Sept. 4, 1956 J. SZUECS 2,761,246 MUSHROOM CULTURE Filed May 19, 1953 Fig. / 2,761,246 United States Patent 0 _, lC€ Patented Sept. 4, 1956 1 2 taste, ?avor and texture; the provision of methods of growing mushroom tissue of the genus Morchella under submerged aerobic conditions in liquid substrates com 2,761,246 prising a wide range of low cost nutrient: material, par MUSHROOM CULTURE ticularly molasses, Jerusalem artichoke and wholly syn thetic mediums; with a wide tolerance to solution con Joseph Szuecs, Yonkers, N. Y. centration and solution contamination. Referring now to the drawing, Application May 19, 1953, Serial No. 355,949 Fig. 1 is a diagrammatic sketch of the apparatus for 16 Claims. (Cl. 47—1.1) carrying out the submerged aerobic growth according to the invention; Fig. 2 is a photolithograph of the pellet-like Hel vellaceae Morchella esculenla product produced in ac cordance with the teachings of ‘the invention. The present invention relates to the growth of edible 15 The apparatus shown in Fig. l is of a size and con mushroom mycelium of the family Helvellaceae in a struction adaptable for the carrying out of the method simple, submerged, aerobic medium. According to the according to the invention on a scale suitable for the invention, a mushroom product is obtained, itself capable quantity of solutions hereinafter given by way of ex of synthesizing growth promoting substances such as ample. It will be obvious to those skilled in the art that vitamins. , 20 larger and more elaborate apparatus can and should be In my prior U. S. patent application Serial No. 21,845 employed for commercial production in accordance with ?led April 19, 1948, I described a method of providing an the invention. edible mushroom mycelium product under submerged Referring to Fig. 1, 1 is an especially constructed ?ask aerobic conditions in a nutrient-containing, liquid sub having a capacity of between 1% to 3 gallons. At the strate. The present invention relates to further improve 25 top or neck of the ?ask,.is a mercury seal 2 through which ments and discoveries pertaining generally to the same passes shaft 3 rotated by an electric motor or other suit ?eld as the aforesaid copending application. able means. As the conditionpinside the flask 1 must be There are more than two thousand species of edible as sterile as possible, the mercurial seal 2 prevents the mushrooms. Of these many species, only three can entrance of air into the ?ask 1 and the stopper 9, con be grown with commercial success. (1) Agaricus necting the mercurial seal to the ?ask, can be a grounded campestris, ordinarily grown in stable manure, is the joint, lubricated with silicon paste. Inlet 4 is provided species employed in the inoculum of my copending ap for the introduction of the liquid nutrient and the in plication; it is susceptible to growth by aerobic sub oculum. Flask 1 has a sintered glass bottom 5. Sterile merged methods as there described. (2) The Chinese air is led through the entrance 6, thence through the mushroom of the genus Volvaria, which may be grown 35 sintered glass bottom 5 and through the liquid substrate. on rice or paddy straw; and (3) the Japanese mushroom ’ Ground outlet 7 is provided for the escape of air after it Shii-take, Cortinellus berkelyanus. The‘ reason that has diifused through the liquid substrate. The shaft 3 these three species are the sole ones in cultivation de is connected to a propeller 8 which in the present case volves upon the fact that propitious circumstances must has a diameter of 11.0 cmnand a blade width of 1.5 cm. exist before other species of mushrooms can grow; these 40 for the 1% gallon ?ask and includes 2 blades. For a 3 circumstances are found only in nature and ordinary gallon ?ask the propeller 8 may have a diameterrof 15.0 methods of cultivation are futile. ‘ ‘ . cm. and a width of 3.4 cm. The propeller dimensions One family of mushrooms especially desirable, inter bear upon the speed at which the shaft is to be rotated. alia, because of their taste and ?avor are of the Helvell aceac, particularly the species Morchella ‘esculenta. 45 THE INOCULUM Clyde M. Christensen'in “Common Edible Mushrooms,” The preparation of the inoculum according to the pres University Press of Minnesota, 1947, ‘page 90 states in ent invention provides a novel two stage operation, al~g reference thereto: “In ?avor and in texture, they sur though conventional methods as described in my copenda pass both the common cultivated species and most other ing application can be employed at a loss. of ef?ciency. wild mushrooms, and attempts have been made and still 50 The preparation of the inoculum may begin with pure are being made to grow them commercially; so far with ' cultures of a chosen mushroom species grown on potato out success . . .” 1 . dextrose agar; the preparation of ‘such cultures being In accordance with the present invention, it haspbeen known ‘and understood by those skilled ‘in the art. ‘I found that mushroom mycelium of the family Hel have found the following species to be particularly de vellaceae, more particularly of the genus Morchella, sirable and adapted to the process hereinafter described. otherwise known as morels, may be grown in a wide 1 Of the genus Morchella: the species esculenia‘; angusticeps variety of nutrients in which Agaricus campestris, the Pk; bispora Sor; conica Pers., delicosa-Fr., horténsis species employed in the process of my copending ap Boud.; crassipes (Vent) Pers.; H. elastz'ca Bull; generally plication cannot grow. Molasses can be used as a nu of the family Helvellaceae; Hélvella laczmosa, Afz; trient in the present process; additionally the nutrient so 60 Gyrometra esculenta (Pers.) Fr.; of the ‘genus Verpa; lutions employed can be of greater concentration. Even conica (Muell) Swartz; bohemica (Kromb) Schroet. Of with such nutrient solutions, the morels provide faster these species, Morchella esculenta has been found by me utilization of carbohydrates giving much greater daily to be the most desirable for carrying out the invention. growth; possess greater tissue forming ability and are In the ?rst stage of the inoculum preparation, the pure capable of employing inexpensive ammonium salts as the 65 cultures grown on potato-dextrose‘ agar are used to inocu only source of nitrogen, none of thesethings are possible ' late 250 cc. Erlenmeyer‘?asks each containing 50 cc. of with Agaricus campestris employed as the inoculating potato-dextrose solution, adjusted to ‘a pH between 4.5 material. ‘ ‘ to 8.0. With lower pH, susceptibility for bacterial con-r The object of the invention includes: the production tamination reduces. It is important that effective distri of edible mushroom tissue of the family Helvellaceae, 70~ bution‘ of the mycelium is here maintained, allowing the inoculum to grow from as many starting. points as pos-_ particularly of the genus Morchella, having a superior s‘ible. A tissue grinder, such as the microhomogenizer de 2,761,246 scribed by Brendler in “Science,” vol. 114, July 20, 1951 A In this event 51 g. of CaSOt is substituted therefor. As at pp. 61-62, is very suitable for effecting such distribu before, the solution is not ?ltered. When inoculated tion. The Erlenmeyer ?asks are agitated on a rotary with 2 g (calculated as dry weight) of Morchella esca shaker at a speed of about 100 R. P. M. Withlower agi lenta submerged culture, there is a yield of approximately tation, the mycelium is inclined to clog or grow together 31.2 g. (calculated as dry weight) of Morchella esculenta. forming few large aggregates; at higher speed, the my On a sugar to Morchella esculenta (calculated as dry cellium may be harmed. After three days of agitation at weight) increase basis, the yield is 48.66%. room temperature, the culture is suf?ciently advanced for After the completion of the 72 hour run specified both transferrence to the succeeding stage of inoculum prepa ' with regard to Solution A, the entire amount of CaCOx ration. 10 is digested; the nutrient solution maintains a constant In the second stage, a larger unit, such as a 3 liter con pH through the 72 hour period of 6.5. tainer similar to the device shown in Fig. 1 having a For the beet molasses suggested in Solution A, there sintered glass bottom for air distribution and a stirring may be substituted an equivalent amount of blackstrap device, is used. Proper agitation of the liquid is main molasses, high test or Cuban inverted molasses, citrus tained at about 100 R. P. M. The growing solution can 15 molasses, and wood molasses. The aforesaid wood mo again be the same potato-dextrose solution adjusted to a lasses is cheaply produced by the hydrolysis of hard wood pH of 4.5. A large number of growing solutions, how chips with dilute sulfuric acid ?ltered and is a by-product ever, are here suitable. They need only have the neces , of the Wilson Dam project of the Tennessee Valley sary mineral requirements for growing fungi; a carbohy Authority. In all these types of molasses dilution may drate source such as many of the common monosaccha 20 be made to a ?nal sugar concentration of 5% with rides, or some of the disaccharides or their hydrolysates; substantially unimpaired growth. Other members than and a nitrogen source such as ammonium salts or amino Morchella esculenta of the Helvellaceae family grow quite acids or any kind of protein hydrolysate. well in nutrient solutions utilizing molasses, giving yields The second stage is carried on for a period of about 3 in sugar to (calculated as dry weight) mycelium increases days at which time the dextrose is consumed. The fungi 25 of up to 60%. Agaricus campestris does not grow on grown will be very light in weight and distributed through molasses as described with the possible exception of high out the solution. Although the tendency is to form test molasses. Even on high test molasses, however, the colonies, the structure of these colonies is loose and growth of Agaricus campestris is slow and and retarded, watery, having a solid content of 0.5 to 2.0%. The the yield being less than 2% Weight increase. inoculum thus grown is removed from solution to a An important and inexpensive nutrient can be used in Buechner funnel for separation. The inoculum shrinks lieu of Solution A by making an extract in boiling water to a plate-like layer with a thickness of l to 2 mm. of the Jerusalem artichoke, Helianthus tuberoszts. Such The inoculum is now ready for addition to the nutrient an extract would have all the needed requirements in— solution and the final development stage giving the de cluding the carbohydrate source, Inulin, in favorable rela sired tissue formation. 35 tive amounts. An important and necessary part for carrying out the THE FINAL NUTRIENT SOLUTION process according to the present invention is the forma tion of colonies and mushroom tissue. These colonies In the tissue forming stage, certain requirements are and tissue form about small particles found in the nutrient to be met by the nutrient solution and apparatus em 40 material; I call these particles particulate support mate ployed. I have found that molasses and ammonium rial. To illustrate the importance of this support mate phosphate are adequate, e?icient and cheap components rial, if instead of the CaCOs or CaSO4 suggested hereto which meet these requirements. Thus beet molasses, one fore, a substitution is made of CaCl2.2H2O, 6.0 g. and of the cheapest and most readily obtainable of materials the solution ?ltered, the nutrient material becomes a can be used as follows: 45 solution without the particulate support material con Solution A tributed by the CaCOa or CaSOa. With the same pH of 6.5 and an inoculation of 2 g. (calculated as dry weight) Grams of Marchella esculenta submerged culture provides a Beet molasses ______.._ 120 50 harvest after 72 hours of 16 g. (calculated as dry weight) (NH4)2I-IPO-1 ______.. 6 of mycelium. Thus, without the use of a particulate CaCOs ______4.8 support material, the yield on the basis of sugar to (calcu K2504 ______1.7 lated as dry weight) Morchella esculenta increase is only MgSO4.7H2O .... 0.7 23.3%, substantially less than the results obtained with Water to 2 1. 55 un?ltered Solution A containing CaCOs and CaSO4 as described. This solution is adjusted to a pH of 6.5 and is not ?ltered, The solution is then inoculated with 2 grams (calculated ' A new and unexpected result according to the inven as dry weight) of submerged culture inoculum, prepared tion is the ability of the family Helvellaceae, more par ticularly Morchella esculenla to synthesize “growth pro by the process heretofore described. 60 The nutrient and inoculum is then transferred to the moting” substances. It has always been considered that apparatus previously described with respect to Fig. 1; “growth promoting" substances are necessary for the agitation is begun at a speed of about 100 R. P. M. The proper cultivation of mushrooms. Cecil Treshchow speed speci?ed is of course dependent upon the shape of (Dansk Botanish Arkiv, vol. XI, No. 6, 1944) has said the agitator employed; the speed given has been found 65 that mushroom cultivation is impossible without “growth suitable for the propeller 8, described. Air is introduced promoting” substances such as vitamins, etc. While, as at a rate per minute equal approximately to the number set forth in my previously cited copending application, it of volumes of the solution in the ?ask. is true that Agaricus campestris requires growth sub After 72 hours of agitation and aeration in the manner stances when used for submerged culture in a purely syn described, assuming inoculum with Morchella esculenta, to thetic medium, I have discovered that mushrooms of the there will be harvested 36.7 g. (calculated as dry weight) family Helvellaceae, particularly Morchella esculenta, of mycelium. The yield, on a basis of sugar to Morchella can be employed in a submerged culture process accord esculenta (calculated as dry weight) increase is 57.8%. ing to the present invention and synthetize entirely the I have obtained successful results substituting growth promoting substances which it requires. CaSO-r.2HzO for the CaCO3 speci?ed in Solution vA. 75 Thus, an inoculum of submerged Morchella esculenta 2,761,246‘

culture is prepared as previously described and is then‘ Helvellaceae," an aqueous solution containing nutrient‘ added to the following nutrient solution: ' ‘ material, applying gentle agitation to the said inoculum and solution su?icient to maintain the said inoculum in Solution B suspension, and simultaneously aerating the aqueous solu Glucose ______g__ 75 tion thereby cultivating tissue of the family Helvellaceae 2(NH4.)HPO4 ______g__ 6.0 in pellet formation while submerged in the said solu MgSO4.7H2O ______g.._ 2.26 tion. ‘ K2504 ______._g.._ 1.7 2. A process for the production of edible mushroom MnSOrAHzO ______mg.._ 50 tissue of the family Helvellaceae comprising the steps of FeCla ______mg_ __ 10 10 inoculating with an inoculum of mushroom of the‘ family CaCOa ______g__ 10 Helvellaceae, an aqueous nutrient solution comprising Water to make 2 1. molasses, applying gentle agitation to the said inoculum and solution suf?cient to maintain the said inoculum in The pH of this solution is adjusted to 6.5. One g. dry suspension, and simultaneously aerating the aqueous solu-' Weight of submerged Morchella esculenta culture ‘is 15 added to Solution B and thence subjected to 72 hours tion thereby cultivating tissue of the family Helvellaceae aeration and agitation in the apparatus as previously de in pellets while submerged in the said solution. scribed. There results a harvest of 44.3 g. mycelium 3. A process of the production of mushroom tissue of (calculated as dry weight). The yield in sugar to (cal the family Helvellaceae as set forth in claim 2 wherein the culated as dry weight) Monchella esculenta mycelium in» nutrient solution comprises beet molasses. crease is 59%. ‘ 4. A process of the production of mushroom tissue of Using synthetic medium without growth substance, the family Helvellaceae as set forth in claim 2 wherein the Solution B with an inoculum of 1 g. submerged Agaricus nutrient solution comprises blackstrap molasses. 5. A process of the production of mushroom tissue of campestris culture, the harvest after 72 hours of aeration the family Helvellaceae as set forth in claim 2 wherein and agitation will be 2.7 g. (calculated as dry weight). 25 The yield in sugar to (calculated as dry weight) Agaricus the nutrient solution comprises citrus molasses. campestris mycelium increase is 2.26% as compared to 6. A process for the production of edible mushroom the 59.0% increase with Morchella esculenta. It is easily tissue of the family Helvellaceae comprising the steps of provable that this negligible harvest of Agaricus campes inoculating with an inoculum of mushroom of the family Helvellaceae, an aqueous nutrient solution. consisting of tris is due to a lack of growth substance in the syn 30 thetic nutrient. Thus, if Solution B is provided with 5.0 the cooled extract in boiling water of the Jerusalem arti g. of yeast extract (Difco) with the pH adjustment, choke, Heliam‘hus tuberosus, applying gentle agitation to the said inoculum sufficient to maintain the said inoculum aeration, agitation and inoculum Agaricus campestris as in suspension, and simultaneously aerating the aqueous before, there is a harvest of 10.8 g. (calculated as dry solution thereby cultivating tissue of the family Hel weight) mycelium. The yield of sugar to (calculated as 35 dry weight) ‘Agaricus campeslrz's increase is 13.0%. vellaceae in ?rm pellet formation while submerged in the While this increase is a poor comparison to the yield ob solution. 7. A process for the production of edible mushroom tained from Morchella esculenta, the larger yield‘of the tissue of the family Helvellaceae comprising the steps of latter is a natural result over Agaricus campestris used‘ as inoculating with an inoculum of mushroom of the family the inoculating mycelium, and is independent of the ab 40 sence or presence of growth substance. Helvellaceae, an aqueous nutrient solution comprising The synthetizing ability of M orchella esculenta is easily the cooled extracts in boiling water of the Jerusalem arti measurable. Out of the purely synthetic Solution B, choke, Helianthus tuberosus, applying gentle agitation to the said inoculum su?icient to maintain the said inoculum where the only organic component is glucose and the only in suspension, and simultaneously aerating the aqueous nitrogen source is ammonium phosphate, tissue can be 45 solution thereby cultivating tissue of the family Helvel produced With 20% to 45% protein content, depending laceae in pellet formation while submurged in the solu~ upon the concentration of ammonium phosphate utilized. tion. An analysis of the vitamins in the tissue present shows the presence of the following: 8. A process for the production of edible mushroom tissue of the family Helvellaceae comprising the steps of Micrograms / grams dry weight 50 inoculating with an inoculum of mushroom of the family Thiamin ______3.92 Helvellaceae, an aqueous nutrient solution comprising a Ribo?avin ______24.6 medium of carbohydrate and a source of inorganic nitro Niacin ______.._ 82.0 gen in the absence of growth promoting substances, ap plying gentle agitation to the said inoculum su?icient to Pyridoxine ______5.8 55 Pantothenic acid ______8.7 maintain the said inoculum in suspension, and simultane Biotin ______0.75 ously aerating the aqueous solution thereby cultivating Folic acid ___ _ 3.48 tissue of the family Helvellaceae in pellet formation while Vitamin B12 ______0.00362 submerged in the said solution. 9. A process for the production of mushroom tissue of Fig. 2 is a reproduction of a typical end result of car 60 the family Helvellaceae as described in claim 8 wherein rying out the teachings of the present invention. When the said carbohydrate and inorganic nitrogen source com properly carried out, the harvest will comprise ball-like prise glucose and ammonium phosphate, respectively. pellets having a diameter comparable to that shown in 10. A submerged process for the production of edible the ?gure and possessing a ?rm, resilient tissue structure. mushroom tissue of the family Helvellaceae comprising The examples heretofore given are included solely for 65 the steps of inoculating with an inoculum of mushroom the purposes of illustration. They are not regarded as of the family Helvellaceae, an aqueous nutrient solution, necessary limitations of the invention but many modi?ca said nutrient solution comprising at least in part a par tions and variations will be apparent to those skilled in ticulate nutrient support material, applying gentle agita the art; these are included as part of the invention except tion to the said inoculum su?icient to maintain the said as they do not come within the scope of the hereinafter 70 inoculum and support material in suspension, and simul appended claims. taneously aerating the aqueous solution thereby cultivat WhatIclaim is: ‘ ing tissue of the family Helvellaceae in pellets while sub 1. A process for the production of edible mushroom merged in the solution. tissue of the family Helvellaceae comprising the steps of 11. A process for the production of mushroom tissue of inoculating with an inoculum of mushroom of the family 7 the family Helvellaceae as described in claim 10 wherein 2,761,246‘ 7 8. the said particulate nutrient support material comprises the family Helvellaceae, an aqueous‘ nutrient solution, calcium carbonate. _ said nutrient solution comprising at least in part a particu 12. A process for the production of mushroom tissue late nutrient support material, applying gentle agitation to of the family Helvellaceae as described in claim 10 where the said inoculum su?icient to maintain the said inoculum in the said particulate nutrient support material comprises 5 and support material in suspension and simultaneously calcium phosphate. aerating the aqueous solution thereby cultivating tissue of 13. A submerged process for the production of edible the genus Morchella in pellets While submerged in the mushroom tissue of the family Helvellaceae comprising solution. ‘ the steps of inoculating with an inoculum of mushroom 16. A submerged process for the production of edible of the family Helvellaceae, an aqueous nutrient solution, 10 mushroom tissue of the family Helvellaceae comprising said nutrient solution comprising at least in part a par the steps of preparing a culture of a species of mushroom ticulate nutrient support material, applying gentle agita of the family Helvellaceae, inoculating the said culture tion to the said inoculum suf?cient to maintain the said in a ?rst aqueous nutrient solution, agitating the said inoculum and support material in suspension, simultane solution to effect distribution of the culture in the ,?rst ously aerating the aqueous solution thereby cultivating 15 said medium at room temperature, applying agitation tissue of the family Helvellaceae in pellets while sub and aeration to the culture in a second aqueous nutrient merged in the solution, and the pH of the said aqueous solution comprising a mineral source, a carbohydrate nutrient solution being maintained at a substantially con source and a nitrogen source at room temperature there stant value. by forming an inoculum material, inoculating with said 14. A submerged process for the production of edible 20 inoculum a third aqueous nutrient solution, said third mushroom tissue of the family Helvellaceae comprising nutrient solution comprising‘at least in part a particulate the steps of preparing a culture of a species of mushroom nutrient support material, applying gentle agitation to of the family Helvellaceae, inoculating the said cultures said inoculum su?icient to maintain said inoculum and in a ?rst aqueous nutrient solution, agitating the solution support material in suspension and simultaneously aerat to effect distribution of the culture in the ?rst said me 25 ing the aqueous solution thereby cultivating tissue of the dium for a period of about three days at room tempera family Helvellaceae in pellets While submerged in the ture, applying agitation and aeration to the culture in a solution. second aqueous nutrient solution comprising a mineral source, a carbohydrate source and a nitrogen source for References Cited in the ?le of this patent a period of about three days at room temperature thereby 30 UNITED STATES PATENTS forming an inoculum material, inoculating with the said 2,005,365 Di Giacinto ______June 18, 1935 inoculum a third aqueous nutrient solution, said third nutrient solution comprising at least in part a particulate 2,693,665 Humfeld ______,______Nov. 9, 1954 nutrient support material, applying gentle agitation to the FOREIGN PATENTS said inoculum su?icient to maintain the said inoculum 35 315,215 Great Britain ______July 10, 1930 and support material in suspension and simultaneously aerating the aqueous solution thereby cultivating tissue of OTHER REFERENCES the family Helvellaceae in pellets while submerged in the Molliard and Fron: Comptes Rendus, vol. 140, pp. solution. 1146-1148, 1187;1189 (1905). 15. A submerged process for the production of edible 40 Lambert: “Principles and Problems of Mushroom Cul mushroom tissue of the genus Morchella comprising the ture,” Botanical Review, vol. 4, pp. 397-426 (July 1938). steps of inoculating with an inoculum of mushroom of