CN114617195A - Method for improving quality of silage by mixing tannin-resistant lactic acid bacteria and tannin - Google Patents
Method for improving quality of silage by mixing tannin-resistant lactic acid bacteria and tannin Download PDFInfo
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- CN114617195A CN114617195A CN202210112339.5A CN202210112339A CN114617195A CN 114617195 A CN114617195 A CN 114617195A CN 202210112339 A CN202210112339 A CN 202210112339A CN 114617195 A CN114617195 A CN 114617195A
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- tannin
- silage
- lactic acid
- acid bacteria
- resistant
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 122
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- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 61
- 238000000034 method Methods 0.000 title claims abstract description 37
- 238000002156 mixing Methods 0.000 title claims abstract description 16
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K30/00—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs
- A23K30/10—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder
- A23K30/15—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K30/00—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs
- A23K30/10—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder
- A23K30/15—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging
- A23K30/18—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging using microorganisms or enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Polymers & Plastics (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Animal Husbandry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Fodder In General (AREA)
Abstract
The invention discloses a method for improving the quality of silage by mixing tannin-resistant lactic acid bacteria and tannin, and particularly relates to the technical field of feed processing. The invention provides a method for improving the quality of silage, which is characterized in that tannin-resistant lactic acid bacteria and tannin are added into the silage, the mixture is uniformly mixed, the seal treatment is carried out, and the silage is stored at room temperature; in addition, the number of escherichia coli in the feed is also obviously reduced, and the dry matter content of the feed is obviously improved. The method can effectively solve the problem that fresh stylosanthes guianensis and whole soybean are not easy to store, realize the large-scale production of the stylosanthes guianensis and the whole soybean, increase feed resources, and be beneficial to solving the problem of seasonal forage gaps of herbivores.
Description
Technical Field
The invention relates to the technical field of feed processing, in particular to a method for improving the quality of silage by mixing tannin-resistant lactic acid bacteria and tannin.
Background
The production requirement of animal husbandry is limited to a certain extent due to insufficient supply of fresh forage grass and shortage of food and feed. The general conditions of the production and supply of feed resources are that the total amount of feed resources is insufficient, the energy feed mainly comprising grains is insufficient, the protein feed mainly comprising grain and oil processing byproducts is seriously in short supply, the coarse feed resources mainly comprising crop straws and the like are redundant, and the whole feed resource supply and demand relationship has the characteristics of concentrated feed shortage, protein feed shortage, green feed shortage and total amount shortage, namely 'three-deficiency-one deficiency'. Therefore, the silage technology is effectively utilized to fully exert the maximum value and the nutritional quality of the feed.
In order to better solve the problem of insufficient supply of the feed, the proper processing and storage mode can effectively help the problem of feed shortage in time and space. Ensiling is the most widely used mode of processing and storing forage grass, and is a processing method for storing forage grass resources for a long time by fermenting fresh forage grass under anaerobic conditions by attached lactic acid bacteria and converting saccharides into organic acids such as lactic acid and the like, and reducing the pH value to further inhibit or kill harmful microorganisms. The silage has important significance for inhibiting the growth and the reproduction of harmful bacteria, improving the nutritive value of the silage, generating fragrance, stimulating the appetite of livestock and improving the feed intake.
The silage additive mainly comprises a biological additive and a chemical additive. Lactic acid bacteria are the most commonly used biological additives, and can rapidly utilize fermentation substrates in the early stage of ensiling to generate lactic acid, reduce the pH value, inhibit the growth of harmful microorganisms and achieve the purpose of preserving feed. The commonly used chemical additives are mainly acids, such as formic acid, citric acid and the like, which are mainly used for rapidly reducing the pH value of the silage so as to achieve the aim of inhibiting fermentation.
Lactic acid bacteria refer to a group of spore-free gram-positive bacteria that can produce lactic acid using soluble sugars as fermentation substrates. Lactic acid bacteria do not contain proteolytic enzymes and do not degrade proteins. The addition of lactobacillus can also improve the aerobic stability of the silage, and has certain promotion effect on the production performances of dry matter feed intake, body weight increment, milk yield and the like of livestock.
Stylosanthes guianensis is a tropical perennial leguminous grass originally produced in south America, is the most important leguminous grass for both grazing and mowing in tropical and subtropical regions, and is suitable for being planted in southern regions of China. The stylosanthes guianensis has rich nutritive value, is rich in various amino acids and vitamins, is excellent leguminous forage grass and high-protein feeding crops in tropical regions, and is excellent feed for livestock and poultry. But the soluble carbohydrate content and the lactic acid bacteria quantity of the stylosanthes guianensis raw material are small, and harmful microorganisms are more, so that harmful or useless metabolism is generated, the loss of nutrient substances is caused, and the silage fermentation is not facilitated. The conventional silage of the stylosanthes guianensis has higher pH value and butyric acid content, lower lactic acid content and poorer quality.
The whole soybean (Glycine max (Linn.) Merr.) has high content of protein and vitamins, the content of crude fiber of the processed grass meal is low, the nutritive value and the utilization rate are high, and the soybean is a good green coarse feed resource for livestock. Meanwhile, the stem and leaf of the whole plant soybean are soft, tender and juicy, the leaf quantity is large, the palatability is good, and the digestibility of animals can be effectively improved. However, the whole soybean has low soluble carbohydrate content and high buffering capacity, which prevents the pH from rapidly decreasing in the ensiling process, has bad smell and high content of butyric acid, and is difficult to succeed in conventional ensiling.
The original name of the Chinese wampee (Neomarickia cadamba) flower (Anthocaulus chinensis), which is Rubiaceae (Rubiaceae), generally contains some anti-nutritional ingredients, such as tannin, saponin, polyphenol, etc. Tannin is a water-soluble phenol secondary metabolite formed by higher plants in a long-term evolution process, is a product of interaction between plants and the environment, has a molecular mass of 500-3000, widely exists in byproducts of sorghum seeds, fruits, vegetables, tea leaves and the like after processing, and is a fourth most abundant plant component following cellulose, hemicellulose and lignin. The tannin is yellow or brownish yellow loose powder, has strong hygroscopicity in air, is easily soluble in water, acetone and ethanol, is insoluble in benzene, ether and chloroform, and can be classified into Hydrolyzed Tannin (HT) and Condensed Tannin (CT) according to their chemical structures and biological activities. Wherein the hydrolyzed tannin is polyphenol compound formed by combining phenolic acid or its derivative with polyalcohol or glucose, and can be further divided into ellagitannin and gallic acid tannin. The condensed tannin is a polymer formed by more than three monomers of hydroxy flavan-3-ol and hydroxy flavan-3, 4-diol, and the flavanol monomers are easy to be split to form a high electrophilic center under the acidic condition of animal digestive tracts, so that covalent bonds are easily formed with nucleophilic groups in proteins. Thus condensed tannins are less susceptible to enzymatic hydrolysis than hydrolyzed tannins.
The main difficulties of the direct ensiling of the stylosanthes guianensis and the whole soybean are that the fresh sample has high water content and low sugar content, the number of the epiphytic lactic acid bacteria is small, the number of harmful bacteria is large, the fiber content of the stylosanthes guianensis is large, the palatability is poor and the digestibility is low.
Disclosure of Invention
In order to solve the technical problems, the invention provides the method for simultaneously adding the tannin-resistant lactic acid bacteria and the tannin as the silage additive for the first time. Compared with the traditional silage additive, the tannin-resistant lactobacillus functional lactobacillus can keep activity under a certain tannin concentration, effectively increase the number of the lactobacillus in the silage, increase the lactic acid content, reduce the pH value, inhibit the propagation of undesirable microorganisms and effectively improve the silage fermentation quality. Under the condition of tannin-resistant lactic acid bacteria, a proper amount of tannin is added, so that the protein degradation of the silage is reduced. The method can effectively solve the problem that fresh stylosanthes guianensis and whole soybean are not easy to store, realize the large-scale production of the stylosanthes guianensis and the whole soybean, increase feed resources, and be beneficial to solving the problem of seasonal forage gaps of herbivores. Wherein, the screening step of the tannin-resistant lactic acid bacteria for addition is simple, and the tannin-resistant lactic acid bacteria are easy to store and transport after freeze drying; the tannin reagent has wide acquisition route and low price, and can effectively enlarge the use area. The method provides a new idea for silage, and helps China to solve the problem of silage to a certain extent. The silage modulation of the leguminous forage is optimized, and a theoretical basis is provided for the large-scale and industrialized production of the leguminous forage.
A first object of the present invention is to provide a method for improving the quality of silage by adding tannin-resistant lactic acid bacteria and tannin to the silage.
Preferably, the silage is stylosanthes guianensis silage or whole soybean silage.
Preferably, the tannin-resistant lactic acid bacteria are added in an amount of 106cfu·g-1The addition amount of the tannin is 1% -2% of the total weight of the silage.
Preferably, the tannin-resistant lactic acid bacteria are in a state of bacteria powder with the water content of 0%, and are in a state of bacteria liquid activated after the bacteria powder is added with pure water when ensiling is added.
Preferably, the preparation of the tannin-resistant lactic acid bacteria comprises the following steps: collecting fresh leaves of yellow-wood, slicing, vacuum packaging, and storing at normal temperature; sampling and diluting, culturing by using an MRS culture medium, selecting a single bacterial colony, separating and purifying, then measuring the pH value, adding the lactobacillus bacterial liquid with high acid yield into a mixed liquid containing glucose, normal saline and tannin, and culturing and separating to obtain the tannin-resistant lactobacillus. Further, activating, enriching and culturing tannin-resistant lactic acid bacteria, centrifuging, washing with sterile normal saline, centrifuging twice, adding skimmed milk, mixing uniformly, pouring into a culture dish, pre-freezing, putting into a vacuum freeze-drying machine, wherein the vacuum degree is less than 60pa, the temperature of a cold trap is less than-55 ℃, and when the temperature of a sample reaches room temperature, finishing drying, and freezing and storing the prepared bacterial powder.
Preferably, the silage is cut into lengths of 2-3cm before the tannin-resistant lactic acid bacteria and tannin are added to the silage.
Preferably, the tannin-resistant lactic acid bacteria and tannin are added into the silage, and then the mixture is uniformly mixed, sealed and stored at room temperature, wherein the sealing treatment is bag sealing, barreling sealing or bundling sealing.
Preferably, the tannin-resistant lactic acid bacteria are plant lactic acid bacteria (Lactobacillus Plantarum); more preferably, the plant lactic acid bacteria are NCL-LP1, which are deposited under the following accession numbers: CGMCC No. 23489.
The second purpose of the invention is to provide the application of the tannin-resistant lactic acid bacteria in the preparation of stylosanthes guianensis and whole-plant soybean silage additives.
Preferably, the tannin-resistant lactic acid bacteria are plant lactic acid bacteria (Lactobacillus Plantarum); more preferably, the plant lactic acid bacteria is NCL-LP1 with the deposit number: CGMCC No. 23489.
Compared with the prior art, the invention has the advantages that:
(1) the mixing and adding of the tannin-resistant lactic acid bacteria and tannin remarkably reduces the pH value, the content of non-protein nitrogen and ammonia nitrogen of the silage columella flowers and grasses and the silage whole plants, and remarkably improves the content of true protein;
(2) the mixing and adding of the tannin-resistant lactic acid bacteria and the tannin remarkably reduces the number of escherichia coli in the whole silage soybean, and particularly has the best effect under the condition of mixing and adding the tannin-resistant lactic acid bacteria and 2% of tannin;
(3) the dry matter content of the ensiling whole plant soybean is obviously improved by mixing and adding the tannin-resistant lactic acid bacteria and the tannin;
(4) the tannin-resistant lactic acid bacteria and tannin are respectively added into the stylosanthes guianensis and the whole-plant soybean silage in a mixing manner, and the silage is mellow in smell, soft, juicy and good in palatability, so that the utilization value of the silage is greatly improved.
The NCL-LP1 of the invention is preserved in China general microbiological culture Collection center (CGMCC) at 26.09.2021, the address is No. 3 Siro-Lu 1 of the Beijing market facing the Yangtze district, and the zip code is as follows: 100101, with a deposit number of: CGMCC No. 23489.
Detailed Description
The present invention will be further illustrated with reference to the following examples, but is not limited thereto.
The experimental procedures in the following examples were carried out in a conventional manner or according to the instructions of the kit unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
General description:
the variety of the stylosanthes guianensis, the whole soybean and the phellodendron amurense are not specially limited, in the experimental process of the invention, the stylosanthes guianensis, the whole soybean and the phellodendron amurense leaves are all collected in the north test field of Qilin forest of the southern China university of agriculture, and watering and fertilizing are not carried out in the material planting process.
The invention has no special limit on the growth stages of the collected stylosanthes guianensis, the whole soybean and the phellodendron amurense, and the stylosanthes guianensis and the whole soybean are respectively harvested in the full-bloom stage and the grain-swelling stage in the experiment.
The freeze-drying procedure of the lactobacillus powder is not particularly limited, and the freeze-drying procedure involved in the invention is as follows: -drying at 25 ℃ for 300 minutes, -drying at 20 ℃ for 60 minutes, -drying at 15 ℃ for 60 minutes, -drying at 10 ℃ for 60 minutes, -drying at 5 ℃ for 60 minutes, -drying at 2 ℃ for 60 minutes, -drying at 0 ℃ for 60 minutes, drying at 5 ℃ for 60 minutes, drying at 10 ℃ for 60 minutes, and drying at 20 ℃ to the end point. The final point of freeze drying is that the sample temperature is consistent with the room temperature, the vacuum degree of the machine is less than 60pa in the freeze drying process, and the temperature of the cold trap is less than-55 ℃.
The invention has no special limit on the processing technology of the stylosanthes guianensis and the whole-plant soybean silage, and the invention can be applied to the processing methods of a bag sealing method, barrel silage, bale silage and the like in the experimental process.
Example 1 screening of tannin-resistant lactic acid bacteria
Collecting fresh leaves of Ficus carica Diels, slicing, mixing, placing in vacuum packaging bag, storing at room temperature for 4 days, taking out, weighing 10g, adding into 90mL sterilized normal saline, diluting layer by layer, and taking 10-2-10-71mL of the gradient diluent is mixed with 15mL of a solid culture medium (Guangzhou Dingguo Biotechnology Co., Ltd.), and after 2 days of culture at 30 ℃, a single colony is selected for separation and purification. The single bacterial colony obtained by the second generation culture is added into an MRS broth culture medium and cultured for 1 day at 30 ℃ to determine the pH value (table 1), 4 bacteria with lower pH values of numbers 3, 4, 10 and 11 are screened, and the bacteria are determined to belong to lactic acid bacteria through taxonomic identification and are used for the tannin resistant detection test.
TABLE 1 acid production ability of lactobacillus screened from natural ensiling of wampee leaf
100uL of the bacterial liquid of the 4 strains of lactic acid bacteria is taken and placed in a sterilized MRS broth culture medium for two days at 30 ℃, 100uL of the bacterial liquid is taken to dilute and coat the plate for 24 hours at 30 ℃, and then the bacterial count is carried out, and the original bacterial liquid count is obtained on the next day. After the bacterial liquid is taken again and cultured for two days at 30 ℃ according to the method, the bacterial liquid is centrifuged for 10min at 4000rpm, and is eluted once by sterilized normal saline, and then is centrifuged again, and then 5mL of sterilized normal saline is added. Adding 100uL of the counted strains of the original bacteria liquid to 1mL of 20 g.L if the number of the counted strains is in the same order of magnitude level (if the number of the counted strains is in the same order of magnitude, dilution is not needed)-1In the mixture of sterilized glucose solution and 4mL of sterilized normal saline (containing 4% tannin, which needs to be sterilized through a 0.22 μm filter membrane), the sterilized normal saline of the control group does not contain tannin, and the other components are consistent, after 24h of culture at 30 ℃, the mixture is diluted and plated, and after 2 days of culture at 30 ℃, the bacteria are counted, and the growth conditions of the lactic acid bacteria under different tannin concentrations are obtained (Table 2). The preparation method of the 4% tannin comprises the following steps: 4g of tannin was weighed into 100mL of physiological saline.
TABLE 2
Note: the difference is not significant (P is more than 0.05) for the same letter or the non-marked letter, the difference is significant (P is less than 0.05) for the different letters, and the letters indicate the difference of the same line.
Strain 4 shown in table 2 was stored and identified: taking 100 μ L of bacteria solution from the above screened lactobacillus, extracting DNA (Tiangen Biochemical technology Co., Ltd., Beijing) with bacteria genome DNA extraction kit, adding 50 μ L of buffer solution B1, sucking, stirring, standing for 5min, shaking, adding 50 μ L of buffer solution B2, sucking, stirring, standing for 5min, shaking, and standing for 1minCentrifuging at 2500rpm for 2min, sucking supernatant 2.5. mu.L, adding into 50. mu.L system for PCR reaction, and using primers 27F (5-AGTTTGATCMTGGCTCAG-3) and 1492R (5-AAGTCGTAACAAGGTAACC-3'). And (3) PCR reaction system: 25. mu.L of mix enzyme, 1.5. mu.L of pre-primer (27F), 1.5. mu.L of post-primer (1492R), and double distilled water (ddH)2O) 19.5. mu.L, cDNA (template DNA) 2.5. mu.L. PCR reaction procedure: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, and 35 cycles; extending for 5min at 72 ℃; the reaction was terminated at 4 ℃.
A gel block was made, and 10. mu.L of PCR amplification product was added to the first well of the gel block in order using a 2000bp mark. Gel electrophoresis reaction procedure: current 200A, voltage 200U, power 50P, time 20 min. The clear bands are then sent to the department of Biotechnology limited for sequencing. The results obtained after the subsequent sequencing sent by a sequencing company are subjected to homology comparison through a blast program of NCBI to determine the strain, the strain belongs to the strain, the strain is determined to be Lactobacillus Plantarum (Lactobacillus Plantarum) through comparison, the strain is named NCL-LP1 and is preserved in China general microbiological culture Collection center (CGMCC) at 26 days 09 and 26 months 2021, the address is No. 3 of the West Lu 1 of the sunward area of Beijing, and the postal code is as follows: 100101, accession number: CGMCC No. 23489.
Example 2 preparation of tannin-resistant Lactobacillus powder
The lactic acid bacteria screened in the example 1 are prepared into bacterial powder by the following steps: activating, enriching and culturing lactic acid bacteria, centrifuging at 4000rpm for 10min, cleaning with sterile normal saline, centrifuging twice, adding 10% sterile skimmed milk, mixing, pouring into a culture dish, pre-freezing, placing into a vacuum freeze-drying machine, wherein the vacuum degree is less than 60pa, the temperature of a cold trap is less than-55 ℃, drying is finished when the temperature of a sample reaches room temperature, and freezing and storing the prepared bacterial powder.
Example 3 tannin-resistant lactic acid bacteria and tannin addition treatment of silage
1. Collecting stylosanthes guianensis and whole soybeans:
the silage materials are cut at the position 3-5cm away from the ground in the full-bloom stage of the stylosanthes guianensis and the full-plant soybean puffing stage, the stylosanthes guianensis and the full-plant soybean are cut into the length of 2-3cm by a manual guillotine, three repeated samples are collected after the stigmata guianensis and the full-plant soybean are uniformly mixed to measure the relevant characteristic indexes of the raw materials, the characteristics of the stylosanthes guianensis and the full-plant soybean raw materials are shown in a table 3, wherein DM is dry matter, TN is total nitrogen, and FM is a fresh sample.
TABLE 3
| Raw material characteristic measurement items | All-grass of Henry Stylosanthes | Whole plant soybean |
| Dry matter (%) | 32.9±0.22 | 28.3±0.97 |
| Crude protein (% DM) | 8.93±0.37 | 24.1±1.62 |
| True protein (% TN) | 73.16±1.84 | 86.58±0.40 |
| Non-protein Nitrogen (% TN) | 26.84±1.84 | 13.42±0.40 |
| Neutral detergent fiber (% DM) | 68.6±1.61 | 40.6±2.46 |
| Acid detergent fiber (% DM) | 52.9±0.87 | 23.4±0.68 |
| Soluble carbohydrate (% DM) | 1.23±0.17 | 2.39±0.43 |
| Lactic acid bacteria (log)10cfu·g-1FM) | 5.48±0.47 | 6.00±0.10 |
| Yeast (log)10cfu·g-1FM) | 4.29±0.08 | 5.00±0.05 |
| Mold (log)10cfu·g-1FM) | 3.40±0.35 | 3.72±0.13 |
| Escherichia coli (log)10cfu·g-1FM) | 6.44±0.20 | 5.98±0.83 |
2. Adding tannin-resistant lactic acid bacteria and tannin to treat silage, and packaging and storing silage bags:
ensiling stylosanthes guianensis: 500g of chopped Stylosanthes guianensis was mixed with 10mL of pure water and divided into 3 polyethylene bags as Control (CK). And taking 500g of chopped stylosanthes guianensis, and carrying out five treatments: (1) adding 10 in proportion6cfu·g-1The plant milkThe bacillus is LP group; (2) adding 1% tannin to obtain 1% TA group; (3) 2% tannin was added to 2% TA group; (4) adding 10 portions of the raw materials according to the proportion6cfu·g-1Lactobacillus plantarum and 1% tannin were LP + 1% TA group; (5) and 2% tannin is LP + 2% TA group, and the groups are evenly mixed and evenly distributed in 3 polyethylene plastic bags to be used as treatment groups. The lactobacillus plantarum is obtained by adding purified water to the bacterial powder described in example 2 and then activating for 20 min. The 1% and 2% tannin are added, wherein the adding amount of the tannin is 1% -2% of the total weight of the silage.
Ensiling whole soybean: the same method as the method for ensiling stylosanthes guianensis.
All the manufactured silage bags are sealed by a miniature vacuum sealing machine, and the silage bags are opened after being stored for one month at room temperature to measure the relevant indexes of silage quality.
3. Ensiling quality determination: after one month storage at room temperature, the silage bag is opened. The dry matter content, pH value, the number of lactic acid bacteria and Escherichia coli, the contents of lactic acid, acetic acid, propionic acid and butyric acid organic acids, the contents of crude protein, true protein, non-protein nitrogen and ammonia nitrogen were measured, and the results are shown in tables 4 and 5. The measurement method for each item is as follows:
and (3) dry matter determination: after opening the bag, uniformly mixing the silage samples, weighing, sampling, placing in a constant-temperature oven at 65 ℃ for about 48h, drying to be completely dry, and calculating the weight ratio before and after drying to obtain the dry matter content.
And (3) pH value measurement: after opening the bag, randomly taking 20g of silage sample according to a five-point sampling method, adding 180mL of distilled water, uniformly mixing, juicing for 1min by a household juicer, sequentially filtering with 4 layers of gauze and medium-speed qualitative filter paper to obtain a leaching solution, and measuring the pH value by using a pH meter (PHS-3C, Shanghai thunder magnetic).
Determination of soluble sugars: the content of the dry matter was determined by a soluble sugar assay kit (sozhou kommy biotechnology limited).
Fiber (neutral detergent fiber and acid detergent fiber) determination: the fiber content was measured using a Ringbio fiber meter (Shandong Ruibobai Instrument).
Determination of Crude Protein (CP) and Ammonia Nitrogen (NH 3-N): measuring crude protein by adopting a Kjeldahl method; the content of ammoniacal nitrogen is measured by a phenol-sodium hypochlorite colorimetric method.
Determination of True Protein (TP) and non-protein nitrogen (NPN): respectively mixing and soaking 15% trichloroacetic acid solution with fermented stylosanthes guianensis and whole plant soybean dry powder for 1h, taking precipitate filtered by qualitative filter paper, drying in a constant-temperature oven at 105 ℃ without damage, and determining the nitrogen content by using a Kjeldahl method to obtain true protein; the non-protein nitrogen is the difference between the crude protein and the true protein.
Determination of organic acids: filtering the leaching solution with 0.22 μ M filter membrane to obtain filtrate, and determining the content of lactic acid, acetic acid, propionic acid and butyric acid with Shimadzu GC-14 high performance liquid chromatograph (chromatographic column: Shodex Rspakc-811 s DVB gel column, Japan; detector: SPD-M10AVP) with mobile phase of 3 mmol.L-1Perchloric acid, flow rate 1 mL/min-1(ii) a The column temperature is 50 ℃; the detection wavelength was 210nm and the amount of sample was 5. mu.L.
And (3) measuring the quantity of lactic acid bacteria and escherichia coli: 20g of samples are randomly sampled according to a five-point sampling method, 180mL of sterilized normal saline is added, the samples are uniformly mixed and then are diluted step by step, and a plate counting method is adopted for measuring the microbial quantity. The lactobacillus culture adopts MRS agar culture medium, and the Escherichia coli culture adopts crystal violet neutral red bile salt agar culture medium (Guangzhoudingguo biotechnology, Co., Ltd.) and is cultured in a 30 ℃ incubator for 2 days at constant temperature.
TABLE 4
Note: the same or no-mark letters in the same item have no significant difference (P > 0.05), the different ones have significant difference (P <0.05), and the letters represent the difference of the same line.
TABLE 5
Note: the same letter or the same letter in the same item has no sign, the difference is not significant (P is more than 0.05), the difference is significant in the different item (P is less than 0.05), and the letter represents the difference of the same line.
As can be seen from tables 4 and 5, the silage quality of stylosanthes guianensis and the whole soybean is obviously improved after the method is adopted. Compared with the silage column flowers and plants and the whole plants of soybeans of the control group, the tannin-resistant lactobacillus and 2% tannin mixed addition group remarkably reduces the pH values, the contents of non-protein nitrogen and ammonia nitrogen of the silage column flowers and plants and remarkably improves the content of true protein; and the number of Escherichia coli in the whole soybean is obviously reduced, and the dry matter content of the whole soybean is obviously improved. Has obvious effect of improving the quality of the stylosanthes guianensis and the silage of the whole soybean and improving the preservation of the nutrient components.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-described preferred embodiment should not be construed as limiting the present invention.
Claims (10)
1. A method for improving the quality of silage, which is characterized in that: tannin-resistant lactic acid bacteria and tannin are added to silage.
2. The method of claim 1, wherein: the silage is stylosanthes guianensis silage or whole soybean silage.
3. The method of claim 1, wherein: the addition amount of the tannin-resistant lactic acid bacteria is 106cfu·g-1The addition amount of the tannin is 1% -2% of the total weight of the silage.
4. The method of claim 1, wherein: the tannin-resistant lactic acid bacteria are in a bacteria powder state with the water content of 0%, and are in a bacteria liquid state activated after the bacteria powder is added with pure water when ensiling is added.
5. The method of claim 1, wherein said tannin-resistant lactic acid bacteria are prepared by the steps of: collecting fresh leaves of yellow-wood, slicing, vacuum packaging, and storing at normal temperature; sampling and diluting, culturing by using an MRS culture medium, selecting a single bacterial colony, separating and purifying, then measuring the pH value, adding the lactobacillus bacterial liquid with high acid yield into a mixed liquid containing glucose, normal saline and tannin, and culturing and screening to obtain the tannin-resistant lactobacillus.
6. The method as claimed in claim 5, wherein the tannin-resistant lactic acid bacteria are subjected to activation enrichment culture, then are centrifuged, are washed and centrifuged twice by sterile physiological saline, are added with skimmed milk, are uniformly mixed and are poured into a culture dish, are put into a vacuum freeze dryer after being prefrozen, have the vacuum degree of less than 60pa and the temperature of a cold trap of less than-55 ℃, and are subjected to freeze preservation when the temperature of a sample reaches the room temperature, namely the drying is finished.
7. The method of claim 1, wherein: before tannin-resistant lactic acid bacteria and tannin are added to the silage, the silage is cut into 2-3cm long.
8. The method of claim 1, wherein: adding tannin-resistant lactic acid bacteria and tannin into silage, uniformly mixing, sealing, and storing at room temperature, wherein the sealing is bag sealing, barreling sealing or bundling sealing.
9. The method of claim 1, wherein: the tannin-resistant lactic acid bacteria are plant lactic acid bacteria (Lactobacillus plantarum) NCL-LP1, and the preservation number is as follows: CGMCC No. 23489.
10. Application of tannin-resistant lactobacillus in preparing stylosanthes guianensis or whole soybean silage additive.
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| CN115992074A (en) * | 2022-11-10 | 2023-04-21 | 江南大学 | Lactobacillus plantarum and application thereof in production of urolithin A |
| CN115992074B (en) * | 2022-11-10 | 2023-11-28 | 江南大学 | Lactobacillus plantarum and application thereof in production of urolithin A |
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