CA2432331A1 - Feed additives for dogs - Google Patents

Abstract

The present invention relates to feed additives for dogs. The invention provides methods for making a biological compositions comprising yeast cells that can improve the immune functions of dogs. The invention also relates to methods for manufacturing the biological compositions, and methods of using the biological compositions as feed additives.

Description

F'E7ED ADDITIV7ES FOF~ DOGS
1. r~IJD O~ TIIE zNV~NTION
The invention relates to biological compositions comprising yeast cells that can improve the immune functions of animals. The invention also relates to methods for manufacturing the biological compositions, and methods of using the biological compositions as animal feed additives.

2. IBAC~~1~OTJND OF THE INVENTION
Antibiotics have been added to animal feed and pet food since the 1940s. It has been reported in 2000 that more than a third of the antibiotics sold in the United States--about 18 million pounds a year- are used in animal husbandry. They are used to treat sick animals; to prevent other animals housed in confined barns or coops from infections; and to make the animals grow faster. In terms of volume, most antibiotics are i 5 used for the first two reasons. ~nly 6.1 percent of the drugs goes toward growth promotion.
However, in terms of the number of animals exposed, the role of growth promotion is huge.
That is because farmers give antibiotics, in low but daily doses, to entire herds or flocks.
Regular low doses of antibiotics not only help keep livestock healthy, but also improve the absorption of nutrients, which helps the.ani~nals grow faster on less feed, and thus increase profits, particularly in intensive farming operations. It has been estimated that 75 percent of the 92 million pigs in the United States routinely intake feed laced with antibiotics. So do about 6 percent of cattle, 25 percent of chickens and half the turkeys.
l~Ithough antibiotics are not prohibited in pet foods in the United States, they have not been included in pet foods as growth promoter. In fact, the use of antibiotics produce a rapid growth rate and dogs often g:e~,xr too rapidly and enceuntered skeletal abnormalities. It is known that excess antibiotics and chemicals including those that are not metabolized by the animals can either remain in the body or be excreted.
First, there is a possibility that these antibiotics and chemicals will contaminate human food products.
Secondly and more importantly, it exposes microorgaxzisms to the antibiotics, allowing ~hbiotic-resistant strains of the microorganisms to develap. If excreted, these antibiotics and chemicals will be released to the environment where they can come into contact with soil microorganisms. It has been hypothesized that over a period of time, commonly-used antibiotics will be render less effective against a range of microorganisms because of the development of antibiotic resistance and the transfer of such resistance to microorganisms '~ including those that cause infections in humans.

There is growing evidence to suggest that the antibiotics widely used an animals are diminishing the power of important antibiotics to treat human infectious diseases especially those caused by food-borne pathogens. Farm animals treated with Iow levels of antibiotics are developing drug-resistant forms of bacteria. In one instance, while Synercid was approved for human use only in I990's, a closely related drug called virginiamycin has been used on livestock since 1974. Tn. fact, since I990 there have been dramatic increase in the occurrence of multiply drug-resistant strains of zoonotic pathogens causing infections in humans in many developed countries. ~f particular note has been the epidemic spread of MR strains of ,Sczlrnonefl~ ty~himuriuayc DT I04, which now appear to have an almost world-wide distribution. Among the DT '.104 strains, the increasing spectrum of resistance is of considerable concern. In many parts of the world, there is an increased incidence of decreased susceptibility to ciprofloxacin. For Cczmpylobacters species, the incidence of ciprofloxacin-resistant organisms is also increasing, with reports of such isolates from numerous countries throughout the world. See Threllfall E.J. et al., Acta Vet 1 ~ Scand Suppl 2000; 93:63-8; Wegener H.C., N Engl 3 Med. 1999 liday 20;
340(20):I525-32;
Smith K.E. et al., N Engl 3 lVled. 1999 lVlay 20; 340(24):1581-2.; Wegener H.C. et al., Acta Vet,Scand Suppl. 1999; 92:51-7.
Because of concerns over the development of drug-resistance in microorganisms that cause human diseases, regulatory authorities in the United States and the European Union has banned or proposed banning the use of certain antibiotics in animal feed as a growth promoter. In defense, farmers and phaaznaceutical makers argued that reducing the use of antibiotics would lead to increased disease and mortality rates and make meat more expensive by increasing the amount of time it takes to get animals to ideal slaughtering weight. The antibiotics help animals to get fatter quicker because they do not waste energy fighting illness. It has been argued that it i.s a ban on growth promoters that could pose the greatest risk to health. ~lithout their protection, animals could face more serious disease. As a result, veterinarians would have to resort to high doses of therapeutic antibiotics. For example, the year following prohibition of antibiotics use in Sweden, an extra 50,000 pigs died of a form of diarrhoea.
It is clear that while the use of antibiotics as prophylactics in animal husbandry is banned or reduced, an urgent need for alternative means to reduce the incidence of infectious diseases in farm animals is emerging. The present invention provides a solution that uses yeasts to improve the immune functions of animals.
The inclusion of fungal cells or fungal fermentation products in animal feed have been in use for some time. Certain bacteria, yeasts and mold preparations, commonly _2_ referred to as probiotics or direct fed microbiais, axe administered orally or added to animal feeds to provide various benefits. ~lowever, the mechanism of action of such preparations are not properly understood but it is believed that they act by altering the gut microfloralmicrobiota of the animals thereby improving the health of the intestinal tract lining and allowing for improved nutrient absorption. In the case of ruminants, the preparation may ameliorate fermentation in the rumen that results in the wasteful production of gases.
For examples of the use of fungal cells and pxoducts in animal feed, see the annual Feed Additive Compendium published by The Miller Publishing Company (Minnetonka, Minn.) or the following patent literature:
United States Patent No. 3,903,307 discloses a process for making animal feed that is based on the fermentation of waste molasses or bagasse by yeasts, such as Candida utilis and Trichoderma vea-ide.
U.S. Patent No. 4.,055,667 discloses a I~qLUd animal feed supplement that comprises a colloidal mixture of spent brewer's yeast in an aqueous alcoholic medium.
U.S. Patent No. 4,582,708 discloses an animal feed supplement that comprises live yeast cells (Saccha~omyces species) having fermenting activity, a texturizing component comprising ground meal, ground legumes or mixtures thereof, a mineral mixture, a liquid binder, a vitamin mixture, and ground montmoriilonite.
U.S. Patent No. 5,624,686 discloses an arrimal feed additive that is prepared by cultivating certain bacteria or yeast species (such as Saccharomyces cerevisiae, Candida utilis) and disrupting the microbial cells such that metabolites of the cells are efficiently made available to the animal.
U.S. Patent No. 6,214,337 discloses an animal feed that comprises yeast glucan which is derived from the cell walls of various yeast species (such as Sa~ehanomyces cerevisiae, Candida utilis).
Citation of documents herein is not intended as an admission that any of the documents cited herein is pertinent prior art, or an admission that the cited documents are considered material to the patentability of the claims of the present application. loll statements as to the date or representations as to the contents of these documents are based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.

3. SLTM '~.' ~~' 31~E INVENTION
The present >slvention relates to biological compositions that can be added to animal feed to reduce the incidence of infeet~.ous diseases in dogs.
In one embodiment, the present invention provides biological compositions comprising a plurality of liv a yeast cells which are capable of improving the immune functions of dogs when ingested. The biological compositions can be used for reducing the incidence of infectious diseases in dogs or optimizing the health of the animal.
In another embodiment, the invention provides methods of malting the biological composition. In particulax, the methods ofthe invention comprise culturing yeast 1~ cells in the presence of a series of electromagnetic fields of defined frequencies and field strengths, such that the yeast cells becomes metabolically active and potent at stimulating an animal's immune system. Up to four different components of yeast oells can be used to form the biological compositions. The yeast cells can also be subjected to a conditioning step to improve its performance. The conditioning step comprises culturing the yeast cells 15 ~ a culture medium comprising canine gastric juice and 'wild hawthorn juice. lVlethods for manufacturing the biological compositions comprising culturing the yeast cells under activation conditions, mixing various yeast cell cultures of the present invention, followed by drying the yeast cells and packing the final product, are encompassed. 3n preferred embodiments, the starting yeast cells are commercially available and/or accessible to the 20 public, such as but not limited to Sacchczromyces cerevzsicxe.
The biological compositions of the invention can be fed directly to animals or used as an additive to be incorporated into regular dog food. Dog food compositions comprising activated yeast cells of the invention and ingredients such as zeolite powder are encompassed by the invention.

4. I~R~EF DESCRIPTION ~F FIG1U12ES
Fig. I Activation and conditioning of yeast cells. 1 yeast cell culture; 2 container; 3 electromagnetic field source; 4 electrode.

5. DETA~ED DESCRIPTION OF'fI-IE INVENTION
The present invention relates to biological compositions that can improve the immune functions of animals, andlar reduce the incidence of infectious diseases. The JS
_4_ present invention provides ~~ethods for manufacturing the biological compositions as well as methods for using the biological compositions as animal feed additives.
The biological compositions of the invention comprise yeasts. Unlike the traditional use of yeasts as a component of the feed, the yeast cells of the invention are not a primary source of nutrients for the animals. The yeast ceps of the invention sere as a supplement to replace or reduce the antibiotics that are now routinely added to livestock feed. The yeast cells are live when administered orally or ingested along with food by the animals. While in the gastrointestinal tract of an animal, the yeast cells are capable of stimulating the immune system and improving the immune functions of the animal, thereby 1 o xeducing the incidence of infectious diseases. The use of the biological compositions of the invention can lower the overall cost of maintaining the health of animals in commercial breeding operations, and make feasible the minimal use of antibiotics.
While the following terms are believed to have well-defined meanings in the art, the following are set forth to define the terms as used herein, and facilitate explanation 15 of the invention.
As used herein, the term "food" or "feed".broadly refers to any kind of material, liquid or solid, that is used for nourishing an animal, and foi sustaining normal or accelerated growth of an animal including newborns and young developing animals.
The term "animal" as used herein refers to dogs (Canis species) generally, 20~ ~d includes all breeds of domestic dogs.
The term "immune functions" as used herein broadly encompasses specific and non-specific immunological reactions of the animal, and includes both hurnoral and cell-mediated defense mechanisms. The immune functions of the animal enable the animal to survive and/or recovex from an infection by a pathogen, such as bacteria, viruses, fungi, 25 protozoa, helminths, and other parasites. The immune functions of the animal can also prevent infections, particularly future infections by the same pathogen after the animal had an initial exposure to the pathogen. Many types of.immune cells are involved in providing the immune functions, which include various subsets of lymphocytes (B cells, T
cells, NK
cells), different types of leukocytes (macrophages, neutrophils, eosinophils, basophils}, 30 ~tigen presenting cells (dendritic cells, endothelial cells) and cells that are found in specialized organs and tissues with immunological activities (bone marrow, lymph nodes, thymus, bursa, Peyer's patch). Details of the immune system of dogs are described in Clinical Tmrr?unology ofthe Dog and Cat by Michael J. Day, Iowa State University Press, November 1999, which is incorporated hexein by reference in its entirety.

rn one embodiment, the present invention provides biological compositions that comprise at Ieast one yeast cell component. Each yeast cell component comprises a population of live yeast cells which have been cultured under a specific set of conditions such that the yeast cells are capable of improving the immune functions of an animal. In preferred embodiments, the biological compositions of the invention comprise up to four yeast cell components.
According to the invention, under certain specific culture conditions, yeasts can be made metabolically active such that t~~rey become effective in stimulating and enhancing the immune functions of an animal which ingested the yeasts. Without being bound by any theory. or mechanism, the inventor believes that the culture conditions activate and/or amplified the expression of a gene or a set of genes in the yeast cells such that the yeast cells becomes highly potent in stimulating the animal's immune system.
It is envisioned that interactions between certain yeast gene products and elements of the animal's immune system is greatly enhanced by the elevated levels of these yeast gene products after the yeast cells have been cultured under the conditions described hereinbelo~.v. These interactions are believed to involve immune cells lining the gastrointestinal tract, lymph nodes, as well as circulating immune cells. As a result of these interactions, the immune functions of the a -ximals, such as responsiveness to and recovery from an.infection, and resistance to diseases, are improved. The animals are protected from.
may hypes of infectious diseases, including parasitic diseases, such as but not limited to those caused by bacteria, viruses; fungi, protozoa, helminths, and the like.
The benefits of using the biological compositions are demonstrated by e:~perzmental results obtained from animals which show resistance to or rapid recovery from. an infectious disease.
In one embodiment, the biological compositions of the invention can be fed dfrectly to an animal. In another embodiment, the biolo~,~eal compositions can be added to the feed. As known to those skilled in the relevant art, many methods and appliances rnay be used to mix the biological compositions of the invention with dog food. In a particular embodiment, a mixture of culture broths of the yeasts of the present invention are added directly to the dog food just prior to feeding the animal. Dried powders of the yeasts can also be added directly to the dog food just prior to feeding the animal In yet another embodiment of the present invention, the yeast cells are mixed with the raw constituents of the dog food with which the yeast cells become physical:Ly incorporated. The biological compositions may he applied to and/or mixed with the dog food by any mechanized means which may be automated.

-6-The amount of biological composition used depends in part on the feeding regimen and the type of dog food, and can be determined empirically_ For example, the useful ratio of biological composition to dog food ranges from 0.1 % to 1 % by dry weight, preferably, 0.3 to 0.8%, and most preferably at about 0.5%. Although not necessary, the biological compositions of the invention can also be used in conjunction or in rotation with other types of supplements, such as but not limited to vitamins, minerals, and vaccines.
Described below in Section 5.1 and 5.2 are four yeast cell components of the invention and methods of their preparation. Section 5.3 describes the manufacture of the biological compositions of the invention which comprises at least one of the four yeast cell components.
5.i. PREPARATIOhT OF THE YEAST' CELL. CT1LTURES
The present invention provides yeast cells that are capable of i_rnproving the immune functions of an animal which ingested the yeast cells. Up to four different yeast i5 cell components can be vomlbined to make the biological compositions.
A yeast cell component of the biological composition is produced by culturing a plurality of yeast cells in an appropriate culture medium in the presence of an alternating electromagnetic field or multiple alternating electromagnetic fields in series over a period of time. The culturing pxocess allows yeast spores to germinate, yeast cells to grow ~d divide, and can be performed as a batch process or a continuous process. As used herein, the terms "alternating electromagnetic field", "electromagnetic field"
or "EM field"
are synonymous. An electromagnetic field useful in the invention can be generated by various means well known in the art. A schematic illustration of exemplary setups are depicted respectively in Fig. 1. An electromagnetic field of a desired frequency and a desired held strength is generated by an electromagnetic wave source (3) which comprises one or more signal generators that are capable of generating electromagnetic waves, preferably sinusoidal waves, and preferably in the frequency range of 1500 MHz MHz. Such signal generators are well known in the art. Signal generators capable of generating signal with a narrower frequency range can also be used. If desirable, a signal amplifier can also be used to increase the output signal, and thus the strength of the EM
field.
The electromagnetic fzeld can be applied to the oulture by a variety of means including placing the yeast cells in close proximity to a signal emitter connected to a source of electromagnetic waves. In one embodiment, the electroma~etic field is applied by signal emitters in the form of electrodes that are submerged in a culture of yeast cells (1). In _7_ a preferred embodiment, one of the electrodes is a metal plate which is placed on the bottom of a non-conducting container (2), and the other electrode comprises a plurality of wires or tubes so configured inside the container such that the energy of the electromagnetic field can be evenly distributed in the cultuxe. For an upright culture vessel, the tips of the wires or tubes are placed within 3 to 30 cm from the bottom of the vessel (i.e, approximately 2 to I O% of the height of the vessel from the bottom). The n~.z~nber of electrode wires used depends on both the volume of the culture and the diameter of the wire. For example, for a culture having a volume of 10 liters or less, two or three electrode wires having a diameter of between 0.5 to 2.0 mm can be used. For a culture volume of 10 liters to 100 liters of culture, the electrode wires or tubes can have a diameter of 3.0 to 5.0 xnm.
For a culture volume of 100 liters to 1000 liters, the electrode wires or tubes can have a diameter of 6.0 to I 5.0 rnm . For a culture having a volume greater than 1000 liters, the electrode wires or tubes can have a diameter of between 20.0 to 25.0 mm.
In various embodiments, yeasts of the genera of,Saccharomyces, Candida, Crebrothecium, Geotrichum, Hansenula, Kloeckera, Lipamyces, Pichia, Rhodosporidium, Rhodotorula Torulopsis, Trichosporon, and ~ickerhamia can be used in the invention.
Non-limiting examples of yeast strains include Saccharomyces cerevisiae Hansen, ACCC2034, ACCC2035, ACCC2036, ACCC2037, ACCC2038, ACCC2039, ACCC2040, ACCC204I, ACCC2042, AS2.1, AS2.4, AS2.I I, AS2.14, AS2.16, AS2.56, AS2.69, AS2.70, AS2.93, AS2.98, AS2.101, AS2.109, AS2.I10, 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, AS2.39S, AS2.396, AS2.397, AS2.398, AS2.399, AS2.400, AS2.406, AS2.408, AS2.409, AS2.4I3, AS2.414, AS2.415, AS2.416, AS2.422, AS2.423, AS2.430, AS2.431, AS2.432, AS2.45I, AS2.452, AS2.453, AS2.458, AS2.460, AS2.463; AS2.467, AS2.486, AS2.501, AS2.502, AS2.503, AS2.504, AS2.5I6, 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.I190, AS2.1364, AS2.1396, IFFI 1001, IFFI 1002, IEFI 1005, IfFI 1006, IFFI I008,1FFI
1009, IFFI 1010, IFFI I0I2, IFFI 1021, IFFI 1027, IFFI 1037, IFFI I042, IFFI 1043, IFFI 1045, BFI 1048, IFFi 1049, IFFI 1050, IFFI 1052, IFFI 1059, IFFI 1060, IFFI I063, IFFI 1202, IFFI 1203, IEFI 1206, IFFI 1209,1FFI 1210, IFFI 1211, IFFI 1212, TFFI 1213, g'FI 1215, IFFI 1220, IFFI 1221, IFFI 1224, IFFI 1247, IFFI 1248, IFFI 1251, IFFI 1270, IfFI 1277, IFFI 1287, IFFI 1289, IFFI 1290, IFFI 1291, IFFI 1292, IFFI 1293, IFFI 1297, IFFI 1300, IFFI 130I, IFFI I302, IFFI 1307, IFFI 1308, IFFI I309, IFFI I3:10, IFFI I3I
I,1FFI 1331, IFFI 1335, IFFI 1336, IFFI 1337, IFFI 1338, IFFI 1339, IFFI 1340, IFFI 1345, IFFI 1348, _g_ 3FFI 1396, IFFI 1397, IFFI 1399, IFFI 1411, IFFI 1413; 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;
Saccharornyces chevalieri Guillermond, AS2.131, AS2.213; Saccharomyces delbrueckii, AS2.2 85; Saccharomyces delbrueckii Lindner var. m.ongolicus 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 Lodder et Kreger Van Rij, AS2.195; Saccharomyces mieroellipsoides Osterwalder, AS2.699;
Saccharomyces oviformis Osterwalder, AS2.100; Saecharomyces rosei (Guilliermond) Lodder et kreger van Rij, AS2.287; Saccharomyces rouxi i Boutroux, AS2.178, AS2.180, AS2.370, AS2.371; Saccharomyces sake Yabe, ACCC2045; Candida arborecx, AS2.566;
Candida Krusei (Castellani) Berkhout, AS2.1045; Candida lambica(Lindner et Genoud) van.Uden et Buckley, AS2.1182; Candida lipolytica {Harrison) Diddens et Lodder, AS2.1207, AS2.1216, AS2.1220, AS2.1379, AS2.1398, AS2.I399, AS2.1400; Candida parapsilosis (Ashford) Langeron et Talice, AS2.590; Candida parapsilosis (Ashford) et Talice Var. intermedia Van Rij et Verona, AS2.491; Candida pulcherriman (Lindner) Wiudisch, AS2.492; Candida rugousa (Anderson) Diddens et Loddeer, AS2.511, AS2.1367, AS2.1369, AS2.1372, AS2.1373, AS2.13 77, AS2.1378, AS2.1384; Candida tropicalis (Castellani) Berkout, ACCC2004, ACCC2005, ACCC2006, AS2.164, AS2.402, AS2.564, AS2:565, AS2.567, AS2.568, AS2_6I7, AS2.1387; Candida utilis Henneberg Lodder et Kreger Van Rij, AS2.120, AS2.281, AS2.1180; Crebrothecium ashbyii (Guillermond) Routein., AS2.481, AS2.482, AS2.1197; (xeotrichum candidum Link, ACCC2016, AS2.361, AS2.498, AS2.616, AS2.I035, AS2.1062, AS2.1080, AS2.1 i32, 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, AS2.635, AS2.782, AS2.794;
Hansenula arabitolgens Fang, AS2.887; Hansenula jadinii Wickerham, ACGC2019;
Hansenula saturnus (Klocker) H et P sydow, ACCC2020; Hansenula schneggii (Weber) Dekker, AS2.304; .Hansenula subpelliculosa Bedford, AS2.738, AS2.740, AS2.760, AS2.761, AS2.770, AS2.783, AS2.790, AS2.798, AS2.866; Kloeckera apiculata (I~eess emend. Klocker) Janke, ACCC2021, ACCC2022, ACCC2023, AS2.197, AS2.496, AS2.71 l, AS2.714; Lipomyces starkeyi Lodder et van Rij, ACCC2024, AS2.1390;
Pichia farinosa (Lindner) Hansen, ACCC2025, ACCC2026, AS2.86, AS2.87, AS2.705, AS2.803;
pichia membranaefaciens Hansen, ACCC2027, AS2.89, AS2.661, AS2.1039;
_9_ Rhodosporidium toruloides Banno, ACCC2028; Rhadotorula glutinis (Fresenius) Harrison, ACCC2029, AS2.280, ACCC2030, AS2.102, AS2.107, AS2.278, AS2.499, AS2.694, AS2.703, AS2.704, AS2.I146; Rhadotorula minuta (Saito) Harrison, AS2.277;
Rhodotorula rubar (Demure) Lodder, ACCC2031, AS2.21, AS2.22, AS2.103, AS2.105, AS2.108, AS2.140, ~-152.166, AS2.167, AS2.272, AS2.279, AS2.282; Saccharomyces carlsbergensis Hansen, AS2.113, ACCC2032, ACCC2033, AS2.312, AS2.116, AS2.118, AS2.121, AS2.132, AS2.162, AS2.189, AS2.200, AS2.216, AS2.265, AS2.377, AS2.417, AS2.420, AS2.440, AS2.441, AS2.443, AS2.444, AS2.459, AS2.595, AS2.605, AS2.638, AS2.742, AS2.745, AS2.748, AS2.1042; Saccharomyces uvarum Beijer, IFFI 1023, IFFI
1032, LFFI 1036, IFFI 1044, IFFI 1072,1FFI 1205, IFFI 1207; Saccharamyces willianzcs 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; Saccharamyces ludwigii Hanssn, ACCC2044, AS2.243, AS2.508; Saccharomyces sinenses Yue, AS2.1395;
Schizosaccharomyces octosporus Beijerinck, ACCC 2046, AS2.I148;
Sc7aizosaccharomyces pombe Linder, ACCC2047, ACCC2048, AS2.248, AS2.249, AS2.255, AS2.257, AS2.259, AS2.260, AS2.274, AS2.994, AS2.1043, AS2.1149, AS2.1178, IFFI 1056; Sporobolomyces roseus Kluyver et van Niel, ACCC 2049, ACCC
2050, AS2.619, AS2.962, AS2.1036, ACCC2051, AS2.261, AS2.262; Torulopsis candida (Saito) Lodder, ACCC2052, AS2.270; Torulopsis~'amta (Harrison) Lodder et van Rij, ACCC2053, AS2.685; Torulopsis globosa (Jlson et Hammer) Lodder et van I~ij, ACCC2054, AS2.202; Torulopsis inconspicua Lodder et van Rij, AS2.75;
Trichosporon behrendii Lodder et Kreger van Rij, ACCC2055, AS2.1193; Trichosporon capitatum Diddens et Lodder, ACCC2056, AS2.13$5; Trichosporon cutaneum(de Beurm et al.)Ota, ACCC2057, AS2.25, AS2.570, AS2.571, AS2.1374; Wickerhamia fluoresens (Soneda) Soneda, ACCC2058, AS2.1388. Yeasts of the Saccharomyces genus are generally preferred. Among strains of Saccharomyces cerevisiae, Saccharomyces cerevisiae Hansen is a preferred strain.
Generally, yeast strains useful fox the invention can be obtained from private or public laboratory cultures, or publically accessible culture deposits, such as the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209 and the China General Microbioloa cal 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.
Although it is preferred, the preparation of the yeast cell components of the 3 $ invention is not limited to starting with a pure strain of yeast. Each yeast cell component may be produced by culturing a mixture of yeast cells of different species or strains. The constituents of a yeast cell component can be determined by standard yeast identification techniques well known in the art.
In various embodiments of the invention, standard techniques for handling, transferring, and storing yeasts are used. Although it is not necessary, sterile conditions or clean environments are desirable when carrying out the manufacturing processes of the invention. Standard techniques for handling animal blood and immune cells, and for studying immune functions of an animal axe also used. Details of such techniques are described in Advances in Laboratory Methods: General Haematology, 2000, Assendelft et al., (Ed.), Arnold, Edward (Publisher); Handbook of Vertebrate Immunology, 1998, Pastoret et al. (Ed.), Academic Press, and Current Protocols In Immunology, 1991, Coligan, et a1.
(Ed), John Wiley & Sons, Tnc., which are both incorporated herein by reference in their entireties.
In one embodiment, the yeast cells of the first yeast cell component are I5 cultured in the presence of at least one alternating electromagnetic (EM) field with a frequency in the range of 7720 MHz to 7740 MHz. A single EM field or a series of EM
fields can be applied, each having a different frequency within. the stated range, or a different field strength within the stated range, or different frequency and field strength within the stated ranges. Although any practical number of EM fields can be used within a series, it is preferred that, the yeast culture be exposed to a total of 2, 3, 4, 5, 6, 7, 8, 9 or 10 different EM~ fields in a series. The EM field(s), which can be applied by any means known in the art, can each have a frequency of 7720, 7721, 7722, 7723, 7724, 7725, 7726, 7727, 7728, 7729, 7730, 7731, ?732, 7733, 7734, 7735, 7736, '7737, 7738, 7739, 7740 MHz.
The field strength of the EM fields) is in the range of 100 to 250 mV/cm preferably at 187 ~ 5.0 mV/cm. The yeast cells can be cultured in the EM field for 52 to 120 hours. The yeast culture can remain in the same container and use the same set of electromagnetic wave generator and emitters when switching from one EM field to another EM field.
The culture process can be initiated by inoculating 100m1 ofmedium with 1mI of an inoculum of the selected yeast strains) at a cell density of about 10j cells/ml.
The starting culture is kept at 35 °C to 37°C for 24 to 48 hours prior to exposure to the EM
field(s). The culturing process may preferably be conducted under conditions in which the concentration of dissolved oxygen is between 0.025 to 0.08 mol/m3, preferably 0.04 mol/m'.
The oxygen Ievel can be controlled by any conventional means known in the art, including but not limited to stirring and/or bubbling.

The culture is most preferably carried out in a liquid medium which contains animal serum and sources of nutrients assimilable by the yeast cells. Table 1 provides an exemplary medium for culturing the fzrst yeast cell component of the invention.
Table 1 l Medium Composition t2uantity Soluble starch 6.0g Glucose 18.0g K2HPO4 0.3g MgSO4 7H20 0.25g NaCI 0.25 g CaS042H20 0.2g CaC03SH20 3.0g Peptone 1.2g Dog serum 30Qm1 Autoclaved water ~ 700m1 In general, carbohydrates sucb as sugars, lbr example, sucrose, glucose, ~ctose, dextrose, maltose, xylose, and the like and starches, can be used eifher alone ox in combination as sources of assimilable carbon in the culture medium. The exact quantity of the carbohydrate source or sources utilized in the medium depends in part upon the other ingredients of the medium but, in general, the amount of carbohydrate usually varies between about 0.1 % and 5% by weight of the medium and preferably between about 0_5%
~d 2%, and roost preferably about Q.8%. l hese carbon sources can be used individually, or several such carbon sources may be combined in the medium. Among the inorganic salts which can be incorporated in the culture media are the customary salts capable of yielding sodium, calcium, phosphate, sulfate, carbonate, and like ions. Non-limiting examples of nutrient inorganic salts are (NII4)2HPO~, CaC03, MgSO~, NaCl, and CaSO4.
The dog serum (from any breed of dog over 10 kg body weight), which is a fraction of blood that comprises white blood cell, can be prepared from whale blood (1000-2000 ml) by standard methods known in the art, such as density gradient centrifugation.
Red blood cells are separated and discarded. The serum. is added to the culture medium after the medium has been autoclaved and cooled to about 40°C.

It should be noted that the composition of the media provided in Table 1 is not intended to be limiting. The process can be scaled up or down according to needs.
Various modifications of the culture medium may be made by those skilled in the art, in view of practical and economic considerations, such as the scale of culture and local supply of media components.
Although the yeast cells will become activated even after a few hours of culturing in the presence of the EM field(s), the yeast cells can be cultured in the presence of the EM fields} for an extended period of time (e.g., two or more weeks). At the end of the culturing process, the yeast cells which constitute the first yeast cell component of the invention may be recovered from the culture by various methods known irz the art, and stored at a temperature below about 0°C to 4°C. The recovered yeast cells may also be dried and stored in powder form.
A non--limiting example of making a first yeast cell component of the invention with Saccharornyces eerevisiae stxain AS2.504 is provided in Section hereinbelow.
In another embodiment, the yeast cells of the second yeast cell component are cultured in the presence of at least one alternating electromagnetic (EM) field with a frequency in the range of 6840 MHz to 6860 MHz. A single EM field or a series of EM
fzel_ds can be applied, each having a different frequency within the stated range, or a .,~., . ...
different field strength within the stated range, or different frequency and field strength within the stated ranges. Although any practical number of EM fields can be used within a series, it is preferred that, the yeast culture be exposed to a total of 2, 3, 4, 5, 6, 7, 8, 9 or 10 different EM fields in a series. The EM field(s), which can be applied by any means known in the art, can each have a frequency of 6840, 6841, 6842, 6843, 6844, 6845, 6846, 6847, 6848, 6$49, 6850, 6851, 6852, 6$53, 6854, 6855, 6856, 6857, 6858, 6859, o_r 6$60 MH?.
The field strength of the EM fields) is in. the range of 55 to 230 mVlcm, preferably to 212 ~ 5.0 mV/cm. The yeast cells can be cultured in the EM field for 48 to I30 hours. The yeast culture can remain in the same container and use the same set of electroma~etic wave generator and emitters when switching from one EM f.eld to another EM field.
The culture process can be initiated by inoculating 100m1 of medium with lml of an inoculum of the selected yeast strains) at a cell density of about 105 cells/ml.
The starting culture is kept at 35°C to 37°C for 24 to 48 hours prior to exposure to the EM
field(s). The culturing process may preferably be conducted under conditions in which the concentration of dissolved oxygen is between 0.025 to 0.08 mollm3, preferably 0.04 mol/m3.

The oxygen level can be controlled by any conventional means known in the art, including but not limited to stirring andlor bubbling.
The culture is most preferably carried out in a liquid medium which contains animal serum and sources of nutrients assimilable by the yeast cells. Table 2 provides an exemplary medium for culturing the second yeast cell component of the invention.
Table 2 Medium Composition Quantity Mannitol 10.08 Sucrose lO.Og K~'04 0.3g MgS047Hz0 0.238 NaCI ~ 0.3g CaC03SH20 4.0g CaS042H20 0.15g Yeast extract 0.4g Dog serum 350m1 Autoclaved water 650mI .

1n general, carbohydrates such as sugars, for example, sucrose, glucose, fructose, dextrose, maltose, xylose, and the like and starches, can be used either alone or in combination as sources of assimilable carbon in the culture medium. The exact quantity of the carbohydrate source or sources utilized in the medium depends in part upon the other 2~ ingreaients of the medium but, in general, the amount of carbohydrate usuaiiy varies between about O.I % and 5% by weight of the medium and preferably between about 0.5 and 2%, and most preferably about 0.8%. These carbon sources can be used individually, or several such carbon sources may be combined in the medium. Among the inorganic salts which can be incorporated in the culture media are the customary salts capable of yielding sodium, calcium, phosphate, sulfate, carbonate, and like ions. Non-limiting examples of nutrient inorganic salts are (NH4)ZHP04, CaC03, MgSOa, NaCI, and CaS04.
The dog serum {from any breed of do g over 10 kg body weight), which is a fraction of blood that comprises white blood cell, can be prepared from whole blood (1000-2000 ml) by standard methods known in the art, such as density gradient centrifugation.

Red blood cells are separated and discarded. The serum is added to the culture medium after the medium has been autoclaved and cooled to about 40 ° C.
It should be noted that the composition of the media provided in Table 2 is not intended to be limiting. The process can be scaled up or down according to needs.
Various modifications of the culture medium may be made by those skilled in the art, in view of practical and economic considerations, such as the scale of culture and local supply of media components.
Although the yeast cells will become activated even after a few hours of culturing in the presence of the EM field(s), the yeast cells can be cultured in the presence of the EM fields) for an extended period of time (e.g., t'wo or more weeks).
At the end of the culturing .process, the yeast cells which constitute the first yeast cell component of the invention may be recovered from the culture by various methods known in the art, and stored at a temperature below about 0°C to 4°C. The recovered yeast cells may also be dried and stored in powder form.
A non-limiting example of making a second yeast cell component of the ,invention with Saccharomyces cer~evisiae strain IFFI1202 is provided in Section 6 hereinbelow.
In yet another embodiment, the yeast cells of the third yeast cell component are cultured in the,presence of at least one alternating electromagnetic (Eiv~
field with a .r frequency in the range of 8220 MHz to 8240 MHz. A single EM .field or a series of EM
fields can be applied, each having a different frequency within the stated range, or a different field strength within the stated range, or different frequency and field strength within the stated ranges. Although any practical number. of EM fields can be used within a series, it is preferred that, the yeast culture be exposed to a total of 2, 3, 4, 5, 6, 7, 8, 9 or 10 different EM fields in a series. The EM field(s), which can be applied by any means known in the art, can each have a frequency of 8220, 8221, 8222, 8223, 8224, 8225, 8226, 8227, 8228, 8229, 8230, 8231, 8232, 8233, 8234, 8235, 8235, 8236, 8237, 8238; 8239, or 8240 MHz.
The field strength of the EM fields) is in the range of 75 to 260 mV/cm, preferably at 243 ~ 5.0 mVlcm. The yeast cells can be cultured. in the EM
field for 36 to I02 hours. The yeast culture can remain in the same container and use the same set of electromagnetic wave generator and emitters when switching from one EM field to another EM field.
The culture process can be initiated by inoculating 100m1 of medium with ~ lml of an inoculum of the selected yeast strains) at a cell density of about 105 cells/ml.

The starting culture is kept at 3 5 ° C to 3'7 ° C for 24 to 48 hours prior to expcsure to the EM
field(s). The culturing process may preferably be conducted under conditions in which the concentration of dissolved oxygen is between 0.025 to 0..0$ mollm3, preferably 0.04 mol/m3.
The oxygen level can be controlled by any conventional means known in the art, including but not limited to stirring andlor bubbling.
The culture is most preferably carried out in a liquid medium which contains animal senun and sources of nutrients assimilable by the yeast cells. Table 3 provides an exemplary medium for culturing the third yeast cell component of the invention.
Table 3 Medium Composition Quantity Mannitol 1 B.Og MgS047H20 0.2g NaCI 0.258 1e CaS042H20 ~ 0.15g CaCO3SH20 4.0g Peptone 1.5 g KZHPO4 0.3g Dog serum 300m1 , Autoclaved water 700m1 Tn. general, carbohydrates such as sugars, for example, sucrose, glucose, fructose, dextrose, maltose, xylose, and the like and starches, can be used either alone or in combination as sources of assimilable carbon in the culture medium. The exact quantity of the carbohydrate source or sources utilized in the medium depends in part upon the other ingredients of the medium but, in general, the amount of carbohydrate usually varies between about 0.1 % and 5% by weight of the medium and preferably between about 0.5%
and 2%, and most preferably about 0.8%. These carbon sources can be used individually, or several such carbon sources may be combined in the medium. Among the inorganic salts which can be incorporated in the culture media are the customary salts capable of yielding sodium, calcium, phosphate, sulfate, carbonate, and like ions. Non-limiting examples of nutrient inorganic salts are (NH4)ZHP04, CaC03, MgS04, NaCI, and CaS04.
The dog serum (from any breed of dog over 10 kg body weight), which is a fraction of blood that comprises white blood cell, can be prepared from whole blood (1000--ls-2000 ml) by standard methods known. in the art, such as density gradient centrifugation.
Red blood cells are separated and discarded. The serum is added to the cultvue medium after the medium has been autoclaved and cooled to about 40°C.
It should be noted that the composition of the media provided in Table 3 is not intended to be li..miting. The process can be scaled up or down according to needs.
Various modifications of the culture medium may be made by those skilled in the art, in view of practical and economic considerations, such as the scale of culture and local supply of media components.
Although the yeast cells will become activated even after a few hours of culturing in the pxesence of the EM field(s~, the yeast cells can be cultured in the presence of the EM fields) for'an extended period of time (e.g., fiwo or more weeks).
At the end of the culturing process, the yeast cells which constitute the $rst yeast cell component of the invention may be recovered from the culture by various methods known in the art, and stored at a temperature below about 0°C to 4°C. The recovered yeast cells may also be dried and stored in powder form.
A non-limiting example of making a third yeast cell component of the invention with Saccharomyces cerevi~icze strain IFFI1345 is provided in Section 6 hereinbelow.
In yet another embodiment, the yeast cells of the fourth yeast cell component axe cultured in the presence of at Ieast one alternating electromagnetic (EM) field with a frequency in the range of 8530 MI~z to 8550 MHz. A single EM field or a series of EM
fields can be applied, each having a different frequency 'within the stated range, or a different field strength within the stated range, or different frequency and field strength within the stated ranges. Although any practical number of EM fields can be used within a series, it is preferred that, the yeast culture be exposed to a total of 2, 3, 4, 5, 6, 7, 8, 9 or 10 different EM fields in a series. The EM field(s), which can be applied by any means known in the art, can each have a frequency of 8530, 8531, 8532, 8533, 8534, 8535, 8536, 8537, 8538, 8539, 8540, 8541, 8542, 8543, 8544, 8545, 8546, 8547, 8548, 8549, or 8550 MHz.
The field strength of the EM fields) is in the range of 60 to 240 mV/cm preferably at 224 t 5.0 mV/om. The yeast cells can be cultured in the EM field for 60 to 134 hours. The yeast culture can remain in the same container and use the same set of electromagnetic wave generator and emitters when switching from one EM field to another EM field.
The culture process can be initiated by inoculating 100m1 of medium with 1mI of an inoculum of the selected yeast strains) at a cell density of about 105 cells/ml.

The starting culture is kept at 35°C to 37°C for 24 to 48 hours prior to exposure to the EM
field(s). The culturing process may preferably be conducted under conditions in which the concentration of dissolved oxygen is between 0.025 to 0.08 mol/m3, preferably 0.04 mol/m3.
The oxygen level can be controlled by any conventional means known in the art, including but not limited to stirring and/or bubbling.
The culture is most preferably carried out in a liquid medium which contains ~:n~mal serum and sources of nutrients assimilable by the yeast cells. Table 4 provides an exemplary medium for culturing the fourth yeast cell component of the invention.
Table 4 Medium Composition Quantity Mannitol 18.0g KzHPO4 . 0.22g MgSO47HZO 0.2g NaCl 0.23g CaS042H20 0.238 CaC03SHZO 2.5g peptone l .5g Dog serum 200m1 Autoclaved water 800m1 In general, carbohydrates such as sugars, for example, sucrose, glucose, fructose, dextrose, maltose, xylose, and the like and starches, can be used either alone or in combination as sources of assimilable ca.Tbon in the cult..:..=e medium. The exact q~.:antity of the carbohydrate source or sources utilized in the medium depends in part upon the other ingredients of the medium but, in general, the amount oil carbohydrate usually varies between about 0.1% and 5% by weight of the medium and preferably between about 0.5%
and 2%, and most preferably about 0.8%. These carbon sources can be used individually, or Several such carbon sources maybe combined in the medium. Among the inorganic salts which can be incorporated in the culture media are the customary salts capable of yielding sodium, calcium, phosphate, sulfate, carbonate, and like ions. Non-limiting examples of nutrient inorganic salts are (NH4)ZHPO4, CaC03, MgS04, NaCI, and CaSO~.
The dog serum (from any breed of dog over 10 kg body weight), which is a fraction of blood that comprises white blood cell, can be prepared from whole blood (1000-_18-2000 ml) by standard methods known in the art, such as density gradient centrifugation.
Red blood cells are separated and discarded. The serum is added to the culture medium after the medium has been autoclaved and cooled to about 40°C.
It should be noted that the composition of the media provided in Table 4 is not intended to be limiting. The process can be scaled up or down according to needs.
Various modifications of the culture medium may be made by those skilled in the art, in view of practical and economic considerations, such as the scale of culture and loeah supply of media components.
Although the yeast cells will become activated even after a few hours of c~~g ~ ~e presence of the EM field(s), the yeast cells can be cultured in the presence of the EM fields) for an extended period of time (e.g., t~vo or moxe weeks).
At the end of the culturing process, the yeast cells which Constitute the first yeast cell component of the invention may be recovered from the culture by various methods known in the art, and stored at a temperature below about 0°C to 4°C. The recovered yeast cells may also be dried and stored in powder form.
A non-limiting example of making a fourth yeast cell component of the invention with.Saccharorycyces cerevtsiae strain IFFI1012 is provided in Section 6 hereinbelow.
5.2. CONDITIONING OF THE ~~EA.ST CEEES
In another aspect of the invention, performance of the activated yeast cells can be optimized by culturing the activated yeast cells in the presence of materials taken from the gastrointestinal tract of the type of animal to which the biological composition will be fed. The inclusion of this conditioning process allows the activated yeast cells to adapt to and endure the acidic environment of the animal's stomach.
According to the invention, activated yeast cells prepared as described in Section 5.1 can be fixrther cultured in a medium with a composition as shown in Table 5.
Table 5 (Per 1000 ml of culture medium) Medium Composition Quantit;~

Canine gastric juice 600m1; stored at 4C

Wild hawthorn juice 400m1 The process can be scaled up or down according to needs.
3~
_19_ The gastric juice of the a dog can be obtained from the stomach content of a freshly slaughtered animal. About 5008 of the content of the stomach is mixed with 2000 mI of water, and the mixture is filtered under sterile conditions to obtain a clear fluid which can be stored at 4°C before use.
The mixture of yeast cells is (approximately I06 per mI) cultured for about 48 to 96 hours in the presence of a series of electromagnetic f gilds. Each electromagnetic field has a frequency that, depending on the strains of yeast included, corresponds to one of the four ranges of frequencies described in Sections 5.1. If all four yeast components are present, a combination of the following four frequency bands can b.e used :

MHz; 6840-6860 MHz; 8220-8240 Mfiz; 8530-8550 MHz. The EM fields can be applied sequentially or simultaneously. Generally, the yeast cells are subjected to an EM field strength in the range from 85mV/cm to 320mV/cm in this process.
~Thile the yeast cell culture is exposed to the EM field(s), the culture is incubated at temperatures that is kept at about 36°C. At the end ofthe culture period, the activated and conditioned yeast cells can be recovered by centrifugation at about 3500 rpm and stored under 4 ° C.
5.3 MAIVICJFfIC'I"URE OF TI~f7~: ~I~~,~G~Cr~i., CO1VYPOSI~'xOIVS
The present invention further provides a method for manufacturing a biological composition that comprises the yeast cells of the invention.
Preferably, the biological compositions of the invention comprise yeast cells activated by the methods described in section 5.1 and which have been subject to conditioning by the method described in section 5.2. Most preferably, the biological compositions comprise all four yeast cell components.
To mass produce the biological compositions of the invention, the culture process is scaled up accordingly starting with activated yeast cell cultures as prepared in Section 5.1 and preferably conditioned as described in Section 5.2. To illustrate the scaled-up process, a method for producing 1000 kg of the biological composition is described as follows:
A stock culture of each of the four activated yeast cell components are added to a culture medium comprising 100 kg starch in 250 liters of water. The activated yeast cells are then cultured at 35 ° to 38 °C in the presence of an EM fields) of the respective frequencies and a field strength within 120 to 450 mV/cm. The culture process is carried out for about 48 to 96 hours, or when the yeast cell number reaches about 2 x 10'°/ml. At this point, the activated yeast cells must be stored at about 15 ° to 20 °C, and if not used immediately, dried for storage within 24 hours. This process is repeated for each of the four yeast cell components. To make a biological composition comprising all four yeast cell components, 250 liters of culture media of each of the four activated yeast cell components (i.e, a total of 1000 liters) are mixed and combined with 600 kg of starch.
Since the activated yeast cells and the biological compositions are not necessarily used immediately, the prepared yeast cells and biological compositions can be dried in a two-stage drying process. wring the fast drying stage, the yeast cells are dried in a first dryer at a temperature not exceeding 65 °C for a period of time not exceeding 10 mutes so that yeast cells quickly become dormant. The yeast cells are then sent to a second dryer and dried at a temperature not exceeding 70 ° C for a period of time not exceeding 30 minutes to further remove water. After the two stages, the water content should be lower than 5%. It is preferred that the temperatures and drying times be adhered to in both drying stages so that yeast cells do not lose their vitality and functions. The dried I S yeast cells are then cooled to room temperature. The dried yeast cells may also be screened in a separator so that particles of a preferred size are selected. The dried cells can then be sent to a bulls bag filler for packing.
6. ~~i7FI~E
The following example illustrates the manufacture of a biological composition that can be used as a dog food additive.
The biological composition comprises the following four components of yeasts: Saccharomyces cerevisiae AS2.504, IFFI1202,1FFII345 and IFFIl0l2. Each component of yeasts is shown to be capable of inhibiting the development of a rabies virus infection and reducing the mortality of infected dogs. The four yeast cell components are prepared and tested separately as follows:
A starting culture containing about 105 cells/ml of AS2.504 is placed into the container (2) as shown in Figure 1 containing a medium with the composition as shown in Table I. Initially, the yeast cells are cultured for about 24 hours at 36 t 1 °C without an EM field. Then, in the same medium, at 36 t 1 °C, the yeast cells are cultured in the presence of a series of four EM fields applied zn the order stated: 7720MHz at I87mV/cm for 52 hrs; 7724MHz at 187mV/cm for 52 hrs; 7731MHz at I87~2.OmV/cm for 8 hrs;
7737MHz at 187~2.OmV/cm for 8 hrs. The yeast cells were conditioned by further culturing in canine gastric juice and hawthorn juice as described in section 5.2, in the presence of a series of two EM f elds: 7720 MHz at 187 mV/cm for 52 hours and MHz at 187 mV/cm for 52 hours. After the last culture period, the yeast cells are either used within 24 hours to make the biological compositions, or dried for storage as described in section 5.3.
The beneficial effect of this first component of yeast cells on animals was tested as follows: The test was conducted with 120 dogs (white Pekinese), a:11 about three months old, with body weight about 2.5 ~ 0.3 kg each. The animals were divided into four groups each with 30 animals. The animals were further divided into three subgroups of animals. The experiments in each group were triplicated. The animals in all four groups were injected intramuscularly with 5 ml of a solution containing 10'2 rabies virus per ml.
10 The first group of animals (Group A) were fed a diet comprising a mixture of antibiotics as shown in Table 6.
Table 6: composition of animal feed containing antibiotics Ingredients Quantities per metricNotes ton Basic Feed 1000 kg supplied by Beijing Pet Food Factory with no added antibiotics bacitracin zinc 50 g 2,000,000 units destomycin 20 g 2,000,000 units colistin sulfate 40 g 600,000,000 units oxytetracycline 50 g 50,000,000 units Roxarsone 150 g The animals of Group B were fed a diet comprising activated AS2.504 yeast cells. The activated yeast cells were present in an additive which was pxepared by mixing dried cells with zeolite powder (less than 200 mesh) at a ratio of about 109 yeast cells per gram of zeolite powder. For every 995 kg of basic feed, 5 kg o:f'the feed additive was added, nelding an improved feed that comprises 0.5% additive by weight or 5 x 10'2 yeast cells.
The third group of animals (Group C) was fed a diet which contains an additive that was prepared identically to that used in Group B except that the AS2.504 yeast cells were not activated. The animals of Group I~ were fed the basic diet with neither antibiotic nor yeast additives. After six months, the health stai:us of the animals in various groups are shown in Table 7 below.

Table 7: Health status of animals fed with different diets Group Total/group Total No. of sick Dead animals animals A 30 (10 x 3) 7 4 B 30 (10 x 3) 4 1 C 30 (10 x 3) 12 6 D 30 (10 x 3) I3 8 To prepare the second component, a starting culture containing about 105 cells/ml of TFFI1202 is placed into the container (2) as shown in Figure 1 containing a medium with the composition as shown in Table 2. Initially, the yeast cells are cultured for about 22 hours at 36 ~ 1 °C without an EM field. Then, in the same medium., at 36 i 1 °C, the yeast cells are cultured in the presence of a series of four EM fields applied in the order I5 stated: 6843MHz at 212~1.OmV/cm for 55 hrs; 6847MHz at 212~1.OmV/cm for 55 hrs;
6855MHz at 212~1.0mV/cm for 10 hrs; and 6858MHz at 212=l.OmV/cm for 10 hrs.
The yeast cells were conditioned by further culturing in canine gastric juice and hawthorn juice as described in section 5.2, in the presence of a series of two EM fields:
6843 MHz at 212 mV/cm for 55 hours and 6847 MHz at 212 mV/cm for 55 hours. After the last culture .
period, the yeast cells are either used within 24 hours to make the biological compositions, or dried for storage as described ~. section 5.3.
The beneficial effect of this component of yeast cells on animals was tested as follows: The test was conducted with 60 dogs (white Pekinese), all about three months old, with body weight about 2.5 ~ 0.3 kg each. The animals were divided into two groups ~ ~d C) each with 30 animals. The animals were further divided into three subgroups of 10 animals. The experiments in each group were triplic;ated. The animals in both groups were injected intramuscularly with 5 ml of a solution containing 1012 rabies virus per ml.
In this experiment, the animals of Group B were fed a diet comprising activated IFFI1202 yeast cells. The activated yeast cells were present in an additive which was prepared by mixing dried cells with zeolite powder (less than 200 mesh) at a ratio of I x 109 yeast cells per gram of zeolite powder. For every 995 kg of basic feed, 5 kg of the additive was added, yielding an improved feed that comprises 0.5% additive by weight.
The second soup of animals (Group C) was fed a diet which contains an additive that was prepared identically to that used in Group B except that the IFFI1202 yeast cells were not activated.
3 5 After six months, the health status of the animals in the two experimental groups are compared to two control groups. For comparisons, also included in the Table below are the data of the two control groups (A* and D*) of animals in Group A (antibiotics}
and D
(blank) as described in the previous experiment with AS2..504.
Table 8: Health status of ani.,vnals fed with different diets Group Total/group Total No. of sick Dead animals animals A* 30 (10 x 3) 7 4 IO B 30 (10 x 3) 3 2 C 30 (10 x 3) 11 5 D* 30 (10 x 3) 13 8 For the third yeast cell component, a starting culture containing about I05 15 cells/ml of IFFII345 is placed into the container (2) as shown in Figure I
containing a medium with the composition as shown in Table 3. Initially, the yeast cells are cultured for about 33 hours at 36 ~ 1 °C without an E:M field. Then, in the same medium, at 36 t I °C, the yeast cells are cultured in the presence o:P a series of four EIVI fields applied in the order stated: 8221MHz at 243mV/cm for 46 hrs; 82251VIFiz at 243mV/cm for46 hrs;
8232MHz 20 at 243mV/cm for 5 hrs; and 8236MHz at 243mV/cm for 5 hrs. The yeast cells were conditioned by further culturing in canine gastric juice and hawthorn juice as described in section 5.2, in the presence of a series of two EM fields: 8221 l~IHz at 243 mV/cm for 46 hours and 8225 MHz at 243 mVlcm for 46 hours. After the last culture period, the yeast cells are either used within 24 hours to make the biological compositions, or dried for 25 storage as described in section 5.3.
The beneficial effect of this component of yeast cells on animals was tested as follows: The test was conducted with 60 dogs (white Pekinese), all about three months old, with body weight about 2.5 ~ 0.3 kg each. The animals were divided into two groups (B and C) each with 30 animals. The animals were fur~er divided into three subgroups of 30 10 gals. The experiments in each group were triplicated. The animals in both groups were injected intramuscularly with 5 ml of a solution containing 1012 rabies virus per ml.
In this experiment, the animals of Group B were fed a diet comprising activated IFFII345 yeast cells. The activated yeast cells were present in an additive which was prepared by mixing dried cells with zeolite powder (less than 200 mesh) at a ratio of I x 109 35 yeast cells per gram of zeolite powder. For every 995 kg of basic feed, 5 kg of the additive was added, yielding an improved feed that comprises 0.5% additive by weight.
The second group of animals (Group C) was fed a diet which contains an additive that was prepared identically to that used in Group B except that the IFFII345 yeast cells were not activated.
After six months, the health status of the animals in the two experimental groups are compared to two control groups. Far comparisons, also included in the Table below are the data of the two control groups {A* and D*) of animals in Group A (antibiotics) and D
(blank) as described in the previous experiment with AS2.504.
Table 9: Health status of animals fed with different diets Group Total/group Total ~fo. of sick Dead animals animals A* 30 (i0 x 3) 7 4 B 30 (10 x 3) 6 3 i5 C 30(10x3) 12 5 D* 30 (10 x 3) 13 8 To prepare the fourth component, a starting culture containing about 105 cells/ml of IFFI1012 is placed into the container (2) as shown in Figure 1 containing a medium with the composition as shown in Table 4. Initially, the yeast cells are cultured for about 35 hours at 36 ~ I °C without an EM field. Then, in the same medium, at 36 ~ 1 °C, the yeast cells are cultured in the presence of a series of four EM fields applied. in the order stated: 853IMHz at 224tI .OmV/cm for 62 hrs; 8535MEIz at 224~1.OmV/cm for 62 hrs;
8540MHz at 224~1.OrnV/cm for S hrs; and 8546MI~z at 22~~I.OmV/crn for S hrs.
The yet cells were conditioned by further culturing in canine gastric juice anal hawthorn juice as described in section 5.2, in the presence of a series of two EM fields:
8531 MHz at 224 mV/cm far 62 hours and 8535 MHz at 224 mV/cm for 62 hours. After the last culture period, the yeast cells are either used within 24 hours to make the biological compositions, or dried for storage as described in section 5.3.
The beneficial effect of this component of yeast cells on animals was tested as follows: The test was conducted with 60 dogs (white Pekinese), alI about three months old, with body weight about 2.5 ~ 0.3 kg each. The animals were divided into two groups {B and C) each with 30 animals. The animals were further divided into three subgroups of 10 animals. The experiments in each group were triplicated. The animals in both groups were injected intramuscularly with 5 ml of a solution containing 101' rabies virus per ml.

In this experiment, the animals of Group B were fed a diet comprising activated IFFI1012 yeast cells. The activated yeast cells were present in an additive which was prepared by mixing dried cells with zeolite pawder (Less than 200 mesh) at a ratio of I x 109 yeast cells per gram of zeolite powder. For every 995 kg of basic feed, 5 kg of the additive was added, yielding an improved feed that comprises 0.5% additive by weight.
The second group of animals (Group C) was fed a diet which contains an additive that was prepared identically to that used in Group B except that the IEFI1012 yeast cells were not activated.
After six months, the health status of the animals in the two experimental groups are compared to two control groups. For comparisons, also included in the Table below are the data of the two control groups {A* and D*) of animals in Group A (antibiotics) and D
(blank) as described in the previous experiment with A~2.504.
Table 10: Health status of animals fed with different diets Group Totallgroup Total No. of sick Dead animals animals A* 30 (10 x 30) 7 4 B 30 (I0 x 30) S 2 C 30 (10 x 30) 12 _. . . _ 6 ~ D* ! 30 (10 x 30) ~ I3 A biological feed additive comprising all four yeast cell components was prepared by mixing dried cells of each component with zeoLite powder (less than 200 mesh) at a ratio of 1 x I O9 yeast cells per gram of zeolite powder. For every 995 kg of basic feed, 5 kg of the yeast and zeolite powder mixture was added, yielding- an additive that comprises 0.5% yeast and zeolite powder by weight. The beneficial effect of the biological feed additive on animals was tested as follows: The test was conducted with 60 dogs (white Pekinese), all about three months old, with body weight about 2.5 ~ 0.3 kg each. The animals were divided into two groups (B and C) each with 30 animals. The animals were her divided into three subgroups of IO animals. The experiments in each group were triplicated. The animals in both groups were infected intramuscularly with 5 ml of a solution containing 10'Z rabies virus per ml.
In this experiment, the animals of Group B were fed a diet comprising the biological feed additive as described above. The activated yeast cells were present in an 3 $ additive which was prepared by mixing dried cells with zeolite powder (less than 200 mesh) at a ratio of 1 x 109 yeast cells per gram of zeolite powder. For every 99S kg of basic feed, 5 kg of the additive was added, yielding an improved feed that comprises 4.5%
additive by weight. The second group of animals (Group C) was fed a diet which contains an additive that was prepared identically to that used in Group B except that none of the yeast cells were activated. After six months, the health status of the animals in the two experimental groups are compared to two control groups. For comparisons, also included in the Table below are the data of the two control groups (A* and D*) of animals in Group A.
(antibiotics) and D (blank) as described in the previous experiment with AS2.504.
Table 11: Health status of animals fed with different diets Group Totallgroup Total No. of sick Dead animals animals A''~ 30 (1C x ~) p I 4 B 30 (10 x 3) 0 0 C 30 (10 x 3) 21 4 D* 30 (10 x 3) 13 ' 8 The above results indicate that the biological composition of_the invention is a valuable aI feed additive that can be used to maintain the health of the animal, and help the animal recover from an infection.
The present inventi~n is not to be limited in scope by the specific embodiments described which are intended as single illustrations of individual aspects of the i_nventl0n, and functionally equivalent methods and components are within the scope of the invention. Indeed various modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

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Claims (24)

What is claimed is:

1. A biological composition comprising at least one of the following yeast cell components:
(a) a first yeast cell component comprising a plurality of yeast cells that are prepared by culturing the yeast cells in an electromagnetic field or a series of electromagnetic fields having a frequency in the range of 7720 to 7740 MHz and a field strength of 100 to 250 mV/cm;
(b) a second yeast cell component comprising a plurality of yeast cells that are prepared by culturing the yeast cells in an electromagnetic field or a series of electromagnetic fields having a frequency in the range of 6840 to 6860 MHz and a field strength of 55 to 230 mV/cm;
(c) a third yeast cell component comprising a plurality of yeast cells that are prepared by culturing the yeast cells in an electromagnetic field or a series of electromagnetic fields having a frequency in the range of 8220 to 8240 MHz and a field strength of 75 to 260 mV/cm; and (d) a fourth yeast cell component comprising a plurality of yeast cells that are prepared by culturing the yeast cells in an electromagnetic field or a series of electromagnetic fields having a frequency in the range of 8530 to 8550 MHz and a field strength of 60 to 240 mV/cm.

2. The biological composition of claim 1 which comprises the yeast cell components of (a), (b), (c) and (d).

3. The biological composition of claim 1 or 2, wherein the yeast cells are cells of Saccharomyces.

4. The biological composition of claim 1 or 2, wherein the yeast cells are cells of Saccharomyces cerevisiae.

5. The biological composition of claim 1 or 2 in which the yeast cells are dried.

6. An animal feed composition comprising the biological composition of claim 1 or 2, and dog food.

7. The animal feed composition of claim 6 wherein the biological composition further comprises zeolite powder at a ratio of about 10 9 yeast cells to 1 g of zeolite powder.

The animal feed composition of claim 7 in which 0.5% by weight is the biological composition of claim 1 or 2.

9. A method for preparing a biological composition, said method comprising culturing a plurality of yeast cells in an electromagnetic field or a series of electromagnetic fields having a frequency in the range of 7720 to 7740 MHz and a field strength of 100 to 250 mV/cm.

10. The method of claim 9, wherein. said method further comprises culturing the plurality of yeast cells in one or more of the electromagnetic fields in a culture medium comprising canine gastric juice, and wild hawthorn juice.

11. A method for preparing a biological composition, said method comprising culturing a plurality of yeast cells in an electromagnetic field or a series of electromagnetic fields having a frequency in the range of 6840 to 6860 MHz and a field strength of 55 to 230 mV/cm.

12. The method of claim 11, wherein said method further comprises culturing the plurality of yeast cells in one or more of the electromagnetic fields in a culture medium comprising canine gastric juice, and wild hawthorn juice.

13. A method for preparing a biological composition, said method comprising culturing a plurality of yeast cells in an electromagnetic field or a series of electromagnetic fields having a frequency in the range of 8220 to 8240 MHz and a field strength of 75 to 260 mV/cm.

14. The method of claim 13, wherein said method further comprises culturing the plurality of yeast cells in one or more of the electromagnetic fields in a culture medium comprising canine gastric juice, and wild hawthorn juice.

15. A method for preparing a biological composition, said method comprising culturing a plurality of yeast cells in an electromagnetic field or a series of electromagnetic fields having a frequency in the range of 8530 to 8550 MHz and a field strength of 60 to 240 mV/cm.

16. The method of claim 15, wherein said method further comprises culturing the plurality of yeast cells in one or more of the electromagnetic fields in a culture medium comprising canine gastric juice, and wild hawthorn juice.

17. A method of making an animal feed composition, said method comprising (a) preparing one or more of the yeast cell components of claim 1, (b) drying the yeast cell components of (a), and (c) mixing the dried yeast cells with zeolite powder and dog food.

18. The method of claim 17, wherein the drying step comprises (l) drying at a temperature not exceeding 65°C for a period of time such that the yeast cells become dormant; and (b) drying at a temperature not exceeding 70°C for a period of time to reduce the moisture content to below 5%.

19. A method for reducing the incidence of infectious diseases in a dog comprising feeding the dog for a period of time an animal feed composition comprising at least one of the following yeast cell components:
(a) a first yeast cell component comprising a plurality of yeast cells that are prepared by culturing the yeast cells in an electromagnetic field or a series of electromagnetic fields having a frequency in the range of 7720 to 7740 MHz and a field strength of 100 to 250 mV/cm;

(b) a second yeast cell component comprising a plurality of yeast cells that are prepared by culturing the yeast cells in an electromagnetic held or a series of electromagnetic fields having a frequency in the range of 6840 to 6860 MHz and a field strength of 55 to 230 mV/cm;

(c) a third yeast cell component comprising a plurality of yeast cells that axe prepared by culturing the yeast cells in an electromagnetic field or a series of electromagnetic fields having a frequency in the range of 8220 to 8240 MHz and a field strength of 75 to 260 mV/cm; and (d) a fourth yeast cell component comprising a plurality of yeast cells that axe prepared by culturing the yeast cells in an electromagnetic field or a series of electromagnetic fields having a frequency in the range of 8530 to 8550 MHz and a field strength of 60 to 240 mV/cm.

20. The method of claim 19, wherein the animal feed composition comprises the yeast cell components of (a), (b), (c) and (d), and zeolite powder.

21. The method of claim 19, wherein said yeast cells are Saccharomyces cerevisiae cells.

22. The method of claim 19, wherein the yeast cell components and zeolite powder comprises 0.5% by weight of the animal feed composition.

23. The composition of claim 1 or 2, wherein the plurality of yeast cells used in preparing the first yeast cell component comprise cells of Saccharomyces cerevisiae AS2.504, wherein the plurality of yeast cells used in preparing the second yeast cell component comprise cells of Saccharomyces cerevisiae IFFI1202, wherein the plurality of yeast cells used in preparing the third yeast cell component comprise cells of Saccharomyces cerevisiae IFFI1345, and wherein the plurality of yeast cells used in preparing the fourth yeast cell component comprise cells of Saccharomyces cerevisiae IFFI1012.

24. The animal feed composition of claim 6, wherein the plurality of yeast cells used in preparing the first yeast cell component comprise cells of Saccharomyces cerevisiae AS2.504, wherein the plurality of yeast cells used in preparing the second yeast cell component comprise cells of Saccharomyces cerevisiae IFFI1202, wherein the plurality of yeast cells used in preparing the third yeast cell component comprise cells of Saccharomyces cerevisiae IFFI1345, and wherein the plurality of yeast cells used in preparing the fourth yeast cell component comprise cells of Saccharomyces cerevisiae IFFI1012.