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Document Type and Number:
WIPO Patent Application WO/2015/041556
Kind Code:
The present invention relates to the use of carbon dioxide, in two different aggregate states, i.e. the use of carbon dioxide in solid state (dry ice), or gaseous state in the process of conserving / ensiling feeds.

IVETIĆ, Aleksandra (Teodosijeva 1, Beograd, 11000, RS)
Application Number:
Publication Date:
March 26, 2015
Filing Date:
September 17, 2014
Export Citation:
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IVETIĆ, Aleksandra (Teodosijeva 1, Beograd, 11000, RS)
International Classes:
A23L3/34; A01F25/16; A23B7/14; A23K3/03
Domestic Patent References:
Foreign References:
Other References:
MIROSHNIKO A Y A ET AL: "Conservation of animal feedstuffs - by breaking up material, loading it into container layer by layer and admitting carbon di:oxide", WPI WORLD PATENT INF, 1980, XP002948969
MUCK RE ET AL., J. OF DIARY SCI., vol. 90, 2007, pages 5115 - 5125
FILIA I. ET AL., JOURNAL OF DIARY SCI., vol. 90, 2007, pages 108 - 5114
CAI ET AL., J. DAIRY SCI., vol. 82, 1999, pages 520 - 526
MOON NJ, J. APPL. BACTERIOL., vol. 55, 1983, pages 453 - 460
HU ET AL., J. DIARY SCI., vol. 92, 2009, pages 3907 - 3914
MUCK ET AL.: "Silage Science and Technology", 2003, pages: 251 - 304
Attorney, Agent or Firm:
PLAVŠA & PLAVŠA D.O.O. (Patentna kancelarija, Strumička 51, Beograd, 11050, RS)
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Patent claims

1. A method of conserving / ensiling feed using carbon dioxide, characterized in that the carbon dioxide is in a solid state (dry ice), or gaseous state and comprises the steps of:

- Insertion of feed in silos and compaction of feed-plants;

- Addition of carbon dioxide;

- Covering the silos.

2. A method according to claim 1 , characterized in that the carbon dioxide is in the solid state in the form of pellets 16 mm in diameter.

3. A method according to claim 1-2, characterized in that the carbon dioxide in solid state is added in layers.

4. A method according to any one of claims 1 to 3, characterized in that the carbon dioxide in solid state is added in the lower layer.

5. A method according to any one of claims 1 to 3, characterized in that the carbon dioxide in solid state is added in the upper layer.

6. A method according to any one of claims 1 to 3, characterized in that the carbon dioxide is added in solid state in the lower and upper layer.

7. A method according to claim 1 , characterized in that the carbon dioxide in solid state sublimes and displaces the air.

8. A method according to any one of claims 1 to 7, characterized in, that after insertion of carbon dioxide in a solid or a gaseous state provide anaerobic conditions in period of 0 to 2 hours.

9. A method according to any one of claims 1 to 8, characterized in that there is slowdown development of undesirable micro-organisms and a faster development of lactic acid bacteria.

10. A method according to claim 1 , characterized in that the carbon dioxide in a solid or gaseous state is added through a perforated tube placed vertically or horizontally, depending on the state of the carbon dioxide, prior to silos closing.

The method and the use of the additives in feed preservation

Technical Field

The present invention relates to the use of carbon dioxide, in two different aggregate states, that is, the application of carbon dioxide in solid form (dry ice) and in gaseous state, in the process of conserving / ensiling feed.

According to International Patent Classification invention has the following labels: A23K and A23K 3/00 3/03.

Technical problem

The problem being solved by present invention is to provide, in a short period of time, anaerobic conditions for the growth and development of LAB (lactic acid bacteria) and lactic acid fermentation in silage with reduced loss of nutrients, as well as adaptation to the conditions in each farm individually depending on its capabilities in a completely new and safe way with regard to human and animal health, as well as on the protection of the environment.

State of the art

The current state of the art is reflected in the use of biological or chemical additives for plants ensiling. On the market are the most common biological additives, which according to the manufacturer's specifications represent different inoculants strains of heterofermentative and homofermentative lactic acid bacteria - LAB. There are a number of such products, which are intended to increase the number of LAB in silo mass and thereby encourage the channeling of silage fermentation to lactic acidic fermentation (Muck RE et al, 2007, J. of Diary Sci., 90: 5115-5125). The use of additives is intended to create the final product of fermentation, especially lactic acid which is used as a preservative of silage, and then the acetic acid which has a useful role in the duration of the aerobic stability (Filia I. et al., 2007, Journal of Diary Sci. 90 : 5108-5114).

In order to efficiently conservation of silage green mass of plants and reducing the loss of nutritional value during aerobic degradation, leading biotech companies produce microbial additives with a very high concentration of lactic acid bacteria. For example, the number of LAB in such products ranges 1x10 11 CFU (colony forming units), and for 10 tons ensiling green forage is necessary to add 10 g of such additives. Due to the successful preservation of silage depends primarily on the ability of lactic acid bacteria to produce enough acid to stop the growth and activity of undesirable microorganisms under anaerobic conditions (Cai et al, 1999, J. Dairy Sci., 82: 520-526). Lactic acid has a weak fungicidal properties, in contrast to acetic and propionic acid. Therefore, the microbiological composition of the additives is different, i.e. that may be inoculants heterofermentative or homofermentative LAB, or a combination thereof. For example, when the inoculants of heterotrophic LAB are added to the green mass to ensile, aerobic stability of the silage is improved in addition to the production of lactic and of acetic acid (Moon NJ, 1983, J. Appl. Bacteriol. 55: 453-460), whereas with specific microbial inoculants, such as L.Buchneri, the concentration of acetic acid is increased (Hu et al, 2009, J. Diary Sci. 92: 3907-3914).

Other present products in the market can be classified into chemical additives, which represent different acids, which act in the first degree as fungicide, preventing the development molds and yeasts. Because of the negative side effects on the nutritional value of the silage, as well as the characteristics of the very aggressive acids during handling, chemical additives are less frequent.

Patents which describe the use of biological additives in the form of inoculants of LAB are : CN101940272 which describes the use of probiotics, JP200805833 describes adding yogurt as a scrap of food industry, CN103060228, CN102994423, CN102851233 relating to the use of homofermentative LAB, and different strains of Lactobacillus plantarum ; WO2008073853, WO2008073848, WO2008073844, WO2008073841 , WO2008073839, WO2008073839, WO2008073835 regarding the use heterofermentative LAB, and different strains of Lactobacillus Buchneri; AT507290, WO2013001862, KR20120126917, US201 1 142991 , NZ591040 relating to the use of a combination of homo-and heterofermentative LAB.

Patents which describe the use of chemical additives are: DE102007008804 describes a combination of various acids, JP2008199943 which discloses the use of amino acids, while the patent US2008317934 describes the use of propionic acid, and WO2013056829 and WO201 1050478 describe the use of enzymes. Patent CN103053808 and CN10305161 relating to the use of an extract from different plant species, whereas in patent UA28261 straw is added, and in the patent GR1007162 molasses is used in the preparation of the silage. Clime is one of the factors that affect the process of ensiling. The increase in temperature of the environment leads to additional heating of the silo mass, which is one of the risks where the transformation of the present sugar and heat generation, resulting in an increase in temperature (Kunkle et al, 2006, UF / The Inst. Of Food and Agric. Sci. (IFAS), SS-AGR- 177). If the temperature reaches 50 ° C during aerobic activity Maillard reaction can occur, which reduces the digestibility of proteins in silo mass (Muck et al, 2003, Silage Science and Technology, Inc., Madison, Wl, USA, p. 251-304). Also, exposure silo mass to the air extends the activity of harmful microorganisms, such as yeasts and molds, and thus the development of the LAB is disposed, which leads to a deterioration in the quality of silage.

WO 2000045645 describes ensiling in silage bales with a high content of SM over 60% and the use of liquid injector for introducing C0 2 into bales. The main disadvantage of the WO 2000045645 patent is uneconomical. Specifically, it is necessary to separate more funds for the purchase of additional equipment and special machinery for harvesting plants, while in the present invention used existing silo facilities on the farm, with no additional investment. Further, in the aforementioned patent, have been used commercially available additives, without specifying the origin, which represents disadvantage because it can not be concluded certain advantages. Also, the difference is in the fact that it uses liquid C0 2 . All this is from an economic point of view, one major drawback and difference, which is solved by the present invention, because represents ease application and the farmer does not invest in additional equipment, i.e. in its current silage production should not change anything.

WO 9418847 uses blocks of 5 kg and 1 kg, whereas in the present invention were used pellets of a diameter of 16 mm and a length of 8 cm, in order to be as small as possible a layer of dry ice, between layers of the plants, because it avoids the formation of air pockets during the sublimation. Also, the amount of dry ice used is 1-6 kg / 1 of plants, while in the present invention uses the percentage of amount which is calculated according to the dimension of the object, the amount of ensiled plants, feeding plants value and its chemical parameters and what is most important is that the application tailored to the specific conditions at each farm and its possibilities. But the main disadvantage of the above prior patent WO 9418847 is used inoculant that masks the independent effect of C0 2 . Also, the addition of inoculants does not exclude the initial phase of respiration where oxygen is still present and trapped between plant parts and to work undesirable microorganisms that consume nutrients and oxygen use. As long as all the oxygen is not consumed by undesirable microorganisms, they work, grow and develop in the silo mass, degrading its quality and what is left of nutrients after them to use inoculants, and at this stage it is idle because it is waiting to anaerobic conditions. Based on the above, we see that he does not teach nor suggests the application only C0 2 in solid or gaseous state.

Brief description of the invention

The present invention relates to the preservation of animal feed and presents an inventive element in the process of ensiling plants. It makes a big difference in the appearance of the commercial application of additives and is an important finding, which essence is reflected in the ease and simple application, effectiveness and safety. The novelty of the invention and application of the new technology is the use of C0 2 in different aggregate states during the ensiling, acts immediately, there is no waiting for the completion phase of aerobic respiration, because in the only 0-2 hours provide anaerobic conditions for growth and development of lactic acid bacteria that are present in the epiphytic micro flora of the plants (on the surface of green plants). In this way, the initial nutritional values of the plants prior to ensiling are preserved during the conservation, whereas the present invention specifically reduces the loss of dry matter during the conservation process. Extremely favorable effect of C0 2 is to highly slowing down the appearance of molds and yeast, and in this way extends the length of preservation of plants in silos. Also, the losses are minimized at the upper corners of the silo, since the time of formation of the initial anaerobic phase rapidly formed and thus eliminated the development of undesirable microorganisms that cause feed poisoning at animals feeding with bad quality of silage. Anaerobic stability of the silage has a prolonged duration time and up to 80% extension comparing with the silage without application of invention technology. Silage has an excellent quality class after 8 and 18 days of opening of the silo, and the silo opening with a routine of the 40th day. This is particularly useful on a farm where, in the case of deficit of feed, preferably silos has to be open earlier than 40 days, which has so far not been possible because of the time needed to conduct quality lactic acid fermentation and stabilization the processes of conservation.

In high summer ambient temperatures and frequent drought conditions, in the period May - September, when the silage (once a year) is preparing, adding C0 2 in the solid state is very useful, because dry ice, whose temperature is -79 ° C cooling and prevents heating of silage and since there is no oxygen prevents auto ignition of silage.

Brief description of the figures

Figure 1 - graphical presentation of addition the dry ice in three layers, each layer in one third of silos during the filling with plants Figure 2 - graphical presentation of addition the dry ice in a single layer on the lower third of silos during the filling with plants

Figure 3 - graphical presentation of addition the dry ice in a one layer on the lower and the upper third of silos during the filling with plants

Figure 4 -graphical presentation of addition the dry ice in a single layer on the upper third of silos during the filling with plants

Figure 5 - graphical presentation of addition the dry ice though a perforated tube placed vertically, at the final stage of filling the silos with plants

Figure 6 - graphical presentation of gas C0 2 insertion through two perforated tubes placed horizontally, at the final stage of filling the silos with plants

Figure 7 - Speed diagram of achieving the anaerobic conditions and fermentation with use of additives and present invention in the preservation of animal feeds

Detailed Description of the Invention

Preserving feed for feeding domestic animals is used in agriculture around the world. Plants after harvest or mowing are transported from the field to the farm, inserted into the silo, compressed to a displacing the air, and then with the special foils covers the silos order to create the conditions for fermentation progress. Silage is created by the conservation of plants with spontaneous lactic acid fermentation. Preservation of plants is done with the acid that is made with activity of LAB during the spontaneous lactic acid fermentation. The basis for successful conservation of plants in the form of silage is an anaerobic environment. This condition is required for activity of LAB, because they produce lactic acid in the course of their metabolism. The produced a sufficient amount of lactic acid is used as the preservative, lowers the pH of the initial value in green plant and helps to maintain the nutritional value of silage plants. If oxygen is present in the middle of silo mass, it favors the development of harmful aerobic microorganisms such as molds and yeasts which cause spoilage and deterioration quality in process of ensiling, due to such animal feeding with bad silage quality results in poisoning of animals.

Aerobic phase starts immediately after harvest and continues until the oxygen is present in ensiled plants. During this initial phase, which is also called the phase of respiration plants, herbal sugar in freshly cut material are decomposed by heat creating. Aerobic microorganisms (yeasts, molds and aerobic bacteria) are present in the chopped plant material and also use the sugars in the initial phase of respiration. Progressive and rapid development of yeasts and molds, during this phase, leading to warming of silo mass, which has a negative consequence on the end of the process, at the opening of the silo and animal nutrition, and result, is the poor quality silage for animal feed. The process of respiration reduces silage quality because in this stage is highly digestible energy and reduces the amount of nutrient materials necessary for the growth and development of beneficial LAB in the conservation process. Therefore, the farmers at the opening of the silo had to throw away spoiled parts of silage. Common for this stage is that plant cells consume 0 2 and simple carbohydrates are transferred into carbon dioxide and water. Changes are followed by heat, which caused heating silage; if the material is poorly compacted with more air it is possible auto ignition and loss in dry matter (DM), which has a negative effect on the fermentation. Silage temperature is always higher in the middle and lower parts of the silo due to greater retention of heat in silo mass as well as due to higher microbial activity in these areas. Such microorganisms (present in aerobic phase) produce a lot of carbon dioxide, but slightly acetic acid.

During the first two days after ensiling aerobic bacteria disappear; if anaerobic environment is achieved, while Gram-negative and coliform bacteria can be grow and development until the end of the first week of the ensiling depending on the increase or decrease of the pH value. Therefore, when the oxygen is present in the silo mass, metabolism (respiration and proteolysis preferably) of undesirables microorganisms followed by the plant cells of plant enzyme activity is extended.

Taking into account the chemical changes in the chopped plant material filling into silos and present microorganisms, there are 6 phases of chemical changes in the silage. Phases of chemical changes during ensiling plants are: 1) respiration of plant material, 2) the creation of acetic acid, 3) the creation of lactic acid, 4) settling the fermentation process, 5) if the ensiling process was not properly implemented - the creation of butyric acid and 6) aerobic stability.

The first phase of respiration begins immediately because oxygen is present, which allows the growth and development of various MO (microorganisms) that are present on the plant and represent competitors to a LAB, using for their metabolism nutrients that is necessary to preserve during conservation. At this stage with the presence of oxygen the most tumultuous changes are happened (which it cannot quite squeeze with traditionally technology of ensiling), so that oxidative processes are enabled. Consumption of air which was trapped in the silo mass during the initial respiratory is the trigger that activates the beginning of anaerobic fermentation phase and is the first phase of the production of acetic acid and immediately after the lactic acid and respectively lactic acid fermentation phase begins. Preferably due to Enterobacteria which are tolerant to an increase in the heat (in the aerobic phase), producing a number of different products. These bacteria ferment carbohydrate soluble in water (WSC) and hexoses (glucose and fructose), producing: Volatile fatty acids (VFA ' s) a short carbon chain - acetic, lactic and propionic, ethanol and C0 2 . Heterotrophic bacteria of the second phase are insufficient fermenters because they produce very little preserving acid in exchange for the loss of nutrients. The ratios of the creation of acetic acid, in this stage depend on the maturity of the plant, content of moisture and the epiphytic population of bacteria. Enterobacteria are sustainable at pH 5-7, and their work formed VFA ' s that reduce the pH value below pH 6. Therefore, heterofermentative bacteria are inhibited with acids they produced. Enterobacteria create a path to a LAB, which is sustainable at a pH below 5. The drop of pH value indicates the end of the initial anaerobic phase, which typically lasts for 24-72 hours.

Phase of lactic acid fermentation is the largest and the most important for the preparation high quality silage and preserving the nutritional value of ensiled plant. In the process of ensiling major and necessary product of microbial activity is lactic acid, which is a preservative in silage. Lactic acid is dominant in silages best quality (more than 60% of the total VFA ' s), and present is at the level of 3-6% dry matter (DM) in silage with good quality. The dominance of LAB (usually containing strain Lactobacillus plantarum) as compared to other Enterobacteria and bacteria that produce acetic acid, the fermentation process are faster, with a more conserved WSC and nutrients, peptides and amino acids. In natural fermentation with epiphytic MO and without additives at ensiling, the duration of this phase is from 8 days to 3 weeks. Such a large time ratio, depend on the buffer capacity of the plants, the moisture content and the maturity of the crop which is to ensile.

LAB can be divided into four main groups: lactobacilli, streptococci, pediococci and leuconostoc. Growth of Lactobacillus in the substrate is often improved by the anaerobic environment comprising 5-10% C0 2 . LAB can tolerate a temperature range of their growth between 2-53°C and most optimum is 30-40°C, are more sensitive to high temperatures than lower in areas where growth. For the isolation of strains of LAB, liquid glycerol is used at a temperature of -80°C and the temperature of the dry ice -79°C, with no side effect on their potency. The optimum pH value for its growth is in the range from pH 6.2 to 5.5 to the lower value. LAB species important for silage

A. Homofermentative lactobacilli: Lb. casei, Lactobacillus coryniformis, Lb. curvatus, Lactobacillus plantarum

B. Heterofermentative lactobacilli: Lb. brevis, Lactobacillus Buchneri, Lb. fermentum, Lactobacillus viridescens

Homofermentative LAB ferments one mole of hexoses (glucose and fructose) and provides two moles of lactic acid, while fermentation of pentose provides one mole of lactic acid, and one mole of acetic acid. Heterofermentative LAB are less efficient in terms of conversion of carbohydrate, fermentation of 1 mole of glucose provides one mol of lactic acid, acetic acid (a part in the aerobic stability), or ethyl alcohol and carbon dioxide.

Fermentation phase of settling, (stable phase) occurs when lactic acid its reached maximum in the silage crop mass, and caused the lowering of pH below 4.2. Further development and biochemical activity of lactic acid bacteria is slowed and almost development of the others anaerobic bacteria completely stops. Aerobic microorganisms cannot develop due to lack of oxygen, which is partially depleted by the respiration of plant tissues, and partly dissipated with development of aerobic and facultative anaerobic microorganisms, in the first and second stage of ensiling. The first two phases have short duration, one to two days, if are applied correct technique of ensiling. The third and fourth phases are longer, 15-20 days, which makes the process of fermentation completed in about three weeks. Absolutely calming is with about six weeks and then the silage can be used as animal feed. Stable phase lasts during storage. This phase is not static due to various processes that can take place, depending on environmental conditions such as air penetration or the number and type of MO present on epiphytic microflora in the plants before ensiling. The amount of fermented substrate stays at the same level and type of fermentation acids produced in the silage.

If the plants for ensiling have moisture content higher than the recommended during ensiling there is a possibility for a subsequent fermentation. Silage with Clostridia (CI. Botulinum) has a higher content of butyric acid, which is a preservative for inhibiting the activity of yeasts and molds, but is not desirable. If the process of conserving silage fails completely and the pH is above 4.6 to 4.7, may be further developed microbial transformation and degradation. A typical " Clostridia silage " has a higher content of butyric acid (more than 5 g / kg DM), high pH (over 5) and a high content of ammonia and amines. The method of ensiling, which quickly and sufficiently lowered pH of silage have a role in the prevention of the development of Clostridia because similar to Enterobacteria, its growth are inhibited at lower pH values. Also, Clostridia are most sensitive to the low value of water activity (a w ) in contrast to the LAB.

Phase of aerobic quality degradation of silage starts immediately when the silos are opened for animals feeding and silage is exposed to air. During the feeding of animals, this stage is inevitable and occurs in all silage regardless of its quality. It consists of two stages. The first is the beginning of the deterioration due to the degradation of protective organic acids. Increasing the pH begins the second phase of decay in which the temperature increases and the speed of growth of microorganisms. Aerobic degradation occurs in the all quality types of silages, which are open and exposed to air. Air (oxygen) is a major cause of deterioration of silage, because it makes possible unwanted chemical and microbiological activity, which leads to deterioration of silage. The air trapped in the silage, as well as the penetration of air into the silo and silage air exposure during feeding are responsible factors that lead to aerobic deterioration of silage. Up to 50% loss of dry matter (DM) is caused by aerobic degradation on the surface of silage. Some indicators of aerobic degradation of silage can be seen during a visit to the farms. For example, color silage smell (moldy smell or not), visible fungus (possibly yeast), a sense of high temperature, wet silage as a result of aerobic instability of some of the parameters that can be easily seen on farms.

Minimizing exposure to air silage is the main exponential factor for good quality of silage. Therefore, each practice that helps to exclude oxygen from the silage is an important factor to avoid the inhibition of the growth and development of yeasts and fungi.

Because of all this facts, on the basis of the present invention there is a solution that leads to changes in the initial phase of respiration by shortening, so as to speed the beginning of anaerobic phase, which contributes to faster development of lactic acid bacteria and reduces the losses. This technology results preservation of nutrients in silage (for animal feeding), that are not consumed in the initial respiratory aerobic phase for development and growth of undesirable microorganisms which cause spoilage and degradation of quality.

The essence of the present invention, which provides the anaerobic conditions, is the addition of carbon dioxide (C0 2 ), in solid (dry ice) or gaseous aggregate state during ensiling feed, during the filling the silos with green plants in silos, compacting and covering the same silos. Addition of C0 2 in the form of dry ice, which immediately sublimed from solid to gaseous state, or by direct insertion of C0 2 in the form of gas, resulting in ejection of air and oxygen, from the silo mass, because the C0 2 gas is 1.67 times denser than air, fall on the bottom of silos, thereby creating an anaerobic environment conducive to the growth and development of LAB. Invention procedure with direct insertion of C0 2 acts immediately to silo weight, because there is no waiting time for growth of LAB and phase of aerobic respiration, but in the course of only 0-2 hours to ensure anaerobic conditions. Invention provides 50 times faster formation the anaerobic conditions in the silo, compared to the traditional technology of ensiling, where anaerobic phase occurs in the silage mass plant for 100 hours on average in good condition, and this fact highlights the difference and present invention advantage, in the time provided for growth and development LAB and securing desirable lactic acid fermentation.

C0 2 from the ecological aspect is especially important because it is a harmless material, as well as all its forms created by dissolving, natural ingredients and as such does not change the quality of animal feed. C0 2 gas is a colorless, odorless and tasteless, does not burn or supports combustion, is present in the atmosphere. Created by the combustion of hydrocarbons and occurs in three states of matter, gaseous, liquid and solid. Solid C0 2 is in the form of dry ice, the freezing point is -79 0 C. C0 2 is obtained by processing natural gas and industrial waste gas, so its price in the market is very low. Dry ice is industrially produced by separating, purifying and compressing C0 2 from waste gases or from natural gas. Ecological environmental good side is reducing the emission of C0 2 into the atmosphere and the greenhouse effect. Transport of C0 2 in the form of dry ice is carried out in specially constructed and insulated containers for storage, which are standardized and are widely used in other industries, where there is minimal loss of weight of dry ice, which is a day only 1-2% sublimation depending on the volume and quantity of dry ice in a container. The equipment is easy to operate and consists of gloves and metal bucket for taking the dry ice from the container. The additional investments on the farm are not necessary in terms of acquisition of equipment or devices for application of C0 2 .

According to the present invention, the influence of C0 2 in solid or gaseous was investigate on the quality of maize silage hybrids PR32D12 (H1) and PR33T56 (H2) which are prepared separately in separate silos with the addition of C0 2 in solid (dry ice), 100 g per silo, with a different type of treatment, with direct or indirect contact with the silage mass of C0 2 . According to the present invention, the influence of C0 2 in gaseous state was investigate on the quality of maize silage hybrid PR33T56 (H2) which are prepared in separate silos with the insertion gas C0 2 . After 8, 18 and 40 days of ensiling, silage silos were opened (with dry ice and with gas C0 2 ) and analyzed the chemical parameters on the quality, i.e. determine the content of dry matter (DM), crude ash, crude protein (CP), crude fat (EE), insoluble fiber content of neutral detergent (NDF) and insoluble fiber in acid detergent (ADF), lignin (ADL) and is also determined the content of lactic, acetic and butyric acid. In all of the experimental silages (other than the control and the treatment with the gas) was added to 100 g of dry ice. It was used in the form of dry ice pellets with a diameter of 16 mm, which were chopped to 0.5 - 3 mm, and added before ensiling. Maize hybrid is also investigated for gas insertion during ensiling. Silage is stored in a temperature of 26 ± 2 ° C, in order to avoid the influence of the outside temperature oscillations.

On the farm, while ensiling plants, pellets are used in their original form, with a diameter of 16 mm and length of 8cm, because it is unnecessary to cutting. On the farm, all silos tanks were exposed to all the temperature changes of the environment, and opened the 8, 18, 40 and 100 days per charge and coverage.

The application of indirect adding dry ice using anaerobic silo with a double wall, 1.5 I internal silos, which was placed in 3 I external silo tank, with leaving the space for dry ice addition, between the internal walls, was investigated. The inner tank is perforated with holes whose diameters were 5 mm on a side. The monitoring of changes during fermentation, 8, 18 and 40 days of H1 was ensiled in 18 plastic anaerobic silo with a double wall, A and B treatment. In treatments A, were added a further 30 g of dry ice, then after 24 hour and whole closing procedure was repeated. Control silage, without dry ice, were placed in the inner tank with no holes. After closing, tanks were placed separately in plastic bags with an aluminum layer. This implementation is not suitable for practical use on the farm because it requires a special adaptation of the existing silos and additional investment. Also, the further adding dry ice after 24 hours have not practical for use on the farm and the achieved results did not indicate a statistically significant difference compared to the addition of dry ice just before covering the silo, in one term single addition of dry ice.

Example 1

One embodiment of the present invention represents direct addition of dry ice in the ensiled green plants, in this case corn hybrid H1 , which is ensiled in 12 separate anaerobic silos (4 treatments in triplicate, 12 per hybrid), volume 1.5 I. Treatments for H1 were: control (no dry ice), adding 3 layers of dry ice (33.33 g in each layer charge). Silos were open at 8 and 18 days, after silos covering. The results of this method are shown in Table 1.

Based on the above, we see that the treatment of dry ice had higher crude protein content which is of great importance in animal nutrition because they reflect its higher nutritional value than the control treatment without dry ice. Table 1 : Chemical parameters of nutritive value of corn silage hybrid PR 32D12 (H1 ) treated with dry ice and the silo opening at the 8 and 18 days

Example 2

Another embodiment of the present invention, is influence of direct addition of dry ice in the ensiled feed, in this case corn hybrid H2, which is ensiled in a separate 12 (four treatments in triplicate, 12 per each hybrid) anaerobic silos, volume 1.51, for 40 days . Treatments for H2 were: control (no dry ice), adding layers of dry ice (33.33 g in each layer charge) three layers, dry ice at the bottom of the jar (100 g) one layer, the addition of dry ice in the bottom and upper third (50 g in each layer of filling) two layers . The results of those methods are shown in Table 2.

Table 2: Chemical parameters of nutritive value of corn silage hybrid PR 33T56 (H2) treated with dry ice and the opening of the silo 40th day

Example 3

Further embodiment of the present invention relates to testing the difference between the application of the C0 2 , in a solid and gaseous state, where the analysis of inserted gas C0 2 into the silo plant mass was performed. It was used 9 anaerobic silos, volume of 1.5 I (three treatments in triplicate, 9 per hybrid). In the treatment of the gas, after filling of the silo with the green maize mass of H2, C0 2 gas is inserted through a perforated tube which is placed horizontally. In the treatment of indirect addition of dry ice through a perforated tube, silos, dry ice was added through perforated tube placed into green mass before next plant filling and closing the silos. In the treatment with a direct application of dry ice on the top of the silos, dry ice was added directly (100 g) in the last third at the top, in one layer, before the closing the silo. The results of those methods are shown in Table 3.

Table 3: Chemical parameters of nutritive value of corn silage hybrid PR 33T56 (H2) treated with dry ice and insertion of C0 2 gas, with the silo opening at the 40th day

Based on the above, i.e. the fact that the present invention decreases the aerobic phase due to sublimation of the solid C0 2 (dry ice) or in the form of gas C0 2 and displaces the air, allowing the formation of anaerobic fermentation, according to the diagram, we can see that the aerobic phase lasts only 0-2 hours, less than a day, and then immediately begins fermentation phase accompanied by anaerobic conditions (indicated by the start arrow), where the curve of LAB growing is exponentially, while the curve pH value decreases exponentially during the first days, which means quick action of C0 2 and its effectiveness in the occurrence of the fermentation stage during the first day.

Analyzed parameters of estimated nutritional value indicate preservation of the nutritional value of the plants during fermentation. Content of ash was not increased compared to the initial content in the fresh plants before ensiling, suggesting that the losses left behind, and all this accompanied by content of DM, with fluctuations of only 0.5%. The contents of crude protein and crude fat were increased in relation to the content in the fresh plant, compared in this case fresh maize plant before ensiling in an oscillation of 0.5-1 % by varying the mode of application of C0 2 in solid or gaseous. Also, the results of the fiber content indicate that there is a degradation and splitting the cell wall of plants in the treated maize silage, because the lignin content in the treated silage decreased by an average of 40% compared to fresh plant before ensiling, which directly reflects increasing its nutritional value, because the indigestible part of the of the plant is lignin. Based on the above, it can be concluded that the application of the present invention increase the preservation and utilization of nutrients and digestible energy of ensiled plants, and thus the present invention can be used in silage which requires opening the silo after only 8 days of ensiling, as is the case in the absence of feed on the farms. Furthermore, silage treated with dry ice or the gas C0 2 has an extremely longer duration of aerobic stability which protects them from degradation in the quality after the silos are opened and exposed to air, which is a great advantage of the present invention.

On the farm metioned embodiments are applied, with a much smaller percentage of the share of dry ice or gaseous C0 2 during the application, because the share amount of plants are larger and heavier that ensile at farms , putting further pressure for the air displacement. An accurate evaluation of the applied amount of C0 2 in a solid state (dry ice), or gaseous state depends on various factors and directly is determined on the farm. The amount to be used in the preservation ranges from 0,001% in the larger silos with a larger amount of plants or from 0.04% in the smaller of the silo, percentage of the amount of entrapped air within the compacted silage. Determination of amount using C0 2 in solid or gaseous state and for specific the plant species, on the particular farm relay on the following factors:

- The weight of the plants that ensilaged;

Dimensions of the silos;

Dry matter content of fresh plants;

The ratio of height of the silo and a maximum height of silage;

Rate of plant filling silage in the silo;

Speed of covering silage;

The duration from the first to the last plants filling in silos;

The weight of the tractor which is used in compression of silage;

Time for compression the silage;

Weather conditions (rain or dry).

Respectively, the present invention is adapted to the specific circumstances on the farm, as well as to the conditions of the outside temperature. In the event of a prolonged period of filling the silos when delivery of the fresh plants from field to farms is prolonged or when due to bad weather conditions ensiling last more than one day, the method of application C0 2 in the gaseous state is the more appropriate. However, in the case of higher content of dry matter in the plant by 40% (ideally 35% dry matter), recommend method is the addition of C0 2 in the solid state in three layers, due to higher lignin content. If there is a weaker compression is used in the silos, then the recommended method of the present innovation is application of C0 2 in the solid state (dry ice), 0.04% of the volume of entrapped air in a one layer in the lower third or in the upper third layer during the filling of the silos with fresh plants. In addition to the above facts, the applied methods of the present inventions will depend also on the manner and mode of covering the silos on the farms.