CS593085A3 - Mixture of microbial cultures for improving efficiency of a fodder utilization for ruminants and process for preparing microbial cultures - Google Patents

Description

IN

IV - 1 -; The present invention relates to a mixture of microbial cultures for improving feed efficiency in ruminants containing carriers, diluents, preservatives used in animal production and nutritional substances commonly administered to ruminants.

The invention further relates to a process for the preparation of microbial cultures of microorganism strains used as active ingredients. In the above-mentioned composition: Compound stomach ruminants, such as sheep / Ovis ariesaries, bovine / Bos primigenium taurus /, goat / Capra hircus and their wild relatives / deer and mouflon etc ./, have an important role to play as a link in the food chain and in the economy. Their special significance is that they live from food that cannot be exploited by other bulls. Pre-stomachs provide anaerobic environment for rumen flora that can digest cellulose and utilize nitrogen or protein in addition to common nutrients. Composition of rumen flora dependent on food ratio and diet adaptation. However, such adaptation lasts for several days and may require several weeks for final stabilization. A sudden change in ratio affects nao-then adverse reception, digestibility, and production, and can cause disease or even death.

It is well known that millions of bacteria live in 1 ml of rumen fluid. Bacteria anaerobic fermentation is very important for normal digestion and food intake. Energy-producing nutrients are fermented with acetic, propionic and butyric acids, which the animal host uses as fatty acids; the bacterial mass passing through the intestines is digested and used as a higher protein source * With regard to milk and meat production, the intake of acetic acid and propionic acid and their proportions is important. a subtle proportion essential. The rumen flora plays? hence an important role in the maintenance and induction of ruminants. and?

After birth, rumen flora develops spontaneously; and keeps his composition after being weaned? However, it is not certain that this random flora is the optimal fermentation system. It would be advantageous to regulate the composition and / or amount of rumen flora according to the invention

economic aspects. L

The increase in volatile fatty acid production in the barium and hence the improvement of food use and together can be used successfully to separate microorganisms. Fractically, any bacterial strain can be labeled with genetic markers, such as the anti-V 'biotic resisten. allows identification of bacteria among other bacteria ·

The inventors of the present invention isolated the rumen bactery, genetically labeled the strains and, after cultivation, reintroduced into the rumen. They then took samples of rumen content periodically, cultivated them in a selective medium, and found that some of the strains that had beneficial fermentation properties for the animal and that were able to grow in vitro could grow in the rumen and stay for a long time if fed the same food as isolation, stimulate digestion and hence use of animal feed. SUMMARY OF THE INVENTION The present invention provides a mixture of microbial cultures for improving feed efficiency in ruminants containing carriers, diluents, preservatives used in animal production, and nutrients commonly administered to ruminants which contain up to 99% by weight of active ingredient. at least one microbial culture from strains of microorganisms deposited in the Hungarian State Collection of Medical Bacteria of the State Institute of Hygiene of 00 287, 00288 and 00289, capable of adjusting the weight ratio of acetic acid to propionic acid to a value of 1.5 to 4.0: 1, and able to grow in the rumen and

for at least 60 days, the remainder to 100 µM. they form known feed components such as hay, fodder and molasses.

If the mixture is to be used to increase production, the active ingredient is preferably a microbial culture capable of adjusting the ratio of acetic acid to propionic acid to 2.0 to 3.5 to 1, e.g. propionic acid for about 3.0 to 1, for maintenance or for about 4.0 to 1 breeding and about 2.0 to 3.0 to 1 for breeding heifers. propionic acid to the optimum value for these purposes. There is some uncertainty in the literature about the desired ratios of acetic acid to propionic acid, the preferred ratio being the ruminant ruminant, the food used and other factors, and its choice by the experts (see, for example, V. Kaufmann, K. Rohr, Influence of Bacterial Fermentation Feeding on Pre-Stomachs, Handbuch derTiernahrung) , 263, 1969 »Parsey, Hamburg, Berlin.

The mixture preferably takes the form of a microorganism paste, a lyophilizate or a suspension. The invention furthermore relates to a process for the preparation of microbial cultures used as active ingredients in the aforementioned compositions, which comprises extracting the rumen of animals fed with food or feed containing cellulose, starch or mono- and disaccharides of the sample, the metabolism of isolated microorganisms being investigated from the in vitro specimen and the inorganic and microorganisms adjusting the acetic acid / propionic acid ratio to a value of 1.5 to 4.0: 1 are cultured in an environment containing the same feed or feed as the carbon or nitrogen source, the growing microorganisms being introduced as antibiotic resistance as a genetic a marker that; allowing selection, genetically labeled strains are cultured, cultures are reintroduced into the rumen of animals fed with the same food or fodder, sampled from the rumen, counted from the genetically labeled strain, separated by strains overlapping for at least 60 days, and these strains they are formulated into acceptable form in the practice of animal production and nutrition.

These strains are optionally made into a form acceptable for the practice of animal production and nutrition.

According to a preferred embodiment of the method according to the invention, the probe samples from the rumen of ruminant feeded with hay, corn flour or molasses, and cultures containing rumen bacteria dissipate into solid media containing nitrogen and carbon sources, inorganic salts, rumen fluid and agar / Bryant and Burkey , D. Dairy

ι: ΐ 1

Sci., 36, 205, 1953] Cultures are incubated under anaerobic conditions, then clones are isolated and cultured in a similar medium as indicated above.

Grown cultures are inoculated into a liquid medium containing a nitrogen source, inorganic salt, rumen fluid, and hay or cellulose as carbon source, or cereal nebomelas in other cases, and incubated until intense growth begins in the medium.

Cells grown on hay / cellulose, cereal flour / starch / or molasses / sugar / as carbon source are applied, cultured and isolated to solid cellulose-containing cellulose / for cell cultures / glucose for hay cells (glucose) for cells grown on cereal flour (or sucrose) for cells grown on molasses / as a carbon source. The cultures obtained are genetically labeled. Any hereditary genetic marker can be used to mark the labeled microbial among other microbes.

In another preferred embodiment, the invention introduces anti-biotics resistance genes into selected cells. If the rumen bacteria are sensitive to certain antibiotics and if X cells are added to the cells, the growth and disappearance of the latter can be easily observed if the samples containing the same antibiotic are applied. In this case, only resistant cells grow. In the transformation (Bergmans et al., J. Bacteol. 146, 564 (1981) introduce the inventors of the plasmid (Simon et al., Proc.). 8th North AmericanEhizobium Conference, Winnipeg, Canada, Univ. of Manitoba Press, 1983, carrying kanamycin resistance genes chloramphenicol to selected cultures that grow cellulose, cereal flour or molasses. Plasmid DNA was isolated from E. coli / Birnboim and Doly, Nucl.Acid cells. Dog. 7, 1513 (1979). The tribe is deposited with the Maastrian Collection of Medical Bacteria of the State Institute of Hygiene, Budapest, under number 00264 ·

After transformation with DNA harboring antibiotic resistance genes, cells containing and expressing new genetic information are selected on a solid medium of the above composition but supplemented with kanamycin.

These resistant strains will be stored. The nitrogen-containing media, inorganic salts, rumen fluid, are seeded with these isolated and stored cultures to give a semi-solid consistency / and

*, '.., ι.tMrAVjf / ^ jk ^ UvtAí. 'IZ ^ řSSÍjZv.V.U ^ JkU.hřA.V-.tífx'j'Jit. The phosphate is then incubated under anaerobic conditions. Developed cults are mixed with the feed of sheep starving for one day. Before and after feeding the animals with bacteria, samples from the rumen are taken daily; the samples are loaded onto the above-described kanamycin-containing solid media, and the antibiotic-resistant and susceptible bacterial cells are counted. It also qualitatively and quantitatively determines the production of volatile fatty acids in the rumen. The use of ruminant food is known to be influenced by the fatty acid ratio (Eskelanda 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, an optimum ratio of acetic acid to propionic acid is considered to be 2.0 to 3.5 to 1, 3 to 3L and 4 to 1 for growth of "milk production and maintenance, as well as viability". Kaufinann and K. Eohr .; Influence of food on bacterial fermentation in pre-stomachs, Hanbuch der Tier- · nahrung, p. 263, Prey, Hamburg-Berlin, 1969 /.

According to a preferred embodiment of the method of the invention, the bacteria are isolated from the rumen samples, and the ability of the isolates to produce volatile fatty acids is preferred. The microorganism is grown under anaerobic conditions in the complete rumen-containing fluid medium described above, then acetic, propionic and butyric acid concentrations are determined in the cultures. Volatile fatty acid producing microbial cells in the desired ratios are genetically labeled and monitored for rumen growth.

Strains capable of growing in rumen-fed animals for at least 60 days and which can ferment dietary carbohydrates in an optimum ratio to volatile fatty acids are selected, cultured, isolated, maintained and deposited, and their cultures are desired. form acceptable to the animal; ruminants are administered orally to develop or control rumen. Three strains, Hh-GY0KI-1-123Sz, Hh-GYOKI-2-14Ab, and Hh-GY0KI-3-81-Me, capable of growing in the rumen of ruminants fed seneir, cereal flour, or melase, which could persist for a long time; in the rumen and favorably digestion, they were deposited in the Hungarian State Collection, Medical Bacteria of the State-of-the-Art of Hygiene under numbers 00287, 00288 and 00289 ·

According to the invention, it is preferable to cultivate microorganisms isolated from the rumen at a temperature of 32 ° C to 37 ° C under anaerobic conditions with exclusion of oxygen, in a medium containing carbon and nitrogen sources, inorganic

l. - 12 - salts, reducing agents and rumen fluid; the latter fluid is a source of growth factors.

As the carbon source, glucose, cellulose, serum, cereal flour or molasses can be used, while inorganic salts, yeast extract, casein and the like are suitable as nitrogen sources.

It is an essential feature of the invention that unicellular organisms are used, preferably: fermenting fed food and dose and capable of remaining in the rumen for a long time, to modulate rumen flora.

Genetic markers are used to select such strains, as described above, for example, genes conferring resistance to antibiotics, enzyme proteins, or other detectable proteins. Auxotrophic cells can also be used.

The genetic marker is introduced into the cell by a DNA strand molecule, for example by means of a plasmid or phage, but selectable characteristics such as antibiotic resistance can also be selected by means of selective selection.

Selected strains having beneficial fermentation characteristics and remaining in the rumen for a long time are used either to increase the development of rumen growth in suckling ruminant pups, or to advantageously modify the rumen flora composition. It is recommended that the product be administered with a potash or a drinking water.

The microorganisms selected for the preparation of the invention are cultured in a medium containing organic carbon sources, organic or inorganic nitrogen sources and inorganic salts, and then isolated in a form suitable for oral administration or trans-port. If desired, the culture of microorganisms is prepared by mixing with solid or liquid carriers or other additives. The preparation can be mixed with feed or drinking water or it can be fed alone. In the case of sheep, for example, 1 to 20 g, preferably 5 g, of the microorganism culture according to the invention is added about 0.5 kg of food. For example, maize mixes, alfalfa hay and livestock feed can be used as feed; the actual ratio is determined by reference to the actual conditions and the desired daily weight gain. Bovine animals are usually administered from 10 to 200 g, preferably? 50 g of a microorganism culture according to the invention, e.g. in a mixture with about 5 kg of the usual food.

The method of improving food use efficiency in ruminants is also within the scope of this invention.

After cultivation in a liquid medium, as described above, the microorganisms are separated by centrifugation or filtration. Pastes, lyophilized formulations or suspensions containing spores can be added with additives acceptable for animal production and nutrition. Other additives such as proteins, amino acids and glycerol can help maintain the viability of microorganisms. Other compounds commonly used in the art, such as antibiotics that stimulate the growth of the host animal (monensin, nigericin, salynomycin, etc.) or increase the residence of the fed microorganisms can also be added to the compositions of the invention used to improve food use in ruminants.

The microbial cultures of the invention allow the formation of a live microbial culture in ruminants, or a convenient modification of established flora. During the sucking period, flora is unable to efficiently ferment the usual food. Florase develops spontaneously and randomly, and her composition is not optimal for the host animal.

Feeding the selected microbial strains - 15 - instead of the slow and spontaneous development of rumen flora, rapid development can be achieved, and rumen flora capable of optimum food use. The advantage of the present process is that fastadaptation can be achieved by the microorganisms prepared in the manner described (e.g. strains 00287, 00288 and 00289); or the development of rumen flora during a change of food or weaning by increasing the growth of microorganisms in the tumor capable of optimal food degradation.

The method can be used, inter alia, in the following cases: - in dairy cows, changes in lactation, in congestion and during seasonal and other changes in dosage, - in cattle at the beginning and end of grazing, when fattening is changed to intensive by fattening flour from cereals and during other changes in the growth-fattening diet, - in sheep during seasonal changes, by feeding, at the beginning and end of the grazing period and during the beginning of intensive growth and fattening.

Similar possibilities are also available for goats · Micro-cultures prepared: the method of the invention can be used in several special cases,

L 1. <- 16 - wildlife, ruminants, game animals and field game.

It should be noted that although the microbial cultures used in the compositions of the present invention preferably come from the rumen, other bacteria which produce acetic acid and / or propionic acid, which may not necessarily come from the rumen, are also suitable. Such bacteria are certain members of the genus Angero-vibrio (lipolytica), Bacteroides, Selenomonas / ruminanticum and Propionibacteria.

The invention will be further explained by means of the following, but non-limiting examples. In detail, the preparation of microbial strains which are capable of utilizing base doses containing mainly cellulose / hay, starch / cereal flour and sucrose / molasses will be described in detail and persisted in the rumen for a long time. However, the use of the composition is described for sheep, but the scope of the invention also relates to organisms capable of growing on other feeds and to the development or modification of rumen flora in other ruminant species. * - 17 - Example 1

Modification of flora in rumen of hay-fed animals and / Isolation of microorganisms capable of growing in hay

The sheep were laparatized and provided with a rumen probe (felled) and fed for 1 month. A rumen sample is taken from the rumen, diluted and loaded with RGCA solid medium of the following composition:

Salt solution I:

Distilled water

Salt solution II: 0.6 g ad 100.0 g.

NaCl 1.2 g 1.2 g KI ^ PO * 0.6 δ CaCl2 0.12 δ MgSO4.7H2O 0.25 δ distilled water ad 100.0 ml / resazurin / 0.1% solution / 0.1 ml agar / Bacto / * 2.5 δ rumen fluid 10.0 ml glucose 0.05 δ cellobiose 0.05 δ hydrochloride hydrochloride monohydrate 0.05 δ - 18 sodium carbonate / 8% solution / 5.0 g distilled water ad 50.0 ml.

Note:

x / Difco Labs. Detroid, USA xx / A sample of rumen content is filtered through several layers of gauze, then the filtrate is stored under carbon dioxide at -20 ° C. The pH of the RCGA medium is adjusted to 6.8 before sterilization under carbon dioxide gas. Sterilization, medium preparation and culture are performed according to Bryant and Burkeye / J. Dairy Sci., 36, 205, 1953 / ·

Rumen fluid is diluted with a sterile mixture of the following composition: saline solution I / see above / 7.5 ml saline solution II / see front / 7.5 ml cysteine hydrochloride monohydrate 0.05 g Na2CO3 0.3 g resazurin / 0, 1% solution / 0.1 ml distilled water and 100.0 ml

This mixture is referred to as HB.

Cultures are incubated under anaerobic conditions at 35 ° C (see Atlas of Rumen Microbiology,

Ogimoto and Tmai, Japan Scientific Soeieties Press, Toky, 1981, for 120 hddin, then individualclones are inoculated into a medium containing hay extract of the following composition: saline solution I / see front / 15.0% saline solution II / see front / 15.0% resazurin / 0.1% solution / 0.1%

Tripton L42 / Oxoid / * 15.0% Yeast Extract / Oxoid / * 0.5% Rumen Liquid * 3 * 10.0%

Na2CO3 0.4% Cysteine hydrochloride monohydrate 0.05% Hay extract *** 10.0%.

Medium designation: RGCF liquid medium (Oxoid Ltd., London, UK) See front rkk For fine hay extraction, hay is cut, the particles are suspended in water, boiled and filtered. The filtrate is added to the medium prior to sterilization. Before sterilization, the pH is adjusted to 6.5 · r z

Tubes containing 5 ml of sterile medium are inoculated with a microbial suspension obtained from individual clones grown on RGCA solid medium and incubated under anaerobic conditions at 35 ° C. Growth of 20 - is checked by microscopic examination and the culturease is applied to solid RGCA medium where 2.0% of Bacto cellulose is replaced by glucose and cellobiose.

The cultures are incubated under anaerobic conditions at 35 ° C for 120 hours, then the resulting individual cellulose cell clones are seeded on RGCA medium containing cellulose.

Thus, rumen microorganisms capable of growing on hay or cellulose can be obtained. b / Genetic labeling of rumen bacteria capable of growing on the hay

Genetic labeling is performed with the plasmid p110Efcoli according to Simon et al. /Why. 8th North Ameri-can Rhizobium Conference, Winnipeg, Canada, Univ. of Manitoba Press, 1983 / ·

Plasmid DNA was isolated from E.coli Birnboim and Doly / Nucl culture. Acid. Res. 7, 1513 (1979) and dissolved in an aqueous solution containing the following components: 75 mM CaGlg 5 mM MgCl 2 10 mM tris. HCl buffer, tris (hydroxymethyl) -aminoethane hydrochloride. - 21 -

Hay-growing microbes isolated from A / Example 1 are grown on RGCF medium and separated by centrifugation under carbon dioxide. Cells are suspended in an aqueous solution containing the following components in 1 liter: 75 mM CaCl 2 mM MgCl 3 10 mM cysteine monohydrate 1 mM sodium thiosulfate o

The suspension should contain 5 x 10 5 cells per ml. The suspension is diluted with an equal volume of solution containing plasmid DNA (0.1 µg / ml) and incubated for 60 min at 4 ° C. Thereafter, incubation is continued at 41 ° C for 2 minutes, then the culture is loaded onto solid medium containing 500 µg / ml kanamycin B and cellulose. as a carbon source. The culture is incubated at 35 ° C for 120 hours under anaerobic conditions and clones capable of growing in the presence of 500 µg / ml kanamycin B are tested.

Plasmid p1011 carries kanamycin and chloramphenicol resistance-determining genes; longer, the replication device includes replication to allow replication to begin in E. coli cells. If it is transferred to other bacteria due to the lack of water source, it is possible to use it. <.- '. 3V <·'. By replication, the plasmid DNA is either eliminated or incorporated into the chromosomes (partially or totally) and genetic recombination is taking place. The resulting gene incorporated into the chromosome is displaced and confers cell resistance to kanamycin and chloromycetin. In the experiments of the present inventors, kanamycin B resistant clones of trans-5 formation frequency of 3x10 · were obtained.

Several resistant clones have been isolated and antibiotic sensitivity has been \ t

* Initial, to kanamycin B resistant / Km / strains. The results obtained with several strains are reported in Table 1.

Table 1

Kanamycin B sensitivity in rumen bacteria and genetically labeled strains and degrading hay

Microbes Smallest effective kanamycin B concentration, yUg / ml rumen fluid 31 initial strains 4.0 to 7.5 genetically labeled Km strains

Hh-GY0KI-l-8 500 - 23 - continued table 1 27 250 91 500 123 1000 142 500

In this way, it is possible to obtain rumen-derived organisms that are able to use hay or cellulose and dehydrate in the presence of high amounts of kanamycin B.

Strain Hh-GYOKI-1-123 / Km® / was applied to RGCAmedium containing cellulose, followed by the addition of kanamycinB at 1000, 5000 and 10,000 µg / ml. Cultures are incubated under anaerobic conditions at 35 ° C for 168 hours, and isolated by strains growing in the presence of 10,000 µg / ml of antibiotic.

Thus, spontaneous selection of highly resistant to kanamycin B is obtained by spontaneous selection. One of these strains was designated Hh-GY0KI-1-123Sz and deposited in the Hungarian State Collection; of medical bacteriaState Hygiene Institute of Budapest under number 00287. C / Reanimation of labeled microbes

The sterile solid medium, designated RGCFα, was inoculated with the Hh-GYOKI-1-123 / Km® strain culture on RGCAI agar at +4 ° C. The composition of RGCFa media - 24 - is as follows: 1. KgHK ^ 0.3% 45 ml of solution 2. / NH4 / 2SO4 0.6% NaCl 0.6% MgSO4.2H2O 0.06% CaCl2.2H2O 0.06% kh2po4 0.3% 45 ml solution mixture 3 · Cellulose / Bacto / * 1.8% fe i - Agar / Bacto / X 3.0% 65 ml mixture solution 4. yeast extract 0.1% 20 ml 5. cysteine solution. HCl.HgO 0.1% 20 ml of solution 6. Sodium thiosulphate 0.1% Na2CO3 0.2% 10 ml of Mixture Solution XX 7. Rumen Liquid 20 ml

Explanations:

x / Difco Labs. Detroit USA xx / see above These seven solutions are prepared separately and mixed in the indicated order.

The culture is carried out in 500 ml Erlenmeyer flask containing 150 ml of medium under anaerobic conditions. Growth is controlled for 48 hours, then mixed with hay-fed feed. g of a 340 ml culture containing 4.7 x 10 bacteria in ml - 25 - was administered orally to sheep. A 50 to 200 ml sample was collected by the rumen probe prior to administration and the following days. Samples are diluted with HB solution and annealed to RGCA medium containing no kanamycin B or other antibiotics. Cultures are anaerobic incubated at 35 ° C for 72 hours, and bacterial clones counted. Results are given in Table 2 »

Table 2

Changes in rumen flora in sheep after administration of the strain

Hh-GYOKI-1-123 / KmR / * ·

Sample Cell number / ml without antibiotics in the presence

1000yUg / mlkanamycin B Before administration 5x10 ° 0 After day 1 administration 5.9x106 2, Ox104 2, day 3.2x107 4, 1x104 day 3 2.4x106 3.1x104 6, day 3.9x107l, 8x104 day 8 9.8x10 6 l, O5x105 Day 15, lx105 3.1x104-26 - It can be seen from the table that the administered microorganisms persist and grow in the rumen.

According to the above method, the inventors also cultured the Hh-GXOKI-1-123Sz strain resistant to 10,000 µg / ml kanamycin H and orally administered the same sheep on day 15.

140 ml of the culture was administered orally.

2x10 bacteria per ml.

The rumen bacteria were cultured and counted as before. The results are shown in Table 3.

Table 3

Changes in floj $ e rumen sheep. after adding 'T?

Hh-GX0KI-l-123Sz / Km /

Sample

Cell number / ml without antibiotics in the presence of 8000 / U / ml

kanamycin B Before administration 1,4x10 After day 1 administration 7,4x10 day 2 1,7x10 0 3,2xl04 l, 3x104 t -27 - continuation of table 3 Day 5 4, Ox106 9, 1x103 7t day Ι, ΙχΙΟ7 2 , 0xl04 14th day 8.0x106 3.1x104 Day 21.7x105 6.2xlG4 Day 28 8.2x10 8, 1x104 Day 35 8.7x106.6.8x10 4 Data from Table 3 show that the administered microorganisms are present in considerable quantities in ba; ΪΙ i ’chora, and because the fluid phase of the rumen content ends; replenishes, there is no doubt that replicas will occur. more

Differences in the number of bacteria in individual j

samples can be explained by changes in the consistency of the rumen content from dense to fluidized state. Example 2! ξ [· her ·

Modification of flock flora of sheep fed fl

Cereal corner f m

'.....) N A / Isolation of microorganisms capable of growing under flour or

H from cereals g> b v

The method described under a / in Example 1 is repeated except that the initial sample is taken from a sheep flask fed with cereal flour, individual - 28 - the isolates are inoculated into RGCF liquid medium / front / containing 2% flour from cereals powdered in a mortar instead of extracted hay, cultured on RGGF liquid medium is applied on RGCA solid medium (see above) and clones derived from cells capable of; use cereal flour to isolate it with a similar medium ·

This provides microorganisms capable of growing on cereal flour as carbon sources. b) Genetic labeling of rumen bacteria using cereal flour

The method described under B / v of Example 1 is repeated except that the strains obtained according to A (in Example 2) are used for transformation with p110 µl plasmid DNA instead of those obtained according to A / in Example 2.

Resistance of several transformed strains

* D and Kin strains to kanamycin B are listed in Table 4 · * - lí 'li - 29 -

Table 4

Sensitivity of rumen bacteria and genetically labeled strains using cereal flour to kanamycin B

Microorganisms

The lowest concentration of acecanamycin inhibiting growth, µg / ml rumen liquid 31 initial strains 1.8

H Genetically Marked Km TribesHh-GYOKI-2-4 250 -14Ab 250 -37 250 -81 125

By performing the above method, rumen-derived microbes are obtained that are capable of using cereal flour as a carbon source and are 18 times more resistant to kanamycim B than rumen.

The strain designated Hh-GY0KI-2-14Ab was deposited in the Hungarian State Collection of Medical Bacteria, State Institute of Hygiene, Budapest, under number 02288. μ; hi - 30 - 0 / Restoration of genetically labeled microorganisms to the rumen

The process described under C / in Example 1 is repeated except that the sterile RGCFα medium containing 1.8% starch instead of 1.8% cellulose (Bacto) is inoculated, mixed with the sheep's food and sonicated daily; Samples were taken prior to administration and after administration of 310 310 ml culture containing 7.1x1010 bacteria / ml, the sheep were administered orally.

Table 5

Changes in rumen flora in sheep after addition of strain

Hh-GY0KI-2-l4Ab / KmR /

Sample Cell number / ml

Antibiotic free In the presence of 250 µg / ml kanamycin B Prior to administration 4i3x106 1,4x1ο1 After administration: Day 4.1.106 l, 8x103 Day 2 3.2x106 2.2x103 3 * day 8, Ox103 Ι, ΙΟχΙΟ3 Day 6 9, lx106 7, lx102 8th day 8,0x106 8, lxl03 - 31 - continuation of the table 5 15 * day 6,8x106 8,7x103 22nd day 5, Ox106 9,8x103 29th day 8, Ox106 Ι, ΙχΙΟ4 36. Day 9, 1x106 7, Ox103, Day 7, Ox106 7.9x10360 &lt; tb &gt; day 6, 1x106-7.0x103 The data in Table 5 show that the administered microorganisms remain and replicate for a long time in the rumen. Example 3

Modification of rumen ovc® fed to molassesA / Isolation of microorganisms capable of growing on molasses

The method described under A / in Example 1 is repeated except that the starting samples are collected from molasses fed sheep, individual isolates are inoculated into RGCF medium containing glucose instead of extracted hay, cultures grown in liquid medium are applied to RGCA medium, clones grown from molasses cells are inoculated into the same RGCA medium. - 32 -

Rumen microorganisms using molasses are thus obtained. B / Genetic labeling of molasses bacteria

The method described under B / in Example 1 is repeated except that the cells prepared according to A (in Example 3) are transformed with the plasmid plasmid DNA instead of the cells obtained according to A / in Example 1.

R

The resistance of some Km strains to kanamycin B is shown in Table 6.

Table 6

Resistance of rumen bacteria and genetically labeled molasses to kanamycin B

Microorganisms

The lowest concentration of cannabin B inhibiting growth of rumen fluid 31 by initial strains of 7.5

R genetically labeled Kin strains' Hh-GYOKI-3-2 250 -14 500 -34 250 - 33 continuation of table 6 81Me 500 -132 500

This yields highly resistant kanamycin B and molasses isolates.

The strain designated Hh-GY0KI-3-81Me is deposited with the Hungarian State Collection of Medical Bacteria of the State Institute of Hygiene in Budapest under number 00289. 0 / Re-introduction of genetically labeled bacteria into h

rumen. Her

The method described under C / Example 1 is repeated except that the Hh-GY0KI-3-81Me strain is seeded into RGCFA medium containing 1.8% glucose instead of 1.8% glucose instead of 1.8% cellulose, and spheres The mixture is mixed with a diet for sheep fed with molasses. 380 ml of a culture containing 1.6x10 6 bacteria / ml is administered orally. One sample is taken daily from the rumen probe before and after administration.

- 34 -

Table 7

Changes in rumen flora sheep after podhhh-GY0KI-3-81Me / KmS /

Sample

Cell number / ml

Without antibiotics In the presence of 500

/ Ug / ml kanamycinB Before administration 5,0x10 ° 0 After administration 1, day Ι, ΙΟχΙΟ6 l, 9xio5 day 2, 8x107 2,5x105 day 3 6,2x106 6, 1x105 6 · day 8, 1x106,7xl05 8th day 6,2x106 9,1x10 15x day l, 3x103 l, 3x105 22nd day 3, Ox106 3, 1x105 29th day 1,1x106 7, Ox105 36th day 8,0x105 9,1x104 43th day. 6,1x106 8,1x105 60th day 6,8x106 δ ', ΙΟχΙΟ ^ MM · _ M - MMMM M «... M MMMM«. MMM MMMMMMMMMMMM · M-WIM - WTtm-WMM ·

Note: x / sample error - 35 The data show that the administered microorganisms remain in the rumen of molasses fed for a long time.

Changes in volatile fatty acid ratios by bacterial preparation

Two sheep are fed complete doses for 14 days and rumen samples are taken from the rumen · The rumen liquid is filtered through several layers of gauze. The appropriate residue is suspended in a liter of phosphate buffer (see below) and mixed as before. Combine the two filtrates, leave to stand for two hours, remove the solid mass on the surface and use the liquid phase for testing.

The composition of the physiological buffer is as follows:

Na2HPO4 0.316 &amp; H KH2P ° 4 0.152 g / 1 NaHCO ^ 2.260 g / 1 KC1 0.375 g / 1 NaCl 0.375 g / 1 MgSO2 0.112 g / 1 CaCl2.H2O 0.050 g / 1 PeSO4.7H2O 0.008 g / 1

1 and 36

MnSO 4 .H 2 O

ZnSO 4 .7H 2 O

CuSO4.5H2O

CoC12.6H20 0.004 S / 10.004 &amp; / 10.002 g / 10.001 g / 1

The pH of the mixture is determined, and if desired, the pH is adjusted to 7.2 with aqueous hydrochloric acid or sodium hydroxide solution (Cheng et al., J.Dairy Sei). 38, 1225, 1955. An equal volume of physiological buffer is added to the obtained mixture and 4 g of the dose are suspended in 1 liter of the diluted mixture. 30 ml of each suspension is poured into a 100 ml Erlenmeyer flask. 200 doses were sterilized and 200 others were not.

Sterile media and media containing live rumen bacteria are inoculated with bacterial strains to be monitored for production; acetic, propionic and butyric acids.

Bacterial strains capable of persisting for a long time in the following are monitored; In addition, bacterial strains isolated from the rumen fluid of the sheep are also fed: fed complete or any dose according to A, B and C of Example 1.

The bacteria to be monitored are cultured on RGC + CG medium (see below) under anaerobic conditions at 37 ° C for 48 hours. RGC + CG medium composition: salt solution I (see Example A Example 1) 15% Saline II Solution / See A Example 1/15% Trace Element Solution x 0.3% Yeast Extract / Oxoid / 0.5% Rumen Filtered Liquid 10.0% Na2CO3 0.4% cy s te in>. HCl. HgO 0.05% sodium thiosulfate 0.008% cellulose / Bacto / 0.3% glucose 2.0% x Composition of trace element solution: ZnClg 40 mg CuC12.2H20 10 mg disodium tetraborate / decahydrate / 10 mg ammonium molybdate / tetrahydrate / 10 mg FeClyoHgO 200 mg MnCl2.4H2O 10 mg deionized water and 1000 ml

The cultures are inoculated into medium prepared by Erlenmeyer flasks. * Two ml of each culture - 38 - are inoculated in two parallel flasks of 50 ml medium.

The flasks are incubated under anaerobic conditions for 40 hours. Growth is stopped by 10% formic acid, and the content of volatile, fatty acids in cultures is monitored.

The cultures were filtered through gauze layers and centrifuged at 4000 rpm. for 15 minutes, then filtered again and loaded onto a Carlo Erba GI-452 gas-liquid chromatograph equipped with a flame ionization detector to determine C 8 -C 14 fatty acids.

Column temperature: 150 ° C

Separation column: 2 m length, 4 mm width / internal diameter / glass tube filled with 10% ethyl glycol adi-pent and 2% o-phosphoric acid on silane silica gel carrier / particle diameter 0.2-0.3 mm /.

Injector temperature: 190 ° C

Nitrogen flow: 50 ml / min

Hydrogen current: 50 ml / min

Air flow: 200 ml / min

Paper movement: 160 cm / hr.

Chromatography duration: 20 min. »

Sample volume: lyophil. - 39 - Three measurements were made for each sample.

The standard solution contains acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid. Of the sheep fed complete doses, the isolates were more than 90 strains of bacteria. The strains were then labeled genetically and the strains were monitored / repeated several times. Representative results are shown in Table 8.

Explanation of the abbreviations used in Table 8: S: vaccinated after sterilization NSi culture containing live rumen flora was vaccinated a / trace amounts of b / Z negative control: content of volatile fatty acids in medium prepared from rumen fluid, physiological buffer and feed used for, piece / average of 12 measurements /; c / positive control: the content of volatile fatty acids of the incubated culture containing the initial: rumen rash, and otherwise prepared in the same way / mean of 12 measurements /

However, 5 ppm of monensin sodium / mean of 6 measurements were added to the medium; however, 10 ppm of sodium sodium / mean of 6 measurements were added to the medium. 41

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Hh-GYOKT-126 S 0.56 0.60 and 0.52 aa 0.93 NS 0.83 1.14 and 1.55 0.11 0.70 0.73 - 44 - Data from Table 8 show that ratios of volatile fatty acids produced by fermentation by rumen flora can be modulated widely by the administration of microbial cultures prepared. according to the invention. For example, the production of propionic acid can be significantly stimulated by a culture prepared from the Hh-GY0KI-48a strain, whereas the Hh-GYOKI-109 strain stimulates the production of acetic acid. Stimulating production. Individual fatty acids were observed in a medium lacking (S / i) containing / NS / rumen vermin. In our experimental system, monensin reduced the acetic acid to propionic acid ratio by 0.1 or 0.2 (d, e).

Microorganisms selected as described above are genetically labeled, administered to ruminants and tested for ruminant growth and persistence by repeating the procedure described in b) in Example 1. Cinemas with advantageous fermentation and long rumen survival will be administered orally for the production of volatile fatty acids. - 45 - 5

Bacterial preparation for oral administration to ruminants

The bacteria to be administered are cultured on RGCA = CO medium (Example 4) under anaerobic conditions as described. After cultivation, cells are separated by filtration or centrifugation.

Separated cells were suspended in physiological buffer (Example 4) and lyophilized. the lyophilized weighing bacterial preparation is stored, suitably adjusted, and orally chewed.

The microorganisms can be cultured in another commonly used medium, for example in a medium containing glucose and starch, etc. as a carbon source and inorganic salt as a source of nitrogen. The composition can be administered easily by mixing it with food or drinking water, alone or together with other biologically active substances, for example antibiotic and vitamin.

Except lyophilized preparation; other products can also be prepared. Microorganisms can also be administered after mixing filtered or skimmed

Terial masses with a suitable carrier or diluents, for example calcium carbonate, concentrates, premixes and others.

The bacterial strain (s) is selected from microorganisms prepared according to the method of the invention and preferably rumen-improving flora, and the amount thereof to be fed is determined in a dose-dependent manner; and animal consumption. If a reduction in the ratio of acetic acid to propionic acid is desired, a culture prepared from the Hh-GYOKI-48a strain may be used, but the administration of the Hh-GYOKI-3-81Me strain is recommended to increase this ratio.

Determining the desired amount of microbial cells cannot be a problem for a person skilled in the art. Do you recommend administering 5x10 ^ to 5x10 cells? microorganisms per ml of rumen fluid. Example6

Administration of Hh-GY0KI-48a and Hh-GY0KI-1-123S strains from sheep

The Hh-GY0KI-48a strain is cultured on RGCA-CG medium (Example 4) in 5 liter fermenters / useful volume / at 37 ° C under anaerobic conditions. Permentacese is started by inoculating 10 milliliters of a culture similar to the composition, and the PQ of the 48 hour culture is centrifuged.

- 47 -. II nímturn / 5000 rpm / and wet sediment weighs! |

- - '': ‘. ; 58 g is mixed carefully with 4 kg of corn-flour. she eats

The mixture is divided into eight: equal parts and orally? ií ί

«Ί L serves eight sheep, which have been starving for 24 hours. The Hh-GYOKI-1-123Sz strain can be used similarly, with the

. · IN terial yield of 53 g · 1? 'Ϊ? In a growth fattening experiment, 23 sheep were fed ad libitum by hay from lean grass and the animals were weighed 5 weeks each week. Eight sheep experimental groups were fed one of the bacterial preparations, each for a single feeding and seven sheep 1 (served as a control. Daily 600 to 900 g of hay plus mineral and vitamin was consumed) pre-mix mixed with maize flour (100 g) The results are shown in Table 9 and Table 51. ia O o »· · v v v:: * * * M M M M M M M M M M M M M M 04 04 04 04 04 04 04 Aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 04 04 m 04 04 04

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- 53 -

Sheep fed Hh-GYOKI-1-123Sz and Hh-GYOKI-48a control groups had an average of 2620, 3875, and 360 g in lean doses over a 35-day experimental period.

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

Table IX

Statistical Evaluation of Final Body Weights Control Hh-GYOKI-1-123SZ Hh-GYOKI- 48a Average / kg / 28.357 30.375 31.6875 Corrected Square Standard Deviation 1.476 3.910 2.281 p /% / <5.0 <0.1

i - 54 -

Table 12

Statistical evaluation of body weight gain. 5 weeks / kc> ntrola Hh-GYOKI-1-123SZ Hh-GYOKI- 48a number of animals total weight. 7 8 8 Ag / 2.5 growth 21.0. 31.0 Maximum Ag Increment / 2.5 4.5 5.0 Ag Increment / Average Increment -2.0 1.0 2.0 In Ag / Corrected Quadrate 0.3571 2.6250 3.8750 Standard Deviation 2.143 1,768 0,839 standard deviation + 1,355 + 1,244 + 0,857 The data show that the compositions manufactured according to the invention can significantly stimulate sheep weight gain, Example 9

The persistence of genetically labeled bacteria in bovine tumors

The method described under C in Example 1: 4 * ΐΧΛ «Λ« Λ> -7Ζ <* ’Μνκηρ ^ &amp; i

-55 is repeated: the difference that the Hh-GY0KI-1-123Sz strain, resistant to 10,000 µg / ml kanamycin, is cultured in 4 liters of RGCTa medium. After reaching the stationary phase (thirty-eighth hour), the culture is isolated by centrifugation (5000 rpm) and the cells are thoroughly mixed with 500 g of corn flour and cows are used. Samples are taken weekly from the sample probe, and the presence of the rumen strain is determined as described in Example 1. The results show that the Hh-GY0KI-1-123 strain grows in the rumen of cattle and can last for at least 40 days. i * 4 ij Η v

Effect of Hh-GY0KI-48a microorganisms on weight gain compared to negative and positive controls

The Hh-GY0KI-48a microorganism is grown in the manner described in Example 6, except that the rumen is not added to the nutrient broth. 41 g of bacteria in the form of wet paste, obtained after centrifugation, are mixed with 1.2 kg of corn meal and the material obtained is divided into 12 equal parts. In the same manner as in Example 6, except that 36 sheep are divided into three groups, positive controls are given 100 g of feed in addition to feed and hay. The results are shown in Table 13 below, in which n is the number of animals in the group. - 57 -

Table 13

Negative control group, n 12 number weight (kg) in week increment in kg: ii animal and 1 2 4 in 4 weeks 1 32,8 31,0 33,2 34,1 1,3 lJ 2 34,8 33, 0 35.4 37.0 2.2 si 3 26.7 27.4 28.5 30.1 3.4 4 32.5 35.4 36.3 35.7 3.2 1 Ě 5 32.6 30 , 6 32,0 34,0 1,4 F 6 35,0 35,1 34,9 35,2 0,2 ϊ 1 Π 3 $ j6 N 7 34,7 36,4 37,7 3,0 4 8 35.8 36.8 37.5 38.1 2.3 il 9 32.0 32.7 '34.0 35.0 3.0 H 10 30.0 30.3 32.0 34.2 4.2 11 34.0 33.9 35.5 36.6 2.6 i * ·, 12 35.8 35.0 36.1 38.0 2.2 i V 1 &amp; Φ Φ total 396.7 397.8 411,8 425,7 29,0 5 «£, t * average 33,06 33,15 34,3 35,475 2,4 her {- 58 -

Positive group, n = 12 number weight (kg) per week increment in kg animal 0 1 2 2 4 4 in 4 weeks 13 35,2 37,6 38,0 39,0 3,8 14 31,6 32,7 33 , 4 35.3 3.7 15 33.3 34.6 36.3 37.0 3.7 16 33.2 34.0 35.6 36.0 2.8 17 34.5 36.8 39.0 39.6 5.1 18 27.3 29.5 31.4 32.4 5.1 19 34.5 36.0 37.4 37.7 3.2 20 31.7 31.6 34.0 35 0 3.3 21 31, o 36.8 38.0 38.7 7.7 22 35.5 37.0 38.9 39.8 4.3 23 34.4 35.5 37.7 38.8 4 , $ 24 34.0 34.9 37.2 37.9 3.9 Total 396.2 417.0 436.9 447.2 51.0 Average 33.0 34.75 36.40 37.266 4.3 - 59 - experimental group, n = 12 • »<i increment of 4 weeks animal number weight (kg) in week 0 1. 2 4 25 33,2 35,9 37,4 • 38,1 4,9 26 33,6 35,7 37,0 37,9 4,3 27 34,7 36,4 37,4 38,0 3 3 28 33.5 35.0 36.8 37.3 3.8 29 28.8 32.0 33.2 34.2 5.4 30 27.6 31.9 33.4 35.0 7.4 31 32,0 32,5 35,5 36,1 4,1 32 30,5 35,6 35,9 38,0 7,5 33 33,4 33,7 34,5 35,3 1,9 34 31 3 36.0 37.3 38.9 7.6 35 34.7 33.5 34.1 36.8 2.1 36 31.6 34.8 36.1 37.2 5.4 Total 384.9 413 , 0 428.6 442.8 57.7 Average 32.075 34.42 35.7 36.98 4.8

MB ί «

- 60 Mathematical Analysis Results, - <;: J- J. and

Comparison between negative and positive control group by positive control and experimental group between negative control and experimental group S = + 1,236 S = + 1,664 S = + 1,613 ú = 1.9 Δ = 0,5 Δ «2,4 t = 3,766 t = 0,736 t = 3,645 p <0, 01 p <0.5 p <0.05 very significant weak significant

Notes: S, 4, t and p are statistical constants.S stands for standard deviation, 4 means difference in values for mean, t means t-sample, p stands for statistical significance. From the above results, the weight of the animals in the microorganism group during the course of 28 days is shown to be as follows: &lt; tb &gt; ______________________________________ &lt; tb &gt; days, calculated per animal exceeded the average weight of control animals by 2.4 kg. The increase in animals in the positive control group was on average 4.3 kg, ie. 0.5 kg less than the average increment of animals treated with microorganisms. This may mean that the administration of the composition according to the invention in the abovementioned amount to lambs while maintaining the increment can be saved per animal of at least 100 grams per day. Appendices 1 and 11

Process for the preparation of the microorganism composition HH-G®0KI-48a A · Method of making the lyophilized composition.

The OE-950 paste obtained by the method of Example 6 after 100 g centrifugation is lyophilized in a lyophilizer

(Labor MIM, Hungary), then 54 g of the product thus obtained are mixed gently with 486 g of wheat germ, and placed in a container or bag, impermeable to bacteria. In this way, a composition containing 10% of the active ingredient is obtained. B. Process for preparing a composition comprising a wheat germ carrier 100 g of wet microorganism paste as obtained by the method of Example 6 are mixed with 100 g or 900 g of wheat germs and then placed in a container or bag which is impermeable to bacteria. In this way, a composition containing 5 or 10% of the active ingredient is obtained. C. Preparation of a composition containing 100 kg of bentonite powder "Veegumneutral" (Lehmann und Voss Co., Hamburg, Germany) as carrierbentonite at 160 ° C for 2 hours, then placed in a mixer at room temperature typeLodige íM 130 (manufacturer: Gebruder Lodige Maschinenbau

I - 63 -

GmbH, Paderborn, NSR) The mixer is brought into the feed and 10 kg of the microorganism suspension obtained by the method of Example 6 is fed through the liquid inlet. The obtained microorganism dispersions and bentonite are dried at 30 ° C until the water content is 6 to 8% and then placed in containers or bags which are not permeable to bacteria. 5 g of the product thus obtained are stored at 4 DEG C., room temperature at 37 DEG C., samples are taken on days 0, 20, 51 and 72, and the composition is suspended in a sterile HB composition with the composition given in Example 1 and after appropriate the dilution is inoculated on RGCA agar, also described in Example 1. The semicroorgahisms are then cultured for 46 hours under anaerobic conditions, and the amount of colony formed is counted. The results obtained are shown in Table 14 below.

- 64 -

+

Taboo 1 to 14,

Stability of the bentonite-containing product as carrier

Temperature Number of bacteria / g of product on day 0 20 51 72 4 ° C - 4.3 x 107 room temperature 2 x 108 6 x 107 37 ° G 3 x 107 2.5 x 108 8 x 108 9 x 106 8 x 107 9 x 106 5 x 10 6 From the data shown in the table it is evident that the bacterial content of the bentonite-containing composition varies very little over the course of the aforementioned measurement. D. Process for the preparation of a composition containing 100 kg of colloidal attapulgite "Pharmasorb HVM" "(Chemie-Mineralien KG, Burden, NSR) is sterilized for 2 hours at 160 ° C, after

Cooling down to room temperature, the Diosna P-25O-A type mixing device (manufacturer: Dierks Sohne Maschinenfabrik, Osnabruck, Germany) is placed at room temperature. The mixer is operated with a liquid inlet of 10 kg of suspensions of microorganisms prepared as described in Example 6.

The thus obtained dispersions of atapulgite and microorganisms are dried at 30 ° C until a water content of 6% is reached and then stored in containers or bags which are impermeable to microorganisms. 5 g of product are stored at a temperature of 4 ° C, at room temperature and at 37 ° C, whereupon samples are taken on days 0, 20, 51 and 72. The composition was suspended in HB-sterile with the composition of Example 1 and seeded on RGGA agate after appropriate dilution, as described in Example 1. Microorganisms were grown for 46 hours under anaerobic conditions and then the amount of colony formed was counted. The results obtained are shown in Table 15 below.

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The stability of the composition according to the invention, which comprises a carrier and / or

Temperature Number of bacteria / g of product on day 0 20 51 72 4 ° <3 2 x 108 1,5 x 107 3 x 107 temperature

7 R room 9 x 10 '3 x 10 9 x 107 5 x 108

37 [deg.] C. 2 x 10 &lt; 8 &gt; x 10 &lt; 8 &gt; x 10 &lt; 8 &gt; K &lt; tb &gt; &lt; tb &gt; &lt; tb &gt; &lt; tb &gt;

The products produced according to this example in paragraphs A to D

They may also be supplemented with other carriers, such as batter flour, crushed maize sticks, ground calcium carbonate, dibasic phosphate, or other substances commonly used in the manufacture of feed additives and other feedstuffs. using it adds to the feed, feed or drinking water.

Claims (4)

preservatives used in animal feed and nutritional substances commonly administered to ruminants, characterized in that it contains up to 99 wt. at least one microbial culture from strains in microorganisms deposited in the Hungarian State Collection of Bacteria of the State Institute of Hygiene under numbers 00287, 00288 and 00289, capable of adjusting the weight ratio of acetic acid to propionic acid to 1.5 to 4.0: 1; growth in the rumen and stay there for at least 60 days, the remainder to 100 wt. they form simple feed components such as hay, forage and molasses. 2. A composition according to claim 1 comprising a microbial culture capable of adjusting the ratio of acetic acid to propionic acid in ruminants to 2.0 to 3.5: 1. 3. A composition according to claim 1 or 2, wherein the composition comprises a paste of microorganisms, a lyophilizate or a suspension. 4. Method for preparing microbial cultures of strains. microorganisms used as active ingredients in the mixture according to items 1 to 3, characterized in that they are removed from the rumen the animals fed with food or feed containing cellulose, starch or mono- and disaccharides samples, investigates the semi-metabolism of microorganisms isolated from the in vitro in vitro sample and the microorganisms adjusting the acid: acetic acid ratio to 1.5. up to 4.0: 1 are cultured in an environment containing the same food or feed as carbon or nitrogen, antibiotic resistance is introduced into the growing microorganisms as a genetic marker, allowing for selection, genetically labeled strains are cultured, cultures are again; they are fed to the rumen by feeding the food or feed, samples are collected from the rumen, the number of cells of the genetically labeled strain is counted, the sections are separated for at least 60 days, and these are treated in a form acceptable in, livestock and nutritional practice. £; j t t E