Apple pomace silage as well as preparation method and application thereof

Abstract

The invention relates to the technical field of feed preparation, in particular to apple pomace silage and a preparation method and application thereof.The preparation method of the apple pomace silage comprises the following steps that apple pomace and wheat bran are mixed according to the mass ratio of 73-80: 20-27, and a silage raw material is obtained; spraying a bacterium-enzyme mixed solution on the silage raw material, and fermenting under a vacuum sealing condition, so as to obtain the apple pomace silage; the mass-volume ratio of the silage raw material to the bacterium-enzyme mixed solution is 1kg: 8mL-10mL; the concentration of the lactobacillus plantarum in the bacterium enzyme mixed solution is 2.0 * 10 < 8 > cfu / mL to 4.0 * 10 < 8 > cfu / mL, and the concentration of the cellulase in the bacterium enzyme mixed solution is 20000 IU / mL to 60000 IU / mL. The water content of the apple pomace is reduced through the wheat bran, and the ensiling fermentation process is promoted, the content of structural carbohydrates is reduced, the production and accumulation of lactic acid are promoted and the ensiling quality is improved by adopting bacterium-enzyme combined fermentation.

Description

Technical Field

[0001] The invention relates to the technical field of feed preparation, and in particular to apple pomace silage and a preparation method and application thereof. Background Art

[0002] Apple pomace is the residue left after crushing and squeezing fresh apples to extract juice. Apple pomace mainly consists of peel, core and residual pulp. It contains rich nutrients such as minerals, soluble sugars, vitamins and cellulose, and is a good feed resource. At present, apple pomace is usually processed into apple pomace dry powder, which is used to prepare complete feed or granular feed as feed for livestock and poultry such as pigs, cattle and sheep.

[0003] The moisture content of fresh apple pomace is about 23% to 30%. During the utilization of fresh apple pomace, it is very easy to deteriorate and needs to be processed immediately. However, since fresh apple pomace is produced seasonally and the output is very concentrated, most of the fresh apple pomace will deteriorate during the preparation of dry powder, resulting in a large amount of waste and failure to fully utilize the fresh apple pomace.

[0004] Fresh apple pomace is rich in non-structural carbohydrates, which can provide sufficient fermentation substrates for lactic acid bacteria, promote lactic acid production and reduce pH, and meet silage conditions. Therefore, the prior art prevents the corruption and deterioration of fresh apple pomace through silage treatment, thereby obtaining apple pomace silage, which is used to fully utilize fresh apple pomace. However, during the silage process, due to the low dry matter content of fresh apple pomace, the quality of the obtained apple pomace silage is low. In addition, the moisture content of fresh apple pomace is relatively high, and a high cost needs to be invested in the moisture adjustment process. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides an apple pomace silage and a preparation method and application thereof, which can effectively improve the quality of the silage and reduce the investment cost of removing moisture from the apple pomace.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows.

[0007] A first aspect of the present invention provides a method for preparing apple pomace silage, comprising the following steps:

[0008] The apple pomace and the wheat bran are mixed in a mass ratio of 73-80:20-27 to obtain a silage raw material;

[0009] Spraying the bacterial enzyme mixture on the silage raw material, performing anaerobic fermentation, and obtaining apple pomace silage;

[0010] The mass volume ratio of the silage raw material to the bacterial enzyme mixed solution is 1kg:8mL-10mL ；

[0011] The bacterial enzyme mixed solution is obtained by mixing cellulase, plant lactobacillus and water;

[0012] The concentration of Lactobacillus plantarum in the bacterial enzyme mixture is 2.0×10 8 cfu / mL~4.0×10 8 cfu / mL, and the concentration of cellulase is 20000IU / mL~60000IU / mL.

[0013] The present invention mixes apple pomace and wheat bran at a mass ratio of 73-80:20-27, and adjusts the moisture in the apple pomace through the wheat bran, thereby reducing the input for moisture adjustment; and during the fermentation process, under the action of the bacterial enzyme mixture, the silage fermentation process can be promoted, the water-soluble carbohydrate content and the effective degradation rate of rumen dry matter can be increased, thereby effectively increasing the dry matter content in the apple pomace silage, and at the same time, the production and accumulation of lactic acid can be promoted, the fermentation effect can be improved, and the pH value can be reduced, thereby effectively improving the quality of the apple pomace silage.

[0014] More importantly, in the present invention, the specific concentration of 20000IU / mL to 60000IU / mL cellulase and the concentration of 2.0×10 8 cfu / mL~4.0×10 8 cfu / mL of plant lactobacillus can reduce neutral detergent fiber and acid detergent fiber, increase water-soluble carbohydrates, promote lactic acid and acetic acid accumulation, reduce pH value, and not increase dry matter loss rate, so that the rumen degradation characteristics of apple pomace silage are significantly improved, thereby improving the feeding value of apple pomace silage. If the types or concentrations of cellulase and plant lactobacillus are replaced, the above-mentioned corresponding technical effects cannot be achieved.

[0015] In another preferred embodiment, the temperature of the anaerobic fermentation is 23°C to 27°C.

[0016] In another preferred embodiment, the anaerobic fermentation time is 20 days to 80 days.

[0017] In another preferred embodiment, the apple pomace is fresh apple pomace after apple juice is squeezed.

[0018] The second aspect of the present invention provides the apple pomace silage.

[0019] The third aspect of the present invention provides the use of the apple pomace silage in improving the rumen fermentation capacity of ruminants.

[0020] In another preferred embodiment, the rumen fermentation capacity of ruminants refers to DPPH antioxidant activity and rumen dry matter degradation rate.

[0021] In another preferred embodiment, the ruminant is a cow.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The present invention uses wheat bran and apple pomace as silage raw materials, and the wheat bran can effectively reduce the moisture in the apple pomace, reduce the cost of moisture removal, and also improve the dry matter quality, which is beneficial to the silage process and improves the silage quality; and in the fermentation process, bacterial enzyme combined fermentation is used to promote the silage fermentation process, reduce the content of structural carbohydrates, promote the production and accumulation of lactic acid, reduce the pH value, and does not increase the dry matter loss rate, thereby improving the silage quality.

[0024] (2) The cellulase in the present invention can reduce the neutral detergent fiber and acid detergent fiber of fresh apple pomace silage, increase the content of water-soluble carbohydrates, promote the production of acetic acid, and reduce the pH value; adding plant lactobacillus can promote the enrichment of g_Lactiplantibacillus, increase the lactic acid content, and reduce the pH value. The bacterial enzyme synergistic treatment can reduce neutral detergent fiber and acid detergent fiber, increase water-soluble carbohydrates, promote the accumulation of lactic acid and acetic acid, reduce the pH value, and do not increase the dry matter loss rate. Adding cellulase and plant lactobacillus can change the rumen degradation characteristics of fresh apple pomace silage, further improve the effective rumen degradation rate of dry matter, neutral detergent fiber, and acid detergent fiber, thereby improving its feeding value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the dry matter weight result graph of different silages at different silage times.

[0026] Figure 2 This is the result graph of DPPH free radical scavenging ability of different silages.

[0027] Figure 3 The following are the results of microbial diversity analysis of different silages; a is the ASV Venn diagram, b is the microbial β diversity analysis diagram, and c is the microbial α diversity analysis diagram.

[0028] Figure 4 Relative abundance diagram of microorganisms in different treatment groups, where a is the relative abundance diagram of microbial composition at the phylum level, b is the relative abundance diagram of microbial composition at the family level, and c is the relative abundance diagram of microbial composition at the genus level.

[0029] Figure 5 Figures 2 and 3 are the LEfSe analysis results of different treatment groups; a is the LEfSe analysis results of CE and CON, b is the LEfSe analysis results of LP and CON, and c is the LEfSe analysis results of LPCE and CON. DETAILED DESCRIPTION

[0030] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0031] In the following examples, cellulase (500,000 IU / g) was purchased from Guangdong Yiduoli Biotechnology Co., Ltd.; Lactobacillus plantarum (Lp194, 1.0×10 11 cfu / g) was purchased from Weikang Probiotics (Suzhou) Co., Ltd.). Apple pomace is the residue left over from the production of apple juice in a juice factory in Shaanxi Province. The dry matter, organic matter and water-soluble carbohydrate contents of fresh apple pomace were 147.8 g / kg (fresh weight basis), 971.9 g / kg and 85.3 g / kg (dry basis), respectively, the neutral detergent fiber and acid detergent fiber were 586.5 g / kg (dry basis) and 525.6 g / kg (dry basis), respectively, and the crude protein was 64.5 g / kg (dry basis).

[0032] Example 1

[0033] A method for fermenting apple pomace silage comprises the following steps:

[0034] S1. Mix apple pomace and wheat bran in a mass ratio of 8:2 to obtain silage raw material.

[0035] S1. Spray 10 mL of the bacterial enzyme mixture on each kilogram of silage raw material, and ferment for 20 days under vacuum sealing conditions at 23°C to obtain apple pomace silage.

[0036] In the bacterial enzyme mixture, the concentration of cellulase was 20000IU / mL, and the concentration of Lactobacillus plantarum was 2.0×10 8 cfu / g.

[0037] In order to further illustrate the effect of bacterial-enzyme combined fermentation, the following experiments were conducted for demonstration.

[0038] 1. Apple pomace silage obtained by different fermentation methods

[0039] Apple pomace and wheat bran were used as silage materials, and different bacterial enzyme reagents were added during the fermentation process. The following four treatments were set up:

[0040] a is the control group, recorded as CON: not inoculated with the bacterial enzyme mixture; b is the cellulase group, recorded as CE: only inoculated with cellulase; c is the Lactobacillus plantarum group, recorded as LP: only inoculated with Lactobacillus plantarum; d is the bacterial enzyme mixture group in Example 1, recorded as LPCE.

[0041] For each treatment, different bacterial enzyme reagents were dissolved in 30 mL of sterile water, mixed evenly, and then sprayed on the silage raw materials. For CON, an equal amount of sterile water was sprayed. The silage raw materials evenly inoculated with bacterial enzyme fermentation agents were divided into 15 cm × 20 cm silage fermentation bags, with 0.5 kg in each bag, which were vacuum-sealed and stored in a dark and dry environment.

[0042] Fermentation was carried out under vacuum sealing at 23℃, and samples were taken at 20d, 40d, and 80d, and the samples were stored at -80℃ for testing. The fermentation parameters, nutritional components, and antioxidant activity of the samples after 20d, 40d, and 80d of ensiling were measured; the microbial composition of the 80d ensiling samples was measured; and the rumen degradation characteristics of the 80d ensiling samples were measured using the nylon bag method. There were 48 bags in this experiment, i.e., 4 treatments × 3 ensiling times × 4 replicates.

[0043] (1) Nutritional composition determination

[0044] Equal amounts of samples were taken from the above four treatment groups and dried at 55°C to constant weight. Subsequently, the four samples were crushed and passed through a 1 mm sieve and stored for testing. The drying method in the AOAC method was used to determine dry matter, the Kjeldahl method was used to determine crude protein, and the ignition method was used to determine organic matter. Neutral detergent fiber and acid detergent fiber were determined using a semi-automatic fiber analyzer. The neutral detergent fiber content was determined by referring to the method of VanSoest et al. (1991), and the acid detergent fiber content was determined according to the method described in the AOAC method. The water-soluble carbohydrate content was determined by the anthrone colorimetric method using the A145-1-1 kit (Nanjing Jiancheng Biotechnology Co., Ltd., Nanjing, China). The results are shown in Tables 1 and Figure 1 shown.

[0045] Table 1 Nutritional composition of wheat bran and apple pomace mixed silage

[0046]

[0047]

[0048] Note: Different letters in Table 1 indicate significant differences compared with the CON group.

[0049] As shown in Table 1, the dry matter content of CE was significantly lower than that of CON, LP, and LPCE at 20, 40, and 80 days of ensiling (P<0.05), and there was no significant difference between LP, LPCE, and CON (P>0.05). At different ensiling times, CE and LPCE significantly reduced the content of neutral detergent fiber and acid detergent fiber (P<0.05), while increasing the content of water-soluble carbohydrates (P<0.05); at 80 days of ensiling, LP significantly reduced the content of water-soluble carbohydrates (P<0.05). There was no significant difference in organic matter and crude protein among different treatments (P>0.05).

[0050] Depend on Figure 1 It can be seen that the dry matter loss rate of CE was significantly higher than that of other treatment groups (P<0.05); there was no significant difference among CON, LP and LPCE (P>0.05).

[0051] (2) Determination of silage fermentation parameters

[0052] Weigh 10 g of the above four samples and prepare the extract according to the following steps: (1) 90 mL of sterile water, homogenize for 1 min, and then extract at 4°C for 24 h; (2) Filter using 4 layers of gauze and double-layer filter paper; (3) Store the extract at -20°C for testing. Immediately measure the pH value, and use the phenol-sodium hypochlorite colorimetric method to determine the ammonia nitrogen content. The volatile fatty acid content was determined by high-performance gas chromatography. The lactic acid content was determined using the A019-2-1 kit (Nanjing Jiancheng Biotechnology Co., Ltd., China), and the results are shown in Table 2.

[0053] Table 2 Fermentation parameters of wheat bran and apple pomace mixed silage

[0054]

[0055]

[0056] Note: The groups with different letters in Table 2 are significantly different from those in the CON group.

[0057] As shown in Table 2, after 20 days of ensiling, there were no significant differences in pH value, lactic acid, butyric acid and ammonia nitrogen content among different treatment groups (P>0.05); the contents of acetic acid and total organic acids in CE and LPCE were significantly higher than those in CON and LP (P<0.05), while the lactic acid / acetic acid ratio was significantly reduced (P<0.05); the propionic acid content in LP and LPCE was significantly higher than that in CON and CE (P<0.05). After 40 days of ensiling, the pH value of CE was significantly lower than that of CON, LP and LPCE (P<0.05); the lactic acid content in LP and LPCE was significantly higher than that in CON and CE (P<0.05), and LP was significantly higher than LPCE (P<0.05); the acetic acid content in CE and LPCE was significantly higher than that in CON and LP (P<0.05), and CE was significantly higher than LPCE (P<0.05); the total organic acid content in CE, LP and LPCE was significantly higher than that in CON (P<0.05); the lactic acid / acetic acid ratio of CON, LP and LPCE was significantly higher than that of CE (P<0.05), and LPCE was significantly higher than that of CON and LP (P<0.05); there was no significant difference in the contents of propionic acid, butyric acid and NH3-H (P>0.05). After 80 days of ensiling, CE and LPCE significantly reduced the pH value (P<0.05), and LPCE was significantly lower than CE (P<0.05); LP and LPCE significantly increased the lactic acid content (P<0.05), and LP was significantly higher than LPCE (P<0.05); CE significantly increased the butyric acid content (P<0.05); the total organic acid content of CE, LP, and LPCE groups was significantly increased (P<0.05), and LPCE was significantly higher than CE and LP (P<0.05); the lactic acid / acetic acid in CON and LP was significantly higher than CE and LPCE (P<0.05), and LP was significantly higher than CON (P<0.05); there was no significant difference in the contents of acetic acid, propionic acid, and ammonia nitrogen (P>0.05).

[0058] (3) Biological activity assay

[0059] Take 20g of the above 4 samples for freeze-drying to retain the active ingredients in the samples. Take 1g of dry matter freeze-dried sample, add 20mL of 80% ethanol, and extract ultrasonically at 40°C for 30min to dissolve the active ingredients in ethanol. Centrifuge the extract suspension at 6000rpm / min for 10min, filter through a 0.45μm filter membrane to obtain the extract, and store it at -20°C for testing. Mix 1mL of the extract with 2mL of DPPH ethanol solution (0.2mM). Incubate the mixture in the dark at room temperature for 30min, use an enzyme reader to measure the absorbance at 517nm, and calculate the DPPH free radical scavenging activity. The results are as follows Figure 2 shown.

[0060] Depend on Figure 2It can be seen that at 20 and 80 days of ensiling, the DPPH antioxidant activity of LP and LPCE was significantly higher than that of CON and CE (P<0.05), while there was no significant difference between CE and CON (P>0.05). At 40 days of ensiling, there was no significant difference in DPPH antioxidant activity among different treatment groups (P>0.05).

[0061] (4) Determination of microbial composition

[0062] The microbial composition of the 80-day silage samples was determined as follows:

[0063] Microbial DNA extraction: The kit uses MN NucleoSpin 96Soi, and the reagents used in the following experimental steps are all from MN NucleoSpin 96Soi.

[0064] Sample transfer: transfer 250 mg of the above different samples to a 2.0 mL imported centrifuge tube, pour the grinding beads in the NucleoSpinBeadTube into the sample tube, add 700 μL of SL2; add 150 μL of Enhancer SX. SL and SX can be mixed in advance at a ratio of 700:150. The mixed solution is prepared before use. If it becomes turbid, it needs to be heated at 65°C for a few minutes to make the mixed solution clear before use. Cover the centrifuge tube tightly and vortex to mix well to ensure that the sample and lysate are fully mixed.

[0065] Lysis samples: Place the above different sample tubes into a vortex shaker and shake at 70°C and 1000 rpm for 10 min to promote sample lysis.

[0066] Precipitation to remove impurities: After the lysis, centrifuge the sample at 12000rpm for 2min, transfer 700μL of the supernatant to a new 2.0mL centrifuge tube, add 150μL of SL3, invert to mix, incubate at 4℃ for 5min; centrifuge at 12000rpm for 2min, paying attention to the order of the samples.

[0067] Filtration to remove inhibitors:

[0068] 1) Insert the MTP / MULTI-96PLATE gasket into the vacuum box with the notched side facing upwards;

[0069] 2) Place the MN Square-well Block on the spacer;

[0070] 3) Close the lid of the vacuum box;

[0071] 4) Place the NucleoSpin Inhibitor Removal Plate into the plate slot on the lid.

[0072] 5) Add about 600 μL of the supernatant in 3) to the plate in order (can be marked), turn on the vacuum pump and adjust the vacuum until all the liquid flows through the plate; adjust the binding conditions, add 250 μL of Buffer SB to the filtrate in 4), and mix by gently pipetting.

[0073] DNA Binding:

[0074] 1) Place the waste liquid tank into the vacuum box;

[0075] 2) Insert the MTP / MULTI-96PLATE gasket into the vacuum box with the notched side facing upwards;

[0076] 3) Place the MN Wash Plate on the gasket;

[0077] 4) Close the lid of the vacuum box;

[0078] 5) Place the NucleoSpin Soil Binding Plate into the plate slot on the lid;

[0079] 6) Add the mixed liquid in 5) to the plate in the corresponding order, turn on the vacuum pump and adjust the vacuum until all the liquid flows through the plate. The liquid flow rate should not exceed 1 to 2 drops / s.

[0080] Cleaning the silicon matrix membrane:

[0081] 1) 1nd wash: Add 500 μL of SB and turn on the vacuum pump until all the liquid flows through the plate;

[0082] 2) 2nd wash, add 550 μL of SW1 and turn on the vacuum pump until all the liquid flows through the plate;

[0083] 3) 3rd wash: Add 700 μL of SW2 and turn on the vacuum pump until all the liquid flows through the plate;

[0084] 4) 4th wash: Add 700 μL of SW2 and turn on the vacuum pump until all the liquid flows through the plate.

[0085] Drying Silicon Matrix Membrane:

[0086] 1) Remove the NucleoSpin Soil Binding Plate and absorb the remaining wash solution at the bottom of the column on absorbent paper;

[0087] 2) Remove the MN Wash Plate;

[0088] 3) Take the waste liquid tank out of the vacuum box;

[0089] 4) Close the lid of the vacuum box;

[0090] 5) Place the NucleoSpin Soil Binding Plate back into the plate slot on the lid;

[0091] 6) Turn on the vacuum pump, adjust the vacuum degree to the maximum value and continue to pump for 15 minutes to ensure that the column membrane is completely dry. The residual ethanol in the washing solution will inhibit the subsequent enzymatic reaction;

[0092] 7) Absorb the remaining wash solution at the bottom of the column on absorbent paper.

[0093] DNA Elution:

[0094] 1) Insert the MICROTUBE RACK gasket into the vacuum box with the notched side facing upwards;

[0095] 2) Place the Rack of Tube Strips on the gasket;

[0096] 3) Close the lid of the vacuum box;

[0097] 4) Place the NucleoSpin Soil Binding Plate back into the plate slot on the lid;

[0098] 5) Add 80 μL of Buffer SE preheated to 70°C to the center of the column membrane and let stand at room temperature for 5 minutes to promote elution;

[0099] 6) Turn on the vacuum pump until all the liquid has flowed through the plate.

[0100] DNA transfer:

[0101] Transfer the DNA to a new 1.5 mL inlet centrifuge tube for later use.

[0102] Illumina Novasek sequencing:

[0103] Sequencing primers 338F-806R were used to amplify the variable region of 16S rRNA gene V3-V4 by PCR. The amplification procedure was as follows: 95℃ pre-denaturation for 3min, 25 cycles (95℃ denaturation for 30s, 50℃ annealing for 30s, 72℃ extension for 40s), then 72℃ stable extension for 7min, and finally stored at 4℃ (PCR instrument: 96well PCR instrument). The PCR reaction system was: KOD FX NeoBuffer 5μL, dNTP (2mM each) 2μL, upstream primer (10μM) 0.3μL, downstream primer (10μM) 0.3μL, genomic DNA 50ng, KOD FX Neo 0.2μL, ddH2O supplemented to 10μL; each sample was repeated 3 times.

[0104] The PCR products of the above steps were mixed according to the quantitative results of electrophoresis using Image J software at a mass ratio of 1:1. After mixing, the samples were purified using an OMEGA DNA purification column. After electrophoresis on a 1.8% agarose gel at 120V for 40 minutes, the target fragment was cut and recovered.

[0105] The original data were spliced ​​(version 1.2.11), the spliced ​​sequences were quality filtered (version 0.33), and chimeras were removed (version 8.1) to obtain high-quality tag sequences. The sequences were clustered at the level of 97% similarity (version 10.0), and ASV was filtered with a threshold of 0.005% of all sequence numbers.

[0106] The 16 samples from the four treatment groups after 80 days of silage fermentation were sequenced by Illumina platform. According to the 97% sequence similarity, the sequences were clustered and 725 ASVs were obtained, such as Figure 3 The results of microbial diversity analysis showed that CE significantly reduced Observed_species (P<0.05); Chao1, Observed_species, and Shannon of LP and LPCE were significantly reduced (P<0.05); compared with the other three groups, Goods_coverage in the LP group was significantly increased (P<0.05). Figure 3 The PCoA results show that, except for LP and LPCE, the other groups are clearly separated, as shown in Figure 3 The Veen diagram shows the common and unique ASVs among different treatment groups. The results show that CON, CE, LP and LPCE groups have 192, 135, 122 and 128 unique ASVs, respectively; the four treatment groups have a total of 148 ASVs, and CON, CE, LP and LPCE groups have 279, 215 and 224 ASVs, respectively. Figure 3 As shown in a in .

[0107] The relative abundance of microorganisms (phylum, family and genus levels) in different treatment groups is shown in Figure 2. Figure 4 At the phylum level, the main microorganisms in wheat bran and apple pomace mixed silage were Firmicutes and Proteobacteria, accounting for more than 90% of the total bacterial abundance. Figure 4 As shown in a in the figure; Lactobacillaceae, Mitochondria, Acetobacteraceae and Chloroplast are the dominant families. Figure 4As shown in b in the figure; at the genus level, the main dominant genera are Lentilactobacillus, Levilactobacillus, Mitochondria, Gluconobacter, Chloroplast, Secundilactobacillus, Acetobacter, Liquorilactobacillus, Lactiplantibacillus, etc. Figure 4 As shown in c.

[0108] LEfSe analysis results are as follows Figure 5 As shown, compared with CON, CE increased the relative abundance of p_Firmicutes, c_Bacilli, o_Lactobacillales, f_Lactobacillaceae, and g_Levilactobacillus (P<0.05), and decreased the relative abundance of p_Proteobacteria, c_Alphaproteobacteria, o_Rickettsiales, f_Mitochondria, f_Fructobacillus, g_Acetobacter, g_Mitochondria, p_Cyanobacteria, c_Cyanobacteriia, o_Chloroplast, f_Chloroplast, and g_Chloroplast (P<0.05). Figure 5 As shown in a; LP increased the relative abundance of p_Firmicutes, c_Bacilli, o_Lactobacillales, f_Lactobacillaceae, g_Levilactobacillus, o_Burkholderiales, g_Oxalobacteraceae, and g_Morganellaceae (P<0.05), and decreased the relative abundance of p_Proteobacteria, c_Alphaproteobacteria, o_Rickettsiales, f_Mitochondria, f_Fructobacillus, g_Streptococcus, p_Cyanobacteria, c_Cyanobacteriia, o_Chloroplast, f_Chloroplast, and g_Chloroplast (P<0.05). Figure 5As shown in b; LPCE increased the relative abundance of p_Firmicutes, c_Bacilli, c_Clostridia, o_Lactobacillales, o_Pseudomonadales, f_Levilactobacillus, f_Pseudomonadaceae, g_Pseudomonas, and g_Schleiferilactobacillus (P<0.05), and decreased the relative abundance of p_Cyanobacteria, c_Cyanobacteriia, o_Chloroplast, f_Chloroplast, and g_Chloroplast (P<0.05). Figure 5 As shown in c.

[0109] 5) Rumen degradation characteristics

[0110] The above four samples after 80 days of silage were dried at 55℃ for 72h, crushed and passed through a 2mm sieve for later use. Four Chinese Holstein cows in good health, with similar dry matter intake and body weight and permanently equipped with fistulas were selected as experimental animals.

[0111] The experiment included a 14-day pre-feeding period and a 3-day rumen incubation and sample collection period. The feeding management remained unchanged during the experiment, with feeding twice a day, at 7 a.m. and 4 p.m., and free access to water. The composition and nutritional level of the total mixed diet used are shown in Table 3.

[0112] Table 3 Composition and nutritional level of total mixed ration

[0113]

[0114]

[0115] Note: The premix provides per kilogram of diet: Vitamin A 7266 IU, Vitamin D 2362 IU, Vitamin E 91 IU, Copper 7.27 mg, Manganese 41.78 mg, Zinc 41.78 mg, Cobalt 0.73 mg.

[0116] About 4.0 g of apple pomace silage sample that has passed through a 2 mm sieve was placed in a nylon bag of a specific size and particle size (pore size 50 μm, specification 8 cm × 10 cm), and the sample weight was accurately recorded. The nylon bags containing the apple pomace silage samples were placed in the rumen of the experimental dairy cows for incubation, with 56 bags per cow (4 samples × 7 time points × 2 repetitions). After incubation in the rumen for 0 h, 4 h, 8 h, 16 h, 24 h, 48 h, and 72 h, the bags were taken out and immersed in ice water to stop fermentation. The nylon bags after incubation were naturally rinsed until the water was clear, then dried at 55 ° C, weighed, and stored in a ziplock bag at room temperature. The dry matter, neutral detergent fiber, and acid detergent fiber content in the samples after degradation were analyzed.

[0117] The degradation rate (P) of dry matter, neutral detergent fiber and acid detergent fiber at different incubation times was calculated using formula (1):

[0118] P = (nutrient content before degradation - nutrient content in sample after degradation) / nutrient content before degradation × 100% formula (1);

[0119] Among them, a certain nutrient is dry matter, neutral detergent fiber or acid detergent fiber.

[0120] The degradation kinetic parameters a, b and c of dry matter, neutral detergent fiber and acid detergent fiber were calculated using formula (2). The calculation formula is as follows:

[0121] P = a + b × (1 - e - ct) Formula (2);

[0122] Wherein, a is the rapidly degradable part (%), b is the slowly degradable part (%), and c is the degradation rate of b (% / h).

[0123] Formula (3) was used to calculate the effective degradation rate ED of each nutrient in the rumen. The calculation formula is as follows:

[0124] ED = a + (b × c) / (c + k) Formula (3);

[0125] In the formula, ED is the effective degradation rate of a nutrient component in the rumen of the sample to be tested (%), k is the outflow rate of the feed raw material in the rumen (h -1 ), in this test, k was set to 0.031% / h.

[0126] Table 4 Rumen degradation characteristics of apple pomace and wheat bran silage dry matter by adding cellulase and Lactobacillus plantarum

[0127]

[0128] Note: CON control group; CE cellulase group; LP plant lactobacillus group; LPCE bacterial enzyme synergistic group. Different letters after the value indicate significant difference (P<0.05), the same letter or no letter indicates no significant difference (P>0.05). a Rapidly degradable component (%); b Slowly degradable component (%); c: Degradation rate of b (% / h); ED effective degradation rate. Same below.

[0129] Compared with CON, the rumen degradation rate of dry matter in CE was significantly increased at 4h, 8h, and 12h of incubation (P<0.05), but the degradation rate of dry matter in 24h of incubation was significantly decreased (P<0.05); LP significantly increased the degradation rate of dry matter in 8h of incubation (P<0.05); LPCE significantly increased the degradation rate of dry matter in 4h, 8h, 12h, and 48h of incubation (P<0.05), but decreased the degradation rate of dry matter in 24h of incubation (P<0.05). Compared with CON, CE and LPCE significantly increased the fast degradation part (P<0.05), while reduced the slow degradation part and the degradation rate of the slow degradation part (P<0.05). However, CE did not change the effective degradation rate of dry matter (P>0.05), while LPCE increased the effective degradation rate of dry matter (P<0.05). There was no significant difference between the rumen degradation parameters of LP and CON (P>0.05).

[0130] Table 5 Rumen degradation of neutral detergent fiber of apple pomace and wheat bran silage

[0131]

[0132]

[0133] As shown in Table 5, compared with CON, CE significantly reduced the degradation rate of neutral detergent fiber at 12h, 24h, 48h and 72h of rumen incubation, and thus reduced the effective degradation rate of neutral detergent fiber (P<0.05); LP significantly increased the degradation rate of neutral detergent fiber at 48h of incubation (P<0.05), but there was no significant difference in rumen degradation parameters (P>0.05); LPCE significantly reduced the degradation rate of neutral detergent fiber and the effective degradation rate of neutral detergent fiber at 24h and 48h of rumen incubation (P<0.05).

[0134] Table 6 Rumen degradation of acid detergent fiber of apple pomace and wheat bran silage

[0135]

[0136] As shown in Table 6, compared with CON, CE significantly reduced the degradation rate of acid detergent fiber at 24h, 48h, and 72h of rumen incubation (P<0.05); LP significantly increased the degradation rate of neutral detergent fiber at 48h of incubation (P<0.05); LPCE significantly increased the degradation rate of acid detergent fiber at 12h of incubation (P<0.05), but reduced the degradation rate of acid detergent fiber at 24h of incubation (P<0.05). CE and LPCE significantly increased the fast degradation part of acid detergent fiber (P<0.05), but reduced the degradation rate of the slow degradation part and the effective degradation rate of acid detergent fiber (P<0.05); LP increased the slow degradation part (P<0.05), but did not affect the effective degradation rate of acid detergent fiber (P>0.05).

[0137] From the above results, it can be seen that the use of mixed silage of wheat bran and apple pomace is a feasible treatment method. The addition of cellulase reduces neutral detergent fiber and acid detergent fiber, increases the content of water-soluble carbohydrates and acetic acid, but increases the dry matter loss rate. In addition, the addition of cellulase can increase the effective degradation rate of rumen dry matter, but reduces the effective degradation rate of neutral detergent fiber and acid detergent fiber. Inoculation of Lactobacillus plantarum alone can promote the production and accumulation of lactic acid, lower pH, and enhance the antioxidant activity of DPPH. The mixed addition of bacterial enzymes has a synergistic effect, which can effectively reduce neutral detergent fiber and acid detergent fiber, increase the content of water-soluble carbohydrates and the effective degradation rate of rumen dry matter, while promoting the production of lactic acid, lowering pH, and enhancing the antioxidant activity of DPPH.

[0138] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0139] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing apple pomace silage, characterized in that: The following steps are involved: The apple pomace and the wheat bran are mixed in a mass ratio of 73-80:20-27 to obtain a silage raw material; Spraying the bacterial enzyme mixture on the silage raw material, performing anaerobic fermentation, and obtaining apple pomace silage; The mass volume ratio of the silage raw material to the bacterial enzyme mixture is 1kg:8mL-10mL; The bacterial enzyme mixed solution is obtained by mixing cellulase, plant lactobacillus and water; The concentration of Lactobacillus plantarum in the bacterial enzyme mixture is 2.0×10 8 cfu / mL~4.0×10 8 cfu / mL, and the concentration of cellulase is 20000IU / mL~60000IU / mL.

2. The method for preparing apple pomace silage according to claim 1, characterized in that: The temperature of the anaerobic fermentation is 23°C to 27°C.

3. The fermentation method of apple pomace silage according to claim 1, characterized in that: The anaerobic fermentation time is 20 days to 80 days.

4. The method for preparing apple pomace silage according to claim 1, characterized in that: The apple pomace is fresh apple pomace after apple juice is squeezed.

5. Apple pomace silage prepared according to the method for preparing apple pomace silage according to any one of claims 1 to 4.

6. Use of the apple pomace silage according to claim 5 in improving the rumen fermentation capacity of ruminants.

7. The use according to claim 6, characterized in that: The rumen fermentation capacity of ruminants refers to the DPPH antioxidant activity and the rumen dry matter degradation rate.

8. The use according to claim 6, characterized in that: The ruminant is a dairy cow.

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