. S. DEPARTMENT OF AGRIC BURLY U OF ANIMAL INDUSTRY. BULLETIN No. 82 A. D. MtLVIN, CHIEF OF BUREAU. SOUTHERN BRANCH, UNIVERSITY OF CALIFORNIA, LISRA GI IN CHEESE RIPENING CAMEMBERT AND ROQUEFORT. BY CHARLES THOM, PH. D., Mycologist in Cheese Investigations > Dairy Division, Bureau of Annual Industry. SJ9AIUfl WASHINGTON: r.oVI-RNMF.NT PRINTING ()l-l 1M IfLKEAL OF ANIMAL A. D. MF.LVIX, IX V. s. intCh'n/: \. M. FAKKIM.TON. I 1 ,. S.. !>. V. M. /Clerk: K. B.JoNBS, LL. M., M. D. ,try Dirixion: Ki>. 11. WKBSTKI;, M. S., chief; CI.AKENCK B. LANK, 15. S.. ;issisi:nit chief. aspect ion Diritiion: KICK 1'. STEWJO.M, V. S., chief. Quarantine Division: IXICIIAHD W. HICKMAX, I'll. Ci., V. M. D., chief. Animal Husbandman: GEOKCE M. ROMMEL, B. S. A. /:;. .IAMKS M. PICKKNS. .b'//,s/: W.S. I). HAINES. Librarian: BEATRICE C. OBEKLY. LABORATORIES. Biochemic Division: MARION DORSET, M. I)., chief. Pathological Division: JOHN K. MOIILKI:, A. M., V. M. D., chief. Zoological Division: BRAYTON H. RANSOM, B. Sc., A. M., scientific assistant in charge. EXPERIMENT STATION. Superintendent: E. C. SCTIROEDER, M. D. V.; expert assistant, W. E. COTTON. MEAT INSPECTION. Inspectors in charge. Austin, Minn. Dr. M. O. Anderson, care George A. Ilormel .V Co. Baltimore, M<1. Dr. H. A. Hedrick, 215 St. Paul street. Bloomington, 111. Dr. Frederick Braginton, care Continental Packing Company. Boston, Mass. Dr. J. F. Ryder, 141 Milk street. Bright wood, Mass. Dr. \Y. J. Murphy, care Springfield Provision Company. Buffalo, N. Y. -Dr. 13. P. Wonde, Live Stock Ex- change P.iilcling, East Buffalo. Pj.pids, Iowa. Dr. T. A. Shipley, care T. M. F^K-iair A: C<. Chicago, 111. Dr. S. E. Bennett, room 316 Ex- change Building, Union Stock Yards. Cincinnati, Ohio. Dr. A. G. G. Richardson, care Union Stock Yards. Cleveland, Ohio. Dr. E. P. Schaffter, care Cleve- land Provision Company. Davenport, Iowa. Dr. E. L. Bertram, caie Henry Kohrs Packing Company. Denver, Colo. Dr. W. E. Howe, care Western Packing Company. Des Moincs, Iowa. Dr. A. B. Morse, care The Agar Packing Company. Detroit, Mich. Dr. L. K. Green, care Hammond, Standish & Co. Eau Claire, Wis. Dr. G. W. Butler, care Drum- mond Brothers. Fort Worth, Tex. Dr. A. II. Wallace, care Swift & Co. Hiitchinson, Kans. Dr. J. E. Blackwell, care Hutchinson Packing Company. 'Msnapolis, Ind. Dr. N. C. Sorenscn, care Kin- "o. N. J. Dr. Julius Huelsen, care The '"k Yard Company. -Dr. L. R. Baker, room 338 K. Rishel, care Cudahy "orge, 507 Johnson , care Macbeth & . F. Price, care -.re Jacob E. Milwaukee, Wis. Dr. A. E. Behnke, room 432 Federal Building. Nashville, Tenn. Dr. W. B. Lincoln, care Ten- nessee Packing and Provision Company. National Stock Yards, 111. Dr. ,1. B. Clancy. Nebraskq, City, Nebr.- Dr. W. II. Gibbs,care Mor- ton-G-regson Company. Newark, N. J. Dr. Thomas Castor, care Swift & Co., Harrison Station. New Haven, Conn. Dr. Albert Long, care Spcrry & Barnes. Xew York, N. Y. Dr. H. N. Waller, 109 West Forty-second street. Ottumwa, Iowa. Dr. Joshua Miller, care John Morrcll & Co. Philadelphia, Pa. Dr. C. A. Schaufler, 134 South Second street. Pittsburg, Pa. Dr. F. W. Ainsworth, ' Stock Yards. Portland, Oreg. Dr. Clarence Loveberry, room 402, custom house (new). Quincy.Ill. Dr. J.S. Kelly, care lilomer ear long, loosely divergent chains of conidia. Basidia 8 to 11 by 2.4 to 3 //; conidia at first cylindrical, then elliptical, and finally globose when ripe, smooth, bluish-green by transmitted light, thin-walled and com- monly guttulate, 4.5 to 5.5 /< in diameter, swelling in germination to 8 to 10 //. Germ- tubes one to several. Cells of mycelium about 5 by 20 to 40 f.i ; liquefies sugar gelatin only under the center of the colony. Changes blue litmus to red strongly at first, then after four to six days begins to turn the red back to blue at the center and continues outward concen- trically until all has become blue. Growing and fruiting period about two weeks. Fruits only upon exposed surfaces of the substrata never produces spores in cavities not very broadly open. Habitat, cheese. a Penicillium camemberti (nomen novum). This species is unquestionably the one referred to by Maze" in his recent papers as P. album Epstein. Professor Maze" was kind enough to show me the cultures. But the name P. album was already used by Preuss some fifty years earlier for a sjx'cies of Penicillium, hence by the rules of nomenclature should not be used again for a species whose identity with /'. album Preuss is not claimed by Epstein. Upon this ground Lindau, in Ralx>nhorst's Kryptogamcnflora, has changed the name of Epstein's fungus to /'. fpxteini Lindau. and extracted from the article written by Epstein a brief and totally insufficient diagnosis. A careful study of the physiological data given by Epstein shows that they differ from the data so far found for this species so materially as to lead to the probability that he was studying another form entirely. I therefore give /'. album Epstein in the list of possible synonymy only, localise the name is accepted by for what I know to be this species. 84 FUNGI IN CHEESE RIPENING. THE ROQUEFORT MOLD (PENICILLIUM ROQUEFORTl). The spores of the Roquefort mold grow very rapidly, often produc- ing new mycelium and ripe spores within thirty-six hours. The colo- nies are white at the very first, but begin to become green at the cen- ter within two days in a rapidly growing colony. Such a colony may become a half inch in diameter in the first two days. The mycelium is mostly submerged, but very close to the surface, and grows rapidly outward from the starting point in a radial manner, w^hich is rendered prominent by certain of the threads lying just under the surface for the most part, but making loops into the air by rising just above the sub- stratum for a little way, then reentering the medium again. This gives a grayish, almost cobwebby (arachnoid) , appearance to the mar- gin of the young colony. The rate of growth is not uniform in the cir- cumference of such a colony, which makes the border of a colony uneven instead of regularly circular, as most species appear. The superficial portion of the Roquefort mold is almost entirely composed of the fruiting hyphae or conidiophores, the vast majority of which arise as branches of submerged hyphae and consequently stand sepa- rately as short, unbranched threads of approximately equal length, which gives the surface a velvety appearance. They are usually 0.2 or 0.3 mm. or less in length, say one seventy-fifth of an inch. Such a colony spreads indefinitely in the substratum, so that the center will be composed of ripe fruit, while the margin is still actively growing. In laboratory culture, however, the development is so rapid that the entire surface is covered within the first few days; then growth ceases. The mycelium here, as in the Camembert mold, produces but a single crop of spores, then dies. These spores are a bright green at first, but in a short time become a dirty-brown color in dry culture. The spores of this fungus are much more resistant than those of the Camembert mold both to heat and to natural exposures. They will retain their viability for months in old cultures under the ordinary conditions of exposure in the laboratory. Upon a cheese this mold produces a bright green area which extends rapidly. Its action can be detected in a few days by the bitter taste of the curd near to the mycelium. A similar taste is, however, produced at least in some measure by other green forms, so that it is not diagnostic except as between this and the Camembert species. A colony upon the surface of a cheese becomes brown in two or three weeks, but colonies growing in the cavities which are so characteristic of the center of this type of cheese retain their bright green color for long periods. This mold is not limited to dairy products, but is widely distributed. It has been sent to the laboratory from the most distant correspond- ents. It has been found in silage, and in laboratory cultures from many substances. It has been found to be the green mold of Stilton, Gorgonzola, and Brinse, as well as in certain tvpes of prepared cheese CAMEMBERT AND KOQUEFORT. 35 purchased in the market. Once in a laboratory it stays and seems to get into everything. In other words, this is one of the cosmopoli- tan and omnivorous species of the genus. One character seems to differentiate this mold from most of the others that is, its power of growing into and fruiting normally within narrow cavities, such as appear in cheese. It appears that this character exerts a sort of automatic (perhaps we may call it a truly "natural") selection which eliminates all other species from the ripening processes of Roquefort and related types of cheese. n PIG. 2. Roquefort Penicillium (P. roqueforti). a, part of conidiophore and of has of fructification, highly magnified, showing the production of basidia on the sides as well as at the apex of the basidiophore; b, c, other types of branching; d, young conidiophore just branching; e, f, basidia and the formation of conidia, highly magnified; g, h,j, diagrams of types of fructification as seen under low power (x 80); k, I, m, n, germination of c-onidia and new conidia produced directly on the first hyphse. TECHNICAL CHARACTERIZATION OF THE ROQUEFORT MOI.D.O A technical characterization is offered of Penicillium roqueforti (fig. 2), as follows: Colonies quickly turning green, becoming a dirty brown in age, velvety strict, indetermi- nately spreading by large main radiating, branching hyphse, giving a somewhat uneven or a Penicillium roqueforti (nomen novum). In offering a new specific name for this well- known fungus, the author is perfectly aware that the mold is often referred to in the litera- ture as P . glaucum. A careful study of the literature fails to disclose a single description which indicates that this is identical with the plant descril>ed as P. glaucum. As a prelimi- nary step, therefore, to the proper determination of the green species of Penicillium which have hitherto l>een collectively referred to as P. glaucum, this very distinct and easily rec- ognized form is named from its universal (x-currence P. roqueforti. 36 FUNGI IN CHEESE RIPENING. indefinite margin, which gets a white, fibrous, almost spider-web appearance from its alter- nation of submerged parts of hyphir with short prostrate aerial loops; reverse of colony yel- lowish white. Conidiophores arising separately and in acropetal succession from the grow- ing parts of submerged hypha* (comparatively few from aerial parts, but some), 200 to 300 /.i septate. Fructification 90 to 120 ;< or at times 160 /< by 30 to 60 // at broadest place, usually appearing double by the divergence of the lowest branch ; branchlets (basidiophores) irregularly verticillate, bearing crowded verticils of appressed basidia 9 to 11 // by 2.5 // with long divergent chains of conidia. Conidia bluish green, cylindrical to globose, smooth, rather firm-walled, 4 to 5 // in diameter, germinating by a straight tube. Colonies do not liquefy sugar gelatin, though they soften it somewhat. The fungus changes litmus from red to blue very rapidly and strongly, almost from the beginning of growth. Fruiting period short, but one crop of spores upon the mycelium. Cosmopolitan and omnivorous, or nearly so. Characteristic of Roquefort and related types of cheese. OIDIUM LACTIS. The mold (fig. 3) variously known as Oidium, or Oospera, lactis is another cosmopolitan organism. This fungus differs widely from the species previously described. Inoculated into any suitable medium it grows with enormous rapidity. A single spore (or oidium) may give rise to several centimeters of mycelium and hundreds of spores in twenty-four hours. It prefers very moist situations, since almost the entire mycelium is developed below the surface of the sub- stratum. It is therefore passed unnoticed many times or produces changes which are attributed by the observer to bacteria. Descrip- tion, therefore, must depend upon microscopic characters. The study of the border of the young colony shows numerous vegetative hyphse radiating outward. Each of these is found to divide dichotomously (fig. 3, a, &), so that the border is a crowded series of forking branches. In the older parts of the mycelium a branch may be produced at each end of eA r ery cell, or several at each end, and these branch indefinitely. The fruiting branches are mostly produced as outgrowths from the dis- tal ends of the cells. These extend upward into the air or remain en- tirely submerged in many cases. From the ends of these outgrowths one to several rows of oblong or cylindrical cells begin to be pinched off. If extending above the surface this gives rise to chains of delicate shim- mering cells appearing as a powdery covering upon the surface, which can be seen with a good lens to be arranged in chains. In some strains of Oidium all of these chains (and some of the chains in all strains) of spores remain submerged and germinate at once, so that they give rise to unintelligible mats of hyphae. Oidium produces a very slight acid reaction to litmus at first, then a strong and continued alkaline reaction. It liquefies sugar gelatin under the colonies, but does not extend the area of liquefaction beyond the edge of the colony. Oidium always and everywhere tested has produced a strong and very characteristic odor. Once familiar with this odor the worker may recognize its presence by its spores or oidia, which are hyaline, CAMEMBERT AND ROQUEFORT. 37 smooth, cylindrical, 3.5 to 5 /* by 6 to 30 /*, varying with the condi- tions and the substratum and perhaps at times exceeding these limits. These swell variously and germinate in many ways, so that no germi- nation characters are definite. Upon some media this mold may be induced to produce a large growth of aerial mycelium, but the limits here denned will include the variations to be found upon the usual culture media. Oidium lactis is described as universally present on milk and its products. Epstein even suggests that experiments upon milk and cheese can not be freed from its presence without sterilizing. The FIG. 3. Oidium lactis. a, b, dichotomous branching of growing hyphac; c. d, (j, simple chains of oidia breaking through substratum at dotted line y-ij, dotted portions submerged; e, f, chains of oidia from a branching outgrowth of a submerged cell; h, branching chain of oidia; k, I, m, n, o, p, s, types of germination of oidia under varying conditions; t, diagram of a portion of a colony show- ing habit of Oidium lactis as seen in culture media. same or almost indistinguishable forms are found upon decaying vege- tables and fruits, which may give reason for the statement that the odor produced by Oidium is that of rotten cabbage. There seems to be good reason for saying that all these forms are but varieties or strains of the same species. Comparison of several of them shows that under uniform conditions the morphology of all these forms is very nearly the same. This is largely true also of their physiological effects. This mold has been much studied and numerous papers dis- cuss its nature and physiological effects as well as its relationships. 38 FUNGI IN CHEESE RIPENING. It will be sufficient to describe here the fungus and to give figures to assist in its recognition. Its relations to the problems of cheese ripen- ing have already been indicated. SUMMARY. CAMEMBERT CHEESE. The acidity of the curd resulting from the action of lactic organ- isms reduces where it does not entirely eliminate the growth of objec- tionable bacteria. Many species of dairy fungi exert in the course of their development the power of changing this reaction to alkaline. The Camembert Penicillium and Oidium lactis possess this power, but not in greater degree than many other species. Many species of fungi possess the ability to change curd to a greater or less extent. The breaking down of curd by fungi is due in the cases studied to the production of enzymes. The texture, appearance, and flavor of curd acted upon by such fungi are different for different species. The Camembert Penicillium (P. camemberti) is the only species so far studied with, which the particular appearance and texture sought in the ripened Camembert can be produced from curd soured by lactic bacteria without producing any objectionable flavor. Oidium lactis is always found upon Camembert cheese and so closely associated with the presence of the flavor as to indicate its agency in flavor production, though only circumstantial proof of such function has been possible thus far. The participation of bacteria in flavor production is not excluded by these results. Other species of fungi have been shown to produce variations in this flavor such as have been often found in certain market cheeses. In this way it is possible to look for the cause of differences in flavor in contamination of the cultures upon the cheeses. This points toward the use of pure cultures for inoculation, with the addition of special organisms if certain variations from what we have regarded as typical flavor are found to be of value in the market rather than dependence upon accidental occurrence of the desired species in the factory. ROQUEFORT CHEESE. In the ripening of Roquefort cheese the only organisms found neces- sary are lactic bacteria and the Roquefort species of Penicillium. The Roquefort Penicillium has been shown to possess the power to reduce the acidity, to digest the curd, and to produce the typical flavor. CAMEMBERT AND ROQUEFORT. 39 OTHER VARIETIES OF CHEESE. The Roquefort species of Penicillium is found in the imported Stil- ton, Gorgonzola, and Brinse, as well as in Roquefort cheese. Oidium lactis alone of the forms studied has been found upon the various brands of Limburger, Brie (American type), Isigny, and related cheeses found in the market. Other species incidentally occur, but not uniformly, and such occurrence is avoided as far as possible by the makers. BIBLIOGRAPHY. (1) CONN, HERBERT WILLIAM; THOM, CHARLES; BOSWORTH, A. W.; STOCKING, W. A., Jr., and ISSAJEFF, T. W. The Camembert type of soft cheese in the United States. Bull. No. 71, U. S. Department of Agriculture, Bureau of Animal Industry. Washington, 1905. Also published as Bull. No. 35 of the Storrs Agricultural Experiment Station, Storrs, Conn., Apr., 1905. (2) CONN, HERBERT WILLIAM. Bacteria in milk and its products. Illus. 306. pp. Phila- delphia, Blakiston's Sons & Co., 1903. See p. 268. (3) EPSTEIN, STANISLAUS. Untersuchungen iiber die Reifung von Weichkasen. Arch. f. Hyg., Bd. 43, Hft. 1, pp. 1-20; Bd. 45, Hft. 4, pp. 354-376. Munich and Leipzig, 1902. (4) JOHAN-OLSEN, OLAV. Die bei der Kasereifung wirksamen Pilze. Cent. f. Bakt., Abt. 2. Bd. 4, No. 5, pp. 162-169. Jena, March 5, 1898. (5) CONSTANTIN, J., and RAY, J. Sur les champignons du fromage de Brie. Compt. rend. Soc. de biol., Paris, ser. 10, t. 5, No. 16, pp. 504-507. Paris, May 13, 1898. (6) ROGER, GEORGES. [Article in] Revue hebdomadaire, v. 7, p. 334. Paris. (7) MARGARET, pseudonym. The practice of cheesemaking at home and abroad. The Creamery Journal, v. 1, No. 11, pp. 313-315. London, July 20, 1905. <8) EPSTEIN, STANISLAUS. See Citation 3, above, p. 373. (9) MAZE, P. Les microbes dans 1'industrie fromagere. Ann. de 1'Inst. Past., ann. 19, No. 6, pp. 378-403, June 25; No. 8, pp. 481-493, August 25. Paris, 1905. (10) SMITH, ERWIN F., and SWINGLE, DEAN B. The dry rot of potatoes, due to Fusarium oxysporum. Bull. No. 55, U. S. Department of Agriculture, Bureau of Plant Industry. Washington, February 16, 1904. LANG, M.. and FREUDENREICH, EDUARD VON. Uber Oidium lactis. Landwirthschaftl. Jahrbuch der Schweiz, Bd. 7, pp. 229-237. Bern, 1893. MARPMANN, G. Beitrage zur Kaseflora. Ztschr. f. angewandte Mikroskopie, Bd. 2, Hft. 3, pp. 68-79. Berlin, June, 1896. TEICHERT, KURT. Beitrage zur Biologic einiger in Molkereiproduction vorkommenden Schimmelpilzen. Milch-Xeitung, v. 32, No. 50, pp. 786-787. Bremen, December 12, 1903. THOM, CHARLES. Some suggestions from the study of dairy fungi. Jrn. of Mycology, v. 2, No. 77, pp. 117-124. Columbus, Ohio, May, 1905. o 1 6 1995 RLF QUARf ER LOAN i lied of sheep and rattle in't he \Vest. Albuquerque. N. Mex. --Dr. Louis Metsker, room 22 X. T. Annijo Building. I >en ver, Colo. Dr. Lowell Clarke, room 320 Quincy Building. 1VN1) ERADICATION OF CONTAGIOUS DISEASES. Inspectors in charge of districts. '{JO Quincy Building. Fargo, X. Dak. Dr. R. II. Tr. irge of eradication of Kansas Cily, Ivans. --Albert Dean', room ;!js Lh Exchange. Salt Lake City, Utah. Dr. George S. Hickox. room 21 Eagle block. INSPECTION OF LIVE STOCK FOK EXPORT. Inspectors in charge. Baltimore, Md. Dr. II. A. Iledrick, 21o St. Paul street. New York, X. Y. Dr. W. IL Rose, is Broadway. ,N 7 orfolk, Va. Dr. G. C. Faville, I 1 . (.). l,i.\ 7%. Philadelphia, Pa. Dr. C. A. Schaufler, 13-1 South Second >t ; Portland, Me. Dr. F. \V. Huntington, U. S. cus- toms oiliee. (Jrand Trunk R. R. wharf. INSPECTION AND QUARANTINE OF IMPORTED ANIMALS. Qjiarantine stations. Athonia, N. J. (for the port of New York). Dr. George \v. Pope, superintendent. Halethorp, Md. (for the port of Baltimore). Wil- liam II. Wade, superintendent. Littleton, Mass, (for the port of Boston). Dr. J. V. Ryder, inspector in charge. Ml Milk 'street, Boston, Mass. Inspectors on the Canadian border. . Me. Dr. II. T. Potter. Carthage, X. Y. Dr. W. S. Corlis. .1 v Mich. Dr. L. K. Green, care Hammond, Standisli ,\ . .'it-field, Me. Dr. F. M. Perry. Malone, N. Y. Dr. II. D. Mavne. Xcwport, Vt. Dr. G. W. Ward. Ogdensburg, X. Y. Dr. Charles Co\vie. Orono, Me. Dr. F. L. Russell. Port, Huron, Mich. Dr. David dimming, 012 Lapeer avenue. St. Alhan-s, \'t. Dr. C. L. Morin. Sault Ste. Marie, Mich. Dr. J. F. Deadman. Inspectors on the Mexican border. Kl I'aso, Tex. Dr. Thomas A. Bray. San Antonio, Tex. Dr. Joseph \V. "Parker. San Diego, Cal. -Dr. Robert Darling, care Charles S. Hardy. VETERINARY INSPECTORS STATIONED ABROAD. Dr. W. H.Wray, 34Streatham Hill, London, S. W., Dr. James Johnston, care U. S consulate 1 iver- Eagland,inchargef or Great Britain and Ireland. pool, Knirlimd. Dr. 'I'. A. (ieddes, care U. S. consulate, London, Dr. V. A. Xiirgaard, Honolulu, Hawaii England. DAIRY INSPECTORS. M. W. Lang, room 23 Marine Building, <: Holier? MeAdam, room 23 Marine Building, Chi- i, 111. K. A. McDonald JS and :>'.> Downs block, Seattle, Wash. B. F. VanValkcnburgh, His Chambers street, New- York, N. Y. Levi \Vells, S|)ring Hill, Pa. G. M. Whitaker P. O. box 1332, Boston, Mass.