CN114591862B - Dzo rumen natural co-culture for improving yellow storage quality of corn straw and application thereof - Google Patents
Dzo rumen natural co-culture for improving yellow storage quality of corn straw and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of microbial probiotic application, and particularly relates to a natural co-culture (Methanobrevibacter soleyae) Cattle-YakTZ1 of Piromobacter and methane brevibacterium of dzo rumen for improving the yellow storage quality of corn straws.
Description
Technical Field
The invention relates to the field of biotechnology renewable energy, in particular to a dzo rumen natural co-culture for improving yellow storage quality of corn straw and application thereof.
Background
The number of yaks in China is more than 90% of the total number of yaks in the world, and about 2000 thousands of yaks are used. The cattle is a local variety in China, and has the advantages of coarse feed resistance, strong adaptability and the like. Dzos are derived from yaks and are fruits of yak population. Dzos are F1 generation hybrids of distant hybridization between yaks and cattle or improved breeds, male cattle are hybridized with female yaks, the hybrids are named as dzos, male yaks are hybridized with female cattle, and the hybrids are named as dzos. No matter the male of dzo and yak is sterile, the production performance of the male of dzo and yak is obviously superior to that of the parental yak in terms of growth, development, meat production, viability and the like, the capability of adapting to the ecological environment in a high-altitude area is higher than that of the parental yak, and the male and female yak has very important significance for improving the living standard of people in pasturing areas, flourishing the economy of pasturing areas and maintaining the ecological balance of the three river source areas. Investigation finds that by 2012, dzos account for 3.5 percent of the total number of cattle, not less than 12 ten thousand of the total number of the cattle, and dzos grow fast and have strong disease resistance, are generally not easy to get ill, are suitable for grazing at high mountains, are used together with meat, have good milk production performance and high milk quality, and show obvious heterosis. Under the same breeding condition, dzos show outstanding heterosis in all aspects of weight, length, height and the like compared with cattle and yaks. The feed conversion rate, meat quality and milk quality of cattle, dzo and yak growing in the same region are different, and the reason for the difference is presumed to be closely related to rumen microorganisms besides genetic difference. The genetic distance of the gastric juice fungal region of yaks and dzos is closer, and the difference of the fungal region of gastric juice of yaks and dzos from the gastric juice fungal region of rumens of cattle is larger. Dzo adapts to the cold ecological condition, and is resistant to rough feeding, severe cold and low oxygen severe natural environment. The rumen of dzos inhabits uniquely, complexly and various, and a large number of microbial communities synergistically and efficiently degrade wild pastures to provide survival energy and nutrient substances for dzos. Research shows that anaerobic fungi in the rumen of ruminant can produce a series of lignocellulose degrading enzymes with high activity for degrading plant cell walls, including cellulase, hemicellulase (mainly xylanase), laccase, esterase and the like, and play an important role in degrading rumen lignocellulose. Substrates that anaerobic fungi can utilize and degrade include soluble sugars, as well as complex structural and storage polysaccharides such as cellulose, xylan, glucan, and the like. In-vitro degradation experiments show that the methanogen can form a stable co-culture with the anaerobic fungi by using metabolic products of the anaerobic fungi, the growth of the anaerobic fungi and the methanogen is promoted in the process, the degradation and utilization of the lignocellulose substrates by the anaerobic fungi are obviously improved, higher-activity lignocellulose degrading enzymes are generated, and a large amount of methane and acetic acid are used as main fermentation end products.
Corn is one of the world's major crops. After the corn ears are harvested, the remaining corn straws are used as byproducts of agricultural crops, have great feeding value, are used as coarse feed resources of herbivorous livestock, and are extremely widely applied to ruminants in particular. As a big agricultural country in China, the corn straws are widely distributed and are in large quantity, the annual corn straw yield is over 2 hundred million tons, but a great part of the straws are incinerated, so that a large amount of resources are wasted, and serious air pollution is caused. The corn straw has rich nutrient components, the average content of organic substances is 15 percent, and the corn straw is a very valuable renewable resource. However, the corn straw is also a natural fiber inferior feed, and the organic substances in the feed directly have low nutritive value. The main components that make up the cell wall of corn stover are cellulose, hemicellulose, lignin and pectic polysaccharides, where crude fiber accounts for 75.4% of dry matter, with lesser amounts of crude protein (6% -7%) and crude fat (1% -2%), but of inferior quality. Relevant researches show that cellulose and hemicellulose in the corn stalks are wrapped by lignin, wherein part of the cellulose and the hemicellulose are combined with the lignin in a covalent form to form a compact structure, so that the utilization rate of the corn stalks is limited. Through proper physical, chemical and biological technical treatment of the straws, the nutritive value and the palatability of the straws are greatly improved. Currently, methods for treating straw feed include: ensiling, yellow storage, ammoniation and hot spraying. Under the background of modern economic development, how to fully utilize crop straws and how to convert resource benefits into economic benefits has very important social, economic and ecological significance. The comprehensive utilization of the corn straw has wide prospect. The soluble carbohydrate and fiber material content of the corn straws is very high, and sufficient substrates can be provided for microorganisms and enzymes, so that the method for treating the straws by biological methods such as ensiling, yellow storage or enzyme pretreatment is a good choice.
The yellow storage is that the moisture content of dry yellow straw, wheat straw and other raw materials (the moisture content is generally 10% -30%) is adjusted to generally reach the moisture content of the silage (65% -75%), so that the pH value in the storage process is reduced through lactic acid, acetic acid and the like generated by lactic acid bacteria fermentation, and the purpose of prolonging the storage period is achieved. In the process of raising dairy cows, yellow corn straws can be used for replacing a part of silage corn straws, the milk yield of the dairy cows is not obviously influenced, the raising cost is reduced, and the problem of silage shortage caused by factors such as climate, season and the like is solved. Scholars at home and abroad try to obtain the fermentation quality of high-quality silage or yellow silage by adding biological and chemical additives such as urea, enzyme preparations, cane sugar, lactic acid bacteria, sodium nitrite, formic acid, propionic acid and the like in a block acidification process to avoid poor fermentation. Researches find that the effects of the cellulase and the xylanase are better than those of other enzyme preparations in the aspect of improving the fermentation quality; the effect of cellulase, xylanase and beta-glucanase is superior to other enzyme preparations in terms of nutritional quality. The cellulase, xylanase and beta-glucanase 3 enzyme preparations are more suitable to be used as additives of whole-plant corn silage or yellow corn silage.
The method is an original, novel and effective means at home and abroad, and is characterized in that corn straws are pretreated by fermentation liquor of a natural co-culture of naturally symbiotic new ostrinia and methane brevibacterium separated from gastric juice of dzo rumen in grazing and then are subjected to yellow storage. The invention unexpectedly separates a natural co-culture (Methanobrevibacter oleyae) Cattle-YakTZ1 of new Verbena and methane brevibacterium from the rumen of dzo, and uses the fermentation liquor of the co-culture which takes corn straw as substrate to pretreat dry yellow corn straw, and then carries out yellow storage of the corn straw, and the experimental result shows that: by the pretreatment of the natural co-culture (Methanobacter olleyae) Cattle-YakTZ1 fermentation liquor of the new Verbena officinalis and the methane brevibacterium, the sensory quality of yellow corn stalks can be obviously improved; the neutral detergent cellulose (NDF) and acid detergent cellulose (ADF) content of the corn straws is reduced; the pH value of the corn straw in yellow storage fermentation is rapidly reduced; the content of lactic acid is obviously improved, the content ratio of lactic acid/acetic acid is obviously higher than that of other groups, and homolactic acid fermentation is adopted; the butyric acid content is low at the later stage of fermentation, which indicates that the propagation activities of putrefying bacteria, mould and butyric acid bacteria are limited; the content of ethanol is obviously improved; the content ratio of ammonia nitrogen to total nitrogen is reduced, and the amount of feed protein converted into ammonia nitrogen is less; the content of crude protein is obviously improved. Namely: obviously improves the sensory quality, the nutrient content and the nutritive value of coarse feed of yellow corn stalk.
Disclosure of Invention
The invention aims to provide a natural co-culture of mebiothrix and methane brevibacterium (Methanobrevibacter oleyae) Cattle-YakTZ1 of dzo rumen for improving the yellow storage quality of corn straw, wherein the natural co-culture of the mebiothrix and the methane brevibacterium (Methanobrevibacter oleyae) Cattle-YakTZ1 is stored in the common microorganism center of China microorganism culture preservation management committee at 11 months and 25 days 2021, the storage number is CGMCC No.23971, the storage address is China academy of sciences microorganism institute No. 3 of Western No.1 on North Chen Yang district in Beijing, and the postal code is 100101; the telephone is as follows: 0 to 64807355; the fax is as follows: 010-64807288.
The IT1S sequence of the neocallimastix in the natural co-culture (Methanobrevibacter oleyae) Cattle-YakTZ1 of the neocallimastix is shown as SEQ ID No. 1; the IT1S sequence of the methane brevibacterium in the natural co-culture (Methanobacter olleyae) Cattle-YakTZ1 of the new Verbena officinalis and the methane brevibacterium is shown as SEQ ID No. 2.
The second purpose of the invention is to provide the method for culturing the natural co-culture of the new Verbena officinalis and the methane brevibacterium (Methanobacter olleyae) Cattle-YakTZ1, which comprises the step of culturing the natural co-culture of the new Verbena officinalis and the methane brevibacterium (Methanobacter olleyae) Cattle-YakTZ1 in an anaerobic environment.
The third purpose of the invention is to provide the fermentation liquor of the natural co-culture of the new Verbena officinalis and the methane brevibacterium (Methanobacter olleyae) Cattle-YakTZ1.
The fourth purpose of the invention is to provide a preparation method of crop straw yellow storage feed, which comprises the step of adding the natural co-culture (methanobacterooleyae) of the new Verbena officinalis and the methane brevibacterium into yellow storage engineering to form bottle-YakTZ 1 fermentation liquor.
The fifth purpose of the invention is to provide a yellow storage bacterium agent, which comprises the natural co-culture (Methanobacter ollenyae) Cattle-YakTZ1 bacterium body of the new Verbena and the methane brevibacterium.
The sixth purpose of the invention is to provide a yellow storage feed additive, the active component of which is natural co-culture (Methanobrevibacter olleyae) Cattle-YakTZ1 of the new Verbena officinalis and the methane brevibacterium.
Preferably, the yellow corn stalk yellow corn silage additive is a corn stalk yellow corn silage additive.
A seventh object of the present invention is to provide a yellow silage comprising the yellow silage additive of claim 6.
The eighth purpose of the invention is to provide the application of the natural co-culture of the new Verbena officinalis and the methane brevibacterium (Methanobrevibacter olleyae) Cattle-YakTZ1 in preparing the yellow storage feed additive.
The ninth purpose of the invention is to provide the application of the natural co-culture of the new Verbena officinalis and the methane brevibacterium (Methanobacter olleyae) Cattle-YakTZ1 in preparing yellow silage.
The invention has the beneficial effects that: the invention firstly provides a fermentation liquor of a natural co-culture (Methanobacter olleyae) of new Verbena and methane Brevibacterium, namely a Cattle-YakTZ1 microbial inoculum, wherein the microbial inoculum fermentation liquor can be used for preparing corn straw yellow storage feed. The microbial inoculum fermentation liquor used for preparing the yellow silage has the advantages that: (1) the sensory quality of yellow corn stalk storage is obviously improved; (2) The neutral detergent cellulose (NDF) and acid detergent cellulose (ADF) content of the corn straws is reduced; (3) The pH value of the corn straw in yellow storage fermentation is rapidly reduced; (4) The content of lactic acid is obviously improved, the content ratio of lactic acid/acetic acid is obviously higher than that of other 2 groups, and homolactic fermentation is taken as the main part; (5) The content of butyric acid is very low at the later stage of fermentation, which indicates that the propagation activities of putrefying bacteria, mould and butyric acid bacteria are limited; (6) the content of ethanol is obviously improved; (7) The ammonia nitrogen/total nitrogen content ratio is the lowest, which shows that the amount of the feed protein converted into ammonia nitrogen is less; (8) The content of crude protein is obviously improved, and the nutritional value of the yellow storage feed is higher than that of other 2 groups. That is to say, the natural co-culture (Methanobrevibacter olleyae) Cattle-YakTZ1 of the new Verbena officinalis and the Brevibacterium methanolica of the invention pretreats dry yellow corn straws and inoculates a common silage bacterial agent to carry out yellow silage fermentation, thereby remarkably improving the yellow silage sensory quality, the nutrient content and the coarse fodder nutrient value of the corn straws and having very wide application prospect.
Drawings
FIG. 1 changes in the sensory quality of straw during fermentation of silage from different pretreatment groups
( a. Yellow storage for 30 days in experimental groups; b. yellow storage for 30d in blank group; c. yellow storage for 30 days in a control group; d. yellow storage for 45 days in experimental group; e. storing the blank group in yellow for 45d; f. yellow storage of control group for 45d )
FIG. 2 dynamic changes in dry matter content during fermentation
FIG. 3 dynamic Change of neutral Wash cellulose during fermentation
FIG. 4 dynamic Change of acid Wash cellulose during fermentation
FIG. 5 pH dynamic changes during fermentation
FIG. 6 dynamic changes in lactic acid during fermentation
FIG. 7 dynamic changes in acetic acid during fermentation
FIG. 8 dynamic ethanol Change during fermentation
FIG. 9 Ammonia Nitrogen/Total Nitrogen dynamics in fermentation Process
FIG. 10 dynamic changes in crude protein content during fermentation
Detailed Description
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise, and it should also be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the features, steps, operations and/or combinations thereof.
The present invention is further illustrated by reference to specific examples, which are intended to be illustrative only and not limiting. If the experimental conditions not specified in the examples are specified, they are generally according to the conventional conditions or according to the conditions recommended by the reagents company; reagents, consumables and the like used in the following examples are commercially available unless otherwise specified.
In the invention, ensiling is a technology for directly storing harvested fresh (green) straws in a cellar, a bag or a bundle for wrapping and storing after being crushed. Compacting and sealing the silage, and carrying out anaerobic fermentation on microorganisms contained in or added to the silage under a proper humidity condition to reduce the pH value in the silage to about 4.5. At this time, most microorganisms stop reproducing, and finally, the lactic acid bacteria are controlled by lactic acid generated by the lactic acid bacteria to stop growing, so that the purpose of ensiling is achieved. The ensiling requires that the moisture content of the straws is above 60% -70%, and the operation cannot be carried out when the moisture content is too low, so that the ensiling is limited in time.
The yellow storage is a technique of using dried (yellow) straw as raw material, mechanically kneading and crushing, adding a proper amount of water and biological bacteria agent, and baling and then bagging for storage. The fermentation liquor of the high-efficiency microbial inoculum added in the yellow storage decomposes partial cellulose, hemicellulose and even some lignin under a proper anaerobic environment and converts the partial cellulose, the hemicellulose and even some lignin into saccharides such as glucose, xylose and the like. The saccharides are converted into lactic acid, acetic acid and propionic acid through organic acid fermentation, and the propagation of harmful bacteria such as butyric acid bacteria and mould is inhibited, and finally the storage effect same as that of silage is achieved.
EXAMPLE I preparation of Natural Co-culture of Verbena neoformans and Brevibacterium methanolicum (Methanobrevibacterium oleyae) Cattle-YakTZ1 microbial inoculum
Inoculating a natural co-culture (Methanobrevibacter oleyae) of new Verbena officinalis and Brevibacterium methanolica into a liquid anaerobic culture medium with the inoculation amount of 10% (v/v), adding 1% (w/v) of dried and crushed corn straws as a substrate, simultaneously adding a compound antibiotic, and placing at 39 ℃ for anaerobic culture for 72h to obtain the high-activity microbial inoculum.
The anaerobic culture medium comprises the following components: yeast extract 1.0g, naHCO 3 5.0g of resazurin (1.0 g/L) 1mL, L-cysteine hydrochloride 1.7g, salt solution I82.5 mL, salt solution II 16.5mL, and distilled water to 1000mL.
Salt solution I comprises 6g of NaCl, (NH) 4 ) 2 SO 4 3g，KH 2 PO 4 3g，CaCl 2 ·2H 2 O 0.4g，MgSO 4 ·2H 2 O0.6 g and distilled water to 1000mL.
The salt solution II comprises 4g of K 2 HPO 4 And distilled water is added to the volume of 1000mL.
After addition of different roughage substrates, oxygen was removed and carbon dioxide was introduced. Sterilizing at high temperature and high pressure.
Preferably, the compound antibiotics are penicillin sodium and streptomycin sulfate, and the concentrations are 1600IU/mL and 2000IU/mL respectively.
Example two, the fermentation liquor of a natural co-culture of new Verbena officinalis and Brevibacterium methanolica (Methanobacter oleyae) Cattle-YakTZ1 pretreats corn stalks and then inoculates an ensilage agent for yellow storage.
Corn stalk: is collected from Dingxi city of Gansu province in 2020, 12 months and winter, and the water content is 5.25% when collected, and the collected material is dry yellow, and is pulverized to 0.5-1cm for use.
Natural co-culture of ruminal Neomeibia and Brevibacterium methanolicum (Methanobrevibacter oleyae) Cattle-YakTZ1 fermentation broth of dzo: inoculating 50mL of natural co-culture (Methanobacter olleyae) of neocallimastix and methane brevibacterium into an anaerobic bottle containing 450mL of anaerobic fungus liquid culture medium with crushed and air-dried corn straw as a substrate, culturing for 72h, collecting fermentation liquor, centrifuging the fermentation liquor at 5000r/min for 10min, and obtaining supernatant for later use.
Commercial xylanase solution: is from silver Sainuo biotechnologies, inc., and is xylanase in powder form with enzyme activity of 60 ten thousand u/g. And dissolving the commercial xylanase in distilled water until the commercial xylanase is saturated, centrifuging, removing the precipitate, and taking xylanase supernatant for later use.
Yellow storage bacterial agent: common lactobacillus forage silage and yellow storage leaven. 1g of strain powder and 10g of brown sugar are added into 1000mL of water, and the mixture is cultured for 20 hours for later use.
The pretreatment experimental design for yellow fermentation is as follows in table 1:
TABLE 1 fermentation pretreatment design for silage
As shown in table 1, the dry yellow corn stalks were divided into 3 pretreatment groups, distilled water was added to the blank group until the moisture content was 65.48%, saturated xylanase solution was added to the control group for pretreatment until the moisture content was 64.72%, and natural co-culture of ruminobactor ruminonotus dzo and methanobrevibacterium (methanobacter oleyae) cat-YakTZ 1 was added to the experimental group for pretreatment until the moisture content was 65.05%. The 3 groups of corn straws are respectively pretreated for 48 hours and then respectively put into fermentation tanks, and are respectively inoculated with common silage and yellow rice straw strain agents, sealed, oxygen-insulated and placed in a room temperature environment (about 25 ℃) for solid state fermentation. Each treatment group was run in 3 replicates. Taking out corresponding fermentation samples at 15d, 30d and 45d respectively, and analyzing and measuring the yellow storage quality of the corn straws from aspects of sensory quality, lignocellulose component, fermentation characteristic, nutrient component and the like to comprehensively evaluate the fermentation quality of yellow storage of the corn straws after the dry yellow corn straws are pretreated by fermentation liquid generated by degrading the corn straws by adopting a natural co-culture (methanobacteria olleyae) of dzo rumen testes and methane brevibacterium.
EXAMPLE III analytical determination of maize straw yellow stock quality
1. Sensory evaluation of corn straw yellow corn silage
After the seal is opened, the sensory quality of the corn straw yellow storage sample after the seal is opened is firstly evaluated according to the green yellow storage quality evaluation standard of German agriculture Association (namely, the green storage feed is evaluated according to the three dimensions of smell, structure and color), and the evaluation standard is shown in Table 2.
TABLE 2 sensory evaluation of silage
2. Corn stalk lignocellulose analysis
2.1 determination of the Dry Matter (DM) content
Crushing a proper amount of sample by using a crusher, sieving by using a 40-mesh sieve, putting the sieved sample into a drying crucible, drying at 105 ℃ for 4h for constant weight, and putting the dried sample into a dryer to cool to room temperature.
M 1 Sample before drying at-105 ℃ and weighing vessel
M 2 Sample dried at-105 ℃ and weighing vessel
M 0 Weighing the sample vessel with constant weight
2.2 determination of neutral detergent cellulose (NDF)
Crushing a proper amount of sample by using a crusher, sieving by using a 40-mesh sieve, putting the sieved sample into a drying crucible, drying at 105 ℃ for 4h for constant weight, and putting the dried sample into a dryer to cool to room temperature. Placing the processed sample into a sample bag, marking, soaking in acetone for 20min, and placing in a ventilated place for 10min to volatilize residual acetone.
Putting the soaked sample into an alpha F6800 type full-automatic fiber determinator, and starting a neutral cellulose washing procedure; after the program is finished, the mixture is put into a drying crucible to be dried for 4h at the temperature of 105 ℃ and the constant weight, and then the mixture is put into a dryer to be cooled to the room temperature.
M 3 Weight of sample + Filter bag after drying
M 2 Initial weight of sample
M 1 Weight of Filter bag
2.3 measurement of acid-washed cellulose (ADF)
Crushing a proper amount of sample by using a crusher, sieving by using a 40-mesh sieve, putting the sieved sample into a drying crucible, drying at 105 ℃ for 4h for constant weight, and putting the dried sample into a dryer to cool to room temperature. Placing the processed sample into a sample bag, marking, soaking in acetone for 20min, and placing in a ventilated place for 10min to volatilize residual acetone.
Putting the soaked sample into an alpha F6800 type full-automatic fiber determinator, and opening an acid cellulose washing program; after the program is finished, the mixture is put into a drying crucible to be dried for 4h at the temperature of 105 ℃ and the constant weight, and then the mixture is put into a dryer to be cooled to the room temperature.
M 3 Weight of dried sample + Filter bag
M 2 Initial weight of sample
M 1 Weight of Filter bag
3. Corn straw fermentation characteristic and nutrient component determination
3.1 Determination of pH value
Taking 50g of a sample, pulping according to the proportion of 1.
3.2 measurement of lactic acid, acetic acid, and butyric acid content
Accurately weighing chromatographic standard substances (purity not less than 99.5%) of lactic acid, acetic acid and butyric acid, diluting with water, diluting to 100mL to obtain mixed standard stock solution, storing at 4 deg.C for use, diluting the stock solution with distilled water to obtain 5, 50, 100, 150, 300, 500mg/L series mixed standard works, filtering with 0.33 μm porous membrane, analyzing on machine, and sucking 200 μ L H 3 PO 4 Dissolving in 1000mL distilled water to obtain organic acid extractive solution, and collecting 1mLH 3 PO 4 Distilled water is used for fixing the volume to 1000mL to prepare a chromatographic mobile phase, and the chromatographic mobile phase is filtered by a 0.33 mu m pore membrane for later use.
A chromatographic column: phenomenex MARS MOA (300mm 7.8mm,10 μm,8% degree of crosslinking), homologous 50mm guard column, mobile phase 0.1% H 3 PO 4 Solution (v/v), flow rate 0.60mL/min, column temperature 57 ℃, differential detector temperature 30 ℃, response time 4s, sample volume 20. Mu.L.
Pulping 50g of a sample according to the proportion of 1. And calculating the content of the target by adopting a peak area external standard method.
3.3 determination of Ethanol (EA) content
Accurately weighing 10g of a sample, adding 100mL of distilled water for homogenate, centrifuging at 8000g and 25 ℃ for 10min, and taking supernatant for later use; according to the instruction of ethanol content determination by a micro method (reagent kit for determining ethanol content by Shanghai Yuanxin biological micro method), 6mL of reagent III is added into the reagent II before the test is started and is fully dissolved for standby.
Sucking 100 mu L of the first reagent, 50 mu L of the second reagent, 10 mu L of the fourth reagent and 40 mu L of the supernatant, uniformly mixing, recording an initial absorbance value A1 at 450nm, and an absorbance value A2 after incubation for 10min in the dark at 37 ℃, wherein delta A = A2-A1.
The regression equation is determined under standard conditions to be y =0.0256x +0.0055R 2 ＝0.9991
x is ethanol content (mu mol/mL), y is absorbance difference delta A
3.4 measurement of Ammonia Nitrogen content
Accurately removing 40 mul of ammoniacal nitrogen standard solutions of 1, 2, 4, 8, 16 and 32mg/100mL, adding the ammoniacal nitrogen standard solutions into a pre-marked glass test tube, adding 40 mul of methanol into each tube, uniformly mixing, adding 2.5mL of phenol color developing agent, uniformly mixing, adding 2.0mL of sodium hypochlorite solution, bathing for 10min at 37 ℃, taking out, standing for 10min at room temperature, using the ultraviolet spectral light at a unique wavelength of 650nm, taking an actual blank as a worn coin, and measuring the absorbance value of the worn coin. And drawing a standard curve by taking the ammoniacal nitrogen concentration (mg/100 mL) as an abscissa and taking the corresponding absorbance value as an ordinate, and performing a simplified first fitting regression equation.
Taking 50g of a sample, pulping according to the proportion of 1. Absorbing 80 μ L of the stock solution, adding into a glass test tube, adding 2.5mL of phenol color developing agent, mixing, adding 2.0mL of sodium transcribe solution, mixing, preserving in water bath at 37 deg.C for 10min, taking out, standing at room temperature for 10min, measuring absorbance at 650nm of ultraviolet spectrophotometer with reagent blank as reference.
Ammoniacal nitrogen concentration (mg/100 mL) = C 1 -C 0
C 1 Ammonia nitrogen concentration in mg/100mL, determined on a standard curve
C 0 -ammonia nitrogen concentration in mg/100mL found in the blank
3.5 crude protein assay
Digestion: placing 3g potassium sulfate, 0.2g copper sulfate, 0.5g sample, 10mL concentrated sulfuric acid and a funnel in a graphite digestion instrument, recording the total digestion volume V, simultaneously making digestion blank test samples (3 g potassium sulfate, 0.2g copper sulfate and 10mL concentrated sulfuric acid), and setting digestion temperature and time: 180 ℃/30 min-250 ℃/3 min-320 ℃/30 min-400 ℃/30min, if not well digested, then digested at 400 ℃ for 40 min-1 h until the blue-green color is digested, and the volume V' of digestion liquid after digestion is recorded.
The digested tubes were placed on a retort while one erlenmeyer flask was connected. Setting automatic parameters, diluting water quantity of 20mL, distilling time of 5min, rinsing water quantity of 20mL, and during empty distillation: adjusting the boric acid and sodium hydroxide to 10mL, and adding the sample: 20mL of boric acid and 40mL of sodium hydroxide. A250 mL Erlenmeyer flask was charged with 2-3 drops of a mix indicator (initially red with boric acid and then blue after absorbing ammonia, if the color is abnormal, the instrument is abnormal.) and the liquid in the Erlenmeyer flask was titrated to colorless with 0.05mol/L HCl and the volume V2/V1 of HCl consumed was recorded.
V2-volume of hydrochloric acid standard titration solution consumed by titration sample
V1-volume of hydrochloric acid standard titration solution consumed for titration of blank
m-sample mass
V-total volume of digestion solution
V' -distillation for removing liquid
4. Data analysis
The basic data are preliminarily arranged and made into a graph through Excel 2010, statistical analysis is carried out on the test data through SPSS 20.0 software, a single-factor ANOVO model is adopted for processing, multiple comparative analysis is carried out on the data through a Duncan method, the measurement result is represented by the mean value plus or minus standard deviation, and P is less than 0.05, which represents that the data have significance difference.
5. Analysis of results
5.1 sensory evaluation of yellow storage
Table 3 shows the results of sensory evaluation during fermentation. As can be seen from Table 3, the results of sensory evaluation in the whole fermentation period of the experimental groups were all "excellent", and the odor was expressed as weak butyric acid odor, strong sour taste, weak aroma, good retention of straw stem and leaf structure, and similar color to the raw material. When the control group is fermented for 0-15 days, sensory evaluation results are all 'excellent', and when the control group is fermented for 30 days, sensory evaluation results are 'good', and differences are mainly reflected in that the structure of the stems and leaves of the corn straws is poor, the color of the stems and leaves is slightly changed, and the odor of butyric acid is slight; the fermentation is poor after 45 days, the yellow storage surface has slight mildew phenomenon, and the butyric acid is fed heavily. When the blank group is fermented for 0d to 15d, sensory evaluation results are good, the odor of butyric acid is weak, the odor is poor when the fermentation is carried out for 30d, the surface of yellow corn silage is mildewed and has serious butyric acid smell, the odor is decayed when the fermentation is carried out for 45d, the surface of yellow corn silage is mildewed seriously, and the yellow corn silage has strong mildewed smell and serious pollution as shown in a graph 1, and the yellow corn straws are dark green.
TABLE 3 sensory evaluation of corn stalk yellow storage
5.2 measurement of lignocellulose in the fermentation Process of yellow silage
5.2.1 nutrient ingredients of raw materials
TABLE 4 nutritional ingredients of corn stover feedstock
5.2.2 dynamic variation of Dry Matter (DM) content
FIG. 2 is a graph showing the dynamic change of Dry Matter (DM) in the solid state fermentation process of corn stover, showing that: along with the extension of the fermentation time, the DM content of the blank group is in a descending trend overall, the DM content of the control group is in a stable trend after being increased, the DM content difference is not significant (P is more than 0.05) at 30d and 45d, the DM content of the experimental group is in a gradually increasing trend, wherein the DM content of the experimental group is significantly higher than that of the other groups (P is less than 0.05) when the experimental group is stored for 30d in yellow, and the DM content of the blank group is relatively lower when the blank group is stored for 45d in yellow.
5.2.3 dynamic Change in neutral detergent cellulose (NDF) content
FIG. 3 is a graph showing the dynamic change in Neutral Detergent Fiber (NDF) content during solid state fermentation of corn stover, showing that: the NDF content of the blank group gradually increases along with the extension of the fermentation time, and the NDF content of the control group and the NDF content of the experimental group generally decrease. The NDF content difference between the control group and the blank group is not significant (P > 0.05) at 30d and 45d, and the NDF content difference between the blank group and the blank group is not significant (P > 0.05). The experimental group had a lower NDF content than the other groups at 45d fermentation.
5.2.4 acid-washed cellulose (ADF) content dynamic Change
Fig. 4 is a dynamic change of acid-washing fiber (ADF) content during solid-state fermentation of corn stalks, and it can be seen that the ADF content of the blank group generally decreases first and then increases with the increase of fermentation time, and the difference between the fermentation time 15d and the fermentation time 30d is not significant (P > 0.05), the ADF content of the control group and the experimental group generally decreases, and the difference between the fermentation time 30d and the fermentation time 45d is not significant (P > 0.05), wherein the ADF content of the experimental group is significantly lower than that of the other groups (P < 0.05) when the experimental group is stored for 45d in yellow.
5.3 measurement of fermentation characteristics and Nutrition ingredients in the fermentation Process of yellow fermentation
5.3.1 Dynamic change of pH value
FIG. 5 is the dynamic change of pH value in the solid-state fermentation process of corn stalk, which can be seen as follows: the blank group showed an overall increasing pH value with increasing fermentation time, and the pH value was significantly higher than that of the other treated groups at 45d (P < 0.05), the control group showed an overall decreasing and increasing pH value with the experimental group, wherein the difference between the control group and the experimental group was not significant at 15d and 30d of yellow storage (P > 0.05), and the pH value of the experimental group at 30d was significantly lower than that of the other treated groups (P < 0.05) throughout the fermentation period.
5.3.2 dynamic variation of Lactic Acid (LA) content
FIG. 6 is the dynamic change of Lactic Acid (LA) content during solid state fermentation of corn stover, which can be seen: along with the extension of fermentation time, the blank group shows a trend of ascending first and then descending, the LA content in the blank group reaches the highest at 30d, the LA contents in the control group and the experimental group both show a trend of gradually ascending, and the lactic acid content reaches the highest at 45d, wherein the LA content in the experimental group is obviously higher than that in other treatment groups at 45d (P < 0.05).
5.3.3 dynamic variation of Acetic Acid (AA) content
FIG. 7 is the dynamic change of Acetic Acid (AA) content during the solid state fermentation of corn stalks, which shows that: along with the extension of the fermentation time, the AA content of the blank group and the control group gradually increases along with the extension of the fermentation time, the AA content of the experimental group gradually decreases and then increases, and reaches the lowest at 30d, wherein the AA content of the control group and the experimental group does not significantly differ at 30d (P > 0.05), and the AA content of the blank group is significantly higher than that of the other groups at 45d (P < 0.05).
5.3.4 dynamic changes in lactic acid/acetic acid ratio and butyric acid content
TABLE 5 dynamic variation of lactic acid/acetic acid ratio and butyric acid content in corn stalk yellow storage fermentation
5.3.5 dynamic Change in Ethanol (EA) content
FIG. 8 is a graph of the dynamic change in Ethanol (EA) content during solid state fermentation of corn stover, showing that: with the extension of the fermentation time, the EA content of the blank group and the control group is in a trend of increasing firstly and then decreasing with the extension of the fermentation time, the EA content reaches the highest value at 30d, the EA content of the experimental group is in a trend of gradually decreasing, and the EA content reaches the lowest value at 45 d.
5.3.6 Ammonia Nitrogen/Total Nitrogen (AN/TN) content dynamic Change
FIG. 9 is the dynamic change of ammoniacal nitrogen/total nitrogen (AN/TN) ratio during the solid state fermentation of corn stover. As can be seen from FIG. 9, the AN/TN ratios of the blank groups generally increased with the increase of the fermentation time, wherein the AN/TN ratios of the blank groups were significantly higher than those of the other groups at 45d of fermentation (P < 0.05), the AN/TN ratios of the control groups and the experimental groups generally increased gradually, and the AN/TN ratios of the white groups and the control groups were not significantly different at 15d of yellow storage (P > 0.05).
5.3.7 dynamic variation of Crude Protein (CP) content
FIG. 10 shows the dynamic change of Crude Protein (CP) in the solid-state fermentation process of corn stalk, and it can be seen that: the CP content of the blank group gradually decreases with the increase of the fermentation time, the CP content of the control group gradually decreases at 15-30 d, and the CP content difference is not significant at 30d and 45d (P > 0.05). The CP content of the experimental group is gradually reduced, the CP content difference is not obvious (P > 0.05) at 30d and 45d, and the CP content of the experimental group is obviously higher than that of the blank group and the control group (P < 0.05) at 30d and 45 d.
Experimental results the statistical results are shown in table 6.
TABLE 6 quality analysis and determination of maize straw yellow storage 45d
In conclusion, after the corn straws are pretreated for 48 hours by the high-efficiency fermentation liquor added with the natural co-culture of dzo rumen neocallimastix and methanobrevibacterium (methanobreyeae) Cattle-YakTZ1, the common yellow storage microbial inoculum is inoculated for yellow storage fermentation of the corn straws for 45d (an experimental group), and the yellow storage quality of the corn straws is not only remarkably superior to the yellow storage quality (a blank group) of directly inoculating the common yellow storage microbial inoculum for yellow storage fermentation of the corn straws for 45d, but also superior to the yellow storage quality (a control group) of adding the commercial feed xylanase for pretreatment of the corn straws for 48 hours, and then inoculating the common yellow storage microbial inoculum for yellow storage fermentation of the corn straws for 45d (a control group). Its outstanding advantages are mainly shown in the following 8 aspects: 1. obviously improves the yellow storage sensory quality of the corn straws; 2. the neutral detergent cellulose (NDF) and acid detergent cellulose (ADF) content of the corn straws is reduced; 3. the pH value of the corn straws in the yellow storage fermentation is rapidly reduced; 4. the content of lactic acid is obviously improved, the content ratio of lactic acid/acetic acid is obviously higher than that of other 2 groups, and homolactic fermentation is mainly used; 5. the content of butyric acid is very low at the later stage of fermentation, which indicates that the propagation activities of putrefying bacteria, mould and butyric acid bacteria are limited; 6. the content of ethanol is obviously improved; 7. the ammonia nitrogen/total nitrogen content ratio is the lowest, which shows that the amount of the feed protein converted into ammonia nitrogen is less; 8. the content of crude protein is obviously improved, and the nutritional value of the yellow storage feed is higher than that of other 2 groups. And comprehensively obtaining the conclusion that: the natural co-culture (methanobacterobacter oleyae) of ruminal neocallimastix and methanobrevibacterium of dzos, which is disclosed by the invention, is used for pretreating dry yellow corn straws and then inoculating a common silage microbial inoculum for yellow storage fermentation, so that the sensory quality, the nutritional ingredients and the nutritional value of coarse feed of the yellow storage of the corn straws are remarkably improved, and the application prospect is very wide.
Sequence listing
<110> institute of biological research of academy of sciences of Gansu province
Dzo rumen natural co-culture for improving yellow storage quality of corn straw and application thereof
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<170> SIPOSequenceListing 1.0
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Dzo <213> (ball)
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tttgttgttt gtaataattt tttttgattt taaaaaaaaa ttatttatgg ttttgtctat 180
ccctaaaatt ggtttgttgt gaaatgaatc aaatttaggg aataggcttt cataaataag 240
ttttttttaa agtcttaaaa gaccttcttt aaaattaaac ttttgtattc atttgtctaa 300
atttttttta aaataattta aaaacaactt ttgacaatgg atctcttggt tctcgcaacg 360
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Dzo <213> (bull)
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tgctcagtaa cacgtggata acctaccctt aggactggga taaccctggg aaactggggc 60
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cctaaggatg ggtctgcggc agattaggta gttggttggg taatggctta ccaagcctat 180
gatctgtacg ggttgtgaga gcaagagccc ggagatggaa cctgagacaa ggttccaggc 240
cctacggggc gcagcaggcg cgaaacctcc gcaatgtgag aaatcgcgac ggggggatcc 300
caagtgccat tcttaacggg atggcttttc ttaagtgtaa aaagcttttg gaataagagc 360
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catctcacca ggagcgacag ctgtatgatt accaggctga tgaccttgtt tgactagctg 900
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Claims (10)
1. A natural co-culture (Methanobrevibacter oleyae) Cattle-YakTZ1 of dzo rumen neomerle and methane brevibacterium for improving yellow storage quality of corn straws is characterized in that the natural co-culture (Methanobrevibacter oleyae) Cattle-YakTZ1 of the mehtiolima and the methane brevibacterium is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.23971.
2. The method for culturing the natural co-culture of the anemarrhena and methanobacterium (methanobacterobacter oleyae) Cattle-YakTZ1 as claimed in claim 1, which comprises the step of culturing the anaerobic fungus and methanobacterium co-culture (N.frontalis + M.oleyae) Cattle-YakTZ1 in an anaerobic environment.
3. The fermentation culture of natural co-culture of Verbena neoformans and Brevibacterium methanolica (Methanobacter oleyae) Cattle-YakTZ1 of claim 1.
4. A preparation method of a crop straw yellow storage feed is characterized by comprising the step of adding the natural co-culture (Methanobrevibacter olleyae) Cattle-YakTZ1 fermentation liquor of the neocallimastix neoformans and the methanobrevibacterium of claim 1 in a yellow storage project.
5. A silage additive comprising a natural co-culture of the bacterium Neocallimastix and Brevibacterium methanolica (Methanobacter oleyae) Cattle-YakTZ1 strain of claim 1.
6. A silage additive, characterized in that the active ingredient of the silage additive is the natural co-culture of neocallimastix and methanobrevibacterium (methanobacterohollyae) cat-YakTZ 1 of claim 1.
7. The silage additive of claim 6, wherein the silage additive is a corn stalk silage additive.
8. A silage characterized in that the silage additive of claim 6 is included.
9. Use of the natural co-culture of neocallimastix and brevibacterium methanolica (methanobacterbeviter olleyae) cat-YakTZ 1 of claim 1 for the preparation of a yellow silage additive.
10. Use of a natural co-culture of neocallimastix and brevibacterium methanolica (methanobacterooleyae) cat-YakTZ 1 according to claim 1 for the preparation of yellow silage.
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| CN106978355A (en) * | 2017-03-16 | 2017-07-25 | 甘肃省科学院生物研究所 | The coculture of a kind of anaerobic fungi and methane backeria and the application in the feeding native enzyme of milk cow |
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