CN103549129B - Enzyme and bacterium complexing agent for degrading crop straws - Google Patents

Abstract

The invention relates to an enzyme and bacterium complexing agent for degrading crop straws. The enzyme and bacterium complexing agent comprises the active components including a microorganism complexing agent and compound enzyme. An experiment result shows that xylogen, neutral detergent fibers, acid detergent fibers, acid detergent xylogen, cellulose, crude protein and the like of the straws treated by the enzyme and bacterium complexing agent are well degraded; the degrading rate of rumens of ruminants to the xylogen of the straws is increased, and the surrounding of the xylogen around other nutritional components is broken through, so that the availability of the straws is improved. The enzyme and bacterium complexing agent disclosed by the invention is convenient to use; the straws are not required to be subjected to physical pretreatment; the fermentation time of the straws is short; an effect of the complexing agent is better than that in the prior art.

Description

An enzyme-bacteria compound agent for degrading crop straw

technical field

The invention relates to the field of processing and utilization of ruminant roughage in the feed industry, in particular to an enzyme-bacteria compound agent for degrading crop straws.

Background technique

my country is a large agricultural country with extremely rich crop straw resources, and the world's annual straw output is as much as 3 billion tons. In 2009, the remaining straw output after crop harvest in my country was as high as 740 million tons, ranking among the top in the world. However, due to the particularity of the physical and chemical properties of crop straw, it has many limitations when used as feed. Straw is relatively rough in texture, poor in palatability, poor in nutritional value, high in lignin and cellulose, and low in digestion and utilization. Only a small part can be used to feed ruminants. Monogastric animals generally cannot use straw, and the digestibility of straw by ruminants is only 20%-30%. How to improve the utilization of straw resources is a long-term concern.

In recent years, a lot of research has been done on how to effectively improve the utilization rate of straw at home and abroad. Biological treatment of straw does not require too complicated equipment and excessive energy consumption, nor does it require high temperature, high pressure, strong alkali and strong acid, etc. Conditions, relying only on the biodegradability of probiotics and enzymes to degrade the anti-nutritional factors in straw, it has the advantages of low energy consumption, little pollution, and easy operation. The research results show that microorganisms and their metabolites have the function of cracking the fiber structure of straw, so the use of biotechnology to improve the utilization rate of straw has attracted more and more attention from domestic and foreign scientific researchers.

The microbial preparations used for the biological treatment of crop straws mainly include bacteria and fungi, among which fungi produce cellulase activity and high lignocellulose decomposition efficiency. Trichoderma, Aspergillus, Penicillium, Verticillium, Rhizopus and other fungi can decompose cellulose and hemicellulose, and have the strongest effect under aerobic medium temperature. Trichoderma viride can produce highly active cellulase. White-rot fungi such as mushrooms, toadstools, poria cocos, polypores, and phloem can degrade lignin. Microorganisms with the strongest ability to degrade lignin use white-rot fungi to biodegrade corn stalks. The degradation rate is increased from 35-40% without adding nutrients to 55-65%.

There are many researches on white rot fungi at home and abroad, and the research mainly focuses on environmental management and pulp and paper industry, and there are many researches on feed industry. The degradation mechanism of white rot fungi to lignin relies on an extracellular degradation system mainly composed of enzymes produced and secreted by cells. It needs oxygen and is activated by the H ₂ O ₂ formed by itself, and a series of free radical chains are triggered by enzymes. reaction to achieve non-specific degradation of the substrate. Studies have found that white rot fungi secrete manganese peroxidase (Mn-peroxidase), lignin peroxidase (Ligninperoxidase), laccase, etc. All these enzymes form a complex enzyme system of white rot fungi and participate in the degradation of lignin together . Among the white-rot fungi, Phanerochaete chrysosporium is the most studied lignin-degrading bacteria, which has the characteristics of strong adaptability, stable enzyme production, and deep delignification ability. The extracellular lignin peroxidase produced by Phanerochaete chrysosporium includes lignin peroxidase, manganese peroxidase, laccase, cellobiose dehydrogenase, etc., in the presence of hydrogen peroxide Lignin degradation can completely decompose lignin into CO ₂ and H ₂ O.

Trichoderma viride can produce a variety of enzymes with biological activity, such as cellulase, chitinase, xylanase, etc. It is one of the strains with the highest cellulase activity. Crop stalks are degradable.

Aspergillus niger secretes cellulase and pectinase, which are generally recognized as safe microorganisms. Fermenting feed with Aspergillus niger is safe, reliable, and does not produce toxins. Moreover, Aspergillus niger grows fast, has strong anti-bacteria ability, and is easy to operate in production. Short, low cost and other advantages.

Penicillium can secrete peroxidase, especially lignin peroxidase, but reports are rare. Compared with white rot fungi, it has the characteristics of fast growth, short cycle and simple nutritional conditions. It is more suitable for industrial production application and experimental research.

There are many biological agents used for roughage such as crop stalks in the current literature and patent applications, generally including bacteria, fungi and enzyme preparations, but there is no combination of Phanerochaete chrysosporium with Aspergillus niger, Penicillium, Trichoderma fungus and wood. A form of organic combination of glycanase and cellulase.

Existing and the enzyme bacterium combination of the present invention closest report is as follows:

CN200710061529.4 (publication number is CN101058792A) discloses a high-efficiency straw decomposition composite flora, which is composed of the following strains by weight: 1.4-2.6 parts of Phanerochaete chrysosporium, 1.4-2.6 parts of Lentinus edodes, Trichoderma harzianum 0.7-1.3 parts, Trichoderma viride 0.7-1.3 parts, Trichoderma korningen 0.7-1.3 parts, Aspergillus niger 0.7-1.3 parts, Bacillus subtilis 0.73-1.24 parts, Feed Bacillus 0.73-1.24 parts, Bacillus megaterium 0.73-1.24 parts 0.14-0.26 parts of Candida, 0.14-0.26 parts of alcoholic yeast, 0.07-0.13 parts of edible yeast, 0.14-0.26 parts of brown spherical nitrogen-fixing bacteria, and 0.35-0.65 parts of high-temperature actinomycetes. When used for straw decomposition, its addition amount is 1-5‰ of straw weight. It can fully degrade lignin, cellulose, hemicellulose and other organic substances in straw.

CN201110441198.3 (CN102424808A) "A compound bacterial agent for straw degradation and its application in ethanol production pretreatment". Its content is that the No. I bacterial agent is composed of one or a mixture of two or more of Phanerochaete chrysosporium, Bacillus and Streptomyces in any proportion, and the No. II bacterial agent is composed of Trichoderma, Penicillium, Aspergillus and Actinomyces In the mixture of one or two or more bacteria in any proportion, in the mixture of No. .

CN201010537830.X (CN102048025A), the title of the invention is "Composite starter of xylanase combined with multi-strain and method for fermenting straw feed", which discloses a compound starter of xylanase combined with multi-strain and fermented straw method of feeding. It consists of 4 parts of xylanase; 2 parts of Lactobacillus acidophilus; 2 parts of propionic acid bacteria; 2 parts of Saccharomyces cerevisiae; 3 parts of Bacillus subtilis; 1 part of Lactobacillus plantarum;

CN201210178963.1 (publication number is CN102690755A) discloses a composite microbial bacterial agent for degrading crop straw, which is composed of Bacillus subtilis, Bacillus cereus, Aspergillus niger, Aspergillus flavus, Trichoderma reesei, Trichoderma longifera, Thermosporidium and Phanerochaete chrysosporium are produced by solid fermentation.

http://www.zgzlwx.com/hgyj/201105/3058337.html, Disclosed in the special CD-ROM of straw degradation technology (3058337-0019-0003) is a method for preparing protein feed by mixing solid-state fermentation and steam-exploding straw, including the following Steps: straw, steam explosion treatment, steam explosion material, preparation of fermentation medium according to the formula, sterilization, insertion of fermentation strains, shallow plate solid-state fermentation, drying, protein feed, wherein: fermentation medium is steam explosion A mixture of straw, bran and inorganic salt solution, the bacteria used for fermentation are a mixture of Penicillium recumbentum, Phanerochaete chrysosporium, and Candida tropicalis; the protein content of the protein feed product can reach more than 20% , the degradation rate of crude fiber is above 60%, which can completely replace the concentrate composed of grain.

Most of the existing patents use the compound action of bacteria and fungi, such as CN200710061529.4, CN201010537830.X, CN201210178963.1, etc. The content of cellulose and lignin in crop straw is high, the cell wall is hard, and it is difficult to degrade. Fungi have a good decomposing effect on cellulose and lignin, and although bacteria also have the corresponding ability to produce cellulase, the degradation effect is not as good as that of fungi, especially in straw fermentation, when straw lignin has not been degraded, bacteria The amount of carbon and nitrogen required for growth and expansion is insufficient to maintain the activity of bacteria. At the same time, the use of too many strains not only increases the cost, but also does not guarantee a high fiber degradation effect.

Existing straw-decomposing bacteria have the problem of long fermentation period, such as CN200710061529.4 needs to be fermented for 15 days, CN201210178963.1 needs to be fermented for 4-25 days; although the fermentation time is shortened, some straws need to be physically pretreated, which increases the cost. For relatively cheap straws, the gains outweigh the losses. For example, although the fermentation time of CN201110441198.3 is 2 days, the straws need to be soaked in ammonia water for 24-48 hours in advance; in the data of 3058337-0019-0003, steam explosion treatment is adopted.

Therefore, it is necessary to explore less strain combinations, adopt convenient and fast fermentation methods, and achieve high-efficiency degradation of straw fibers.

Contents of the invention

The purpose of the present invention is to adopt the method of combining enzyme activity, cellulose and lignin content measurement with the actual measurement of lignocellulose degradation rate by rumen nylon bag method to screen out the best and more refined enzyme-bacteria compound agent. The method is used for degrading lignin and cellulose in crop stalks and improving the utilization rate of the stalks.

An enzyme-bacteria compound agent for degrading crop stalks provided by the invention contains the following active ingredients: microbial compound agent and compound enzyme.

Specifically, the active ingredient contains the following components by weight: 20-60 parts of microbial complex agent, and 5-20 parts of complex enzyme.

Preferably, the active ingredient contains the following components by weight: 30-50 parts of microbial complex agent, and 8-15 parts of complex enzyme.

Further preferably, the active ingredient contains the following components by weight: 45 parts of microbial complex agent, 10 parts of complex enzyme.

Among the above-mentioned enzyme-bacteria compound agents:

The parts by weight can be μg, mg, g, kg and other well-known weight units in the field of medicine, and can also be multiples thereof, such as 1/10, 1/100, 10 times, 100 times, etc.

The microbial composite agent contains the following components by weight: 10-45 parts of Phanerochaete chrysosporium, 20-35 parts of Aspergillus niger, 20-35 parts of Penicillium, 0-30 parts of Trichoderma viride, and each part of bacteria contains 25ml of culture solution, the number of viable bacteria in the culture solution is 10 ⁸ /mL.

Preferably, the microbial complex agent contains the following components by weight: 25-45 parts of Phanerochaete chrysosporium, 25-33 parts of Aspergillus niger, 20-33 parts of Penicillium, and 5-33 parts of Trichoderma viride.

Further preferably, the microbial complex agent contains the following ingredients in parts by weight: 25 parts of Phanerochaete chrysosporium, 25 parts of Aspergillus niger, 25 parts of Penicillium, and 25 parts of Trichoderma viride.

The compound enzyme is composed of the following enzyme preparations in parts by weight: 25-40 parts of xylanase, 10-30 parts of cellulase and 5-20 parts of pectinase.

Preferably, 30-38 parts of xylanase, 15-25 parts of cellulase, and 8-15 parts of pectinase.

Further preferably, the compound enzyme is composed of the following enzyme preparations in parts by weight: 35 parts of xylanase, 20 parts of cellulase, and 10 parts of pectinase.

The carrier is a mixture of glucose and calcium chloride, the weight ratio of the two is 1-5:1, and the amount used in the mold complex agent is 20-75 parts, preferably 30-65 parts, more preferably 45 parts. The weight ratio of glucose and calcium chloride is preferably 3:1.

The present invention also provides a preparation method of the above-mentioned enzyme-bacteria compound agent, the method comprising the following steps:

1) Cultivate each single bacterial preparation in PDA medium until the number of spores reaches 10 ⁸ /mL;

2) Take Phanerochaete chrysosporium, Aspergillus niger, Penicillium, and Trichoderma viride according to the proportion, mix them evenly, and set aside;

3) Centrifuge the fermented bacteria at 3000-5000rpm for 50-60min at high speed, collect the bacteria, and make a microbial compound agent;

4) Weigh glucose and calcium chloride in proportion and mix them to make a carrier;

5) Mix the microbial compound agent and the carrier evenly in proportion, and dry it to make a powder;

6) Weigh xylanase, cellulase, and pectinase in proportion, mix them evenly to make a compound enzyme, and mix the compound enzyme with the powder obtained in 5) according to the weight ratio.

In the above method:

The PDA medium is: 1 L of potato extract, 20 g of glucose, 3 g of KH ₂ PO ₄ , 1.5 g of MgSO ₄ , 10 mg of vitamin B ₁ , 15 g of agar, pH 6.0. Among them, the potato extract is: Weigh 200g of potatoes, wash, peel, cut into small pieces, add 1000mL of water, boil for 30min, filter with gauze to obtain clear liquid, add water to 1000mL.

The invention also provides the application of the enzyme-bacteria compound agent in the degradation of crop straws.

The application refers to adding straws of crops (corn, wheat, rice) in an amount of 1-3% of the weight of the straws, preferably 2%. The compound agent is first mixed with 10 times of water, and evenly sprayed in the straw mixture.

For example: mix 100kg of straw with 20-30kg of small bran, add 30g of magnesium sulfate, 140g of diammonium sulfate, 200g of potassium dihydrogen sulfate, 25g of ferrous sulfate monohydrate, 8g of manganese sulfate heptahydrate, and 7g of zinc sulfate heptahydrate; Cobalt 10g, water 100L. Weigh the above-mentioned enzyme-bacteria compound agent, inoculate it into the straw mixture with 20% inoculum amount, compact it, and ferment for 7-10 days at room temperature under airtight conditions.

An enzyme-bacteria compound agent for degrading crop straws provided by the present invention has the following advantages:

1. The enzyme-bacteria compound agent provided by the invention is composed of a microbial compound agent and a compound enzyme.

The content of cellulose and lignin in crop straw is high, the cell wall is hard, and it is difficult to degrade. Fungi have a good decomposing effect on cellulose and lignin, while bacteria also have the corresponding ability to produce cellulase, but the degradation effect is not as good as that of fungi. Therefore, the present invention designs a microbial compound agent of fungal combination to ensure the effective degradation of lignocellulose and shorten the fermentation time.

Cellulose, hemicellulose and lignin exist in the cell walls of higher plants and fill between microfibrils, which play a role in strengthening and bonding plant tissues. The structure is complex and difficult to degrade. Among them, the content of lignin is second only to cellulose and hemicellulose. It contains various complex combinations that are biochemically very stable in its structure, which is difficult to be decomposed by general microorganisms and cannot be digested and absorbed by animals. Organic substances such as cellulose and hemicellulose polysaccharides are easily decomposed by microorganisms or enzymes. However, lignin and hemicellulose are blended together to tightly wrap the cellulose inside and become a peripheral matrix to protect the cellulose from microbial attacks and degradative enzymes, and the rumen microorganisms of herbivores lack lignin-degrading enzymes. Therefore, the nutrients in straw cells cannot be released, which limits the degradation and utilization of cellulose and hemicellulose by animals, resulting in low digestibility of straw. The higher the degree of lignification, the lower the digestibility. Therefore, the degradation of lignin must precede the degradation of cellulose, and the key to improving straw digestibility is to degrade lignin.

White rot fungi are currently discovered microorganisms that can completely degrade lignin. They can secrete extracellular enzymes such as lignin peroxidase (Lip), manganese peroxidase (Mnp), and laccase (Lac) to degrade lignin. . Phanerochaete chrysosporium is a typical representative of white rot fungi. It secretes lignin-degrading enzymes mainly Lip and Mnp. Lip catalyzes the single-electron oxidation of aromatic substrates and breaks C _α -C _β . Mnp catalyzes the oxidation of Mn(II) to form Mn(III), which can oxidize a series of organic substrates.

Penicillium is one of the fungi that can not only secrete lignocellulose-degrading enzymes with complete composition and high enzyme activity, but also have the advantages of easy cultivation and fast growth.

The invention uses Phanerochaete chrysosporium and Penicillium to degrade the lignin of the straw to the greatest extent, open the peripheral matrix surrounding cellulose and hemicellulose, and then supplement with the secretion of cellulase, chitinase and xylanase Aspergillus niger and Trichoderma viride, such as pectinase, achieve the purpose of decomposing straw cellulose step by step, and improve the degradation efficiency.

The product of the present invention adds compound enzyme while degrading lignin etc. with microbial compound agent, traces it to its cause:

Cellulase can degrade cellulose to generate glucose, and hydrolyze β-1,4 glucosidic bonds to convert a part of cellulose in the straw into usable cellobiose or glucose. Xylanase can further destroy lignin components in straw. Pectinase can destroy the protective layer of the plant surface, and probiotics can enter the plant tissue and decompose the fiber components, increasing the degradation rate of lignocellulose. The above three enzymes act on cellulose, lignin, and the pectin layer of plants respectively, so that probiotics can enter the plant cells in time to play a role; destroy the structure of plant fibers and convert cellulose into usable carbohydrates, thereby further improving Degradation rate of lignocellulose.

2. Select the best combination ratio through animal experiments

The simple combination of different cellulose-degrading bacteria and lignin-degrading bacteria often fails to achieve satisfactory results, and the effect of some combinations on degrading straw is not obvious. Through a series of experimental studies, the present invention confirms that the lignin degradation rate of the straw under the combination of Phanerochaete chrysosporium + Aspergillus niger + Penicillium + Trichoderma is higher than that of single bacteria pure culture fermentation.

3. The experimental results show that after the straw is treated with the enzyme-bacteria compound agent provided by the present invention, the lignin, neutral detergent fiber, acid detergent fiber, acid detergent lignin, cellulose, hemicellulose, etc. of the straw have a good degradation effect , improve the degradation rate of straw lignocellulose by rumen of ruminants, break the encirclement of other nutrients by lignin, and thus improve the utilization rate of straw. The enzyme-bacteria compound agent of the invention is convenient to use, does not need to carry out physical pretreatment on the straw, has shorter fermentation time of the straw, and has better effect than the prior art.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

The Phaerochaete chrysosporium 5.776 was purchased from the Culture Collection Center of Microorganisms, Chinese Academy of Sciences.

The Aspergillius niger, Penicillium sp., and Trichoderma viride were screened and preserved by the Livestock Nutrition and Feed Laboratory of Feed Research Institute, Chinese Academy of Agricultural Sciences, and can also be purchased from the market.

Described cellulase, 50,000 U/g, the product of domestic and foreign markets.

The xylanase, ≥50,000 U/g, is a product in domestic and foreign markets.

Described pectinase, 100,000 U/g, the product of domestic and foreign markets.

The composition of the PDA medium is: 1 L of potato extract, 20 g of glucose, 3 g of KH ₂ PO ₄ , 1.5 g of MgSO ₄ , 10 mg of vitamin B ₁ , 15 g of agar, and pH 6.0. The preparation method of the potato extract comprises the following steps: weighing 200g of potatoes, washing, peeling, cutting into small pieces, adding 1000mL of water, boiling for 30min, filtering with gauze to obtain a clear liquid, adding water to 1000mL.

Other carriers, feed additives, and raw materials can be purchased from the feed market.

Example 1: An enzyme-bacteria compound agent for degrading crop stalks

1. Composition: 45 parts of microbial compound agent, 10 parts of compound enzyme, 45 parts of carrier. in:

Microorganism complex agent: 25 parts of Phanerochaete chrysosporium, 25 parts of Aspergillus niger, 25 parts of Penicillium, 25 parts of Trichoderma viride; each part of bacteria contains 25ml culture solution, and the number of viable bacteria in the culture solution is ¹⁰⁸ / mL.

Compound enzyme: 35 parts of xylanase, 20 parts of cellulase, 10 parts of pectinase;

Carrier: 75 parts of glucose, 25 parts of calcium chloride.

2. Preparation method:

1) Propagate each single strain with PDA medium until the number of viable bacteria in the culture medium is ¹⁰⁸ /mL, and set aside;

2) Weigh the medium of Phanerochaete chrysosporium, Aspergillus niger, Penicillium, and Trichoderma viride according to the proportion, mix evenly, centrifuge the fermented strains at 4000rpm for 50min at high speed, collect the precipitate of the bacteria, and set aside;

3) Weigh the raw materials according to the ratio of glucose: calcium chloride = 3:1, mix the two raw materials evenly as the carrier, and set aside;

4) Mix the bacteria according to the ratio, mix with the carrier evenly, and dry to make a powder;

5) Weigh xylanase, cellulase, and pectinase in proportion, mix them uniformly initially, add them to the above powder in proportion, and mix them evenly again.

Example 2: An enzyme-bacteria compound agent for degrading crop stalks

1. Composition: 30 parts of microbial compound agent, 15 parts of compound enzyme, and 55 parts of carrier.

Microorganism complex agent: 33 parts of Phanerochaete chrysosporium, 33 parts of Aspergillus niger, 33 parts of Penicillium; each part of bacteria contains 25ml of culture solution, and the number of viable bacteria in the culture solution is 10 ⁸ /mL.

Compound enzyme: 40 parts of xylanase, 10 parts of cellulase, 20 parts of pectinase;

Carrier: 50 parts of glucose, 50 parts of calcium chloride.

2, preparation method: with embodiment 1.

Example 3: An enzyme-bacteria compound agent for degrading crop stalks

1. Composition: 55 parts of microbial compound agent, 5 parts of compound enzyme, 30 parts of carrier.

Microorganism complex agent: 45 parts of Phanerochaete chrysosporium culture fluid, 30 parts of Aspergillus niger culture fluid, 20 parts of Penicillium culture fluid, 5 parts of Trichoderma viride, each portion of bacterium contains 25ml, and the number of viable bacteria is 10 ⁸ individual/mL.

Compound enzyme: 25 parts of xylanase, 30 parts of cellulase, 15 parts of pectinase;

Carrier: 80 parts of glucose, 20 parts of calcium chloride.

2, preparation method: with embodiment 1.

Experimental example: verification of the effect of enzyme-bacteria compound agent on straw degradation

1. Test material

1.1 Corn stalks: sourced from Tongzhou, Beijing, dried naturally, crushed through a 40-mesh sieve, and stored in ziplock bags. Mix 100kg of straw with 25kg of bran, add an equal volume of magnesium sulfate 30g, diammonium sulfate 140g, potassium dihydrogen sulfate 200g, ferrous sulfate monohydrate 25g, manganese sulfate heptahydrate 8g, zinc sulfate heptahydrate 7g; cobalt sulfide 10g, water 100L.

1.2 Enzyme-bacteria compound agent: see Examples 1, 2, and 3 for its composition, and see Example 1 for the specific preparation method.

1.3 Experimental animals: 3 Du-Han hybrid F1 sheep with permanent rumen fistula were selected for the experiment, weighing 50±2kg. The feed was carried out according to the level of 1.3 times the maintenance nutritional requirement, and the ratio of the diet to coarse feed was 4:6. The experimental sheep were reared in a single pen, fed twice a day at the same amount at 6:00 and 18:00, and had free access to water.

1.4 Nylon bag: sewn from 300-mesh nylon cloth. The size is 6cm×10cm, the two corners of the bottom of the bag are blunt and round, with double stitching. Burn the loose sides of the nylon bags with a candle and number them with a marker that does not fade in the rumen. The new nylon bag should be placed in the rumen for 72 hours, taken out, washed, and dried in an oven at 65°C before use.

2. Test groups

Control group: corn stalks without adding any foreign enzymes and bacteria preparations.

Group A: corn stalks + enzyme-bacteria compound agent of Example 1.

Group B: corn stalks + enzyme-bacteria compound agent of Example 2.

Group C: corn stalks + the enzyme-bacteria compound agent of Example 3.

Group D: corn stalks + composite bacteria (Phanerochaete chrysosporium: Aspergillus niger: Trichoderma viride = 1:1:1).

Group E: corn stalks + composite bacteria (Phanerochaete chrysosporium: Penicillium: Trichoderma viride = 1:1:1).

3. Measuring indicators and methods

3.1 Straw fermentation conditions and measurement indicators: Add 2% of the enzyme-bacteria compound agent to the straw treated in 1.2 (the compound agent is first mixed with 10 times of water, and evenly sprayed in the straw mixture), compacted, and the air is removed. Normal temperature fermentation 10d.

Straw samples were taken every day during the fermentation process to determine filter paper carbohydrase (FPA), carboxymethyl cellulase (CMCase), dextranase, xylanase, manganese peroxidase (Mnp), lignin peroxidase (Lip) activity. After 10 days, the samples were taken out, dried at 65°C for 2 days, and the contents of neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL), cellulose and hemicellulose were determined. Calculate the reduction ratio of the above indicators in the samples before and after 10 days of fermentation.

3.2 Determination of rumen degradation rate

Air-dry the unfermented stalks and the stalk samples fermented with the enzyme-bacteria complex agent of Examples 1, 2, and 3 for 10 days for later use.

Accurately weigh 3g of the sample to be tested (accurate to 0.0001g) and put it into the treated nylon bag, and tie the mouth of the bag tightly with a nylon rope. Three replicates of each sample were placed in the rumens of three fistula sheep, and two nylon bags were used for each replicate. Clip the nylon bag to one end of a semi-soft plastic tube about 50 cm long, and tie it tightly with a rubber band. On the plug of the fistula, take out the nylon bag 72 hours after putting the bag in, put the nylon bag and hose in cold water immediately to stop the action of microorganisms, and then rinse with tap water until the water is clear. After rinsing, put the nylon bag and sample into an oven at 65°C, dry to constant weight, rehydrate for 24 hours, and weigh. Mix the residues in two nylon bags from the same sheep at the same time point, put them into a ziplock bag, and set aside.

Determine the content of dry matter (DM), NDF, ADF, ADL, cellulose, hemicellulose, and crude protein in the sample before and after being placed in the rumen, and calculate the degradation rate. The formula is: nutrient degradation rate = (nutrient content before degradation Mass - mass of nutrients in the residue after degradation)/mass of nutrients before degradation × 100%.

4. Data statistics: The data were analyzed using SAS software.

5. Results

5.1 Relevant enzyme activities of straw after fermented by enzyme-bacteria compound agent: see Table 1

Table 1-1: Enzyme activity of corn stalks fermented with enzyme-bacteria complex agent in Example 1

Table 1-2: Enzyme activity of corn stalks fermented with enzyme-bacteria complex agent in Example 2

Table 1-3: Enzyme activity of corn stalks fermented with enzyme-bacteria complex agent in Example 3

Table 1-4: Enzyme activity of corn stalks treated with compound agent fermentation of group D

Table 1-5: Enzyme activity of corn stalks treated with compound agent fermentation of group E

In Table 1-1, 1-2, 1-3, 1-4, 1-5: FPA, CMCase, and glucanase activities can represent the cellulose decomposition ability of bacteria, and xylanase activity can be used as a comparison of hemicellulose The index of decomposition ability, the activity of manganese peroxidase (Mnp) and lignin peroxidase (Lip) indicates the lignin degradation ability of the fungus. As can be seen from Table 1, the enzyme-bacteria compound agent Mnp of embodiment 1 is higher, can reach 449.58U/mL in the time of 5d, and three embodiment compound agents all have higher Mnp simultaneously; Embodiment 2 glucanase The activity is higher, and the activity of Example 3 Lip is higher. It shows that the composite agents of Examples 1, 2, and 3 may have advantages in lignin and cellulose decomposition capabilities.

The results show that: the enzyme-bacteria compound agent provided by the invention has a high degrading ability to lignin and cellulose. From the analysis of the changes in the activity of each enzyme, it is suggested that the fermentation time can be 7-10 days.

5.2 Changes in the lignocellulose content of corn stalks after fermentation with the enzyme-bacteria compound agent: see Table 2

Table 2: Lignocellulose content of fermented corn stover (DM,%)

project NDF ADF ADL Cellulose Hemicellulose control 62.70±0.29 ^a 36.68±0.85 ^a 10.07±0.36 ^a 37.87±0.73 ^a 26.28±0.56 ^a Group A 54.73± ^0.25c 31.27± ^0.64c 7.72± ^0.23c 31.71±0.35 ^bc 23.46± ^0.78c Group B 55.86± ^0.35bc 30.86± ^0.71c 8.52± ^0.33ab 31.19± ^0.44cd 24.99± ^0.97ab Group C 55.18± ^0.95bc 31.18± ^0.65c 8.98± ^0.76ab 30.88± ^0.28c 24.10± ^0.32bc Group D 59.17± ^0.33ab 34.39± ^0.97b 8.04± ^0.89ab 33.75± ^0.77b 24.77± ^0.84ab

Group E 61.08±1.22 ^a 35.54± ^0.83ab 9.66± ^0.64a 34.67± ^0.53b 25.54±0.60 ^a

Note: There is a significant difference between the data in the same column with different letters on the shoulder (P<0.05).

The results in Table 2 show that after 10 days of fermentation, the contents of five indicators (NDF, ADF, ADL, cellulose, and hemicellulose) in straw treated with microorganisms were lower than those in untreated straw, especially the difference in ADL and cellulose content Obvious (P<0.05).

Compared with groups D and E, the contents of NDF, ADF, ADL and cellulose in group A were all decreased, and the contents of NDF, ADF and cellulose in groups B and C were lower.

The results show that: after the straw is fermented with the enzyme-bacteria compound agent provided by the invention, the lignin and cellulose parts of the straw are degraded, which is better than using microorganisms and biological enzymes alone.

5.3 Changes in the ruminal degradation rate of corn stalks after fermentation of the enzyme-bacteria compound agent: see Table 3

Table 3: 72h rumen degradation rate (%) of fermented corn stalks

Note: There are significant differences between peers with different letters on their shoulders (P<0.05).

The results in Table 3 show that after digesting in the rumen of mutton sheep for 72 hours, the degradation of DM, NDF, ADF, ADL, cellulose, hemicellulose, and crude protein in the straw treated with the enzyme-bacteria compound agent was lower than that of the untreated straw The rates were significantly increased (P<0.05). Compared with groups D and E, after treatment with the enzyme-bacteria compound agent A, B and C of the present invention, the degradation rate of the fibrous material of the stalks was improved, and there was an increase in the degradation rate of NDF, ADF, ADL, cellulose and crude protein. Certain advantages.

The results show that: the enzyme-bacteria compound agent provided by the invention can relatively destroy straw lignocellulose, improve the degradation rate of straw lignin in the rumen of ruminants, and break the encirclement of lignin to other nutrients, thereby improving the yield of straw. The availability is better than using microorganisms or biological enzymes alone.

Straw is one of the main roughages for ruminants. The enzyme-bacteria compound agent provided by the invention improves the degradation rate of straw lignocellulose, can supply more carbohydrates to rumen microorganisms of ruminants, increases the production of microbial protein, promotes the growth of animals, and reduces feeding cost.

Summary: After the straw is treated with the enzyme-bacteria compound agent provided by the invention, the lignin, neutral detergent fiber, acid detergent fiber, acid detergent lignin, cellulose, hemicellulose, etc. The degradation rate of lignin in straw by animal rumen breaks the encirclement of lignin on other nutrients, thereby improving the availability of straw. The enzyme-bacteria compound agent of the invention is convenient to use, does not need to carry out physical pretreatment on the straw, has shorter fermentation time of the straw, and has better effect than the prior art.

Although, the present invention has been described in detail with general description, specific implementation and test above, but on the basis of the present invention, some modifications or improvements can be made to it, which will be obvious to those skilled in the art . Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (12)

1. An enzyme-bacteria composite agent for degrading crop stalks, characterized in that its active ingredient is composed of the following components by weight: 20-60 parts of microbial composite agent, 5-20 parts of composite enzyme, and the microbial composite agent Contains the following components by weight: 10-45 parts of Phanerochaete chrysosporium, 20-35 parts of Aspergillus niger, 20-35 parts of Penicillium, 0-30 parts of Trichoderma viride, and each part of bacteria contains 25ml of culture solution, The number of viable bacteria in the culture solution is ¹⁰⁸ /mL; the compound enzyme is composed of the following enzyme preparations in parts by weight: 25-40 parts of xylanase, 10-30 parts of cellulase, 5-30 parts of pectinase 20 servings. 2. The enzyme-bacteria compound agent according to claim 1, characterized in that, the active ingredient is composed of the following components by weight: 30-50 parts of microbial compound agent, and 8-15 parts of compound enzyme. 3. The enzyme-bacteria compound agent according to claim 1, characterized in that, the active ingredient is made up of the following components by weight: 45 parts of microbial compound agent, 10 parts of compound enzyme. 4. according to the described enzyme bacterium compound agent of any one of claim 1-3, it is characterized in that, described microbial compound agent contains the composition of following weight portion: 25-45 parts of Phanerochaete chrysosporium, 25-45 parts of Aspergillus niger 33 parts, Penicillium 20-33 parts, Trichoderma viride 5-33 parts. 5. enzyme bacteria compound agent according to claim 4, is characterized in that, described microorganism compound agent contains the composition of following weight portion: 25 parts of Phanerochaete chrysosporium, 25 parts of Aspergillus niger, 25 parts of Penicillium, green 25 parts of Trichoderma. 6. The enzyme-bacteria compound agent according to any one of claims 1-3, characterized in that, the compound enzyme is composed of the following enzyme preparations in parts by weight: 30-38 parts of xylanase, 15 parts of cellulase -25 parts, pectinase 8-15 parts. 7. The enzyme-bacteria compound agent according to claim 6, characterized in that, the compound enzyme is composed of the following enzyme preparations in parts by weight: 35 parts of xylanase, 20 parts of cellulase, 10 parts of pectinase . 8. The enzyme-bacteria compound agent according to any one of claims 1-3, characterized in that it also contains a carrier, the carrier is a mixture of glucose and calcium chloride, and the weight ratio of the two is 1-5:1, Its dosage in the enzyme-bacteria compound agent is 20-75 parts. 9. The enzyme-bacteria compound agent according to claim 8, characterized in that, the carrier is a mixture of glucose and calcium chloride, and the consumption in the enzyme-bacteria compound agent is 30-65 parts. 10. The enzyme-bacteria compound agent according to claim 8, characterized in that, the carrier is a mixture of glucose and calcium chloride, and the consumption in the enzyme-bacteria compound agent is 45 parts. 11. The application of the enzyme-bacteria compound agent described in any one of claims 1-10 in the degradation of crop straws. 12 . The application according to claim 11 , characterized in that, the application refers to adding to crop straws, and the addition amount is 1-3% of the weight of the straws.