Abstract:
The invention provides compositions comprising a plurality of yeast cells, wherein said plurality of yeast cells are characterized by their ability to decrease liver collagen level or formation of liver fibrous tissue or to normalize serum γ-globulin level, and therefore are useful for ameliorating and/or preventing liver cirrhosis in a subject, said ability resulting from their having been cultured in the presence of an alternating electric field having a specific frequency and a specific field strength. Also provided are methods of making and using these compositions.

Description:
FIELD OF THE INVENTION  
       [0001]     The invention relates to yeast compositions that can ameliorate or prevent liver cirrhosis and are useful as a dietary supplement or medication. These compositions contain yeast cells obtainable by growth in electromagnetic fields with specific frequencies and field strengths.  
       BACKGROUND OF THE INVENTION  
       [0002]     Liver cirrhosis, or cirrhosis, is a chronic liver disease in which fibrous tissue and nodules replace normal tissue, interfering with blood flow and normal functions of the organ. Cirrhosis can be caused by, e.g., chronic alcoholism, chronic viral hepatitis (types B, C, and D), cystic fibrosis, severe reactions to prescribed drugs, prolonged exposure to environmental toxins, etc.  
         [0003]     Cirrhosis causes irreversible liver damage. If untreated, liver and kidney failure and gastrointestinal hemorrhage can occur, sometimes leading to death. In the United States, cirrhosis results in about 25,000 deaths annually. Apart from a vegetable protein-rich diet, abstinence from alcohol and rest, common medication includes vitamin B, vitamin E, vitamin C, etc. But these treatments are less than satisfactory. There remains a need for an effective method for treating liver cirrhosis.  
       SUMMARY OF THE INVENTION  
       [0004]     This invention is based on the discovery that certain yeast cells can be activated by electromagnetic fields having specific frequencies and field strengths to produce substances beneficial for the liver and therefore improving liver health. Compositions comprising these activated yeast cells can be used as dietary supplement for alleviating and/or preventing liver cirrhosis.  
         [0005]     This invention embraces a composition comprising a plurality of yeast cells that have been cultured in an alternating electric field having a frequency in the range of about 7700-12800 MHz (e.g., 7800-8000 or 12150-12750 MHz), and a field intensity in the range of about 240-500 mV/cm (e.g., 260-280, 270-290, 300-330, 310-340, 320-350, 330-370, 340-370, 350-380, 400-440, or 430-470 mV/cm). The yeast cells are cultured in the alternating electric field for a period of time sufficient to substantially increase the capability of said plurality of yeast cells to produce substances beneficial for the liver (e.g., for treating cirrhosis). In one embodiment, the frequency and/or the field strength of the alternating electric field can be altered within the aforementioned ranges during said period of time. In other words, the yeast cells can be exposed to a series of electromagnetic fields. An exemplary period of time is about 40-160 hours (e.g., 60-150 hours).  
         [0006]     Also included in this invention is a composition comprising a plurality of yeast cells that have been cultured under acidic conditions in an alternating electric field having a frequency in the range of about 12150-12750 MHz (e.g., 12550-12750 MHz) and a field strength in the range of about 280 to 420 mV/cm (e.g., 320-380 mV/cm). In one embodiment, the yeast cells are exposed to a series of electromagnetic fields. An exemplary period of time is about 30-100 hours (e.g., 40-74 hours).  
         [0007]     Included in this invention are also methods for making the above compositions.  
         [0008]     Yeast cells that can be included in this composition can be derived from parent strains publically available from the China General Microbiological Culture Collection Center (“CGMCC”), China Committee for Culture Collection of Microorganisms, Institute of Microbiology, Chinese Academy of Sciences, Haidian, P.O. BOX 2714, Beijing, 100080, China. Useful yeast species include, but are not limited to  Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces rouxii, Saccharomyces sake, Saccharomyces uvarum, Saccharomyces  sp.,  Schizosaccharomyces pombe , and  Rhodotorula aurantiaca . For instance, the yeast cells can be of the strain  Saccharomyces cerevisiae  Hansen AS2.562 or AS2.69,  Saccharomyces  sp. AS2.311,  Schizosaccharomyces pombe  Lindner AS2.994,  Saccharomyces sake  Yabe ACCC2045,  Saccharomyces uvarum  Beijer IFFI1044,  Saccharomyces rouxii  Boutroux AS2.180,  Saccharomyces cerevisiae  Hansen Var. ellipsoideus AS2.612,  Saccharomyces carlsbergensis  Hansen AS2.377, or  Rhodotorula rubar  (Demme) Lodder AS2.282. Other useful yeast strains are illustrated in Table 1.  
         [0009]     Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting. Throughout this specification and claims, the word “comprise,” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.  
         [0010]     Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0011]      FIG. 1  is a schematic diagram showing an exemplary apparatus for activating yeast cells using electromagnetic fields. 1: yeast culture; 2: container; 3: power supply.  
         [0012]      FIG. 2  is a schematic diagram showing an exemplary apparatus for making yeast compositions of the invention. The apparatus comprises a signal generator (such as models 83721B and 83741A manufactured by HP) and interconnected containers A, B and C. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0013]     This invention is based on the discovery that certain yeast strains can be activated by electromagnetic fields (“EMF”) having specific frequencies and field strengths to produce agents useful for treating liver cirrhosis. Yeast compositions containing activated yeast cells can be used as medication, or as a dietary supplement in the form of health drinks or dietary pills.  
         [0014]     In certain embodiments, the yeast compositions of this invention inhibit the synthesis and secretion of collagen in liver. In other embodiments, the yeast compositions inhibit the formation of intra- and inter-molecular cross-linking of collagen molecules. In further embodiments, the yeast compositions reduce the level of serum γ-globulin.  
         [0015]     Since the activated yeast cells contained in these yeast compositions have been cultured to endure acidic conditions (pH 2.5-4.2), the compositions are stable in the stomach and can pass on to the intestines. Once in the intestines, the yeast cells are ruptured by various digestive enzymes, and the bioactive agents are released and readily absorbed.  
         [0000]     I. Yeast Strains Useful in the Invention  
         [0016]     The types of yeasts useful in this invention include, but are not limited to, yeasts of the genera of  Saccharomyces, Rhodotorula , and  Schizosaccharomyces.    
         [0017]     Exemplary species within the above-listed genera include, but are not limited to, the species illustrated in Table 1. Yeast strains useful in this invention can be obtained from laboratory cultures, or from publically accessible culture depositories, such as CGMCC and the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209. Non-limiting examples of useful strains (with the accession numbers of CGMCC) are  Saccharomyces cerevisiae  Hansen AS2.562 and AS2.69,  Saccharomyces  sp. AS2.311,  Schizosaccharomyces pombe  Lindner AS2.994,  Saccharomyces sake  Yabe ACCC2045,  Saccharomyces uvarum  Beijer IFFI1044,  Saccharomyces rouxii  Boutroux AS2.180,  Saccharomyces cerevisiae  Hansen Var. ellipsoideus AS2.612,  Saccharomyces carlsbergensis  Hansen AS2.377, and  Rhodotorula rubar  (Demme) Lodder AS2.282. Other non-limiting examples of useful strains are listed in Table 1. In general, preferred yeast strains in this invention are those used for fermentation in the food and wine industries. As a result, compositions containing these yeast cells are safe for human consumption.  
         [0018]     The preparation of the yeast compositions of this invention is not limited to starting with a pure strain of yeast. A yeast composition of the invention may be produced by culturing a mixture of yeast cells of different species or strains.  
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 TABLE 1                       Exemplary Yeast Strains                      Saccharomyces cerevisiae  Hansen            ACCC2034   ACCC2035   ACCC2036   ACCC2037   ACCC2038       ACCC2039   ACCC2040   ACCC2041   ACCC2042   AS2.1       AS2.4   AS2.11   AS2.14   AS2.16   AS2.56       AS2.69   AS2.70   AS2.93   AS2.98   AS2.101       AS2.109   AS2.110   AS2.112   AS2.139   AS2.173       AS2.174   AS2.182   AS2.196   AS2.242   AS2.336       AS2.346   AS2.369   AS2.374   AS2.375   AS2.379       AS2.380   AS2.382   AS2.390   AS2.393   AS2.395       AS2.396   AS2.397   AS2.398   AS2.399   AS2.400       AS2.406   AS2.408   AS2.409   AS2.413   AS2.414       AS2.415   AS2.416   AS2.422   AS2.423   AS2.430       AS2.431   AS2.432   AS2.451   AS2.452   AS2.453       AS2.458   AS2.460   AS2.463   AS2.467   AS2.486       AS2.501   AS2.502   AS2.503   AS2.504   AS2.516       AS2.535   AS2.536   AS2.558   AS2.560   AS2.561       AS2.562   AS2.576   AS2.593   AS2.594   AS2.614       AS2.620   AS2.628   AS2.631   AS2.666   AS2.982       AS2.1190   AS2.1364   AS2.1396   IFFI1001   IFFI1002       IFFI1005   IFFI1006   IFFI1008   IFFI1009   IFFI1010       IFFI1012   IFFI1021   IFFI1027   IFFI1037   IFFI1042       IFFI1043   IFFI1045   IFFI1048   IFFI1049   IFFI1050       IFFI1052   IFFI1059   IFFI1060   IFFI1062   IFFI1063       IFFI1202   IFFI1203   IFFI1206   IFFI1209   IFFI1210       IFFI1211   IFFI1212   IFFI1213   IFFI1214   IFFI1215       IFFI1220   IFFI1221   IFFI1224   IFFI1247   IFFI1248       IFFI1251   IFFI1270   IFFI1277   IFFI1287   IFFI1289       IFFI1290   IFFI1291   IFFI1292   IFFI1293   IFFI1297       IFFI1300   IFFI1301   IFFI1302   IFFI1307   IFFI1308       IFFI1309   IFFI1310   IFFI1311   IFFI1331   IFFI1335       IFFI1336   IFFI1337   IFFI1338   IFFI1339   IFFI1340       IFFI1345   IFFI1348   IFFI1396   IFFI1397   IFFI1399       IFFI1411   IFFI1413   IFFI1441   IFFI1443              Saccharomyces cerevisiae  Hansen Var.  ellipsoideus  (Hansen) Dekker            ACCC2043   AS2.2   AS2.3   AS2.8   AS2.53       AS2.163   AS2.168   AS2.483   AS2.541   AS2.559       AS2.606   AS2.607   AS2.611   AS2.612              Saccharomyces chevalieri  Guilliermond            AS2.131   AS2.213                          Saccharomyces delbrueckii              AS2.285                              Saccharomyces delbrueckii  Lindner ver.  mongolicus  (Saito) Lodder et van Rij            AS2.209   AS2.1157                          Saccharomyces exiguous  Hansen            AS2.349   AS2.1158                          Saccharomyces fermentati  (Saito) Lodder et van Rij            AS2.286   AS2.343                          Saccharomyces logos  van laer et Denamur ex Jorgensen            AS2.156   AS2.327   AS2.335                      Saccharomyces mellis  (Fabian et Quinet) Lodder et kreger van Rij            AS2.195                              Saccharomyces mellis Microellipsoides  Osterwalder            AS2.699                              Saccharomyces oviformis  Osteralder            AS2.100                              Saccharomyces rosei  (Guilliermond) Lodder et Kreger van Rij            AS2.287                              Saccharomyces rouxii  Boutroux            AS2.178   AS2.180   AS2.370   AS2.371                  Saccharomyces sake  Yabe            ACCC2045                              Candida arborea              AS2.566                              Candida lambica  (Lindner et Genoud) van. Uden et Buckley            AS2.1182                              Candida krusei  (Castellani) Berkhout            AS2.1045                              Candida lipolytica  (Harrison) Diddens et Lodder            AS2.1207   AS2.1216   AS2.1220   AS2.1379   AS2.1398       AS2.1399   AS2.1400              Candida parapsilosis  (Ashford) Langeron et Talice Var.  intermedia  Van Rij et Verona            AS2.491                              Candida parapsilosis  (Ashford) Langeron et Talice            AS2.590                              Candida pulcherrima  (Lindner) Windisch            AS2.492                              Candida rugousa  (Anderson) Diddens et Lodder            AS2.511   AS2.1367   AS2.1369   AS2.1372   AS2.1373       AS2.1377   AS2.1378   AS2.1384              Candida tropicalis  (Castellani) Berkhout            ACCC2004   ACCC2005   ACCC2006   AS2.164   AS2.402       AS2.564   AS2.565   AS2.567   AS2.568   AS2.617       AS2.637   AS2.1387   AS2.1397              Candida utilis  Henneberg Lodder et Kreger Van Rij            AS2.120   AS2.281   AS2.1180                      Crebrothecium ashbyii  (Guillermond)       Routein ( Eremothecium ashbyii  Guilliermond)            AS2.481   AS2.482   AS2.1197                      Geotrichum candidum  Link            ACCC2016   AS2.361   AS2.498   AS2.616   AS2.1035       AS2.1062   AS2.1080   AS2.1132   AS2.1175   AS2.1183              Hansenula anomala  (Hansen)H et P sydow            ACCC2018   AS2.294   AS2.295   AS2.296   AS2.297       AS2.298   AS2.299   AS2.300   AS2.302   AS2.338       AS2.339   AS2.340   AS2.341   AS2.470   AS2.592       AS2.641   AS2.642   AS2.782   AS2.635   AS2.794              Hansenula arabitolgens  Fang            AS2.887                              Hansenula jadinii  (A. et R Sartory Weill et Meyer) Wickerham            ACCC2019                              Hansenula saturnus  (Klocker) H et P sydow            ACCC2020                              Hansenula schneggii  (Weber) Dekker            AS2.304                              Hansenula subpelliculosa  Bedford            AS2.740   AS2.760   AS2.761   AS2.770   AS2.783       AS2.790   AS2.798   AS2.866              Kloeckera apiculata  (Reess emend. Klocker) Janke            ACCC2022   ACCC2023   AS2.197   AS2.496   AS2.714       ACCC2021   AS2.711              Lipomycess starkeyi  Lodder et van Rij            AS2.1390   ACCC2024                          Pichia farinosa  (Lindner) Hansen            ACCC2025   ACCC2026   AS2.86   AS2.87   AS2.705       AS2.803              Pichia membranaefaciens  Hansen            ACCC2027   AS2.89   AS2.661   AS2.1039                  Rhodosporidium toruloides  Banno            ACCC2028                              Rhodotorula glutinis  (Fresenius) Harrison            AS2.2029   AS2.280   ACCC2030   AS2.102   AS2.107       AS2.278   AS2.499   AS2.694   AS2.703   AS2.704       AS2.1146              Rhodotorula minuta  (Saito) Harrison            AS2.277                              Rhodotorula rubar  (Demme) Lodder            AS2.21   AS2.22   AS2.103   AS2.105   AS2.108       AS2.140   AS2.166   AS2.167   AS2.272   AS2.279       AS2.282   ACCC2031              Rhodotorula aurantiaca  (Saito) Lodder            AS2.102   AS2.107   AS2.278   AS2.499   AS2.694       AS2.703   AS2.1146              Saccharomyces carlsbergensis  Hansen            AS2.113   ACCC2032   ACCC2033   AS2.312   AS2.116       AS2.118   AS2.121   AS2.132   AS2.162   AS2.189              Saccharomyces uvarum  Beijer            IFFI1023   IFFI1032   IFFI1036   IFFI1044   IFFI1072       IFFI1205   IFFI1207              Saccharomyces willianus  Saccardo            AS2.5 AS2.7   AS2.119   AS2.152   AS2.293           AS2.381   AS2.392   AS2.434   AS2.614   AS2.1189              Saccharomyces  sp.            AS2.311                              Saccharomycodes ludwigii  Hansen            ACCC2044   AS2.243   AS2.508                      Saccharomycodes sinenses  Yue            AS2.1395                              Schizosaccharomyces octosporus  Beijerinck            ACCC2046   AS2.1148                          Schizosaccharomyces pombe  Lindner            ACCC2047   ACCC2048   AS2.214   AS2.248   AS2.249       AS2.255   AS2.257   AS2.259   AS2.260   AS2.274       AS2.994   AS2.1043   AS2.1149   AS2.1178   IFFI1056              Sporobolomyces roseus  Kluyver et van Niel            ACCC2049   ACCC2050   AS2.19   AS2.962   AS2.1036       ACCC2051   AS2.261   AS2.262              Torulopsis candida  (Saito) Lodder            AS2.270   ACCC2052                          Torulopsis famta  (Harrison) Lodder et van Rij            ACCC2053   AS2.685                          Torulopsis globosa  (Olson et Hammer) Lodder et van Rij            ACCC2054   AS2.202                          Torulopsis inconspicua  Lodder et Kreger van Rij            AS2.75                              Trichosporon behrendii  Lodder et Kreger van Rij            ACCC2056   AS2.1193                          Trichosporon capitatum  Diddens et Lodder            ACCC2056   AS2.1385                          Trichosporon cutaneum  (de Beurm et al.) Ota            ACCC2057   AS2.25   AS2.570   AS2.571   AS2.1374              Wickerhamia fluorescens  (Soneda) Soneda            ACCC2058   AS2.1388                  
 
 II. Application of Electromagnetic Fields 
 
         [0019]     An electromagnetic field useful in this invention can be generated and applied by various means well known in the art. For instance, the EMF can be generated by applying an alternating electric field or an oscillating magnetic field.  
         [0020]     Alternating electric fields can be applied to cell cultures through electrodes in direct contact with the culture medium, or through electromagnetic induction. See, e.g.,  FIG. 1 . Relatively high electric fields in the medium can be generated using a method in which the electrodes are in contact with the medium. Care must be taken to prevent electrolysis at the electrodes from introducing undesired ions into the culture and to prevent contact resistance, bubbles, or other features of electrolysis from dropping the field level below that intended. Electrodes should be matched to their environment, for example, using Ag—AgCl electrodes in solutions rich in chloride ions, and run at as low a voltage as possible. For general review, see Goodman et al.,  Effects of EMF on Molecules and Cells , International Review of Cytology, A Survey of Cell Biology, Vol. 158, Academic Press, 1995.  
         [0021]     The EMFs useful in this invention can also be generated by applying an oscillating magnetic field. An oscillating magnetic field can be generated by oscillating electric currents going through Helmholtz coils. Such a magnetic field in turn induces an electric field.  
         [0022]     The frequencies of EMFs useful in this invention range from about 7700-12800 MHz (e.g., 7800-8000 or 12150-12750 MHz). Exemplary frequencies include 7886, 7907, 12224, 12646, and 12662 MHz. The field strength of the electric field useful in this invention ranges from about 240-500 mV/cm (e.g., 260-280, 270-290, 300-330, 310-340, 320-350, 330-370, 340-370, 350-380, 400-440, or 430-470 mV/cm). Exemplary field strengths include 274, 278, 311, 324, 337, 347, 355, 364, 368, 413, and 442 mV/cm.  
         [0023]     When a series of EMFs are applied to a yeast culture, the yeast culture can remain in the same container while the same set of EMF generator and emitters is used to change the frequency and/or field strength. The EMFs in the series can each have a different frequency or a different field strength; or a different frequency and a different field strength. Such frequencies and field strengths are preferably within the above-described ranges. Although any practical number of EMFs can be used in a series, it may be preferred that the yeast culture be exposed to a total of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or more EMFs in a series. In one embodiment, the yeast culture is exposed to a series of EMFs, wherein the frequency of the electric field is alternated in the range of about 7800-8000, 12150-12300, and 12550-12800 MHz.  
         [0024]     Although the yeast cells can be activated after even a few hours of culturing in the presence of an EMF, it may be preferred that the activated yeast cells be allowed to multiply and grow in the presence of the EMF(s) for a total of 40-160 hours.  
         [0025]      FIG. 1  illustrates an exemplary apparatus for generating alternating electric fields. An electric field of a desired frequency and intensity can be generated by an AC source (3) capable of generating an alternating electric field, preferably in a sinusoidal wave form, in the frequency range of 5 to 20,000 MHz. Signal generators capable of generating signals with a narrower frequency range can also be used. If desired, a signal amplifier can also be used to increase the output. The culture container (2) can be made from a non-conductive material, e.g., glass, plastic or ceramic. The cable connecting the culture container (2) and the signal generator (3) is preferably a high frequency coaxial cable with a transmission frequency of at least 30 GHz.  
         [0026]     The alternating electric field can be applied to the culture by a variety of means, including placing the yeast culture (1) in close proximity to the signal emitters such as a metal wire or tube capable of transmitting EMFs. The metal wire or tube can be made of red copper, and be placed inside the container (2), reaching as deep as 3-30 cm. For example, if the fluid in the container (2) has a depth of 15-20 cm, 20-30 cm, 30-50 cm, 50-70 cm, 70-100 cm, 100-150 cm or 150-200 cm, the metal wire can be 3-5 cm, 5-7 cm, 7-10 cm, 10-15 cm, 15-20 cm, 20-30 cm, and 25-30 cm from the bottom of the container (2), respectively. The number of metal wires/tubes used can be from 1 to 10 (e.g., 2 to 3). It is recommended, though not mandated, that for a culture having a volume up to 10 L, metal wires/tubes having a diameter of 0.5 to 2 mm be used. For a culture having a volume of 10-100 L, metal wires/tubes having a diameter of 3 to 5 mm can be used. For a culture having a volume of 100-1000 L, metal wires/tubes having a diameter of 6 to 15 mm can be used. For a culture having a volume greater than 1000 L, metal wires/tubes having a diameter of 20-25 mm can be used.  
         [0027]     In one embodiment, the electric field is applied by electrodes submerged in the culture (1). In this embodiment, one of the electrodes can be a metal plate placed on the bottom of the container (2), and the other electrode can comprise a plurality of electrode wires evenly distributed in the culture (1) so as to achieve even distribution of the electric field energy.  
         [0000]     III. Culture Media  
         [0028]     Culture media useful in this invention contain sources of nutrients that can be assimilated by yeast cells. Complex carbon-containing substances in a suitable form (e.g., carbohydrates such as sucrose, glucose, dextrose, maltose, xylose, cellulose, starch, etc.) can be the carbon sources for yeast cells. The exact quantity of the carbon sources can be adjusted in accordance with the other ingredients of the medium. In general, the amount of carbohydrate varies between about 1% and 10% by weight of the medium and preferably between about 1% and 5%, and most preferably about 2%. These carbon sources can be used individually or in combination. Amino acid-containing substances such as beef extract and peptone can also be added. In general, the amount of amino acid containing substances varies between about 0.1% and 1% by weight of the medium and preferably between about 0.1% and 0.5%. Among the inorganic salts which can be added to a culture medium are the customary salts capable of yielding sodium, potassium, calcium, phosphate, sulfate, carbonate, and like ions. Non-limiting examples of nutrient inorganic salts are (NH 4 ) 2 HPO 4 , CaCO 3 , KH 2 PO 4 , K 2  HPO 4 , MgSO 4 , NaCl, and CaSO 4 .  
         [0000]     IV. Electromagnetic Activation of Yeast Cells  
         [0029]     To activate or enhance the ability of yeast cells to produce agents useful for treating cirrhosis, these cells can be cultured in an appropriate medium under sterile conditions at 20-35° C. (e.g., 28-32° C.) for a sufficient amount of time (e.g., 60-150 hours) in an alternating electric field or a series of alternating electric fields as described above.  
         [0030]     An exemplary set-up of the culture process is depicted in  FIG. 1  (see above). An exemplary culture medium contains the following per 1000 ml of sterile water: 18 g of mannitol, 50 μg of Vitamin B 6 , 80 μg of Vitamin B 12 , 50 μg of Vitamin H, 100          g of Vitamin E, 35 ml of fetal bovine serum, 0.2 g of KH 2 PO 4 , 0.25 g of MgSO 4 .7H 2 O, 0.3 g of NaCl, 0.2 g of CaSO 4 .2H 2 O, 4 g of CaCO 3 .5H 2 O, and 2.5 g of peptone. Yeast cells of the desired strain(s) are then added to the culture medium to form a mixture containing 1×10 8  cells per 1000 ml of culture medium. The yeast cells can be of any of the strains listed in Table 1. The mixture is then added to the apparatus shown in  FIG. 1 .  
         [0031]     The activation process of the yeast cells involves the following steps: (1) maintaining the temperature of the activation apparatus at 24-33° C. (e.g., 28-32° C.), and culturing the yeast cells for 24-30 hours (e.g., 28 hours); (2) applying an alternating electric field having a frequency of 7886 MHz and a field strength of 260-280 mV/cm (e.g., 274 mV/cm) for 11-17 hours (e.g., 15 hours); (3) then applying an alternating electric field having a frequency of 7907 MHz and a field strength of 300-330 mV/cm (e.g., 311 mV/cm) for 31-37 hours (e.g., 35 hours); (4) then applying an alternating electric field having a frequency of 12224 MHz and a field strength of 320-350 mV/cm (e.g., 337 mV/cm) for 39-45 hours (e.g., 43 hours); (5) then applying an alternating electric field having a frequency of 12646 MHz and a field strength of 340-370 mV/cm (e.g., 355 mV/cm) for 33-39 hours (e.g., 37 hours); and (6) then applying an alternating electric field having a frequency of 12662 MHz and a field strength of 270-290 mV/cm (e.g., 278 mV/cm) for 13-19 hours (e.g., 17 hours). The activated yeast cells are then recovered from the culture medium by various methods known in the art, dried (e.g., by lyophilization) and stored at 4° C. Preferably, the concentration of the dried yeast cells is no less than 10 10  cells/g.  
         [0000]     V. Acclimatization of Yeast Cells to the Gastric Environment  
         [0032]     Because the yeast compositions of this invention must pass through the stomach before reaching the small intestine, where the effective components are released from these yeast cells, it is preferred that these yeast cells be cultured under acidic conditions to acclimatize the cells to the gastric juice. This acclimatization process results in better viability of the yeast cells in the acidic gastric environment.  
         [0033]     To achieve this, the yeast powder containing activated yeast cells can be mixed with a highly acidic acclimatizing culture medium at 10 g (containing more than 10 10  activated cells per gram) per 1000 ml. The yeast mixture is then cultured first in the presence of an alternating electric field having a frequency of 12646 MHz and a field strength of 350-380 mV/cm (e.g., 368 mV/cm) at about 28 to 32° C. for 40 to 50 hours (e.g., 45 hours). The resultant yeast cells can then be further incubated in the presence of an alternating electric field having a frequency of 12662 MHz and a field strength of 320-350 mV/cm (e.g., 324 mV/cm) at about 28 to 32° C. for 16 to 24 hours (e.g., 20 hours). The resulting acclimatized yeast cells are then dried and stored either in powder form (≧10 10  cells/g) at room temperature or in vacuum at 0-4° C.  
         [0034]     An exemplary acclimatizing culture medium is made by mixing 700 ml fresh pig gastric juice and 300 ml wild Chinese hawthorn extract. The pH of the acclimatizing culture medium is adjusted to 2.5 with 0.1 M hydrochloric acid (HCl) and 0.2 M potassium hydrogen phthalate (C 6 H 4 (COOK)COOH). The fresh pig gastric juice is prepared as follows. At about 4 months of age, newborn Holland white pigs are sacrificed, and the entire contents of their stomachs are retrieved and mixed with 2000 ml of water under sterile conditions. The mixture is then allowed to stand for 6 hours at 4° C. under sterile conditions to precipitate food debris. The supernatant is collected for use in the acclimatizing culture medium. To prepare the wild Chinese hawthorn extract, 500 g of fresh wild Chinese hawthorn is dried under sterile conditions to reduce water content (≦8%). The dried fruit is then ground (≧20 mesh) and added to 1500 ml of sterilized water. The hawthorn slurry is allowed to stand for 6 hours at 4° C. under sterile conditions. The hawthorn supernatant is collected to be used in the acclimatizing culture medium.  
         [0000]     VI. Manufacture of Yeast Compositions  
         [0035]     To manufacture the yeast compositions of the invention, an apparatus depicted in  FIG. 2  or an equivalent thereof can be used. This apparatus includes three containers, a first container (A), a second container (B), and a third container (C), each equipped with a pair of electrodes (4). One of the electrodes is a metal plate placed on the bottom of the containers, and the other electrode comprises a plurality of electrode wires evenly distributed in the space within the container to achieve even distribution of the electric field energy. All three pairs of electrodes are connected to a common signal generator.  
         [0036]     The culture medium used for this purpose is a mixed fruit extract solution containing the following ingredients per 1000 L: 300 L of wild Chinese hawthorn extract, 300 L of jujube extract, 300 L of  Schisandra chinensis  (Turez) Baill seed extract, and 100 L of soy bean extract. To prepare hawthorn, jujube and  Schisandra chinensis  (Turez) Baill seed extracts, the fresh fruits are washed and dried under sterile conditions to reduce the water content to no higher than 8%. One hundred kilograms of the dried fruits are then ground (≧20 mesh) and added to 400 L of sterilized water. The mixtures are stirred under sterile conditions at room temperature for twelve hours, and then centrifuged at 1000 rpm to remove insoluble residues. To make the soy bean extract, fresh soy beans are washed and dried under sterile conditions to reduce the water content to no higher than 8%. Thirty kilograms of dried soy beans are then ground into particles of no smaller than 20 mesh, and added to 130 L of sterilized water. The mixture is stirred under sterile conditions at room temperature for twelve hours and centrifuged at 1000 rpm to remove insoluble residues. To make the culture medium, these ingredients are mixed according to the above recipe, and the mixture is autoclaved at 121° C. for 30 minutes and cooled to below 40° C. before use.  
         [0037]     One thousand grams of the activated yeast powder prepared as described above (Section V, supra) is added to 1000 L of the mixed fruit extract solution, and the yeast solution is transferred to the first container (A) shown in  FIG. 2 . The yeast cells are then cultured in the presence of an alternating electric field having a frequency of 12646 MHz and a field strength of about 400-440 mV/cm (e.g., 413 mV/cm) at 28-32° C. under sterile conditions for 32 hours. The yeast cells are further incubated in an alternating electric field having a frequency of 12662 MHz and a field strength of 330-370 mV/cm (e.g., 347 mV/cm). The culturing continues for another 12 hours.  
         [0038]     The yeast culture is then transferred from the first container (A) to the second container (B) which contains 1000 L of culture medium (if need be, a new batch of yeast culture can be started in the now available first container (A)), and subjected to an alternating electric field having a frequency of 12646 MHz and a field strength of 430-470 mV/cm (e.g., 442 mV/cm) for 24 hours. Subsequently the frequency and field strength of the electric field are changed to 12662 MHz and 350-380 mV/cm (e.g., 364 mV/cm), respectively. The culturing continues for another 12 hours.  
         [0039]     The yeast culture is then transferred from the second container (B) to the third container (C) which contains 1000 L of culture medium, and subjected to an alternating electric field having a frequency of 12646 MHz and a field strength of 310-340 mV/cm (e.g., 324 mV/cm) for 24 hours. Subsequently the frequency and field strength of the electric field are changed to 12662 MHz and 260-280 mV/cm (e.g., 274 mV/cm), respectively. The culturing continues for another 12 hours.  
         [0040]     The yeast culture from the third container (C) can then be packaged into vacuum sealed bottles for use as dietary supplements, e.g., health drinks, or medication in the form of pills, powder, etc. If desired, the final yeast culture can also be dried within 24 hours and stored in powder form. The dietary supplement can be taken three to four times daily at 30-60 ml per dose for a three-month period, preferably 10-30 minutes before meals and at bedtime.  
         [0041]     In some embodiments, the compositions of the invention can also be administered intravenously or peritoneally in the form of a sterile injectable preparation. Such a sterile preparation can be prepared as follows. A sterilized health drink composition is first treated under ultrasound (20,000 Hz) for 10 minutes and then centrifuged for another 10 minutes. The resulting supernatant is adjusted to pH 7.2-7.4 using 1 M NaOH and subsequently filtered through a membrane (0.22 μm for intravenous injection and 0.45 μm for peritoneal injection) under sterile conditions. The resulting sterile preparation is submerged in a 35-38° C. water bath for 30 minutes before use. In other embodiments, the compositions of the invention may also be formulated with pharmaceutically acceptable carriers to be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, suspensions or solutions.  
         [0042]     The yeast compositions of the present invention are derived from yeasts used in food and pharmaceutical industries. The yeast compositions are thus devoid of side effects associated with many pharmaceutical compounds.  
       VII. EXAMPLES  
       [0043]     The following examples are meant to illustrate the methods and materials of the present invention. Suitable modifications and adaptations of the described conditions and parameters which are obvious to those skilled in the art are within the spirit and scope of the present invention.  
         [0044]     The activated yeast compositions used in the following experiments were prepared as described above, using  Saccharomyces cerevisiae  Hansen AS2.562 cells cultured in the presence of an alternating electric field having the electric field frequency and field strength exemplified in the parentheses following the recommended ranges listed in Section IV, supra. Control yeast compositions were those prepared in the same manner except that the yeast cells were cultured in the absence of EMFs. Unless otherwise indicated, the yeast compositions and the corresponding controls were administered to the animals by intragastric feeding.  
       Example 1  
     Effects of Yeast Compositions on Fibrous Tissue Formation and Collagen Level in Liver  
       [0045]     Fibrous tissue formation as a result of liver cell regeneration and high collagen level are characteristics of liver cirrhosis. To test the ability of the yeast composition containing EMF-treated AS2.562 cells to ameliorate or prevent cirrhosis, the composition&#39;s effects on liver fibrous tissue formation and collagen level were examined in Wistar rats with liver cirrhosis induced by subcutaneous injection of CCl 4 . The activated yeast composition of this invention was shown to significantly alleviate these symptoms of cirrhosis. This result was obtained as follows.  
         [0046]     Forty Wistar rats (half male, half female, 6-9 months old, and 250-280 g in weight) were divided randomly into four groups of ten rats each: AY, for treatment with activated yeast composition; NY, for treatment with control yeast composition (unactivated yeast); CK1, control group for treatment with saline; and CK2, normal control without induction of cirrhosis for treatment with saline.  
         [0047]     To induce cirrhosis, on day one of the nine-week experiment the AY, NY, and CK1 groups of rats were each administered 5.0 ml/kg (body weight) CCl 4  by subcutaneous injection. Each rat was then injected with 3 ml/kg of CCl 4  containing 40% plant oil (such as peanut oil). For the first two weeks, the rats&#39; diet contained 79.5% corn flour, 20% lard, and 0.5% cholesterol, and their drinking water contained 30% alcohol. From the third week to the end of the ninth week, the diet contained 99% corn flour and 1% cholesterol, and the drinking water contained 30% alcohol.  
         [0048]     Starting from the second day of the experiment, each AY rat was administered 1.5 ml per 100 g body weight of the activated yeast composition twice daily till the end of the experiment; rats in groups NY and CK1 were given the control yeast composition and saline at the same dosage, respectively. The fourth group of rats, CK2, were not challenged with CCl 4  but were fed normally and provided normal drinking water during the nine-week period. They were given 1.5 ml of saline twice daily starting from the second day of the experiment. The four groups of rats were otherwise maintained under the same conditions.  
         [0049]     At the end of the ninth week, each rat was sacrificed and the left lobe of the liver was fixed in 10% formaldehyde. Paraffin sections were prepared and stained with HE (hematoxylin-eosin) and/or VG (van Gieson), and fibrous tissue formation was examined under the microscope. The rest of the liver sample was immersed first in 95% ethanol for 12 hours and then in acetone for 48 hours to extract fat. The liver was then dried at 110° C. and ground into powder.  
         [0050]     To measure the liver hydroxyproline (“Hyp”) level, 40 mg of the liver powder was added to 3 ml of 6 M HCl and incubated at 125° C. to hydrolyze for five hours. The sample was then cooled down to room temperature and its pH adjusted to 6.0 with 6 M NaOH. The volume was brought up to 50 ml with de-ionized water. After filtration, 2 ml of the resulting solution was mixed with 1 ml of chloramine-T and incubated at room temperature for twenty minutes. One milliliter of perchloric acid was subsequently added. Five minutes later, 1 ml of 10% p-dimethylaminobenzaldehyde was added and the reaction was incubated in a 60° C. water bath for 20 minutes for color to develop. Optical densities of the samples were then measured at 550 nm. Hyp levels (Y) of the samples were obtained based on a proline standard curve. The proline standard curve was made by assaying proline solutions of several different concentrations following the procedure as described above. Since every microgram (μg) of Hyp corresponds to about 7.46 microgram (μg) of collagen in the liver, the liver collagen level (X) was calculated by the following formula: 
 
 X =[(7.46×50)/40 ]×Y= 9.325 ×Y  (mg per gram liver dry weight). 
 
         [0051]     The data from the above experiments are summarized in Table 2 below.  
                                                                                               TABLE 2                                       Fibrous tissue formation in liver   Collagen                #                   Average   (mg/g dry       Group   rats   −*   +   ++   +++   (%)**   liver)                    AY   10   8   2   0   0   0.4   16.7 ± 6.2       NY   10   0   0   3   7   2.9    37.8 ± 18.3       CK1   10   0   0   2   8   3.1    38.6 ± 17.4       CK2   10   10   0   0   0   0   15.3 ± 5.5                 *“-”: no fibrous tissue; “+”: 0-0.25%, fibrous tissue volume v. total liver volume;            “++”: 0.25-2.5%; “+++”: 2.5-5.0%.            **Average fibrous tissue volume as percent of total liver volume.             
 
         [0052]     As shown in Table 2 above, the CK1 rats developed severe cirrhosis, indicating the success of cirrhosis induction by CCl 4 . The AY rats, like the healthy control CK2 rats, had significantly less fibrous tissue formation or collagen in the liver compared to CK1 rats, while the NY rats were similar to CK1 rats in terms of the severity of cirrhosis. These data demonstrate that the activated yeast composition can significantly alleviate the symptoms of liver cirrhosis, e.g., decrease liver collagen level and the formation of liver fibrous tissue, as compared to the control yeast composition.  
       Example 2  
     Effects of Yeast Compositions on the Serum γ-Globulin Level  
       [0053]     Serum proteins are generally classified into albumin and globulins. Globulins are roughly divided into α, β, and γ globulins, which can be separated and quantitated by electrophoresis and densitometry. The γ-globulins include the various types of antibodies, such as immunoglobulins M, G, and A. When the liver tissue is damaged as in cirrhosis, serum γ-globulin levels increase because B cells secret more antibodies as a result of, inter alia, the saturated phagocytosis capability of the Kuffer cells and inadequate T-cell function. Thus, serum γ-globulin level is one of the important indicators of liver functions.  
         [0054]     To evaluate the effects of the activated yeast composition of this invention on serum γ-globulin levels, rats with CCl 4 -induced liver cirrhosis were treated with the yeast compositions according to the procedure described in Example 1. The rats were sacrificed at the end of the ninth week. Blood samples were drawn from each of the sacrificed rats and sera were prepared. To determine the relative serum y-globulin level, the sera were subjected to standard serum globulin electrophoresis. After the electrophoresis was completed, the electrophoresis membrane was stained in amido black 10 B solution for 10 minutes, and then destained to get rid of background staining. Each of the albumin or globulin bands was then excised. The membrane containing albumin was soaked in 6 ml of 0.4 M NaOH in a test tube, and the globulin bands were each soaked in 3 ml of 0.4 NaOH. All tubes were incubated at room temperature for an hour with agitation to elute the dye from the membrane. The optical density of each sample was measured at 580 nm, using 0.4 M NaOH for calibration. The relative proportion of each protein fraction was calculated using the following formulae: 
 
Total serum protein=Σ E= 2 ×E   A   *+E   α1   +E   α2   +E   β   +E   γ 
 
albumin (%)=[(2 ×E   A )/Σ E]× 100 
 
α1 globulin (%)=( E   α1   /ΣE )×100 
 
α2 globulin (%)=( E   α2   /ΣE )×100 
 
β globulin (%)=( E   β   /ΣE )×100 
 
γ globulin (%)=( E   γ   /ΣE )×100 
 
  *E: optical density; A: albumin.  
 
         [0055]     The average serum y-globulin level (as percent of total serum protein) for the different groups of rats were shown in Table 3 below.  
                                   TABLE 3                                               γ-globulin           Group   # rats   Treatment   level (%)                           AY   10   cirrhosis rat with activated   13.9 ± 2.1                   yeast comp           NY   10   cirrhosis rat with control   25.9 ± 4.3                   yeast comp           CK1   10   cirrhosis rat with saline   26.6 ± 4.5           CK2   10   healthy rat with saline   15.7 ± 3.3                      
 
         [0056]     The data demonstrate that the activated yeast composition was effective in maintaining normal serum γ-globulin levels in rats with cirrhosis, while the control yeast composition was not.  
         [0057]     While a number of embodiments of this invention have been set forth, it is apparent that the basic constructions may be altered to provide other embodiments which utilize the compositions and methods of this invention.