Compound microbial fermentation preparation for broussonetia papyrifera silage and application of compound microbial fermentation preparation
By using a compound microbial fermentation agent to improve the quality of paper mulberry silage, the problem of nutrient loss in paper mulberry silage was solved, and the quality of paper mulberry silage was significantly improved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHIHEZI UNIVERSITY
- Filing Date
- 2023-12-19
- Publication Date
- 2026-04-24
AI Technical Summary
There is limited research on paper mulberry silage in existing technologies, and traditional lactic acid bacteria preparations have limited effectiveness, resulting in nutrient loss and insufficient quality improvement in paper mulberry silage.
A compound microbial fermentation preparation, composed of Lactobacillus plantarum MZ008357, Lactobacillus brevis, and Enterococcus faecalis, supplemented with molasses and urea, is used for mulberry silage fermentation to form a highly efficient fermentation system that preserves dry matter and crude protein, reduces neutral detergent fiber content, and minimizes protein spoilage.
It significantly increases the dry matter and crude protein content of paper mulberry silage, reduces the neutral detergent fiber content, improves fermentation quality, reduces protein spoilage, and enhances the quality of silage.
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Figure CN121910099A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial fermentation products technology, and in particular to a compound microbial fermentation preparation for mulberry silage and its application. Background Technology
[0002] With the continuous development of the livestock industry and the rise in grain prices, developing high-quality, safe, and inexpensive forage has become an inherent requirement for transforming the economic growth model of the livestock industry. Paper mulberry (Broussonetia papyrifera) is a native tree species in my country rich in nutrients and highly adaptable. After harvesting, it can be used as livestock feed. Paper mulberry raw materials are characterized by high crude protein content, high buffering energy value, low soluble sugar content, and soft, juicy texture, making it an excellent livestock feed ingredient. Compared to the traditional method of processing paper mulberry into hay, the silage fermentation method can slow down the loss of nutrients in paper mulberry and effectively improve the palatability and digestibility for livestock.
[0003] Currently, many technologies disclose conventional forage silage methods. However, due to the nutritional characteristics of paper mulberry itself, few of the existing publicly available technologies or methods involve paper mulberry silage, and the silage effect is not ideal. The main problems are: First, there is a scarcity of relevant research on paper mulberry silage; Second, there are few microbial preparations currently used for paper mulberry silage fermentation. Some methods use homogeneous lactic acid bacteria Lactobacillus plantarum and heterogeneous lactic acid bacteria Lactobacillus bruneri, either alone or in combination, supplemented with sugar sources or cellulase. However, the lactic acid bacteria used in these methods are the conventional lactic acid bacteria commonly used by the public, not the lactic acid bacteria naturally attached to paper mulberry silage. Ultimately, while these microbial preparations have some effect on paper mulberry silage, their improvement on the quality of paper mulberry silage products is very limited.
[0004] Given the problems with lactic acid bacteria additives used for paper mulberry silage and the nutritional characteristics of paper mulberry itself, existing technologies cannot effectively complete paper mulberry silage production, ensure the silage's effectiveness, or maximize the retention of its nutrients. Summary of the Invention
[0005] The purpose of this invention is to provide a compound microbial fermentation preparation for mulberry silage and its application. When applied to mulberry silage, the compound microbial fermentation preparation can preserve the dry matter and crude protein content of mulberry silage, reduce the content of neutral detergent fiber in the fermentation system, reduce protein spoilage, and improve fermentation quality.
[0006] To achieve the above objectives, the present invention provides a compound microbial fermentation preparation for mulberry silage, wherein the compound microbial fermentation preparation is prepared from the following raw materials in parts by weight:
[0007] Lactobacillus plantarum MZ008357 0.5-1.5 parts, Lactobacillus brevis (purchased from China General Microbiological Culture Collection Center CGMCC: 1.2028) 0.5-1.5 parts, Enterococcus faecalis (purchased from China General Microbiological Culture Collection Center CGMCC: 1.2025) 0.5-1.5 parts, molasses 0.3-1.7 parts, urea 0.5-1.5 parts;
[0008] The *Lactobacillus plantarum* MZ008357 was deposited by the China General Microbiological Culture Collection Center (CGMCC) on November 13, 2023, with accession number CGMCC No. 28968.
[0009] Preferably, the viable count of the compound microbial fermentation preparation is 10. 8 ~10 10 CFU / mL.
[0010] Preferably, the 16S rDNA sequence of the *Lactobacillus plantarum* MZ008357 is shown in SEQ ID NO.1.
[0011] This invention also provides the application of a compound microbial fermentation preparation in improving the quality of paper mulberry silage.
[0012] Preferably, the compound microbial fermentation agent is sprayed onto the mulberry silage for fermentation treatment.
[0013] Preferably, the spraying dosage of the compound microbial fermentation agent is 5 × 10⁻⁶. 5 ~2×10 6 CFU / Paper Tree.
[0014] Preferably, the fermentation temperature of the fermentation treatment is 15-25℃, the storage environment temperature is -30-35℃, and the storage time does not exceed 3 years.
[0015] Therefore, the present invention adopts the above-mentioned compound microbial fermentation preparation for mulberry silage and its application. The compound microbial fermentation preparation is suitable for the fermentation process of mulberry silage, and can preserve the dry matter and crude protein content in mulberry silage, reduce the content of neutral detergent fiber in the fermentation system, and reduce protein spoilage.
[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description
[0017] Figure 1 This invention relates to a compound microbial fermentation preparation for mulberry silage and the Gram staining results of lactic acid bacteria in its application. Detailed Implementation
[0018] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.
[0019] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.
[0020] Preparation process
[0021] I. Isolation and identification of dominant lactic acid bacteria strains attached to natural silage paper mulberry:
[0022] a. Raw material preparation: When the paper mulberry trees reach a height of about 1.5m, harvest the entire tree using a forage harvester, cut the raw material into short pieces of 1-2cm, and prepare silage using the bale-wrapping method, with a bale density of 500kg / m³. 3 The packaging dimensions are: radius 50cm, height 100cm, packaging film thickness 0.1mm, packaging layers 8-10. After fermentation at 18-23℃ for 60 days, samples are taken (raw materials from 5 randomly selected packages) for the isolation and identification of lactic acid bacteria.
[0023] The MRS agar medium formula is as follows: peptone 10.0 (g / L), beef extract 8.0 (g / L), yeast extract 4.0 (g / L), glucose 20.0 (g / L), dipotassium hydrogen phosphate 2.0 (g / L), diammonium hydrogen citrate 2.0 (g / L), sodium acetate 5.0 (g / L), magnesium sulfate 0.2 (g / L), manganese sulfate 0.04 (g / L), agar 14.0 (g / L), Tween 80, pH 6.5±0.2. Sterilize at 121℃ for 15 min.
[0024] The M17 broth culture medium formula is as follows: soybean peptone 5.0 (g / L), peptone 2.5 (g / L), casein peptone 2.5 (g / L), yeast extract 2.5 (g / L), beef extract 5.0 (g / L), lactose 5.0 (g / L), sodium ascorbate 0.5 (g / L), sodium β-glycerophosphate 19.0 (g / L), magnesium sulfate 0.25 (g / L), pH 7.2±0.2.
[0025] Isolation and purification of lactic acid bacteria: Take 20g of the prepared silage from each package, and seal it in an Erlenmeyer flask containing 180mL of sterile physiological saline. Place the flask on a shaker at 120rpm for 2 hours (37℃), then remove and let it stand. In a clean bench, perform serial dilutions with sterile physiological saline, and spread four suitable gradients (10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸) onto plates, with three replicates for each gradient. Use the plate spreading method and anaerobic incubate at 37℃ for 48–72 hours on MRS M17 medium (containing calcium carbonate). Afterward, observe the colony morphology, size, color, and luster. Select typical colonies with a calcium-dissolving zone for Gram staining, oil immersion microscopy, and catalase contact test. Figure 1 As shown, all Gram-positive and catalase-negative strains were preliminarily identified as lactic acid bacteria. They were isolated and purified by streak plating (four-zone streak method) on M17 medium 2-3 times. They were then enriched with M17 liquid medium (30℃, 24h), mixed with an equal volume of glycerol, packaged, and stored at -80℃ for later use.
[0026] Identification of *Lactobacillus c.*: DNA was extracted from the purified lactic acid bacteria culture broth according to the kit instructions. PCR amplification was performed using universal bacterial primers FA-27F (5′-GCAGAGTTCTCGGAGTCACGAAGAGTTTGATCCTGGCTCAG-3′) and RA-1495R (5′-AGCGGATCACTTCACACAGGACTACGGGTACCTTGTTACGA-3′). The reaction volume (50 μL) was: 5 μL DNA template, 1 μL each of primers 27F and 1495R (10 μmol / L), 25 μL 2×Master Mix, and ddH2O to a final volume of 50 μL. The reaction program was: 95℃ for 10 min, 95℃ for 30 s, 60℃ for 30 s, 72℃ for 45 s, for 30 cycles. The PCR amplification products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Based on the homology analysis of the 16S rDNA gene sequence (see Table 1), the accession numbers of Bacillus plantarum MZ008357, G7, and G9 in GenBank are MZ008357-MZ008359.
[0027] Table 1. Results of 16S rDNA gene sequence analysis of lactic acid bacteria strains.
[0028]
[0029] II. Identification of acid production and growth rate of isolated lactic acid bacteria strains
[0030] The lactic acid bacteria suspension selected in the first step was inoculated at a rate of 3% into MRS and M17 liquid media (both media were initially adjusted to pH 6.2) and incubated at 37°C for 24 hours. Using the liquid media without bacterial suspension as a blank, samples were taken every 2 hours, and the OD of each sample was measured using a UV-Vis spectrophotometer (Hitachi, U-2910). 600nmThe values were plotted as follows: culture time (h) on the x-axis and corresponding absorbance (OD) on the y-axis, to create a statistical graph. The pH of the fermentation broth was measured using a pH meter (Shanghai Leici, PHSJ-3F), and the acid production rate curve was plotted based on the changes in the average pH of the fermentation broth at different fermentation times (h). The results showed that *Lactobacillus plantarum* strain MZ008357 had a strong acid production capacity, with the pH value dropping below 5 at 12h and below 4.5 at 24h, indicating that its acid production capacity was superior to other strains. *Lactobacillus plantarum* strain MZ008357 entered a rapid growth phase at 8h and a stable growth phase at 12h, with its growth capacity at 24h being superior to other strains. The correlation between the growth rate and acid production rate results indicates that *Lactobacillus plantarum* strain MZ008357 was generally superior to other isolated strains.
[0031] Acid production rate and growth rate are parameters used to measure the fermentation ability of microorganisms to utilize resources under the same conditions, and are also important indicators for screening superior lactic acid bacteria strains. In this invention, all lactic acid bacteria showed no obvious lag phase during fermentation, indicating that they grew and multiplied rapidly in the culture medium, which could rapidly increase the number of lactic acid bacteria, enhance fermentation, and quickly reduce the pH value.
[0032] The preferred strain in the mulberry silage compound microbial fermentation preparation of the present invention is Lactobacillus plantarum MZ008357, which was selected after judging the acid production rate and growth rate of the strains initially isolated and identified.
[0033] The 16S rDNA sequence of Lactobacillus plantarum MZ008357 is shown in SEQ ID NO.1;
[0034] The *Lactobacillus plantarum* MZ008357 of this invention exhibits both strong growth rate and acid-producing capacity.
[0035] Lactobacillus plantarum MZ008357 was deposited by the China General Microbiological Culture Collection Center on November 13, 2023, with accession number CGMCC No.28968.
[0036] III. Preparation of Compound Microbial Fermentation Agents
[0037] Activated *Lactobacillus plantarum* MZ008357 was inoculated onto a culture medium and cultured to obtain a *Lactobacillus plantarum* MZ008357 bacterial suspension. The *Lactobacillus plantarum* MZ008357 bacterial suspension, *Lactobacillus brevis* (purchased from the China General Microbiological Culture Collection Center CGMCC: 1.2028), and *Enterococcus faecalis* (purchased from the China General Microbiological Culture Collection Center CGMCC: 1.2025) were mixed in a 1:1:1 ratio according to the number of live bacteria to obtain a compound microbial fermentation preparation. This invention does not limit the activation method; any method well known to those skilled in the art can be used.
[0038] The *Lactobacillus plantarum* MZ008357 of the present invention was inoculated onto M17 medium; the pH value of the medium was 6.5; the medium was used after sterilization; the sterilization temperature was 121°C; and the sterilization time was preferably 15 min.
[0039] The compound microbial fermentation preparation for mulberry silage of this invention can efficiently promote the fermentation of mulberry silage, thereby obtaining mulberry silage of better quality. Furthermore, this compound microbial fermentation preparation is more suitable for the mulberry silage fermentation process. Therefore, the compound microbial fermentation preparation of this invention can be used for mulberry silage fermentation and to improve the quality of mulberry silage.
[0040] Example 1
[0041] Formulation 1 of the paper mulberry compound microbial fermentation preparation: includes 1 part of Lactobacillus plantarum MZ008357, 0.5 parts of molasses, and 1.5 parts of urea.
[0042] Example 2
[0043] Formulation 2 of the paper mulberry compound microbial fermentation preparation includes 1 part of Lactobacillus brevis (CGMCC: 1.2028), 0.5 parts of molasses, and 1.5 parts of urea.
[0044] Example 3
[0045] Formulation 3 of the paper mulberry compound microbial fermentation preparation includes 1 part of Enterococcus faecalis (CGMCC: 1.2025), 0.5 parts of molasses, and 1.5 parts of urea.
[0046] Example 4
[0047] Formulation 4 of the paper mulberry compound microbial fermentation preparation includes 1 part of Lactobacillus plantarum MZ008357 + 1 part of Enterococcus faecalis (CGMCC: 1.2025), 0.5 parts of molasses, and 1.5 parts of urea.
[0048] Example 5
[0049] The mulberry compound microbial fermentation preparation consists of 1.5 parts of Lactobacillus plantarum MZ008357, 1 part of Lactobacillus brevis (CGMCC: 1.2028), 0.5 parts of molasses, and 1.5 parts of urea.
[0050] Example 6
[0051] The compound microbial fermentation preparation of paper mulberry consists of 1 part of Lactobacillus plantarum MZ008357, 1 part of Lactobacillus brevis (CGMCC: 1.2028), 1 part of Enterococcus faecalis (CGMCC: 1.2025), 0.5 parts of molasses, and 1.5 parts of urea.
[0052] Comparison Example
[0053] Only 0.5 parts molasses and 1.5 parts urea are added.
[0054] Chop the paper mulberry into 1-2cm pieces, dissolve formulas 1-6 and the control in water or physiological saline, activate at room temperature for 1-3 hours, mix thoroughly with the chopped paper mulberry raw material, pack into silage containers, press and seal, and ferment at room temperature for 60 days.
[0055] The fermentation quality of all samples obtained from the examples and control examples was evaluated.
[0056] In this invention, the main nutritional components measured are dry matter (DM), crude protein (CP), soluble carbohydrates (WSC), neutral detergent fiber (NDF), and acid detergent fiber (ADF). DM content was determined using a 105°C drying method; CP content was determined using the Kjeldahl method; WSC content was determined using the anthrone colorimetric method; and NDF and ADF content were determined using the Van der Waals method. Fermentation characteristics were mainly measured for pH, ammonia nitrogen (NH3-N), lactic acid (LA), acetic acid (AA), propionic acid (PA), and butyric acid (BA). pH was measured using a pH meter; LA, AA, PA, and BA contents were determined using liquid chromatography; and NH3-N content was determined using the phenol-hypochlorous acid colorimetric method.
[0057] The microbiological assay primarily determined the number of aerobic bacteria (AB), lactic acid bacteria (LAB), yeast, and mold. AB, LAB, yeast, and mold were cultured on nutrient agar, MRS medium, maltose syrup agar, and high-salt Czapek's medium, respectively. After culturing, plate counts were performed, and the microbial counts were calculated.
[0058] The results measured in this invention are as follows:
[0059] Table 2. Effects of compound microbial fermentation preparations on the nutritional quality of Broussonetia papyrifera silage.
[0060] index Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Comparison Example pH 5.28 5.16 6.45 4.85 4.15 4.75 6.42 DM / % 36.10 35.35 36.67 35.26 35.81 36.69 35.01 CP / %DM 18.52 17.22 18.94 18.04 17.50 19.12 17.88 NDF / %DM 38.91 38.46 39.39 40.57 39.64 39.93 42.78 ADF / %DM 21.04 20.68 20.49 19.43 19.33 18.88 20.16 WSC / %DM 6.11 6.30 6.11 6.81 6.77 6.55 7.42
[0061] As shown in Table 2, in this invention, the DM content in Examples 1 and 6 is higher than that in the control example. The NDF content in the other examples, except for Example 4, is significantly lower than that in the control group. There is no significant difference in ADF content among the experimental groups. The CP content in Examples 3 and 6 is significantly higher than that in all other groups except Example 1.
[0062] In this invention, except for Example 3, the pH value of all other groups was lower than that of the control group; the NH3-N content of each example was significantly lower than that of the control group.
[0063] Table 3. Effects of compound microbial fermentation preparations on organic acids in Broussonetia papyrifera silage.
[0064] index Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Comparison Example LA / %FM 6.66 6.27 5.09 5.77 5.52 6.01 5.41 AA / %FM 1.89 2.13 1.58 1.82 2.07 2.12 1.73 PA / %FM 0.14 0.12 0.28 0.25 0.18 0.23 0.23
[0065] Note: Butyric acid was not detected in any of them.
[0066] As shown in Table 3, in this invention, the LA content in Example 1 is significantly higher than that in the control example. The AA content in Examples 2 and 6 is significantly higher than that in the control example, the PA content in Example 3 is significantly higher than that in the control example, and the PA content in Examples 1, 2, and 5 is significantly lower than that in the control example.
[0067] Table 4. Effects of compound microbial fermentation preparations on the number of microorganisms in Broussonetia papyrifera silage.
[0068] index Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Comparison Example LAB(logCFU / gFM) 8.71 8.60 8.77 8.56 8.88 8.69 8.52 Yeast(logCFU / gFM) 3.72 3.54 3.88 3.71 3.62 3.65 3.80 Mold(logCFU / gFM) 3.71 ＜2.00 ＜2.00 ＜2.00 ＜2.00 ＜2.00 4.15 AB(logCFU / gFM) 7.53 7.40 7.45 7.41 7.71 7.57 7.86
[0069] As shown in Table 4, in this invention, the number of LLABs in all embodiments except for those treated in Examples 2 and 3 was significantly higher than that in the control example. The number of Yeasts in the Example 3 treatment was the highest, significantly higher than in the other embodiments except the control. The number of Yeasts in the Example 2, Example 5, and Example 6 treatments was significantly lower than that in the control example. Except for the control example and Example 1 treatment, the number of molds in the other treatment groups was <2 log CFU / g FM. The number of ABs in each embodiment was significantly lower than that in the control example, with the number of ABs in the Example 5 and Example 6 treatments being significantly higher than the other four embodiments.
[0070] In summary, the combination of different bacterial strains can generate a large amount of lactic acid in the fermentation system, enabling it to quickly enter an acidic environment in the early stages of fermentation, reducing nutrient loss, and inhibiting the growth and reproduction of harmful bacteria. Example 6 showed the best results. This invention can provide a reference for the silage technology industry and has broad application value and prospects.
[0071] Therefore, the present invention adopts the above-mentioned compound microbial fermentation preparation for mulberry silage and its application. The compound microbial fermentation preparation is suitable for the fermentation process of mulberry silage, can preserve the dry matter and crude protein content in mulberry silage, reduce the content of neutral detergent fiber in the fermentation system, reduce protein spoilage, and improve fermentation quality.
[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.
Claims
1. A compound microbial fermentation preparation for mulberry silage, characterized in that, The compound microbial fermentation preparation is prepared from the following raw materials in parts by weight: Lactobacillus plantarum MZ008357 0.5-1.5 parts, Lactobacillus brevis 0.5-1.5 parts, Enterococcus faecalis 0.5-1.5 parts, molasses 0.3-1.7 parts, urea 0.5-1.5 parts; The *Lactobacillus plantarum* MZ008357 was deposited by the China General Microbiological Culture Collection Center (CGMCC) on November 13, 2023, with accession number CGMCC No. 28968.
2. The compound microbial fermentation preparation for mulberry silage according to claim 1, characterized in that, The viable count of the compound microbial fermentation preparation is 10. 8 ~10 10 CFU / mL.
3. The compound microbial fermentation preparation for mulberry silage according to claim 1, characterized in that, The 16S rDNA sequence of the *Lactobacillus plantarum* MZ008357 is shown in SEQ ID NO.
1.
4. The application of the compound microbial fermentation preparation according to any one of claims 1 to 3 in improving the quality of paper mulberry silage.
5. The application according to claim 4, characterized in that, The compound microbial fermentation preparation described in any one of claims 1 to 3 is sprayed onto the silage of paper mulberry trees for fermentation treatment.
6. The application according to claim 5, characterized in that, The application rate of the compound microbial fermentation agent is 5×10. 5 ~2×10 6 CFU / Paper Tree.
7. The application according to claim 5, characterized in that, The fermentation temperature for the fermentation treatment is 15-25℃, the storage environment temperature is -30-35℃, and the storage time does not exceed 3 years.