CN103549129B - Enzyme and bacterium complexing agent for degrading crop straws - Google Patents
Enzyme and bacterium complexing agent for degrading crop straws Download PDFInfo
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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
Technical field
The present invention relates to feed industry ruminant roughage processing and utilization field, be specifically related to a kind of enzyme bacterium complexing agent for agricultural crop straw of degrading.
Background technology
China is a large agricultural country, and have extremely abundant agricultural straw resource, annual whole world stalk output has 3,000,000,000 tons more than.After China's crop harvestings in 2009, remaining stalk output is up to 7.4 hundred million tons, and position is at the forefront in the world.But due to the particularity of agricultural crop straw physicochemical property, it has a lot restricted as feed.Stalk quality is more thick and stiff, palatability is bad, nutritive value is poor, lignin and the high and digestive utilization ratio of content of cellulose lower etc., only have a little part can be used to raise ruminant.Nonruminant generally can not utilize stalk, and ruminant also just only has 20%-30% to the digestibility of stalk.The utilization how improving stalk resource is a thing be concerned for a long time.
In recent years, large quantifier elimination has been carried out both at home and abroad to how effectively improving straw utilization rate, with stalk of carrying out a biological disposal upon, neither need the consumption such as too complicated equipment and too much energy, also do not need the conditions such as high temperature, high pressure, highly basic and strong acid, only the biodegradability of dependence probio and enzyme preparation carrys out the ANFs in degrading straw, has energy consumption low, pollute little, be easy to the advantages such as operation.Result of study shows, microorganism and metabolite thereof have the function cracking stalk fibre structure, therefore utilizes biotechnology raising straw utilization rate to be more and more subject to the attention of domestic and international scientific researcher.
Microorganism formulation for farm crop straw organism process mainly contains bacterium and fungi etc., the fungi that wherein cellulase-producing is active, lignocellulose degradation efficiency is higher.The fungies such as wood is mould, aspergillus, mould, Verticillium sp, head mold, energy decomposition of cellulose and hemicellulose, and act on the strongest in aerobic under temperature, Trichoderma viride can produce highly active cellulase.The ocean white-rot fungi such as mushroom, terrible agaric, Poria cocos, bracket fungus, bast bacterium can lignin degrading, the microorganism that lignin degrading ability is the strongest, utilize whiterot fungi to carry out biodegradation pretreatment to maize straw, the degradation rate of lignocellulosic brings up to 55-65% by the 35-40% originally not adding nutriment.
More to the research of white-rot fungi both at home and abroad, its research mainly concentrates on environmental improvement and pulping and papermaking industry, also many in the research of feed industry.The degradation mechanism of white-rot fungi to lignin is, rely on one primarily of cell produce secretion enzyme system composition extracellular degraded system, aerobic and by self formed H
2o
2activate, triggered by enzyme and start a series of radical chain reaction, realize substrate without specific degraded.Research finds white rot fungus to excrete manganese peroxidase (Mn-peroxidase), lignin peroxidase (Ligninperoxidase), laccase etc., all these enzymes form the complex enzyme system of white-rot fungi, participate in the degraded activity of lignin together.Phanerochaete chrysosporium in white-rot fungi is the lignin-degrading bacteria of most study, has strong adaptability, and enzyme system produces stable, has the features such as extended delignification ability.The outer lignin peroxidase system of the born of the same parents that Phanerochaete chrysosporium produces comprises lignin peroxidase, manganese peroxidase, laccase, cellobiose dehydrogenase etc., carries out lignin degradation in the presence of hydrogen peroxide, lignin thoroughly can be decomposed into CO
2and H
2o.
Trichoderma viride can produce multiplely has bioactive enzyme system, and as cellulase, chitinase, zytase etc., be one of institute's cellulase-producing the highest active bacterial strain, the cellulase produced has degradation to crop material.
Aspergillus niger eccrine fiber element enzyme and pectase are generally regarded as safe microorganisms, with fermentation of Aspergillus niger feed safety, reliably, do not produce toxin, and Aspergillus Niger Growth are fast, press down miscellaneous bacteria ability strong, easy to operate aborning, have the cycle short, low cost and other advantages.
Mould can secrete peroxidase, especially lignin peroxidase, but reports actually rare.Compared with white-rot fungi, there is the growth fast i.e. cycle short, the features such as nutritional condition is simple.More adapt to suitability for industrialized production application and experimental study.
Have a lot for the biologic product of the roughages such as agricultural crop straw in current document and patent application, generally comprise bacterium, fungi and enzyme preparation, but there is no with Phanerochaete chrysosporium coordinate aspergillus niger, Penicillium notatum, Trichoderma fungi combination with zytase, cellulase organic assembling form.
Existing and enzyme bacterium of the present invention combines as follows close to reporting the most:
CN200710061529.4(publication number is CN101058792A) disclose a kind of highly effective straw decomposition composite flora, be made up of the bacterial classification of following weight portion: Phanerochaete chrysosporium 1.4-2.6 part, Lenlinus edodes 1.4-2.6 part, Trichoderma harzianum 0.7-1.3 part, Trichoderma viride 0.7-1.3 part, the mould 0.7-1.3 part of healthy and free from worry wood, aspergillus niger 0.7-1.3 part, bacillus subtilis 0.73-1.24 part, feed bacillus 0.73-1.24 part, bacillus megaterium 0.73-1.24 part, Candida 0.14-0.26 part, distillery yeast 0.14-0.26 part, food yeast 0.07-0.13, aztobacter sp 0.14-0.26 part, thermophilic actinomycete 0.35-0.65 part.During for Straw decomposing, its addition is the 1-5 ‰ of stalk weight.Lignin, cellulose, hemicellulose and other organic substances in the comprehensive degrading straw of energy.
CN201110441198.3(CN102424808A) " a kind of straw-degrading composite microbial inoculum and the application in alcohol production pretreatment thereof ".Its content is, I microbial inoculum is made up of the mixture of one or more arbitrary proportions in Phanerochaete chrysosporium, bacillus and streptomycete, II microbial inoculum is mould by wood, the mixture of one or more arbitrary proportions in mould, aspergillus and actinomyces forms, in I microbial inoculum and II microbial inoculum mixture, mass percent shared by I microbial inoculum is 50%-70%, II microbial inoculum is surplus.
CN201010537830.X(CN102048025A), denomination of invention is " the zytase associating composite ferment of multi-cultur es and the method for fermented stalk feed ", discloses a kind of zytase associating composite ferment of multi-cultur es and the method for fermented stalk feed.It is by zytase 4 parts; Lactobacillus acidophilus 2 parts; Bacterium acidi propionici 2 parts; Saccharomyces cerevisiae 2 parts; Bacillus subtilis 3 parts; Lactobacillus plantarum 1 part; Bacillus coagulans 1 part composition.
CN201210178963.1(publication number is CN102690755A) disclose a kind of complex micro organism fungicide of crop material of degrading, be that, sporotrichum thermophile mould by bacillus subtilis, Bacillus cercus, aspergillus niger, aspergillus flavus, trichoderma reesei, long handle wood and Phanerochaete chrysosporium are made through solid fermentation.
Http://www.zgzlwx.com/hgyj/201105/3058337.html, the method that one (3058337-0019-0003) mixed solid fermentation and straw puffing prepares protein feed is disclosed in degrading straw technology thematic information CD, comprise the following steps: stalk, steam explosive-crushing process, vapour is disclosed, fermentation culture medium is made by formula, sterilizing, access fermented bacterium, tray solid state fermentation, dry, protein feed, wherein: fermentation culture medium is the mixture of steam puffed stalk and wheat bran and inorganic salt solution, the bacterial classification that fermentation adopts is penicillium decumbens, Phanerochaete chrysosporium, with the mixture of candida tropicalis, obtained protein feed product protein content can reach more than 20%, lignocellulose degradation rate more than 60%, the fine fodder that can grain be replaced completely to form.
Existing patent uses the compound action such as bacterium, fungi mostly, as CN200710061529.4, CN201010537830.X, CN201210178963.1 etc.Crop branch fiber element, content of lignin are higher, and cell membrane is hard, degrades more difficult.Fungi has good decomposition to cellulose, lignin, and though bacterium also has the ability of corresponding cellulase-producing, but degradation effect is not as fungi, especially in stalk fermentation, when straw lignin is not yet degraded, bacterial growth expands numerous required carbon nitrogen quantity deficiency, can not maintain the active of bacterium.Adopt multi-cultur es simultaneously, not only added cost, and do not ensure higher fiber degradation effect.
Existing Straw decomposing bacterium also exists the long problem of fermentation period, as CN200710061529.4 needs fermentation 15d, CN201210178963.1 at 4-25d; Although the fermentation time reduction had, need to carry out physics pretreatment to stalk, increase cost, for relative low price stalk lose more than gain, though as CN201110441198.3 fermentation time be 2d, need to soak stalk 24-48h in advance with ammoniacal liquor; What adopt in 3058337-0019-0003 data is steam explosive-crushing process.
Therefore need to explore less strain combination, adopt conveniently fermentation process, reach the method for high efficiency degrading straw fiber.
Summary of the invention
The object of the invention is to adopt enzyme work, cellulose and content of lignin mensuration to survey ligocellulose degradation with Rumen Nylon Bag and lead the method combined, filter out the enzyme bacterium complexing agent of the best, comparatively refining, adopt simple method, for lignin in agricultural crop straw and the cellulose of degrading, improve straw utilization rate.
A kind of enzyme bacterium complexing agent for agricultural crop straw of degrading provided by the invention, its active component contains following composition: microorganism complex, complex enzyme.
Concrete, described active component contains the composition of following weight portion: microorganism complex 20-60 part, complex enzyme 5-20 part.
Preferably, described active component contains the composition of following weight portion: microorganism complex 30-50 part, complex enzyme 8-15 part.
Further preferably, described active component contains the composition of following weight portion: microorganism complex 45 parts, complex enzyme 10 parts.
In above-mentioned enzyme bacterium complexing agent:
Described weight portion can be the known unit of weights of field of medicaments such as μ g, mg, g, kg, also can be its multiple, as 1/10,1/100,10 times, 100 times etc.
Described microorganism complex contains the composition of following weight portion: Phanerochaete chrysosporium 10-45 part, aspergillus niger 20-35 part, Penicillium notatum 20-35 part, Trichoderma viride 0-30 part, and the nutrient solution containing 25ml in every part of bacterium, the viable count in nutrient solution is 10
8individual/mL.
Preferably, described microorganism complex contains the composition of following weight portion: Phanerochaete chrysosporium 25-45 part, aspergillus niger 25-33 part, Penicillium notatum 20-33 part, Trichoderma viride 5-33 part.
Further preferably, described microorganism complex contains the composition of following weight portion: Phanerochaete chrysosporium 25 parts, aspergillus niger 25 parts, Penicillium notatum 25 parts, Trichoderma viride 25 parts.
Described complex enzyme, is made up of by weight following enzyme preparation: zytase 25-40 part, cellulase 10-30 part, pectase 5-20 part.
Preferably, zytase 30-38 part, cellulase 15-25 part, pectase 8-15 part.
Further preferably, described complex enzyme, is made up of by weight following enzyme preparation: zytase 35 parts, cellulase 20 parts, pectase 10 parts.
Described carrier is the mixture of glucose and calcium chloride, and the two weight ratio is 1-5:1, and its consumption in mould complexing agent is 20-75 part, is preferably 30-65 part, more preferably 45 parts.The weight ratio of glucose and calcium chloride is preferably 3:1.
Present invention also offers the preparation method of above-mentioned enzyme bacterium complexing agent, the method comprises the following steps:
1) often kind of single bacteria preparation is reached 10 with PDA medium culture to spore count
8individual/mL;
2) Phanerochaete chrysosporium, aspergillus niger, Penicillium notatum, Trichoderma viride culture medium is taken according to proportioning, after mixing, for subsequent use;
3) by the bacterial classification high speed centrifugation 50-60min under 3000-5000rpm after fermentation, collect thalline, make microorganism complex;
4) take glucose, calcium chloride in proportion, be mixed and made into carrier;
5) microorganism complex, carrier are mixed in proportion, dry and make pulvis;
6) taking zytase, cellulase, pectase in proportion, mix, make complex enzyme, by complex enzyme and 5) gained pulvis mixes by weight proportion.
In said method:
PDA culture medium is: potato leachate 1L, glucose 20g, KH
2pO
43g, MgSO
41.5g, Cobastab
110mg, agar 15g, pH6.0.Wherein potato leachate is: take 200g potato, cleans, removes the peel, be cut into small pieces, boil 30min filtered through gauze and become clear liquid, add water to 1000mL after the 1000mL that adds water.
Present invention also offers the application of enzyme bacterium complexing agent in agricultural crop straw degraded.
Described application refers to and is added into crops (corn, wheat, paddy rice) stalk, and addition is the 1-3% of stalk weight, is preferably 2%.Complexing agent is first converted with the water of 10 times punching, is evenly sprayed in stalk mixture.
Such as: stalk 100kg is mixed with little wheat bran 20-30kg, adds magnesium sulfate 30g, sulfuric acid two ammonium 140g, potassium dihydrogen sulfate 200g, ferrous sulfate monohydrate 25g, seven water manganese sulfate 8g, white vitriol 7g; Cobalt sulfide 10g, water 100L.Take above-mentioned enzyme bacterium complexing agent, the inoculum concentration with 20% is seeded in stalk mixture, compacting, normal temperature fermentation 7-10d under airtight condition.
A kind of enzyme bacterium complexing agent for agricultural crop straw of degrading provided by the invention has the following advantages:
1, enzyme bacterium complexing agent provided by the invention is made up of microorganism complex and complex enzyme.
Crop branch fiber element, content of lignin are higher, and cell membrane is hard, degrades more difficult.Fungi has good decomposition to cellulose, lignin, though and bacterium also has the ability of corresponding cellulase-producing, but degradation effect is not as fungi.Therefore the present invention devises the microorganism complex of fungi combination, guarantees effective degraded of lignocellulosic, shortens fermentation time.
Cellulose, hemicellulose and lignin are present in the cell membrane of higher plant, are filled between microfibril, play reinforcing and cementation, complex structure to plant tissue, are difficult to degraded.Wherein the content of lignin is only second to cellulose and hemicellulose, and it containing various complex combination forms very stable in biochemistry, is difficult to, by general microbial decomposition, to be digested and assimilated by animal in the structure.Cellulose, Hemicellulose Polysaccharide type organic matter are then easily decomposed by microorganism or enzyme.But lignin and hemicellulose are blended together; cellulose is tightly wrapped in the inside; become surrounding matrix; protection cellulose exempts from microorganism and attacks and digestive enzyme attack, and the rumen microorganism of plant-eating animal lacks the enzyme of lignin degrading, so the intracellular nutriment of stalk can not discharge; thus limit animal to the degraded of the composition such as cellulose and hemicellulose and utilization; cause stalk digestibility low, degree of lignification is higher, and its digestibility is also just low.Therefore, the degraded of lignin must prior to cellulose degradation, and the key improving stalk digestibility is lignin degrading.
White-rot fungi be find at present can the microorganism of thorough lignin degrading, it can secrete the ectoenzyme lignin degradings such as lignin peroxidase (Lip), manganese peroxidase (Mnp), laccase (Lac).Phanerochaete chrysosporium is the Typical Representative of whiterot fungi, and secretion lignin-degrading enzymes is mainly Lip, Mnp, and the one-electron oxidation of Lip catalysis aromatic substrate, makes C
α-C
βfracture.Mnp then catalysis Mn (II) oxidation forms Mn (III), the oxidable a series of organic substrates of Mn (III).
Penicillium is that some in fungi can not only secrete that composition is complete, enzyme lives higher lignocellulolytic enzymes system, and has and easily cultivate and grow fast advantage.
The present invention adopts Phanerochaete chrysosporium and mould, the at utmost lignin of degrading straw, open the surrounding matrix surrounding cellulose and hemicellulose, be aided with aspergillus niger and the Trichoderma viride of eccrine fiber element enzyme, chitinase, zytase, pectase etc. again, reach the cellulosic object of decomposing straw step by step, improve degradation efficiency.
The while that product of the present invention being with microorganism complex lignin degrading etc., with the addition of complex enzyme, tracing it to its cause is:
Cellulase biodegradable fiber element generates glucose, and a part of cellulose conversion in stalk is the cellobiose or glucose that can be utilized by hydrolysis β-Isosorbide-5-Nitrae glycosidic bond.Zytase can destroy the lignin component in stalk further.Pectase can make the protective layer on plant top layer be damaged, and probio can enter plant tissue and decompose fibre composition, improves the degradation rate of lignocellulosic.Above-mentioned 3 kinds of enzymes act on the pectin layer of cellulose, lignin, plant respectively, are convenient to probio and enter in plant cell in time and play a role; Destroy the structure of string, make cellulose conversion be utilizable carbohydrate, thus improve the degradation rate of lignocellulosic further.
2, studied by animal experiment, screening best of breed ratio
Different cellulose-degrading bacterias and lignin-degrading bacteria are simply combined, and often do not reach satisfied effect, and the effect of some combination degrading straws is also not obvious.The present invention by series of experimental research, determines the Lignin degradation rate of stalk under the mould combination of Phanerochaete chrysosporium+aspergillus niger+mould+wood and ferments higher than single bacterium pure culture.
3, experimental result shows: after enzyme bacterium complexing agent Treating straw provided by the invention, the lignin, neutral detergent fiber, acid detergent fiber, acidic cleaning lignin, cellulose, hemicellulose etc. of stalk have good degradation, improve the degradation rate of ruminant tumor gastric to straw lignocellulose, break the encirclement of lignin to other nutritional labelings, thus improve the availability of stalk.Enzyme bacterium complexing agent of the present invention is easy to use, and without the need to carrying out physics pretreatment to stalk, the stalk fermentation time is shorter, and effect is better than prior art.
Detailed description of the invention
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
Described Phanerochaete chrysosporium (Phaerochaete chrysosporium5.776), is purchased from Chinese Academy of Sciences's Culture Collection.
Described aspergillus niger (Aspergillius niger), Penicillium notatum (Penicillium sp.), Trichoderma viride (Trichoderma viride) are screened by Institute of Feeds,China Academy of Agriculture Sciences's livestock nutrition and feed research department and are preserved, and also buy by market.
Described cellulase, 50,000 U/g, the product of domestic and international market.
Described zytase, >=5 ten thousand U/g, the product of domestic and international market.
Described pectase, 100,000 U/g, the product of domestic and international market.
Consisting of of described PDA culture medium: potato leachate 1L, glucose 20g, KH
2pO
43g, MgSO
41.5g, Cobastab
110mg, agar 15g, pH6.0.Wherein the preparation method of potato leachate comprises the following steps: take 200g potato, cleans, removes the peel, be cut into small pieces, boil 30min filtered through gauze and become clear liquid, add water to 1000mL after the 1000mL that adds water.
Other carriers and feed addictive, raw material all can be bought from feed market and obtain.
Embodiment 1: a kind of enzyme bacterium complexing agent for agricultural crop straw of degrading
1, form: microorganism complex 45 parts, complex enzyme 10 parts, 45 parts, carrier.Wherein:
Microorganism complex: Phanerochaete chrysosporium 25 parts, aspergillus niger 25 parts, Penicillium notatum 25 parts, Trichoderma viride 25 parts; Containing 25ml nutrient solution in every part of bacterium, the viable count in nutrient solution is 10
8individual/mL.
Complex enzyme: zytase 35 parts, cellulase 20 parts, pectase 10 parts;
Carrier: glucose 75 parts, 25 parts, calcium chloride.
2, preparation method:
1) by each single culture with PDA culture medium expand numerous to viable count in nutrient solution for 10
8individual/mL, for subsequent use;
2) take Phanerochaete chrysosporium, aspergillus niger, Penicillium notatum, Trichoderma viride culture medium according to proportioning, after mixing, by the bacterial classification high speed centrifugation 50min under 4000rpm after fermentation, collect bacterial sediment, for subsequent use;
3) according to glucose: the ratio of calcium chloride=3:1 takes raw material, 2 kinds of raw materials are mixed as carrier, for subsequent use;
4) according to proportioning, thalline is mixed, mix with carrier, dry and make pulvis;
5) take zytase, cellulase, pectase respectively in proportion, tentatively mix, add in proportion in above-mentioned pulvis, again mix.
Embodiment 2: a kind of enzyme bacterium complexing agent for agricultural crop straw of degrading
1, form: microorganism complex 30 parts, complex enzyme 15 parts, 55 parts, carrier.
Microorganism complex: Phanerochaete chrysosporium 33 parts, aspergillus niger 33 parts, Penicillium notatum 33 parts; Containing 25ml nutrient solution in every part of bacterium, the viable count in nutrient solution is 10
8individual/mL.
Complex enzyme: zytase 40 parts, cellulase 10 parts, pectase 20 parts;
Carrier: glucose 50 parts, 50 parts, calcium chloride.
2, preparation method: with embodiment 1.
Embodiment 3: a kind of enzyme bacterium complexing agent for agricultural crop straw of degrading
1, form: microorganism complex 55 parts, complex enzyme 5 parts, carrier 30 as one kind part.
Microorganism complex: Phanerochaete chrysosporium nutrient solution 45 parts, aspergillus niger nutrient solution 30 parts, Penicillium notatum nutrient solution 20 parts, Trichoderma viride 5 parts, containing 25ml in every part of bacterium, described viable count is 10
8individual/mL.
Complex enzyme: zytase 25 parts, cellulase 30 parts, pectase 15 parts;
Carrier: glucose 80 parts, 20 parts, calcium chloride.
2, preparation method: with embodiment 1.
Experimental example: the compliance test result of enzyme bacterium complexing agent degrading straw
1, test material
1.1 maize straws: derive from Tongzhou, Beijing, naturally dry, pulverized 40 mesh sieves, were put in valve bag and preserved.Stalk 100kg is mixed with little wheat bran 25kg, adds isopyknic magnesium sulfate 30g, sulfuric acid two ammonium 140g, potassium dihydrogen sulfate 200g, ferrous sulfate monohydrate 25g, seven water manganese sulfate 8g, white vitriol 7g; Cobalt sulfide 10g, water 100L.
1.2 enzyme bacterium complexing agent: its composition is shown in embodiment 1,2,3, and concrete preparation method is shown in embodiment 1.
1.3 experimental animal: 3 Du-cold first familiar generation wethers that permanence lymphoma stomach fistulization pipe is housed are selected in test, body weight 50 ± 2kg.Raise according to the maintenance nutritional need level of 1.3 times, daily ration essence coarse fodder ratio is 4:6.Test sheep separated raising, every day feeds 2 times in 6:00 and 18:00 equivalent respectively, freely drinks water.
1.4 Nylon Bags: 300 order nylon cloths are made and formed.Specification 6cm × 10cm, bottom bag, two jiaos is blunt round, and two-wire is sewed up.With the loose limit of the burned Nylon Bag of candle, and be used in the marking pen numbering of not easily fading in cud.New Nylon Bag will be placed on 72h in cud, takes out, cleans, after oven dry, can use in 65 DEG C of baking ovens.
2, test grouping
Control group: maize straw, does not add any external enzyme, bacteria preparation.
A group: maize straw+embodiment 1 enzyme bacterium complexing agent.
B group: maize straw+embodiment 2 enzyme bacterium complexing agent.
C group: maize straw+embodiment 3 enzyme bacterium complexing agent.
D group: maize straw+compound bacteria (Phanerochaete chrysosporium: aspergillus niger: Trichoderma viride=1:1:1).
E group: maize straw+compound bacteria (Phanerochaete chrysosporium: mould: Trichoderma viride=1:1:1).
3, testing index and method
3.1 stalk fermentation condition and testing indexs: stalk after 1.2 process is added the enzyme bacterium complexing agent (complexing agent is first converted with the water of 10 times punching, is evenly sprayed in stalk mixture) of 2%, compacting, deaeration, normal temperature fermentation 10d under airtight condition.
In sweat, every day gets the activity that straw sample measures filter paper carbohydrase (FPA), carboxymethylcelluloenzyme enzyme (CMCase), dextranase, zytase, manganese peroxidase (Mnp), lignin peroxidase (Lip).After 10d, take out sample, dry 2d for 65 DEG C, measure neutral detergent fiber (NDF), acid detergent fiber (ADF), acidic cleaning lignin (ADL), cellulose, hemicellulose level.Calculate the reduction ratio of These parameters in sample before and after fermentation 10d.
3.2 rumen digestibility measure
By unleavened stalk and through embodiment 1,2,3 enzyme bacterium complexing agent fermentation 10d straw sample air-dry for subsequent use.
Accurately take sample 3g(and be accurate to 0.0001g) put into processed good Nylon Bag, with nylon rope, sack is tightened.The repetition of 3, each sample, puts into the cud of 3 Fistulated sheep respectively, each repetition 2 Nylon Bags.Nylon Bag is clipped in half flexible plastic tube one end that 1 about 50cm is long, and tighten with rubber band, before early raising, the Nylon Bag clipped is put into cud abdomen Nang Chu, the other end nylon wire of flexible pipe is fixed on fistula stopper, after putting bag, 72h takes out Nylon Bag, the Nylon Bag taken out connects flexible pipe and puts into cold water immediately to stop the effect of microorganism, then uses tap water, till water is clear.After flushing, Nylon Bag and sample are put into 65 DEG C of baking ovens, dry to constant weight, moisture regain 24h, weighs.By residue mixing in 2 Nylon Bags of same the same time point of sheep, load in zip lock bag, for subsequent use.
Measure the content putting into the dry (DM) of sample before and after cud, NDF, ADF, ADL, cellulose, hemicellulose, crude protein, and calculating degradation rate, formula is: quality × 100% of nutriment before nutriment degradation rate=(quality of the rear residue Middle nutrition material of quality-degraded of the front nutriment of degraded)/degraded.
4, data statistics: data acquisition SAS software carries out statistical analysis.
5, result
The related enzyme activity of stalk after 5.1 enzyme bacterium complexing agent fermentations: in table 1
Table 1-1: the enzyme of embodiment 1 enzyme bacterium complexing agent fermentation process maize straw is lived
Table 1-2: the enzyme of embodiment 2 enzyme bacterium complexing agent fermentation process maize straw is lived
Table 1-3: the enzyme of embodiment 3 enzyme bacterium complexing agent fermentation process maize straw is lived
The enzyme of table 1-4:D group complexing agent fermentation process maize straw is lived
The enzyme of table 1-5:E group complexing agent fermentation process maize straw is lived
In table 1-1,1-2,1-3,1-4,1-5: FPA, CMCase, dextranase activity can represent the cellulose-decomposing ability of bacterium, xylanase activity is as the index comparing hemicellulose capacity of decomposition, and manganese peroxidase (Mnp), lignin peroxidase (Lip) activity illustrate the lignin degradation ability of bacterium.As can be seen from Table 1, the enzyme bacterium complexing agent Mnp of embodiment 1 is higher, can reach 449.58U/mL when 5d, and three embodiment complexing agents all have higher Mnp simultaneously; Embodiment 2 dextranase activity is higher, and embodiment 3Lip activity is higher.Illustrate that embodiment 1,2,3 complexing agent likely has advantage on lignin, cellulose-decomposing ability.
Result shows: enzyme bacterium complexing agent provided by the invention is higher to lignin, cellulosic degradation capability, and from each Enzyme activities situation analysis, suggestion fermentation time can at 7-10d.
5.2 the change of maize straw wood fibre cellulose content after the fermentation of enzyme bacterium complexing agent: in table 2
Table 2: the wood fibre cellulose content (DM, %) of fermentation process maize straw
| Project | NDF | ADF | ADL | Cellulose | Hemicellulose |
| Contrast | 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 |
| A group | 54.73±0.25 c | 31.27±0.64 c | 7.72±0.23 c | 31.71±0.35 bc | 23.46±0.78 c |
| B group | 55.86±0.35 bc | 30.86±0.71 c | 8.52±0.33 ab | 31.19±0.44 cd | 24.99±0.97 ab |
| C group | 55.18±0.95 bc | 31.18±0.65 c | 8.98±0.76 ab | 30.88±0.28 c | 24.10±0.32 bc |
| D group | 59.17±0.33 ab | 34.39±0.97 b | 8.04±0.89 ab | 33.75±0.77 b | 24.77±0.84 ab |
| E group | 61.08±1.22 a | 35.54±0.83 ab | 9.66±0.64 a | 34.67±0.53 b | 25.54±0.60 a |
Note: with the different alphabetical person's significant difference (P<0.05) of column data shoulder mark.
Table 2 result shows: after fermentation 10d, content through microbiological treating straw five kinds of indexs (NDF, ADF, ADL, cellulose, hemicellulose) is lower than undressed straw content, and especially on ADL and content of cellulose, difference is comparatively obviously (P<0.05).
Compared with D, E group, the NDF of A group, ADF, ADL, content of cellulose all decrease, and the NDF of B, C group, ADF, content of cellulose are lower.
Result illustrates: after enzyme bacterium complexing agent fermentation process stalk provided by the invention, lignin and the cellulosic sections of stalk are degraded, and are better than being used alone microorganism and biology enzyme.
The change of maize straw rumen digestibility after 5.3 enzyme bacterium complexing agent fermentations: in table 3
Table 3: the 72h rumen digestibility (%) of fermentation process maize straw
Note: the different alphabetical person's significant difference (P<0.05) of colleague's data shoulder mark.
Table 3 result shows: digest 72h in mutton sheep cud after, than undressed stalk, the degradation rate of DM, NDF in the stalk after the process of enzyme bacterium complexing agent, ADF, ADL, cellulose, hemicellulose, crude protein all significantly improves (P<0.05).Compared with D, E group, after A, B, C enzyme bacterium complexing agent of the present invention process, the degradation rate of the fiber substance of stalk increases, and NDF, ADF, ADL, cellulose and crude protein degradation rate have certain advantage.
Result shows: enzyme bacterium complexing agent provided by the invention to straw lignocellulose have compared with destruction, improve the degradation rate of ruminant tumor gastric to straw lignin, break the encirclement of lignin to other nutritional labelings, thus improve the availability of stalk, be better than being used alone microorganism or biology enzyme.
Stalk is one of main roughage of ruminant, enzyme bacterium complexing agent provided by the invention improves straw lignocellulose degradation rate, can supply the more carbohydrate of ruminant tumor gastric microorganism, improves microprotein output, promote the growth of animal, reduce feeding cost.
Sum up: after enzyme bacterium complexing agent Treating straw provided by the invention, the lignin, neutral detergent fiber, acid detergent fiber, acidic cleaning lignin, cellulose, hemicellulose etc. of stalk have good degradation, improve the degradation rate of ruminant tumor gastric to straw lignin, break the encirclement of lignin to other nutritional labelings, thus improve the availability of stalk.Enzyme bacterium complexing agent of the present invention is easy to use, and without the need to carrying out physics pretreatment to stalk, the stalk fermentation time is shorter, and effect is better than prior art.
Although above with general explanation, detailed description of the invention and test, the present invention is described in detail, and on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (12)
1. the enzyme bacterium complexing agent for agricultural crop straw of degrading, it is characterized in that, its active component is grouped into by the one-tenth of following weight portion: microorganism complex 20-60 part, complex enzyme 5-20 part, described microorganism complex contains the composition of following weight portion: Phanerochaete chrysosporium 10-45 part, aspergillus niger 20-35 part, Penicillium notatum 20-35 part, Trichoderma viride 0-30 part, the nutrient solution containing 25ml in every part of bacterium, the viable count in nutrient solution is 10
8individual/mL; Described complex enzyme, is made up of by weight following enzyme preparation: zytase 25-40 part, cellulase 10-30 part, pectase 5-20 part.
2. enzyme bacterium complexing agent according to claim 1, it is characterized in that, described active component is grouped into by the one-tenth of following weight portion: microorganism complex 30-50 part, complex enzyme 8-15 part.
3. enzyme bacterium complexing agent according to claim 1, it is characterized in that, described active component is grouped into by the one-tenth of following weight portion: microorganism complex 45 parts, complex enzyme 10 parts.
4. the enzyme bacterium complexing agent according to any one of claim 1-3, it is characterized in that, described microorganism complex contains the composition of following weight portion: Phanerochaete chrysosporium 25-45 part, aspergillus niger 25-33 part, Penicillium notatum 20-33 part, Trichoderma viride 5-33 part.
5. enzyme bacterium complexing agent according to claim 4, it is characterized in that, described microorganism complex contains the composition of following weight portion: Phanerochaete chrysosporium 25 parts, aspergillus niger 25 parts, Penicillium notatum 25 parts, Trichoderma viride 25 parts.
6. the enzyme bacterium complexing agent according to any one of claim 1-3, is characterized in that described complex enzyme is made up of by weight following enzyme preparation: zytase 30-38 part, cellulase 15-25 part, pectase 8-15 part.
7. enzyme bacterium complexing agent according to claim 6, is characterized in that described complex enzyme is made up of by weight following enzyme preparation: zytase 35 parts, cellulase 20 parts, pectase 10 parts.
8. the enzyme bacterium complexing agent according to any one of claim 1-3, is characterized in that, also containing carrier, described carrier is the mixture of glucose and calcium chloride, and the two weight ratio is 1-5:1, and its consumption in enzyme bacterium complexing agent is 20-75 part.
9. enzyme bacterium complexing agent according to claim 8, is characterized in that, described carrier is the mixture of glucose and calcium chloride, and the consumption in enzyme bacterium complexing agent is 30-65 part.
10. enzyme bacterium complexing agent according to claim 8, is characterized in that, described carrier is the mixture of glucose and calcium chloride, and the consumption in enzyme bacterium complexing agent is 45 parts.
The application of enzyme bacterium complexing agent described in 11. any one of claim 1-10 in agricultural crop straw degraded.
12. application according to claim 11, is characterized in that, described application refers to and is added in agricultural crop straw, and addition is the 1-3% of stalk weight.
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| CN108651694A (en) * | 2018-04-20 | 2018-10-16 | 常州市蒽盗钟情生物科技有限公司 | A kind of method of stalk fermentation |
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| CN118291277B (en) * | 2024-06-06 | 2024-08-09 | 南京农业大学三亚研究院 | Low-temperature straw degrading fungus cladosporium fungi NJAU-M3a1 and application thereof |
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