JPH08332032A - Peroral enzyme preparation for animal and its use - Google Patents

Abstract

PURPOSE: To obtain an enzyme preparation capable of imparting a vegetable fiber digesting function to a land animal such as pig, chicken, cow, horse, dog, cat and silkworm by the addition of a small amount of enzyme by using an enzyme having activity to isolate and decompose lignin in a vegetable fiber material. CONSTITUTION: This enzyme preparation contains an enzyme capable of isolating and decomposing lignin in vegetable fiber material such as hemicellulase (e.g. xylanase, mannanase or pectinase) or Bacillus sp. SD902 (FERM BP-4508).

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the fields of foods, feeds, drugs, etc. using a plant fiber material as a raw material, and utilizes an enzyme agent containing an enzyme that efficiently releases or decomposes lignin and the enzyme. The present invention relates to the provision of oral animals.

[0002]

BACKGROUND OF THE INVENTION It has been widely practiced since ancient times to produce agricultural materials, foods or food processed products using plants as raw materials.
Among them, many attempts have been made to process plant fiber substances in plants using various techniques. For example, in the field of feed, plant fiber substances such as grass (ryegrass),
Grains (corn, wheat, etc.), beans (alfalfa, etc.), etc. have been used as feed crops for storage, and natural and artificial processing techniques such as silage adjustment have been processed and stored before use.

On the other hand, with the recent increase in awareness of global environment protection, agricultural waste such as rice straw, rice husk, straw, vegetable waste, and bamboo, molasses molasses, yeast meal, fermented meal, soybean meal, okara,
Effective use of food waste such as fruit peel and fruit meal is required,
One of them is being considered for use in the feed field. In addition to grasses, grains, and beans that have been conventionally used as a feed for storage or compounding, this agricultural waste or food waste is used alone or in combination with grasses. There are various methods for preparing this feed, such as being self-produced by a farmer using silage, or prepared in advance by a feed company. For example, silage preparation involves lactic acid fermenting grass in silage. Generally, the pH is 4.5, and sometimes drops to 4, and the inside temperature becomes high due to fermentation, which suppresses the growth of undesirable yeasts and bacteria other than lactic acid bacteria. This has led to the preservation, palatability and nutritional value of silage-regulated grasses. However, depending on the material, climate, and storage conditions, it may cause deterioration due to oxidation or deterioration of nutritional value due to poor fermentation.

Further, as one of the methods for improving feed value, there is a method in which enzymes, particularly cell wall digestive enzymes such as cellulase and hemicellulase are added to grasses to promote the decomposition of plant cell wall (Japanese Patent Publication No. 3-34909). ). When these enzymes act on the plant cell wall of grass, the cell wall is decomposed and the water-soluble carbohydrate in the cytoplasm is released to the outside. During the preparation of feed such as silage, these cytoplasms and decomposed cell wall components are present in the grass or serve as nutrients for the growth of microorganisms added from the outside, contributing to the high-quality lactic acid fermentation of the grass during storage, and other stored grass. Has the effect of promoting the further decomposition of However, since the inside becomes acidic in silage adjustment, the enzyme to be added is one which has a strong activity under acidic conditions and has a characteristic of exerting a synergistic effect with lactic acid bacteria. As described above, the current enzyme treatment is performed only by using an enzyme that can adapt to some limited environmental conditions such as acidic conditions during lactic acid fermentation and conditions that are close to room temperature in terms of temperature.

When these enzymes are used as enzyme treatment conditions under severe conditions, for example, in a process using a heating type granulator such as an extruder or pelleter, the high temperature condition becomes a destabilizing factor for these enzymes. The enzyme is inactivated. In order to overcome such conditions and obtain a sufficient effect, a large amount of enzyme has to be added, which is difficult to achieve in terms of cost. Japanese Patent Laid-Open No. 63-157938 proposes to prepare an enzyme premix composed of an enzyme and a carrier to stabilize the enzyme against heat and mix it with feed. This is a new method for preparing the premix. It requires a different manufacturing process and is disadvantageous in cost. Even with this premix, the usage conditions are 65 ° C or lower, preferably 4
It is 5 ° C or lower.

[0006] In addition, as a method of using the enzyme in the feed, there is known a method in which one or several kinds of enzymes are added to the feed to feed. For example, Japanese Examined Patent Publication No. 58-32575 describes a method for producing an enzyme composition comprising a plurality of plant tissue-disintegrating activities by culturing a basidiomycete belonging to the genus Phomitopsis or the genus Irpex. In particular, commercially available enzyme compositions include dolicerase (Kyowa Hakko Co., Ltd.), which includes cellulase, laminarinase,
It is characterized by being composed of a plurality of enzymes including xylanase, pectinase, amylase, protease, etc., and the effect is exhibited by the interaction of these plurality of enzymes. However, it is unknown what effect each enzyme has on its own. See you again (Bull. Azabu Un
iv. , Vet. Med. 11, 45-49 (199
According to 0)), the glycerase composed of a plurality of enzymes has no effect of adding an enzyme to plant fiber substances having a high lignin content, such as rice straw.

Further, in the case of a multi-enzyme preparation, for example, in the case of containing cellulase, the value of feed is reduced because cellulose in the fiber material is strongly decomposed. In other words, cellulose degraded by cellulase is a major nutritional element of feed for livestock and its value as an energy source decreases. In addition, in order to maintain good rumination especially in rumen tissues of the rumen of cattle, a certain amount of fibrous substance is required, but the fact that cellulose, which is its main component, is decomposed before feeding is a function of feed. It is incompatible with high quality feed. In addition, the inclusion of proteases leads to the degradation of other useful enzymes, which reduces the value as an enzyme composition, and the inclusion of lipases degrades lipid, which is a high energy source, and impairs the nutritional value of feed. In addition, as an example of using the enzyme alone, there is Tokuhyo 5-501657, which is 50000 U / kg in order to obtain a sufficient effect.
It was necessary to add a large amount of xylanase (feed weight) or more.

[0008] As described above, attempts to hydrolyze hardly-decomposable plant fibers in plants such as various grasses and legumes and to use them in feeds and saccharified and fermented products have been made in the feed field for silage adjustment and other foods. In the field, various methods such as fermentation have been conventionally performed. However, these plant fibers generally have a robust structure in which crystalline cellulose, lignin,
The structure around lignin was generally difficult to decompose due to the mixture of ash. In order to improve these points, JP-A-49-117255, JP-A-53-69178,
There are physical or chemical methods such as Japanese Patent Publication 60-13659 and Japanese Patent Publication No. 29329. The structure of the plant fiber substance is destroyed to swell the contained cellulose to expose its amorphous part, and physical treatment or chemical treatment is performed to solubilize the lignin embedded in the plant fiber. It has been applied according to the target plant and usage. However, these methods have the drawbacks that they require high temperature and high pressure, acid / alkali, organic solvent and the like, and have high energy consumption, potential danger and complicated steps in processing. On the other hand, the enzyme can suppress energy consumption to a low level under relatively mild conditions, but no enzyme capable of efficiently releasing or degrading lignin has been found.

One of the methods for decomposing lignin in plant fiber substances is to utilize microorganisms such as white rot fungi, but the decomposition of lignin by microorganisms requires a relatively long period of time, and therefore it is rapid and effective. Release lignin,
It cannot be disassembled. In addition, plants that conventionally contain a large amount of lignin have a poor digestion efficiency, so that most of them tend not to be used as feed. However, looking at the recent circumstances surrounding the livestock sector, the productivity of livestock is approaching the limit, and in certain areas and countries from the viewpoint of environmental response, the types and amounts of livestock feed are restricted, and livestock excretion is limited. The environment is becoming more and more severe year by year due to restrictions on substances and their components (such as nitrogen and phosphorus). Therefore, higher efficiency in animal feed production tends to be required in terms of cost and nutrition, and the appearance of high-quality feed compositions superior to conventional products is strongly desired all over the world.

For example, dairy cows are highly lactated, and accordingly, it is necessary to feed high nutritional value feedstuffs. As a result, the amount of milk produced increased, but there were many breeding problems due to the high supply of concentrated feed, and the momentum for conversion to the roughage feeding method is increasing. However, it is also difficult to obtain the same effects as the concentrated feed by only using the conventional feeding method. Therefore, in order to improve feed efficiency, digestion efficiency, milk quality, etc., various enzymes have been added to the feed for feeding. However, even in that method, there is still a large amount of non-digested fraction in the feed, and there is a lot of room for improvement in feed digestibility or feed efficiency.

On the other hand, in the fields of foods and pharmaceuticals, it has recently been recognized that dietary fiber (dietary fiber) in plant fiber substances is very effective in maintaining biological function, and a substance having functionality As a result, many attempts have been made to positively use it. In particular, the development of products in which dietary fiber is added to beverages, confectionery, etc. is remarkable. Further, as disclosed in JP-A-3-227933, it has also been practiced to extract useful components such as rice bran and wheat bran, which were conventionally used mainly for feed, and to use them as one drug component. It is desired to develop a method for directly using slag without going through a processing step.

[0012]

DISCLOSURE OF THE INVENTION The present invention relates to a natural product, a processed product in an agricultural / food processing industry, or an agricultural waste or a food waste made of a plant fiber material containing lignin, which is a food product, a feed, a drug. And the like, and a method of leading to a shape that can be effectively used as a raw material thereof. Since the lignin in the plant fiber material generally forms a strong bond with hemicellulose, its release is very difficult by the conventional technique. Physical or chemical methods also involve high energy consumption, potential hazards and complicated processes. It is required to process these under relatively mild conditions to suppress the energy consumption to a low level and to further improve the effect and reduce the processing cost. Many conventional enzyme agents used for efficient treatment of plant fiber substances are compositions of a plurality of enzymes, and the synergistic effect of the interaction of the plurality of enzymes plays a large role. On the other hand, however, it is also known to reduce its value by containing an undesirable enzyme component. In addition, during processing, a heat load is applied depending on the equipment used, and general enzymes are deactivated. Currently, many enzymes cannot be used in high temperature processes because their use temperature is limited to about 60 ° C or lower. Most of the applied pH has strong activity on the acidic side like the enzymes currently used for silage,
It may be desirable to apply the enzyme under neutral or alkaline conditions.

[0013]

Means for Solving the Problems In the present invention, as a result of extensive studies to solve the above problems, an enzyme having an activity of liberating or degrading lignin in producing an oral animal product containing plant fiber is included. Enzymes are effective, and the enzyme resistance, especially heat resistance, is excellent and the working pH is high.
The inventors have found that they have a surprising effect when they have a wide range, and completed the invention. That is, the present invention provides the following.

1) An enzyme preparation for oral animals containing an enzyme having an activity of releasing or decomposing lignin in a plant fiber material. 2) The above 1) containing at least one hemicellulase
The enzyme preparation for animal oral products described. 3) The enzyme preparation for animal oral substance according to the above 2), wherein the hemicellulase is xylanase, mannanase or pectinase.

4) The enzyme preparation for animal oral administration according to 1), which contains at least one lignin-degrading enzyme. 5) The enzyme preparation for animal oral administration according to 4), wherein the lignin-degrading enzyme is ligninase, manganese-requiring peroxidase or laccase. 6) The above-mentioned 1), wherein the enzyme having an activity of releasing or decomposing lignin in plant fiber material is an enzyme having heat resistance
To 5) the enzyme preparation for oral administration to animals.

7) The enzyme preparation for oral administration of animals as described in 1) to 6) above, wherein the enzyme having an activity of releasing or decomposing lignin in the plant fiber material is an enzyme derived from a bacterium of the genus Bacillus. 8) An enzyme having an activity of releasing or decomposing lignin in plant fiber material is Bacillus sp. SD902 (FER
MBP-4508), Bacillus sp. SD401 (F
ERM P-10527), Bacillus sp. SD402
(FERM BP-3431), Bacillus sp. SD4
03 (FERM P-11647), Bacillus sp. S
D516 (FERM P-10427), Bacillus s
p. SD771 (FERM P-11455), or Bacillus sp. SD772 (FERMP-11456)
The enzyme preparation for animal oral preparations according to 1) to 6), which is an enzyme that can be obtained from

9) An oral animal product, which comprises the enzyme agent for oral animal product described in 1) to 8) above and vegetable fiber. 10) The animal oral product according to 9), wherein the enzyme-degrading enzyme for an oral animal product has a cellulolytic activity of 1 U or less per 1 kg of plant fiber. 11) A feed composition comprising the enzyme preparation for animal oral substance according to 1) to 8) and vegetable fiber. 12) The feed composition as described in 11) above, wherein the enzyme preparation for oral administration of animals has a cellulolytic activity of 1 U or less per 1 kg of plant fiber.

13) A method for producing an oral animal product, which comprises the step of releasing or decomposing lignin corresponding to at least 1% of a plant fiber substance with the enzyme agent for oral animal product according to 1) to 8). Method. 14) A method for producing a feed composition, comprising the step of releasing or decomposing lignin corresponding to at least 1% of a plant fiber substance with the enzyme preparation for animal oral substance according to 1) to 8). 15) A method for producing a feed composition, which comprises a step of mixing the enzyme agent for animal oral substance according to 1) to 8) above and vegetable fiber and then heating to 70 ° C. or higher with a heating type device. .

The present invention will be described in detail below. In the present specification, the term “oral animal product” refers to food products, feeds, drugs, etc., which are ingested through the mouth of animals including humans, fish, birds and the like, and precursors thereof. The nutritional value of feed, which is one of the oral products of animals, is determined by the nutritional value and digestive absorption rate of its constituents. Constituent elements are plant cell contents (C
C) and the cell wall substance (CW), and the organic matter of the cell wall substance is a fraction (Oa) with higher digestibility and a fraction (O) with lower digestibility.
It is classified into b). For example, ruminants digest CC and Oa by almost 90 to 100%, but the digestibility of Ob is low. This is related to the fact that the digestion of the cellulose fraction in the plant fiber is good, but the digestibility of lignin, which is an indigestible substance, and hemicellulose accompanying it is low due to the microorganisms in the rumen. Thus, the digestibility is said to have a negative correlation with the lignin content, and according to Kondo (Tohoku Agricultural Res. Inst., 85,103-144 (1992)), the digestibility is about 5% as the lignin content increases by about 1%. %descend.

Although the composition of the plant body is diverse, it is mainly composed of the three major components of cellulose, hemicellulose and lignin. In addition, it contains fats and oils, small amounts of proteins, and ash such as silica. It is not always clear in what state these constituent components are bound to each other, but generally, hemicellulose mainly forms a strong bond with lignin, and its release has been considered to be very difficult in the prior art. It was As an example, for a lignin of a certain straw, as a result of comparatively examining three kinds of lignin of alkali-extracted lignin extracted from the straw, finely-ground extracted lignin, and enzyme-extracted lignin, the lignin in the straw is arabinoglucurose which is hemicellulose. It is presumed that it has a strong bond with xylan, and the bond is also a strong cross-linking bond via ferulic acid.

(Enzyme) The enzyme used in the present invention liberates lignin from the above rigid structure or decomposes lignin itself. In the former, if hemicellulose in the raw material plant fiber tissue can be sufficiently decomposed, lignin in the embedded form can be easily released. For this, hemicellulases such as xylanase, mannanase and pectinase can be used. Further, in this case, since the main hemicellulose is often xylose, it is more preferable to use xylanase as its degrading enzyme. Xylanase is an enzyme having an action of hydrolyzing a polysaccharide xylan mainly composed of β-1,4-bonded xylose to produce xylose which is a constituent component thereof and low molecular weight xylooligosaccharide, and is widely present in animals and plants. Some microorganisms produce xylanase, and yeast, fungi, bacteria, actinomycetes, and other enzymes have been known so far.

The latter directly decomposes lignin and aims to disassemble the complex structure, and lignin-degrading enzymes such as ligninase, manganese-requiring peroxidase, and laccase can be used. Further, it is known that the constituent components of the plant body change depending on the growing season, and in some cases, it is desirable to add other degrading enzymes accordingly.

It is desirable that the enzyme used in the present invention has a characteristic that the enzyme activity remains sufficiently even under severe conditions such as high temperature and long time. As a result, it can be used and treated in a heating type device. For example, a pellet machine, an extruder, or a device such as a dryer can be used for molding. When using such equipment, the treatment temperature is 0 to 120 ° C., generally 60 ° C. or higher. Such a thermostable enzyme is an enzyme derived from an animal or a plant or a microorganism, and has an enzyme activity of 50% or more after treatment for at least 60 ° C for 24 hours, preferably 70% or more under the same conditions, or 70 ° C. An enzyme having an enzyme activity of 60% or more after treatment for 10 minutes, preferably an enzyme having an enzyme activity of 80% or more under the same conditions, for example, Bacillus sp. SD771 (consignment number FERM P-114
55), SD772 (accession number FERM P-1145)
6), SD902 (accession number FERMBP-4508)
Enzymes found in culture products such as
-261750). Further, some of these enzymes have an optimum temperature of 65 ° C or higher, and can exert a remarkable effect in treatment at a temperature higher than 60 ° C.

When a microorganism is used as a method for producing an enzyme, an ordinary bacterial culture method such as liquid culture or solid culture can be used as a culture method. From the economical point of view, an aerobic liquid culture method is preferable, and examples thereof include aeration stirring culture method and shaking culture method under aerobic conditions. As the medium, any medium component can be used as long as the strain to be used can grow, but for example, as the carbon source, any assimilable carbon compound or a compound containing this can be used, for example, in the case of the production of xylanase,
Use of xylan such as various xylan, wheat bran, pulp waste liquor, saccharified meal or rice straw, or various raw materials containing xylan-based polysaccharides, starch hydrolyzate such as glucose, starch or liquefied starch, and sugars such as molasses, either alone or in combination. Can be done.

Further, for example, the nitrogen source may be any assimilable nitrogen compound or one containing the same, and the organic nitrogen-containing substance may be various amino acids, corn steep liquor, malt extract, peptone, soybean flour, defatted soybean flour and the like. However, as the inorganic nitrogen compound, ammonium chloride,
Ammonium salts such as ammonium sulfate can be used alone or in combination. In addition, various organic substances and inorganic substances necessary for the growth of bacteria and enzyme production or those containing them, for example, salts such as phosphates, magnesium salts, calcium salts, manganese salts, vitamins, yeast extract, etc. are appropriately added. You can also

The culture temperature and the culture pH may be in the temperature range in which the strain can grow, but generally 10 to 90 ° C., pH 3 to
A range of 11 is used. The culturing time may be any time as long as each enzyme is accumulated, but the culturing is preferably terminated when the accumulated amount reaches the maximum. This culture broth may be used as an enzyme, but a crude purified product or a purified product that has undergone a purification step may also be used. The purification process includes operations such as centrifugation, filtration, precipitation, concentration, and drying, which can be appropriately selected and used.

When a plant is used, the enzyme is obtained by extraction from the whole plant or a part of its organs, or from a culture solution or cells obtained by a plant culture method such as liquid culture or solid culture. Can be done. In the case of animals, the target enzyme can be obtained from a part of the organs or a culture solution of animal cell culture. Any medium can be used as the medium for the culture as long as the cultured cells grow and produce the target enzyme. Regarding these extractions, generally, a buffer solution is added at a low temperature such as ice-cooling to homogenize the tissue, and a supernatant obtained by centrifugation or a filtrate obtained by filtering is used as a crudely purified enzyme solution. Further, operations such as centrifugation, filtration, precipitation, concentration and drying may be appropriately selected for purification, and the purified enzyme solution may be used.

(Method of measuring enzyme activity) In the case of xylanase, the presence or absence of xylan-decomposing activity of the enzyme solution can be examined by the following method as a simple method. That is, commercially available oat-derived xylan (manufactured by SIGMA) was suspended in an aqueous solution adjusted to an arbitrary pH so as to have a concentration of 1% by weight, and agar was added so as to have a concentration of 2% by weight. Heat to 100 ° C. to make agar plates. The enzyme solution is appropriately diluted or concentrated, spotted on an agar plate, and then incubated at an arbitrary temperature. After 24 hours, the agar plate is observed, and if a clear zone is observed around the spot, it can be determined that it has xylan-decomposing activity. For other enzymes, if an agar plate containing an appropriate substrate is prepared, its degrading activity can be similarly determined by the presence or absence of a clear zone around the spot.

The xylanase activity was measured by using autospert xylan (Sigma) in 50 mM sodium acetate (pH 5.0) as a substrate at 39 ° C. and pH 3.5.
The amount of enzyme that produces 1 μmol of xylose per minute was set to 1 unit (U), but many of the enzymes used in the present invention are characterized by their properties in the high temperature range. According to 6-261750, using a birch xylan (birch-derived xylan) in a 0.1 M phosphate buffer (pH 7.0) as a substrate and reacting at 50 ° C. and pH 7.0, the reducing sugar produced is known. Method 3,
It is measured with 5-dinitrosalicylic acid (DNS), and the amount of enzyme that produces 1 μmol xylose per minute is 1 unit (S
U).

The method for measuring the cellulolytic activity is as follows. Carboxymethyl cellulase (CMC) activity is 0.05M sodium carbonate-0.05M as a substrate.
CMC in boric acid-potassium chloride buffer (pH 9.0)
The resulting reducing sugar was measured by DNS after reacting at 30 ° C. and pH 9 by using, and the amount of the enzyme that forms the reducing sugar corresponding to 1 μmol glucose per minute was 1 U. In addition, the avicelase activity is 0.05M sodium carbonate-0.05M boric acid-potassium chloride buffer solution (pH
9.0), using Avicel (Merck) at 30 ° C, p
After reacting with H9, the reducing sugar produced was measured by DNS, and the amount of enzyme producing a reducing sugar corresponding to 1 μmol of glucose per minute was 1 U.

(Enzyme preparation for animal oral preparation) The enzyme preparation for modifying animal oral preparation of the present invention contains at least one of the above-mentioned enzymes, and the purity of the enzyme may be selected according to the situation. Further, other enzymes can be mixed, and they may be in a form in which they act simultaneously or a form in which they act sequentially in a form such as sustained release. For example, an oil-degrading enzyme or a protein-degrading enzyme can be mixed in order to decompose the oil-fat or protein contained in the plant fiber material. However, the effect may be reduced, so the conditions should be carefully examined. For example, in the case of a cellulolytic enzyme, it is not preferable because it may decompose the fibers of the plant itself and impair the nutrients, but 1 kg of the plant fiber substance at the time of use
It is possible to enhance the effect by mixing so as to be within the range of 1 U or less and acting partially or minutely. In addition, other components such as a stabilizer can be mixed if necessary.

The content of the enzyme having the activity of releasing or decomposing lignin in the enzyme preparation for oral animals of the present invention is not particularly limited as long as it is an amount that produces an effect at the time of use. Therefore, if the amount of the enzyme agent added is large at the time of use, the enzyme content in the enzyme agent may be small, and it also depends on the specific activity of the enzyme. Also, depending on the physical form of the enzyme preparation, it is usually 0.1 SU / ml for liquids.
Or more, preferably 1 SU / ml or more, more preferably 1
0 SU / ml, even more preferably 100 SU / ml
Although it is above, it may be 0.1 SU / ml or less. In the case of a solid, it is usually 0.01 SU / mg or more, preferably 0.1 SU / mg or more, and more preferably 1 SU / mg.
It is at least mg, more preferably at least 10 SU / mg, but may be at most 0.01 SU / mg.

There is no limitation on the physical form of the enzyme preparation of the present invention, and it may be in the form of powder, granules, liquid or the like. The enzyme preparation of the present invention has sufficient lignin releasing activity or degrading activity at high temperatures, and preferably has sufficient activity at 50 ° C. The method for producing the enzyme preparation of the present invention can be appropriately selected depending on the component, the form and the application. For example, in the case of a multi-component powdery form, each component may be prepared into a powdery form and then mixed, or may be mixed in a solution state and then dried to form a powdery form. In the case of a liquid, the enzyme solution obtained in a liquid state may be used as it is, or a powdered product may be dissolved and used. In the case of granules, the granulation method used therefor is not limited, and conventionally known methods can be used.

(Plant Fiber Material) As the plant fiber material in the present invention, for example, in the case of feed, it is processed not only in conventional feed crops but also in agricultural waste and food industry which are discarded without being used after harvesting. Food waste or the like that is later discarded can be used as the material. Regarding plant fiber substances used in other than feed, plant fiber has recently been attracting attention for its functionality, such as wheat bran, corn fiber, beet fiber, okara, etc., pulp treated from wood treated with alkali, ether. Modified carboxymethyl cellulose (CMC) is widely used in the field of food products such as cereals, confectionery and beverages and pharmaceuticals. These functions are expected to have various effects such as intestinal regulation, cholesterol lowering, blood sugar level elevation, and fatty liver formation suppression.

The plant fiber material may be one which has not been subjected to any pretreatment, but in order to make the enzyme action more effective, JP-A-49-117255, JP-A-53-69178, JP-B-60-13659, As described in JP-B-4-29329, the raw material itself is subjected to a physical method such as cutting, shredding, crushing, wetting, and swelling before the enzyme treatment, and an acid / alkali agent, a surfactant, etc. It is possible to apply a chemical method such as permeating or immersing the above chemicals. For example, in order to make the fiber tissue of the plant soft, add ammonia water, which is an alkaline agent, in a wet state containing a large amount of water,
The effect can be enhanced by an attempt to widen the site of contact with the fibrous tissue from the outside. Also in this case, it is preferable to conduct a preliminary experiment in advance to set the optimum conditions and also to study the inhibitory factors and the removal of chemicals from the product in consideration of the addition of the enzyme.

(Treatment of Plant Fiber Material) In the treatment of the plant fiber material with the enzyme agent of the present invention, the effect tends to be higher as the pH and temperature conditions are closer to the optimum conditions for the decomposition activity of the enzyme. There is no need to be particular about, and the effect may be sufficient as long as the enzyme acts. pH
Is usually 2 to 12, preferably 3 to 11. Temperature is 0-12
The temperature is 0 ° C., generally 20 ° C. or higher, preferably 50 ° C. or higher, more preferably 60 ° C. or higher. The treatment time is affected by pH, temperature, and amount of enzyme, but is usually 0.5 hour to 1 week, preferably 1 to 48 hours, more preferably 2 to 24 hours.
Time. The treatment can be performed by standing or with appropriate agitation. In any case, the enzyme treatment conditions may be determined with due consideration of practical constraints, and it is preferable to conduct preliminary experiments in advance to set the optimum conditions. Also, a plurality of enzyme agents can be used simultaneously or sequentially.

By the above-mentioned treatment of vegetable fiber with an enzyme agent, modified vegetable fiber, that is, vegetable fiber having a reduced lignin content, is obtained, and it becomes an animal oral substance and its material.
Considering the digestibility, the reduction amount of the lignin content is preferably 1% or more of the plant fiber material. It should be noted that it is not necessary that the enzyme activity of the enzyme preparation remains in the animal oral substance produced by the method including this treatment, but if the enzyme activity remains, it may be consumed especially in feed. Because it can be expected to have an effect of supporting digestion in the body of livestock (generally, the ability to digest and decompose hemicellulose is low),
As a positive effect, it is likely that the result of improved digestibility is obtained. When used in food products, it is not necessary for the enzyme activity to remain. The reason for ingesting plant fiber substances as foods is that dietary fibers (so-called diet fibers) are functional (mainly intestinal regulating action), and improve digestive degradation / absorption efficiency as in feed applications. This is because it has no purpose.

(Enzyme-containing Animal Oral Product) The enzyme-containing animal oral product of the present invention contains the enzyme agent of the present invention and vegetable fiber. In the production method, good results can be obtained by mixing the enzyme preparation directly with the target plant fiber material, but to the plant fiber material which has been previously treated with alkali, acid, wet, swelling or the like. You may mix. The amount of the enzyme preparation in the oral animal product of the present invention is not particularly limited as long as the effect can be obtained. Orally, from the viewpoint of enzyme activity, 1 kg of oral animal products
Therefore, it is preferably 10 SU or more, more preferably 10 SU or more.
It is suitable to mix an enzyme agent corresponding to 0 SU or more, and more preferably 1000 SU or more. The amount of vegetable fiber in the oral animal product of the present invention is not particularly limited. For example, in the case of a mixed feed such as birds and pigs, it is usually 2 to 10
%, And grasses, cereals, and blister are often mixed in such a range. Also, for example, in the case of cattle, grasses, cereals, and blister are usually used as the plant fiber-containing substance. The plant fiber contained therein depends on the growth state and harvest time, but is generally 10 to 50.
%, For example, if the feed is about 100% forages, the plant fiber content is 10 to 50%, and if it is half, it is 5 to 25%.
%.

In addition, although the effect can be sufficiently obtained only by mixing and mixing in molding, in consideration of the ingestibility as feed, food, and medicine, it seems to be more granular (granules or pellets) than powder. It may be in the form of a solid product, an agar-like (jelly-like) product, a kneaded-like semi-solid product, or a product obtained by dipping, adhering, or coating an enzyme on a plant fiber material.

The enzyme preparation of the present invention exhibits an excellent effect even when a heating type apparatus is used in the production of an oral animal product containing the enzyme agent. Examples of the apparatus include a pellet mill, a pellet machine, an extruder and an extruder. Molding machine, etc.
Generally, there is a molding device that can be heated to be used for pelletizing feed, and a dryer for molded products, such as a fluidized bed granulation coating device, a centrifugal fluidized coating granulator, a centrifugal fluidized granulator, A spray dry type granulator, a rotary disc type fluidized bed granulator, a centrifugal rolling type granulator, a floating fluidized granulator, a swirling fluidized granulator and the like can also be appropriately used according to the application.

The enzyme in the animal oral substance produced according to the above method has sufficient lignin-releasing or decomposing activity, and acts during storage and use to release or decompose lignin in plant fibers. To do. Regarding the measurement of the release or decomposition of lignin in the plant fiber material in the present invention, Morison et al. (J. Sci. FoodAg
ric. 23,455-463, (1972)).

[0042]

The present invention will now be described in more detail by way of examples, which are merely examples and do not limit the present invention. [Example 1] 1.0% birch xylan, 0.1% yeast extract, 1.0% polypeptone, 0.5% dipotassium hydrogen phosphate, 0.05% magnesium sulfate, 0.002% iron sulfate, 0 Liquid medium consisting of 0.05% sodium chloride (p
H7.0) Put 2L into a 5L jar mentor,
Sterilized at 121 ° C. for 20 minutes. Bacillus sp. SD902 (FERM BP-450
The cells of 8) were inoculated and aerobically agitated and cultured at 55 ° C. for 48 hours. After culturing, the culture solution was centrifuged at 6000 rpm to remove bacterial cells. Next, the supernatant obtained by the above method was concentrated by ultrafiltration to obtain xylanase activity 200
A solution A in which SU / ml was observed was obtained.

[Example 2] 1.0% birch xylan,
0.1% yeast extract, 1.0% polypeptone, 0.5%
Dipotassium hydrogen phosphate, 0.05% magnesium sulfate,
2 L of a liquid medium (pH 7.0) consisting of 0.002% iron sulfate and 0.05% sodium chloride was placed in a 5 L jar fermenter and sterilized at 121 ° C. for 20 minutes. Bacillus sp. SD902 (FERM
The cells of BP-4508) were inoculated and aerobically agitated and cultured at 55 ° C. for 48 hours. After culturing, add 60
The cells were removed by centrifugation at 00 rpm. Next, ammonium sulfate was added to 700 ml of the supernatant obtained by the above method so as to be 60% saturated, and salting out was performed. The enzyme precipitate obtained by salting out was treated with 50 mM phosphate buffer (p
H7.0) and dialyzed overnight. The enzyme solution after dialysis was subjected to anion exchange chromatography (DEAE-Cellulofine, manufactured by Seikagaku Corporation), and the flow-through fraction was subjected to cation exchange chromatography (CM-Cellulofine, manufactured by Seikagaku Corporation) to give 0 to 0. 6M sodium chloride 1
Fractionation was performed by elution with a linear concentration gradient of 000 ml. A fraction B in which a xylanase activity of 500 SU / ml was observed was obtained.

[Example 3] 2.0% soybean powder, 0.5% yeast extract, 0.5% sodium chloride, 0.1% dipotassium hydrogen phosphate, 0.02% magnesium sulfate, 0.5%
2 L of liquid medium consisting of maltose, 0.1% CMC and 0.3% sodium carbonate was placed in a 5 L jar famentor and sterilized at 121 ° C. for 20 minutes. Bacillus sp. SD401 (FERM P-1
0527), SD402 (FERMBP-3431),
SD403 (FERM P-11647), Bacillus
Licheniformis SD516 (FERM P-1042
Each of the cells of 7) was inoculated and aerobically agitated culture was carried out at 35 ° C. for 35 hours. After culturing, add 6000
The cells were removed by centrifugation at rpm. Next, the supernatants obtained by the above method were concentrated by ultrafiltration to obtain solutions C, D, E and F, respectively. Concentration rate of C, D, E is about 10
F was about 8 times.

[Example 4] 3.0% peptone, 1.0%
From water-soluble starch, 1.0% DE-50, 1.0% yeast extract, 1.0% wheat bran, 0.5% dipotassium hydrogen phosphate, 0.05% magnesium sulfate, 0.5% sodium carbonate 2 L of the liquid medium was placed in a 5 L jar-famenter and sterilized at 121 ° C. for 20 minutes. Bacillus sp. SD771 (FERM
P-11455), SD772 (FERM P-114
The cells of 56) were inoculated and aerobically agitated and cultured at 55 ° C. for 48 hours. After culturing, add 600 to each culture
The cells were removed by centrifugation at 0 rpm. Next, the supernatant obtained by the above method was concentrated about 10 times by ultrafiltration, and calcium chloride was added thereto to a final concentration of 2 mM to obtain solutions G and H, respectively.

[Example 5] 5 rice straws having a dry weight of 100 g were used.
After cutting into cm length, it was immersed in 16.5 L of water and left at room temperature all day and night. After that, the rice straw was taken out and washed thoroughly with water to remove deposits such as mud. Further, this was subjected to centrifugal dehydration, and then 5 L of water was added to obtain a raw material liquid adjusted to pH 6.0. To this, 50 ml of the enzyme solution A prepared in Example 1 was added and a non-added section was prepared, and the mixture was stirred at 50 ° C. for 24 hours.
After the reaction, rice straw was taken out, pulverized with a mixer, and washed with water.
According to the method of Morrison et al., It was solubilized in acetyl bromide and the absorbance at 280 nm was measured to measure the change in the lignin content relative to the non-added section.
The lignin content was reduced by 2.3%.

[Example 6] 5 rice straws having a dry weight of 100 g were used.
After cutting into cm length, it was immersed in 16.5 L of water and left at room temperature all day and night. After that, the rice straw was taken out and washed thoroughly with water to remove deposits such as mud. Further, this was subjected to centrifugal dehydration, and then 5 L of water was added to obtain a raw material liquid adjusted to pH 6.0. The enzyme solution A prepared in Example 1 was added thereto in an amount of 0.5 to 5
0 ml addition and non-addition sections were prepared and kept at 40 ° C. for 7 days. After the reaction, rice straw was taken out, pulverized with a mixer, and washed with water. This was solubilized in acetyl bromide according to the method of Morrison et al., And the absorbance at 280 nm was measured to measure the change in the lignin content relative to the non-added section.
The results are shown in Table 1.

[Table 1]

[Example 7] 5 rice straws having a dry weight of 100 g were used.
After cutting into cm length, it was immersed in 16.5 L of water and left at room temperature all day and night. After that, the rice straw was taken out and washed thoroughly with water to remove deposits such as mud. Further, this was centrifugally dehydrated, and then 2 L of water was added to obtain a raw material liquid adjusted to pH 7.0. 0.2 to 2 of the enzyme solution A prepared in Example 1 was added thereto.
0 ml addition and non-addition sections were prepared and kept at 60 ° C. for 3 days. After the reaction, rice straw was taken out, pulverized with a mixer, and washed with water. Similarly, the change in the lignin content with respect to the non-addition group was measured. Table 2 shows the results.

[Table 2]

[Embodiment 8] 5 rice straws having a dry weight of 100 g were used.
After cutting to cm length, 10% sodium hydroxide aqueous solution 1
It was immersed in 6.5 L and left at room temperature all day and night. Thereafter, the rice straw was taken out and washed thoroughly with water to remove sodium hydroxide, eluate, and mud and other deposits. Further, this was subjected to centrifugal dehydration, and then 2 L of water was added to adjust the pH to 7.0. 20 m of each of the enzyme solutions A and B prepared in Examples 1 and 2 was added thereto.
1 was added and the mixture was kept at 60 ° C. for 3 days. After the reaction, rice straw was taken out, pulverized with a mixer, and washed with water. Similarly, the change in the lignin content of untreated rice straw was measured. The results are shown in Table 3.

[Table 3]

Example 9 Three straws having a dry weight of 500 g were used.
After cutting to cm length, 5% sodium hydroxide aqueous solution 33
It was immersed in L and left at room temperature for one day. After that, take out the straw and wash it thoroughly with water to remove the deposits such as sodium hydroxide, eluate, and mud, and then centrifuge and dehydrate it.
Of water was added to adjust the pH to 7.0 and 50 ° C. 50 to each of the enzyme solutions prepared in Examples 1 to 4 and the mixture thereof.
ml was gently added and stirred for 24 hours. After the reaction, the straw was taken out, pulverized with a mixer and washed with water. Similarly, the change in the lignin content with respect to the non-addition group was measured. The results are shown in Table 4.

[Table 4]

Example 10 Straws having a dry weight of 500 g were cut into a length of 3 cm, and then a 5% aqueous sodium hydroxide solution 3 was used.
It was immersed in 3 L and left at room temperature all day and night. After that, take out the straw and wash it thoroughly with water to remove the deposits such as sodium hydroxide, eluate and mud, and then centrifuge and dehydrate it.
L of water was added to adjust the pH to 7.0 and 65 ° C. To this, 5 parts of each enzyme solution prepared in Examples 1 to 4 and the mixture thereof were added.
0 ml was gently added and stirred for 24 hours. After the reaction, the straw was taken out, pulverized with a mixer and washed with water. Similarly, the change in the lignin content with respect to the non-addition group was measured. The results are shown in Table 5.

[Table 5]

Example 11 Straws having a dry weight of 500 g were cut into a length of 3 cm, and then a 5% aqueous sodium hydroxide solution 3 was used.
It was immersed in 3 L and left at room temperature all day and night. After that, take out the straw and wash it thoroughly with water to remove the deposits such as sodium hydroxide, eluate and mud, and then centrifuge and dehydrate it.
L of water was added to adjust the pH to 5.8 to 8.0 and 65 ° C. To this, the enzyme solution A prepared in Example 1 was gently added so that the xylanase activity was 1000 U / kg dry straw, and the mixture was stirred for 24 hours. After the reaction, the straw was taken out, pulverized with a mixer and washed with water. Similarly, the change in the lignin content with respect to the non-addition group was measured. The results are shown in Table 6.

[Table 6]

[Example 12] Rice straw having a dry weight of 500 g was cut into a length of 3 cm, immersed in 33 L of water, and allowed to stand overnight at room temperature. After that, take out the rice straw, wash it thoroughly with water to remove the deposits such as mud, centrifuge and dehydrate it, and then
L of water was added to adjust the pH to 6.0 and 70 ° C. To this, the enzyme solution B prepared in Example 2 had a xylanase activity of 1
000 to 10,000 U / kg was gently added so as to obtain dried rice straw, and the mixture was stirred for 4 hours. After the reaction, the rice straw was taken out, crushed with a mixer and washed with water. Then, the change in the lignin content of untreated rice straw was measured. The results are shown in Table 7.

[Table 7]

[Example 13] Corn 22%, barley 19%, beet pulp 18%, soybean 14%, soybean meal 6
%, Cottonseed meal 6%, corn gluten 3%, hay cube 8
%, Amino acid mix 0.05%, vitamin mix 0.04% as the base (1), and 1% addition group (2) and 0.1% addition group (3) of the enzyme solution A prepared in Example 1 thereto. ) And a non-addition group (4) were prepared. (2) to (4) were crushed, thoroughly mixed with a mixer, and then pelletized by a pelletizer. The molded products of (2) to (4) were dried with a dryer at a temperature of 80 ° C to produce pellet feed. (1) to (4) were crushed, adjusted to a 10% aqueous solution, and stirred in warm water at 50 ° C. for 24 hours. After washing this with water, the lignin content was measured. The lignin content of (2) to (4) is 20.3%, and
It decreased by 4.1% and 3.5%. Since lignin is contained in straw and the like in an amount of 10 to 20%, the lignin in the target plant fiber material was removed or decomposed by at least 1.0% by the enzyme.

[Example 14] Corn 35%, wheat 10%, grain sorghum 15%, fish meal 8%, soybean meal 8
%, Bran 15%, alfalfa meal 3%, calcium 5%, salt 0.3%, vitamin premix 0.3
% Was used as a base (1), and 1% addition section (2) and non-addition section (3) of each enzyme solution prepared in Examples 1 to 4 were prepared. After crushing (2) and (3) and thoroughly mixing them with a mixer, pellets were molded with a pelletizer. This molded product was dried at a temperature of 80 ° C. with a dryer to produce pellet feed. (1) to (3) were crushed, adjusted to a 10% aqueous solution and stirred in warm water at 50 ° C. for 24 hours. After washing this with water, the lignin content was measured. (1) to (2)
Table 8 shows the reduction rate of the lignin content of the above. The reduction rate of the lignin content in (3) was 3.3% with respect to (1).

[Table 8] In each case, 1% or more of the lignin in the target plant fiber material was released or decomposed and removed.

[0056]

By using the enzyme preparation of the present invention, the effect can be improved with a small amount of the enzyme preparation, as compared with the conventional treatment of plant fiber with an enzyme having a low activity of releasing or decomposing lignin. Also, in the past, it was necessary to add a large amount of enzyme at the start of the treatment because the enzyme activity decreased during the treatment, but by using a resistant enzyme, the same effect as the conventional product can be obtained with a smaller amount of enzyme added. Obtainable. In addition, by using a resistant enzyme, the time during which high-temperature treatment is possible has dramatically increased, and long-term treatment under these conditions has become possible.

The oral animal product produced by the method of the present invention and the oral animal product containing the enzyme of the present invention can exert great effects in the fields of food, feed and pharmaceuticals. Especially in the field of feed, it is used as a feed substance for land animals such as pigs, chickens, cows, horses, dogs, cats, and silkworms. It is expected that the feeding efficiency and the meat quality will be improved with the increase in feed intake. In addition, since feed digestion efficiency is also improved in poultry rearing, improvement in feed supply efficiency is expected. Further, the method of the present invention enables efficient production of the above-mentioned oral animal product.

Front page continuation (72) Inventor Shinobu Ito 1-13-9 Shibadaimon, Minato-ku, Tokyo Showa Denko KK

Claims (15)

[Claims] 1. An enzyme preparation for oral administration of animals, which comprises an enzyme having an activity of releasing or decomposing lignin in a plant fiber substance. 2. The enzyme preparation for animal oral administration according to claim 1, which contains at least one hemicellulase. 3. The enzyme preparation for oral administration of animals according to claim 2, wherein the hemicellulase is xylanase, mannanase or pectinase. 4. The enzyme preparation for animal oral substance according to claim 1, which contains at least one lignin-degrading enzyme. 5. The enzyme preparation for an oral animal product according to claim 4, wherein the lignin-degrading enzyme is ligninase, manganese-requiring peroxidase or laccase. 6. The enzyme preparation for oral animal products according to claim 1, wherein the enzyme having an activity of releasing or decomposing lignin in the plant fiber substance is an enzyme having heat resistance. 7. The enzyme preparation for oral administration of animals according to claim 1, wherein the enzyme having an activity of releasing or decomposing lignin in the plant fiber material is an enzyme derived from a bacterium of the genus Bacillus. 8. An enzyme having an activity of releasing or decomposing lignin in plant fiber material is Bacillus sp. SD902
(FERM BP-4508), Bacillus sp. SD4
01 (FERM P-10527), Bacillus sp. S
D402 (FERM BP-3431), Bacillus s
p. SD403 (FERM P-11647), Bacillus sp. SD516 (FERM P-10427), Bacillus sp. SD771 (FERM P-1145
5), or Bacillus sp. SD772 (FERM P
-11456), which is an enzyme that can be obtained from (11456). 9. An animal oral product comprising the enzyme agent for animal oral product according to any one of claims 1 to 8 and vegetable fiber. 10. The animal oral product according to claim 9, wherein the enzyme-degrading enzyme for an oral animal product has a cellulolytic activity of 1 U or less per 1 kg of plant fiber. 11. A feed composition comprising the enzyme preparation for animal oral substance according to any one of claims 1 to 8 and vegetable fiber. 12. The cellulolytic activity of the enzyme preparation for animal oral substance is 1 U or less per 1 kg of plant fiber.
The feed composition described. 13. A method for producing an oral animal product, which comprises the step of releasing or decomposing lignin corresponding to at least 1% of a plant fiber substance with the enzyme agent for oral animal product according to any one of claims 1 to 8. Method. 14. A feed composition comprising the step of liberating or decomposing lignin corresponding to at least 1% of a plant fiber substance with the enzyme preparation for animal oral substance according to any one of claims 1 to 8. Production method. 15. After mixing the enzyme preparation for animal oral substance according to any one of claims 1 to 8 with vegetable fiber, the mixture is heated with a heating type device.
A method for producing a feed composition, comprising the step of heating to 0 ° C. or higher.