IL29621A - Process for preparing a microbial rennin - Google Patents

Description

PATENTS FORM NO. 3. ^ PATENTS AND DESIGNS ORDINANCE SPECIFICATION PROCESS FOR PREPARING A MICROBIAL RENNIN" We, BAXTER LABORATORIES, INC. a company incorporated under the laws of the State of Delaware, United States of America, of 6301 North Lincoln Morton Grove, Illinois, United States of America, DO HEREBY DECLARE the nature of this invention and in what manner the same is to be performed, to be particularly ascertained in and by the following statement : - This invention relates to a method of making a microbial rennin and to a method of making cheese with said rennin.

The conventional method of making cheese involves the use of rennet for coagulating milk. Rennet is an enzyme -containing composition prepared from the fourth stomach of milk-fed calves. In the process of making cheese, the rennet is added to milk and the enzyme, rennin, exerts a mildly proteolytic action on the casein and other proteins present in the milk. This breakdown of the proteins causes the milk to coagulate or to form solid curds. These curds are separated from the whey, which is predominantly an aqueous suspension of low solids content. The curds are then mixed with salt, etc. , and formed into blocks or rounds and cured to form cheese.

Because of the particular animal origin of rennet, the supply and quality of rennet are subject to wide fluctuations. In view of these variable factors, investigators in the field have attempted to find cheese making substitutes for rennet. Numerous vegetable and microbial enzyme preparations have been investigated in the search for suitable substitute materials having the milk coagulating properties of rennet. Among the microorganisms which have been disclosed as useful for this ur ose are for exam le, the various mic oor anisms disclosed in U . S. P. 1 , 391 , 210, the Kndoth ia parasitica disclosed in U. S. P. 3, 275, Γ> and the M cor pusiUus disclosed in U. S. P. \s 3, 1 51 , 030 and 3, 2.1 2, 905.

In accordance with the present invention a microb ial renn in having high m ilk coagulating activity and low proteolytic acl!ivity i s prepared by inoculating a medium containing available carbonj nitrogen and trace nutrients with a culture of Mucor michci, fermenting under aerobic conditions until a substantial amount of said microbial rennin is procluced, and thereafter recovering the microbial rennin from the fermentation medium. The rennin thus prepared can be used in the process of making cheese during the milk coagulating step as a substitute for part or all of the rennet normally used.

A description of the general characteristics of the organism Mucor rn.ieh.c i is provided by Cooney and Emerson, "Thermophilic JAingi", pp. 11- 21 (1964), published by W. 11. Freeman and Co.- ?.

San Francisco and London.

A culture of a preferred strain of the Mucor miehei used in accordance with the present invention is on deposit with the Nortliern Regional Research Laboratories, Peoria, Illinois, under the code designation NRRL 3169. Other strains of Muco michci which can be used to obtain the present microbial rennin are obtainable from the same laboratories under the designations: NRRL 2543, A 7772, A 13, 042, and A .1 3, 131. It will be understood that still other strains of Mucor mi chci can be used, and the invention is not limited to the specific strai ns set forth above.

The microbial rennin of the p resent invention preferably is prepared by growing' a culture of ucor miehci in a suitable medium in the presence of air at temperatures of from about 30°C to about 55°C for periods of time of from about 2 to about 14 days. The fe mentatio medium generally has a pH of from about 3 to about 8 and preferably a pH of from about 4 to about 7. Submerged aerobic fermentation methods, for example, deep fermentation in commercial ferrncntation tanks or fermentation in flasks on a rotary shaker, as well as various methods of surface aerobic fermentation can be used to prepare the microbial rennin of the present invention.

Recovery of the microbial rennin from the filtered ferm entation medium can be carried out by various methods conventionally used in the separation of proteins from mixtures, for example, solvent precipi tation, salt precipitation, chromotography and other such methods of protein recovery. Examples of solvents that c n be used for the precipitation of the microbial rennin are ketones such as acetone and methyl ethyl ketone, alcohols such as methanol, ethanol and isopropano and other such organic solvents. Illustrative of the salts which can be employed are ammonium sulfate, sodium sulfate and the like mineral salts. Still other methods of recovery of the microbial rennin can be used, for example dialysis, electrophoresis, freeze- drying and the like Suitable fermentation media can be prepared from carbohydrate materials such as whey, degraded corn starch, Cerelose, wheat bran and other organic sources of available carbon, and from nitrogenuous materials such as brewers' yeast, soya protein, casein, urea, arn onr. salts, nitrates and other such organic or inorganic sources of available nitrogen. The sources of these ferm entation media ingredients may be with the m ajor fermentation media ingredients, such that the separate addition of trace nutrients is generally not required. For example, trace amounts of inorganic salts such as metal chlorides, sulfates, phosphates and nitrates are generally present in the fermentation media in association with the carbonaceous and nitrogenous ingredients.

Proteases, in general, coagulate milk. However, most proteases cause considerable protein digestion. When these proteases, of animal, plant and microbial origin, are employed for the coagulation of milk, the resulting curds develop xmdesirable off-flavors due to proteolysis and thus are not well suited for cheese making.

A principal advantage of the present invention is that the rennin prepared from Mucor rniehei unexpectedly has a very low proteolytic activity associated with high milk coagulating activity. The milk proteolysis curves obtained with the Mucor rniehei of the present invention are identical to those obtained with rennet, indicating a very low proteolytic activity. The Mucor rniehei rennin also appears to be substantially less proteolytic than the Muco pusill s disclosed in U. S. P. 3, 151, 039. Using the Mucor rniehei rennin, less than 10% of the total Kjeldahl nitrogen has been found in. whey whereas 20% Kjeldahl nitrogen transfers into the whey according to U. S. P. 3, 151, 039 (col. 4 lines 58- 61).

Although the inventor is not to be bound by any particular theory, it is believed that the cheeses made with the microbial rennin of the present invention are essentially free from any off -flavors due in a large measure to the low proteolytic activity in the rennin product. free from any lipase whereas in the production of certain other cheeses it may be desirable to use a microbial rennin obtained from ucor mic ci in which the rennin contains lipase. It will be understood that- both of the aforesaid microbial rennins are included within the scope . of the present invention.

The following examples will further illustrate the present invention although the invention is not limited to these specific examples. All percentages expressed herein are on a weight basis unless otherwise specified.

Example 1 v.cor mie ei, NRRL 3160, is transferred from an ga slant under sterile conditions into a one liter Fernbach flask containin; 200 ml of the following aqueous medium : Whey 32% Degraded corn starch . . .. . . . 5% Brewers' yeast 1% Cerelose (glucose) 1% , Water . . . 11% 10G% The flask is incubated on a rotary shaker at 37 C for 163 hours.

The fermentation broth is filtered and the filtrate shows an activity equal to 12, 680 meg F rennet/ml. To the chilled filtrate (<5° C) is added two volumes of cold acetone. The resulting precipitate is filtered and washed with acetone, yielding a fine white powder with an activity of 968 mg NF rennet /g.

The activity" of rennin containing material as described herein is determined in the following'manner: To 9 ml of fresh homogenized milk (pH adjusted to 6. 2 with lactic acid) at 35° C1 is added one ml of aqueous solution of rennin sample. The sample is agitated periodically and the time required for the first appearance of fiocculation is observed. The same procedure is conducted simultaneously using several levels o: NF rennet. A standard curve is prepared for fiocculation time versus level of NF rennet. The fiocculation time for the test sample is then related to the standard curve to determine the activity of the test sam le in terms of NF rennet. the making of ripened cheeses such as, for example, Cheddar typ cheeses, Swiss cheese and other such so-called hard and soft cheeses, and for any other types of cheeses. wherein rennet pre¬ parations have heretofore been used.

Example 2 • t" The procedure of Example 1 is repeated, substituting" for Mucor miehei, RP.L 3169, the strain of Mucor miehei designate NRRL 2543 by the Northern Regional Research Laboratories. Rennin activity for the production of a satisfactory cheese is obtained.

Example 3 The procedure of Example i is repeated, substituting for the aqueous ferm entation medium, the following media: (a) Whey 97% (b) Degraded corn starch . . . 5% Brewers' yeast 1% Cerelose (glucose) . . . . i% Whey ■ . . 0 - S0% Water 13 - 93% · 100% (c) Whey 50% Γ Degraded corn starch. 5% Cerelose (glucose) 1% Nitrogen source*. . . 0. 5 - 5% Water 39 - 43. 5% 100% #Casein, soya protein, peptone, enzymaticaily degraded casein, oi corn steep liquor.

The above fermentation media are further examples ox suitable media containing available carbon, nitrogen and trace nutrients for the preparation of a microbial rennin of relatively high milk coagulating activity and relatively low proteolytic activity in accordance with the procedure described herein! j Example 4 1 In place of the submerged fermentation in liquid media of Exam ucor raiohci rennin is prepared in (a) still and (b) agitated orme: tio s on semi-solid media comprising 25% yheat, 25% sugar beet pulp and 50% whey. These rennin products can be used to prepare satisfactory hard and soft cheeses.

Example 5 A Cheddar cheese is made by substituting for the renne preparation ordinarily used in the setting step, an equivc/ient amour of the microbial rennin product prepared in Example 1. In this one percent by weight of a commercial lactic acid starter solution is added. The microbial rennin is then added to the milk at a rate of 3 ounces per 1000 pounds of milk. The mixture is agitated until a curd of satisfactory firmness is obtained. The curd is cut into cubes and then cooked at 100°F for several hours. The curd is separated from the whey and layered into slabs. The milled curd is then salted with 3% by weight of cheese salt. The salted curd is transferred to hoops, pressed, and then placed in a curing room. The cheese made from the microbial rennin of this example is sampled periodically after several days of curing and is found to possess excellent qualities and to be essentially free, rom off- flavors, Other conventional' ethods of making Cheddar cheese, "for example, those generally described by Prescott and Dunn, "Industrial Microbiology", Chapter 21 (3d ed. 1959), McGraw-Hill Book Co. , Inc. , New York, and references cited therein, can be used in the practice of the present invention by substitution for the rennet pre-parations ordinarily employed in said cheese m aking, an equivalent amount of the microbial rennin from Mucor Miehei of Example 1 I ■ herein, to produce good quality cheese. | As will be readily apparent to those skilled in} the art, other examp] j of the herein- defined invention can be devised after reading the foregoin specification and claims appended hereto by various modifications and adaptations without departing from the spirit and scope of the invention.

Claims (10)

Having now particularly described and ascertained the nature of ir said invention and in what manner the same is to be performed, e declare that what we claim is : -

1. . The process of preparing a microbial rennin having relatively high milk coagulating activity and relatively low proteolytic activity , which comprises inoculating a medium containing available carbon, nitrogen and trace nutrients with a culture of ucor miehei , fermenting under aerobic conditions until a substantial amount of microbial rennin is produced and thereafter recovering the microbial rennin from the fermentation medium .

2. . The proces s of claim 1 , wherein the Mucor miehei is a strain selected from the group consisting of NRRL numbers: 3169 , A 7772 , A 13 , 131 , A 13 , 042 and 2543.

3. . The process of claim 1 or 2 , wherein the fermentation is carried out under submerged aerobic conditions at a temperature of from about 30°C to about 55°C for about 2 days to about 14 days .

4. The process of preparing a microbial rennin substantially as herein described with particular reference to any one of these specific examples .

5. . microbial rennin product having relatively high milk coagulating activity and relatively low proteolytic activity obtained by the process of any of claims 1 to 4.

6. . The product of claim 5 which is relatively free from lipase .

7. . The process of making chees e in which curds are prepared from milk , which comprises substituting for at least part of the rennet normally us ed in said step the microbial rennin produced by a strain of Mucor miehei . as claimed in claim 5. 3,SBAE¾

8. The process of claim 7, wherein the strain of Mucor miehel is selected from the group consisting of NRRL numbers: 3169, A 7772, A 13,131, A 13,042 and 2543.

9. The process of making cheese substantially as herein described with particular reference to any one of the specific examples.

10. The product obtained by the process of claims 7, 8 or 9. BATED the 11th March, 1968