CA1276084C - Composition and method for improving the efficiency of ruminant feed utilization - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
The invention relates to a composition for improving the efficiency of ruminant feed utilization, which comprises as active ingredient one or more microbial cultures capable of adjusting the weight ratio of acetic acid to pro-pionic acid in the rumen to an optimum value, of from 1.5-4.0 : 1, and of growing in the rumen and persisting there at least for 60 days. The composition optionally includes one or more carriers, diluents or preserving agents conven-tionally used in animal husbandry and nutritive and/or other substances conventionally administered to ruminants. A
process for the preparation of microbial cultures used as active ingredient in the compositions is also disclosed as well as a process for the preparation and use of the compo-sitions.

Description

1;Z76~34 -- 1 ~
The invention relates to a composition for improving the efficiency of ruminant feed utilization.
More particuIarly, the invention concerns a compo-sition which comprises as active ingredient one or more micro-S bial cultures capable of adjusting the weight ratio of aceticacid to propionic acid in the rumen to an optimum value of from 1.5-4.0 : 1, and of growing in the rumen and persisting there for at leas-t 60 days. The composition optionally includes orie or more carriers, diluents or preserving agen-ts conventionally used in animal husbandry as well as nutritive and/or other substances conven-tionally administered to ruminants. The invention further relates to the preparation of microbial cultures used as active ingredient in the above compositions and to the use of the compositions.
Ruminants possessing a composite stomach, such as sheep (Ovis aries aries?, cattle (~os primigenius taurus), goat (Capra hircus), and their wild relatives (deer and muflon, etc.) have an important role in the nutrien-t chain and in the economy. Their special impor-tance evolves from the fact that they live on feedstuffs which cannot be utilized by other herbivores. The forestomachs provide an anaerobic environment for the rumen flora, which are able to digest cellulose and utilize non-proteinaceous nitrogen as well as the COmTnOn nu-trients. The composition of rumen flora largely depends on the ration fed and adapts -to the diet.
This adaption, however, takes several days and the final ~ ,~

a.;Z7~ 4 stabilization may require several weeks. An abrupt change of the ration adversely af~ects intake, digestibility and production, and may cause illness or even death.
It is well known that millions of bacteria live and grow in a single ml of rumen liquor. Anaerobic fermenta-tion by bacteria is of high importance for the normal diges-tion and feed intake. Energy producing nutrients are fermented to acetic, propionic and butyric acids, which are ; used by the host animal as fat-ty acids; the bacterial mass passing to the intestines will be digested and uséd as a protein source. With respect to the milk and meat production the supply of acetic acid and propionic acid and their mutual proportion play an essential role. Therefore, the rumen flora fulfills an important role in the maintenance and pro-duction of the ruminant farm animals.
After birth, the rumen flora develops spontaneouslyand attalns its adult composition after weaning to solid food. It is not sure, however, -that this accidental flora would represent the optimum fermentative system. I-t would 20 be advantageous to have a process for -the modulation of the composition and/or of the number of rumen flora according to economic interests.
The increase of volatile fa-tty acid production in the rumen and thereby the improvement of feed utilization 25 and, accordingly, meat or milk production constitutes a long felt want in animal husbandry. Certain . .~

~i, ....

76~134 .

results have been obtained with manensin [2-[5-ethyl-tetrahydro-5- {tetrahydro-3-methyl-5-[tetrahydro-6--hydroxy-6-(hydroxymethyl)-3,5-dimethyl-2H-pyran-2--yl]-2-furyl} -2-furyl]-9-hydroxy-~-methoxy-~,~,2,8--tetramethyl-1,6-dioxaspirol4.5]decane-7-butyric acid~
originally used as coccidios-tat (see e.g U.S. paten-t specfication No. 4,085,255). Experimental use of other related polyethers, such as salinomycin, lasalocid, etc., phthalide derivatives (U.S. pa-tent specification No.
4,333,923) and glycopeptides, such as avoparcin, actaplanin and the like (Ingle et al., Abstr. Am. Soc. Anim. Sci. 424 [1978]) has also been reported. The effect observed is, however, strongly differen-t for animals fed on various feedstuffs and is not altogether substantial (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, ~bstr. Am. Soc.
~ Anim. Sci., 410 [1978]). There is no method known in the ; 20 art by which the microbial cuIture present in the rumen of rum~ants could be directly influenced to provide significant results.
During our experiments we have found that genetic recombination methods can be successfully used for the separation of microorganisms. Practically any bacterial strain can be labelled by genetic markers, e.g. by an antibiotic resistance factor that allows identification of the bacterium among other bacteria.

r .,.~ .. .

I

"~ ~2~6~

We isolated rumen bacteria, gene-tically labelled the strains and a~ter culturing we reintroduced them into the rumen. Then we took samples of rumen con-tent periodi-cally, cultured them in selective media, and found that some S of the strains, tha-t had fermentative characteristics advan-tageous for the host animal and that were able to grow in vitro, couId grow in the rumen and persist there for a long time, if the same feedstuff was fed as during isolation, and they stimulated diges-tion and -thereby feed utilization 10 by the host animal.
Accordingly, the present invention relates to a composition for improving the efficiency of ruminent feed utilization, which comprises as active ingredien-t one or more microbial cultures capable of adjusting the weight ratio 15 of acetic acid to propionic acid in the rumen to an optimum value of from 1.5-4.0 : 1, and of growing in the rumen and persisting there at least 60 days. The composition optionally includes one or more carriers, diluents or preserving agents conventionally used in animal husbandry and nutritive and/or 20 other substances conventionally administered to ruminants.
If the compositions are to be used to increase meat production, the active ingredient is preferably a microbial culture capable of adjusting the ratio of acetic acid to propionic acid in the rumen to 2.0-3.5 : 1, e.g.
25 2:1. For milk production -the optimum acetic acid to pro-pionic acid ra-tio is abou-t 3.0:1, for subsistence or ~,' ~716~34 yestation abou-t 4.0:.1, and for heifer breeding about 2.0-3.0:1. It is therefore, advisable to use compositions capable of adjusting the acetic acid to propionic acid ratio in the rumen to the optimum va~ue for these purposes. In 5 the literature there is some uncertainty as to the most aesired acetic acid to propionic acid ratios, the preferred ratio being a function of the ruminan-t, the feedstuff employed and other factors, and its selection is a task for those skilled in the art ~see e.g. Kaufmann, W. and Rohr, K., ~er 10 Einfluss des Futters auf die bakterielle Fermentation in Vormagen, Handbuch der Tiernahrung, 263, 1969, Parey, ; Hamburg, ~erlinl-: The inven-tion further relates to a process for the preparation of the microbial cultures used as active 15 ingreaient in the above compositions, which comprises taking samples from the rumen of animals fed on a given feedstuff or ration, examining in vitro and/or in vivo, the me-tabolism ; of microorganisms isolated from the sample and cultivating microorganisms with advantageous metabolism characteristics . 20 in media containing the same feedstuff or ration as carbon-or nitrogen-source, introducing into the growing micro-organisms a genetic marker, ~hich makes selection possible, cul-tivating the genetically labelled s-trains, reintroducing the cultures into the rumen of the animals fed on the same 25 feedstuff or ration, taking samples from the rumen, counting the cell number of -the genetically labelled strain, and separating strains persisting for at least 60 days and able ,~

~Z~76~34 to adjust ~he acetic acid to propionic acid ratio to an optimum value of 1.5-4.0 : 1.
If desired, these strains are -thereafter formulated into a form acceptable for the practice of animal husbandry 5 and nutrition.
According to a preferred emodiment of the process of the invention, 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 10 solid media containing N- and C-sources, inoryanic salts, rumen liquor and agar (Bryant and Burkey, J. Dairy Sci., 36, 205, 1953). The cultures are incubated under anaerobic conditions, then the clones are isolated and cultured in similar media as above.
The cultures grown out are inoculated into liquid media containing a nitrogen-source, inorganic salts, rumen liquor, and as a carbon-source, hay or cellulose, or in other ; cases cereal meal or molasses, and they are incubated until in-tensive growth begins in the media.
Cells grown on hay (cellulose), cereal meal (starch) or molasses ~sucrose) as carbon sources are spread, cultured and isolated on solid media containing cellulose (for cells grown with hay), glucose (for cells grown with cereal meal) ,. ~

or sucrose ~for cells grown with molasses) as carbon source.
The cultures obtained are labelled genetically.
For labelling purposes, any inheritable genetic marker can be used that allows the identiEication of the labelled 5 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 10 are mi~ed with them, growth and death of the latter microbes may be followed easily if the samples are spread on media containing -the same antibiotic. In this case only the resistant cells will grow.
; By transformation (Bergmans et al., J. Bacteriol.
15 146, 564 [19811) plOll plasmid (Simon et al., Proc. 8th North ` American Rhizobium Conference, Winnipeg, Canada, Univ. of Manitoba Press, 1983) carrying kanamycin and chloramphenicol resistant genes was introduced into selected cultures which grow on cellulose, cereal meal or molasses. Plasmid DNA
20 was isolated from ~. coli cells (Birnboim and Doly, Nucl.
Acia. Res. 7, 1513 [1979]). The strain is deposited in the Hungarian National Collection of Medical Bacteria of the National Institute of Hygiene, Budapest, under No. 00264.

: .,., ,. - - ., ;.

27~

After transformation with DNA carrying antibiotic resis-tant genes, cells containing and expressing the new genetic information are selected on solid media with the above-mentioned composition, but supplemented with kanamycin.
5 The resistant strains will be stored.
To the isolated and stored cultures, media containing a nitrogen-source, inorganic salts, rumen liquor and agar (to achieve semi-solid consistency) are inoculated and incubated under anaerobic conditions. The developed cultures are mixed with the feed of sheep starved for one day. Before and after feeding bacteria to the animal, rumen samples are taken daily, the samples are spread on the above-` described solid media containing kanamycin, and bacterial cells resistant and sensitive to the antibiotic are counted.
Ruminal production of volatile fatty acids is also determinedqualitatively and quantitatively. It is known that feed utilization by ruminants is affected by the ratio of volatile fatty acids (Eskeland et al., J. Anim. Sci 33, 282 [1971];
Church et al., Di~estive Physiology and Nutrition of Ruminants, Vol. 2, pp. 622-625, 1971). As men-tioned above, the optimum ratio of acetic acid to propionic acid is considered to be 2.0-3.5:1, 3:1, and 4:1, respectively, for growing, milk production, and maintenance as well as pregnancy t resp.
(Kaufmann, W. and Rohr, X.: Der Einfluss des Futters auf die bakterielle Fermentation in Vormagen. In: Handbuch der Tierernahrung, p. 263, Parey, Hamburg-Berlin, 1969).

~ .

76~8~

g According to a preferrea embodiment of the process of the invention bacteria are isola-ted from rumen samples and the capacity of the isolates to produce volatile fatty acids is examined. The microorganism is cultured under anaerobic conditions in the described complete media con-taining rumen llquor, then the acetic, propionic and bu-tyric acid concentrations of the cultures are determined. Microbial cells producing volatile fatty acids in the required ratios are labelled genetically and their ruminal growth are examined.
Strains that are able to grow in the rumen of the animal fed on the described feedstuff for at least 60.days and that can ferment dietary carbohydrates to form volatile fatty acids in optimum ratios, are selected, grown, isolated, maintained and stored, and, if desired, their cultures, in a form acceptable for animal husbandry, will be orally administered to ruminants, for developing or modulating the rumen flora.
Three strains, Hh-GYOKI-1-123Sz, Hh-GYOKI-2-14Ab and Hh-GYOKI-3-81Me, capable of growing in the rumen of animals fed on hay; cereal meal or molasses, respectively, that can persist in the rumen for a long time and which affected digestion advantageously, have been deposited in the Hungarian National Collection of Medical Bacteria of the Na-tional Institute of Hygiene under Nos. 00287, 00288 : 25 and 00289.
According to the invention i-t is preferred to cul.ture the microorganisms of rumen origin at a temperature t~,'.~, ., .~ :~, '; ~ ' -- 10 .~
between 32C and 37C, under anaerobic conditions, with the exclusion of oxygen, in media containing carbon- and nitrogen-sources, inorganic salts, reducing agents and rumen liquor; the latter provides growth factors. As carbon source, glucose, cellulose, hay, cereal meal or molasses can be used, while inorganic sal-ts, yeast extract, casein and similar additives are suitable N-sources.
An essential feature of the invention is that unicellular organisms advantageously fermenting the fed feed-stuff or ration and capable of persisting in the rumen fora long period are used for the modulation of ruminal flora.
For the selection of such strains genetic marks are used, as described above, e.g. genes coding antibiotic resistance, enzyme proteins or other detectable proteins. Auxotrophic cells can also be used.
The genetic marker is introduced in-to the cell by a vector DNA molecule, e.g. by a plasmid or phage, but ; selectable characteristics, e.g. resistance to an antibiotic, can also be chosen by spontaneous selection.
The selected strains, which have advantageous fermentative characteristics and persis-t in the rumen for a long period, are used either -to enhance the development of rumen flora in suckling ruminants or to modify advantage-ously the composition of the established rumen flora. It is recommended to administer the preparation with the feed or drinking wa-ter.

'7~i~8~

The microorganism selected for maki~g the prepara-tion according to the invention is cultured in a medium con-taining an organic carbon source, organic or inorganic nitro-gen source 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 formu-lated by mixing it with solid or liquid carriers or other additives. The preparation can be mixed with the feed or drinking water, or can be fed alone.
In case of sheep, for example 1 to 20 g, preferably 5 g, of microorganism cuIture according to the invention are added to about 0.5 kg of feed. As a feedstuff e.g. a mixture of corn-meal, lucerne hay and beef cattle feed can be used/ and -the actual proportions should be determined in view of the actual conditions and the desired daily gain in weight. Cattle are generally administered 10 to 200 g, preferably 50 g, of a microorganism culture according to the invention per day, e.g. in admixture with about 5 kg of a conventional feedstuff.
The method of improving the efficiency of feed utilization of ruminants is also wi-thin the scope of the invention.
After culturing in liquid media, as mentioned hereinbefore, the microorganisms are separted by centrifuga-tion or filtra-tion. Pastes, freeze dried preparations or suspensions containing spores or vegetative forms, etc.
may be prepared and additives acceptable for animal husbandy ~h 76~

and nutrition may be added. Other additives, e.g. proteins, amino acids or glycerol, may help to keep the microorganisms viable. To the compositions according to the invention used to improve fe~d utilization in ruminants~ other substances conventionally used in practice may also be added, e.g.
antibiotics that stimulate the growth of the host animal (monensin, nigericin, salynomycin etc.) or enhance the persistency of the microorganisms fed.
Thus, the microbial cultures according to the invention enable the formation of a living microbial culture in the rumen or the advantageous modificatiQn or an estab-lished flora.
~ During -the suckling period, the rumen flora is ;~ unable effectively to ferment the common feeds-tuffs. The lS flora develops spon-taneously and accidentally, and it is by no means cer-tain -that its composition is optimal for the host animal.
By feeding the selected microbial strains, instead of a slow and spon-taneous development of the rumen flora, a rapid development may be achieved, and -the rumen flora ; will be capable of optimally utilizing the feed.
The advantage of the instant process is that with the microorganisms prepared in the described way (e.g. with the strains 00287, 0028~ and 00289) we can promote the rapid adap-tion or development of rumen flora during feed change or weaning, by enhancing ruminal growth of microorganisms , ~.~ .~..

~ ;~76~!84 capable of optimal degradation of the feed.
The process can be used, inter alia, in the following cases:
- for dairy cows during changes of lactation, at the end of the pregnancy and during seasonal and other changes ; of the ration;
- for beef cattle a-t the beginning and end of the grazing period, at the change of fattening with roughages to an intensive fattening with cereal meal, and during other 10 changes of the growing-fattening diet; and - for sheep during the seasonal changes of feeding at the beginning and end of the grazing period and during the commencement of an intensive growing and fat-tening.
The possibilities are also similar in goat husbandry.
15 Microbial cultures prepared by the process of -the invention ~ may also be used in several special cases, e.g. for wild-f living ruminants, in game preserves and for fallow-deer.
It should be noted that, although the microbial cultures used in the compositions according to the invention 20 are preferably of rumen origin, other acetic acid and/or propionic acid producing bacteria, which do not necessarily originate from the rumen, are also suitable. Such bacteria include cer-tain members of the genus ~ngerovibrio (lipolytica), Bacteroides, ~.2~

Selenomonas ~ruminanticum) and Propionibacteria~
'~he invention will further be illustratsd by the aid of the following, non limiting Examples.
~he preparation of microbial strai.ns which are capable of utilizing basic rations containing mainly cellulose (hay), starch (cereal meal) or sucrose (molasses) and persist in the rumen for a long period will be described in detail. The use of the preparation is described :Eor sheep, but the scope of protection.extends to micro-organisms capable of growing on other feedstuffs andto the development or modification of the rumen flora of other ruminant species as well.

Example 1 Mod_fication of the _umen flora of a mals fed on~
:~ A) Isolation of microorganisms capable of : growing on hay Sheep are laparatomized fitted with rumen fistula and fed on hay for a month. Rumen sample is taken through the fistula, diluted and spread on RGCA
solid media of following composition:
Salt solution I:
K2HP0~ o.6 g distilled waterad lOOoO g Sal-t solution II:
NaCl 10 2 g (NH4)2S04 1.2 g .

~276~8~
, . .

2P4 0.6 g CaC12 0.12 g g 4 7 2 0.25 g distilled water ad 100.0 ml 5 Resazurin (0.1 % solution) 0~1 ml Agar (Bacto)X 2.5 g Rumen liquorXx lOoO ml : Glucose o5 g Cellobiose o,o5 g 10 Cys-tein.HCl monohgdrate 0~05 g Sodium carbonate (8 % solution)5.0 g Distilled water ~ ad 50.0 ml x) Difco Labs, Detroit, USA
~15 xx) The sample of rumen con-tsnt is filtered through ; several layers of gauze, then the filtrate is stored under carbon dioxide at -20 C.
~ efore sterilization under C02 gas, the pH
of the media RGCA is adjusted to 6.8. Steriliza-tion, ~ 20 preparation of the media and cultivation are performed : according -to Bryant and Burkey (J. Dairy Scio 1 36, 205 ~1953]).
. rrhe rumen liquor is diluted wi-th a sterile mix-ture havlng -the Eollowing composition:
salt solution I (see above)7.5 ml salt solution II (see above) 7.5 ml cystein.HCl monohydrate 0.05 g Na2C3 0-3 g`

1~6~

resazurin (0.1 % solu-tion) 0.1 ml distilled water ad lOOoO ml he sign of thls mix-ture is HB.
~he cultures are incubated unaer anaerobic conditions at 35 C (see Atlas of Rumen Microbiology, Ogimoto and Imai, Japan Scientific Societies Press, rrokyo, 1981) for 120 hours, then the individual clones are inoculated into media, containing extracted hay, of -the ~o}lowing composition: .
salt solutiorl I (see above) 1500 %
sal-t solution II (see above) 15.0 %
~ resazurin (0.1 % so}ution) 0.1 %
- }5 r~ripton ~2 (Oxoid)X 15. %
yeast extract (Oxoid)X 005 %
~ rumen liquorXx 10.0 %
., ; Na2C03 0.~ %
. cystein.HC} monohydrate0.05 %
extracted hay xx 10.0 %

Sign of the media: RGC~ liquid media x) Oxoid ~td., London, UK.
xx) See abo~e.
; 25 xxx) ~or preparing-the extracted hay, finely cut hay particles are suspended in water, boiled and filtered.
The filtration residue is added -to the media before sterilization.
~ Before sterilization, the pH oE the media is ~7~

adjus-ted to 6.5.
Test tubes containing 5 ml of sterile media are inoculated with the microbial suspension obtained from indi-vidual clones grown on RGCA solid media and incubated under anaerobic conditions at 35C. The growth is checked by microscopic examination and the cultures are spread on RGCA
solid media where 2.0~ Bacto cellulose are substituted ~or the glucose and the cellobiose.
The cultures are incubated under anaerobic con-ditions at 35~C for 120 h, then individual clones are madeup of cells utilizing the cellulose are inoculated onto RGCA media containing cellulose.
In this way, ruminal microorganisms capable of growing on hay or cellulose can be ob-tained.

B) Genetic labelling of rumen bacteria capable .
of growin~ on hay - .
Genetic labelling is performed with E. coli plOll plasmid, according to Simon et al. (Proc. 8th Nor~h American Rhizobium Conference, Winnipeg, Canada, Univ. of Manitoba Press, 1983).
The plasmid DNA is isolated from an E. coli culture according -to Birnboim and Doly ~Nucl. Acid Res. 7, 1513 [1979]) and is dissolved in an a~ueous solution containing the following components.
75 mM CaC12 5 mM MgC12 10 mM tris.~Cl bufferX~ p~l 7.5 x) tris (Hydroxyme-thyl) aminomethane hydrochloride - 18.-Microbes capable of growiny on hay and isolatedaccording to item A) of Example 1 are cultured on RGCF media and separated by centrifugation under C02. The cells are suspended in an aqueous solution containing in 1 litre the following components:
75 mM CaC12 5 mM MgC12 10.mM tris.HCl:buffer, ph:7.5 1 mM cystein.HCl monohydrate 1 mM sodium thiosulfate wherein the suspensions should contain 5xlO9 cells per ml.
The suspension is diluted with the same volume of solution containing plasmid DN~ ~0.1 /ug/ml~ and is incubated for 60 min. at 4C. Then the incubation is continued at 41C
for 2 min., a~ter which the culture is spread on a solid medium containing 500./uy/ml of kanamycin B, and cellulose as a carbon source. The culture is incubated at 35C ~or 120 h under anaerobic conditions and clones able to grow in the presence of 500:/ug/ml o kanamycin B are examined.
Plasmid plOll carries genes determining resistance to kanamycin and chloramphenicol; furthermore, it contains replication origin allowing the start of replication in E.
coli cells. If transformed into other bacteria, owing to its lack of suitable origin allowing replication, the plasmid DN~ is either eliminated or incorporated into -the chromosome (partly or completely) ana genetic recombina-tion takes place.
Eventually the ~Z76Q~

gene incorporated into the chromosome is expressed and endows the cells with kanamycin and chloramphenicol resistance.
In our experiments we obtained kanamycin B
resistant clones with transformational frequency of 3xlO 5.
Several resistant clones were isolated and we determined the sensitivity to antibiotic of the initial and kanamycin B resistant (KmR) strains. Results ob-tained with several strain`s are shown in ~able 1.

~able 1 ~55~LL~L{i~ a~S~g_B of rumen bacteria and ofstrains labelle _ genetically_and ~ din~_ h~

Microbe~east effective concentration of kanamycin B, /ug/ml Rumen liquor 31 20 Initial strains 4.0 to 7.5 Genetically labelled KmR strains Hh-GYOKI-1-8 500 : -91 500 ; 25 -123 1000 _ _ _ _ _ _ So we can obtain microorganisms of rumen origin ` ~276~4 ~ 20 -that are able to utili~e hay or cellulose and to ~row in the presence of high amounts of kanamycin ~.
The s-train ~-GYOKI-l 123 (~nR) is spread on RGCA media containing cellulose, whereafter kanamycin ~ in concentrations of 1000, 5000 and 109000 /ug/ml is added. ~he cultures are incubated under anaerobic conditions at 35 C for 168 h and -the strains growing in the presence of 10,000 /ug/ml antibiotic are isolated.
So, with spontaneous selection, we ob-tain spontaneous mutants highly resistant to kanamycin B. One of these . s-trains has been designa-ted Hh-GYOKI-1-123Sz and deposited in the ~ungarian National Collection of Medical Bacteria : ; of the National Institu-te of Hygiene, ~udapest~ under No. 002870 : 15 C) ReintIoduction of labelled~ to the rumen ; Sterile, solia media ~amed RGC~a are inoculated with the oulture of strain Hh-GYOKI-1-123 (KmR) s-tored on RGC~ slants at ~4 C. ~he composition of RGC~a media is as f~llows:
1. K2HP0~ 0.3 % 45 ml solution 2- (NH4)2S4 o.6 %
NaCl o.6 ~
25 g 4 2 oO6 %
CaC12 2H20 0 .o6 %
KH PO 0.3 % 45 ml solution of the 2 4 mi~ture ~ 21 -3. ~ellulose ~Bacto)X 1.8 %
Agar (Bacto)X 300 % 65 ml solution of the mix-ture 4. Yeast extract 0.1 % 20 ml solution 5. C~steinOHCl.E~20 0.1 % 20 ml solu-tion 5 6. Sodium thiosulfate 0.1 %
Ma C0 0.2 % 10 ml solution of the 2 3 mixture 7. Rumen liquor~ 20 ml x) Difco Labs, Detroit, USA
10' Xx) See above The 7 solutions are prepared separately and ; mixed in the given sequence.
Cultivation is performed in 500 ml Erlemneyer flasks containing 150 ml of media, under anaerobic con-ditions. Growth is ,checked after 48 hours 9 then theculture is mixed with the feed of a hay-fed sheep.
;Three hundred and for-ty ml of culture con-taining 4.7x106 bacteria per ml were orally adminis-tered to the sheep, Before administration and on consecutive days, 50 to 200 ml samples are taken through the rumen fistula.
The samples are diluted with HB solution and spread on RGCA media lacking kanamycin B or other antibiotics.
~he cultures are incubated under anaerobic conditions at 35 C for 72 hours and the bacterial clones are counted.
, 25 Results are shown in Table 2.

... ....

~ 6~34 Table 2 C~anges of _m n flora of a ~he_e_~reated w _h strain Hh-GYOKI-1-123_~KmR) _ _ _ _ _ _ ~ _ _ ._ _ _ _ _ Sample Cell count/ml Withou-t antibiotics In the presence oi' 1000 /ug/ml of kanamycin B
__ _ __ ___ .___ Before 6 10 administration 5xlO O
A~ter administration day 1 5.9x106~ 2.0x104 day 2 302x107 4.1x104 day 3 2.4~10 3.1x104 day 6 3.9x107 1.8x104 day 8 . 9.8x106 1.05x105 day 15 8.1x105 301x104 _ _ _ _ _ _ _ _ _ __ . _ _ It is seen ~rom Table 2 that the micro-organism administered to -the animal persists and grows in the rumen.
According to the abo~e-mentioned process we also cu}-tivated the strain Hh-GYOKI-1-123Sz resistant to 10,000 /ug/ml kanamycin B, and orally administered it to the same sheep on the 15th day.
140 ml cultures containing 2X108 bac-teria per ml were administered orally.

~2~

Bac-teria of rumen samples were cul tiva ted and counted as before. Resul-ts are shown in Table 3.

Table 3 .
5 _hanges in the rum r flor_of a she._E~ated~ with strain Hh-GYOKI-1-123Sz_KmR ) Sample Cell count/ml Wi-thout antibio-tics In the presence of 8000 /ug/l~ll of kanamycin B

adminis tra t ion 1. 4x106 0 Af t er 15 administratiOn day 1 7 . 4X106 3 . 2x104 day 2 1. 7xlO 5 1. 3x104 day 5 4 . 0x106 9 .lx103 day 7 1. lx107 2 . Ox104 day 14 8 . 0x106 3 0 lx104 day 21 7, lx105 6 . 2x104 day 28 . 8 . 2X106 8 .lx104 day 35 8.7x106 1.8x104 , . __ __ __ 'The data of 'i'able 3 indicate that -the micro organisrn adrninistered i3 present ir~ significant quan-tities in the rumen and, because the fluid phase of the rurnen content is continuously emptied, it clearly replicates.
~. ~
.

` ~76&~
. ., Differences of bacterium counts between samples may be explained by variations in the consistency of rumen content from thick to fluid.

.. ..
EXample 2 Modlflca-tlon ~f rumen ~ora in~a shéeF fed on cereal meal , ~ A) IsGlation of microo~ganisms capable of . . . . . . .. . . . . . . . . . . . .
g~owing on ce~eal meal The process described under item A) of Example 1 is repeated with the difference that the initial sam~le : is taken from the rumen of a sheep fed on cereal meal, the individual isolates are inoculated into RGCF liquid media (see above) containing 2~ of cereal meal puIverized in a mortar, instead of extracted hay, cuI-tures grown iD RGCF
liquid media are spread on RGCA solid media (see above) and clones developed from cells capable of utilizing cereal meal are isolated on similar media.
In this way microorganisms capable of growing on cereal meal as a carbon source are obtained.

. . . . . . . . . . . . .
B) Genetic labelliny of ~e~ bacteria utiliz-- in~ cereal meal The process described under item B) of Example 1 is repeated with -the difference that strains obtained according to item A) of Example 2 are used for transforma-tion by plOll plasmid DNA, instead of thase ob-tained ~, according to item A)of E~ample 2.
Resistance to kanamycin B of several tran~
formed and KmR strains is shown in Table 4.

Table 4 Sensi~ am~cin B of rumen 'oacteria and of strains labelled ~eneticall~ and utilizin~

1 0 __ ~ _ __ Micrcorgani~m~ Lowest kanamycin B concentra-tion inhibiting growth, Jug/ml Rumen liquor 31 Initial strains 1.8 Genetically labelled KmR 3 trains Hh-GYOKI-2-4 250 -14Ab 250 ~37 250 ~ -81 125 By performing the above-mentioned process, microbes of rumen origin are obtained that are capable of u-tilizing cereal meal as carbon source and are eight times more resistant to kanamycin B than the rumen flora.
The strain designated a~ ~-GYOKI-2-14Ab has been deposited in the Hurgarian National Collection ~'` 3L276~

of Medical Bacteria of the Mational Institute of Hygiene~ Budape~t, under No. 00288.

C~_R ~ rgani~m~ labelled ~eneticallv into the rumen The process described undeI item C) of 3xample i3 repeated with the difference that 9 terile RGCFa media containing 1.8 % of ~tarch, in3-tead of 1.8 % of ceIluLose (Bacto), is inoculated, mi~ed into the feed ~ of the sheep and sample~ are taken daily through the fistula before administration and after a~lmini3tration.
310 ml of cul-ture con-taining 7.1x105 bacteria/ml was administered orally to the sheep.

Table 5 __ : Changes in the rumen flora of sheep treated w h strain Hh~GYOKI-2-14Ab (~nR) Sample Cell number/ml Without anti- In the pre~ence of biotic~ 250 /ug/m} o~
kanamycin B
Before administration 4.3x106 1.4~10 25 After admini~tration day 1 4.1x106 1.8~103 day 2 3.2xlO 2~1x103 day 3 8.0~103 1.1~10 ;. ..

Table 5 (contd.) _ _ _ _ ___ _ Sample Cell number/ml Without anti- In the pre~ence of biotics 250 /ug/ml of kanamycin B
__ day 6 9.12106 7.1X102 : day 8 8.0x106 3.1~103 day 15 6.8x106 3.7;{103 day 22 5.0x106 9.~103 day 29 8~0X106 l.lx104 day 36 9~1xlO 7.0~103 day 43 7~0~106 7.9x103 day 60 6.1x106 7vOx103 ~

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

Modification of the rumen flora of a sheep fed on molasses A) Isolatio~ of microorganisms capable of growing on molas5es The process described under item A) of E~ample 1 is repeated with the difference that the initial samples are taken from a sheep fed with ~olasses, -the individual isolates are inoculated onto RGCF media containing glucose instead of extracted hay, the cultures grown in liquid ~ 6~8~
- 2~ -media are spread on RGCA media and clone~ grown from cells utilizing molaqsss are inoculated on-to the same RGCA media.
AS a result, microorganisms of rumen origin utili~ing molasses are obtained.

B) Genet_c_labellin~ of bacteria utilizin~_ mola~se 9 -'~he process described under item B) of E~ample 0 1 i9 repeated with the di~ference that cells prepared according to item A)o~ Example 3 are t~an~formed by plOll pla~mid DNA, in~tead of tho3e obtained according to i-tem ~ of Example 1.
The re~i~tance to kanamycin B o~ several KmR
strains i~ shown in '~able 6.

lable 6 Resi~tance to kanam cin B of rum_n bacteria and of genetica~_led ~trains utilLzing mola~es , Microor~anism~ ~owe~t concentration o~
kanamycin B inhibiting growth Rumen liquor 31 25 Initial strains 7~5 Genetically labelled KmR ~trains Hh-GYOKI-3-2 250 ,, .~, .~
- ...

` 31.Z7~84 - 29 - .

Table 6 (co~td. ) Microorganism~ Lowest concentration of kanamycin B inhibiting growth ~34 250 -81Me 500 In thi3 way,isolates hi~hly reqi3tant to kanamycin B and utilizing mola~ses are obtained~
The strain designa-ted E~h-GYOKI-3-81Me has heen deposited in the EIungarian National Collection of Medical Bacteria of the National Institute o~ Hygiene, Budape~t under No. 00289.

C) Rein ~ ~eneticall~ labelled bacteria in~to the rumen The proce~s described under item C) of E~ample 1 i~ repeated with the difference that the ~train Hh-GYO~I-3-81Me is inoculated onto RGCFa media containing 1.8 % of gluco~e in~tead o~ 1.8 % o~ cellulose, and the culture ie mixed with the feed of a sheep fed on molasse~ 380 ml o a cul-ture contain- -ing 1.6~107 bacteria per ml were orally admini~tered.
One sample of each is taken daily before and after administration through a rumen fistula.
,, ", ., 7~Q~4!

Table 7 Chang~s in the ~rumen flora of a sheep treated w _~

~___ _ _ Sample Cell number/ml Without anti- In the presence o~
biotics 500 /ug/ml of kanamycin B
10 Be~ore admini~tration 5.0x106 0 After admini~tration day 1 l.lx106 l~9x105 day 2 1.82107 2.5x105 day 3 6.2xlo6 6~1~105 day 6 8~12106 8.72105 day 8 6.2glO6 9.1~105 day 15x 1.3~103 103X102 day 22 3.0x106 3.1x105 day 29 l.lx106 7.0x105 day 36 s.o~lo5 9.1x104 day 43 601~106 8.1~105 day 60 6.8x106 6~4x105 x) Sampling error The aats indicate that the microorgani~m admini~tered persists for a long period in the rumen of ~heep fed on mola~ses.

76~8~ , ExamE~e 4 c_~ ~ a~
as a result of treatment with a bacterial preparation Two sheep are fed on a ~omplete ration for 14 days, then rumen samples are taken -throu~h a fistula.
Two liters of rumen liquor are filtered through several : layers of gauze. The particulate residue is suspen~ed in 1liter of physiological buffer (ses below), mixed and filtered as before. The -two iltr~tes are mi~ed, left to stand for an hour, ~olid~ floating on the ~urface are discarded and the liquid phase is used for the examination.
The composition of the physiological buffer 15 is as follow~:
Na2HP04 0~316 g/l 2P4 0,152 g/l NaHC03 2.260 g/l KCl 0~375 g/l 20 NaCl oO375 g/l MgS04 0.112 g/l CaC12-H20 o5 g/l ~eS04 .7E~20 . . O oO08 g/l MnS04.H20 00004 g/l Z 4 7 2 0.004 g/l CuSO4 n 5H20 0.002 g/l CC12-6H2 OoOOl g/l ~7~;~8~

The pH of the mixture is checked and, if required, adjus-ted to pH 7.2 with an a~ueous HC1 or Na~H solution (Cheng e-t al.: J. Dairy Sci. 38, 1225 [1955]).
To the mixture obtained, the same volume of physiological buffer is added and in 1 liter of the diluted mixture 4 g of the ration is suspended. Thirty ml each of the suspension is poured into Erlenmeyer flasks of 100 ml volume. 200.doses are s-terilized and another 200 are not.
Sterile media and media containing living rumen bacteria are inoculated with bacterial strains to be examined for producing acetic, propionic and butyric acids.
Bacterial strains proven to be capable of persisting in the rumen for a long time after in vitro cultivation will be examined. In addition, bacterial strains isolated from the rumen liquor o~ a sheep fed on a comple-te ration according to items A, B and C of Example 1 are also examined.
Bacteria to be examined are cultivated on RGC+CG
: ; media ~see below) under anaerobic conditions at 37C for 48 hours.
20 Composition of RGC+CG media:
salt solution I (see item A of Example 1) 15%
salt solution II (see item A of Example 1) 15%
trace element solutionX 0.3%
yeast ex-tract (Oxoid) ~ 0.5%

'' ' ` 1 ~76~84 filtered r~en liquor 10.0 %
2C3 Q.4 %
cystein.HGl.H20 o~o5 %
sodium thiosulfate 00008 %
cellulose (~acto) o.3 %
; glucose 2.0 %

XComposition of the trace element solution:
ZnC12 40 mg Cual2.2H20 10 mg disodium tetraborate dekahydrate 10 mg ammonium molybdenate tetrahydrate 10 mg ~eCl30 2 200 mg MnC12.~H~0 10 mg deionized water ad 1000 ml Cultures are inoculated into media prepared in Erlenmeyer flasks. ~wo ml of each culture is inoculated into 50 ml of media, in two parallel flasks. Non-sterile 20 ~ultures are also inocula-ted.
The flasks are incubated under anaerobic con-ditions for 40 hours. The growth is stopped with 10 %
formic acid solution and the volatile fatty acid content of the cul,tures is e~amined.
The cultures are filtered through gauze layers and centrifuged at 4000 re~. per min. for 15 min~, then filtered again and brought into the separation column of a Carlo Erba ~I-452 gas-liquid chromatograph9 fitted with flame ionization detector, for determining the C2-C5 fatty acid~.
~emperature o~ the column: 150 C
5 Separation column: 2 m long, 4 mm wide (inner diameter) gla3s tube filled with 10 % of ethylene glycol adipate and 2 % of o-pho~phoric acid on a 3ilansted ~ilica gel carrier (0.2 to 0.3 mm particle diameter).
~emperature of the injector: 190 C.
10 N2 ~tream rate: 50 ml/min.
H2 stream rate: 50 ml/min.
Stream rate of the air: 200 ml/minn Paper movement: 160 cm/hour~
Dh~ration of chromatography: 20 min.
Sample volume: 1 ~ul~
~riplicate measurements are made ~rom each sampleO The ~tandard solution contains acetic acid, propionic acid, isobutyric acid, butyric acid, i~o-valeric acid and valeric acid.
More than 90 ~train~ o~ bacteria were isolated ~rom a sheep fed on a complete ration. ~hen the ~trains were labelled genetically and examined ~the positive strain3 were examined several times). Representative results are shown in Table a.
Explanation of the ~igns u~ed in Table 8:
S: inoculated after sterilization NS: culture containing living rumen flora was inoculated a) trace amounts;

~ ~76~a .. ~

b) negative control: volatile fatty acid content of media prepared from rumen li~uor, phy~iological buffer and feed used for the experiment (~verage o~ 12 measure~
ments~;
c) positive control: volatile fatty acid content of the : incubated culture containing the initial ~umen ~ac-teria and otherwi~e prepared by the same proce~s (average of 12 mea3urements);
d) as c) but 5 ppm monen~in Na were added to the media (average of 6 determinations);
e) as c), but 10 ppm monensin Na wa~ added to the media (average o~ 6 determinationg ) e ;

~ ~ ' . i ~276~84 ,` l l rl ?~ ' , c~ ~rl r~ o 0 r ~ ~ c ~ o oo ~o t I ~ Ll~ a~ GO
c~ rl o o o r i t ~ t~\ I c~J r~ ~i ~ I o o o o r-l ~ l I
3 l I
rl r~ I I 1 0 1 1 ~ 0 o 0 0 1 1 0 ~0~ l l rl l l 3 ~ ~ 0 ~ 0 o ~ 0 1 0 o ~ o ~ o 1 0 ~:S l l rl r-i aS 0 ~ o o o o I o o o o I o o o o F~ ~ I i ~1 ¦ o l l l ~rl, I .1 rl rl e 0 0 ~ 0 P I r~ l O r~~ O O I O O r~ O ~ J
r ~ e ~ g a~ ~ ~ ~ ~ I ~ w a~ ~ ~ l O ~i'. ~i ~ I ~i ~i cq ~e u~ æ Ul æl u~ æ I h I u~ æ ~ æ

~ I i i 0 0 . . I 1~ ~

~rl ~ ~ ~O q~ ~ O O O
~a >~
, ~ ~ h V r~ Vr~ ~1 CO I I I I 1 1 0 ~ ~ r~

37 ~Z76~
. ~
~.~

. d~ u~ o N ~ ,~
0 ~ o a~ ~ ~ c a" Ir~ N 11~ ~I CS~
~) rl u~ C~ ~ C\l C\J C~i ~--; N l-~ CO O O
o C~ ~
o ~ o, ~0~

h o bD 0 ~ 0 ~ I I I I 1 1 0 'o r-i ~ O N U~
~ 0 ~ O' V O O' O O O O O O O ~t I --~ :~ ~ ~ 0 0 0 0 0 0 aS 0 0 0 0 0 ~ 0 bD
~ .
, C~' ~ ~ ~ ~ O ~ o t~ D O ~
h 0 ~ O O O OO O O OO O O ~i rl ~ ~~ O ~I r-5 ~ C~~ O ~ cJ~ Ll~ C~
3 ~ C~J N C~J C~l ~i ~i r i C~J C~l C~l u~ æ u, æ u~ æ u, æ u~ æ u, æ
,n ~ 0 ~ " ~

¦ ~ h ¦ O V ~ C~ C1 V
m U~

~7 EiQ8~
,.

rrhe data of Table 8 indicate that the ratio~
of volatile fatty acid~ produced by the fermentative function of the rumen flora can be modified over a wide range by the administration of microbial culture~ pre-pared accordiLg to the inventionO Eogo production ofpropionic acid can be significantly stimulated with a culture prepared from strain Hh-GYOKI-48a, while ~train Hh-GYOKI-109b ~timulate~ production of acetic acid.
Stimulation of production of individual fatty acids was ob~erved both on media lacking (S) or containing tNS) living rumen microbe~. In our experimental sy~-tem monen-~in Na de¢rea~ed the ratio of acetic acid to propionic acid by 0.1 or 0~2 (d; e)O
Microorgani~ms cho~en by the above-mentioned proce~s are labelled genetically~ admini~tered to ruminant~ and e~amlned for ruminal growth and persistence by repeating the proce~s de~cribed in item B)of Example 1. Strain~ with an advantageou~ fermentative pattern and long ~ minal persistence will be orally adminis-tered for modifying the production of volatile fattyacid~O

. .
Bacterial preparation for_oral admini~tra-tion to ruminants Bacteria to be admini~tered are cultured on RGCA+CG medi~ (E~ample 4) under anaerobic condi-tions, by the described process. After cultivation, the cells are ~eparated by filtration or centrifugation.
~,~

Separated cells are suspended in physiological buffer ~Example 4) and freeze dried. The lyophilized ~acterial preparation i~ stored1 ~uitably formulated and adminis-tered to ruminant 9 orally~
Microorganisms may be cultivated ln other conventionally u~ed media a~ well, e.g. in media con-taining ~lucose and ~taxch etc. a~ carbon source and inorganic salts as N-qource.
The preparation can be easily administered by mixing it with feed or drinking water, alone or together wlth other biologically active agents, e.g. with anti-; biotics and vltaminsO
I~ addition to the freeze-dried preparation o-ther products can also be prepared. The micro-organi~m~ may al~o be administered after mixing the filtered or centrifuged bacterial mass with suitable carrier or diluting ~ubstances, e.g CaC03, concentrates, ; premixe~ or other Peedstuffs.
~he bacterial strain(~) are chosen from the '~ 20 mlcroorgani~ms, prepared by the process of the i~ven-tion ~nd advantageou~ly modifying the rumen flora, and the quAntity to be fed i~ determined depending on the ra-tion and -the intended use of the animal. If a decrease of the acetic ~cid to propionic acid r~tio i~ required, we may u~e e.g. a culture prepared from strain Hh-GYOKI--48a, but for an increase of the ratio the administra-tion oP str~i~ Hh-GYOKI-3-81Me is recommended.
Determination of the required microbial cell . . .
~ 1 ` ' " ' ` ' - ` ~276(~

number should not involve any difficulty for those skilled in the ar-t. It is recommended that the cells be administered in a quan-tity resulting in 5X102 to 5x107 cultivated mirco-organisms per ml of rumen liquor.

EXample 6 Administràtion of st~ain5 Hh-GYOKI-48a and .................
Hh-GYOKI-1-123Sz to sheep Hh-~YOKI-48a strain is cultivated on RGCA~CG
media tExample 4) in two 5-liter fermentors (useful vo:Lume) at 37C, under anaerobic conditions. ~ermentation is commenced by inoculation with a 10 ml culture of similar composition. After 4~ hours of cultivation the cells are ; separated by centrifugation (5000 r.p.m.) and the wet sediment weighing 58 g is mixed carefully with 4 kg of corn meal.
The mixture i.s divided into eight equal parts and orally administered to eight sheep previously starved for 24 hours.
Strain Hh-GYOKI-1-123Sz may be used similarly, with a bacterial harvest of 53 g.
In a growing-fattening experiment 23 sheep were ad libitum fed on poor grass hay and the animals were weighed every week for 5 weeks. The experimental groups consisting of eight sheep were fed with one of the bacterial preparations each for a single feeding and seven sheep served 25 as control. 600 to 900 g of hay were consumed per day per animal, plus mineral and vitamin premix mixed with corn meal (100 g). The results are shown in Table 9.

.

~7~
`~ `

Table 9 W~. ~

5 Serial Initial l~t 2nd 3rd 4th 5th n~
C ontrol 1 ~Y.~ 29.5 30.0 29.5 29.5 29.5 2 27.0 27.5 2~.5 27.0 2~.5 28.0 3 28.5 27.0 27.0 27.5 26.5 26.5 10 4 3000 30.5 30.0 30.5 30.0 29.0 29~5 30.0 29.0 30.5 29.5 30.0 6 25.0 25.5 26.5 26.0 26.0 27.5 7 27.0 27.0 27.5 28.0 28.0 28.0 ~reated wL-th ~trai Hh-GYOKI-1-123Sz ~ 15 8 29.5 32.0 32.5 3300 33.5 34.0 ,~ 9 25.5 26.5 27.0 2805 29.5 29.0 25.0 25.5 25.0 28cO 2900 28.5 11 25.5 24.5 25.5 27.5 27.0 28.0 12 29.0 28.0 29.0 28.5 29.0 30.0 2013 29.5 30.0 30.5 29.5 2905 30.5 14 29.5 28.5 29.5 30.0 30~5 31.0 28.5 29.0 30.5 30.0 30.5 32.0 ~reated with ~train Hh-GYOKI-48a 16 29.5 30.0 30.5 3100 32.0 33.5 2517 26.5 25.0 26.5 27.0 29.0 30.0 18 27.0 27.5 28.5 29.5 30.5 31.0 19 26.0 26.0 27.0 28.0 29.0 30.5 29.5 30.5 31.0 31.5 32.5 33.0 : - ., ,. ~ . , , ", Table 9 (contd.) -- .
Serial Initial l~t 2nd 3rd 4th 5th 5 number weight w e e k _ _ _ 21 28.0 28.0 28.0 29.0 29.0 3000 22 29.0 30.5 33.2 30.0 32~8 33.5 ~3 27.0 26.0 27.5 29.0 31.0 32.0 - _ _ The average daily weight g~ins are calculated from the data o~ Table 9 and are ~ho~Nn in Table 10.

;~ Table 10 Avera~e_wei ht and dail~ wei~ht ~ain of experL-mental a d control sheep ~ . .

Initial 1st 2nd 3rd 4th 5th weight w e e k _ _ _ ~
Control Mean body weight (kg) 28.00 28.14 28.36 28.43 28.00 28.36 Mean daily weight gain (g) -~20 +31 +10 -61 +51 Treated with ~train Hh-GYOKI-1~123Sz Mean body weight (kg) 27.75 28.00 28.69 29.31 29.81 30.37 Mean daily weight gain (g) +31 +86 +77 +62 +70 Treated with ~train Hh-GYOKI-48a Mean body weight (kg) 27.81 27.94 29.02 29.37 30.73 31.69 Mean d~ily weight gain (g) +16 +135 +44 +170 +120 -- . _ .,,, ,~
.. .

2~

Sheep treated with strain3 E~-GYOKI-1-123Sz and Hh-GYOKI-48a and the control group gained on the poor ration on average 2620, 3875 and 360 g, resp0ctively, during the 35-day e~perimental periodO
Initial body weight~ did not differ signifi~
cantly between group~, but significant di~ferences ; were found in the final body weights (Table 11) and in the daily gains (Table 12).

Table 11 Stati~tical evaluation of final bod Nei~hts Control E~-GYOKI-1-123Sz E~h-GYOKI-48a Mean (kg) 28.357 30~375 31.6875 Corrected quadrate o~
~tandard deviation 1.476 3.910 2.281 P (%) ~ 5~0 ~ O 1 Table 12 Stati~-tical evaluation o~ bod~ weig~t gain~
~ ks~

Control E~-G~OKI-1-123Sz Hh-GYOKI-48a .
- Number of animal~ 7 8 8 Total weight gain of the group (kg) 2.5 21.0 31.0 _ {..~

f``~ ~7~

~able 12 (contd.) Control Hh-GYOKI-1-123Sz ~-GYOKI-4 Maximum gain (kg) 2.5 4.5 5.0 Nlinim~un gain (kg) -2.0 1.0 2.0 Mean gain per ~heep (kg) 0.3571 2.6250 3.8750 Corrected quadrate of standard de~iation 2.143 1.768 0.839 Standard deviation+1,355 +1.244 +0.857 The data indicate that preparations made according to the invention may markedly stimulate weight 15 gain in sheep.

- Example_~
Persi~tance oî n~l~r labelled bacteria lhe proces~ described under item C) of Example 1 is repeated with the di~ference that the strain Hh--GYOKI-1-123Sz re~istant to 10,000 /ugtml kanamycin i9 cultivated in 4 liter o~ RGC~a medi~s. After reaching the stationary phase (38th hour) the culture i~ harvested by centrifugation (5000 rOp.m.) and the cell~ thoroughly mi~ed with 500 g of corn meal are fed to a cow. Weekly sample~ are taken through a Iistula, and ruminal per-sistence of the strain administered is determined ~ ~27~
, according to item C) o~ E~ample l.
Re3ult~ indicate that 3train Hh GYOKI-1-123Sz growq in the bovine rumen and it can per~i~t there ~or at lea3t 40 days.

Claims (16)

1. A composition for improving the efficiency of ruminant feed utilization, which comprises as active ingredient one or more microbial cultures capable of adjusting the weight ratio of acetic acid to propionic acid in the rumen to an optimum value of from 1.5 to 4.0:
1, and of growing in the rumen and persisting there for at least 60 days.

2. A composition as claimed in claim 1, and including one or more carriers, diluents, or preserving agents conventionally used in animal husbandry and nutritive and/or other substances conventionally administered to ruminants.

3. A composition as claimed in claim 1 or 2, which comprises at most 95% by weight of active ingredient.

4. A composition as claimed in claim 1 or claim 2, which contains a microbial culture capable of adjusting the acetic acid to propionic acid ratio in the rumen to a value of from 2.0-3.5 : 1.

5. A composition as claimed in claim 1 or 2, which contains a microbial culture selected from the micro-organism strains deposited in the Hungarian National Collection of Medical Bacteria of the National Institute of Hygiene under Nos. 00287, 00288 and 00289.

6. A composition according to claim 1 or 2, in the form of a microorganism paste, lyophilizate or suspen-sion.

7. A process for the preparation of a microbial culture used as active ingredient in a composition according to claim 1, which comprises taking samples from the rumen of animals fed on a given feedstuff or ration, examining in vitro and/or in vivo, the metabolism of microorganisms isolated from the sample and cultivating microorganisms with advantageous metabolism characteristics in media containing the same feedstuff or ration as carbon-or nitrogen-source, introducing into the growing microorganisms a genetic marker, which makes selection possible, cultivating the genetically labelled strains, reintroducing the cultures into the rumen of the animals fed on the same feedstuff or ration, taking samples from the rumen, counting the cell number of the genetically labelled strain, and separating strains persisting for at least 60 days and able to adjust the acetic acid to propionic acid ratio to an optimum value of 1.5-4.0 : 1.

8. A process as claimed in claim 7, wherein the selected strains are thereafter formulated into a form acceptable for the practice of animal husbandry and nutrition.

9. A process as claimed in claim 7 or 8 in which antibiotic resistance is used as genetic marker.

10. A process as claimed in claim 7, in which microorganisms are isolated from the rumen of animals which have been fed a cellulose-containing feedstuff, a starch-containing feedstuff, or a mono- and/or disaccharide-con-taining feedstuff, and the genetically labelled strains are reintroduced into the rumen of animals fed on one of the above feedstuffs for testing, and the suitable microbial cultures are cultivated and then isolated.

11. A process as claimed in claim 10, wherein the cultures obtained are thereafter formulated into a suitable form for the practice of animal husbandry and nutrition.

12. A process is claimed in claim 10 or 11, wherein a cellulose-containing feedstuff is hay.

13. A process as claimed in claim 10 or 11, wherein a starch-containing feedstuff is fodder.

14. A process as claimed in claim 10 or 11, wherein a mono-and/or disaccharide-containing feedstuff is molasses.

15. A process as claimed in claim 7, 8 or 10, wherein the microorganism cells are separated in a lyophilized form.

16. A process for the preparation of a compo-sition for improving the efficiency of ruminant feed utilization, which comprises formulating a microbial culture prepared according to claim 7, 8 or 10 alone or in admixture with one or more carriers, diluents, and/or preserving agents conventionally used in animal husbandry and one or more nutritive and/or other substances conventionally adminis-tered to ruminants as an oral formulation.