GB2163650A - Compositions comprising microbial cultures - Google Patents

Abstract

The invention relates to a composition for improving the efficiency of ruminant feed utilization, which comprises one or more microbial cultures, which are capable of adjusting the weight ratio of acetic acid to propionic acid produced during fermentation of energy-producing nutrients in the rumen of an animal to an optimum value, preferably to 1.5-4.0:1, and of growing in the rumen and persisting there at least for 60 days. The cultures may optionally be in admixture with carriers, diluents, and preserving agents conventionally used in animal husbandry and nutritive and/or other substances conventionally administered to ruminants. According to another aspect of the invention there is provided a process for the preparation of microbial cultures used as active ingredient in the above composition. The invention further relates to a process for the preparation and use of said compositions.

Description

SPECIFICATION Composition and method for improving the efficiency of ruminant feed utilization The invention relates to a composition for improving the efficiency of ruminant feed utilization.

The invention further relates to the preparation of microbial cultures used as active ingredient in the above compositions and to the application of said compositions.

Ruminants possessing a composite stomach, such as sheep (Ovis aries aries), cattle (Bos primigenius taurus), goat (Capra hircus), and their wild relatives (deer and muflon, etc.) have an important role in the nutrient chain and in the economy. Their special importance is that they live on feedstuffs which cannot be utilized by other herbivores. The forestomachs provide an anaerobic environment for the rumen flora, which enables digestion of cellulose and utilization of nonprotein nitrogen in addition to the common nutrients. The composition of rumen flora largely depends on the fed ration and adapts to the diet. This adaption, however, takes several days and the final stabilization may require several weeks. Abrupt change of the ration adversely affects intake, digestibility and production, and it may cause illness or even death.

It is well known that in 1 ml of rumen liquor millions of bacteria live and grow. Anaerobic fermentation by bacteria is of high importance for normal digestion and feed intake. Energyproducing nutrients are fermented to acetic, propionic and butyric acids, which are used by the host animal as fatty acids; the bacterial mass passing to the intestines will be digested and used as 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. The rumen flora therefore has an important role in the maintenance of the ruminant farm animals and in what they produce.

After birth the rumen flora of a ruminant animal develops spontaneously and attains its adult composition after weaning to solid food. It is not certain, however, that this accidental flora represents the optimum fermentative system. It would be advantageous to have a process for the modulation of the composition and/or of the number of the rumen flora according to economic interests.

The increase of volatile fatty acid production in the rumen and, accordingly, meat or milk production is a long felt want in animal husbandry. Certain results have been obtained with monensin [2-[5-ethyitetrahydro-5-ltetrahydro-3-methyl-5-[tetrahydro-6-hydroxy-6-(hydroxymethyl)- 3 ,5-dimethyl-2H-pyran-2-yl]-24uryl-24uryl]-9-hydroxy-fl-methoxy-a,y,2,8-tetramethyl 1 ,6-dioxaspiro[4.5]decane-7-butyric acid] originally used as coccidiostat (see e.g. U.S. patent specification No. 4,085,255). Experimental use of other related polyethers, such as salinomycin, lasalocid, phthalide derivatives (US. patent 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 however, varies greatly in 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, Abstr. Am.

Soc. Anim. Sci., 410 /1978/). There is no method known in the art by which the microbial culture present in the rumen of ruminants can be directly influenced to provide significant results.

We have now found that genetic recombination methods can be successfully used for the separation of microorganisms. Bacterial strains can be labelled by genetic markers, e.g. by an antibiotic resistance factor that allows identification of the bacterium among other bacteria.

We have isolated rumen bacteria, genetically labelled the strains and after culturing have reintroduced them into the rumen. We have taken samples of rumen content periodically, cultured them in selective media, and have found that some of the strains, that had fermentative characteristic advantageous for the host animal and that were able to grow in vitro, can grow in the rumen and persist there if the same feed-stuff was fed as during isolation. Such strains stimulated digestion and thereby utilization of feed by the host animal.

According to one aspect of the invention, we provide a composition which comprises a microbial culture which is capable of adjusting the weight ratio of acetic acid to propionic acid produced during fermentation of energy-producing nutrients in the rumen of an animal to an optimum value of 1.5-4.0 : 1, and of growing in the rumen and persisting there at least 60 days. Such composition may also contain carriers, diluents, preserving agents conventionally used in animal husbandry and nutrition and/or other substances conventionally administered to ruminants.

If such a composition is desired to be used to increase meat production, the active ingredient preferably is a microbial culture capable of adjusting the ratio of acetic acid to propionic acid to 2.0-3.5 : 1, e.g. 2:1. For milk production, however, the optimum acetic acid to propionic acid radio is about 3.0:1, for subsistence or gestation about 4.0:1, and for heifer breeding about 2.0-3.0:1. It is, therefore, preferred to use compositions capable of adjusting the acetic acid to propionic acid ratio to the optimum value for these purposes. In the literature there is some uncertainty as to the most desirable acetic acid to propionic acid ratios for particular functions, the preferred ratio being a function of the ruminant, the feedstuff employed and other factors and its selection is the task of those skilled in the art (see e.g.Kaufmann, W. and Rohr, K., Der Einfluss des Futters auf die bakterielle Fermentation in Vormägen, Handbuch der Tiernährung, 263, 1969, Parey, Hamburg, Berlin).

The invention further relates to a process for the preparation of microbial cultures able to be used as active ingredient in the above compositions, which comprises, taking a sample from the rumen of an animal fed on a given feedstuff or ration, identifying microbes with desired metabolic characteristics and cultivating such microbes in media containing the same feedstuff or ration as a carbon- or nitrogen-source, introducing a genetic marker, to facilitate such identification into the growing microbes; cultivating genetically-labelled strains; reintroducing the culture into the rumen of an animal fed on the same feedstuff or ration;; samples are taken from the rumen, the cell number of the genetically labelled strain is counted, and separating any strains which persist for at least 60 days and which adjust the ratio of acetic acid to propionic acid formed during fermentation of energy-producing nutrients in the rumen of said animal to an optimum value preferably to 1.5-4.0 : 1.

These strains may be formulated in a form acceptable for the practice of animal husbandry and nutrition.

According to a preferred embodiment 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 solid media containing N- and C-sources, inorganic salts, rumen liquor and agar (Bryant and Burkey, J. Diary Sci., 36, 305, 1953). The cultures are incubated in anaerobic conditions, then the clones are isolated and cultured in similar media as above.

The cultures grown 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 intensive 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, any inheritable genetic marker can be used that allows the identification of the labelled microbe among other microbes.

According to a further preferred embodiment of the invention, antibiotic resistance 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 them, the growth and death of the latter microbes may easily be followed 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. 146, 564 /1981/) we introduce p101 1 plasmid (Simon et al., Proc. 8th North American Rhizobium Conference, Winnipeg, Canada, Univ.

of Manitoba Press, 1983) carrying kanamycin and chloramphenicol resistance genes into the selected cultures which grow on cellulose, cereal meal or molasses. Piasmid DNA was isolated from E. coli cells (Birnboim and Doly, Nucl. Acid. Res. 7, 1513 /1979/). The strain was deposited in the Hungarian National Collection of Medical Bacteria of the National Institute of Hygiene, Budapest, under No. 00264 on 19 April 1983.

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

With 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 in 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 to the antibiotic are counted. Ruminal production of volatile fatty acids is also determined qualitatively 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., Digestive Physiology and nutrition of Ruminants, Vol. 2, pp. 622-625, 1971/. As mentioned above, the optimum ratios of acetic acid to propionic acid are considered to be 2.0-3.5:1, 3:1, and 4:1, respectively, for growth, milk production, and maintenance as well as pregnancy, resp.

(Kaufmann, W. and Rohr, K.: Der Einfluss des Futters auf die bakterielle Fermentation in vormä- gen. In: Handbuch der Tierernährung, p. 263, Parey, Hamburg-Berlin, 1969).

According to a preferred embodiment of the process of the invention, bacteria are isolated from rumen samples and the capacity of the isolates to produce volatile fatty acids is examined.

The microorganisms are cultured in anaerobic conditions in the described complete media con taining rumen liquor. Then, the concentrations of acetic acid, propionic acid and butyric acid in the cultures are determined. Microbial cells producing volatile fatty acids in required ratios labelled genetically and their ruminal growth is examined.

Strains that are able to grow in the rumen of the animal fed on the described feedstuff at least for 60 days and that can ferment dietary carbohydrates to volatile fatty acids in optimum ratios, are selected, grown, isolated, maintained and stored. If desired, their cultures, in a form acceptable for animal husbandry, may be orally administered to ruminants, for developing or modifying 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 persist in the rumen for long time and affect digestion advantageously, have been deposited in the Hungarian National Collection of Medical Bacteria of the National Institute of Hygiene on 26 June 1984 under Nos. 00287, 00288 and 00289, respectively.

In a further embodiment, it is preferred to culture the microorganisms of rumen origin between 7 32"C and 37"C, 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 salts, yeast extract, casein and similar additives are suitable N-sources.

An essentiai feature of the invention is that unicellular organisms advantageously fermenting the fed feedstuff or ration and capable of persisting in the rumen for a long period are used for the modification of ruminal flora. For the selection of such strains genetic markers are used, as described above, e.g. genes coding antibiotic resistance, enzyme proteins or other detectable proteins. Auxotroph cells may also be used.

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

The selected strains, which have advantageous fermentative characteristics and persist in the rumen for a long period, may be used either to enhance the development of rumen flora in suckling ruminants or to modify advantageously the composition of the established rumen flora.

It is recommended to administer the preparation with the feed or drinking water.

The microorganism selected for making the preparation according to the invention is cultured in media containing organic carbon source, organic or inorganic nitrogen 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 formulated by mixing it with solic or liquid carriers or other additives. The preparation can be mixed to the feed or drinking water, or can be fed alone.

In the case of sheep, for example, 1 to 20 g, preferably 5 g, of microorganism culture according to the invention may be added to about 0.5 kg of feed. As a feedstuff, for example, a mixture of corn-meal, lucerne hay and beef cattle feed can be used. 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 pro 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 within the scope of the invention.

After culturing in a liquid medium, as mentioned hereinbefore, the microorganisms may be separated by centrifugation or filtration. Pastes, freeze dried preparations or suspensions containing spores or vegetative forms may be prepared and additives acceptable for animal husbandry 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 feed 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, or salynomycin) or enhance the persistency of the microorganisms fed.

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

During the suckling period, the rumen flora is unable effectively to ferment the common feedstuffs. The flora develops spontaneously and accidentally, and it is by no means certain that its composition is optimal for the host animal.

By feeding the selected microbial strains, instead of a slow and spontaneous 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 microorganisms obtained according to the invention (e.g. with the strains 00287, 00288 and 00289) the rapid adaptation or development of rumen flora during feed change or weaning can be promoted by enhancing ruminal growth of microorganisms capable of optimal degradation of the feed.

The process can be used, among others, in the following cases: -for dairy cows during changes of lactation, at the end of pregnancy and during seasonal and other changes of their rations; -for beef cattle at 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 changes of the growingfattening diet; -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 fattening.

The possibilities are similar in goat husbandry. Microbial cultures prepared by the process of the invention may be used also in several special cases, e.g. for wild-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 preferably are 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 certain members of the genus Angerovibrio (lipolytica), Bacteroides, Selenomonas (ruminanticum) and Propionibacteria.

The invention will further be illustrated by the aid of the following, non-limiting Examples. The preparation of microbial strains 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 for sheep, but the scope of protection also extends to microorganisms capable of growing on other feedstuffs and to the development or modification of the rumen flora of other ruminant species.

Example 1 Modification of the rumen flora of animals fed on hay (AJ Isolation of microorganisms able to grow 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: K2HPO4 0.6 g distilled water ad 100.0 g Salt solution II:: NaCI 1.2 g (NH4)2S04 1.2 g KH2PO4 0.6 g CaC12 0.12 g MgSO4.7H20 0.25 g distilled water ad 100.0 ml Resazurin (0. 1 % solution) 0.1 ml Agar (Bacto)x 2.5 g Rumen liquor 10.0 ml Glucose 0.05 g Cellobiose 0.05 g Cystein.HCI monohydrate 0.05 g Sodium carbonate (8% solution) 5.0 g Distilled water ad 50.0 ml (x) Difco Labs, Detroit, USA (xx) The sample of rumen content is filtered through several layers of gaze, then the filtrate is stored under carbon dioxide at -200C.

Before sterilization under CO2 gas, the pH of the media RGCA is adjusted to 6.8. Sterilization, preparation of the media and cultivation are performed according to Bryant and Burkey (J. Dairy Sci., 36, 205 /1953/).

The rumen liquor is diluted with a sterile mixture of the following composition: salt solution I (see above) 7.5 ml salt solution II (see above) 7.5 ml cystein.HCI monohydrate 0.05 g Na2C03 0.3 g resazurin (0.1% solution) 0.1 ml distilled water ad 100.0 ml The sign of this mixture is HB.

The cultures are incubated in anaerobic conditions at 35"C (see Atlas of Rumen Microbiology, Ogimoto and Imai, Japan Scientific Societies Press, Tokyo, 1981) for 120 hours, then the individual clones are inoculated into media, containing extracted hay, of the following compo sition: salt solution I (see above) 15.0% salt solution II (see above) 15.0% resazurin (0.1% solution) 0.1% Tripton L42 (Oxoid)x 15.% yeast extract (Oxoid)" 0.5% rumen liquor"" 10.0% Na2CO3 0.4% crystein.HCI monohydrate. 0.05% extracted hay""" 10.0% Sign of the media: RGCF liquid media (x) Oxoid Ltd., London, UK.

(xx) See above (xxx) For preparing 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 of the media is adjusted to 6.5.

Test tubes containing 5 ml of sterile medium are inoculated with the microbial suspension obtained from individual clones grown on RGCA solid media and incubated under anaerobic conditions at 35"C. The growth is checked by microscopic examination and the cultures are spread on RGCA solid media where 2.0% Bacto cellulose are substituted for the glucose and the cellobiose.

The cultures are incubated in anaerobic conditions at 35"C for 120 h, then individual clones made up of cells utilizing cellulose are inoculated onto RGCA media containing cellulose.

This way ruminal microorganisms able to grow on hay or cellulose can be obtained.

(B) Genetic labelling of rumen bacteria able to grow on hay Genetic labelling is performed with the E. coli plasmid p1011, according to Simon et al. (Proc.

8th North 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 aqueous solution containing the following components: 75 mM CaCI2 5 mM MgCl2 10 mM tris.HCI buffer', pH 7.5 (x) tris-(Hydroxymethyl)-aminomethane hydrochloride Microbes able to grow on hay and isolated according to item (A) of Example 1 are cultured on RGCF media and separated by centrifugation under CO2. The cells are suspended in an aqueous solution containing in 1 litre the following components: 75 mM CaCI2 5 mM MgCl2 10 mM tris.HCl buffer, pH 7.5 1 mM cystein.HCI nonohydrate 1 mM sodium thiosulfate wherein the suspensions should contain 5X109 cells per ml.The suspension is diluted with the same volume of solution containing plasmid DNA (0.1 ,ug/ml) and is incubated for 60 min. at 4"C. Then the incubation is continued at 41"C for 2 min., then the culture is spread on solid media containing 500,ug/ml of kanamycin B, and cellulose as a carbon source. The culture is incubated at 35"C for 120 h under anaerobic conditions and clones able to grow in the presence of 500 Hg/ml of kanamycin B are examined.

Plasmid p101 1 carries genes determining resistance to kanamycin and chloramphenicol; furthermore, it contains a replication origin enabling replication in E. coli cells. If transformed into other bacteria, owing to its lack of suitable origin allowing replication, the plasmid DNA is either eliminated or incorporated into the chromosome (partly or completely) and genetic recombination takes place. Eventually the 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 3X10-5.

Several resistant clones were isolated and we determined the sensitivity to antibiotic of the initial and kanamycin B resistant (KmR) strains. Results obtained with several strains are shown in Table 1.

Sensitivity to kanamycin B of rumen bacteria and of strains labelled genetically and degrading hay Microbe Least effective concentration of kanamycin B, /ug/ml Rumen liquor 31 Initial strains 4.0 to 7.5 Genetically labelled KmR strains Hh-GYOKI-1-8 500 -27 250 -91 500 -123 1000 -142 500 So we can obtain microorganisms of rumen origin that are able to utilize hay or cellulose and to grow in the presence of high amounts of kanamycin B.

The strain Hh-GYOKI-1-123 (KmR) is spread on RGCA media containing cellulose, whereafter kanamycin B in concentrations of 1000, 5000 and 10,000 ,ug/ml are added. The cultures are incubated in anaerobic conditions at 35"C for 168 h and the strains growing in presence of 10,000 iig/ml antibiotic are isolated. So, with spontaneous selection, we obtain spontaneous mutants highly resistant to kanamycin B. One of these strains has been designated Hh-GYOK I-1-123Sz and deposited in the Hungarian National Collection of Medical Bacteria of the National Institute of Hygiene, Budapest, under No. 00287.

(C) Reintroduction of labelled microbes into the rumen Sterile, solid media named RGCFa are inoculated with the culture of strain Hh-GYOKI-1-123 (KmR) stored on RGCA slants at +4 C. The composition of RGCFa media is as follows: 1. K2HPO4 0.3% 45 ml solution 2. (NH4)2S04 0.6% NaCI 0.6% MgSO4.2H2O 0.06% CaCl2.2H2O 0.06% KH2P04 0.3% 45 ml solution of the mixture 3. Cellulose (Bacto)x 1.8% Agar (Bacto)" 3.0% 65 ml solution of the mixture 4. Yeast extract 0.1% 20 ml solution 5. Cystein.HCl.H2O 0.1% 20 ml solution 6. Sodium thiosulfate 0.1% Na2C03 0.2% 10 ml solution of the mixture 7. Rumen liquor" 20 ml (x) Difco Labs, Detroit, USA (xx) See above The 7 solutions are prepared separately and mixed in the given sequence.

Cultivation is performed in 500 ml Erlenmeyer flasks containing 150 ml of media, under anaerobic conditions. Growth is checked after 48 hours, then the culture is mixed to the feed of a hay-fed sheep.

340 ml of culture containing 4.7X 106 bacteria per ml were orally administered to the sheep. Before administration and on the 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. The cultures are incubated in anaerobic conditions at 35"C for 72 hours and the bacterial clones are counted. Results are shown in Table 2.

Table 2 Changes of rumen flora of a sheep treated with strain Hh-GYOKI-1-123 (KmR) Sample Cell count/ml Without antibiotics In the presence of 1000 /ug/ml of kanamycin B Before 6 administration 5xlO 0 After administration day 1 5.9x106 2.0x104 day 2 3.2x107 4.1x104 day 3 2.4x106 3.1x106 day 6 3.9x1n7 l.8x164 day 8 2.8x106 1.05x105 day 15 8.1x105 3.1x104 It is seen from Table 2 that the microorganism administated to the animal persists and grows in the rumen.

According to the above-mantioned process, we also cultivated the atrain Hh-GYOKI-1-123Sz resistant to 10,000 g/ml kanamycin B, and orally administered it to the same sheep on the 15th day.

140 ml culture containing 2X108 bacteria per ml were administered orally.

Bacteri of rumen samples were cultivated and counted as before. Results are shown in Table 3.

Table 3 Changes in the rumen flora of a sheep treated with strain Hh-GYOKI-1-123Sz (KmR) Sample Cell count/ml Without antibiotics In the presence of 8000 /ug/ml of kanamycin B Before 6 administration 1.4x106 0 After administration day 1 7.4x106 3.2x104 day 2 1.7x105 1.3x104 day 5 4.0x106 9.1x103 day 7 1.1x107 2.0x104 day.14 8.0x106 301x104 day 21 7.1x105 6.2x104 day 28 8.2x106 8.1x104 day 35 8.7x106 1.8to04 The data of Table 3 indicate that the microorganism administered is present in significant quantities in the rumen and, because the fluid phase of the rumen content is continuously emptied, it surly replicates.Differences of bacterium counts between samples may be explained by variations of the consistency of rumen content from thick to fluid.

Example 2 Modification of rumen flora in a sheep fed on cereal meal (A) Isolation of microorganisms able to grow on cereal meal The process described under item (A) of Example 1 is repeated with the difference that the initial sample 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 powderized in a mortar, instead of extracted hay, cultures grown in RGCF liquid media are spread on RGCA solid media (see above) and clones developed from cells able to utilize cereal meal are isolated on similar media.

In this way microorganisms able to grow on cereal meal as a carbon source are obtained.

(B) Genetic labelling of rumen bacteria utilizing 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 transformation by p1011 plasmid DNA, instead of those obtained according to item (A) of Example 2.

Resistance to kanamycin B of several transformed and KmR strains is shown in Table 4.

Table 4 Sensitivity to kanamycin B of rumen bacteria and of strains labelled genetically and utilizing cereal meal Microorganisms Lowest kanamycin B concentra tion inhibiting growth, /ug/ml Rumen liquor 31 Initial strains 1.8 Genetically labelled KmR strains FIli-GYOKI -2-4 250 -14Ab 250 -37 250 -81 125 By performing the above-mentioned process microbes of rumen origin are obtained that are able to utilize cereal meal as carbon source and are eight times more resistant to kanamycin B than the rumen flora.

The strain designated as Hh-GYOKI-2-14Ab has been deposited in the Hungarian National Collection on Medical Bacteria of the National Institute of Hygiene, Budapest, under No. 00288.

(C) Reintroduction of microorganisms labelled genetically into the rumen The process described under item (C) of Example 1 is repeated with the difference that sterile RGCFa media containing 1.8% of starch, instead of 1.8% of cellulose (Bacto), is inoculated, mixed into the feed of the sheep and samples are taken daily through the fistula before administration and after administration. 310 ml culture containing 7.1 X 105 bacteria/miwere ad.

ministered orally to the sheep.

Table 5 Changes in the rumen flora of the a sheep treated with strain Hh-GYOKI-2-14Ab (KmR) Sample Cell number/ml Without anti- In the presence of biotics 250 /ug/ml of kanamycin B Before administration 4.3x106 1.4x10 After administration day 1 4.1x106 1.8x10 day 2 3.2x106 2.1x10 day 3 8.0x10 1.1x10 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - day 6 9.1x106 7.1x10 day 8 8.0x106 8.1x103 day 15 6.8x106 8.7x103 day 22 5.0x106 9.8x103 day 29 8.0x106 1.1x104 day 36 9.1x106 7.0x103 day 43 7.0x106 7.9x103 day 60 6.1x106 7.0x103 The data of Table 5 indicate that the microorganism administered persists and replicates in the rumen for a long time.

Example 3 Modification of the rumen flora of a sheep fed on molasses (A) Isolation of microorganisms able to grow on molasses The process described under item (A) of Example 1 is repeated with the difference that the initial samples are taken from a sheep fed on molasses, the individual isolates are inoculated to RGCF media containing glucose instead of extracted hay, the cultures grown in liquid media are spread on RGCA media and clones grown from cells utilizing molasses are inoculated on the same RGCA media.

So microorganisms of rumen origin utilizing molasses are obtained.

(B) Genetic labelling of bacteria utilizing molasses The process described under item (B) of Example 1 is repeated with the difference that cells prepared according to item (A) of Example 3 are transformed by p1011 plasmid DNA, instead of those obtained according to item (A) of Example 1.

The resistance to kanamycin B of several KmR strains is shown in Table 6.

Table 6 Resistance to kanamycin B of rumen bacteria and of genetically labelled strains utilizing molasses Microorganisms Lowest concentration of kanamycin B inhibiting growth Rumen liquor 31 Initial strains 7.5 Genetically labelled KmR strains Hh-GYOKI-3-2 250 -14 500 -34 250 -81Me 500 -132 500 In this way isolates highly resistant to kanamycin B and utilizing molasses are obtained.

The strain signed Hh-GYOKI-3-81Me has been deposited in the Hungarian National Collection of Medical Bacteria of the National Institute of Hygiene, Budapest under No. 00289.

(C) Reintroduction of genetically labelled bacteria into the rumen The process described under item (C) of Example 1 is repeated with the difference that the strain Hh-GYOKI-3-81Me is inoculated onto RGCFa media containing 1.8% of glucose instead of 1.8% of cellulose, and the culture is mixed with the feed of a sheep fed on molasses. 380 ml of a culture containing 1.6X107 bacteria per ml were orally administered. One sample each will be taken daily before and after administration through a rumen fistula.

Table 7 Changes in the rumen flora of a sheen treated with strain Hh-GYOKI-3-81Me (KmR) Sample Cell number/ml Without anti- In the presence of biotics 500 lug/ml of kanamycin B Before administration 5.0x106 0 After administration day 1 1.1x106 119x105 day 2 1.8x107 2.5x105 day 3 6.2x106 6.1x105 day 6 8.1x106 8.7x105 day 8 6.2x106 9.1x105 day 15x 1.3x103 103x102 day 22 3 .OxlO6 3.1z105 day 29 1.1x106 7.0x105 day 36 8.0x105 9.1x104 day 43 6.1x106 8.1x105 day 60 6.8x106 6.4x105 (X) Sampling error The data indicate that the microorganism administered persists for a long period in the rumen of sheep fed on molasses.

Example 4 Changes in ratios of volatile fatty acids owing to treatment with the bacterial preparation Two sheep are fed on a complete ration for 14 days and then a rumen sample is taken through a fistula. Two liters of rumen liquor are filtered through several layers of gauze. The particulate residue is suspended in 11 of physiological buffer (see below), mixed and filtered as before. The two filtrates are mixed, left to stand for an hour, solids floating on the surface are discarded and the liquid phase is used for the examination.

The composition of the physiological buffer is as follows: Na2HPO4 0.316 g/l KH2PO4 0.152 g/l NaHCO3 2.260 g/l KCI 0.375 g/l NaCI 0.375 g/l MgSO4 0.112 g/l CaCl2.H20 0.050 g/l FeSO4.7H20 0.008 g/l MnSO4.H20 0.004 g/l ZnSO4.7H20 0.004 g/l CuS04.5H20 0.002 g/l cOCl2.6H2o 0.001 g/l The pH of the mixture is checked and, if required, adjusted to pH 7.2 with an aqueous HCI or NaOH solution (Cheng et al.: J. Dairy Sci. 38, 1225 /1955/).

To the resulting mixture, the same volume of physiological buffer is added and in 1 liter of the diluted mixture 4 g of the ration is suspended. 30 ml each of the suspension is poured into Erlenmeyer flasks of 100 ml volume. 200 doses are sterilized 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 able to persist in the rumen for a long time after an an vitro cultivation will be examined. In addition, bacterial strains isolated from the rumen liquor of a sheep fed on complete or any ratio according to items A, B and C of Example 1 are examined, too.

Bacteria to be examined are cultivated on RGC+CG media (see below) in anaerobic conditions at 37"C for 48 hours.

Composition of RGC+CG media: salt solution I (see item A of Example 1) 15% salt solution Il (see item A of Example 1) 15% trace element solutionx 0.3% yeast extract (Oxoid) 0.5% filtered rumen liquor 10.0% Na2CO3 0.4% cystein.HCI.H20 0.05% sodium thiosulfate 0.008% cellulose (Bacto) 0.3% glucose 2.0% "Composition of the trace element solution: ZnCI2 40 mg CuC12.2H20 10 mg disodium tetraborate dekahydrate 10 mg ammonium molybdenate tetrahydrate 10 mg FeCl3.6H20 200 mg MnCI2.4H20 10 mg deionized water ad 1000 ml Cultures are inoculated into media prepared in Erlenmeyer flasks. 2 ml of each culture is inoculated into 50 ml of media, in two parallel flasks. Non-sterile cultures are also inoculated.

The flasks are incubated under anaerobic conditions for 40 hours. The growth is stopped with 10% formic acid solution and the volatile fatty acid content of the cultures is examined.

The cultures are filtered through gauze layers and centrifuged at 4000 rev. per min. for 15 min., then filtered again and brought onto the separation column of a Carlo Erba Gl-452 gas liquid chromatograph, fitted with flame ionization detector, for determining the C2-Cs fatty acids.

Temperature of the column: 150"C Separation column: 2 m long, 4 mm wide (inner diameter) glass tube filled with 10% of ethylene glycol adipate and 2% of o-phosphoric acid on a silanated silica gel carrier (0.2 to 0.3 mm particle diameter).

Temperature of the injector: 190"C.

N2 stream rate: 50 ml/min.

H2 stream rate: 50 ml/min.

Stream rate of the air: 200 ml/min.

Paper movement: 160 cm/hour.

Duration of chromatography: 20 min.

Sample volume: 1 ,ul.

Triplicate measurements are made from each sample. The standard solution contains acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid.

More than 90 strains of bacteria were isolated from a sheep fed on a complete ration. Then the strains were labelled genetically and examined (the positive strains were examined several times). Representative results are shown in Table 8.

Explanation of the signs 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 of media prepared from rumen liquor, physiolog ical buffer and feed used for the experiment (average of 12 measurements); (c) positive control: volatile fatty acid content of the incubated culture containing the initial rumen bacteria and otherwise prepared by the same process (average of 12 measurements); (d) as (c) but 5 ppm monensin Na were added to the media (average of 6 determinations); (e) as (c), but 10 ppm monensin Na was added to the media (average of 6 determinations). Table 8 Bacterium Remark Acetic Propionc i-Butyric Butyric i-Valeric Valeric Acetic acid/ strain acid acid acid acid acid acid /propionic acid g/ml g/ml g/ml g/ml g/ml g/ml Hh-GYOKI S 1,71 1,87 a 0,35 a - 0,91 -1-123Sz NS 2,00 2,60 a 0,50 a - 0,77 Hh-GYOKI S 0,38 0,58 a 0,35 a - 0,65 -2-14Ab NS 1,95 1,36 a 0,52 0,12 a 1,43 Hh-GYOKI- S 2,21 0,67 a a a - 3,29 -3-81Me NS 2,81 0,81 a a a - 3,47 - b 1,51 0,74 a 0,39 a a 2,04 - c 1,68 0,91 a 0,51 0,11 0,09 1,84 - d Control 1,69 1,08 a 0,41 a a 1,67 - e 1,61 0,91 a 0,37 a a 1,77 Hh-GYOKI-48a S 1,26 1,60 a 0,32 a - 0,79 NS 1,21 2,38 a 0,34 0,48 a 0,51 Hh-GYOKI-50a S 1,44 1,56 a 0,34 a - 0,92 NS 1,73 2,01 a 0,35 0,15 - 0,86 Table 8 (contd.) Bacterium Remark Acetic Propionc i-Butyric Butyric i-Valeric Valeric Acetic acid/ strain acid acid acid acid acid acid /propionic acid g/ml g/ml g/ml g/ml g/ml g/ml Hh-GYOKI-51a S 2,37 0,47 a 0,36 a - 5,04 NS 2,02 0,70 a 0,35 0,15 - 2,89 Hh-GYOKI-55a S 2,10 0,51 a 0,34 a - 4,12 NS 2,18 0,78 a 0,37 0,14 - 2,79 Hh-GYOKI-113 S 1,39 0,67 a 0,34 - - 2,07 NS 1,77 0,60 a 0,27 - - 2,95 Hh-GYOKI-122 S 1,31 0,87 a 0,31 - - 1,50 NS 2,02 0,91 a 0,31 - - 2,22 Hh-GYOKI-109b S 2,49 0,45 a 0,29 - - 5,53 NS 2,92 0,36 a 0,30 - - 8,11 Hh-GYOKI-126 S 0,56 0,60 a 0,52 - a 0,93 NS 0,83 1,14 a 1,55 0,11 0,70 0,73 The data of Table 8 indicate that the ratios of volatile fatty acids produced by the fermentative function of the rumen flora can be modified in a wide range by the administration of microbial cultures prepared according to the invention. The production of propionic acid can be significantly stimulated with a culture prepared from strain Hh-GYOKI-48a, while strain Hh-GY OKI-109b stimulates production of acetic acid. Stimulation of production of individual fatty acids was observed both on media lacking (S) or containing (NS) living rumen microbes. In our experimental sysem monensin Na decreased the ratio of acetic acid to propionic acid by 0.1 or 0.2 (d, e).

Microorganisms chosen by the above-mentioned process are labelled genetically, administered to ruminants and examined for ruminal growth and persistence by repeating the process described in item (B) of Example 1. Strains with an advantageous fermentative pattern and long ruminal persistence will be orally administered for modifying the production of volatile fatty acids.

Example 5 Bacterial preparation for oral administration to ruminants Bacteria to be administered are cultured on RGCA+CG media (Example 4) under anaerobic conditions, by the described process. After cultivation, the cells are separated by filtration or centrifugation. Separated cells are suspended in physiological buffer (Example 4) and' freeze dried.

The lyophilized bacterial preparation is stored, suitably formulated and administered to ruminants orally.

Microorganisms may be cultivated in other conventionally used media as well, e.g. in media containing glucose and starch etc. as carbon source and inorganic salts as N-source.

The preparation can be easily administered by mixing it to feed or 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 be prepared as well. The microorganisms may also be administered after mixing the filtered or centrifuged bacterial mass with suitable carrier or diluting substances, e.g. CaCO2, concentrates, premixes or other feedstuffs.

The bacterial strain(s) are chosen from the microorganisms, prepared by the process of the invention and advantageously modifying the rumen flora, and their quantity to be fed is determined depending on the ration and the use of the animal. If a decrease of the acetic acid to propionic acid ratio is required, we may use e.g. a culture prepared from strain Hh-GYOKI-48a, but for an increase of the ratio the administration of strain Hh-GYOKI-3-81Me is recommended.

Determination of the required microbial cell number may not mean any difficulty for those skilled in the art. It is recommended to administer the cells in a quantity to make 5 X 102 to 5X107 cultivated microorganisms per ml of rumen liquor.

Example 6 Administration of strains Hh-GYOKI-48a and Hh-GYOKl- 1- l23Sz to sheep Hh-GYOKI-48a strain is cultivated on RGCA+CG media (Example 4) in two 5-liter fermentors (useful volume) at 37"C, under anaerobic conditions. Fermentation is commenced by inoculation with a 10 ml culture of similar composition. After 48 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 is divided to 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 by one of the bacterial preparations each for a single feeding and seven sheep served as control. 600 to 900 g of hay were consumed per day and animal, plus mineral and vitamin premix mixed with corn meal (100 g). The results are shown in Table 9.

Table 9 Weight gain in sheep fed ad libitum on grass hay Serial Initial 1st 2nd 3rd 4th 5th number weight w e e k Control 1 29.O 29.5 30.0 29.5 29.5 29.5 2 27.0 27.5 28.5 27.0 26.5 28.0 3 28.5 27.0 27.0 27.5 26.5 26.5 4 30.0 30.5 30.0 30.5 30.0 29.0 5 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 Treated with strain Hh-GYOKI-1-123Sz 8 29.5 32.0 32.5 33.0 33.5 34.0 9 25.5 26.5 27.0 28.5 29.5 29.0 10 25.0 25.5 25.0 28.0 29.0 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 13 29.5 30.0 30.5 29.5 29.5 30.5 14 29.5 28.5 29.5 30.0 30.5 31.0 15 28.5 29.0 30.5 30.0 30.5 32.0 Treated with strain kIh-GY0KI-48a 16 29.5 30.0 30.5 31.0 32.0 33.5 17 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 20 29.5 30.5 31.0 31.5 32.5 33.0 Table 9 (contd.) Serial Initial 1st 2nd 3rd 4th 5th number weight w e e k 21 28.0 28.0 28.0 29.0 29.0 30.0 22 29.0 30.5 33.2 30.0 32.8 33.5 23 27.0 26.0 27.5 29.0 31.0 32.0 The average daily weight gain is calculated from the data of Table 9 and are shown in Table 10.

Table 10 Averse weight and daily weight gain of experi- mental and control sheep Initial 1st 2nd 3rd 4th 5th weight w e e k Control Mean body weight (keg) 28.00 28.14 28.36 28.43 28.00 28.36 Mean daily weight gain (g) +20 +31 +10 -61 +51 Treated with strain 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 strain Hh-GYOKI-48a Mean body weight (kg) 27.81 27.94 29.02 29.37 30.73 31.69 Mean daily weight gain (g) +16 +135 +44 +170 +120 Sheep treated with strains Hh-GYOKI-1-123Sz and Hh-GYOKI-48a and the control group gained on the poor ration in average 2620, 3875 and 360 g, respectively, during the 35-day experimental period.

Initial body weights did not differ significantly between groups, but significant differences were found in the final body weights (Table II) and in the daily gains (Table 12).

Table 11 Statistical evaluation of final body weights Control Hh-GYOKI-1-123Sz Hh-GYOKI-48a Mean (kg) 28.357 30.375 31.6875 Corrected quadrate of standard deviation 1.476 3.910 2.281 p (%0) < 5.0 < 0.1 Table 12 Statistical evaluation of body weight Rains (5 weeks) Control Hh-GYOKI-1-123Sz Hh-GYOKI-48a Number of animals 7 8 8 Total weight gain of the group (kg) 2.5 21.0 31.0 Maximum gain (kg) 2.5 4.5 5.0 Minimum gain (kg) -2.0 1.0 2.0 Mean gain per sheep (kg) 0.3571 2.6250 3.875G Corrected quadrate of standard deviation 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 gain in sheep.

Example 9 Persistance of genetically labelled bacteria in the bovine rumen The process described under item (C) of Example 1 is repeated with the difference that the strain Hh-GYOKI-1-123Sz resistant to 10,000 ,ug/ml kanamycin is cultivated in 4 liter of RGCFa media. After reaching the stationary phase (38th hour) the culture is harvested by centrifugation (5000 r.p.m.) and the cells thoroughly mixed with 500 g of corn meal are fed to a cow. Weekly samples are taken through a fistula, and ruminal persistence of the strain administered is determined according to item (C) of Example 1.

Results indicate that strain Hh-GYOKI-1-123Sz grows in the bovine rumen and it can persist there for at least 40 days.

Claims (17)

1. A composition for improving the efficiency of ruminant feed utilization, which comprises a microbial culture which is capable of adjusting the weight ratio of acetic acid to propionic acid produced during fermentation of energy-producing nutrients in the rumen of an animal to an optimum value of 1.5 to 4.0 : 1, and of growing in the rumen and persisting there at least for 60 days.

2. A composition as claimed in claim 1 wherein the culture comprises at most 95% by weight of the composition.

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

4. A composition as claimed in any one of claims 1 to 3, which comprises as a microbial culture one or more members selected from the group consisting of the microorganism strains deposited in the Hungarian National Collection of Medical Bacteria of the National Institute of Hygiene under Nos. 00287, 00288 and 00289.

5. A composition according to any one of claims 1 to 4 in the form of a microorganism paste, lyophilizate or suspension.

6. A process for the preparation of a microbial culture able to be used as active ingredient in a composition according to iny one of claims 1 to 5, which comprises taking a sample from the rumen of an animal fed on a given feedstuff or ration, identifying microbes with desired metabolic characteristics and cultivating such microbes in media containing the same feedstuff or ration as a carbon- or nitrogen-source, introducing a genetic marker, to facilitate such identification in the growing microbes; cultivating genetically-labelled strains; reintroducing the culture into the rumen of an animal fed on the same feedstuff or ration;; samples are taken from the rumen, the cell number of the genetically labelled strain is counted, and separating any strains which persist for at least 60 days and which adjust the ratio of acetic acid to propionic acid formed during fermentation of energy-producing nutrients in the rumen of said animal to an optimum value.

7. A process as claimed in claim 6, in which antibiotic resistance is used as genetic mai'ker.,

8. A process as claimed in claim 6 or claim 7, in which microorganisms are isolated from the rumen of animals fed on a cellulose-containing feedstuff, a starch-containing feedstuff, or a mono- and/or disaccharide-containing feedstuff, and the genetically labelled strains are reintroduced into the rumen of animals fed on one of the above feedstuffs for testing, the suitable microbial cultures are cultivated and then isolated and, if desired, the cultures obtained are formulated in a suitable form.

9. A process as claimed in any of claims 6 to 8, which comprises separating the microorganism cells in a lyophilized form.

10. A process for the preparation of a composition for improving the efficiency of ruminant feed utilization, which comprises formulating a microbial culture as defined in claim 1 either along or optionally in admixture with a carrier, diluent, or preserving agent conventionally used in animal husbandry and nutritive and/or other substances conventionally administered to ruminants as an oral formulation.

11. A process for improving the efficiency of ruminant feed utilization, which comprises orally administering said animals an effective amount of one or more microbial cultures capable of adjusting the weight ratio of acetic acid to propionic acid produced during fermentation of energy-producing nutrients in the rumen of an animal to an optimum value of 1.5-4.0 : 1 and of growing in the rumen and persisting there at least for 60 days.

12. A process as claimed in claim 11, which comprises using as a microbial culture one or more members selected from the group consisting of the microorganism strains deposited in the Hungarian National Collection of Medical Bacteria of the National Institute of Hygiene under Nos.

00287, 00288 and 00289.

13. A composition as claimed in claim 1 substantially as hereinbefore described.

14. A composition as claimed in claim 1 substantially as hereinbefore described with reference to any of the Examples.

15. A process as claimed in claim 6 substantially as hereinbefore described.

16. A process as claimed in claim 6 substantially as hereinbefore described with reference to any of the Examples.

17. A microbial culture as defined in claim 1.