NL8502260A - PREPARATION AND METHOD FOR IMPROVING THE USEFUL EFFECT OF THE USE OF FEED FOR Ruminants. - 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

> NL 33.021 Kp / vdM ** '1 - 1 -

Preparation and method for improving the beneficial effect of the utilization of feed for ruminants.

The invention relates to a composition for improving the useful effect of the use of feed for ruminants. More particularly, the invention relates to a composition containing, as active ingredient, one or more microbial cultures capable of optimizing the weight ratio of acetic acid to propionic acid, preferably to a value of 1 , 5-4.0: 1, and have the ability to grow and remain in the ruminants for at least 60 days, optionally mixed with carriers, diluents, preservatives commonly used in animal husbandry, as well as nutrients and / or other substances normally administered to ruminants. The invention further relates to the preparation of microbial cultures, which are used as active ingredients in the above mentioned preparations, as well as to the use of such preparations.

Gorges that have a compound stomach, such as sheep (Ovis aries aries), cattle (Bos primigenius 20 taurus), goats (Capra hircus) and their wild relatives (deer and muflons, etc.) play an important role in the food chain and in the economy. Their special interest is that they live on nutrients that cannot be used for other herbivores. The pre-stomach provides an anaerobic environment for the rumen flora, which is able to digest cellulose and uses non-protein nitrogen in addition to the normal nutrients.

The composition of the rumen flora largely depends on the feed ratio and adapts to the diet. However, this adjustment takes several days, while the final stabilization can take several weeks. Abrupt change of the ratio adversely affects absorption, digestion and production and can lead to illness and even death.

S302 2SC

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It is known that millions of bacteria live and grow in 1 ml of rumen liquid. Anaerobic fermentation by bacteria is of great importance for the normal digestion and feed intake. Energy producing nutrients are fermented into acetic acid, propionic acid and butyric acid, which are used by the host animal as fatty acids; the bacterial mass, which traverses the intestinal tract, will be digested and used as a protein source. With regard to milk and meat production, the supply of acetic acid and propionic acid, as well as their mutual ratio, play an essential role. As a result, the rumen flora plays an important role in the preservation and production of the livestock.

After birth, the rumen flora develops spontaneously and reaches its adult composition after switching to solid food. However, it is not certain that this random flora forms the optimal fermentative system.

It would be beneficial to have a method for changing the composition and / or number of the rumen flora according to economic interests.

The increase in volatile fatty acid production in the rumen, and thereby the improvement of feed utilization and consequently the meat or milk production, is something which has been pursued in livestock farming for a long time. Certain results were obtained using monensin / 2- / 5-ethyltetrahydro-25 5- {tetrahydro-3-methyl-5- / tetrahydro-6-hydroxy-6- (hydroxymethyl) -3,5-dimethyl-2H-pyran -2-yl7-2-furyl} -2-furyl7 "9-hydroxy - $ - methoxy-α, γ, 2,8-tetramethyl-2,6-dioxaspiro / 4,57" decane-7-butyric acid7, which substance was originally used as a coccidio state (see, e.g., U.S. Patent No. 4,085,255). Experimental use of other related polyethers, such as salinomycin, lasalocide, etc., phthalide derivatives (U.S. Patent No. 4,333,923) and glycopeptides, such as avoparcine, actaplanin, etc. (Ingle et al., Abstr. Am. Soc. Anim. Sci. 424 / 1 * 9787) was also published. However, the observed effect is very different in food with various nutrients and in all it is not essential (Chalupa, W., Chemical Control of Rumen Microbial Metabolism, Digestive Physiology and Metabolism in Ruminants, 8 * 02260 ♦ 4 <'- 3 - MTP Press, Lancaster, England, 1980 and Chalupa, W. et al., Manipulating Ruinen Fermentation with Monensin and Amicloral, Abstr. Am. Soc. Anim. Sci., 410/19787). There is no known method in the art by which the microbial culture present in the ruminant rumen could be influenced such that significant results could be obtained.

According to the invention, however, it has been found that genetic recombination methods can be successfully used to separate ant organisms. Virtually any strain of bacteria can be labeled via genetic markers, e.g. by an antibiotic resistance factor, which allows identification of the bacteria between other bacteria.

According to the invention, rumen bacteria were isolated, the strains were genetically labeled, and after breeding they were reintroduced into the ruminants. Thereafter, rumen content samples were taken from time to time, followed by culturing them in selective media, finding that some of the strains had fermentative properties that were beneficial to the host animal and capable of growing in vitro , could grow and exist for a long time in the ruminants if the same nutrient was administered as during isolation and that they stimulate digestion and also promote feather utilization in the host animal.

The present invention relates to a composition containing as the active ingredient one or more microbial cultures capable of adjusting the weight ratio of acetic acid to propionic acid to an optimal value of 1.5-4.0: 1 and have the ability to grow and persist in the rumen for at least 60 days, optionally mixed with carriers, diluents, preservatives commonly used in animal husbandry and nutrients and / or other substances commonly administered to ruminants .

If the preparations are used to increase meat production, the active ingredient is preferably a microbial culture which is able to adjust the weight ratio of acetic acid to propionic acid to a value of 8502280 '- 4 - Λ I'. , 0-3.5: 1, e.g. 2: 1. For milk production, the optimum acetic acid to propionic acid ratio is approx.

53.0: 1, about 4.0: 1 for maintenance or pregnancy and about 2.0-3.0: 1 for aquaculture. It is therefore advisable to use preparations which are capable of Propionic acid ratio to an optimum value for these purposes. There is some uncertainty in the literature as to the most favorable acetic acid to propionic acid ratios, the preferred ratio being a function of the ruminant, the feed used and other factors, leaving the choice to the person skilled in the art (see eg Kaufmann, W. and Rohr, K., Der Einfluss des Futters 15 auf die bacterielle Fermentation in VormSgen, Handbuch der Tiernahrung, 263, 1969, Parey, Hamburg, Berlin).

The invention further relates to a method for the preparation of microbial cultures, which are used as active ingredient in the above preparations, in which samples are taken from the rumen of the animals fed on a given nutrient or ration. by examining metabolism of the microbes isolated from the sample in vitro and / or in vivo, followed by culturing the microbes with appropriate metabolic properties in media containing the same nutrient or ration as carbon or nitrogen source, after which a genetic marker, allowing selection, is introduced into the growth microbes, cultivating the genetically labeled strains, re-culture the animals in the rumen of the animals, which are fed with the same nutrient or ration introduced, followed by sampling from the rumen, after which the number of cells from the genetically labeled strain is counted, 8502 2 6 0% - 5 - «* where the strains, which persist for at least 60 days, and where the acetic acid to propionic acid ratio is adjusted to an optimal value, preferably to 1.5-4.0: 1, are separated and finally these trunks are, if desired, transferred into a form suitable for the practice of animal husbandry and feeding.

According to a preferred embodiment of the present method, samples are taken from the rumen of a fistulated cattle, fed with hay, cereal flour or molasses, after which the cultures with the rumen bacteria therein are spread over solid media containing N and C sources, inorganic salts, rumen fluid and agar (Bryant and Burkey, J. Dairy Sci., 36, 205, 1953). The cultures are incubated under anaerobic conditions, after which the clones are isolated and grown in similar media as above.

The grown cultures are inoculated in liquid media containing nitrogen source, inorganic salts, rumen liquor and hay or cellulose carbon source, or in other cases cereal flour or molasses, while incubating until intensive growth in the media can be observed. .

Cells growing on hay (cellulose), grain meal (starch) or molasses (sucrose) as carbon sources are dispersed, grown and isolated on solid media containing cellulose (for cell growth with hay), glucose (for cell growth with grain meal) or sucrose ( for cell growth with molasses) as the carbon source.

The cultures obtained are genetically labeled.

Any hereditary genetic marker, which allows identification of the labeled microbes between other microbes, can be used for the labeling.

According to another preferred embodiment of the invention, antibiotic resistant genes are introduced into the selected cells. If the rumen bacteria are sensitive to a particular antibiotic and mix resistant cells with it, growth and death of the last microbes can be easily monitored if the 8502260 * - 6 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.

5 146, 564 / T981_7), pi 011 plasmid is introduced (Simon et al., Proc. 8th North American Rhizobium Conference, Winnipeg, Canada, Univ. Of Manitoba Press, 1983), carrying kanamycin and chloramphenicol resistant genes, introduced into the selected cultures , which grow on cellulose, grain flour or 10 molasses. Plasmid DNA was isolated from E. colli cells (Bimboim and Doly, Nucl. Acid. Res. 1_, 1513/19797)

The strain is deposited with the Hungarian National Collection of Medical Bacteria of the National Institute of Hygiene, Budapest, under number 00264.

After transformation with DNA carrying antibiotic resistant genes, the cells containing and propagating the new genetic information are selected on solid media of the above composition, but supplemented with kanamycin. The resistant strains are preserved.

With the isolated and preserved cultures, media containing a nitrogen source, inorganic salts, rumen liquid and agar (to obtain a semi-solid consistency) are inoculated and incubated under anaerobic conditions. The developed cultures are mixed with the sheep feed, which was fasting for one day. Before and after feeding the bacteria to the animals, rumen samples are drawn daily, spread on the solid media described above containing kanamycin, and the bacterial cells, which are resistant and sensitive to antibiotics, are counted. The ruminal production of volatile fatty acids is also determined qualitatively and quantitatively. Feed utilization in ruminants is known to be affected by the weight ratio of volatile fatty acids (Eskeland et al., J. Anim. Sci. 33_, .282 / T971 ^ 7; Church 35 et al., Digestive Physiology and Nutrition of Ruminants, Vol. 2, biz. 622-625, / 19717). As mentioned above, the optimum ratio of acetic acid to propionic acid is 2.0-3.5: 1, 3: 1 and 4 1 1, for respectively. growth, milk production and maintenance 85 0 2 2 6 0% - 7 - • <as well as pregnancy (Kaufmann, W. and Rohr, K .: Der Einfluss des Futters auf die bakterielle Fermentation in VormSgen. In: Handbuch der Tieremahrung , p. 263, Parey, Hamburg-Berlin, 1969).

In a preferred embodiment of the present method, bacteria are isolated from rumen samples and the capacity of the isolates to produce volatile fatty acids is investigated. The microorganism is grown under anaerobic conditions in the described complete media containing rumen liquid, after which the acetic acid concentration, propionic acid concentration and butyric acid concentration of the cultures are determined. The volatile fatty acid producing microbial cells in the required proportions are genetically labeled and their ruminal growth examined.

Strains capable of fermenting feed carbohydrates into volatile fatty acids in optimal proportions in the rumen of the animal fed the described nutrient for at least 60 days are selected, cultured, isolated, preserved and stored and if desired, their cultures are administered orally to the ruminants in a form suitable for animal husbandry to develop or modify the rumen flora.

There are three strains, Hh-GYOKI-1-123Sz, Hh-GYOKI-2-14Ab and Hh-GYOKI-3-81Me, capable of growing in the rumen of animals fed with hay, grain meal or molasses and which can exist in the rumen for a long time and favorably affect digestion, deposited with the Hungarian 30 National Collection of Medical Bacteria of the National Institute of Hygiene under resp. the numbers 00287, 00288 and 00289.

According to the invention, it is preferable to cultivate the rumen origin ant organisms between 32 and 37 ° C, under anaerobic conditions, excluding oxygen in media containing carbon and nitrogen sources, inorganic salts, reducing agents and rumen fluid; the latter of which provides the growth factors. As the carbon source, use can be made of glucose, cellulose, hay, cereal flour or molasses, while inorganic salts, yeast extract, casein and similar additives are suitable N sources.

An essential aspect of the invention is that use is made of unicellular organisms which suitably ferment the supplied nutrient or ration and are able to reside in the rumen for a long period of time, which are used to modify the rumen flora. For the selection of such strains, genetic markers are used, as described above, eg genes encoding antibiotic resistance, enzyme proteins or other detectable proteins. Auxotrophic cells can also be used.

The genetic marker is introduced into the cell using a vector DNA molecule, eg, via a plasmid or phage, but the selectable properties, eg resistance to an antibiotic, can be selected via spontaneous selection.

The selected strains, which have beneficial fermentative properties and can reside in the rumen for a long time, are used to either promote the development of rumen flora in ruminant ruminants, or to favorably modify the composition of the existing rumen flora. It is recommended to administer the preparation together with the feed or drinking water.

The microorganism selected for the preparation of the present composition is grown in media containing an organic carbon source, an organic or inorganic nitrogen source and organic and inorganic salts, followed by isolation in a form suitable for oral administration or transport. If desired, the microorganism culture is formulated by mixing with solid or liquid carriers or other additives. The preparation can be mixed with the feed or drinking water, or it can be administered as such.

In the case of sheep, for example, 1-20 g, preferably 5 g, of the microorganism culture according to the invention is added to about 0.5 kg of feed. As a nutrient 8502 260% - 9 - • *, for example, a mixture of maize meal, lucemehay and cattle feed can be used, while the actual proportions can be determined in view of the current conditions and the desired daily weight gain. Cattle are generally administered 10-200 g, preferably 50 g, of a microorganism culture according to the invention per day, eg mixed with about 5 kg of a conventional nutrient.

The method of improving the useful effect of feed utilization in ruminants is also within the scope of the invention.

After culturing in liquid media, as mentioned above, the ant organisms are isolated by centrifugation or filtration. There may be pastes, freeze-dried preparations, or suspensions, which contain spores or vegetative forms, etc.

15, while additives acceptable for animal husbandry and nutrition can be added. Other additives, eg proteins, amino acids or glycerol can help keep the microorganism alive. To the compositions of the invention used to promote the utilization of the feed in ruminants, other substances commonly used in practice, eg antibiotics, which stimulate the growth of the host animal (monensin, nigericin, salynomycin) , etc.) or that promote the persistence of the administered microorganisms.

The microbial cultures according to the invention therefore allow the formation of a living microbial culture in the rumen or the favorable modification of an already existing flora.

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

By administering the selected microbial strains, rapid development can be achieved instead of the slow and spontaneous development of the rumen flora, while the rumen flora is able to make optimum use of the feed.

The advantage of the present process is that 8502260-10. * with the microorganisms prepared in the manner described (e.g. with strains 00287, 00288 and 00289) the rapid adaptation or development of the rumen flora during feed change or weaning can be promoted by the ruminal growth of the 5 microorganisms, which are capable of to achieve optimal degradation of the feed.

The method can be applied, inter alia, in the following cases: - for dairy cows during change of lactation at the end of pregnancy and during seasonal and other changes of the ration; - for beef cattle at the beginning and at the end of the grazing period, at the change of the fat fattening with roughage to an intensive fattening with grain flour and during other changes of growth-fattening diet; for sheep during the seasonal changes of feed, at the. beginning and at the end of the grazing period and during the beginning of an intensive growth and fattening period. .

20 The options are also similar in goat farming. The microbial cultures prepared by the present method can also be used in various special cases, e.g., for wild ruminants, game reserves and fallow deer.

It is noted that although the microbial cultures which can be used in the compositions of the invention are preferably of rumen origin, other acetic and / or propionic acid producing bacteria not necessarily derived from the rumen are also suitable.

Such bacteria are certain representatives of the genus Angerovibrio (lipolytica), Bacterorden Selenomonas (ruminanticum) and Propionibacteria.

The invention is now further illustrated by the following non-limiting examples. The preparation of microbial strains, which are able to utilize basic rations containing mainly cellulose (hay), starch (grain meal) or sucrose (molasses) and which can remain in the rumen for a long time, will be further described 3502260 «* Live. The use of the composition has been described for sheep, however the scope of the description extends to microorganisms capable of growing on other nutrients and for the development or modification of the rumen flora of other ruminant species.

EXAMPLE I

Modifying the rumen flora of animals fed hay as food.

a) Isolation of the microorganisms that can grow on hay 10.

Sheep were paratomized, provided with rumen fistula and fed with hay for one month. A rumen sample was drawn through the fistula, diluted and spread over RGCA solid medium of the following composition: saline 1: K2HPO4 0.6 g distilled water ad 100.0 g saline 2:

NaCl 1.2 g 20 (NH4) 2 SO4 1.2 g KH2PO4 0.6 g

CaCl2 0.12 g

MgS04.7H20 0.25 g distilled water ad 100.0 ml 25 Resazurin (0.1% solution) 0.1 ml *

Agar (Bacto) 2.5 g

Rumen fluid 10.0 ml

Glucose 0.005 g

Cellobiose 0.005 g Cysteine.HCl Monohydrate 0.05 g

Sodium carbonate (8% solution) 5.0 g

Distilled water ad 50.0 ml *) Difco Labs, Detroit, United States of America **) The rumen sample was filtered through several layers of gauze and the filtrate was stored at -20 ° C under carbon dioxide.

Prior to sterilization under CO2 gas, the pH of the RGCA medium was adjusted to 6.8. Sterilization, medium preparation and cultivation were performed according to the method of Bryant and Burkey (J. Dairy Sci., 36, 205/1953).

The rumen fluid was diluted with a sterile mixture of the following composition: saline 1 (see above) 7.5 ml of saline 2 (see above) 7.5 ml of cysteine. HCl monohydrate 0.05 g

Na2 CO3 0.3 g 10 resazurin (0.1% solution) 0.1 ml distilled water ad 100.0 ml

This mixture is indicated by HB.

The cultures were incubated under anaerobic conditions at 35 ° C (see Atlas of Rumen Microbiology, 15 Ogimoto and Imai, Japan Scientific Societies Press, Tokyo, 1981) for 120 hours, after which the individual clones were inoculated in the media, in which extracted hay is present wash, with the following composition: saline 1 (see above) 15.0% 20 saline 2 (see above) 15.0% resazurin (0.1% solution) 0.1% • k

Tripton L42 (Oxoid) 15.% * yeast extract (Oxoid) 0.5% • rumen liquid kit 10.0% 25 Na2CO3 0.4% cysteine.HCl monohydrate 0.05% • β »β * extracted hay 10.0 %

This medium is called: RGCP liquid medium.

*) Oxoid Ltd., London, UK 30 **) See above ***) For the preparation of extracted hay, finely cut hay particles were suspended in water, boiled and filtered. The residue left after filtration was added to the medium before sterilization.

Prior to sterilization, the pH of the medium was adjusted to 6.5.

Test tubes containing 5 ml of sterile medium were inoculated with the microbial suspension obtained from individual clones grown on RGCA solid medium and incubated under anaerobic conditions at 35 ° C. Growth was monitored by microscope, after which the cultures were spread on RGCA solid medium, while 2.0% Bacto cellulose was used in place of the glucose and cellobiose.

The cultures were incubated under anaerobic conditions at 35 ° C for 120 hours, after which the individual clones obtained from cells that had used cellulose were inoculated on RGCA medium containing cellulose.

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

b) Genetic labeling of rumen bacteria, which can grow on hay.

Genetic labeling was performed using E. coli pi 011 plasmid, according to the method of Siion ion et al. 20 (Proc. 8th North American Rhizobium Conference, Winnipeg, ^ Canadat, Univ. Of Manitoba Press. 1983).

The plasmid DNA was isolated from an E. coli culture according to Bimboim and Doly (Nucl. Acid Res. 7, 1513/19797), followed by dissolving in an aqueous solution with the following components: 75 mM CaCl2 5 mM MgCl0 Δ * 10 mM tris.HCl buffer, pH 7.5 *) tris- (hydroxymethyl) -aminomethane.hydrochloride.

Microbes capable of growing on hay and isolated according to a) of Example I were grown on RGCF medium and separated by centrifugation under CO2. The cells were suspended in an aqueous solution containing per liter the following components: 75 mM CaCl 2 5 mM MgCl 2 10 mM tris.HCl buffer, pH 7.5 1 mM cysteine. HCl monohydrate 1 mM sodium thiosulfate 85 02 260 - 14 - 9 ••• The suspensions should contain 5 x 10 cells per ml. The suspension was diluted with the same volume of plasmid DNA solution (0.1 µg / ml), which suspension was incubated at 4 ° C for 60 min. Then incubation was continued for 2 min at 41 ° C, after which the culture was spread on solid medium containing 500 µg / ml kenamycin B and cellulose as a carbon source. The culture was incubated for 120 hours at 35 ° C under anaerobic conditions, while the clones capable of growing in the presence of 10 500 µg / ml kanamycin B were examined.

Plasmid p1011 carries genes resistant to kanamycin and chloramphenicol; in addition, the plasmid contains replication origin, which allows the initiation of replication in E. coli cells. When transferred to other bacteria due to the lack of suitable origin to allow replication, the plasmid DNA was either eliminated or incorporated into the chromosome (partial or complete) and genetic recombination occurred. Optionally, the gene contained in the chromosome is expressed and confers resistance to the cells against kanamycin and chloramphenicol.

According to the tests carried out, kanamycin B resistant clones were obtained with a transformation frequency -5 of 3 x 10.

Several resistant clones were isolated, with the antibiotic susceptibility of the original

R

strains and kanamycin B resistant (Km) strains were determined. The results obtained with various strains are listed in Table A.

30 3502260

- 15 TABLE A

Susceptibility to kanamycin B from rumen bacteria and genetically labeled strains and to break down hay smallest effective concentration of microorganisms:> kanamycin B, µg / ml rumen fluid 31 original strains 4.0 to 7.5

R

genetically labeled Km strains

Hh-GYOKI — 1-8 500 10 -27 250 -91 500 -123 1000 -142 500

In this way microorganisms of ruin -15 origin can be obtained, which are able to utilize hay or cellulose and grow in the presence of large amounts of kanamycin B.

The strain Hh-GYOKI-1-123 (KmR) was spread on RGCA medium containing cellulose and kanamycin B 20 was added at concentrations of 1000, 5000 and 10,000 µg / ml. The cultures were incubated under anaerobic conditions for 168 hours at 35 ° C, after which the strains growing in the presence of 10,000 µg / µl antibiotic were isolated. Thus, under spontaneous selection, spontaneous rautants, which were very resistant to kanamycin B, were obtained. One of these strains was designated Hh-GYOKI-1-123Sz and deposited with the Hungarian National Collection of Medical Bacteria of the National Institute of Hygiene, Budapest, under number 00287.

C) Re-introduction of the labeled microbes into the rumen.

A sterile solid medium, designated RGCPa, was inoculated with the culture of the strain Hh-GYOKI-1-123 (Km), which was stored on RGCA slants at + 4 ° C. The composition of the RGCA medium was 8502260-16: 1. K ^ HPO ^ 0.3% 45 ml solution 2. (NH4) 2 SO4 0.6%

NaCl 0.6% 5 MgS04.2H20 0.06%

CaCl2.2H20 0.06% KH2PO4 0.3% 45 ml solution of the mixture * 3. cellulose (Bacto) 1.8% * 10 agar (Bacto) 3.0% 65 ml solution of the mixture 4. yeast extract 0, 1% 20 ml solution 5. cysteine.HC1.H20 0.1% 20 ml solution 6. sodium thiosulfate 0.1% 15 Na2 CO2 0.2% -10 ml solution of the mixture 7. rumen liquid 20 ml *) Difco Labs, Detroit , USA

**) See above.

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

Culturing was performed in 500 ml Erlenmeyer flasks. containing 150 ml of medium under anaerobic conditions. Growth was checked after 48 hours, after which the culture was mixed into the feed intended for a hay-fed sheep. 340 ml of a culture containing 4.7 x 10 bacteria per ml was administered orally to the sheep. 50-200 ml samples were taken from the rumen fistula prior to administration and on the following days. The monsr. 30 ters were diluted with HB solution and spread over RGCA medium containing no kanamycin B or other antibiotics. The cultures were incubated at 35 ° C for 72 hours under anaerobic conditions, after which the bacterial clones were counted. The results are reported in Table B.

8502260 - 17 -

TABLE B

Changes in rumen flora in a sheep treated with a trunk

Hn-GYOKI-1-123 (KmR) cell count / ml 5 in the presence sample without antibiotics of 1000 jig / ml

Kanijrycine B

before administration 5 x 10 ^ 0 after administration 10 day 1 5.9 x 106 2.0 x 104 day 2 3.2x10 ^ 4.1x104 day 3 2.4 x 106 3.1 x 104 day 6 3.9 x 107 1.8 x 104 day 8 9.8 x 106 1.05 x 105 15 day 15 8.1 x 105 3.1 x 104, Table B clearly shows that the microorganism administered to the animal continues to exist and grow in the rumen .

According to the above method, the strain Hh-GYOKI-1-123Sz was also grown, which was resistant to 10,000 µg / ml kanamycin B. This strain was administered orally to the same sheep on the 15th day.

O

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

Bacteria from rumen samples were grown and counted as previously described. The results are shown in Table C.

30

Ss 0 2 2 δ ö - 18 -

TABLE C

Changes in rumen flora in a sheep treated with the strain Hh-GYOKI-1-123Sz (KmR) cell count / ml 5 in the presence of a sample without antibiotics of 8000 µg / ml

kanamycin B

6 before administration 1.4 x 10 0 after administration 10 day 1 7.4 x 106 3.2 x 104 day 2 1.7 x 105 1.3 x 104 day 5 4.0 x 106 9.1 x 103 day 7 - 1.1 x 107 2.0 x 104 day 14 8.0 x 106 3.1 x 10415 day 21 7.1 x 105 6.2 x 104 day 28 8.2 x 106 8.1 x 104 day 35- 8.7 x 106 1.8 x 104

The data from Table C clearly show that the administered ant organism is present in significant amounts in the runes and because the liquid phase from the rumen content is continuously removed, the microorganism multiplies readily.

The differences of bacterial counts between samples can be explained by the variations of the consistency of the rumen content from thick to liquid. EXAMPLE II

Modification of the rumen flora in sheep fed grain flour.

a) Isolation of microorganisms capable of growing on grain flour.

The method described under a) of Example I was repeated with the difference that the original sample was taken from the rumen of a sheep fed with grain meal, after which the individual isolates were inoculated in RGCF liquid medium (see above) containing 3502260 * - 19 - 2% grain flour, crushed in a mortar, instead of extracted hay, the cultures grown in RGCF liquid medium were spread over RGCA solid medium (see above), after which the clones, developed from the cells, containing grain flour 5 on the same medium were isolated.

In this way, microorganisms capable of growing on grain flour as a carbon source were obtained.

b) Genetic labeling of rumen bacteria, which use grain flour.

The method described under b) of Example I was repeated with the difference that the strains obtained under a) of Example II were used for transformation by p1011 plasmid DNA, instead of those obtained in a) of Example II.

The resistance to kanamycin B from various transformed Km strains is shown in Table D.

TABLE D

Susceptibility to kanamycin B from rumen bacteria and genetically labeled strains and utilization of cereal flour 20 lowest kanamycin B concentrate organisms that inhibit growth, µg / ml rumen fluid 31 original strains 1.8 £ genetically labeled Km strains 25 Hh-GYOKI 2-4 250 -14Ab 250 -37 250 -81 125

In carrying out the above method 30, microbes of the rumen origin were obtained, which were able to use grain flour as a carbon source and were eight times more resistant to kanamycin B than the rumen flora.

The strain, designated Hh-GYOKI-2-14Ab, was deposited with the Hungarian National Collection of Medical 35 Bacteria of the National Institute of Hygiene, Budapest, 8502 250 m - 20 - under number 00283.

c) Re-introduction of genetically labeled micro-organisms into the rumen.

The method 5 described under c) of Example 1 was repeated with the difference that the sterile RGCFa medium containing 1.8% cellulose instead of 1.8% cellulose (Bacto) was inoculated, mixed with sheep feed and previously samples were taken daily through the fistula prior to administration and after administration. Sheep 10 was normally administered 310 ml of culture containing 7.1 x 10 bacteria / ml.

TABLE E

Changes in the rumen flora of the sheep treated with the strain Hh-GYOKI-2-14Ab (KmR) 15 cell number / ml in the presence of a sample without antibiotics of 250 µg / ml

. kanamycin B

before administration 4.3x106 1.4x10 ^ 20 after administration day 1 4.1 x 106 1.8 x 103 day 2 3.2 x 106 2.1 x 103 day 3 8.0 x 103 1.1 x 103 day 6 9.1 x 106 7.1 x 102 25 day 8 8.0 x 106 8.1% x 103 day 15 6.8 x 106 8.7 x 103 day 22 5.0 x 106 9.8 x 103 day 29 8.0 x 106 1.1 x 104 day 36 9.1 x 106 7.0 x 103 30 day 43 7.0 x 106 7.9 x 103 day 60 6.1 x 106 7.0 x 103

The results listed in Table E clearly show that the administered ant organism in the rumen persists for a long time and multiplies.

8502260 * Λ - 21 -

EXAMPLE III

Modification of the rumen flora in a sheep fed with molasses.

a) Isolation of ant organisms / capable of growing on molasses,

The procedure described under a) of Example I was repeated except that the original samples were taken from a sheep fed with molasses, after which the individual isolates were inoculated in an RGCF medium, which instead of extracted hay contained glucose, after which the cultures grown in a fluid medium were spread on an RGCA medium, while the clones generated from the cells using molasses were inoculated on the same RGCA medium.

In this way, microorganisms of rumen origin using molasses were obtained.

b) Genetic labeling of bacteria that use molasses.

The method 20 described under b) of Example I was repeated except that the cells prepared according to a) of Example III were transformed by p1011 plasmid DNA, instead of those obtained according to a) of example I.

The resistance to kanamycin B of various £ 25 Km strains is shown in Table F.

TABLE F

Resistance to kanamycin B from rumen bacteria and genetically labeled strains that utilize molasses.

30 lowest concentration of kanamycin microorganisms B that prevents growth rumen fluid 31 original strains 7.5 T3 genetically labeled Km strains 35 Hh-GYOKI-3-2 250 -14 500 -34 250 -81Me 500 -132 500 83TZTT5: - - 22 -

In this way isolates were obtained which were very resistant to kanamycin B and which also used molasses.

The strain designated Hh-GYOKI-3-81Me was deposited with the Hungarian National Collection of Medical Bacteria of the National Institute of Hygiene, Budapest under number 00289.

c) Introduction of genetically labeled bacteria into the rumen.

The procedure described under c) of Example I was repeated with the proviso that the strain Hh-GYOKI-3-81Me was inoculated on an RGCFa medium containing 1.8% glucose instead of 1.8% cellulose. after which the culture was mixed with the feed for the sheep fed on molasses. Orally, 380 ml of a culture of 1.6 x 10 bacteria / ml was administered. A sample was taken daily through a rumen fistula before and after administration.

TABLE G

Changes in rumen flora in a sheep treated with the strain Hh-GYOKI-3-8iMe (KmR) cell number / ml in the presence of a sample without antibiotics of 500 µg / ml

kanamycin B

. .....- '.....

25 before administration 5.0 x 10 0 after administration day 1 1.1 x 106 1.9 x 105 day 2 1.8 x 107 2.5 x 105 day 3 6.2x106 6.1x105 3 0 day 6 8.1 x 106 8.7 x 105 day 8 6.2 x 106 9.1 x 105 day 15 * 1.3 x 101 1.3 x 102 day 22 3.0 x 106 3.1 x 105 day 29 1.1 x 106 7.0 x 105 35 day 36 8.0 x 105 9.1 x 104 day 43 6.1x106 8.1x105 day 60_6.8 x IQ6_6.4 x 1Q5_ 8502260 - 23 - *) sampling error.

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

EXAMPLE IV

Changes in the ratio of volatile fatty acids as a result of treatment with the bacterial preparation.

Two sheep were fed a complete ration for 14 days, rumen samples being taken through a fistula. 2 liters of the rumen liquid were filtered through various layers of gauze. The particle-containing residue was suspended in 1 liter of physiological buffer (see below), mixed and filtered as previously described.

The two filtrates were mixed and then left to stand for one hour. The surface floating solids were removed while the liquid phase was used for the study.

The composition of the physiological buffer is as follows:

Na2HPO4 0.316 g / l KH2PO4 0.152 g / l

NaHCO 3 2.260 g / 1 KCl 0.375 g / 1 NaCl 0.375 g / 1

MgSO 4 0.112 g / l

CaCl2.H2 O 0.050 g / l

FeS04.7H20 0.008 g / l

MnS04.H20 0.004 g / l ZnS04.7H20 0.004 g / l

CuSO4.5H20 0.002 g / l

CoC12.6H20 0.001 g / 1 1 5 * 3 0 $ Λ t. t- "ia ^ ^ - 24 -

The pH of the mixture was checked and adjusted to 7.2 if desired using an aqueous HCl or NaOH solution (Cheng et al .: J. Dairy Sci.

38, 1225/19557).

The same volume of physiological buffer was added to the mixture thus obtained, after which 4 g of the ration was suspended in 1 liter of the diluted mixture. 30 ml of the suspension were each transferred to 100 ml Erlenmeyer flasks. 200 doses were sterilized and another 200 were not.

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

Bacterial strains, which were able to persist in the rumen for a long time, were tested after cultivation in vitro. In addition, bacterial strains isolated from the rumen fluid of a sheep fed on a complete or other ration according to a), b) and c) of Example 1 were also examined.

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

Composition of RGC + CG medium: 25 saline 1 (see a from example I) 15% saline 2 (see a from example I) 15% spore element solution * 0.3% yeast extract (Oxoid) 0.5% filtered rumen liquid 10.0% 30 Na2CO3 0.4% cysteine.HCl.H20 0.05% sodium thiosulfate 0.008% cellulose (Bacto) 0.3% glucose 2.0% 35 * The composition of the trace element solution:

ZnCl2 40 mg

CuCl2.2H20 10 mg disodium tetraborate decahydrate 10 mg 8502 260 -25- ammonium molybdenate tetrahydrate 10 mg

FeCl3.6H20 200 mg

MnCl2.4H2 O 10 mg deionized water ad 1000 ml 5 Cultures were inoculated into media prepared in Erlenmeyer flasks. 2 ml of culture were inoculated in 50 ml of medium in 2 parallel flasks. Non-sterile cultures were also inoculated.

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

The cultures were filtered through gauze layers and then at 4000 rpm. Centrifuged for 15 min. Then they were filtered again and then transferred to a separating column of a Carlo Erba GI-452 gas-liquid chromatograph equipped with a flame ionization detector to determine the C 1 -C 2 fatty acids. Temperature of the column: 150 ° C.

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

Injector temperature: 190 ° C.

Nitrogen flow rate: 50 ml / min.

H2 flow rate: 50 ml / min.

Air flow rate: 200 ml / min.

30 Paper speed: 160 cm / h.

Chromatography time: 20 min.

Sample volume: 1 µl.

Triplicate measurements were made on each sample. The standard solution contained 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 4 2 4 £ δ € -. .............

Strains were genetically labeled and tested (positive strains were tested several times). Representative results are presented in Table H.

Explanation of the symbols used in Table H: 5 s: inoculated after sterilization NS: culture containing live rumen flora was inoculated a) trace amounts; b) negative control: the volatile fatty acid content of a medium prepared from rumen fluid, physiological buffer and 10 feed, used for the test (average of 12 measurements); c) positive control: volatile fatty acid content of the incubated culture containing the initial rumen bacteria and otherwise prepared in the same way (average of 12 measurements); D) as c), however, 5 ppm of monensin Na was added to the medium (mean 6 determinations); e) as c) with the proviso that 10 ppm of monensin Na was added to the medium (6 determinations on average).

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The data from Table H show that the proportions of volatile fatty acids produced by the rumen flora fermentative function can be modified over a wide range by the administration of microbial cultures prepared according to the invention . For example, the production of propionic acid can be significantly stimulated using a culture prepared from the strain Hh-GYOKI-48a, while the strain Hh-GYOKI-109b stimulates the production of acetic acid. Stimulation of the production of 10 separate fatty acids was investigated both on media in which no (S) was present, and on media containing (NS) live rumen microbes. According to the present experimental system, monensin Na was found to decrease the ratio of acetic acid to propionic acid by a factor of 0.1 or 0.2 (d, e).

Microorganisms chosen according to the above method are genetically labeled, then administered to ruminants and examined for ruminal growth and persistence by repeating the method described in b) of Example I. Strains with a favorable fermentative pattern and long ruminal persistence were administered orally to modify the production of volatile fatty acids.

EXAMPLE V

Bacterial preparation for oral administration to chewers.

The bacteria to be administered were grown on RGCA + CG medium (Example IV) under anaerobic conditions according to the method described above. After cultivation, the 30 cells were isolated by filtration or centrifugation. The isolated cells were suspended in a physiological buffer (Example IV) and then lyophilized. The lyophilized bacterial preparation was stored, formulated appropriately and then administered orally to ruminants.

The microorganisms can be grown on other conventional media, e.g., in a medium containing glucose and starch, etc. as a carbon source, as well as inorganic 8502260 V * - 30 salts as an N source.

The preparation can be easily administered by mixing it with the feed or drinking water, alone or together with other biologically active agents, eg anti-5 biotics and vitamins.

In addition to the lyophilized preparation, other products can also be prepared. The ant organisms can also be administered after mixing the filtered or centrifuged bacterial mass with a suitable carrier or diluent, eg CaCO 3, concentrates, premixes or other nutrients.

The bacterial strains are selected from the ant organisms, which have been prepared according to the present method and which are capable of modifying the rumen flora, while their quantity to be administered depends on the ration and the application of the animal. If a decrease in the acetic acid to propionic acid ratio is desired, e.g.

use is made of a culture prepared from the strain

Hh-GYOKI-48a, while for the increase of the ratio the administration of the strain Hh-GYOKI-3-81Me is recommended.

Determination of the required microbe cell number does not present difficulties for the skilled person. It is recommended that 12 cells be administered in an amount of 5x10 to 7 5x10 cultured ant organisms per ml of rumen fluid.

EXAMPLE VI

Administration of strains Hh-GYOKI-48a and Hh-GYOKI-1-123SZ to sheep.

Hh-GYOKI-48a strain is grown on RGCA-CG medium (Example IV) in two 5-liter fermenters (useful volume) at 37 ° C under anaerobic conditions. The fermentation was started by inoculation with a 10 ml culture of a similar composition. After 48 hours of culture, the cells were isolated by centrifugation (5000 rpm) while the wet sediment weighing 58 g was gently mixed with 4 kg of corn flour. The mixture was divided into 8 equal parts and then administered orally to 8 sheep previously fasting for 24 hours. Strain Hh-GYOLI-1-123Sz can also be used in a bacterial crop of 53 g.

8502 2 60 - 31 -

In a growth-fattening experiment, 23 sheep were fed ad libitum * with poor grass hay, the animals being weighed every week for 5 weeks. The experimental groups, consisting of 8 sheep, were fed using one of the bacterial preparations, each for a single feed, while 7 sheep served as controls. 600 to 900 g of hay per animal was consumed per animal, while the mineral and vitamin premix was mixed with the corn flour (100 g).

The results are shown in Table I.

1 ° TABLE I

Weight gain in sheep, fed ad hibitum with grass hay series- initially 1st 2nd 3rd 4th 5th number weight week control 15 1 29.0 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 20 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 the 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 25 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 30 15 28.5 29.0 30.5 30.0 30.5 32.0

Treated with strain Hh-GYOKI-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 35 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 8502 260 r »k - 32 - TABLE I (continued) series - initially 1st 2nd 3rd 4th 5th issue poem week control 5 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 mean daily weight gain was calculated from the data listed in Table I and is shown in Table J.

TABLE J

Average weight and daily weight gain of experimental and control sheep.

initial 1st 2nd 3rd 4th 5th 15th week weight control average body weight (kg) 28.00 28.14 28.36 28.43 28.00 28.36 average daily 20 weight gain (g) +20 +31 +10 -61 - 51 treated with the 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 averaged 2620, 3875 and 360 g, respectively, in the bad ration. in weight during the 35 day experiment: 8502260 4 - 33 -

The original body weights did not show significant differences between the groups, however significant differences were seen in the end body weights (Table K) and in the daily weight gains (Table L).

TABLE K

Statistical evaluation of the end body weights.

control Hh-GYOKI-1-123Sz Hh-GYOKI-48a mean (kg) 28,357 30,375 31,6875 10 corrected square of standard deviation 1,476 3,910 2,281 p (%) <5.0 <0.1

TABLE L

15 Statistical evaluation of body weight gain.

(5 weeks) control Hh-GYOKI-1-123Sz Hh-GYOKI-48a number of animals 78 8 total weight-20 increase in group (kg) 2.5 21.0 31.0 maximum weight gain (kg) 2.5 4.5 5.0 Minimum weight gain (kg) -2.0 1.0 2.0 Average weight gain per sheep (kg) 0.3571 2.6250 3.8750 Adjusted quarter of the standard deviation 2.143 1,768 0.839 standard deviation ± 1.355 ± 1.244 ± 0.857

The data show that the compositions prepared according to the invention can noticeably stimulate weight gain in sheep.

8502260 - 34 -

EXAMPLE IX

Persistence of genetically labeled bacteria in the bovine rumen.

The method 5 described under c) of Example I was repeated, except that the strain Hh-GYOKI-1-123Sz, resistant to 10,000 µg / ml kanamycin, was grown in 4 L RGCFa medium. After reaching the stationary phase (38th hour), the culture was harvested by centrifugation (5000 rpm), after which the cells were thoroughly mixed with 500 g cornmeal and then fed to cows. Samples were taken weekly through a fistula and the ruminal persistence of the administered strain was determined according to c) of Example I.

The results found show that the strain Hh-GYOKI-1-123Sz grows in the bovine rumen and can persist therein for at least 40 days.

20 850 2 2 60

Claims (12)

1. A preparation for improving the useful effect of the utilization of feed by ruminants, characterized in that the preparation as active ingredient contains one or more microbial cultures capable of reducing the weight ratio of acetic acid to propionic acid. set to an optimum value of 1.5-4.0: 1 and grow and persist in the rumen for at least 50 days, optionally mixed with carriers, diluents, preservatives commonly used in animal husbandry and nutrients and / or other substances commonly administered to ruminants. 2. Preparation according to claim 1, characterized in that it contains a maximum of 95% by weight of the active ingredient. The composition according to claim 1 or 2, characterized in that the composition contains a microbial culture capable of adjusting the acetic to propionic acid ratio in the rumen to 2.0-3.5: 1. Preparation according to any one of the preceding claims 1 to 3, characterized in that as a microbial culture, one or more of the microorganisms deposited with the Hungarian National Collection of Medical Bacteria of the National Institute of Hygiene, under numbers 25 00287, 00288 and 00289. Preparation according to any one of the preceding claims 1-4, in the form of a microorganism paste, a freeze-dried preparation or a suspension. 6. Process for the preparation of microbial cultures for use as active ingredient in a preparation according to any one of the preceding claims 1-5, characterized in that samples are fed from the rumen of animals fed with a certain nutrient or ration are taken, in which the metabolism of microorganisms isolated from the sample is investigated in vitro and / or in vivo, and the mine organisms with favorable metabolic properties are cultivated in a medium which is 85 02 2 50 - 36 - whether nitrogen source contains the same nutrient or the same ration, a genetic marker is introduced in the growing microorganisms, which allows selection, the genetically labeled strains are grown, the cultures are re-rumened in the animals, which were given the same nutrient or the same ration are introduced, samples are taken from the rumen, the cell count of the genetically labeled strain is counted, the strains which persist for at least 60 days and which adjust the acetic acid to propionic acid ratio to an optimum value of 1.5-4.0: 1 are sounder and these strains are optionally formulated in a the practice of animal husbandry and nutrition acceptable form. 7. A method according to claim 6, characterized in that the antibiotic resistance is used as a genetic marker. 8. A method according to claim 6 or 7, characterized in that the microorganisms from the animal rumen are fed on a cellulose-containing nutrient, preferably hay, a starch-containing nutrient, preferably dry feed or a mono and / or disaccharide-containing nutrient, preferably molasses, are isolated and the genetically labeled strains are reintroduced into the rumen of the animals, fed with any of the above nutrients, are tested, the appropriate microbial cultures are grown and isolated and the cultures obtained, if desired, formulated in a suitable form. Method according to any one of the preceding claims 6-8, characterized in that the microorganism cells are separated in a lyophilized form. 10. A process for the preparation of a preparation for improving the beneficial effect of the utilization of the feed by ruminants, characterized in that the microbial cultures prepared according to any one of the preceding claims 6-9, alone or optionally mixed with carriers, diluents, preservatives commonly used in animal husbandry and nutrients and / or other substances commonly administered to ruminants are formulated into an oral preparation. Method for improving the beneficial effect of the use of feed for ruminants, characterized in that the animals are administered an effective amount of one or more microbial cultures capable of reducing the acetic acid propionic acid ratio to an optimum value of 1.5-4.0: 1 and grow and ruminate in the rumen for at least 60 days. Method according to claim 11, characterized in that as a microbial culture one or more representatives of the group of the microorganism strains deposited with the Hungarian National Collection of Medical Bacteria of the National Institute of Hygiene under the numbers 00287, 00288 and 00289 are used. 25 8502260