claim: 1. A process for pre@t)aring novel varieties of tempeh suitable both for direct consumption and for the preparation of deep fried flakes, chips, and wafers comprising inoculating a pre-boiled and hydrated but - ungelatinized 50 substrate selected from the group consistin.- of a cracked cereal grain and a 1:3 to 3:1 mixture of a preboiled cracked cereal grain and pre-boiled soybean grits with an aqueous suspension of spores of Rhizopus oligosporus, NRRL 2710, incubating the inoculated substrate at about 55 mixture of one part of pre-boiled cracked rice and three parts of pre-boiled soybean grits. 6. Tempeh produced by the process of claim 4. 7. As a novel article of manufacture, deep fried wafers of the tempeh of claim 4. No references cited. A. LOUIS MONACELL, Primary Examiner. S. J. BAICKER, Assistant Examiner.

3 1 2 4 3 , 3 0 1 Un'l,ted States Patent Office Patented Mar. 29, 1966 3,243,301 CEREAL-CONTAINING VARIETMS OF TEMPEH ANID PROCESS THEREFOR CUfford W. HesseItine and Mabel L. Smith, Peoria, 111" 5 assignors to the United States of America as represented by the Secretary of Agriculture No Drawing. Filed Apr. 3, 1964, Ser. No. 357,330 7 Claims. (Cl. 99-80) A nonexclusive, irrevocable, royalty-free license in the 10 invention herein described, throu.-hout the world for all purposes of the Un@ted States Governinent, with the power to grant sublicenses for such purposes, is hereby granted to the Government of the United States of America. This invention relates to novel varieties of the Indone15 sion highly molded soybean food called tempeh, and to the process for making the novel varieties. More specifically, this invention relates to the preparation of novel varieties of tempeh not exclusively from soybeans as in the prior art but more particularly bY 20 subjecting exclusively either a pretreated ceral grain substrate such as wheat, rye, oats, rice, or barley or a substrate comprising an intimate mixture of a said cereal grain and soybean -rits to the action of the mold Rhizopus oligosporits, NRRL 2710, which mold is characterized by 25 the presence of large amourts of both proteolytic and lipolytic enzymes but little or no amylolytic enzyme.' Although rice, for example, is very widely used in the Far East alon.- with soybeans for the preparation of fermented foods such as miso and soy sauce and is freely 30 available in Indonesia, tempeh is prepared exclusively from soybeans, apparently because the conventional molds contain large amounts of amylolytic enzymes which would hydrolyze any cereal starch in the substrate to sugars that would then be fermented to disagreeable organic 35 acids, and it has not been possible prior to the instant invention to make acceptable ter@ipeh from a cereal-containin@ medium. ,@i In view of the constant introduction of additional exotic 40 specialty foodstuffs, it would be advantageous to prepare improved varieties of tempeh that nutritionally complement soybean protein with cereal grain components. Such products would, of course, utilize some of our cereal grain surplusage, and otir invention would also provide the Far 45 East with a sorely needed flexibility of substrates for the preparation of difft.rently flavored varieties of tempeh. In addition, our process comprises a greatly shortened culture time for growing mycelium than has until now been possible on a cereal grain-based substrate, and this Nvould 50 -materially lower the cost and thus aid the commercialability of the finished product. The principal object of oiir invention is the preparation -of novel grain-based varieties of tempeh and deep-fried versions thereo,'. Another object is a process for prepar55 ing bland and completely acceptable varieties of tempeh usin,@ cereal grains such as wheat, rye, rice, barley, and oats or mixtures of the same alone or in combination with pretreated soybean grits. Still another object is a 60 process in which the required growth of rnycelium is completed in not more than about 24 to 25 hours. The above and related objects will be more clearly understood by a reading of the following specification. 2 Tempeh, until now made by the action of molds exclusively on soybeans, is a bland tasting, off-white or cream colored, somewbat cheeselike solid that can be eaten as is or, pref,-rably for our tastes, in the form of deep fried flakes or chips. The mild mushroom-like to popcorn flavors of the fresh or fried tempeh contra@,t greatly with the e.xtremely strong flavors of soy sauce and miso, which require extensive dilution and are used as condiments rather than as principal foods. In accordance with the objects of the present invention we have now discovered that novel varieties of tempeh can be obtained from a pretreated cereal grain or mixed soybean-cereil grain substrate when the molding organism is limited to a fungus that produces large amounts of both proteolytic and lipolytic enzymes and but little if any amylolytic enzyme, since any appreciable ariount of the latter class of enzyme would break down the cereal grain starch to simpler sugars that would then b.- fermented to disagreeably tasting organic acids and highly colored materials. Thus, we have found that Rhizopus oligosporous, NRRL 2710, is completely operative for the purpose of our invention whereas the closely related fungus Rhizopus oryzae and the mold Aspergillus aryzae on substrates comprising a cereal grain produce darkly colored, ill-smelling, and utterly unacceptable products. Although it would not be apparent that any pretreatment of th,- cereal grain could be important where the mold does not contain or produce amylolytic enzymes, we have found that only a very limited number of the myceliumdeveloping from the germinating spores succeed in penetrating an unruptured seed coat and that pre-cracking of the cereal grain kernels ard a fully hydrated state are required if the mold is to grow extensively and quickly enough to avoid the development of adverse flavors, the expedited growth of the mold apparently resulting also from an increased accessibility to the nutrients of the gralin below the seed coat. It will be seen from the examples that the principal steps of our process may be summarized as follows: (1) Boil pre-washed cracked cereal grain or soybean grits for at least 12 minutes to hydrate and sterilize at 100' C. (2) Af' er removal of excess water, inoculate the desired grain or mixed grain-soybean medium with an aqueous suspension of spores of Rhizopus oligosporus, NRRL 27 10. (3) Incubate at 31' C. under aerobic conditions for 18-24 hours. (4) Kill spores and enzymes with steam or by frying. EXAMPLE I Two 1,000-gram lots of Hard Red Winter wheat, difiering only in that the kern--Is of one lot had been lightly cracked by mechanical means, were treated in an identical manner, as fgllows. Each lot was covered with water and allowed to soak at about 25' C. for 20 hours, drained, washed in tliree changes of water, recovered with water, boiled at atrnospheric pressure for 10 minutes in the case of the cracked wheat and for 30 minutes in the case of the uncracked (whole) wheat, cooled, inoculated with 30 ml. of a sterile aqueous suspens.lon. of Rhizopus oligosporous, NRRL 2710, spores obtained from a 5-day potato dex-

3,243,301 3 4 trose agar slant lherepf, tran.sferred to petri dishes and Table I incubated at 31' C. for 25 hours. The dishes containing the uncracked (whole) wheat showed a very liniited and Amount of incoinplete growth that was not significantly increased by Substrate Form of substrate boiling at 10G, C. extendin.- the incubation to 36 hours, at -v@hich time the 5 (minutes) slightly molded product was still very friable and could not be properly sliced for fryin.-. The cracked wheat Soybeaiis --------------- Dehulled, coarse grits ------- 25 Wheat (Hard Red Sprini) --- Cracked --- --------------- -- 12 tempeh at 25 hours consisted of a solid cake in which the White wheat ---------------- ----- do ---------------------- 12 Barley ----- --------------- -- Dehulled and cracked --- ---- 12 cracked wheat was held firmly together by the extensive Oats - ---------------------- ----- do ------- --------------- 8-10 white mycelia of the mold, thus permitting slicing into 10 Rye ----- -------------------- Cleaned and cracked -------- 12 Corn -------- --------------- - Cracked --- --------------- -- 25 wafers having a thickness of less than 5 mm. The fresh Sorghum -------------------- Cleaned and cracked -------- 25 wafers having a pleasant yeasty odor were dipped in salt Peanuts --------------------- Roasted, dehulled and 25 sliced into small pieces. water for several minutes and then deep fried in vege- Rice ---- -------------------- Polished and cracked ------- 10 table oil to provide a crisp chip item having a flavor resembling that of popcom. 15 It will be appreciated that the pre-fermentation boiling period should be adjusted for the particular legume or cereal so as to avoid, gelati.nization that would leave inadequate space for the mycelium to develop betweei 20 the particles of substrate. Table I shows boiling times that we have found operative. EXAMPLE 2 Oth@-r cereal grains, nel barley, oats, ry am , y e, corn, sorghum and rice that were lightly cracked and boiled for the time shown i n Table I -were otherwise treated precisely as the cracked wheat of Example I excepting that the fermentation was terminated at 22 hours instead of 25 hours. The results are sbown in Tables 11 and 111. Table II Fresli product .A.bility of substrate, to Substrate hold tog,?tber and Fermentation- be sliced development of Odor Rhizopus @oyb e.anp ------------------ Exceu ent -------- Pleasa nt ---------- Firm solid and sliced Whea t --------------- Gooc T ----------- Y a@gtn-e ------- d p Virin, slice satisfae@ White wheat -------------- ---- do ----------- torily. ----- do ------------- Do. Barley --------- -------- ---- do - ------- ----- do ---- Like A,he,,it. Dat ---------------------- ----- do ----------- ----- do ---- Do' RYe ---------------------- ----- do ----------- Faint ------------- Do. Corn -------------------- - - F@06r ------------- F@int to none ----- Poor-slieu bre4ic, $orgh um--------- --- ----- do - - - Ye@ @iiiie --------- ------ ---- --- - Do. Peanu ts-- --------- -- ----- do Ranci d peanuts -- Poor. Rice ---------------- ----- Good ----- I------ Sweet, fragr@nt slices I well. Ri6e (3 parts) and soy- Excellent -------- ------ do --------- --- Slices very well. b-ean gr-its (1 part). Wheat (3 parts) ajid soy- Good -------- Do. --- Slightly yeasty,---bedn gtits (I part). Table III Substrate Deep fried product Appearance Taste and acceptability C olor of food Odor Wheat -------------------- Brown ------------------ Pleasant ---------------- Slices bat surfaces Excellent-tastes like rough @ popcorn. White wheat -------------- ----- do- -- ----- do -------------------- Like wheat ------------ Crunchy but acceptable, Barley ------------------- ,----- d6-: -------- :: ------- Noiae -------------------- ----- do ----------- ---- Crunchy and like wheat, Oats- Light broNvi2 ------------ Pleasant ----------------;----- do - ----------------- Very faint flavor. -------------------- ]@ye ---------------------- Dar.k broivn ------------- ----- do ------------------- ExceEent -------------- Like wheat but more f l a v o r . Corn --------------------- @ YelloNv ---------- ------- Faint ------------------- Breakes into, pieces ---- Faiiatly like popcorn., Sorghum --------- ------- Irregular in color -------- Slight -------------------- As corn --------------- Poor. Peanuts ----------------------- ao ------------------- ----- do ------------------- Poor ----------I-------- Do. Rice --------------------- (,oldeia brown ---------- Faint to none ----------- Excellent -------------- Ver,y acceptable@ Rice (3 parts) and soy- Darker in, color thaii Exceuent-somevibat ----- do ----------------- Do. bean (I part). rice. like soybean tempeh-. Wheat (3 parts) and. soy- Speelled ---- ------------ Like soybean tempoh --- ----- do ----------------- Very acceptable-supebean (1 part). rior to wheat alone.

3,243,301 5 6 EXAMPLE 3 31' C. for about 18-22 hours in the presence of availVarieties of tempeh from substrates consisting of 3:1 able oxygen, and steaming to kill the spores and enzymes. to 1: 3 m@ ixtures of soybean grits that had been boiled 2. The process of claim I wherei.1 the substrate is for 30 minutes and either cracked wheat preboiled for preboiled cracked wheat. .12 minutes or cracked rice preboiled for 8 minutes were 5 3. The process of claim I wherein the substrate is preeach inoculated with spore - suspensions of Rhizopus b oiled cracked rice. oli,@osporus, NRRL 2710, and incubated in petri dishes 4. The process of claim I wherein the substratp- conat 31' C. for 22 hours. The - characteristics of the obsists of equal parts of pre-boiled cracked wheat and pretained fresh and deep fried tempeh products are given boiled soybean grits. in Table IV. 10 S. The process of claim I wherein the substrate is a Table IV Substrate Before cooking Ability to slice Odor Appearance Wheat ------------------ Yeasty, fragrant ------------ Brownish-gray Avith spores- Poor. Soybean grits ----------- Usual, shghtly a=onical- - -Uniformly white Nvith Good. spores at edge. Rice -------------------- Yeasty ---------------------- -Uniforrnly white, ilo spores-- Like soybeans. Wheat (I part), Soy- Like soybean teinpoh ------- Speck-led -------------------- Good. bean (3 parts). Wheat (2 paxts), Soy- Yeasty and like soybea n ----- do ------------ ----------- Excellent. bean (I part). tempeh. Wheat (3 parts), Soy - ----- do ------- ---------------- Darker ---------------------- Somewhat like wheat. bean (1 part). Rice (I part), Soybean Soybean tempoh and some- Uniformly white with Good. (3 parts). what yeasty. spores at edge. Rice (2 parts), Soybean Yeasty and like soybean Nearly uniform with Do. (2 T)arts). tempoh. spores at edge. Ried(3 parts), Soybean Yeasty ----------- ----------- Uniformly white with Do. (I part). spores at edge. Substrate After cooking Acceptability Color Taste Wheat ------------------ Dark brown ---------------- Like popcorn --------------- Good. Soybean grits ----------- Golden tan ------------------ Pleasant -------------------- Do. Rice -------------------- Light yehow ---------------- Slightly sour ---------------- Very acceptable. Wheat (I part), Soy- Speckled -------------------- ExceRent -------------------- Do. bean (3 parts). Wheat (2 parts), Soy - ----- do ------- ---------------- ----- do ----------------------- Do. bean (2 parts). Wheat (3 parts), Soy- Brownish ------------------- ----- do ----------------------- Good. bean (1 part). Rice (1 part), Soybean YeRow to tan --------------- ----- do ----------------------- Very accpptable. (3 parts). Rice (2 parts), Soybean Goldeia tan ----------------- ----- do -- -------- Do. (2 parts). @ Goo Rice (3 parts), Soybean Yellow to tan ---- ----------- Not as good as (,ther r;,@. d. (I part). combinations; crunchy. We