Abstract:
Yeast cells that express rennin or prorennin are used in disrupted cellular form to clot milk without the need for purification of the rennin or prorennin. Cheese making is simplified and reduced in cost by the avoidance of substantial purification techniques to obtain rennin or prorennin from living cells which produce these materials.

Description:
BACKGROUND OF THE INVENTION 
     A large variety of cheeses are produced by clotting raw or pasteurized milk using acid, rennin or both. In the past, the rennin used is often obtained from rennet. This rennin is in nature an enzymatic protein used for coagulating milk casein in cheese making. It also acts as a cheese ripening material in the latter stages of cheese making through its proteolytic activity. Rennet has in the past been obtained from butchered milk-fed calves&#39; stomachs processed in accordance with known techniques. 
     More recently, rennin or prorennin has been formed by recombinant DNA techniques, see for example European patent publication No. 82 110 124.5 filed Jan. 8, 1982 Publication No. 0057,350,A3 dated Aug. 11, 1982 Bulletin 82/32. 
     In the past, it has been suggested that where rennin or prorennin is produced from cellular materials, purification should be carried out to obtain the purified enzymatic material capable of activation to have milk clotting activity. 
     SUMMARY OF THE INVENTION 
     According to the invention, a method of clotting milk to form cheese comprises providing an active rennin enzyme along with disrupted yeast cells from which the rennin has been obtained, in a milk-based mixture to clot the milk. Preferably, the milk is clotted in the presence of an acid such as lactic acid. In the most preferred form, the yeast cells are those which produce a prorennin material which is activated to have rennin activity. 
     A resulting cheese product contains disrupted yeast cellular material obtained from yeast cells that express rennin or prorennin. The cellular material as well as the rennin remain in the cheese without substantially adversely affecting the edible nature of the cheese. 
     In some forms of the invention, the cell membranes can be removed from the yeast cells after they are disrupted while in other cases, the entire disrupted cell is employed by addition to the cheese making batch. 
     It is a feature of this invention that no separation of rennin or prorennin is required with the producer cells themselves capable of being added directly to the cheese making process. The disruption of the cells aids in freeing the rennin and prorennin materials. The materials can be activated to have rennin enzymatic activity in the presence of the yeast cells with or without the addition of acid. Taste and edibility of resulting cheese can be good with no adverse effects imparted to cheese made employing the methods and materials of the present invention. 
     It is an object of this invention to provide a means and method for clotting milk in a cheese making process using disrupted cellular material having contained therewith active rennin enzyme from said cellular material. 
     Still another object of this invention is to provide a method of clotting milk in an efficient manner with minimized separation of material having rennin activity from yeast cells which produce said material. 
     Still another object of this invention is to provide a cheese producting containing disrupted yeast cellular material from yeast cells that express rennin or prorennin. 
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The yeast cells which are used in the process of clotting milk in cheese making in accordance with the present invention can be any yeast cells which can be transformed with DNA as for example species of Saccharamyces such as Saccharomyces cerevisiae, Saccharomyces carlsbergensis and Saccharomyces uvarum, species of Saccharomycopsis such as Saccharomycopsis lipolytica and species of Schizosaccharomyces such as Schizosaccharomyces pombe. Transformable yeasts are those into which can be introduced foreign vectors with any known recombinant DNA and which produces yeast progeny that carry the vectors. Any such yeast cells which contain recombinant material that expresses rennin or prorennin, and clots milk can be used in connection with the method and products of this invention. 
     Such yeast cells contain DNA materials as for example that shown in Table 1 where the rennin gene is shown at nucleotide 379 to 1350. Several precursors thereof are broadly referred to as &#34;prorennin&#34; herein wich is defined here as precursor forms of rennin having additional amino acids at the amino terminus of rennin, which can be removed to yield an active rennin enzyme which clots milk. Preferably the plurality of nucleotide bases before the 5&#39; end of the rennin are in the range of from 1 to 174 bases. &#34;Prorennin&#34; as used herein is shown in Table 1 at nucleotide 205 to 1350. 
     
                                           TABLE 1__________________________________________________________________________                                 30AAG CTT GOG OGA GOG  AGG GGT AGG OCA TCC  OCA GGA TCC CGT OGA           60                                        90ATT OGG CAT AGG TGA  AGA CGT OCC OGG GCT  OCT GGG TGC TCA GGC                                120CTA CTG TCT GCT GGA  TGT OCA CAA TGT TGG  AGA CAG TGA OGG TGT           150                                       180CAT AGC OCA GGA TGC  OCT GCA TGC TGC CTG  TCC OGT AGT GGA TAG                                210ACA GOG GCT GGA COC  AGA TCC AAG ATG AGG  TGT CTC GTG GTG CTA                            MET ARG  CYS LEU VAL VAL LEU           240                                       270CTT GCT GTC TTC GCT  CTC TCC CAG GGC GCT  GAG ATC ACC AGG ATCLEU ALA VAL PHE ALA  LEU SER GLN GLY ALA  GLU ILE THR ARG ILE                                300OCT CTG TAC AAA GGC  AAG TCT CTG AGG AAG  GOG CTG AAG GAG CATPRO LEU TYR LYS GLY  LYS SER LEU ARG LYS  ALA LEU LYS GLU HIS           330                                       360GGG CTT CTG GAG GAC  TTC CTG CAG AAA CAG  CAG TAG GGC ATC AGCGLY LEU LEU GLU ASP  PHE LEU GLN LYS GLN  GLN TYR GLY ILE SER                                390AGC AAG TAC TCC GGC  TTC GGG GAG GTG GOC  AGC GTG COC CTG AOCSER LYS TYR SER GLY  PHE GLY GLU VAL ALA  SER VAL PRO LEU THR           420                                       450AAC TAC CTG GAT AGT  CAG TAC TTT GGG AAG  ATC TAC CTC GGG ACCASN TYR LEU ASP SER  GLN TYR PHE GLY LYS  ILE TYR LEU GLY THR                                480OCG COC CAG GAG TTC  ACC GTG CTG TTT GAC  ACT GGC TOC TCT GACPRO PRO GLN GLU PHE  THR VAL LEU PHE ASP  THR GLY SER SER ASP           510                                       540TTC TGG GTA COC TCT  ATC TAC TGC AAG AGC  AAT GOC TGC AAA AACPHE TRP VAL PRO SER  ILE TYR CYS LYS SER  ASN ALA CYS LYS ASN                                570CAC CAG OGC TTC GAC  OCG AGA AAG TOG TOC  ACC TTC CAG AAC CTGHIS GLN ARG PHE ASP  PRO ARG LYS SER SER  THR PHE GLN ASN LEU           600                                       630GGC AAG COC CTG TCT  ATC CAC TAC GGG ACA  GGC AGC ATG CAG GGCGLY LYS PRO LEU SER  ILE HIS TYR GLY THR  GLY SER MET GLN GLY                                660ATC CTG GGC TAT GAC  ACC GTC ACT GTC TOC  AAC ATT GTG GAC ATCILE LEU GLY TYR ASP  THR VAL THR VAL SER  ASN ILE VAL ASP ILE           690                                       720CAG CAG ACA GTA GGC  CTG AGC ACC CAG GAG  COC GGG GAC GTC TTCGLN GLN THR VAL GLY  LEU SER THR GLN GLU  PRO GLY ASP VAL PHE                                750ACC TAT GOC GAA TTC  GAC GGG ATC CTG GGG  ATG GOC TAC COC TOGTHR TYR ALA GLU PHE  ASP GLY ILE LEU GLY  MET ALA TYR PRO SER           780                                       810CTC GOC TCA GAG TAC  TOG ATA COC GTG TTT  GAC AAC ATG ATG AACLEU ALA SER GLU TYR  SER ILE PRO VAL PHE  ASP ASN MET MET ASN                                840AGG CAC CTG GTG GOC  CAA GAC CTG TTC TOG  GTT TAC ATG GAC AGGARG HIS LEU VAL ALA  GLN ASP LEU PHE SER  VAL TYR MET ASP ARG           870                                       900AAT GGC CAG GAG AGC  ATG CTC ACG CTG GGG  GOC ATC GAC CCG TCCASN GLY GLN GLU SER  MET LEU THR LEU GLY  ALA ILE ASP PRO SER                                930TAC TAC ACA GGG TOC  CTG CAC TGG GTG OCC  GTG ACA GTG CAG CAGTYR TYR THR GLY SER  LEU HIS TRP VAL PRO  VAL THR VAL GLN GLN           960                                       990TAC TGG CAG TCC ACT  GTG GAC AGT GTC ACC  ATC AGC GGT GTG GTTTYR TRP GLN PHE THR  VAL ASP SER VAL THR  ILE SER GLY VAL VAL                                1020GTG GOC TGT GAG GGT  GGC TGT CAG GCC ATC  CTG GAC ACG GGC ACCVAL ALA CYS GLU GLY  GLY CYS GLN ALA ILE  LEU ASP THR GLY THR           1050                                      1080TOC AAG CTG GTC GGG  COC AGC AGC GAC ATC  CTC AAC ATC CAG CAGSER LYS LEU VAL GLY  PRO SER SER ASP ILE  LEU ASN ILE GLN GLN                                1110GOC ATT GGA GOC ACA  CAG AAC CAG TAC GAT  GAG TTT GAC ATC GACALA ILE GLY ALA THR  GLN ASN GLN TYR ASP  GLU PHE ASP ILE ASP           1140                                      1170TGC GAC AAC CTG AGC  TAC ATG COC ACT GTG  GTC TTT GAG ATC AATCYS ASP ASN LEU SER  TYR MET PRO THR VAL  VAL PHE GLU ILE ASN                                1200GGC AAA ATG TAC OCA  CTG ACC COC TCC GOC  TAT ACC AGC CAG GACGLY LYS MET TYR PRO  LEU THR PRO SER ALA  TYR THR SER GLN ASP           1230                                      1260CAG GGC TTC TGT ACC  AGT GGC TTC CAG AGT  GAA AAT CAT TCC CAGGLN GLY PHE CYS THR  SER GLY PHE GIN SER  GLU ASN HIS SER GLN                                1290AAA TGG ATC CTG GGG  GAT GTT TTC ATC OGA  GAG TAT TAC AGC GTCLYS TRP ILE LEU GLY  ASP VAL PHE ILE ARG  GLU TYR TYR SER VAL           1320                                      1350TTT GAC AGG GCC AAC  AAC CTC GTG GGG CTG  GOC AAA GOC ATC TGAPHE ASP ARG ALA ASN  ASN LEU VAL GLY LEU  ALA LYS ALA ILE                                1380TCA CAT OGC TGA OCA  AGA ACC TCA CTG TOC  OCA CAC ACC TGC ACA           1410                                      1440CAC ACA TGC ACA CAT  GTA CAT GGC ACA TGT  GCA CAC ACA CAG ATGAGG TTT OCA GAC OCA  AGC TT__________________________________________________________________________ 
    
     Yeast cells which can be used in accordance with the present invention include microorganisms such as Saccharomyces cerevisiae identified as Accession No. 20623 deposited in the American Type Culture Collection of 12301 Park Warren Drive, Rockville, Md. 20852 by Collaborative Research, Inc. 
     The preferred yeast microorganism useful in this invention insofar as known to date is yeast cells deposited as Accession No. 20662 which is a strain of Saccharomyces cerevisiae deposited in the American Type Culture Collection of 12301 Park Warren Drive, Rockville, Md. 20852 in February of 1983 which expresses prorennin which can be activated to rennin. The prorennin is expressed in an amount of about 0.2% by weight of all soluble protein expressed by the cells. 
     The yeast cells can be grown in conventional yeast media under conventional yeast growing conditions. For example, common laboratory media known for producing yeast under relatively uniform conditions can be used. Usual growth requirements of yeast include: 
     (a) organic carbon compound for carbon and energy; 
     (b) organic or inorganic nitrogen for the synthesis of proteins and nucleic acids; 
     (c) various minerals (including compounds furnishing trace elements); and 
     (d) frequently a mixture of vitamins. 
     Such growth requirements are met by yeast nitrogen base (YNB, obtained from Difco Company of Detroit, Mich.), a chemically defined medium which contains a number of trace elements, 9 vitamins, trace amounts of amino acids to stimulate growth of certain fastidious yeasts and the principal minerals, potassium phosphate, magnesium sulfate, sodium chloride, and calcium chloride. The nitrogen source is ammonium sulfate. The desired carbon source must be added and is normally at a concentrations of 0.5-3% by weight. Additions can be made to this medium to fit particular strain requirements. The pH range of the medium is usually from pH 3-8. The preferred range is pH 4.5-6.5. 
     The yeast cells are grown under standard conditions as for example temperatures of from 20° to 35° C. as known in the art. The yeast cells can be grown in the culture media to cell densities consistent with maximizing production from the media involved. Often it is found that cells can be grown in the media to densities of from 30 to 100 Klett units corresponding to cells per ml of from 1 to 4×10 7  cells/ml. Preferably the cell density is from 50 to 70 Klett. The time periods of cells growth can vary greatly from two hours to several days as desired. Double times of from 2 to 10 hours are known for most yeast cells. 
     After the cells are grown in the media, they are preferably first separated from the media and resuspended in buffer prior to use in clotting milk in a cheese making operation. However, in some cases the media itself along with the cells can be treated to break the cell walls by mechanical agitation or enzymatic disruption with the resulting disrupted cells in the media itself added to milk in a cheese making operation. 
     Preferably, the cells are first removed from the media as by centrifugation harvesting at speeds at times which do not break the cells as for example rpms of from 1000 to 10,000 at times of from about 30 minutes to about 5 minutes respectively. The cells can also be separated from the media by filtration processes if desired. 
     The cells removed from the media can be stored in a pellet form or used immediately. Pellet forms can be frozen at -70° and stored for indefinite times. 
     Yeast cells containing the recombinant DNA material for expressing rennin or prorennin and preferably prorennin, are preferably suspended in a buffer prior to disruption of the cells. The buffer can be any conventional buffer including: sodium or potassium phosphate, Tris, sodium or potassium acetate. The buffer used where the cells are to be treated with acid for activation of the yeast-synthesized prorennin to obtain rennin activity is preferably in the range of from about pH 4 to about pH 9 and preferably pH 4.7 to 8.7. Where the cells are to be used without addition of acid, the buffer is preferably selected to have a pH in the range of 3.7 to 5.0. 
     The method of breaking the cells walls to form disrupted cells in accordance with the invention can vary. Preferably the cell walls and membranes of the yeast cells are broken by mechanical means although enzymatic disruption of the cell wall can be used. For example, vigorous agitation in the presence of glass beads of 250-450 micrometers in diameter (M. Rose, M. J. Casadaban &amp; D. Botstein, 1981, Proc. Nat&#39;l. Acad. Sci. USA, 78, 2460-2464) can be used to disrupt the yeast cells. The agitation can be by vortexing, centrifuging, stirring or otherwise agitating. Digestion with enzymes such as glusulase (L. Gaurente &amp; M. Ptashne, 1981, Proc. Nat&#39;l Acad. Sci. USA, 78, 2199-2203), lyticase (P. Novick, S. Ferro &amp; R. Schekman, 1981, Cell, 25, 461-469) or zymolase (K. Kitamura &amp; Y Yamamoto, 1981, Agric. Biol. Chem. (Japan), 45, 1761-1766) followed by transfer to low osmotic strength solution (K. Kitamura &amp; Y. Yamamoto, supra) will disrupt yeast cells generating a crude extract. 
     The disrupted cell material includes cytoplasmic and nuclear material and the disrupted cell membranes and walls. However, in many cases, after mechanical breakage, centrifugation or filtering can be used to remove the cell debris, and the disrupted cellular material is used to clot milk with the cell debris, i.e., walls and membranes, removed. Commonly centrifugation at from 10,000 to 30,000 rpm for 30 minutes to 60 minutes will allow removal of the cell walls and membranes as a pellet from the disrupted cellular solutions. The terms &#34;disrupted cellular material&#34; and &#34;disrupted yeast cells&#34; as used herein mean and include all of the cellular material with the cell membranes and walls broken and include such materials with the cell membranes and walls removed as by filtration or centrifugation. 
    
    
     The following examples are illustrative of the invention showing the activation of prorennin made in yeast to given rennin activity in a milk clotting process for use in cheese making. 
     EXAMPLE I 
     Yeast strain CGY 461 (American Type Culture Collection Accession Number 20662) was grown from a single colony on an S-galactose plus leucine agar plate (some composition of liquid media below but also containing 1.5% agar) at 30° C. in 10 ml of S-galactose plus leucine liquid media (having per liter: 6.7 grams Difco yeast nitrogen base without amino acids which was sterilized by autoclaving at 120° C., 15 lbs. pressure for 30 minutes and then supplemented with 100 ml of 20% D-galactose obtained from Sigma Chemical Co. of St. Louis, Mo. and 33 mg L-leucine both of which had been autoclaved separately) to a density of Klett 100-200. A 500 ml culture of the same liquid media was then inoculated at a level of 2% with the smaller overnight culture above and allowed to grow about 15 hours at 30° C. in the liquid media described above until it had reached a Klett of 70. The yeast culture was then harvested in 50 ml aliquots by centrifugation at 7000 rpm for 10 minutes, washed once with distilled water and stored as a cell pellet frozen at -70° C. 
     The prorennin in the yeast cells was activated to rennin as follows. Each frozen cell pellet, representing the cells from 50 ml of S-galactose plus leucine culture, was resuspended in 250 μl of buffer containing 50 mM Tris-Hcl ph 8.7, 10% glycerol, 4 mM (EDTA) ethylenediaminetetraacetic acid and the cells were disrupted by vortexing at top speed in a Genie Vortex Mixer made by Fisher Scientific Company of St. Louis, Mo., with 0.4 grams of nitric acid-washed and water-rinshed glass beads, having average diameters of 250 μm, for four one-minute periods with cooling on ice between cycles. The mass of disrupted cellular material was removed from the glass beads by pipette and transferred to another tube. Within a few minutes, 20 μl of 4M lactic acid was added to 200 μl of the crude cell extract, and the mixture was incubated at 25° C. for 60-90 minutes. After this incubation, which activated the prorennin to rennin, 3.3 μl of 6M NaOH was added to neutralize the mixture. This activated crude disrupted cellular material displays about 1.3 milliunits of milk clotting activity by a modification of the method of Foltmann (B. Foltmann, Methods in Enzymology, 19 421-436). The assays were done at 1/10 scale of Foltmann. Activated extracted (100-200 μl) was added to 1 ml of dried milk (12 g/100 ml) reconstituted in 10 mM CaCl 2  and incubated at 30° C. with shaking. One unit of rennin clots 1 ml milk in 10 seconds by this modified assay. The same procedure performed on the cells of the parent yeast strain containing no prorennin gene results in no detectable milk clotting activity. The buffer used can vary from a pH of 4.7 to 8.7 while the glycerol and EDTA can be eliminated. When the glass beads are changed, mechanical breakage can be carried out at various size beads but preferably the size is in the range of from 200 to 400 μm. The lactic acid used which is about pH 2, may be 4M to 6.5M and other acids can be used so long as the pH is about 2 to permit activation to the rennin activity desired. 
     EXAMPLE II 
     In a second specific example, yeast cells of strain CGY 461 (American Type Culture Collection Accession No. 20662) were grown, harvested and stored as in Example I above. 
     The prorennin in the yeast cells was activated to rennin without the addition of acid here. Each frozen cell pellet, representing the cells from 50 ml of S-galactose plus leucine culture was resuspended in 250 μl of 30 mM sodium acetate buffer at pH 4.7 and the cells were disrupted by vortexing at top speed in a &#34;Genie Vortex Mixer&#34; of Fisher Scientific Co. of St. Louis, Mo., with 0.4 grams nitric acid washed and water rinsed glass beads of average diameter 250 μm for 4 one-minute periods with cooling on ice between cycles. The crude disrupted cells were then removed from the glass beads by pipette and transferred to another tube. After incubation at 25° C. for one hour, 200 μl of the activated crude disrupted cellular material displayed 2 milliunits of milk clotting activity using a modificaton of the method of Foltmann as before (B. Foltmann, Methods in Enzymology, 19, 421-436). A similar extract from yeast cells containing no prorennin gene displayed no detectable milk clotting activity. Here the buffer used can range in pH from 3.7 to 5.0. 
     In a theoretical example of making cheese, cheddar cheese is made by substitution for the rennet preparation ordinarily used in a setting step, an equivalent amount of the disrupted cellular product prepared as in Example I. In this procedure, pasteurized whole milk is adjusted to 86°-88° F. and 1% by weight of a commercial lactic acid starter solution is added. The disrupted cellular material as obtained in Example I from 25 liters of culture is added for 10 liters 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 can be transferred to hoops, pressed and then placed in a curing room. Cheese made from the rennin of this example can be sampled periodically after several days of curing and can be found to possess excellent qualities and be essentially free of unwanted flavors. 
     While specific embodiments of the present invention have been shown and described, many variations are possible. Other conventional methods of making cheddar cheese, for example, those described by Prescott and Dunn in &#34;Industrial Microbiology&#34;, chapter 21 (3rd edition 1959), McGraw-Hill Book Co., Inc., New York and the references cited therein can be used in the practice of the present invention by substitution for the rennet preparations ordinarly employed in cheese making, an amount of the disrupted cellular material of the present invention containing an equivalent amount of rennet to produce quality cheese. The cheeses can be cheddar or any of the roughly eighteen varieties known. 
     The amount of disrupted cellular material used is preferably that which carries rennin activity units of rennin or prorennin in an amount equivalent to the rennin activity units of conventional rennin conventionally added to milk in cheese making procedures. For example, when making cheddar cheese, 130 Foltmann units of rennin activity in a volume of 50 ml to each 10 liters of milk is customarily used. However, the volume of Foltmann units can vary depending upon the particular cheese, the hardness of the cheese and other values in the final product desired. When the rennin activity units of the yeast cell used is maintained at about the level of conventional rennet, rennin activity levels as used in various cheese making operations, similar results can be obtained. For example, in making cheddar cheese, sufficient disrupted cells containing about 130 Foltmann units in each 50 ml of buffer for each 10 liters of milk to be clotted are used and yield desirable results are obtained in cheese making. 
     The cells of Example I when disrupted contain sufficient genetic material to provide at least 0.2% of the protein produced by such cells being prorennin with 0.025% rennin activity. However, desirably, yeast cells for use in the present invention have at least 0.5% of the protein produced by the yeast cells having rennin activity or being prorennin which can be activated to that level of rennin activity. Where the yeast cells used do not themselves have sufficient rennin activity as may be desired in a particular cheese making procedure, additional or supplementary naturally occurring or otherwise required rennin can be added to the process to enhance cheese making. 
     In the embodiment shown, cells from 16 liters of culture grown as in Example I are sufficient for clotting 10 liters of milk when all of the prorennin of the cells is activated to have rennin activity. When cells having 0.5% of protein with rennin activity are used, cells from 6.5 liters as grown in Example I clot 10 liters of milk. When the cells of Example I are used with only 0.025% of protein having rennin activity, the cells from 65 liters should be used to clot 10 liters of milk or supplementary rennin can be added along with small quantities of cells. Of course the amounts of cellular material and rennin used can vary greatly depending on the type of cheese desired and other parameters used and materials added in the cheese making operation.