NO164152B - PREPARATION FOR ORAL ADMINISTRATION FOR DRUGS FOR AA IMPROVE THE EFFECTIVENESS OF THESE RECYCLING. - Google Patents

Description

The present invention relates to a preparation for

oral administration to ruminants to improve their utilization efficiency.

Ruminants that have a compound stomach such as sheep (Ovis aries aries), cattle (Bos primigenius taurus),

goat {Capra hircus) and their wild relatives (deer and mouflon) play an important role in the food chain and in the economy. Their special importance is that they live on nutrients that cannot be utilized by other herbivores. The rumen provides an anaerobic environment for the rumen flora which is capable of digesting cellulose and utilizes non-protein nitrogen alongside the usual nutrients. The composition of the rumen flora largely depends on the feed ration and is adapted to the diet. However, this adaptation takes several days and the final stabilization may require several weeks. Abrupt changes in the ration adversely affect intake, digestibility and production, and can cause illness or even death.

It is well known that millions of bacteria live and grow in 1 ml of rumen juice. Anaerobic fermentation by bacteria is of great importance for normal digestion and intake. Energy-producing foodstuffs are fermented into acetic acid, propionic acid and butyric acid, which are used by the host as fatty acids, and the bacterial mass that passes to the intestines is digested and used as a source of protein. When it comes to milk and meat production, the supply of acetic acid and propionic acid and their mutual relationship will play a significant role. Therefore, the rumen flora plays an important role in the maintenance and production of ruminants in agriculture.

After birth, the rumen flora develops spontaneously and assumes the adult composition after adaptation to solid food. However, it is not certain that this random flora

will represent the optimal fermentation system. It would thus be advantageous to have a method for modulating the composition of and/or the number in the rumen

the flora according to economic interests.

Increasing the production of volatile fatty acid in the rumen and thereby improving utilization and consequently meat or milk production is a long-felt need in livestock farming. Certain results have been obtained with monensin-£2-^5-ethyl-tetrahydro-5-{tetrahydro-3-methyl-5-./tetrahydro-6-hydroxy-6-(hydroxymethyl)-3,5-dimethyl-2H -pyran-2-yl7-2-furyl}-2-furyl7-γ-hydroxy-Jα-methoxy-α,γ,2,8-tetramethyl-1,6-dioxaspiroZ"4,57decane-7-butyric acidJ used as an agent against spp. similar (Ingle et al., Abstr. Am. Soc. Anim. Sei. 424

(1978)) have also been reported. The observed effect

is, however, very different in animals fed on different precursors and is overall not significant (Chalupa,

W., Chemical Control of Rumen Microbial Metabolism, Digestive Physiology and Metabolism in Ruminants, MTP Press, Lancaster, England, 1980 and Chalupa, W. et al., Manipulating Rumen Fermentation with Monensin and Amicloral, Abstr. Am. Soc. Animation Sci., 410 (1978)). The

no method is known in the art by which the microbial culture present in the rumen of ruminants can be directly influenced to produce significant results.

During the present experiment, it has been found that genetic recombinant methods can be successfully used for the separation of microorganisms; Virtually any bacterial strain can be tagged with genetic markers, e.g. by an antibiotic resistance factor that enables identification of the bacterium among other bacteria.

Rumen bacteria have been isolated, the strains have been genetically marked, and after cultivation these were reintroduced into the rumen.i Samples of the rumen contents were periodically taken and cultured in selective media, and it was found that some of the strains that had fermentative characteristics that were beneficial for the host animal and which was able to grow in vitro, could grow in the rumen and stay there for a long time if the same nutrients were added as during the isolation, and these stimulated digestion and thereby nutrient utilization

to the host animal.

The present invention thus relates to a preparation for oral administration to ruminants to improve the efficiency of utilization for them, which is characterized by the fact that it contains a microbial culture selected from the group consisting of microorganisms deposited at the National Collection of Agricultural and Industrial Microorganisms under the deposit designations NCAIM B (P) 000287, 000288 and 000289, which are able to adjust the weight ratio between acetic acid and propionic acid, formed during fermentation of energy-producing nutrients in the rumen of an animal, to an optimal value of 1.5 to 4.0:1 , and which are able to grow in the rumen and remain there for at least 40 days.

If the preparation is used to increase meat production, the active ingredient is preferably a microbial culture which is able to adjust the ratio between acetic acid and propionic acid to 2.0 - 3.5:1, e.g. 2:1.

For milk production, the optimum ratio between acetic acid and propionic acid is approx. 3.0 : 1, for maintenance or pregnancy approx. 4.0 : 1, and for heifer breeding approx. 2.0 - 3.0 : 1. It is therefore advisable to use preparations that are i

able to adjust the ratio between acetic acid and propionic acid to the optimum value for this purpose. In the literature, there is some uncertainty regarding the most desirable ratio between acetic acid and propionic acid,

with the preferred ratio being a function of the ruminant, the precursor used and other factors and its selection being a task for the person skilled in the art (see, e.g., Kaufmann, W. and Rohr,

K., Der Einfluss des Futters auf die bakterielle Fermentation in Vormagen, Handbuch der TiernShrung, 263, 1969, Parey, Hamburg, Berlin.

The microbial cultures used as an active ingredient in the above-mentioned preparation are produced by a method where

samples are taken from the rumen of animals sheep with a given

nutrient or a specific ration, the metabolism of microbes isolated from the sample is investigated in vitro and/or in vivo, and microbes with advantageous metabolic characteristics are grown in media containing the same nutrient or the same ration as the carbon or nitrogen source,

a genetic marker that enables selection is introduced

in the adult microbes, the genetically marked strains are cultured, the cultures are reintroduced into the rumen of the animals fed the same nutrient or ration,

samples are taken from the rumen,

the cell number of the genetically marked strain is counted,

strains which persist for at least 60 days and which adjust the ratio of acetic acid to propionic acid to an optimal value, preferably 1.5 - 4.0 : 1, are separated, and

if desired, these strains are formulated in a form acceptable for use in animal husbandry and nutrition.

According to a preferred embodiment of the method, samples are taken from the rumen

of a fistulated ruminant fed on hay, cereal meal or molasses, and the cultures containing rumen bacteria are spread on a solid medium containing N and C sources, inorganic salts, rumen juice and agar (Bryant and Burkey, J. Dairy Sei., 36, 205 , 1953). The cultures are incubated under anaerobic conditions after which the clones are isolated and grown in a similar medium as indicated above.

The grown cultures are inoculated in liquid media containing a nitrogen source, inorganic salts, rumen juice,

and as a carbon source, hay or cellulose, or in other cases cereal flour or molasses, and incubated until intensive growth begins in the media.

Cells grown on hay (cellulose), cereal flour (starch) or molasses (sucrose) as carbon sources are spread, grown and isolated on solid media containing cellulose (for cells grown with hay), glucose (for cells cultured with cereal flour) or sucrose ( for cells grown with molasses) as carbon sources.

The cultures obtained are genetically marked. For labeling, any heritable genetic marker can be used

which enables the identification of the labeled microbe among other microbes.

According to a further preferred embodiment of the invention, antibiotic-resistant genes are introduced into the selected cells. If the bacteria living in the rumen are sensitive to a certain antibiotic and resistant cells are mixed with these, growth and death of the latter microbes can easily take place if the samples are spread on media containing the same antibiotic. In this case, only resistant cells will grow.

Upon transformation (Bergmans et al., J.Bacteriol. 146, 564 (1981)), plOll plasmid (Simon et al., Proc-8th North American Rhizobium Conference, Winnipeg, Canada, Univ. of Manitoba Press, 1983) which carrying kanamycin- and chloramphenicol-resistant genes introduced into the selected cultures growing on cellulose, cereal flour or molasses. Plasmid DNA was isolated from E. coli cells (Birnboim and Doly, Nucl. Acid. Res. 7, 1513 (1979)). The strain is deposited in the National Collection of Agricultural and Industrial Microorganisms, Budapest, under the designation NCAIM B (P) 000264.

After transformation with DNA-carrying antibiotic-resistant genes, cells containing and expressing the new genetic information were selected on solid media with the above composition but supplemented with kanamycin.

The resistant strains were stored.

With the isolated and stored cultures, media containing nitrogen source, inorganic salts, juice and agar (to obtain a semi-solid consistency) were inoculated and incubated under anaerobic conditions. The developed cultures were mixed with feed for sheep fasted for one day. Before and after feeding the bacteria to the animal, rumen samples were taken daily, the samples were spread out on the above-described solid medium containing kanamycin, and bacterial cells that were resistant and sensitive to the antibiotic were counted. Rumen production of volatile fatty acids is also determined qualitatively and quantitatively. It is known that the pre-utilization of ruminants is affected by the ratio of volatile fatty acids (Eskeland et al., J. Anim. Sei. 33, 282 (1971); Church et al., Digestive Physiology and Nutrition of Ruminants, Vol. 2, p. 622-625, 1971). As stated above, the optimal ratio between acetic acid and propionic acid is considered to be 2.0 - 3.5 : 1, 3:1 and 4:1 for growth, milk production and maintenance respectively as well as pregnancy, (Kaufmann, W. and Rohr, K.: Der Einfluss des Futter auf die bakterielle Fermentation in Vormagen.

In: Handbuch der Tieremahrung, p. 263, Parey, Hamburg-Berlin, 1969).

According to a preferred embodiment of the method, bacteria are isolated from

rumen samples and the capacity of the isolates in terms of volatile fatty acid production are investigated. The microorganism is grown in anaerobic conditions in the described complete media containing rumen juice, after which the acetic acid, propionic acid and butyric acid concentrations of the cultures are determined. Microbial cells that produce volatile fatty acids in the required conditions are genetically marked and their rumen growth is investigated.

Strains which are capable of growing in the rumen of animals fed the described ffirstoff for at least 60 days and which can ferment carbohydrates to volatile fatty acids in optimal conditions are selected, grown, isolated, maintained and stored and, if desired, their cultures in a form acceptable to livestock is administered orally to ruminants for the development or modulation of the rumen flora.

Three strains, Hh-GYOKI-1-123Sz, Hh-GYOKI-2-14Ab and Hh-GYOKI-3-81Me, which are capable of growing in the rumen of animals fed hay,1 cereal meal or molasses and which can remain in the rumen for a long time and affect digestion beneficially, have been deposited at the National Collection of Agricultural and Industrial Microorganisms under the deposit designations NCAIM B (P) under 00028 7, 00028 8 and 0002 89.

The microorganism Hh-GYOKI-1-123 Sz that was isolated from rumen biosynthesizes propionic acid. The bacterium is gram-positive and rod-shaped. It ferments glucose and starch into propionic acid and acetic acid, while some butyric acid and carbon dioxide are formed. The cells, which are rod-shaped in the early stage on complete culture medium, will later change to a pleomorphic form without a cilium, and often expand at the ends and take on an irregular shape and size. It grows readily under anaerobic and also under semi-anaerobic conditions, and is a facultative anaerobe.

On the basis mentioned above, the strain Hh-GYOKI-1-123 Sz can be classified in the genus Propioniumbacterium in the family Propionibacteriaceae. (The microorganism Hh-GYOKI-48a which derives from Hh-GYOKI-1-123 Sz can likewise be classified in the genus Propioniumbacterium).

The microorganism Hh-GY0KI-2-14Ab isolated by the inventors from rumen produces propionic acid and acetic acid, it is immobile and spherical in shape, it is gram-negative and does not produce dye. It grows easily also under anaerobic conditions at a temperature of 37°C. The spherical cells are precipitated in large quantities next to each other,

and the average size is 0.3 - 0.4 ym. It ferments acid and carbon dioxide from carbohydrates and produces hydrogen sulphide on complete culture medium. It liquefies-does not gelatinize and does not hemolyze. On the basis of the above-mentioned features, the microorganism Hh-GYOKI-2-14Ab can be classified in the little-known genus Weillonella.

The microorganism Hh-GYOKI-3-8lMe produces acetic acid and is a facultative anaerobe. The cells do not move, are rod-shaped and the ends are flattened. They are gram-positive.

In addition to acetic acid, they produce lactic acid from glucose. On different cultivation media, the staves are rarely found individually, but are often in a chain. Production of pigments cannot be observed.

Based on the above-mentioned facts, the microorganism Hh-GYOKI-3-8lMe can be classified in the family Lactobacteria-ceae of the genus Bifidobacteria.

The species regulations have been made in accordance with

the following literature: Bergey's Manual of Determinative

Bacteriology; Breed, Murray and Hitchens, The Williams and Wilkins Company, Baltimore, 1948, 8th ed.

It is preferred to cultivate the microorganisms from the rumen at between 32 and 37°C under anaerobic conditions and under the utilization of oxygen, in media containing carbon and nitrogen sources, inorganic salts, reducing agents and rumen juice, the latter providing growth factors. Glucose, cellulose, hay, cereal flour or molasses can be used as a carbon source, while inorganic salts, yeast extract, casein and similar additives are suitable N sources.

An essential feature is that unicellular organisms which advantageously ferment the supplied feed or the supplied ration, and which are able to remain in the rumen for a long time, are used for modulation of the rumen flora. For the selection of such strains, genetic markers are used as described above, eg genes that code for antibiotic resistance, enzyme proteins or other detectable proteins.Auxotropic cells can also be used.

The genetic marker is introduced into the cell by a vector DNA molecule, e.g. with a plasmid or phage, but the selectable characteristics, e.g. resistance to an antibiotic can be selected by spontaneous selection.

The selected strains which have beneficial fermentative characteristics and which remain in the rumen for a long time are used either to increase the development of the rumen flora in lactating ruminants or to advantageously modify the composition of the established rumen flora. It is recommended to administer the preparation with food or with drinking water.

The selected microorganism for the preparation of the preparation according to the invention is cultivated in media containing organic carbon sources, organic or inorganic nitrogen sources and organic and inorganic salts, and is then isolated in a form suitable for oral administration or for transport. If desired, the microorganism culture is formulated by mixing it with solid or liquid carriers or other additives. The preparation can be mixed into the feed

or drinking water, or can be fed alone.

In the case of sheep, for example 1 to 20 grams, preferably 5 grams, of a preparation according to the invention is added to approx. 0.5 kg for. For example, a mixture of maize flour, blue alfalfa hay and beef cattle fat can be used, and the relevant proportions are determined in light of the relevant conditions and the desired daily weight gain.

Cattle are generally administered in 10 to 200 g, preferably

50 g of a preparation according to the invention per day, e.g. in a mixture with approx. 5 kg of conventional fuel.

After cultivation in liquid media as described above, the microorganisms are separated by centrifugation or filtration. Eastas, freeze-dried preparations or suspensions containing spores or vegetative forms can be prepared, and additives acceptable for animal husbandry and nutrition can be added. Other additives, for example proteins, amino acids or glycerol can help to keep the microorganisms alive. To the preparations according to the invention used to improve utilization in ruminants, other substances that are conventionally used in the field can also be added, e.g. antibiotics that stimulate the growth of the host animal (monensin, nigericin, salynomycin etc) or increase the durability of the added microorganisms.

The preparations according to the invention thus enable the formation of a living microbial culture in the rumen or the beneficial modification of an established flora.

During the lactation period, the rumen flora is capable of effectively fermenting normal forage. The flora develops spontaneously and randomly, and it is by no means certain that its composition is optimal for the host animal.

When cultured with the selected microbial strains

instead of the slow and spontaneous development of the rumen flora, a rapid development can be achieved, and the rumen flora will be able to optimally utilize the feed.

The advantage of the present method is that with the microorganisms produced in the described manner (for example with the strains 000287, 000288 and 000289) one can activate a rapid adaptation or development of the rumen flora during pre-change or habituation, by increasing the rumen growth of microorganisms that are in able to optimally break down the feed.

The procedure can, among other things, be used in the following cases: - for dairy cows during changes in the milking period, at the end of pregnancy and during seasonal and other changes in the sheep ration; - for beef cattle at the beginning and end of the grazing period, when changing the fattening with roughage to an intensive fattening with ceral meal, and during other changes of the growth-fattening diet; - for sheep during seasonal changes in flocking, at the beginning and end of the grazing period and during the start of intensive growth and fattening.

The possibilities are similar in goat households. Microbial cultures produced by the method described above can also be used in several special cases, e.g. for wild ruminants, in game care and for fallow deer.

It should be noted that although the microbial cultures used in the preparations according to the invention are preferably of rumen origin, other acetic acid- and/or propionic acid-producing bacteria which do not necessarily originate from the rumen can also be used. Such bacteria include certain members of the genera Angerovibrio (lipolytica), Bacteroides, Selenomonas (ruminanticum) and Propionibacteria.

The invention is further illustrated in the following examples. "The Making of

microbial strains that are able to utilize primarily rations containing mainly cellulose

(hay), starch (ceral flour) or sucrose (molasses) and which remain in the rumen for a long time, will be described in detail. Use of the preparation is described for sheep, but the scope of the invention also applies to preparations with microorganisms that are able to grow on other sheep matter and provide development or modification of the rumen flora in other ruminants.

chewers.

Example 1

Modification of the rumen flora in animals fed on hay

A) Isolation of microorganisms capable of growing on hay

Sheep were laparomed and fitted with a rumen fistula and fed on hay for 1 month. Rumen samples taken through the fistula were diluted and spread on RGCA solid medium with the following composition:

Before sterilization under CO 2 ~ gas, the pH of RGCA medium was adjusted to 6.8. Sterilization, preparation of the medium and cultivation were carried out as described by Bryant and Burkey (J. Dairy Sci., 36, 205 (1953)).

The rumen juice was diluted with one sterile mixture of the following composition:

The indications for this mixture are HB.

The cultures were incubated in anaerobic conditions

at 35° C (see Atlas of Rumen Microbiology, Ogimoto and Imai, Japan Scientific Societies Press, Tokyo, 1981) for 120 hours, after which individual clones were inoculated and media containing hay extracted, with the following composition:

Before sterilization, the pH of the medium was adjusted to 6.5. Test tubes containing 5 ml" of sterile medium were inoculated with the microbial suspension obtained from individual clones grown on RGCA solid medium and incubated under anaerobic conditions at 35° C. Growth was checked by microscopic examination, and the cultures were spread on RGCA solid medium in which 2.0% Bacto-cellulose was used

instead of the glucose and cellobiose.

The cultures were incubated in anaerobic conditions at 35°C for 120 hours, after which individual clones of cells utilizing cellulose were inoculated onto RGCA media containing cellulose.

In this way, rumen microorganisms capable of growing on hay or cellulose can be obtained.

B. Genetic labeling of rumen bacteria capable of growing on hay

Genetic labeling was performed with the E. coli plOll plasmid, according to Simon et al. (Proe. 8th North American Rhizobium Conference, Winnipeg, Canada, Univ. of Manitoba Press, 1983).

Plasmid DNA was isolated from an E. coli culture

according to Birnboim and Doly (Nucl. Acid Res. 7, 1513

(1979)) and was dissolved in an aqueous solution containing the following components:

Microbes capable of growing on hay and isolated according to section A) of Example 1 were cultured on RGCF media and separated by centrifugation with CC>2. The cells were suspended in an aqueous solution containing in 1 liter the following components:

in which the suspension must contain 5 x 10 9 cells per ml. The suspension was diluted with the same volume of solution containing plasmid DNA (0.1 µg/ml) and incubated for 60 minutes at 4° C. The incubation was then continued at 41° C for 2 minutes, after which the culture was spread on solid medium containing 500 ug/ml kanamycin B, and cellulose as carbon source. The culture was incubated at 35°C for 120 hours under anaerobic conditions and clones able to grow in the presence of 500 µg/ml kanamycin B were examined.

Plasmid plOll carries genes that determine resistance to kanamycin and chloramphenicol, and further contains an origin of replication that enables the start of replication in E. coli cells. If the plasmid is transformed in other bacteria, the plasmid DNA will, due to its lack of suitable invention enabling replication, either be eliminated or incorporated into the chromosomes (partially or

in

complete) and genetic recombination takes place. Optionally, the gene is expressed incorporated into the chromosome and equips the cell with kanamycin and chloramphenicol resistance.

In the present experiment, kanamycin B resistant clones were obtained with a transformation frequency of 3 x 10<-5>

Several resistant clones were isolated and the sensitivity to antibiotics of the original strain and the kanamycin B-resistant (Km ) strain was determined. The results obtained with several strains are shown in Table 1.

One can thus obtain microorganisms of rumen origin which are able to utilize hay or cellulose and grow in the presence of large amounts of kanamycin B.

The strain Hh-GYOKI-1-123 (Km<R>) was spread on the RGCA medium containing cellulose, after which kanamycin B

in concentrations of 1000, 5000 and 10,000 ug/ml were added. Cultures were incubated in anaerobic conditions at 35° C i

168 hours, and strains growing in the presence of 10,000 ug/ml antibiotic were isolated. With spontaneous selection, spontaneous mutants were obtained that were highly resistant to kanamycin B. One of these strains has been designated Hh-GYOKI-1-123Sz and is deposited at the National Collection of Agricultural and Industrial Microorganisms, Budapest, under the designation NCAIM B ( P) 000287.

C) Reintroduction of labeled microbes into the rumen

Sterile solid medium designated RGCFa was inoculated with the culture of strain Hh-GYOKI-1-123 (Km<R>) stored on RGCA slant agar at +4° C. The composition of the RGCFa medium was as follows:

The 7 solutions were prepared separately and mixed in the specified order.

The cultivation was carried out in 500 ml Erlenmeyer flasks containing 150 ml medium, under anaerobic conditions. Growth was checked after 48 hours, after which the culture was mixed into the feed of a hay-fed sheep. 340 ml culture containing 4.7 x 10^ bacteria per ml was administered orally to the sheep. Before administration and on the following days, 50 to 200 ml samples were withdrawn through the rumen fistula. The samples were diluted with HB solution and spread on RGCA medium lacking kanamycin B or other antibiotics. The cultures were incubated under anaerobic conditions at 35°C for 72 hours, and the bacterial clones were counted. The results are shown in Table 2.

It appears from Table 2 that the microorganisms administered to the animal persist and grow in the rumen.

According to the above-described method, the strain Hh-GYOKI-1-123Sz resistant to 10,000 ug/ml kanamycin B was also cultivated, and it was orally administered to the same sheep on the 15th day.

q

140 ml culture containing 2 x 10 bacteria per ml was administered orally.

Bacteria in rumen samples were cultured and counted as previously indicated. The results are shown in Table 3.

The data in Table 3 indicate that the administered microorganism is present in significant quantities in the rumen, and because the liquid phase of the rumen content is continuously emptied, this is replicated. Differences in bacterial counts between samples can be explained by variations in the consistency of the rumen contents from a thick consistency to a liquid consistency.

Example 2

Modification of rumen flora in sheep fed on cereal flour

A) Isolation of microorganisms capable of growing on cereal flour

The procedure described in section A) in Example 1 was repeated with the difference that the original sample was taken from the rumen of a sheep fed on cereal flour, and the individual isolates were inoculated in RGCF liquid medium (see above) containing 2% powdered cereal flour in a mortar, instead of extracted hay, the cultures grown in RGCF liquid medium were spread on RGCA solid medium (see above)

and clones developed from the cells capable of utilizing cereal flour were isolated on similar medium.

In this way, microorganisms were obtained which are able to grow on cereal flour as a carbon source.

B) Genetic labeling of rumen bacteria that utilize cereal flour

The procedure described under section B) in Example 1 was repeated with the difference that the strains obtained according to section A) in Example 2 were used for transformation with plOll plasmid DNA instead of those obtained according to section A) in Example 1.

Resistance to kanamycin B for several transformed and Km strains is indicated in Table 4.

By carrying out the above procedure, microbes of rumen origin are obtained which are able to utilize cereal flour as a carbon source and which are eight times more resistant to kanamycin B than the rumen flora.

The strain designated as Hh-GYOKI-2-14Ab has been deposited at the National Collection of Agricultural and Industrial Microorganisms, Budapest, under the designation NCAIM B (P) 000288.

C). Reintroduction of genetically labeled microorganisms in the rumen

The procedure described under section C) in Example 1 was repeated with the difference that sterile RGCFa medium containing 1.8% starch instead of 1.8% cellulose (Bacto) was inoculated, mixed in the precursor for sheep, and samples were taken daily through the fistula before administration and after administration. 310 ml's culture containing 7.1 x 10<5> bacteria/ml was administered orally to the sheep.

The data in Table 5 indicate that the administered microorganism stays and replicates in the rumen for a long time.

Example 3

Molification of the rumen flora in a sheep fed on molasses

A) Isolation of microorganisms that are starrid to grow on molasses

The procedure described in section A) of Example 1 was repeated with the difference that the original samples were taken from a sheep fed on molasses, the individual isolates were inoculated on RGCF medium containing glucose instead of extracted hay, the cultures grown in liquid medium were spread on RGCA medium and clones grown from cells utilizing molasses were inoculated on the same RGCA medium.

Microorganisms of rumen origin which utilize molasses were thus obtained.

B) Genetic labeling of bacteria that utilize molasses

The procedure described in section B) in Example

1 was repeated with the difference that cells prepared according to section A) of Example 3 were transformed by plO11 plasmid DNA instead of those obtained according to section A) of Example 1.

The resistance to kanamycin B for several Km R strains is shown in Table 6.

In this way, isolates were obtained which are highly resistant to kanamycin B and which utilize molasses.

The strain designated as Hh-GYOKI-3-81Me has been deposited at the National Collection of Agricultural and Industrial Microorganisms, Budapest, under the designation NCAIM B (P) 000289.

C) Reintroduction of genetically marked bacteria into the rumen

The procedure described in section C) in Example 1 was repeated with the difference that the strain Hh-GYOKI-3-81Me

were inoculated on RGCFa medium containing 1.8% glucose

instead of 1.8% cellulose, and the culture was mixed with the feed of a sheep fed on molasses. 380 ml of a solution containing 1.6 x IO<7> bacteria per ml was administered orally. A sample was taken daily before and after administration through a rumen fistula.

The data indicate that the administered microorganism persists for a long time in the rumen of a sheep fed on molasses.

Example 4

Changes in the ratio of volatile fatty acids due to treatment with a bacterial preparation

Two sheep were fed a complete ration for 14 days, after which the rumen was sampled through a fistula

2 liters of rumen juice were filtered through several layers of gas. The particulate residue was suspended in 1 liter of physiological buffer (see below), mixed and filtered as before. Two filtrates were mixed, allowed to stand for 1 hour, solid material that floated to the surface was discarded, and the liquid phase was examined.

The composition of the physiological buffer was as follows:

The pH of the mixture was controlled and, if necessary, adjusted to pH 7.2 with an aqueous HCl or NaOH solution (Cheng et al.: J. Dairy Sci. 38, 1225 (1955)).

To the mixture obtained was added the same volume of physiological buffer, and in 1 liter of the diluted mixture 4 g of the ration was suspended. 30 ml each of the suspension was poured into Erlenmeyer flasks with a volume of 100 ml. 200 doses were sterilized and 200 were not sterilized.

Sterile media and media containing live rumen bacteria were inoculated with the bacterial strains to be investigated with regard to the production of acetic acid, propionic acid and butyric acid.

Bacterial strains that proved to be able to remain in the rumen for a long time after an in vitro culture were investigated. In addition, bacterial strains isolated from the rumen juice and a sheep fed on complete ration or any forage according to sections A, B and C of Example 1 were also examined.

The bacteria to be examined were grown on RGC+CG medium (see below) in anaerobic conditions at 37°C for 48 hours.

Composition of RGC+CG medium:

Cultures were inoculated into media prepared in Erlenmeyer flasks. 2 ml each of culture was inoculated into 50 ml medium, in two parallel flasks. Non-sterile cultures were also inoculated.

The flasks were incubated under anaerobic conditions

for 40 hours. Growth was stopped with 10% formic acid solution, and the volatile fatty acid content of the cultures was examined.

The cultures were filtered through gas layers and centrifuged at 4000 rpm. per my. for 15 minutes, and was then again filtered and transferred to the separation column of a Carlo Erba GI-452 gas-liquid chromatograph equipped with a flame ionization detector, for the determination of C2-C1 fatty acids.

The temperature of the column: 150° C.

Separation column: 2 meter long, inner diameter 4 mm glass, tubes filled with 10% ethylene glycol adipate and 2% o-phosphoric acid on a silanated silica gel support (0.2 to 0.3 mm particle diameter).

Temperature of the injector: 19 0° C.

N2 flow rate: 50 ml/min.

H2 flow rate: 50 ml/min.

Air flow rate: 200 ml/min.

Paper speed: 160 cm/hour.

Duration of chromatography: 20 min.

Sample volume: 1 yl.

Triplicate measurements were made for each sample. The standard solution contained acetic acid, propionic acid, iso-butyric acid, butyric acid, isovaleric acid and valeric acid.

More than 90 strains of bacteria were isolated

from a sheep fed on a complete ration. The strains were then genetically labeled and examined (the positive strains were examined several times). Representative results are given in Table 8.

Explanation of the symbols used in Table 8:

S: inoculated after sterilization

NS: culture containing living rumen flora was inoculated

a) trace amounts

b) negative control: volatile fatty acid content in media prepared from rumen juice, physiological buffer and precursor

used for the experiment (average of 12 measurements)

c) positive control: volatile fatty acid content of incubated culture containing original rumen bacteria and otherwise

produced by the same process (average of 12

measurements)

d) as c)> but 5 ppm monensin-Na was added to the medium (average of 6 determinations) e) as c) but 10 ppm monensin-Na was added to the medium (average of 7 determinations).

The data in Table 8 indicate that the ratio between volatile fatty acids formed by the fermentative function of the rumen flora can be modulated within a wide range by administering preparations according to the invention

Nelson. For example, production of propionic acid can be significantly stimulated with a culture prepared from strain Hh-GYOKI-48a, while strain Hh-GYOKI-109b stimulates production of acetic acid. Stimulation of production of individual fatty acids was observed both on media lacking (S) or containing (NS) live rumen microbes. In the present experimental system, monensin-Na decreased the ratio between acetic acid and propionic acid by 0.1 or 0.2 (d, e).

Microorganisms selected by the above procedure are genetically marked, administered to ruminants and examined with regard to rumen growth and residence time by repeating the procedure described in section B) of Example 1. Strains with an advantageous fermentative pattern and which remain in the rumen for a long time are administered orally for to modify the production of volatile fatty acids.

Example 5

Bacterial preparation for oral administration to ruminants

Bacteria to be administered were grown on RGCA+CG media (Example 4) under anaerobic conditions by the described process. After cultivation, the cells were separated by filtration or centrifugation. Separated cells were suspended in physiological buffer (Example 4) and freeze-dried. The lyophilized bacterial preparation is stored, formulated and administered orally to ruminants.

Microorganisms can also be grown on other conventionally used media, e.g. in media containing glucose and starch as carbon source and inorganic salts as N source.

The preparation can be easily administered by mixing with the precursor or the drinking water, alone or together with other biologically active agents, e.g. with antibiotics and vitamins.

In addition to the freeze-dried preparation, other products can also be produced. The micro-organisms can also be administered after mixing the filtered or centrifuged bacterial mass with suitable carrier or dilution substances, for example calcium carbonate, concentrates, premixes or other substances.

The bacterial strains are selected from the microorganisms, produced by the method described above, and which advantageously modify the rumen flora, and the amount of these to be administered is determined depending on the ration and use of the animal. If a reduction of the ratio between acetic acid and propionic acid is required, a culture prepared from the strain Hh-GYOKI-48a can be used, for example, while to increase the ratio administration of the strain Hh-GYOKI-3-81Me is recommended.

Determining the required microbial cell count will not pose any problems for the person skilled in the art. It is recommended to administer the cells in an amount to provide 5 x 10 2 to 5 x 10<7> cultured microorganisms per ml rumen juice.

Example 6

Administration of the strains Hh-GYOKI- 48a and Hh-l- 123Sz to sheep

The Hh-GYOKI-48a strain was grown on RGCA+CG media (Example 4) in two 5-liter fermenters at 37°C under anaerobic conditions. The fermentation was started by inoculation with a 10 ml culture of similar composition. After 48 hours of cultivation, the cells were separated by centrifugation (5000 rpm) and the wet sediment weighing 58 g was carefully mixed with 4 kg of cornmeal. The mixture was divided into eight equal parts and administered orally to eight sheep that had previously fasted for 24 hours. The strain Hh-GYOKI-1-123Sz can be used in a similar manner with a bacterial harvest of 53 g.

In a growth-fattening experiment, 23 sheep were fed ad lib on poor hay and the animals were weighed every week for 5 weeks. Experimental groups consisting of eight sheep were fed one of the bacterial preparations each in a single feed, and seven sheep served as controls. 600 to 900 g of hay was consumed per day and animal, plus a mineral and vitamin premix mixed with maize flour (100 g). The results are shown in Table 9.

The average daily weight gain was calculated from the data given in Table 9 and is shown in Table 10.

Sheep treated with strain Hh-GYOKI-1-123Sz and strain Hh-GYOKI-4 8a and the control group increased their weight by feeding the poor ration an average of 2620, 3875 and 360 g during the 35-day experimental period.

The initial body weight did not differ significantly between the groups, but significant differences were found in the final body weights (Table 11) and in the daily increase (Table 12).

The data indicate that preparations according to

the invention markedly stimulates weight gain in sheep.

Example 9

Durability of genetically marked bacteria in cattle rumen

The procedure described in section C) of Example 1 was repeated with the difference that the strain Hh-GYOKI-1-123Sz, which was resistant to 10,000 µg/ml kanamycin, was grown in 4 liters of RGCFa medium. After the stationary phase was achieved (38 hours), the culture was harvested by centrifugation (5000 rpm) and the cells were thoroughly mixed with 500 g of cornmeal and fed to a cow. Weekly samples were taken through a fistula and the rumen durability of the administered strain was determined as described in section c) of Example 1.

The results indicate that strain Hh-GYOKI-1-123Sz grows in the rumen of cattle and can remain there for at least 40 days.

Claims (3)

1. Preparation for oral administration to ruminants to improve the efficiency of utilization for them, characterized in that it contains a microbial culture selected from the group consisting of microorganisms deposited at the National Collection of Agricultural and Industrial Microorganisms under the deposit designations NCAIM B (P) 000287, 000288 and 000289, which are capable of adjusting the weight ratio of acetic acid to propionic acid, formed during the fermentation of energy-producing nutrients in the rumen of an animal, to an optimal value of 1.5 to 4.0:1, and which is capable of to grow in the rumen and stay there for at least 40 days. 2. Preparation according to claim 1, characterized in that it comprises a maximum of 95% by weight active ingredient. 3. Preparation according to claim 1 or 2, characterized in that it comprises a microbial culture which is able to adjust the ratio between acetic acid and propionic acid in the rumen to 2.0 - 3.5: 1.