugar stored for any length of time in warehouses under unfavorable conditions deteriorates. Conclusions. 1. It has been shown that certain yeasts are capable of causing deterioration of sugar. 2. Sugar should be stored in a cool dry place. r.30.. BIBLIOGRAPHY Beijerinck, w. 1896. §p§, octosporous. Cent. Bakt. gs. Browne, C. A. 1918. The Deterioration of Raw Cane Sugar. A Problem in Food Conservation. Jr. Ind. Eng. Chem. 1Q. 178. Browne, 0. A., and Church, M. 1928. The Growth of Micro~ Organisms on Sucrose Crystals, with Special Reference to Certain Phases of Sugar Deterioration. The 1928 Reference Book of the Sugar Industry of the World. Church, M. B., Paine, H. 8., and Hamilton, John, 1927. Sugaerolerant Yeasts in ChocolateHCoated Creams. Jr. Ind. Eng. Chem. 12, 353. Fabian, F. W., and Quinet, R. I. 1928. A Study of the Cause of Honey Fermentation. Tech. Bul. No. 92. Rich. Agr. Esp. Sta. Fabian, F. W., and Hall, H. H. 1933. Yeasts Found in Fermented Maple Syrup. Cent. Bakt., II Abt., Bd. 82. 31. Guilliermond, A. 1901. Recherches histologique sur la sporulation des Schizosaccharomycetes. Compt. Rendus. Acad. Sciences. 121, Guiliermond, A. 191“. Monographie des Levures Papportees d'Afrique Occidentale par la Mission Chevalier. Annales des Sciences Naturelles Botaniques. l2, (See also Guilliermond, A. 1920. The Yeasts p. 261.) Guilliermond, A. 1920. The Yeasts. Trans. by F. W. Tanner, John Wiley and Sons, New York. N. Y. Hall, H. H., and LothrOp, R. E. 193A. The Use of Clarified Honey in Culture Media. Jr. Bact. 21, 399. Hall, H. H., James, L. H. and Stuart, L. S. 1933. Yeast Growth Stimulants in White Sugars. Jr. Ind. Eng. Chem. g5. 1052. Kepeloff, Nicholas, and Kopeloff, Lillian, 1919. The Deterioration of Cane Sugar by Fungi. Bulletin. No. 166. La. Exp. Sta. Lochhead, A. G., and Heron, D. A. 1929. Microbial Studies of Honey. Bul. 116. N. S. Canadian Dept. of Agr. Marvin, G. E., Peterson, W. H., Fred, E. 8., and Wilson, H. F. 1931. Some of the Characteristics of Yeasts Found in Fermenting Honey. Jr. Agr. Res. 43. 121. Official and Tentative Hethods of Analysis. 1930. Association of Official Agricultural Chemists. 1 Owen, W. L. 1911. The Bacterial Deterioration of Sugars. Bul. No. 12’. La. Exp. Sta. Owen, W. L. 1918. The Deterioration of Cane Sugars in Storage; Its Causes and Suggested Heasures for its Control. -Bu1. No. 162. La. Exp. Sta. Owen, W. L. 1926. U. 8. Patent No. 1,579,387. Process for the Preservation of Sugar. Payen, 1851. Compt. rend. 33, 323, (Cited from Browne, C. A. and Church, a. B. 1928.) Sacchetti, D. M. 1932. Intorno ad alcuni “Zygosaccharomyces“. Rend. della R. accad. Naz dei Lincei. 15. 762. Saito, K. 1906. Mikrcbiologische Studien uber Soyabereitung. Cent. Bakt. ll- '1 Sanders, K. 1933. The Dampness cf Refined Goods; the Hygroscopicity of Sugar. Listy Cukrovar. 51, 314. c. A. 21. 35146. Schutt, D. B. 1925. Yeast Contamination as a Source of Explosion in Chocolate Coated Candies. Scientific Agriculture. 6, 118. StellingnDckker, N. M. 1931. Die Hefesammlung des ”Centraalbureau voor Schimmelcultures." Beitrage zu einer MonogrOphie der Hefsorten. Erster Teil. Die sporogenen Hefen. Verhandelingen der Koninklijke Akademie van Wetenschappen ts Amsterdan. Afdeeling Natuurkunde (Tweede Sectie) Deal 28. No. 1. VII + 5“? pp. Svatek, R. 1932. The Cause of Dampening of Crystalline Sugar. Listy Cukrovar. 5g. 430. c. A. g. (1932) 11971;. Vondrak, J. 1932. The Adsorption of Water by Sugar Crystals in Storage Chambers. Z. Zuckerind. Cechoslavmt. Rep. 51. 159. c. A. 21(1933) 2327. Wilson, R. E. 1921. Humidity Control by Means of Sulfuric Acid Solutions with Critical Compilation of Vapor Pressure Data. Jr. Ind. Eng. Chem. 13, 326. H 33 H Hi“? ’f.'.,.....~.._. _, si ROOM USE ONLY Mar 5 I4 9 KW. r' h ll}! III/7W1! 1minWWI/1W1firm/7W . 3 1 93 01730 3763