CN117179153A - Composite biological agent for fermentation of silage of broussonetia papyrifera and application of composite biological agent - Google Patents
Composite biological agent for fermentation of silage of broussonetia papyrifera and application of composite biological agent Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 48
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- 239000003124 biologic agent Substances 0.000 title claims abstract description 44
- 239000002131 composite material Substances 0.000 title claims abstract description 44
- 241000933832 Broussonetia Species 0.000 claims abstract description 104
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
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- Fodder In General (AREA)
Abstract
The invention provides a composite biological agent for fermentation of paper mulberry silage and application thereof, belonging to the technical field of animal feed. A composite biological agent for fermenting silage of Broussonetia papyrifera comprises lactobacillus and tannase; the ratio of the number of viable bacteria of the lactic acid bacteria to the enzyme activity of the tannase is (1-10) x 10 5 CFU:0.1U; the ratio of the viable count of the lactobacillus plantarum to the lactobacillus paracasei in the lactobacillus is (1-3): (0.8-1.2). The composite biological agent is applied to the preparation of the silage of the paper mulberry, can effectively improve the nutrition quality, flavonoid, total phenol and lactic acid content of the feed, simultaneously reduce the content of tannin, acetic acid and ammoniacal nitrogen, reduce the pH value, provide a new means for improving the meat quality of livestock, and have important significance for the utilization and popularization of the paper mulberry.
Description
Technical Field
The invention belongs to the technical field of animal feeds, and particularly relates to a composite biological agent for fermentation of paper mulberry silage and application thereof.
Background
The broussonetia papyrifera is a deciduous arbor of broussonetia genus of Moraceae family, has fast growth speed and strong adaptability, and can be widely used as a livestock and poultry feed raw material in the south of China. The high-temperature high-humidity high-protein pasture such as alfalfa in the southern area of China is difficult to cultivate and utilize, the protein content of the whole broussonetia papyrifera strain with the plant height of 0.8-1.2 m is more than 18%, the protein content of broussonetia papyrifera leaves is up to 20-30%, the rumen degradation rate of broussonetia papyrifera protein is more than 90%, and the broussonetia papyrifera protein can be used as protein feed to replace alfalfa to be added into daily ration of rumen animals such as cattle and sheep, and can solve the problem of protein feed shortage in the southern area of China. Meanwhile, the paper mulberry is rich in various nutrient substances such as amino acids, vitamins, carbohydrates, minerals and the like, and the ingredients such as flavonoids, lignans and the like with antioxidant and antibacterial effects have remarkable effects on improving the pork quality. The paper mulberry has low requirements on the growth environment, the cultivation management technology is simple, the ecological environment is improved, the defect of grain feed can be overcome by developing paper mulberry feed, the contradiction between people and livestock competing for grain is solved, and the paper mulberry feed has huge economic benefit and ecological benefit.
Fresh and tender paper mulberry freshly cut has the characteristics of high water content, low content of soluble carbohydrate, high protein content, strong buffering capacity and the like, the final pH value is higher after direct silage fermentation, clostridium is propagated in a large quantity, and harmful substances such as ammonia, butyric acid and the like are easy to produce, so that the silage quality of paper mulberry is reduced. Meanwhile, tannin contained in the paper mulberry silage is an anti-nutritional factor, can inhibit degradation of protein in rumen, reduces protein utilization rate, and influences livestock production performance.
Disclosure of Invention
In view of the above, the invention aims to provide a composite biological agent for fermentation of paper mulberry silage, which not only can effectively reduce the tannin content and the ammonia nitrogen ratio, but also can improve the content of nutritional and bioactive components such as starch, total phenol, flavonoid and the like in the paper mulberry silage, and can improve the content of lactic acid so as to reduce the pH value of the feed.
The invention provides a composite biological agent for fermentation of paper mulberry silage, which comprises lactobacillus and tannase;
the ratio of the number of viable bacteria of the lactic acid bacteria to the enzyme activity of the tannase is (1-10) x 10 5 CFU：0.1U；
The lactobacillus comprises lactobacillus plantarum and lactobacillus paracasei;
the ratio of the viable count of the lactobacillus plantarum to the lactobacillus paracasei is (1-3): (0.8-1.2).
Preferably, the ratio of the viable count of the lactic acid bacteria to the enzyme activity of the tannase is 5×10 5 CFU：0.1U。
Preferably, the ratio of the viable count of the lactobacillus plantarum to the lactobacillus paracasei is 2:1.
the invention provides application of the composite biological agent in preparing silage of paper mulberry.
Preferably, the application comprises at least one of:
the nutritional quality of the silage of the paper mulberry is improved;
reducing the tannin content in the silage of the broussonetia papyrifera;
increasing flavonoid and total phenol content in the silage of broussonetia papyrifera;
increasing the lactic acid content in the silage of the broussonetia papyrifera;
and reducing the pH, acetic acid, and ammoniacal nitrogen content in the silage of the paper mulberry.
Preferably, the raw materials for preparing the silage of the paper mulberry comprise one or more of paper mulberry leaves, paper mulberry roots, paper mulberry barks and paper mulberry trunks.
Preferably, the dry matter mass percentage of the whole broussonetia papyrifera strain is 27% -34%.
Preferably, when the paper mulberry silage is prepared, the addition amount of the lactobacillus in the composite biological agent is (1-10) multiplied by 10 5 CFU/kg feedstock;
the addition amount of tannase is 80-120U/kg of raw material.
Preferably, when the paper mulberry silage is prepared, the fermentation temperature of the paper mulberry silage is 15-35 ℃.
Preferably, the fermentation time of the paper mulberry silage is 1 year.
The invention provides a composite biological agent for fermentation of paper mulberry silage, which comprises lactobacillus and tannase; the ratio of the number of viable bacteria of the lactic acid bacteria to the enzyme activity of the tannase is (1-10) x 10 5 CFU:0.1U; the ratio of the viable count of the lactobacillus plantarum to the lactobacillus paracasei is (1-3): (0.8-1.2). Experiments prove that the silage for the paper mulberry is prepared by silaging the composite biological agent, so that the tannin content of the anti-nutritional factors in the paper mulberry can be effectively reduced, and the reduction rate is more than 20%. The compound biological agent can also remarkably improve the content of flavonoid and total phenol in the silage of the paper mulberry, thereby being beneficial to improving the antioxidation activity and the antibacterial effect of the feed and improving the quality of fed animals; the composite biological agent can also obviously improve the nutrition quality in the paper mulberry silage, for example, compared with a blank control group, the starch content is obviously improved, the crude protein reduction rate is not obvious, and compared with other additives such as corncob, rice hull powder, molasses and the like, the aNDF content is obviously reduced; the composite biological agent can also obviously improve the fermentation quality of the paper mulberry silage, obviously reduce the pH value of the silage, reduce the acetic acid content in the silage and reduce the ammonia nitrogen content in the paper mulberry silage. Although the tannase alone also has good reduction of the tannin content in the fermentation, the active ingredient is produced, the nutritional quality and the fermentation quality are less effective than the complex biological formulation. The use amount and cost of the tannase fermentation are obviously lower than those of the compound cellulase group, so that the compound biological preparation can achieve the effect of improving the nutrition quality while reducing the cost. Therefore, the composite biological preparation provides an ideal fermentation preparation in the aspects of preparing silage with low content of harmful substances, high nutrition and fermentation quality.
Drawings
FIG. 1 shows the pH measurements of broussonetia papyrifera silage in groups 1-12;
FIG. 2 is an acetic acid measurement of broussonetia papyrifera silage in groups 1-12;
FIG. 3 shows lactic acid measurements of broussonetia papyrifera silage in groups 1-12;
FIG. 4 is a graph showing the results of the determination of the ammonia nitrogen/total nitrogen ratio of the silage of Broussonetia papyrifera in groups 1 to 12;
FIG. 5 is a graph showing the starch content measurement of broussonetia papyrifera silage in groups 1-12;
FIG. 6 shows the crude protein content measurements of broussonetia papyrifera silage in groups 1-12;
FIG. 7 shows the results of aNDF content measurements of broussonetia papyrifera silage in groups 1-12;
FIG. 8 is a graph showing flavonoid content measurements of broussonetia papyrifera silage in groups 1-12;
FIG. 9 is a graph showing the total phenol content measurement of broussonetia papyrifera silage in groups 1-12;
FIG. 10 shows the results of tannin content measurement of broussonetia papyrifera silage in groups 1 to 12.
Detailed Description
The invention provides a composite biological agent for fermentation of paper mulberry silage, which comprises lactobacillus and tannase; the ratio of the number of viable bacteria of the lactic acid bacteria to the enzyme activity of the tannase is (1-10) x 10 5 CFU:0.1U; the ratio of the viable count of the lactobacillus plantarum to the lactobacillus paracasei is (1-3): (0.8-1.2).
In the present invention, the ratio of the number of viable bacteria of the lactic acid bacteria to the enzyme activity of the tannase is preferably 5X 10 5 CFU:0.1U. The ratio of the viable count of the lactobacillus plantarum to the lactobacillus paracasei is preferably 2:1. the strains of Lactobacillus plantarum and Lactobacillus paracasei are not particularly limited in the present invention, and Lactobacillus plantarum and Lactobacillus paracasei known in the art may be used. In the examples of the present invention, the lactobacillus plantarum and lactobacillus paracasei were purchased from the chinese collection of typical cultures, respectively. The tannase was purchased from Zhongsheng Biotechnology Co., ltd and was 97% pure.
In the invention, the composite biological agent can effectively improve the fermentation quality of the silage of the broussonetia papyrifera. The improvement of the fermentation quality of the paper mulberry silage comprises the steps of reducing the content of ammonia nitrogen, reducing the pH value of the feed, improving Lactic Acid (LA) and reducing the content of Acetic Acid (AA). The reduction of the ammonia nitrogen content is beneficial to reducing the content of harmful substances in the feed, simultaneously reducing the degradation rate of protein and keeping higher protein content, and simultaneously reducing the pH value of the feed is beneficial to reducing the propagation of mixed bacteria and improving the quality of the feed. The increase of the lactic acid content is beneficial to lowering the pH value of the feed.
In the invention, the composite biological agent can effectively improve the nutrition quality of the silage of the paper mulberry. The improvement of the nutritional quality of the paper mulberry silage preferably comprises the improvement of the dry matter content, the improvement of the starch content and the like. The effect of the single tannase group and the compound biological agent group on improving the starch content is equivalent to that of the plant lactobacillus plus compound cellulase group, but the effect of the group 9 on improving the nutrition quality while reducing the cost is only inferior to that of the plant lactobacillus and the cellulase combination because the using amount and the cost of the tannase are obviously lower than those of the compound cellulase group. In addition, the present invention also analyzes the effect of complex biological agent treatment on crude protein and structural carbohydrate content in paper mulberry silage, including neutral detergent fiber (aNDF, amylase treated) and Acid Detergent Fiber (ADF). The results show that the crude protein in all the treated feeds is reduced to a certain extent, but the reduction of the crude protein in the composite biological agent group is not more than 10%. The acidic washing fiber in the compound biological agent group feed is not obviously improved, but the neutral washing fiber is obviously reduced.
In the present invention, the complex biological agent is capable of affecting the number of microorganisms in the silage of broussonetia papyrifera. The microorganisms preferably include lactic acid bacteria, yeasts and molds. The composite biological agent can greatly reduce the pH value in the feed, so that the growth of lactic acid bacteria is inhibited. Meanwhile, the compound biological agent ensures that the quantity of saccharomycetes in the feed has no obvious difference from that of a blank control group. The compound biological agent ensures that no mould is detected in the feed, and meets the requirement of feed safety.
In the invention, the compound biological agent can effectively improve the content of active ingredients and the content of anti-nutritional factors. The active ingredients preferably include flavonoids and total phenols. The anti-nutritional factor comprises tannin. The complex biological agent can effectively improve the content of flavonoids and is higher than that of a single lactobacillus compounding group (LAB) and a tannase group. The compound biological agent can effectively reduce the content of tannin, and has better effect than that of a single lactobacillus compound group (LAB) and is equivalent to that of a single tannase group. In terms of total phenol content, the composite biological agent can greatly improve the total phenol content, has obvious difference compared with a control group, and the improvement degree is not as good as that of a single lactobacillus compound group (LAB), but better than that of a single tannase group.
In combination with the fermentation effect, the composite biological agent provided by the invention has excellent performances in reducing the tannin content, the ammoniacal nitrogen content and the pH value, and improving the flavonoid content, the total phenol, the lactic acid, the starch content and the like, so that the composite biological agent is used as a starter to prepare the paper mulberry silage, the quality of the feed can be effectively improved, and the quality of fed animals can be improved.
The invention provides application of the composite biological agent in preparing silage of paper mulberry.
In the present invention, the complex biological agent preferably has at least one of the following applications:
the nutritional quality of the silage of the paper mulberry is improved;
reducing the tannin content in the silage of the broussonetia papyrifera;
increasing flavonoid and total phenol content in the silage of broussonetia papyrifera;
increasing the lactic acid content in the silage of the broussonetia papyrifera;
and reducing the pH, acetic acid, and ammoniacal nitrogen content in the silage of the paper mulberry.
In the invention, the raw materials for preparing the silage of the paper mulberry preferably comprise one or more of paper mulberry leaves, paper mulberry roots, paper mulberry barks and paper mulberry branches. In the embodiment of the invention, the whole broussonetia papyrifera strain is used as a raw material to prepare the silage of the broussonetia papyrifera. The dry matter mass percentage of the whole broussonetia papyrifera strain is preferably 27% -34%, more preferably 28% -32%, and most preferably 30%.
In the present invention, the amount of lactobacillus added in the preparation of the paper mulberry silage is preferably (1-10). Times.10 5 CFU/g of raw material, more preferably 3 to 8X 10 5 CFU/g feed, most preferably 5X 10 5 CFU/kg feedstock. The addition amount of tannase is preferably80 to 120U/kg of raw material, more preferably 90 to 110U/kg of raw material, and most preferably 100U/kg of raw material. The addition mass percentage of the tannase is preferably 0.2%.
In the present invention, the fermentation temperature of the paper mulberry silage is preferably 15 to 35 ℃, more preferably 20 to 30 ℃, and most preferably 25 ℃ when the paper mulberry silage is prepared. The fermentation time of the paper mulberry silage is preferably 1 year.
The present invention provides a composite biological agent for fermentation of paper mulberry silage and application thereof, which are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Preparation of composite biological agent (LAB+DE) for fermentation of paper mulberry silage and method for manufacturing paper mulberry silage
1. Activation of Lactobacillus plantarum and Lactobacillus paracasei
The viable count of the freeze-dried powder of the lactobacillus plantarum and the lactobacillus paracasei is 2.67 multiplied by 10 respectively 10 CFU/g and 1.33X10 10 CFU/g, according to inoculum size 3.33X10 respectively 8 CFU/ml and 1.67×10 8 The CFU/ml ratio is respectively inoculated into each 1ml of sterilized skim milk powder solution, and activated for 2 hours at room temperature to obtain lactobacillus plantarum bacterial liquid and lactobacillus paracasei bacterial liquid.
Respectively measuring viable count of lactobacillus plantarum bacterial liquid and lactobacillus paracasei bacterial liquid to obtain viable bacteria concentration of lactobacillus plantarum bacterial liquid of 3.33X10 8 CFU/ml, live bacteria concentration of Lactobacillus paracasei bacterial liquid is 1.67×10 8 CFU/ml. Then the ratio of the viable count of the lactobacillus plantarum to the lactobacillus paracasei is 2:1, respectively collecting lactobacillus plantarum bacterial liquid 1ml, lactobacillus paracasei bacterial liquid 1ml, mixing, adding distilled water to 20ml to obtain total concentration 5×10 8 The mixed bacterial liquid of CFU corresponds to 1kg of silage raw material.
2. Treatment of silage from Broussonetia papyrifera
The silage raw material of the paper mulberry is properly treated to ensure that the dry matter content of the silage raw material is between 27 and 34 percent. The harvested paper mulberry silage raw material is uniformly cut into small sections with the length of 2-3 cm by a guillotine and fully and uniformly mixed, and is divided into three parts by a quartering method, wherein each part is 1kg.
3. Fermentation
According to the total inoculation amount of 5 multiplied by 10 5 The mixed bacterial liquid is sprayed on silage raw materials of the paper mulberry according to the quantity of CFU/kg, tannase is sprayed according to the addition quantity of 0.2%, after the tannase is fully and uniformly homozygous, each 1kg part is uniformly divided into three parts, and the three parts are placed into silage bags, are vacuumized and sealed, and are stored as three repetitions of each treatment, and the room-temperature storage time is 1 year. The storage temperature is 15-35 ℃.
Comparative example 1
Preparation of microbial preparation (LAB) for fermentation of paper mulberry silage and method for manufacturing paper mulberry silage
The addition of tannase was omitted from the silage based on example 1, and the other operations were identical.
Comparative example 2
Preparation of composite enzyme preparation (E) for fermentation of paper mulberry silage and method for manufacturing paper mulberry silage
Silage from paper mulberry was prepared as in example 1. The cellulase, xylanase, glucanase and pectinase are respectively weighed according to the addition amounts of 10U/g raw materials, 120U/g raw materials, 40U/g raw materials and 20U/g raw materials, and 18ml distilled water is added for full dissolution. Wherein the enzyme activity of the cellulase is 230000U/g, the enzyme activity of the xylanase is 300000U/g, the enzyme activity of the glucanase is 1000000U/g, the enzyme activity of the pectinase is 30000U/g, and the 4 enzymes are all available in pure enzyme form and are all purchased from Guangdong Yiduoli biotechnology Co.
Before silage fermentation, enzyme liquid is sprayed into the raw materials and fully and uniformly stirred. Broussonetia papyrifera silage was then prepared according to the fermentation method of example 1.
Comparative example 3
Method for preparing silage of broussonetia papyrifera by using tannin enzyme (DE)
Silage from paper mulberry was prepared as in example 1. Spraying tannase to the silage raw material of the paper mulberry according to the addition amount of 0.2%, and preparing paper mulberry silage according to the fermentation method of the example 1 after fully and uniformly homozygously.
Comparative example 4
Method for preparing paper mulberry silage by using rice hull powder (DKF)
Silage from paper mulberry was prepared as in example 1. Adding the rice hull powder into the silage raw material of the paper mulberry according to the amount of 5 percent of the addition amount, and fully and uniformly stirring. Broussonetia papyrifera silage was then prepared according to the fermentation method of example 1.
Comparative example 5
Method for preparing paper mulberry silage by using corncob (YMX)
Silage from paper mulberry was prepared as in example 1. Adding corncob into the silage raw material of the paper mulberry according to the amount of 5% of the adding amount, and fully and uniformly stirring. Broussonetia papyrifera silage was then prepared according to the fermentation method of example 1.
Comparative example 6
Method for preparing paper mulberry silage by using molasses (M)
Silage from paper mulberry was prepared as in example 1. The molasses is added into the silage raw material of the paper mulberry according to the amount of 1 percent of the addition amount, and the mixture is fully and uniformly stirred. Broussonetia papyrifera silage was then prepared according to the fermentation method of example 1.
Comparative example 7
Method for preparing silage of broussonetia papyrifera by using composite microbial agent and composite enzyme preparation (LAB+E)
Silage from paper mulberry was prepared as in example 1. A composite microbial bacterium was inoculated into a silage raw material of Broussonetia papyrifera according to the method of comparative example 1 while a composite enzyme preparation was added according to the method of comparative example 2, and then a silage of Broussonetia papyrifera was prepared according to the fermentation method of example 1.
Comparative example 8
Method for preparing paper mulberry silage by using composite microbial agent and tannase (LAB+DE)
Silage from paper mulberry was prepared as in example 1. A broussonetia papyrifera silage was prepared by inoculating a composite microorganism into a broussonetia papyrifera silage raw material according to the method of comparative example 1, adding tannase according to the method of comparative example 3, and then fermenting according to the method of example 1.
Comparative example 9
Method for preparing paper mulberry silage by utilizing composite microbial agent and rice hull powder (LAB+ DKF)
Silage from paper mulberry was prepared as in example 1. Inoculating composite microorganism bacteria into paper mulberry silage raw material according to the method of comparative example 1, adding rice hull powder according to the method of comparative example 4, and preparing paper mulberry silage according to the fermentation method of example 1.
Comparative example 10
Method for preparing paper mulberry silage by using composite microbial agent and corncob (LAB+YMX)
Silage from paper mulberry was prepared as in example 1. The broussonetia papyrifera silage was prepared by inoculating the composite microorganism into the broussonetia papyrifera silage raw material according to the method of comparative example 1 while adding the corncob according to the method of comparative example 5, and then by the fermentation method of example 1.
Comparative example 11
Method for preparing paper mulberry silage by using composite microbial agent and molasses (LAB+M)
Silage from paper mulberry was prepared as in example 1. The silage of broussonetia papyrifera was prepared by inoculating the silage of broussonetia papyrifera with the compound microorganism according to the method of comparative example 1 and adding molasses according to the method of comparative example 6.
Comparative example 12
Method for manufacturing paper mulberry silage based on natural fermentation
Silage from paper mulberry was prepared as in example 1. To the silage raw material of broussonetia papyrifera, 2ml of sterilized skim milk powder+18 ml of distilled water was added, and mixed well, and then the silage of broussonetia papyrifera was prepared according to the fermentation method of example 1.
Example 2
The silage from paper mulberry prepared in example 1 and comparative examples 1 to 12 was numbered, organized and evaluated comprehensively in terms of nutritional quality, fermentation quality, microbial count and bioactive ingredient, and anti-nutritional factor content, respectively
1. Detection of fermentation quality of broussonetia papyrifera silage
After unsealing, uniformly mixing each repeated silage of the paper mulberry, adding 180ml of distilled water into 20g of silage by a five-point sampling method, shaking uniformly by hand, putting into a refrigerator at 4 ℃ for leaching for 24 hours, and filtering with 4 layers of gauze and qualitative filter paper to obtain leaching liquor.
The pH value of the silage was measured by means of a pH meter from Metrele company.
The extract was filtered through a 0.22 μm disposable water filter, and the filtrates were subjected to high performance liquid chromatography to determine Lactic Acid (LA) and Acetic Acid (AA), respectively. The chromatographic conditions were as follows: high performance liquid chromatograph: agilent 1260 (Agilent technologies Co., ltd.); a detector: a Variable Wavelength Detector (VWD); chromatographic column: shodex RSpak KC-G (6.0 mm. Times.50 mm) +Shodex RSpak KC-811S-DVB gel C (8.0 mm. Times.30 cm, shimadzu corporation); sample injection volume 5. Mu.L; mobile phase: 3mM HClO 4 The method comprises the steps of carrying out a first treatment on the surface of the Flow rate: 1mLmin -1 The method comprises the steps of carrying out a first treatment on the surface of the Column temperature: 60 ℃; detection wavelength: 210nm; run time was 20min.
The ratio of ammoniacal nitrogen to total nitrogen was determined using the phenol-sodium hypochlorite method.
The results are shown in Table 1 and FIGS. 1 to 4.
TABLE 1 Effect of different biological formulations on Broussonetia papyrifera silage fermentation quality
As can be seen from Table 1, the pH of silage was significantly reduced in all groups compared with the blank group 1, and the pH reduction ranges in the feeds from group 2 to group 12 were 6.93%, 9.78%, 8.70%, 7.07%, 8.22%, 8.36%, 10.46%, 10.94%, 7.54%, 8.76% and 9.38%, respectively. Of these groups 8 and 9 performed best, the pH was reduced by more than 10%.
The lactic acid content of group 2, group 3, group 4, group 6, group 7, group 9, group 10, group 11, group 12 was increased by 14.31%, 17.56%, 29.88%, 5.58%, 24.41%, 15.39%, 14.12%, 3.23%, 29.61%, respectively. The highest increase was for groups 4 and 12, and groups 7 and 3.
All of groups 2 to 12 reduced the acetic acid content of silage by more than 20% compared to control group 1, including group 6, group 12, group 10, group 5, group 7 and group 11, with groups 7 and 11 being most effective and acetic acid content reduced by 29.26% and 28.86%, respectively.
All groups 2 to 12 significantly reduced the ammonia nitrogen/total nitrogen ratio of silage compared to control group 1 by 39.21%, 31.14%, 34.37%, 37.29%, 36.08%, 49.18%, 36.10%, 40.68%, 36.05%, 35.04%, 40.68%, respectively, with group 7 being the best and groups 12 and 9 being the next least effective. Group 9 had the lowest pH of 4.37 and there was no significant difference between the ammonia nitrogen/total nitrogen ratio and the most effective group 7.
2. Detection of nutritional quality of paper mulberry silage
The remaining about 250g of the sample was deactivated at 105℃for 30min and dried to constant weight at 65℃and the dry matter content (DM) was calculated.
The samples were crushed (1 mm sieve) and filled into self-sealing bags for nutrition quality determination. Wherein the total nitrogen content is measured by Kjeldahl nitrogen determination instrument of goss company, and the crude protein content of silage is calculated by total nitrogen multiplied by 6.25.
Neutral washing fiber (aNDF, amylase treated) and acid washed fiber (ADF) were tested using an Ankom200i automated fiber analyzer from angom corporation.
The content of soluble sugar is determined by adopting an anthrone colorimetric method, and the content of starch is determined by adopting a perchloric acid hydrolysis anthrone colorimetric method.
The tannin, flavonoid and total phenol content are detected by using a kit, namely a tannin content detection kit (BC 1395-100 tubes/96 samples), a plant flavonoid content detection kit (BC 1335-100 tubes/48 samples) and a plant Total Phenol (TP) content detection kit (BC 1345-100 tubes/48 samples), and the brand is Solarbio, which are purchased from Nanjin Kendeda Biotechnology Co.
The effect of different fermenters on broussonetia papyrifera silage dry matter and soluble sugar and starch content is shown in table 2 and fig. 5 and 6.
TABLE 2 Effect of different biological formulations on Broussonetia papyrifera silage dry matter and unstructured carbohydrate content
Group 5, group 6, group 10, group 11 had an increase in dry matter content due to the addition of air-dried rice hull meal and corncob compared to the placebo group, but the other treatment groups were not significantly different from group 1 except that group 11 was significantly higher than the placebo group.
From the perspective of the soluble sugar content, the residual soluble sugar in the paper mulberry silage is lower than 1%, the residual soluble sugar content in the paper mulberry silage is obviously lower than that in the blank control group, and the compound additive groups such as group 10, group 11 and group 12 are obviously lower than that in the blank control group, because more soluble sugar is consumed by the use of lactobacillus, and the soluble sugar in other groups is reduced but not obvious compared with that in the blank control group.
The starch content of group 2, group 3, group 4, group 5, group 7, group 8, group 9 and group 12 was increased by 26.05%, 21.94%, 34.87%, 5.33%, 25.24%, 44.39%, 30.13%, 17.65%, respectively, compared to the blank (group 1). Of these, group 8 performed best, and groups 4 and 9 performed next to each other, but there was no significant difference from group 8. The amount and cost of tannase is significantly lower than that of the complex cellulase group, so that the effect of group 9 on improving the nutritional quality while reducing the cost is inferior to that of the lactobacillus plantarum and cellulase combination.
The effect of different biological agents on crude protein and structural carbohydrates of paper mulberry silage is shown in table 3 and fig. 7.
TABLE 3 Effect of different biological formulations on Broussonetia papyrifera silage crude protein and structural carbohydrate content
The crude protein content was reduced to some extent in all treatment groups compared to the blank (group 1), wherein the crude protein reduction amounts in groups 2, 12, 8, 9, 4, 3, and 7 were not more than 10%, and the minimum reduction was 1.83% for group 2.
ADF content was increased in all treatment groups, except the control group, groups 8 and 9 had the lowest ADF content. The aNDF content of group 2 and group 3 was reduced by 2.03% and 0.4% respectively, and the aNDF content of group 8 and group 9 was reduced by 8.54% and 10.04% respectively, compared to the blank group 1.
From the crude protein content, the decrease of crude protein content of group 5, group 6, group 10 and group 11 is most obvious, and the aNDF and ADF are also increased to a certain extent, which indicates that the addition of corncob and rice hull powder has a certain negative effect on the silage of the paper mulberry. Meanwhile, the aNDF content is reduced by more than 8% under the condition that the crude protein content is reduced by not more than 10%, and the method is obviously superior to the use of rice hull powder and corncobs.
The content of lactobacillus (MRS medium, culture at 37deg.C for 48 h), yeast and mould (Bengalum red medium, culture at 28deg.C for 3-7 days) in the silage of Broussonetia papyrifera is detected by plate counting method. The results are shown in Table 4.
TABLE 4 Effect of different biological formulations on the microbial numbers of Broussonetia papyrifera silage
After silage fermentation for 1 year, fermentation tends to be stable in an anaerobic state, the influence of low pH value on lactic acid bacteria per se can be obviously inhibited, the number of lactic acid bacteria in the group 8, the group 9 and the group 12 is the lowest, and the number of lactic acid bacteria in the group 8 and the group 9 is obviously less than that in the control group due to the lowest pH value. The broussonetia papyrifera silage had a lower number of yeasts, with group 12 having the lowest number of yeasts and group 6 having the highest number of yeasts. In most cases, no mold was detected, and only a small amount of mold was repeatedly detected in groups 1, 6, 10 and 11. It is possible that the external sources such as corncob, rice hull powder and the like are not completely removed after the fermentation of the mold is completed.
The results of the measurement of the content of the bioactive components and the anti-nutritional factors of the silage of the paper mulberry are shown in table 5 and fig. 8-10.
TABLE 5 Effect of different biological formulations on Broussonetia papyrifera silage bioactive ingredient and anti-nutritional factor content
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Compared with the blank control group 1, the flavonoid content in the silage of the paper mulberry is increased by all groups, the increase range is more than 200 percent, the effect of the group 5 is the best, and no obvious difference exists among other treatment groups.
Compared with the blank group 1, the total phenol content is increased in all groups, the effect of the group 2 is best, the total phenol content is increased by 50.25%, the effects of the group 12 and the group 9 are inferior, and the total phenol content is respectively increased by 38.02% and 34.47%.
All groups reduced tannin content by more than 20% compared to control group 1, with group 8, group 9, group 4, group 7, group 11 and group 10, with group 10 being the most effective, with a reduction of 28.92%.
The increase in flavonoid content for group 5 and group 6 and the decrease in tannin content for group 10 and group 11 were mainly due to the increase in flavonoid content and decrease in tannin content caused by exogenously added rice hull meal and corncob, but also resulted in a decrease in crude protein content and an increase in fiber content.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A composite biological agent for fermenting silage of broussonetia papyrifera is characterized by comprising lactobacillus and tannase;
the ratio of the number of viable bacteria of the lactic acid bacteria to the enzyme activity of the tannase is (1-10) x 10 5 CFU：0.1U；
The lactobacillus comprises lactobacillus plantarum and lactobacillus paracasei;
the ratio of the viable count of the lactobacillus plantarum to the lactobacillus paracasei is (1-3): (0.8-1.2).
2. The composite biological agent for fermentation of paper mulberry silage according to claim 1, wherein the ratio of the number of viable bacteria of the lactic acid bacteria to the enzyme activity of the tannase is 5 x 10 5 CFU：0.1U。
3. The composite biological agent for fermentation of paper mulberry silage according to claim 1 or 2, wherein the ratio of viable count of lactobacillus plantarum to lactobacillus paracasei is 2:1.
4. use of a complex biological agent according to any one of claims 1 to 3 for the preparation of silage from broussonetia papyrifera.
5. The application of claim 4, wherein the application comprises at least one of:
the nutritional quality of the silage of the paper mulberry is improved;
reducing the tannin content in the silage of the broussonetia papyrifera;
increasing flavonoid and total phenol content in the silage of broussonetia papyrifera;
increasing the lactic acid content in the silage of the broussonetia papyrifera;
and reducing the pH, acetic acid, and ammoniacal nitrogen content in the silage of the paper mulberry.
6. The use according to claim 4, wherein the raw materials for preparing the silage of broussonetia papyrifera comprise one or more of broussonetia papyrifera leaves, broussonetia papyrifera roots, broussonetia papyrifera barks and broussonetia papyrifera stems.
7. The use according to claim 6, wherein the dry matter content of the whole broussonetia papyrifera strain is 27-34% by mass.
8. The use according to claim 6, wherein the amount of lactic acid bacteria added in the composite biological agent is (1-10) x 10 when the paper mulberry silage is prepared 5 CFU/kg feedstock;
the addition amount of tannase is 80-120U/kg of raw material.
9. The use according to claim 4, wherein the fermentation temperature of the paper mulberry silage is 15-35 ℃.
10. The use according to any one of claims 4 to 9, wherein the fermentation time of the paper mulberry silage is 1 year.
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