Microbiology of Meat Curing I. The Occurrence and Significance of a Motile Microorganism of the Genus Lactobacillus in Ham Curing Brines R. H. DEIBEL AND C. F. NIVEN, JR.' Division of Bacteriology, American Meat Institute Foundation, Chicago, Illinois In contrast with earlier curing methods, the commercially produced modern American ham is subjected to a relatively light cure of short duration. The hams are pumped vascularly to approximately 10 per cent of their weight with a curing solution containing sucrose or glucose, sodium nitrate, sodium nitrite, and sodium chloride. The concentration of these constituents may vary within limits from one establishment to another, but in most instances the sodium chloride concentration ranges from 60 to 70 per cent of saturation. After pumping, the hams are generally submerged in a curing brine of similar composition and held at 3 to 4 C for a time ranging from a few days to 4 weeks. The hams are then removed, washed, trimmed, placed in stockinettes, and smoked and thermally processed to an internal temperature of 60 C or higher. Because of the relatively rapid methods of commercial curing of the modern ham, a study was initiated to determine what role microorganisms might play in the development of the characteristic ham flavor. In the course of this study, a unique, motile species of Lactobacillus was recognized as comprising a major proportion of the bacterial flora in ham curing brines toward the end of the curing period. The characteristics and significance of the motile Lactobacillus species are presented in this paper. MATERIALS AND METHODS Samples of curing brines of various ages, and cured but not smoked hams, were obtained from local packing plants. In addition, samples were examined from a number of hams and curing brines that had been cured experimentally in the laboratory. These samples were plated quantitatively in APT agar' (Evans and Niven, Received for publication February 10, 1958 1 Journal paper no. 153, American Meat Institute Foundation. This research was undertaken in cooperation with the Quartermaster Food and Container Institute for the Armed Forces, and has been assigned number 821 in the series of papers approved for publication. The views or conclusions contained in this report are those of the authors. They are not to be construed as necessarily reflecting the views or indorsements of the Department of Defense. 2APT medium contains per L: tryptone, 10 g; yeast extract, 5 g; NaCI, 5 g; K2HP04, 5 g; sodium citrate, 5 g; glucose, 10 g; Tween 80, 1 g; MgSO4-7H20, 0.8 g; MnCl2.4H20, 0.14 g; and FeS04-7H20, 0.04 g. For plating, add 1.5 per cent agar. 323 1951) and incubated at 30 C. Various temperatures of incubation, media, and concentrations of sodium chloride in the medium were used in preliminary experiments. The use of APT agar and incubation at 30 C resulted in the highest total counts. The physiological characteristics of the lactobacilli were determined in a basal medium containing tryptone (Difco), 10 g; yeast extract (Difco), 5 g; K2HPO4, 2 g; glucose, 0.5 g; bromeresol purple, 0.04 g; distilled water to 1 L; ph 7.0 to 7.2. The inoculum consisted of 1 drop of a 24-hr culture grown in APT broth. Carbohydrates were autoclaved separately and added aseptically to the basal medium. All cultures were reisolated to insure purity. Testing for the presence of catalase was accomplished using the YTG medium' of Felton et al. (1953). The temperature limits of growth and tolerance to sodium chloride were determined using APT broth as the basal medium. Final ph values were determined in a 2 per cent glucose-apt broth. Esculin hydrolysis was detected by the addition of ferric ammonium citrate to broth cultures. The hydrolysis of sodium hippurate was determined by the addition of 0.5 ml of 1:1 sulfuric acid to 1 ml of supernatant aliquots of the broth cultures and the detection of benzoic acid crystals. Some difficulty was encountered in determining the optical rotation of the lactic acid produced from the fermentation of glucose by motile lactobacilli. All cultures were grown in a 2 per cent glucose broth. These cultures had to be neutralized frequently to avoid a cessation of fermentation and a consequent diminished lactic acid yield. The lactic acid was converted to its zinc salt prior to determination of its optical rotation. The water of hydration of the zinc lactate was also determined, and the values obtained agreed with the optical activity of the respective samples. Motility was determined by conventional microscopic methods using 18- to 24-hr cultures. The cultures tended to lose their motility upon prolonged incubation. A semisolid agar medium, as described by Clark and Carr (1951), was also used for determining motility. The 3 YGT agar consists of: tryptone, 1.0 per cent; yeast extract, 0.5 per cent; NaCl, 0.5 per cent; KHP04, 0.2 per cent; glucose, 0.05 per cent; agar, 1.5 per cent.
324 R. H. DEIBEL AND C. F. NIVEN [VOL. 6 most suitable agar concentration to demonstrate motility among the lactobacilli appeared to be 0.2 per cent. Occasional strains were found which had been designated as nonmotile by the direct microscopic method but which proved to be motile in the semisolid agar medium. RESULTS The total bacterial population of the ham curing brines examined in this study ranged from 50,000 to 36,000,000 per ml, with a median of approximately 1,000,000. The brines had been in use for 3 to 30 days at the time of obtaining the samples. The higher counts were noted in the older brines. In most instances, the brines at the end of the cure contained 1,000,000 bacteria per ml or less. The lactobacilli isolated in this study were the most common contaminants encountered in commercial as well as experimental hams and curing brines. All of the strains of the genus Lactobacillus were gram-positive, catalase-negative, nonsporeforming rods that did not reduce nitrate to nitrite. All strains were homofermentative. Optimum temperature of growth was approximately 30 C for the strains studied in detail. A relatively large number of the Lactobacillus strains isolated in this study presented a striking morphological picture upon primary isolation (figure 1). Some of the cells as observed by microscopic examination were straight rods, however, the majority were curved in appearance. Studies to ascertain the environmental conditions which produce the curved state of these bacteria have been unsuccessful. Although the occurrence of these curved rods was associated initially with a high salt content of the growth medium, successive transfers, with or without sodium chloride, eventually resulted in the loss of this characteristic morphological feature. Incubation over a wide range of temperatures in varying concentrations of sodium chloride failed to retain the cultures as curved rods. Occasionally, the straight rods showed a sporadic tendency to revert to the curved state. The finding of curved lactobacilli has been reported by previous investigators. Orla-Jensen (1919) observed curved rods in cultures of Lactobacillus casei and Lactobacillus plantarum. More recently, Harrison and Hansen (1950a) reported the tendency of a strain of L. plantarum to show curved forms. The motile ham lactobacilli varied in size over a rather wide range, even in a single culture. The mean length of the rods was approximately 3.5,u. As demonstrated in figure 1, there was a tendency for the ends of a pair of rods to meet to form a doughnut-likc appearance. Seventeen of the motile lactobacilli were selected for Downloaded from http://aem.asm.org/.a Juk. VW.W.- -Ag: ""M..... ik.a_. on September 10, 2018 by guest.- bw- _.~~~~~~~~~~~~~~A L.0i' ww Fiue1 hrceitcapaac ftemtl acoail npiayioainfo uigbie and1unrcse hm
1958] MICROBIOLOGY OF MEAT CURING 325 detailed physiological study. A summary of these studies is presented in table 1. These studies indicate that the motile lactobacilli comprise a rather unique group of microorganisms. As indicated in the table, they form a relatively homogeneous group with the exception of variations among the fermentation tests. In addition to their motility and peculiar morphology at the time of primary isolation, they are characterized by their rather weak fermentation capacity, their ability to grow at low temperatures and in high salt concentrations, and the production of the l(+)-isomer of lactic acid from the fermentation of glucose. In his description of L. plantarum and L. casei, Orla- Jensen (1919, 1943) differentiated these two species in that L. plantarum fermented melibiose and frequently fermented raffinose, inulin, and the pentoses. The lactic TABLE 1 Physiological characteristics of the motile lactobacilli fr ont hamii curing brines CO2 from glucose Catalase produced Nitrate reduced Acetylmethylearbinol produced Litmus milk Final ph, glucose broth Hydrolysis: Sodium hippurate Esculin Arginine Gelatin Starch Growth: 10 C 40 C 45 C 10% NaCI broth Fermentation: Xylose Arabinose Lactose Raffinose Inulin Glycerol (aerobic) Glycerol (anaerobic) Mannitol Sorbitol Melibiose Rhamnose Sorbose a-methyl-d-glucoside Lactic acid isomer from glucose Slightly acid or no change 4.9 to 5.3 13+; 4-15+; 2-3+; 14-11+; 6-9+; 4-4+; 9- + 6+; 11-4+; 11-1+; 14-11+; 4- I(+) All strains fermented mannose, fructose, galactose, maltose, sucrose, trehalose, salicin, and cellobiose. None of the strains fermented adonitol, melezitose, dulcitol, inositol, or a-methyld-mannosi de. acid produced from glucose was usually optically inactive, although some strains produced an excess of the dextro-rotatory isomer. More rarely, pure dextrorotatory lactate was produced. In contrast, L. casei did not ferment melibiose and "only exceptionally" fermented pentoses, raffinose, and inulin. Dextro-rotatory lactic acid was usually produced by this species. In view of their inability to ferment melibiose, as well as the characteristic production of pure dextrorotatory lactic acid, it would seem that the motile lactobacilli from hams are related to L. casei. However, due to their high final ph value, pentose and inulin fermentation, and inability to hydrolyze sodium hippurate, the taxonomic identity of the motile lactobacilli remains obscure at the present time. Rogosa et al. (1953) demonstrated that L. casei characteristically hydrolyzes sodium hippurate. In the course of this study a number of lactobacilli were isolated from ham curing brines which had characteristics identical to the motile strains with the exception of motility. Therefore, it would seem that motility itself is not a satisfactory primary characteristic for species delineation. However, this character, coupled with other unique features, may well serve to delineate a species. Attempts to further identify the motile lactobacilli by use of serological methods met with failure. Rabbits were injected intravenously (5 days per week) with suspensions of heat-killed cells over a period of 4 weeks. Three representative cultures were employed for these injections. When extracts prepared by the conventional Lancefield technique were tested against the rabbit sera, no positive precipitin reactions were obtained, even among the homologous antigens. Therefore, further attempts along these lines were abandoned. Further investigations of the physiological characteristics of the motile lactobacilli revealed that virtually all cultures produced and accumulated measurable quantities of hydrogen peroxide when grown aerobically in broth culture. Consequently, when streaked upon the cut surface of frankfurters, the motile cultures were able to discolor the cured meat pigment. DISCUSSION The occurrence of motile lactobacilli has beeii reported previously in the literature. Cunningham and Smith (1940) recorded the presence of motile microorganisms in silage which appeared to be lactobacilli. Harrison and Hansen (1950a, b) isolated a motile culture of L. plantarum from turkey cecal feces. Hays and Reister (1952) reported the occurrence of motile L. plantarum in spoiled, frozen, concentrated orange juice. Mann and Oxford (1954) isolated three motile homofermentative, mannitol-fermenting lactobacilli from the calf rumen, and which could not be identified as to species. More recently, Vankova (1957) noted
4326 R. H. DEIBEL AND C. F. NIVEN [VOL. 6 the occurrence of motile, catalase-positive strains of Lactobacillus delbruckii that were used in commercial fermentations. With the possible exception of strains of Lactobacillus pentoaceticus, described by Fred et al. (1919) and Weinstein and Rettger (1932), it should be noted that all motile lactobacilli reported thus far belong to homofermentative species. During the course of this study, three motile cultures from orange juice were obtained from Hays and Reister, and the turkey cecum culture from Harrison and Hansen. These cultures were included for comparative purposes. Table 2 presents a few differential characteristics among the motile strains studied. Included in this table are the results reported by Mann and Oxford (1954) on the three motile strains from the calf rumen. The cultures from the turkey cecum and calf rumen have characteristics much in common and probably should be considered one species, as suggested by Mann and Oxford. On the other hand, the orange juice strains, as well as the strains from curing brines, are distinct within themselves. Therefore, it would seem that motile lactobacilli may be represented among a number of different species. In the light of the fact that motile, homofermentative lactobacilli appear to be rather common in nature, that catalase-positive lactobacilli are known to exist (Dacre and Sharpe, 1956; Vankova, 1957), and, indeed, some are able to reduce nitrate (Costilow and Humphreys, 1955), it would seem to be necessary to broaden the concept of the generic delineation now recognized for this group of lactic acid bacteria. In spite of the fact that the motile lactobacilli usually comprised the majority of the flora in ham curing brines, it is highly doubtful that they influence the curing or final characteristics of the ham. In fact, it is TABLE 2 Comlparison of homofermlentative, miotile lactobacilli isolated fr'omt1 variouts souirces Present Harrison Hays and Mann and restu Study and Hansen Reister Oxford (1950a, b) (1952) (1954) Spoiled Source of cultures... Hams and Turkey concentra- Calf rumen curing brines cecum ted orange juice Number of strains... 17 1 3 3 Final ph.... 5.1 3.9 4.0 4.0 Growth: 10c... 40C... 45C... 10% NaCl... + Lactic acid isomer from glucose... I d1* dl dl* * An exces;s of l(+) lactic acid is produ]ed byi these strains. difficult to conceive that any microorganism is involved in the manufacture and development of the characteristic flavor of the modern commercial ham. The streamlined curing techniques now employed, accompanied by rather prompt thermal processing in the smokehouse, generally precludes the possibility of high bacterial populations in any stage of their manufacture. Other studies have demonstrated a consistently low bacterial population in the interior of the intact ham throughout the curing and smoking procedures. The surface contaminants, largely originating from the curing brine and subsequent handling, are greatly reduced in numbers in the smokehouse before they are given a chance to grow extensively. Experiments have been conducted in the laboratory in which the brines used for pumping and covering the hams deliberately were inoculated with large numbers of the motile lactobacilli. The subsequent smoking schedule of these hams was adjusted to encourage extensive growth of the inoculated bacteria throughout the hams. In spite of the high populations achieved, the resulting organoleptic characteristics usually were not very different from uninoculated control hams processed in a similar manner. In a few instances the inoculated hams developed undesirable flavors and odors. ACKNOWLEDGMENTS The authors wish to express their appreciation to Drs. A. }'. Harrison, Jr., and G. T. Hays for cultures of motile lactobacilli used in this study for comparative purposes. Dr. C. Edith Weir conducted the taste panel tests on the incubated hams employed in this study. SUMMARY Among the microorganisms detected in ham curing brines and on the surface of cured unprocessed hams, a motile, homofermentative species of Lactobacillus was found to be one of the most common contaminants. This species of Lactobacillus was characterized by its peculiar curved appearance upon primary isolation, its high salt tolerance, its weak fermentative ability, and the production of the l(+)-isomer of lactic acid from glucose fermentation. The motile lactobacilli possessed characteristics in common with both Lactobacillus casei and Lactobacillus plantarum, but appeared to be distinct from either one of these. 'Nonmotile strains of Lactobacillus were isolated that otherwise were identical to the motile variety. Evidence indicates that the motile lactobacilli are of no significance in the curing of hams. REFERENCES CLARK, F. E. AND CARR, P. H. 1951 Motility and flagellation of the soil corynebacteria. J. Bacteriol., 62, 1-6.
19581 SEPARATION OF BACTERIAL SPORES BY FOAM FLOTATION327 327 COSTILOW, R. N. AND HUMPHREYS, T. W. 1955 Nitrate reduction by certain strains of Lactobacillus plantarum. Science, 121, 168. CUNNINGHAM, A. AND SMITH, A. M. 1940 The microbiology of silage made by the addition of mineral acids to crops rich in protein. II. The microflora. J. Dairy Research, 11, 243-265. DACRE, J. C. AND SHARPE, M. E. 1956 Catalase production by lactobacilli. Nature, 178, 700. EVANS, J. B. AND NIVEN, C. F., JR. 1951 Nutrition of the heterofermentative lactobacilli that cause greening of cured meat products. J. Bacteriol., 62, 599-603. FELTON, E. A., EVANS, J. B., AND NIVEN, C. F., JR. 1953 Production of catalase by the pediococci. J. Bacteriol., 65, 481-482. FRED, E. B., PETERSON, W. H., AND DAVENPORT, A. 1919 Acid fermentation of xylose. J. Biol. Chem., 39, 347-384. HARRISON, A. P., JR., AND HANSEN, P. A. 1950a A motile Lactobacillus from the cecal feces of turkeys. J. Bacteriol., 59, 444-446. HARRISON, A. P., JR., AND HANSEN, P. A. 1950b Lactobacilli from turkeys. J. Bacteriol., 60, 543-555. HAYS, G. L. AND REISTER, D. W. 1952 The conitrol of "offodor" spoilage in frozen concentrated orange juice. Food Technol., 6, 386-389. MANN, S. 0. AND OXFORD, A. E. 1954 Studies of some presumptive lactobacilli isolated from the rumens of young calves. J. Gen. Microbiol., 11, 83-90. ORLA-JENSEN, S. 1919 The lactic acid bacteria. Mem. Acad. RoV. Sci., Danemark, Sect. Sci., 8 ser., 5, 81-197. ORLA-JENSEN, S. 1943 The lactic acid bacteria. Ergainzungsband. Mem. Acad. Roy. Sci., Danemark, Sect. Sci. Biol., 2, (3), 1-145. ROGOSA, M., WISEMAN, R. F., MITCHELL, J. A., DISRAELY, M. N., AND BEAMAN, A. J. 1953 Species differentiation of oral lactobacilli from man including descriptions of Lactobacillus salivarius nov. spec. and Lactobacillus cellobiosus nov. spec. J. Bacteriol., 65, 681-699. VANKOVA, J. 1957 Motile catalase-producing strains of Lactobacillus delbruckii. Nature, 179, 204. WEINSTEIN, L. AND RETTGER, L. F. 1932 Biological and chemical studies of the Lactobacillus genus with special reference to xylose fermentation by Lactobacillus pentoaceticu.s. J. Bacteriol., 24, 1-28. Separation and Concentration of Bacterial Spores and Vegetative Cells by Foam Flotation Selective methods for removing and/or concentrating bacterial spores and vegetative cells from the growth medium are essential in studies on spore physiology and antigenicity, phagocytosis, and similar problems in which cell preparations free of cellular debris are desired. Development of a method to obtain "clean" preparations followed the observation that masses of material collected above the liquid level in the head of foam when Bacillus anthracis was grown in aerated deep cultures. When this material was smeared and stained, microscopic observation indicated that the material was composed of essentially clean spores. Frequently, on those occasions when masses of the material collected or when foam was incompletely controlled and lost through the air vent, the spore count of the culture was low. These observations led to the conclusion that a collection process could be developed that would separate B. anthracis spores from vegetative cells and cellular debris contained in the culture medium. Such a method of purification by flotation was developed and is reported in this paper. While this manuscript was being edited, it was learned that similar independent observations had been made and the same conclusions I Deceased. W. A. BOYLES1 AND R. E. LINCOLN U. S. Army Chemical Corps, Fort Detrick, Frederick, Maryland Received for publication February 12, 1958 drawn by Black et al. (1958) who observed the loss of Bacillus cereus spores from deep culture fermentations after uncontrolled foaming. In the present study, simple glass cylinders equipped with spargers or diffusers were used as foaming chambers. Serratia marcescens cells were first successfully separated and collected and, with the development of equipment and a working procedure, cells of Brucella suis and Pasteurella (Bacterium) tularensis and spores of B. anthracis and Bacillus subtilis var. niger (B. globigii) were processed. Although major emphasis has been placed on the results obtained with B. anthracis, data on several other species are given to supply additional information on the flotation of cells or spores. THEORY AND BIOLOGICAL APPLICATION OF FOAM PRODUCTION Production of foam requires a lowering of the surface tension and a certain degree of heterogeneity of molecules at the gas-liquid interface. High viscosity and presence of long-chain molecules aid foam formation. Foam stability depends upon concentration of foamproducing agents, whereas the amount of foam is a function of the method by which it is formed and the