FI112161B - Increasing nutritional value of fibrous crops with cellulase - Google Patents

Abstract

The nutritional value, to animals, of a crop is improved by treating with a cellulolytic enzyme compsn. (A) having no enzymatic activities deleterious to the crop. Pref. (A) has been fractionated on an anion exchange resin (AER) to remove the detrimental enzyme components. (A) are produced from commercial cellulase enzyme mixts., e.g. from Trichoderma longibrachiatum, by fractionation on Q-Sepharose AER at pH 8 and conductivity equiv. to 20 mM Tris buffer. The material fraction A) not retained under these conditions was further fractionated on CM-Sepharose; it had esterase; carboxymethylcellulose (CMC); xylosidase; arabinosidase; glucosidase (cellobiose); and xylanase activities.

Description

112161

Enzyme products suitable for use in improving the nutritional value and durability of fiber plants

TECHNICAL FIELD The present invention relates to an improvement in the nutritional value of fiber plants for use in ruminants and monogastric animals, which is achieved by treatment with novel enzyme preparations. More particularly, the invention relates to the fractionation of cellulase enzymes derived, for example (but not necessarily) from Trichoderma species, to increase their efficacy in improving the energy and microbial energy content of the digestive tract and / or the digestive processes of monogastric animals and ruminants; monogastric animals do not have an enzyme system capable of utilizing fiber. In addition, said novel enzyme products can be used to treat fiber crops during conservation with beneficial effects on the silage production process, as well as for the treatment of preserved fiber plants and non-fibrous by-products such as citrus pulp, cotton, etc., etc. ·· forage crops. The invention also relates to processes for the preparation of said enzyme products.

Background of the Invention

As fiber plants mature, the energy production per unit area per unit area increases, but the utilization of energy, i.e., cellulose and hemicellulose, decreases due to the plant lignification process whereby the cellulose chains of plant cell walls are converted to lignin through complex cross-linking. This results in the degradation of the dry matter components in the animal's digestion '. significantly reduced. Thus, even though economically 35 harvest should be harvested at maturity when dry> * * »· material yield is at its peak, the 2 112161 constraint on energy recovery forces the harvest to be harvested at a more immature stage, resulting in reduced dry matter yield and possibly less due to. By cleaving 5 these cross-linking enzymes between the cellulose units can allow harvesting at a later stage of maturity and thus increase energy production without the usual degradation of digestive degradation. The same process can also make sugars available to feed microbes, thereby improving the resistance of plants with low dry matter content to adverse weather conditions.

preservations

Preservation of fiber plants (grasses, legumes, cereals -15 maize, sorghum, etc.) for future use as animal feed is basically either by dehydration of water (hay, etc.) or by preventing air from contacting the pulp and acidifying the pulp (epi-20 physical) polluting microorganisms (yeasts, molds and bacteria) and

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• * the enzymatic activity of the material. In practice, a pH lower than about 4.2 is required, which is achieved either by adding acids to the plant or by the acids produced by the fermentation of the epiphytic Γήινο ι · 25 organisms.

: · Current environmental concerns make the addition of acid unsatisfactory. Environmental concerns are increased by commonly used acids such as formic acid.

. These acids increase the production of acidic effluent from 30 wet fiber plants.

*; * The alternative is to resort to natural fermentation. However, natural fermentation produces mixed results which are sometimes inadequate. For example, unwanted epiphytes may dominate the desirable lactic acid bacteria in fiber plants containing small amounts of sugar substrates. Desirable lactic acid bacteria, i.e., homolactic bacteria, produce mainly lactic acid. This lactic acid production lowers the pH without significantly damaging the value of the feed, especially the quality of the protein. In contrast, with undesirable epiphytes 5 predominantly, the pH only decreases slowly and may not reach a sufficiently low value. This consequence for feed quality is then detrimental to animal behavior.

The situation can be improved by adding enough desirable lactic acid bacteria to the fiber plant 10 to dominate the epiphytes. However, this procedure is not helpful when the level of sugar in the plant is low. Water-soluble carbohydrates required by lactic acid bacteria can be added to the plant. For example, molasses, starch or sugars such as glucose, lactose or sucrose could be added. However, this approach causes other problems. Generally, a large amount of sugar needs to be added, i.e..

10 - 20 kg sugar / tonne and 35 - 45 kg molasses / - 20 tonne. It is difficult to apply such large amounts of said viscous agents to the plant. It is also difficult ··: to add evenly dry matter. Some additives, such as '# ί glucose, lactose and sucrose, are too expensive to use *' ··. Besides, lactic acid bacteria do not use V 25 starch effectively unless amylase is present: * to convert starch to sugar.

Alternatively, enzymes may be used to break down the complex structural carbohydrates present in fiber plants into simple sugars.

\.! Lactic acid bacteria are able to use * · released sugars and dominate fermentation. U.S. Patent 4,751,089 to Heikonen et al. Discloses a method for preserving feed and cereals by the addition of glucose oxidase. Glucose oxidase produces soluble carbon * * · I ». 35 glucose-containing gluconic acid. Accumulation of gluconic acid lowers the pH. Other enzymes such as cellulase, hemicellulase and β-glucosidase can be added to increase glucose production, according to the aforementioned U.S. Patent No. 4,112,161.

The use of cellulose-degrading (cellulolytic) enzymes for preserving and improving the nutritional value of a preservative feed, and for improving palatability, digestibility, and rate of digestion of processed feed in ruminant digestion is also disclosed in U.S. Patent Application 5,106,2006. The enzyme composition preferably contains at least one of the enzymes pectinase, cellulase, xylanase, amylase, arabinosidase, cutinase, lipase and esterase and may be used in combination with homolactic bacteria.

However, cellulose-degrading enzymes can have undesirable side effects when added to fiber plants which contain little (i.e. less than 25%) dry matter, such as immature plants. Cellulose-degrading enzymes can increase the amount of wastewater and its harmfulness. The effluent stream contains cells »I» ·! soluble materials from. The ingredients in question '. give high effluent BHK and can cause environmental problems. Wastewater also reduces the plant's: · 25 nutritional values. The said enzymes can also increase ·· lactic acid values and alter the structure of the fiber to the extent. ''. that it weakens the function of the animal. In addition, higher levels of sugar in the enzymes may result in better yeast and mold growth.

Increased yeast and mold growth can reduce aerobic stability and produce harmful mycotoxins.

>: In addition, cellulases release sugars, which act as substrates and also for spoilage bacteria, especially '. clostridial species that useful lactic acid bacteria.

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Feed efficiency In ruminants, the efficiency with which the host animal utilizes the fiber depends on the efficient functioning of the rumen microbial population. The composition of said microbial population 5 depends on the feed. The end result of microbial action on energy sources is the generation of volatile fatty acids, which act as precursors within the host tissue to supply energy to metabolic processes and produce animal products such as milk, meat and wool. The efficiency with which the latter products are produced depends on the relative proportions of the volatile fatty acids, in particular acetic, propionic, butyric and valeric acids. Feeds with a high starch and / or sugar content promote the synthesis of butyric and propionic acid, whereas the fiber promotes the formation of acetic acid. The desired type of rumen fermentation depends on the desired animal product. Therefore, the ability to modify the substrate is of fundamental economic importance, especially the ability to modify the reactivity of the fiber in this regard, since the latter is the cheapest form of energy.

> «· ·· .: For monogastric animals, the fiber is not a complete source of * · ·, '·· energy, but is present in most starch sources, such as grains. Fiber is also important in maintaining normal bowel function. The latter is related to the reactivity of the fiber, for example the cation exchange capacity. The ability to release energy from the fiber portion of the diet and improve its reactivity is ... of great importance to the nutrition and health of monogastric animals.

There are two major problems with preserving low-solids feed and enzyme products to improve feed utilization. First of all • · »*. in an efficient silage production process, the enzyme should achieve the desired pH and hydrate content of lactic acid and carbohydrate, while minimizing the generation of wastewater and the growth of contaminating organisms.

Treatment of the feed with a ready mix of cellulose-degrading enzymes reduces the silage fiber content by rendering the polymeric carbohydrates soluble.

Effective digestion leads to the complete collapse of cell walls and, as a result, the generation of high sugar wastewater. During the storage of the feed, fermentation is accelerated, lactic acid builds up and the pH drops. Under these 10 conditions, bacterial growth is inhibited, but enzymes prevail over the production of monomeric carbohydrates, some of which are lost in waste water. Silage with high levels of lactic acid and readily fermentable sugars can be detrimental to the ruminant by causing lactic acidosis and indigestion.

Second, the amount and type of sugars available to the microbes present in the rumen should be optimized to ensure efficient function of the rumen and efficient utilization of the feed by ruminants.

The object of the present invention is to provide for use ..! · an enzyme preparation suitable for various plants, maturity and dry matter content, · lacking in known deficiencies. Specifically, it is an object of the present invention to provide • combinations of enzymes that provide useful alterations in the structure of plant cell walls that provide i · only the effective preservation of feed. sugars do not increase wastewater generation and do not promote the growth of clostridia, yeasts or molds, but> · '···' that are capable of altering the structure of plant polymers: they are more susceptible to hydrolysis in the rumen by enzymes and are more easily digested ' .

Another object of the present invention is to provide methods for preparing said enzyme products.

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Summary of the Invention

It has now surprisingly been found that the adverse effects of commercial products are due to the presence of certain enzyme combinations in the commercial grade 5 cellulases used in said products. By fractionating said commercial enzymes in accordance with the present invention, novel novel enzyme products have been obtained, each containing a plurality of different enzymes, each having its own characteristics based on which of the most suitable fractions for each particular use can be selected.

Separation methods useful in the practice of this invention include methods for dividing commercial cellulase compositions based on the ion exchange properties of the proteins. For example, chromatographic methods such as ion exchange chromatography are useful. Suitable resins are Q-Sepharose and Mono-Q (both available from Pharmacia).

Cellulase fractions, in various combinations, are useful for preserving fibrous fodder and improving feed utilization in ruminants and monogastric animals. Each cellulase fraction contains a characteristic set of enzymes, and each has its own · different effects on the fiber plant during feed storage as well as in the rumen and small intestine after ingestion. The fractions may be used alone or in combination and / or in combination with suitable beneficial microorganisms such as bacteria or yeasts to achieve various effects for each specific use. The products can also be used: in fortified feeds just before feeding.

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BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows a commercial cellulase distribution

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The absorbance of the eluate from 35 anion exchange columns at 280 nm.

8 112161

Figure 2 shows the isoelectric points of the proteins present in the commercial cellulase and in each of its four major fractions obtained by anion-exchange chromatography separation.

Figure 3 shows the absorbance at 280 nm of eluate from a commercial cellulase split and its four major anion exchange chromatography fractions on an analytical Mono-Q anion exchange column.

Figure 4 shows a commercial cellulase fraction A

absorbance of eluate from CM-Sepharose (Pharmacia) cation exchange column at 280 nm.

DETAILED DESCRIPTION OF THE INVENTION The invention relates to a method of improving the nutritional value of a plant in an animal, characterized by treating the plant with a cellulose-degrading enzyme composition, a fraction of Cytolase-123 that does not bind Q-Sepharose anion exchange resin at pH 8 and s with a conductivity similar to that of 20 mM tris buffer.

The invention also relates to a cellulose-degrading enzyme composition characterized in that the plant is treated with a cellulose-degrading enzyme composition, a non-binding fraction of Cytolase-123! · Q-Sepharose anion exchange resin at pH 8 and electrical conductivity equivalent to that of 20 mM Tris buffer.

; ··. Mixtures of cellulose-degrading enzymes which • · * ..! 30 are commercial grade (such as those sold by Genencor under the tradename Cytolase-123) typically contain a large amount of ·, · / enzymes. Commercially important cellulose-degrading enzymes include, for example, Trichodermat. enzymes from species such as Trichoderma longibrachiatum, i *, 35. Other mixtures of cellulose-degrading enzymes, such as those from bacterial or fungal sources, have similar properties.

The effect of enzyme fractions on treated feed and silage or activity in the animal, alone or in several different combinations, has been investigated by measuring a large number of parameters. The most important parameters for animal action are the amount and type of sugar released and changes in fiber structure that regulate the fermentation process and digestion in the rumen. By measuring the ability of a fiber to release sugars before and after the silage production process, the reduction in feed value during storage can be determined. If a significant amount of the structural carbohydrates is converted to acid during the preservation process 15, the conditioning is well done. On the other hand, it reduces the energy available to microbes in the rumen and thus limits the value of the feed. The ideal combination would be a product that only liberates enough sugar to ensure good feed preservation, and is a type of sugar that most rotting microbes are unable to utilize, such as xylose, while still: • improving the function of the rumen. Another important feature is also the wastewater production potential associated with • s- * · sugar release activity.

: * 25 Each of the resulting enzyme fractions contains a karakic terrestrial set of enzymes, and each has different effects on fiber structure, sugar release, * foraging, and wastewater generation.

Based on these characteristics, it is possible to choose the most appropriate fraction for each specific application. For fiber plants, i | should be used fractions which give a pH of about ***: 4.0 within 48 hours. Selected fractions that • t * can also be used. improve the feed value of fresh or hardened fiber plants. 35 before feeding. The wastewater problem can be solved by • »10 112161 avoiding fractions that cause the sugar to be released too effectively.

Particularly for the preservation of low solids fiber plants, the enzyme fractions B and C, 5 individually or in combination, would be the preferred choice, since they provide a sufficient level of sugar to promote favorable preservation fermentation without increasing the effluent stream. Fraction A increases, while causing a small increase in the effluent stream, the number of free ends 10 in the sugar chains and makes the fiber more susceptible to further enzymatic hydrolysis by microorganisms present in the rumen or in the digestion process of monogastric animals. Fraction A, alone or in combination with Fraction B or C or Fractions B and C, improves the microbial sugar intake of rumen 15. Fraction A also improves the reactivity of the fiber, alone or in combination with Fraction B or C or with Fractions B and C. Thus, although fractions B, C, and BC are preferred for low fiber dry plant resistance without wastewater effects, the addition of fraction A further improves the reactivity and feed value of the fiber.

• t · *: Where wastewater is not a problem, for example '·: if the dry matter content of preserving fiber crops is higher

, ·· 25% may be used in fraction A, alone or in fraction B or C

| 25 or in combination with fractions B and C, to improve both endurance * and animal performance. Mentioned. the combinations can also be used to improve the nutritional value and stability of a fresh or sustained fiber plant by pre-feeding treatment.

♦ · 30 For low dry fiber plants, the problem of wastewater can be solved by deleting • J! fractions A or D or by avoiding them. Verse D is the primary responsibility for the harmfulness of waste water. Fraction D also has deleterious effects on the release of sugar in that it converts? 1, 35 too much of the complex carbon structure to sugar. In addition, fraction D greatly reduces the activity of the fiber.

Where it is desirable to achieve a rapid improvement in sugar release, for example, prior to feeding the animal, Fractions A and D, and combinations thereof, are advantageous for treating fibrous plants for use in ruminants, particularly monogastric animals.

Thus, the enzyme products of the present invention offer several advantages. By using appropriate enzyme fractions, either alone or in suitable combinations (in combination with potentially useful exogenous microorganisms), major improvements are achieved in both fiber plant preservation and feed utilization and animal function. Four points are particularly worth highlighting: plant durability is improved, waste water problem can be solved, sugar release value can be improved, and fiber reactivity value can be improved using the enzyme products of this invention.

Example 1

Fractionation of Cytolase-123 »♦ •: Fractionation was performed on a 2 liter column containing Q-Sepharose anion exchange resin (Pharmacia).

• ·· For each run, 1 liter of Cytolase-123 (Genencor) containing: * 25 containing approximately 200 g of protein (based on absorbance at 280 nm ···) and pH 5.2 was adjusted to pH 8 and 20 mM Tris buffer solution by conductive ultrafiltration followed by dilution with water. The adjusted enzyme solution was fed to • 30 columns. The unabsorbed material (fraction A) was rinsed from the ·; * column with approximately 10 liters of 20 mM tris buffer.

The absorbed enzymes were eluted with NaCl solution (linear gradient 0 to 0.5 mol / liter) with a total amount of j. was about 10 liters. The eluate exiting the column. 35 were recovered. Bound fractions were eluted with 20 mM Tris-buffer (12121616 D). (Henceforth, these fractions are referred to as A, B, C, and D, respectively). Fractionation results are plotted in Figure 1.

Fractions A, B, C and D contained the following amounts of protein (based on absorbance at 280 nm): A, 27 g; B, 18.3 g; C, 47.6 g; D, 73g.

It is believed that the remainder of fraction D (i.e., the eluted portion of the buffer containing at least 0.3 mol NaCl / l) contained only insignificant amounts of protein material and that the absorbance observed downstream was caused by some low molecular weight UV light absorber.

The fractions were analyzed by isoelectric focusing in gels to separate proteins with 15 isoelectric points from 3 to 9. The results were used as fingerprints to compare the fractions obtained from different separation processes. The results obtained are shown graphically in Figure 1. The major fractions were also analyzed by analytical liquid chromatography on a Mono-Q column with excellent protein separation. The results are shown graphically in Figure 3.

4 ·: The material not absorbed into the Q-Sepharose resin (fraction A) was fractionated using a CM-Sepharose cation exchange column, ···. The results are shown in Fig. 4. Cytolase and Q 'S were detected by Q-Sepharose into 4 enzyme fractions, each containing several different proteins and enzymes. The distribution of the various activities in Cytolase and in the 4 fractions mentioned is shown in Table 1. The results show that Cytolase-123 is a very complex enzyme mixture and that even • · *.,! 30 fractions are multi-enzyme preparations. If desired, fractions • · *; can be further purified, for example, by chromatography '! > or by precipitation.

* i>: Alternatively, enzymes can be eluted • · ». using various combinations of pH, salinity, and buffers, concentration, and type. For example, 20 mM tris-

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the use of buffer at pH 7.6 resulted in fractions B, C and D

13 112161 for looser binding to the column so that they eluted at 20 mmol / l (fraction B), 115 mmol / l (fraction C) and 500 mmol / l (fraction D). However, altering said parameters did not change the order in which the fractions eluted from the column after fraction A was flushed. In an alternative procedure, the enzyme may be eluted using a similar but stepwise gradient of pH, buffer concentration, and salinity.

10 Unfractionated Cytolase and fractions A, B, C and D

was analyzed by analytical liquid chromatography using a Mono-Q column (Pharmacia). The results show that fractions B, C and D each contain more than one component. Refer to Figure 3.

Cellobiohydrolase I, cellobiohydrolase II, and endo-glucanase I were identified using antisera against cellobiohydrolase I, cellobiohydrolase II and endoglucanase I (see Table 1).

The material not absorbed into the Q-Sepharose resin (fraction A) was fractionated from a CM-Sepharose cation exchange column. using. Fractionation of Q-Sepharose fraction A on a CM-Sepharose column revealed (based on enzyme activity assays) that the material eluted at 4.1 ··· and 4.2 had esterase activity; the material eluting at points 4.3, 4.4 and 4.5 had CMC activity; : 1 material eluting at points 4.4 and 4.5 also had: xylosidase activity; the material, which eluted at approximately 4.4, also had arabinoidase activity; the material which eluted at * 1,30 at 4.6 also had glucosidase activity 'l' (cellobase activity); and the material eluting at 4.7 had xylanase activity.

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• HM O * * -X * 3µ <υ no * * * * (dfi cd i »-x * * -x * En Ed ι-j ucm uo 15 112161 FPU = filter paper unit - releases 1 micromole of reducing sugars from 50 mg Whatman No. 1 filter paper at 50 ° C and pH 4.8 for 2 minutes with a reaction volume of 2 ml.

5 CMC = carboxymethylcellulose unit - liberates 1 micromole of reducing sugars from 10 mg of carboxymethylcellulose at 50 ° C and pH 4.8 for 2 minutes at a reaction volume of 2 ml.

Cellobase unit - liberates 1 nanomolar of glucose in 10 seconds from 1.8 micromoles of 4-nitrophenyl-B-glucopyranoside at 50 ° C and pH 4.8 with a reaction volume of 2 ml.

Xylanase Unit - liberates 1 micromole of reducing sugars from 10 mg xylan per minute at 15 ° C and pH 4.2 with a reaction volume of 2 ml.

Arabinosidase unit - liberates 1 nanomolar of p-nitrophenol per second from 3.6 micromoles of p-nitrophenyl-α-L-arabinofuranoside at 50 ° C and pH 4.0 with a reaction volume of 2 ml.

20 Xylosidase unit - releases 1 nanomole of p-nitrophenol per second from 3.6 micromoles of p-nitrophenyl-B-D-xyloxy-pyranoside at 40 ° C and 5 reaction volumes | with 2 ml.

Esterase unit - liberates 1 nanomolar of p-nitrophenol in 25 seconds at 1.8 micromoles of p-nitrophenyl acetate at 50 ° C and pH 4.8 with a reaction volume of! 2 ml.

* * · * ** CBH I is cellobiohydrolase I, CBH II is cellobiohydrolase II and EG I is endoglucanase identified using anti-• * ·> * 30 serum.

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· ... · Fractions A, B, C and D were tested so that their relative concentrations were the same as in the original • I). ··· in Cytolase.

a «» 16 112161

Example 2

Effect of Cytolase-123 Enzyme Fractions on Fiber Structure and Chemical Composition of Tank Fodder 5 Tetraploid granules (fertilization at 120 kg N / ha) were manually cut using aseptic technique, transported cold, and the experiment started within 5 hours of surgery. The solids content was 13% by weight. The hay was cut with scissors into 1 cm pieces and placed in 10 plastic bags which then each contained 80 g of fresh hay. Duplicate samples were treated with all combinations of Cytolase-123 fractions A, B, C and D. After enzyme treatment, the hay was packed in 25 g portions into serum vials (160 ml), which were rinsed for 5 minutes with 15 anoxic gas and sealed with butyl rubber plugs. The flasks were incubated for 6 weeks at 30 ° C and then the fiber was extracted with neutral detergent:

20 ml of water were added to the hay flasks, shaken for 1 hour at room temperature, and the contents filtered through a glass sinter. The filtrate was used for many different assays and the precipitate was further extracted. Extraction of the precipitate with neutral detergent is effected. (I) Quantitatively suspended in 100 ml of a solution (pH 7) (pH 7) · 19.6 g of Titriplex 111, 3.6 g of Na2B407, 4.6 g

Na 2 HPO 4, 30 g of sodium dodecyl sulfate and 10 ml of ethylene glycol monoethyl ether, and the suspension was stirred for 40 min at 60 ° C; (ii) the extraction temperature I · j was raised rapidly to 75 ° C and the suspension was filtered hot using a glass sinter; (iii) the precipitate was then washed with 100 ml of water by shaking with water for 15 min at 60 ° C, raising the temperature rapidly to 75 ° C and filtering the suspension; this extraction with water »». 35 were repeated; (iv) the precipitate was then extracted with 100 ml of brine containing 3.64 g of KNO3 per liter, 17112161 of 1.76 g of KCl, 0.17 g of NaNO3, 1.92 g of Ca (NO3). ) 2-4-20 and 1.96 g Mg (NO3) 2-6H2O; (v) after the temperature dropped from 60 ° C to 40 ° C, the suspension was filtered; (vi) extraction with water (step iii) was repeated twice; (vii) the precipitate was extracted with 75 ml of acetone for 15 min and filtered; the latter was repeated Four times; and (viii) the resultant fiber (NDF) extracted with neutral detergent was dried over vacuum with a sintered glass sinter.

1. Effects of Enzyme Fractions on the Composition of Liu-10 Silage Components The D and L-lactic acid was analyzed using the enzyme assay equipment manufactured by Boehringer Mannheim GmbH and the protocol proposed by the manufacturer. Ammonium was analyzed from alkaline samples using an ammonia-specific electrode.

The soluble protein was analyzed by the Lowry method. Soluble sugars were analyzed as trimethylsilyl derivatives by GLC using a capillary column and temperature programming. Two internal standard standards (erythritol and phenyl-β-D-glucopyranoside) were used.

»T ·: Table 2 summarizes the results obtained.

• · 1 · *

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* · »I ·> t t I · f 1 · • · • · · 112161 18

Table 2

Effects of enzyme fractions, alone and in combination, on the composition of soluble silage constituents 5

Enzyme Residual Ammonium Soluble Lactic Acid Mixture of Sugar (pmol / g DM) Protein Form D (mg / g DM) Proportion (%) in Silo D + L 10 (mg / g DM)

Zero 0 140 50 46 ABCD 20 119 49 48 ABC 5 110 58 51 15 ABD 21 119 60 54 ACD 15 156 58 51 BCD 44 151 57 45 AB -7 154 54 48 AC 2 172 53 48 20 AD 22 186 57 50 BC 22 174 58 46 BD 45 147 54 55 CD 13 151 57 47 ". A 2 149 64 46 • t!:: 25 B -2 172 52 49»: 'C 0 177 51 48 D 52 179 51 46 a: Residual sugar values are stated with the untreated control, positive values for elevated levels and negative values for monosaccharides and di-. because of differences in reception and / or growth potential.

All silages were well preserved, had a pH of about 4 or lower, and had a high lactic acid content. Regarding the lactic acid form, it can be mentioned that the ratio of the two forms of lactate was not significantly affected by the enzyme treatment. In all cases, about 50% of the lactate was in the D form.

10 The remaining amount of sugar is higher for ABD, BCD, AD, BC and BD and fraction D than for ABCD. The choice of enzyme fractions has a clear effect on residual glucose, xylose, fructose and arabinose.

15 Their concentration correlates with other concentrations; the treatment with the highest glucose content also results in the highest levels of other sugars, etc. Fraction D was the most effective apparent sugar producer in silage. In fraction B and in combination AB

The values for 20 apparent sugar production were negative.

The soluble protein content of all: · 50 to 60 mg / g dry hay for treatments; thus, the quality of the silage was not impaired by the enzyme treatment.

· 25 Ammonium concentrations also did not correlate with the presence of any ·· enzyme and yeast.

2. Effect of enzyme fractions on fiber structure Analysis of NDF structure

The NDF obtained was weighed and the dry matter percentage of the starting material was reported.

Sensitivity to enzymatic hydrolysis was determined by hydrolysis of NDF with a ready mixture of cellulose-degrading enzymes Cytolase-123 (50,000 HEC / g). Before '. use of the enzyme preparation was passed through a Sephadex G-25 gel permeation chromatography column to remove any low molecular weight compounds that could interfere with the assay for reducing sugars. 20 mg of NDF was weighed into a tube and 9.6 ml of Sörensen's phosphate buffer (pH 7) was added. After 15 hours incubation at room temperature, enzyme (0.4 mL) was added and the initial rate of sugar release was determined. One milliliter sample was taken at 0, 1, 2.5 and 5 hours. Each sample was immediately mixed with 4 ml of dinitrosalicylic acid (DNS), a reagent used for analysis of reducing sugars. Said reagent py-10 immediately suppresses the enzymatic reaction.

The samples were then heated for 5 minutes in a boiling water bath, rapidly cooled in an ice bath, and the absorbance at 540 nm was measured. The results are expressed as the molar amount of reducing sugars released per gram of NDF per hour.

NDF was determined by the number of reducing end groups by modified DNS assay. 20 mg of NDF was weighed into a tube and 1 ml of water was added, the mixture was incubated for 1 hour at room temperature, 4 ml of 20 DNA was added, the mixture was then heated in a boiling water bath for 5 minutes, rapidly cooled in an ice bath and filtered. · Absorbance at 540 nm. The results are expressed in moles per gram of NDF.

The number of reactive hydroxyl groups was determined by analysis by the following acetylation procedure; the number of acetyl groups attached. Three replicates of 20 mg of NDF were mixed with 2 ml of pyridine. 200 μΐ of [14 C] acetic anhydride 30 (specific activity 1.04.109 dpm / mol) was added and the mixture was incubated in small, tightly closed flasks for 30 min at 70 ° C. The filtrate was removed and the precipitate was washed twice with fresh pyridine and then Four times with water using an ultrasonic bath other than washing the covalently bonded acetate to remove the fiber. The final wash solution was assayed for radioactivity that did not exceed background. The acetylated fiber was hydrolyzed with 2 ml of 4N HCl solution for 5 hours at 90 ° C and finally the radioactivity was measured from the neutralized filtrate. The incorporation of acetyl groups is reported in micromoles per gram of NDF.

The results are summarized in Table 3.

Table 3 10 Effects of enzyme fractions, alone and in different combinations, on fiber structure

Ent-NDF Relative Acetyl Reducing Sugar DM Length (%) Release Groups * (pmol / g 15 termination rate NDF) groups [pmol / g (pmol / g (NDF) h] NDF)

Zero 35 0 0 0 ABCD 18 -3 -144 17 ABC 24 2 45 -13 ABD 20 -3 -143 55: 25 ACD 20 -3 -139 17 BCD 23 -6 -140 29 AB 27 2 58 -62: T: AC 27 2 92 -13 AD 21 -3 -121 55: ·· *: 30 BC 28 0 -49 -2. ··. BD 25 -6 -138 116 CD 23 -6 -141 29 Jul A 30 2 173 -62 B 32 0 -40 0 35 C 31 0 -53 -2 D 26 -6 -125 116 22 112161

Values are expressed relative to the value of the control samples at zero.

DM stands for dry matter.

NDF values decreased when enzymes were used in silage production. Treatment with the enzyme mixture ABCD reduced the amount of NDF from 35% achieved in the case of untreated silage to 18% (DM). The drop in NDF was less when the enzyme fraction D was omitted. Enzyme fraction D had the greatest effect on solubility of 10 NDF. When fraction D was combined with other fractions, the fiber dissolved very efficiently. This also explains why the number of reducing end groups decreased in the presence of fraction D. It is believed that fraction D systematically breaks down the fiber, thereby rendering the pulp soluble. The number of free end groups in the untreated control sample fell below the level observed. Also, fraction A, combined with fractions B and C, was effective in solubilizing NDF. Fraction A also greatly improves reactivity, possibly by increasing the number of reducing end groups.

It is desirable to convert the fiber during storage, • · ··! for easier access to microbes present in the rumen * »·. : access it and make it more reactive to the digestive • '·. channels of. The first to access it in the rumen ': · 25 cellulose-degrading microbes and their enzymes. Therefore, the initial rate of enzymatic release of sugar was determined as a measure of how susceptible the NDF fractions are to the said attack. The values are compared with that of an enzyme-free comparator. sample value (zero), and positive values indicate • · 30 increased sugar intake for microbes in the rumen • φ '·; * and negative values indicate that too much sugar :: has been converted to acids in the feed preservation process. It can be seen that enzyme fraction A, alone or in combination, is less suitable for use in the preservation process due to its high sugar release power. Fraction A had a strong • »» «· positive effect on the initial speed of sugar production. 95% of the 23,112,161 released sugars were glucose and cellobiose. When both fraction A and D were present, fraction D was dominant. Fraction A, alone and in combination with B, C or BC, improves the sugar intake of rumen microbes and is preferred for that use.

Acetylation of NDF fractions from enzyme-treated silage showed that removal of the carbohydrate component of cell wall material does not reduce the amount of reactive hydroxyl groups. In contrast, carbohydrate-efficient enzyme combinations increased the incorporation of [14 C] acetyl groups into the treated fiber. The most efficient solubilizing enzyme combinations and Cytolase itself thus increased the amount of unprotected hydroxyl groups. The greatest effect on the number of hydroxyl groups 15 was in fraction D. The unprotected hydroxyl groups were probably substituents on phenolic lignin. Fraction C appeared to be an antagonist of Fraction D. The effect of section A was also opposite to that of section D.

Thus, the invention makes it possible to modify the insoluble portion of silage 20 in a variety of ways using various enzyme combinations. The most noticeable effects are achieved by using the verses A and D, either,! alone or combined, or by deleting them when it does

; ,, is appropriate. Feed preservation of enzyme fraction D

Any combination in the presence of 25 results in an insoluble fraction which cannot be hydrolyzed with the further used enzyme mixture.

• Fraction A has the opposite effect; silage prepared using fraction A but not fraction D is significantly more sensitive to further enzymatic hydrolysis than the untreated control.

. Although not all verses and combinations of verses exist

I · I

It is understood that all possible combinations of · · · are within the scope of this invention.

• · 24 112161

In a subsequent experiment on maize silage, fractions A and D resulted in a significant reduction in NDF, whereas fractions AD and ABCD had no significant effect. However, only fraction D and combination 5 ABCD significantly reduced ADF.

In the case of alfalfa silage containing clover and timothy, the following NDF and ADF values were achieved using the following fractions: 10

NDF ADF

A 41.97 34.11 15 B 42.40 35.00 c 40.74 35.74 D 41.29 33.50 20 AD 41.33 34.51; BC 40.80 33.40 25 ABC 40.69 33.14 BCD 39.19 32.05 * 1 * * ABCD 40.88 33.09 ί 30: Comparison 41.73 34.22 •> ♦ • j * 25 112161

Only in the case of Fraction B was the NDF value not significant. However, only fraction D and combinations BC and BCD had a significant effect on ADF. Example 3 Effect of Cytolase-123 enzyme fractions on waste water generation and chemical composition of silage made from tetraploid grass

Tetraploid granules (fertilization at 120 kg N / ha) were manually cut using aseptic procedures, transported cold, and the experiment started within 5 hours of surgery. The solids content was 13% by weight. The hay was cut with scissors into 1 cm pieces and placed in plastic bags which then each contained 80 g of fresh 15 hay. Duplicate samples were treated with several combinations of Cytolase-123 fractions A, B, C and D. After enzyme treatment, the hay was packaged in 5 g portions loosely in anaerobic cultivation tubes (20 ml) which were rinsed for 5 minutes with anoxic gas, the grass was packed more tightly and the tubes were sealed with butyl rubber plugs. Each one. 9 replicates were prepared for treatment.

• ♦

Tubes were incubated at 30 ° C and replicates were opened after 1, 2 and 4 weeks. Total contents of tubes: '· (5 g) was used as a sample in each assay.

25 Determining wastewater. The contents of one tube (5 g) were completely packaged in a balanced perforated plastic tube placed in a 50 ml centrifuge tube with a 2 cm spacer at the bottom. The tubes were centrifuged in a Sorvali SS-34 rotor for 20 minutes at 1000 rpm. The perforated inner tubes containing the precipitate were re-equilibrated and the ·, ·, · wastewater volume was calculated (volume of wastewater = 5 g - mass of the precipitate).

Acid production was determined in several ways.

t [,; The amount of wastewater from silage was measured at 1, 2 • ·. After 35 and 4 weeks of incubation. At the same time, the total amount of titratable acids was determined. Acids that were in the form of a salt at pH (4) of the effluent were not included in this analysis. Lactic acid is one of the acids with a pKa lower than the initial pH of the titration. Lactic acid and acetic acid were analyzed separately by HPLC using a column in H + form and UV detection at 210 nm.

Titration of acid. To a tube containing 5 g of silage was added 15 ml of distilled water. The tube was shaken at room temperature for 1 hour and its contents filtered through a 0.2 µm filter and divided into three equal portions. Two samples were used for organic acid analysis and one for acid titration. The sample was diluted with water and titrated with 0.05 M NaOH to neutral using phenolphthalein as indicator.

15 The results are summarized in Table 4.

* *! · * »- 1 f · • · I« · • »• 1 1 t · •» • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •

• · I

I M 'I

• I t »• · 27 112161

Table 4

Effect of Cytolase and enzyme fractions on the composition of wastewater and silage

Ent- Waste-pH- pH- Milk- Milk- Titrate- Titration enzyme water day 7 day 28 acid acid-day-day day-day-month 28 7 28 lynx ca. day day 7 28 ml / g ymol / g ymol / g

Zero 0.45 4.04 4.02 145 171 107 135 ABCD 0.65 3.96 3.95 164 196 138 173 15 ABC 0.53 4.15 4.06 148 175 117 147 ABD 0.63 3.99 3.91 184 223 138 173 ACD 0.61 4.05 3.96 171 206 123 154 BCD 0.56 4.12 4.04 155 184 116 146 20 AB 0.52 4.11 4.03 156 186 117 147

Ac 0.49 4.17 4.08 154 183 104 131 * M, 0.59 4.08 4 176 206 123 154 • j Bc 0.46 4.11 4.03 155 184 113 141 BD 0.55 4, 09 4 159 190 116 146 i 'CD 0.56 4.09 4 139 193 111 139. i * A 0.48 4.2 4.12 150 177 104 131 \; B 0.45 4.08 3.99 151 179 113 141 c 0.46 4.07 3.99 161 193 107 135; · '3 ° D 0.54 4.12 4.04 153 181 111 139 35 * Values are based on the mass of fresh feed.

28 112161

The above results indicate that the silage was well preserved, had a pH of about 4 or lower and had a high lactic acid content.

The production of wastewater from wet plants is a function of enzyme 5 fractions A and D, the latter of which is particularly effective. The enzyme fraction D is responsible for the harmfulness of the effluent stream, especially when combined with other fractions. The effluent stream from fraction A stops after about 14 days, while fraction D produces effluent continuously throughout the storage of the feed.

Example 4

Compositions similar to the combinations ABC, ACD, AC and ABD and other combinations are believed to be produced by genetic manipulation of a cellulase source. For example, in Trichoderma species, the gene encoding cellobiohydrolase I, cellobiohydrolase II and endo-glucanase I, as well as combinations of genes encoding said enzymes, may be deleted. Cellulase isolated from such a gene-deficient species is expected to have a similar effect on fiber plants as against. combinations of cellulosic material fractions from the anion exchange column.

It is also believed that combinations of purified or enriched enzymes present in the cellulase material would have similar effects on plants as combinations of cellulase material fractions derived from the corresponding ion exchange column.

Example 5: V. Silage may be added to plants to produce * · t **> t 30 fractions B or C or combination BC. The said fractions include * regulate the release of xylose.

Alternatively, silage may be added to the plant in the preparation of enzymes which produce the main activity of said fractions, for example xylanase. 35 cellobiohydrolase II and endoglucanase I. For example, xylanase 29 112161 cellobiohydrolase II and endoglucanase I can be added to the plant ton (dry mass). For example, xylanase (about 50 to 50,000 units) may be added per tonne (dry weight) of plant; the ratio of reducing sugars and sugar chains released by xylanase to mono-5-mer xylose should be less than about 100), cellobiohydrolase II (about 2.5-5,000) 000 FPUs and about 50-50,000,000 CMC units), endoglucanase I (about 2.5-5,000,000 FPUs and 50-50,000,000 CMC units) or a combination thereof.

If a xylose-releasing enzyme is used during feed storage, said enzyme may be combined with a microbe which produces organic acids or other silage-stabilizing metabolites, such as sugar alcohols, especially xylitol. Microbes such as lactic acid bacteria, propionic acid bacteria, yeasts and combinations thereof are useful. More useful organic acid producing microbes are those that use xylose as their primary substrate.

In addition, each fraction,, combination, or individual enzyme used in this process should be substantially free of the arabinosidase and cellobiohydrolase i · '·: I activity.

: "Example 6! * 25 In the case of starch-based plants such as i * maize, sorghum and cereal silage, etc., the total animal nutritional mixture (TMR) may be supplemented with fractions A and D or AD, typically about 2 days before the animal is fed. For grasses and forage grasses such as alfalfa, clover and grasses, fraction B, C or D or combinations thereof may be added. When TMR contains cereal feed and

'mixtures of leguminous compound feedingstuffs with fractions A, B, C and D'

ti't> The use of certain combinations depends on the composition of the TMR.

i ·. 35 TMR and fraction (s) are maintained at ambient temperature.

* I * i »30 112161

Alternatively, enzymes which provide the main activity of said fractions, for example arabinosidase, xylanase and cello-biohydrolase I, may be added to the TMR. For example, arabinosidase (about 2.5-5,000,000 units), xylanase (about 50-50,000,000 units), cellobiohydrolase I (about 2.5-5,000,000 FPU, and ca. 50 to 50,000,000 CMC units) or a combination thereof.

The xylanase added to the complete nutritional composition should not release monomeric xylose.

In another alternative, fraction A is used instead of fraction A which does not contain an enzyme activity (e.g. esterase) that causes the release of uronic acid.

Example 7 24 Friesian cows were fed, for the first 150 days of lactation, TMR based on high-moisture corn silage, alfalfa hay fodder and maize starches balanced by protein-mineral supplementation. The TMR was stirred daily and was available for 24 hours.

. ; The TMR feed component was mixed alternately with either 10 liters of water (daily for 3 days) or 25 liters of enzyme solution (daily for 4 days). Enzyme fractions were used in the order ABCD, AB, A, C, BD, D, AD, and CD, respectively, in amounts corresponding to the fraction of the respective fraction in ABCD (approximately 6,000,000 HEC).

Milk data were collected daily and a summary of these is shown in the following table: «t t •» * * I »31 112161 Treatment Milk volume Response1 [kg / d (lb / d)]

Comparison 34,455 75.96 5 ABCD 34,809 76.74 100.35

Comparison 34.913 76.97 AB 33.861 74.65 97.49

Comparison 34,550 76.17 A 28,254 69,291 94.07 10 Comparison 32,722 72,14 C 32,427 71,491 101.42

Comparison 31,221 68.83 BD 31,479 69,401 100.89

Comparison 31.184 68.75 15 D 29.910 65.94 97.14

Comparison 30,395 67.01 AD 28,037 61,811 93,16

Comparison 29,792 65,68 CD 29,901 65,92 20 1 Response = milk production divided by the average of the reference periods limited to production periods.

These data indicate that the addition of these fractions affects the utilization of cows' TMR.

25 ·

Claims (4)

112161 A method for improving the nutritional value of a plant in an animal, characterized in that the plant 5 is treated with a cellulose-degrading enzyme composition which is a fraction of Cytolase-123 which does not bind to Q-Sepharose anion exchange resin at pH 8 and conductivity of 20 mM. n electrical hazard of tris buffer solution. Process according to Claim 1, characterized in that the solution of the cellulose-degrading enzyme composition is added to the growing plant. 3. The method of claim 1, wherein the solution of the cellulose-degrading enzyme composition is added to the harvest. Cellulose-degrading enzyme composition, characterized in that it is a fraction of Cytolase-123 which does not bind to the Q-Sepharose anion exchange resin at pH 8 and has an electrical conductivity similar to that of 20 mM Tris buffer. * t t t »<{* <·. i» f! t 1 I '«lii 112161