Two-cut mulberry twig fermented feed and application thereof in chicken raising

Abstract

The invention relates to the field of microbial feeds, in particular to a two-cut ramulus mori fermented feed and application thereof in chicken raising, two-cut ramulus mori is high in yield and low in collection cost, and feed cost can be greatly reduced. According to the method, silage conditions are optimized for ramulus mori raw materials, firstly, bacterial strains are screened, a bacterial strain XMS03 and a bacterial strain XMS01 are selected for silage of the two-cut ramulus mori, and the two-cut ramulus mori fermented feed is obtained. The method effectively improves the ensiling efficiency of the ramulus mori, assists the ensiling of the two-cut ramulus mori by optimizing an auxiliary agent and adding cellulase and molasses, improves the quality of the ramulus mori ensiling feed, effectively improves the application range of the waste two-cut ramulus mori, can change wastes into valuables to the greatest extent, and meanwhile, due to the fact that the used raw material is the two-cut ramulus mori, the ensiling efficiency of the ramulus mori is greatly improved. Compared with the whole-plant ramulus mori reported in the prior art, the method is simpler in obtaining mode and lower in cost, and compared with the whole-plant ramulus mori reported in the prior art, the nutritional ingredients of the fermentation substrate are more stable, and the application range of the two-cut ramulus mori is widened.

Description

[Technical field]

[0001] The invention relates to the field of microbial feed, and mainly comprises optimization of a two-cut mulberry branch fermentation feed process and application thereof in chicken farming. [Background technology]

[0002] In order to improve the yield and quality of mulberry leaves, mulberry trees are trimmed and pruned in summer and winter every year. The sum of the pruned branches and leaves is called two-cut mulberry branches, which is one of the largest by-products of the sericulture industry. In 2022, the area of ​​mulberry gardens in Guangxi reached 189,400 hm2. 2 It is the largest province in the country for mulberry planting and silkworm breeding. Mulberry branches, which account for more than half of the biological output in mulberry orchards, are not effectively utilized. Based on the annual output of nearly 1 ton of mulberry branches per mu, the annual output of fresh mulberry branches in Guangxi can reach 3 million tons, and the stock is huge.

[0003] At present, there are not many means of development and utilization of two-cut mulberry branches. A small part is processed into mushroom growth sticks, and most of them are discarded in the fields or burned as firewood, which has caused resource waste and environmental pollution, which is an urgent problem to be solved. Generally speaking, two-cut mulberry branches contain 5.44% crude protein, 51.88% cellulose, 18.18% lignin, 23.02% hemicellulose, and ash about 1.57%. Its general nutritional composition is not bad, the collection cost is low, and it is rich in polysaccharides, flavonoids and other active compounds, which have high value for animal health and meat quality improvement. This makes two-cut mulberry branches have the potential to be used as a high-quality roughage resource for livestock and poultry. Guangxi is currently vigorously adjusting the livestock breeding structure, reducing the proportion of grain-consuming livestock and increasing the proportion of grain-saving high-quality livestock. If two-cut mulberry branches can be developed as a source of roughage, it can not only extend the value chain of the mulberry silkworm industry to a greater extent, but also alleviate the problem of forage resource shortage to a certain extent, and promote the sustainable development of animal husbandry production. Therefore, the development and utilization of its feed has profound practical needs and economic value.

[0004] The most common ways to utilize fresh green roughage resources include fresh feeding, drying and crushing, and processing for silage. Mulberry trees are perennial woody plants with flexible fibrous bark and hard lignified components, which greatly restricts the use of two-cut mulberry branches as fresh feed or drying. Two-cut mulberry branches have a large yield, low collection cost, and relatively low nutritional content. They are rich in flavonoids, polysaccharides and other effective ingredients that have antioxidant and immune-enhancing properties. At the same time, their fiber components are also a good source of dietary fiber, which makes them potential for development into feed with certain functions. However, the soluble sugar content of two-cut mulberry branches is low, and the effect and efficiency of their natural silage are poor, which is very easy to cause nutritional loss and corruption. At present, there are two main problems that need to be solved in the utilization of two-cut mulberry branches for silage: (1) Using conventional crushing technology, the degree of crushing of two-cut mulberry branches is not enough, the degree of fermentation is limited, and the lignified components with larger particle size are prone to form thorns, which affects processing and animal palatability. (2) Due to the low nutritional quality of two-cut mulberry branches themselves, the soluble carbohydrate (WSC) content is insufficient. Moreover, using microorganisms attached to the body for fermentation has certain limitations and other problems, which makes silage difficult to succeed. [Summary of the invention]

[0005] The present invention aims at the problems existing in the silage of twice-cut mulberry branches, studies how to optimize the silage conditions of twice-cut mulberry branches, thereby improving the application scope of the waste twice-cut mulberry branches, and can maximize the waste into treasure. By optimizing the microbial agent and the auxiliary agent, it is expected that the fermentation efficiency and fermentation degree of the silage of twice-cut mulberry branches can be targetedly improved, the nutritional value and palatability thereof can be improved, and the quality of the silage feed of twice-cut mulberry branches can be improved.

[0006] A composite bacterial agent for silage, the composite bacterial agent comprising Aspergillus oryzae XMS01 and Lactobacillus plantarum XMS03, the strains were screened by the inventor of the research group during his tenure at the Guangxi Zhuang Autonomous Region Animal Husbandry Research Institute, and have been disclosed in the prior patent application: 201910366868.6;

[0007] Among them, the Aspergillus oryzae XMS01 has a deposit number of CCTCC: M2018425, and is deposited at China Center for Type Culture Collection, Wuhan University, Wuhan, China, and the deposit date is July 2, 2018; the Lactobacillus plantarum XMS03 has a deposit number of CCTCC: M2019003, and is deposited at China Center for Type Culture Collection, Wuhan University, Wuhan, China, and the deposit date is January 2, 2019.

[0008] Furthermore, the composite bacterial agent is used to ferment twice-cut mulberry branches.

[0009] The present invention also includes silage containing the composite bacterial agent.

[0010] Furthermore, the silage also contains molasses and cellulase.

[0011] The present invention also includes a method for preparing the silage, which comprises: crushing two cut mulberry branches, adding probiotics and additives, mixing and silaging;

[0012] The probiotics are composed of Aspergillus oryzae and Lactobacillus plantarum; the Aspergillus oryzae is Aspergillus oryzae XMS01, with a deposit number of CCTCC: M2018425, deposited in: China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, and a deposit date of July 2, 2018; the Lactobacillus plantarum is Lactobacillus plantarum XMS03, with a deposit number of CCTCC: M2019003, deposited in: China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, and a deposit date of January 2, 2019. The strain was screened by the inventor of the research group during his tenure at the Guangxi Zhuang Autonomous Region Animal Husbandry Research Institute, and has been disclosed in the prior patent application: 201910366868.6;

[0013] The additives include molasses and / or cellulase.

[0014] Furthermore, the added amount of the molasses is 4% of the substrate mass and the enzymatic activity of the cellulase is greater than or equal to 5000 U / g.

[0015] Furthermore, the viable count of Aspergillus oryzae is greater than or equal to 6.3×10 7 CFU / g; the viable count of the plant lactic acid bacteria is greater than or equal to 3.1×10 7 CFU / g.

[0016] Furthermore, the silage is stored for 45 days.

[0017] The present invention also includes the use of the silage or the silage prepared by the method in chicken farming, wherein the use is: adding the silage in an amount of 6% by mass to a basic material to feed the chickens.

[0018] The present invention also includes the use of the silage or the silage prepared by the method in reducing the abdominal fat content of chickens.

[0019] The present invention has the following beneficial effects:

[0020] 1. Adding Aspergillus oryzae and lactic acid bacteria to non-grain raw materials for silage processing can effectively improve their nutritional value and fermentation quality. Aspergillus oryzae is an aerobic probiotic that produces complex enzymes such as cellulase, amylase, and saccharifying enzymes, which can assist in the decomposition of cellulose, degrade complex organic matter in feed, promote digestion and absorption, and improve feed utilization. In addition, its early aerobic fermentation can quickly consume the remaining oxygen, allowing the environment to quickly enter the anaerobic fermentation stage. Lactobacillus plantarum is a homotypic fermentative lactic acid bacterium that can effectively enhance the dominant role of lactic acid bacteria in silage, reduce the pH of micro-storage feed, inhibit the growth of harmful bacteria, maintain the balance of intestinal flora, and promote the absorption of nutrients. Cellulase can catalyze the degradation of cellulose and its derivatives in silage, release easily digestible and absorbable nutrients such as intracellular starch and protein, and improve the utilization of intestinal nutrients. As a by-product of the sucrose industry, molasses is rich in soluble sugars and has a moderate cost. It is very suitable as an energy source and carbon source for microbial fermentation, especially lactic acid bacteria fermentation.

[0021] 2. Based on the low nutritional content of two-cut mulberry branches and the difficulty of natural silage, the present application firstly carried out strain screening by optimizing the silage conditions, and screened out the strain with high enzyme activity in the mulberry branch base material: Aspergillus oryzae XMS01, and then found that Lactobacillus plantarum XMS03 had high enzyme activity against Aspergillus oryzae through optimization screening. oryzae)XMS01's laccase and lignin peroxidase have a promoting effect, so the strains XMS03 and XMS01 were selected to jointly ensilage the two-cut mulberry branches, which effectively improved the ensilage efficiency of the mulberry branches. At the same time, the adjuvant was further optimized. Cellulase and molasses were added during the ensilage process to improve the decomposition of mulberry branches in the silage. The nutritional value and fermentation quality of the two-cut mulberry branch silage were improved to varying degrees. Through the detection of microbial diversity, it was found that the relative abundance of Firmicutes and Lactobacillus was increased, while the relative abundance of Proteobacteria, Klebsiella, Pantoea, and Enterobacter was reduced, butyric acid fermentation was effectively inhibited, the silage fermentation quality was improved, and the palatability of the two-cut mulberry branches was better. Through breeding experiments, it was found that the mulberry branch silage of the present application can reduce the abdominal fat content of chickens.

[0022] 3. The nutritional composition and physical structure of two-cut mulberry branches are quite different from those of common forage grasses. Therefore, in response to the problems existing in the silage of two-cut mulberry branches, this research team started from the above four research experiences, and studied the effects of increasing the crushing particle size, adding Aspergillus oryzae, Lactobacillus plantarum, molasses, cellulase, etc. on the nutritional components, fermentation quality and microbial composition of two-cut mulberry branch silage, in order to improve the fermentation efficiency and degree of two-cut mulberry branch silage in a targeted manner, and improve its key technical issues such as nutritional value and palatability. It effectively improves the application scope of waste two-cut mulberry branches, and can turn waste into treasure to the greatest extent. At the same time, because the raw materials used are two-cut mulberry branches, the method of obtaining them is simpler and the cost is lower. Moreover, compared with the whole mulberry branches reported in the prior art, the nutritional components of the fermentation substrate are more stable, which improves the application scope of two-cut mulberry branches.

Brief Description of the Drawings

[0023] Figure 1 This is a diagram showing the effects of different additive combinations on the community composition at the taxonomic level of two-cut mulberry branch silage.

[0024] Figure 2 This is a diagram showing the effects of different additive combinations on the community composition at the taxonomic level of two-cut mulberry branch silage.

[0025] Figure 3 This is a schematic diagram of the visceral fat content after feeding on different mulberry branch silages; in the figure, A is the control group fed with a basic diet, and B is the mulberry branch silage of the experimental group. [Specific implementation method]

[0026] The present invention will be further described below in conjunction with the accompanying drawings, examples and tests.

[0027] This embodiment uses 3 self-selected microorganisms for experiment. The strains were selected by the inventors of the research group during their tenure at the Guangxi Zhuang Autonomous Region Animal Husbandry Research Institute and have been disclosed in the prior patent application: 201910366868.6. Specifically, Aspergillus oryzae XMS01 (hereinafter referred to as XMS01), with a deposit number of CCTCC: M2018425, deposited in: China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, and a deposit date of July 2, 2018; Lactobacillus plantarum XMS02 (hereinafter referred to as XMS02), with a deposit number of CCTCC: M2019002; deposited in: China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, and a deposit date of January 2, 2019; Lactobacillus plantarum XMS03 (hereinafter referred to as XMS03), the deposit number is CCTCC: M2019003; it is deposited in: China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, and the deposit date is January 2, 2019.

[0028] All materials and reagents used in this embodiment are as follows: Two-cut mulberry branches were collected from farmers' mulberry gardens in Yizhou District, Hechi City, Guangxi Zhuang Autonomous Region in mid-December 2023. In order to improve the yield and quality of mulberry leaves, the mulberry garden cuts and prunes the 30-50 cm above the ground of the whole mulberry tree every summer and winter. The discarded mulberry branches pruned for the first and second times are called two-cut mulberry branches. After the fresh two-cut mulberry branches are cut, they are crushed to 1-0.5 cm and 0.3 cm or less using a pulverizer with a kneading powder function. Aspergillus oryzae culture (6.3×10 7 CFU / g and plant lactic acid bacteria liquid (3.1×10 7 CFU / g) was provided by the Animal Nutrition Laboratory of the Animal Husbandry Research Institute of Guangxi Agricultural Vocational and Technical University, and the composite microbial preparations used in feed were commercial bacteria (containing: Pediococcus acidilactici ≥ 5 × 10 7 CFU / g, Saccharomyces cerevisiae ≥ 25×10 7 CFU / g, cellulase ≥200U / g, xylanase ≥100U / g) was purchased from a certain biotechnology company (making silage according to the instructions), and molasses and cellulase (≥5000U / g) were purchased from a certain chemical reagent company.

[0029] Embodiment 1:

[0030] 1. Experimental design:

[0031] (1) Characteristics of the raw materials of two-cut mulberry branches

[0032] The nutritional components of the raw materials are shown in Table 1. The DM content of the two-cut mulberry branch raw material is relatively high, which is 51.1%; the CP content is 5.12%; the ADL, ADF and NDF contents are relatively high, which are 14.45%, 53.58% and 64.65% respectively; the CT content is 3.91 mg / g, the polysaccharide content is 20.11 mg / g, the total flavonoids content is 0.87 mg / g, and the WSC content is relatively low, which is 5.32%.

[0033] Table 1 Nutritional composition of two-cut mulberry branches (dry matter basis)

[0034]

[0035] From the table above, we can see that although two-cut mulberry branches are a common and easily available organic waste, compared with corn stalks, mulberry leaves and other forage and leaf wastes, two-cut mulberry branches have lower nutritional quality and insufficient soluble carbohydrate (WSC) content. There are certain limitations in fermentation using microorganisms attached to them, which makes silage difficult to succeed. Moreover, even if some microorganisms with good silage functions are reported in some existing technologies, they may not be successful in silage fermentation of mulberry branches. Therefore, we need to screen microorganisms specifically based on the nutritional characteristics of two-cut mulberry branches.

[0036] (2) Screening of strains:

[0037] In long-term research, we found that some molds can produce laccase during fermentation, thereby accelerating the decomposition of lignin. However, there is no report in the prior art that lactic acid bacteria can produce laccase. For this reason, we conducted relevant experiments on the probiotics screened by our laboratory, and finally found that: Aspergillus oryzae XMS01 screened by the applicant can produce laccase and lignin peroxidase, and the combined use of Lactobacillus plantarum XMS03 can further enhance the secretion of laccase. The specific experiments are as follows:

[0038] ①Preliminary screening of culture medium:

[0039] The 20 bacterial strains preserved in the laboratory were cultured on the primary screening medium, and 3 mold strains were screened from the primary screening medium. At the same time, fading circles or / and color development circles appeared in the secondary screening medium (guaiacol-PDA medium and aniline blue-PDA medium). The fading circles or color development circles appeared faster and had larger diameters, indicating that they had strong laccase and peroxidase production capabilities.

[0040] ②Mulberry branch re-screening:

[0041] In order to screen out the cultured bacteria with effective decomposition effect on mulberry branches, the three selected fungi were re-screened. Specifically, the three strains were respectively inoculated into the initial solid enzyme-producing culture medium with mulberry leaves as the sole carbon source or mulberry branches as the sole carbon source to screen out the strains that can efficiently degrade mulberry branches. The results showed that among the three strains, only XMS01 had degradation activity on the mulberry branch culture medium. The specific ligninase activity is shown in Table 2.

[0042] Table 2 Ligninase activity of lignin-degrading bacteria

[0043]

[0044] Note: Data in the same column with the same lowercase letters or no letters indicate no significant difference (P>0.05), while data with different lowercase letters indicate significant difference (P<0.05).

[0045] As shown in Table 1, the screened XMS01, XMS61, and XMS83 can secrete laccase and lignin peroxidase in the mulberry leaf carbon source medium, and the secretion amount is XMS01>XMS61>XMS83, and the difference is significant (P<0.05). However, after changing to the mulberry branch carbon source medium, due to the change of carbon source, the amount of laccase and lignin peroxidase secreted by XMS01 decreased, and the difference was significant compared with the mulberry leaf carbon source (P<0.05). XMS61 and XMS83 could not secrete laccase and lignin peroxidase, indicating that the nutritional quality of mulberry branch is low and some microorganisms are not suitable for fermenting mulberry branch.

[0046] In addition, the identification of Lactobacillus plantarum XMS02 and Lactobacillus plantarum XMS03 found that these two strains of lactic acid bacteria could not produce laccase or lignin peroxidase related to lignin degradation.

[0047] In practical application, in order to improve the diversity of microorganisms, enhance the fermentation capacity of mulberry branches and the palatability and health care function of feed, the inventors considered using molds and lactic acid bacteria in combination. Therefore, the inventors used lactic acid bacteria and Aspergillus oryzae XMS01 in combination to prepare a composite bacterial agent to study the effect of lactic acid bacteria on lignin-related enzyme activities, as follows:

[0048] Group 1: Lactobacillus plantarum XMS02 and Aspergillus oryzae XMS01 were mixed at a mass ratio of 1:1 to prepare a composite probiotic, which was then added at a mass percentage of 5% to an initial solid enzyme-producing medium with mulberry branches as the sole carbon source, and the enzyme activity was determined.

[0049] Group 2: Lactobacillus plantarum XMS03 and Aspergillus oryzae XMS01 were mixed at a mass ratio of 1:1 to prepare a composite probiotic, which was then added at a mass percentage of 1% to an initial solid enzyme-producing medium with mulberry branches as the sole carbon source to determine the enzyme activity.

[0050] Control group: Aspergillus oryzae XMS01 was added at a mass percentage of 1% to the initial solid enzyme production medium with mulberry branches as the sole carbon source, and the enzyme activity was measured. The results are shown in Table 3.

[0051] Table 3 Effects of different bacterial agents on ligninase activity in mulberry culture medium

[0052]

[0053] As shown in Table 3, the laccase and lignin peroxidase activities secreted by group 1 were not significantly different from those of the control group (P>0.05), while the laccase and lignin peroxidase activities secreted by group 2 were significantly improved compared with those of the control group (P<0.05), with improvement rates of 68.47% and 15.67%, respectively. This shows that the composite bacterial agent prepared by mixing Lactobacillus plantarum XMS03 and Aspergillus oryzae XMS01 can improve the enzyme activities of laccase and lignin peroxidase. However, our previous studies found that the use of Lactobacillus plantarum XMS03 alone did not produce laccase and lignin peroxidase. The combined use of Lactobacillus plantarum XMS03 and Aspergillus oryzae XMS01 enhanced the activity of laccase and lignin peroxidase. This is probably because Lactobacillus plantarum XMS03 produced certain substances during fermentation that promoted the enzyme production of Aspergillus oryzae XMS01, while Lactobacillus plantarum XMS02 did not have this effect.

[0054] To this end, we selected Lactobacillus plantarum XMS03 and Aspergillus oryzae XMS01 as composite bacterial agents for subsequent mulberry branch fermentation experiments.

[0055] Embodiment 2:

[0056] The Aspergillus oryzae and Lactobacillus plantarum used in Examples T1-T3 are Aspergillus oryzae XMS01 and Lactobacillus plantarum XMS03, respectively. The composite bacterial agent of T4 is commercially available.

[0057] 1. Use single factor experimental design to calculate the amount of each additive based on the fresh weight of mulberry branches.

[0058] The control group CK was added with pure water, and the other four groups were experimental groups T1, T2, T3, and T4. Three replicates were set for each treatment. The specific experimental design is shown in Table 4. The silage raw materials were fully mixed with the added bacterial agent, packed into silage bags with one-way exhaust valves, and sealed with a hand-operated sealer. Each bag contained 3 kg of sample, and the silage was unsealed at room temperature for 45 days. Each bag of silage was mixed evenly and sampled for analysis.

[0059] Table 4 Experimental design

[0060]

[0061] (1) Determination of nutrient composition: Take about 200g of silage sample, place it on a porcelain plate and flatten it in a forced air drying oven, adjust the temperature to 65℃, dry it for 48 hours, and then take it out for nutrient composition determination. Dry matter DM, crude protein CP, acid detergent lignin ADL, acid detergent fiber ADF and neutral detergent fiber NDF shall be determined according to the "Feed Analysis and Material Quality Testing Technology". Ammonia nitrogen (NH3-N) determination method: phenol-sodium hypochlorite colorimetric method. Soluble carbohydrate (WSC) content is determined by anthrone colorimetric method. Tannin content refers to: GB / T 27985-2011 Determination of tannin in feed--spectrophotometric method. Total flavonoids refer to SN / T4592-2016 Determination of total flavonoids in exported food. Polysaccharide content refers to GB / T 40632-2021 Detection method of polysaccharides in bamboo leaves.

[0062] (2) Determination of pH and fermentation quality: Take 20 g of silage sample, add 180 mL of distilled water, stir evenly, squeeze the juice with a juicer for 1 min, filter with 4 layers of gauze and then filter with medium-speed qualitative filter paper to obtain the extract, and measure the pH with a pH meter.

[0063] Determination method of organic acids: Dionex Ultimate 3000 liquid chromatograph, LPG-3400 pump, VWD-3400 UV detector, Thermo Hypersil BDS C18 column (250mm×4.6mm, 5μm) were used to determine the concentrations of lactic acid (LA), acetic acid (AA), propionic acid (PA) and butyric acid (BA).

[0064] (3) Determination of microbial diversity: After ensiling, each bag of silage was mixed evenly, sampled with sterile forceps and quickly placed in a sterilized centrifuge tube, frozen in liquid nitrogen, and stored in a -80°C refrigerator. Sequencing and bioinformatics analysis were commissioned to Guangxi Nanning Aomeike Biotechnology Co., Ltd.

[0065] (4) Statistical analysis

[0066] Excel software was used to record and organize the data, SPSS17.0 was used to perform one-way ANOVA on the data, and Duncan's method was used for multiple comparisons between the treatment data.

[0067] 2. The results are as follows:

[0068] (1) Effects of different additive combinations on the nutritional components of two-cut mulberry branch silage:

[0069] Table 5 Effects of different additive combinations on the nutritional components of two-cut mulberry silage (dry matter basis)

[0070]

[0071] Note: 1. Data in the same row with the same lowercase letters or no letters indicate no significant difference (P>0.05), while data with different lowercase letters indicate significant difference (P<0.05). The same applies to the following tables.

[0072] As shown in Table 5, compared with the CK group, the addition of molasses + cellulase (T3) on the basis of the T1 group with Aspergillus oryzae and Lactobacillus plantarum significantly increased the CP and WSC contents. This shows that the mixed fermentation effect of adding molasses and cellulase on the basis of Aspergillus oryzae and Lactobacillus plantarum is better. This may be because aerobic microorganisms such as Aspergillus oryzae reproduce and grow, and secrete and accumulate non-starch polysaccharides and some proteins with more complex structures, which quickly consume the oxygen in the silage space of the raw materials and provide an anaerobic growth environment for lactic acid bacteria. The addition of molasses can increase the WSC content of the two-cut mulberry branches. Cellulase also degrades the cellulose in the plant cell wall into monosaccharides or disaccharides by acting on the B-1,4-glucosidic bond, and also increases the WSC content. The increase in WSC content promotes the gradual increase in the number of lactic acid bacteria, shortens the silage fermentation to enter the stable stage, and reduces the consumption of fermentation substrates by proteins and other miscellaneous bacteria, thereby reducing the NDF and ADF content in the silage, and relatively increasing the CP content. The ADL, ADF and NDF contents of T1, T2 and T3 groups were significantly lower than those of CK and T4 groups (P < 0.05), indicating that the addition of Aspergillus oryzae and Lactobacillus plantarum to T1, T2 and T3 groups all had the effect of reducing the ADL, ADF and NDF contents of two-cut mulberry branches. Aspergillus oryzae degrades macromolecular substances such as cellulose in the raw materials into small molecular monosaccharides, and fiber degradation improves the fermentation quality of the raw materials. Studies of the prior art have found that the addition of Lactobacillus plantarum to silage of sugarcane tails significantly reduced the ADF and NDF contents. This experiment found that the ADL, ADF and NDF contents of the T3 group were significantly lower than those of the T1 and T2 groups, indicating that the addition of cellulase on the basis of T2, the interaction effect between the bacterial agents reduced ADL, ADF and ADF, and the T3 group had the best effect in degrading fiber, which improved the palatability and nutritional value of the silage mulberry branches, thereby increasing the animal feed intake and digestibility. Compared with the CK group, the tannin content of each test group decreased significantly (P < 0.05), and the tannin content of T3 was significantly lower than that of other groups (P < 0.05), indicating that the strains Lactobacillus plantarum XMS03 and Aspergillus oryzae XMS01 both had different degrees of decomposition of mulberry tannins, and the combined use had a better effect. The polysaccharide content of the T3 group was higher than that of the CK group, indicating that the polysaccharide content of the fermentation product was increased after the two-cut mulberry branches were ensiled with Aspergillus oryzae + Lactobacillus plantarum + molasses + cellulase. It may be that the hardness of the two-cut mulberry branches was reduced after ultrafine grinding, and the surface area was further increased, and there was a good synergistic fermentation effect between the selected bacterial agents Aspergillus oryzae, lactic acid bacteria, cellulase and molasses, which retained the medicinal ingredients and promoted the growth and development of the animal body. The flavonoid content of each experimental group was significantly increased (P < 0.05), among which the flavonoid content of T1, T2 and T3 groups was significantly higher than that of CK group and T4 group (P < 0.05), indicating that strains Lactobacillus plantarum XMS03 and Aspergillus oryzae XMS01 have different degrees of effect on improving the precipitation of flavonoids from mulberry branches, and the combined use effect is better. This also shows that Lactobacillus plantarum XMS03 and Aspergillus oryzae XMS01 have better fermentation and silage effects on mulberry branches raw materials.

[0073] In addition, the degree of feed crushing in groups T1-T4 was lower than that in group CK, which indicates that the finer the green feed is crushed, the more its structure is damaged, the more nutrients are released, and the larger the specific surface area of ​​contact between the substrate and microorganisms, which greatly improves the efficiency and effect of silage fermentation.

[0074] (2) Effects of different additive combinations on the quality of two-cut mulberry branch silage

[0075] Table 6 Effects of different additive combinations on the quality of two-cut mulberry branch silage

[0076]

[0077] ND: Not Detected

[0078] As shown in Table 6, the pH of groups T1, T2 and T3 was significantly lower than that of groups CK and T4 (P < 0.05), and there was no significant difference among the three groups (P > 0.05). Generally, the pH of high-quality silage is 3.8-4.2. In this experiment, the pH of groups CK and T4 was 4.30 and 4.22, respectively. The pH of groups T1, T2 and T3 was lower than that of groups CK and T4 to varying degrees, ranging from 3.8 to 3.95. All three groups reached high-quality grades. It is speculated that this was mainly due to the addition of lactic acid bacteria in T1, T2 and T3. With the continuous fermentation of silage, the lactic acid bacteria content accumulated and became dominant, which reduced the pH value. The NH3.N content of all experimental groups was lower than that of the CK group, and that of T3 was significantly lower than that of the control group (P < 0.05). This was mainly due to the slow pH decrease of the natural silage CK group, and harmful microorganisms consumed protein for nutritional proliferation, which produced more NH3.N. The addition of Aspergillus oryzae + Lactobacillus plantarum + molasses + cellulase (T3 group) strengthened the dominant role of lactic acid bacteria in silage, and the rapidly formed lower pH environment inhibited the activity of plant proteases and the growth of harmful microorganisms, reduced the degradation of CP and WSC, and produced NH 3.N was less, which improved the fermentation quality of the two-cut mulberry branch silage. The LA content of T1, T2 and T3 groups was significantly higher than that of CK and T4 (P < 0.05), and that of T3 group was significantly higher than that of T1 and T2. T3 added molasses and cellulase to provide more fermentation substrate WSC for lactic acid bacteria and produce more organic acids, so a large amount of LA was produced during fermentation, which in turn limited the proteolytic activity and the growth of undesirable microorganisms, reduced the abundance of undesirable microorganisms, and reduced the ammonia nitrogen content. No AA and BA were detected in T1, T2 and T3. Both CK and T4 groups contained a small amount of butyric acid, indicating that no clostridial and butyric acid fermentation occurred in the three groups of silage, which was speculated to be due to the fact that the three groups of two-cut mulberry branches increased the solubility of the effective ingredients after ultrafine grinding, and the low pH formed by the added bacterial agent, and the rapid acidification inhibited the proliferation of harmful microorganisms, thereby improving the fermentation quality of silage. At the same time, it may also be related to the fact that the two-cut mulberry branches themselves have active substances such as polysaccharides, flavonoids, and alkaloids, and the antibacterial effect of polysaccharides and flavonoids active substances.

[0079] (3) Effects of different additive combinations on the microbial diversity of two-cut mulberry branch silage

[0080] ① Effects of different additive combinations on the Alpha diversity of microorganisms in two-cut mulberry branch silage: observed_otus and chao1 indexes represent species richness, and Shannon and simpson indexes represent the diversity of bacterial flora. See Table 7 for details.

[0081] Table 7 Analysis of the Alpha diversity of microorganisms in silage of two-cut mulberry branches by different additive combinations

[0082]

[0083] The observed_otus and chao1 indices represent the number of species. The higher the value, the richer the species. The Shannon and simpson indices represent the diversity of bacterial flora. The higher the value, the higher the diversity. The reduction of microbial diversity is usually regarded as a sign of successful silage fermentation. As shown in Table 7, the chao1 and observed_otus indices of CK, T1, T2 and T3 groups were significantly lower than those of T4 group (P<0.05); the Shannon index and Simpson index of CK and T4 were significantly higher than those of T1, T2 and T3 (P<0.05). This shows that the observed_otus, chao1 and Shannon indices are the most abundant in the number of species in the composite microbial preparation for feed (T4) and the diversity level is high. The Shannon and Simpson indices of T1, T2 and T3 groups are significantly lower than those of T4 and CK groups, and the flora diversity is reduced. This shows that different combinations of bacterial agents have a significant effect on the Alpha diversity of the microbial flora of two-cut mulberry branch silage. This indicates that the addition of a combined bacterial agent based on Aspergillus oryzae and Lactobacillus plantarum is beneficial to the two-cut mulberry branch silage. Lactobacillus can quickly dominate the entire fermentation process and become the dominant bacterial community, showing a state of decreased microbial diversity. The species richness and diversity of the CK and T4 groups are relatively high. It is speculated that the lack of acidification conditions in natural silage or added bacterial agents leads to slow degradation of the speed and degree of acidification of the raw materials, and Lactobacillus does not become dominant, which promotes the proliferation of harmful bacteria, resulting in high microbial richness and increased diversity. The coverage index of the five groups is 1, indicating that the sample sequencing fully covers the core composition of microorganisms.

[0084] ②Effects of different additive combinations on the composition of microbial communities at the level of two-cut mulberry branch silage:

[0085] like Figure 1 and as shown in Table 8.

[0086] Table 8 Effects of different additive combinations on the relative abundance (top 10) of the taxonomic level of two-cut mulberry silage

[0087]

[0088] From Table 8 and Figure 1It can be seen that the sum of the relative abundances of Firmicutes and Proteobacteria was greater than 95%, which was the dominant phylum in each group. The relative abundance of Firmicutes was significantly higher in T1, T2 and T3 than in CK and T4 (P<0.05), and the T3 group was significantly higher than the T1 and T2 groups (P<0.05). The relative abundance of Firmicutes in the T3 group was the highest (93.27%), and the relative abundance in the CK group was the lowest (11.81%), indicating that the addition of Aspergillus oryzae + Lactobacillus plantarum (T1), Aspergillus oryzae + Lactobacillus plantarum + molasses (T2), and Aspergillus oryzae + Lactobacillus plantarum + molasses + cellulase (T3) to the two cut mulberry branches can increase the proportion of Firmicutes in silage and reduce the proportion of Proteobacteria. Firmicutes mainly include mycoplasmas, non-spore-forming and spore-forming bacteria, which can degrade macromolecules such as protein, starch, cellulose, etc. Since most lactic acid bacteria belong to Firmicutes, it means that the three groups T1, T2 and T3 contain high content of beneficial organisms, and the content of beneficial organisms in group T3 is the highest, which can better inhibit the reproduction of harmful microorganisms during silage fermentation, thereby reducing the potential risk of pathogenic bacteria to the health of animals eating silage. At the same time, the relative abundance of Proteobacteria was reduced, among which the Proteobacteria of T1, T2 and T3 were significantly lower than those of CK and T4 (P<0.05). The relative abundance of Proteobacteria in CK group was the highest (87.04%), and the relative abundance in T3 group was the lowest (4.17%). Proteobacteria included most pathogens such as Escherichia coli and Salmonella, indicating that pathogenic microorganisms in natural silage CK and T4 groups increased in value and competed with lactic acid bacteria for substrates, which would lead to an increase in ammonia nitrogen content and a decrease in CP content, an increase in pH, a small amount of butyric acid, and a decrease in the quality of silage fermentation. This explains the reasons for the increase in ammonia nitrogen content and pH, the decrease in CP content and the production of butyric acid in CK and T4 groups.

[0089] ③Effects of different additive combinations on the composition of microbial communities at the genus level in two-cut mulberry silage:

[0090] like Figure 2 and as shown in Table 9.

[0091] Table 9 Effects of different additive combinations on relative abundance (top 10) of genus classification in two-cut mulberry silage

[0092]

[0093] From Table 9 and Figure 2It can be seen that the CK group is mainly composed of non-lactic acid bacteria, including Pantoea, Klebsiella, Pediococcus, and Enterobacter, with relative abundances of 50.58%, 26.56%, 7.98%, and 5.88%, respectively, and the total abundance reaches 91%, while the relative abundance of Lactiplantibacillus is extremely low, only 0.85%, indicating that the natural silage of two-cut mulberry branches is not easy to succeed. Other genera are non-lactic acid bacteria, including Pantoea, Klebsiella, Pediococcus, and Enterobacter, which are absolutely dominant, and spoilage bacteria are not inhibited. T1, T2, and T3 groups are mainly composed of Lactiplantibacillus, with relative abundances of 88.19%, 87.67%, and 90.52%, respectively. Compared with the Lactiplantibacillus in the CK and T4 groups, the relative abundance of Lactiplantibacillus in the T1, T2, and T3 groups increased significantly (P<0.05), and the T3 group was significantly higher than the T1 and T2 groups (P<0.05), indicating that after adding Aspergillus oryzae and Lactobacillus plantarum as the basic combination of bacterial agents to the two silages, the lactic acid production capacity was further enhanced, the silage pH gradually decreased, the relative abundance of Lactobacillus plantarum increased significantly, and the relative abundance of harmful bacteria such as Klebsiella and Enterobacter decreased significantly, inhibiting the reproduction of harmful microorganisms, thereby improving the fermentation quality and nutritional components of the T1, T2, and T3 groups. The relative abundance of Lactobacillus plantarum increased significantly after the addition of cellulase to the T3 group, which is consistent with the results of Ren Haiwei et al. that the relative abundance of lactic acid bacteria increased significantly after adding cellulase to the mixture of straw and cabbage, and the abundance of harmful bacteria Enterobacter decreased significantly. The relative abundance of Enterobacter in group T4 was the highest (8.68%), followed by group CK (5.88%). Non-lactic acid bacteria, including Pantoea, Klebsiella, Pediococcus, Enterobacter and other spoilage bacteria, were not inhibited, preventing the growth of lactic acid bacteria, resulting in a similar downward trend in the quality and nutrition of the two silages. It is speculated that the low nutritional quality of the raw materials themselves and the lack of synergistic effect of the added microbial agents may lead to insufficient fermentation substrates. The insufficient WSC content reduces the value-added of lactic acid bacteria. Although the composite microbial preparation for feeding contains microbial agents such as lactic acid Pediococcus, Saccharomyces cerevisiae, cellulase and xylanase, it is widely used in various types of straw raw materials, but it is not a universal silage composite microbial agent. It may be related to the differences in the physical and nutritional characteristics of the raw materials themselves. The composite microorganisms do not play a synergistic role, and the silage effect is also different. The specific reasons need further study.

[0094] The above conclusions show that natural silage of two-cut mulberry branches is not easy to succeed, the relative abundance of lactic acid bacteria is extremely low, and spoilage bacteria are not inhibited. Adding molasses or molasses + cellulase on the basis of adding Aspergillus oryzae + Lactobacillus plantarum can improve the nutritional value and fermentation quality of two-cut mulberry branch silage to varying degrees, and can increase the relative abundance of Firmicutes and Lactobacillus, while reducing the relative abundance of Proteobacteria, Klebsiella, Pantoea, and Enterobacter, effectively inhibiting butyrate fermentation, improving the silage fermentation quality, and making the two-cut mulberry branches more palatable. Adding composite microbial preparations for feeding silage has the richest number of species, high flora diversity, and spoilage bacteria are not inhibited. Taking all factors into consideration, the best effect is to add Aspergillus oryzae + Lactobacillus plantarum + molasses + cellulase to two-cut mulberry branches. It can be used in two-cut mulberry branch silage.

[0095] Embodiment 3:

[0096] The silage feed is used to raise Fufeng Ma chicken, as follows:

[0097] 80-day-old Fufeng hemp chickens were selected for breeding, and the breeding was mainly free-range. The Fufeng chickens were fed according to conventional management methods. During feeding, the basic feed consisted of the following components by mass percentage: 65% corn, 16% soybean meal, 7% second powder, 5% corn DDGS, 4% puffed soybean, 1% soybean oil, and 2% premix; the control group was fed with a basic diet, and then 0%, 3%, 6% and 9% of the two-cut mulberry branch silage prepared by the T3 group or T4 group of Example 2 were added to the basic feed for feeding, and each group had 3 parallels, and each parallel was 50. After feeding for 80 days, the experiment was terminated, and the feeding experiment was terminated. Ten experimental chickens with similar body weight were extracted from each group, and the slaughter test was carried out after fasting weighing, and the slaughter rate and abdominal fat rate were determined. The survival of the chickens was recorded during the feeding process, and the survival rate and the feed-to-weight ratio were calculated according to the feed loss and weight gain after the feeding was completed, wherein the feed-to-weight ratio was the ratio of the average daily feed intake to the average daily weight gain.

[0098] Table 10 Effects of different feeds on chicken slaughter performance, survival rate and feed-to-weight ratio

[0099]

[0100] Note: 1. Data in the same column with the same lowercase letters or no letters on the shoulders indicate no significant difference (P>0.05), while data with different lowercase letters on the shoulders indicate significant difference (P<0.05).

[0101] As shown in Table 10, in terms of slaughter rate, the slaughter rates of the two experimental groups (6%, 9%) of T3 double-cut mulberry branch feed were significantly higher than those of the other experimental groups and the CK group (P<0.05).

[0102] From the perspective of abdominal fat rate, the abdominal fat rates of the two experimental groups fed with (6% and 9%) T3 twice-cut mulberry branches feed were significantly lower than those of the other experimental groups and the CK group (P<0.05). Compared with the two groups, the abdominal fat rate of the (9%) T3 twice-cut mulberry branches feed experimental group was significantly lower than that of the (6%) T3 twice-cut mulberry branches feed experimental group (P<0.05). However, the abdominal fat rate was too low and the taste of the chicken was not good. Overall, the feeding effect of the (6%) T3 twice-cut mulberry branches feed experimental group was more in line with commercial value.

[0103] In terms of survival rate, the survival rates of the three experimental groups fed with T3 twice-cut mulberry branches (3%, 6%, 9%) were significantly higher than those of the other experimental groups and the CK group (P<0.05).

[0104] In terms of feed-to-weight ratio, the feed-to-weight ratio of the (6%) T3 two-cut mulberry branch feed group was significantly lower than that of the other experimental groups and the CK group (P<0.05).

[0105] Based on the above experimental results, we can see that the feed prepared by fermenting two cuts of mulberry branches with the self-selected strains Lactobacillus plantarum XMS03 and Aspergillus oryzae XMS01 has a significantly better breeding effect than the feed (T4) prepared by adding commercially available strains, and its meat production performance is improved, the fat content is significantly reduced, and the survival rate is significantly improved. Combined with the nutritional component analysis in Table 5, we believe that the fermentation of two cuts of mulberry branches with the self-selected strains Lactobacillus plantarum XMS03 and Aspergillus oryzae XMS01 has precipitated more active ingredients such as mulberry branch flavonoids and polysaccharides, which can significantly improve the immunity of chickens and improve the survival rate of chickens. In addition, the feed-to-weight ratio of the T3 feed group is less than that of the control group. Combined with the nutritional component analysis in Table 5, we believe that the tannin content in the mulberry branch feed is significantly reduced after the mulberry branches are fermented with Lactobacillus plantarum XMS03 and Aspergillus oryzae XMS01, the palatability is significantly improved, the nutritional value is improved, the chickens are more likely to eat, and the production and breeding performance of the chickens is improved. However, the addition of T3 fermented mulberry branch feed is not necessarily better. Considering the taste, commercial value and other factors, we believe that 6% by mass is the most suitable addition amount.

[0106] In addition, the addition of two-cut mulberry branches to feed can significantly reduce the visceral fat content of Fufeng Ma chicken, meeting consumers' demand for chicken fat content. For details, please refer to Figure 3 As shown, Figure 3 Figure B shows the chicken viscera of the experimental group with two-cut mulberry branches added (6%), and Figure A shows the chicken viscera of the control group (CK). It can be seen that the visceral fat of the chickens in the control group is significantly more and thicker than that in the experimental group.

[0107] In summary, according to the characteristic that natural silage of two-cut mulberry branches is not easy to succeed, this application optimizes the silage conditions, first screens strains, and selects strains suitable for mulberry branch silage. Then, by further optimizing the use of adjuvants: molasses and cellulase, the decomposition of mulberry branches in silage is further improved. The nutritional value and fermentation quality of two-cut mulberry branch silage are improved to varying degrees. Through the detection of microbial diversity, it is found that: the relative abundance of Firmicutes and Lactobacillus can be increased, while the relative abundance of Proteobacteria, Klebsiella, Pantoea, and Enterobacter can be reduced, butyric acid fermentation can be effectively inhibited, the quality of silage fermentation can be improved, and the palatability of two-cut mulberry branches is better. After breeding, it can reduce the abdominal fat rate of chickens, make the taste of chicken relatively fresh and tender, and play a positive role in improving the quality of chicken.

[0108] The above description is a detailed description of the preferred feasible embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modified changes completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. A composite bacterial agent for silage, characterized in that: The composite bacterial agent comprises Aspergillus oryzae XMS01 and Lactobacillus plantarum XMS03; The Aspergillus oryzae XMS01 has a deposit number of CCTCC: M2018425; The Lactobacillus plantarum XMS03 has a deposit number of CCTCC: M2019003.

2. The composite bacterial agent for silage according to claim 1, characterized in that: The composite bacterial agent is used for fermenting twice-cut mulberry branches.

3. Silage comprising the composite bacterial agent according to any one of claims 1 or 2.

4. The silage according to claim 3, characterized in that: The silage also contains molasses and cellulase.

5. A method for preparing silage according to any one of claims 3 or 4, characterized in that: The method comprises: crushing two cut mulberry branches, adding probiotics and additives, mixing and silaging; The probiotics are composed of Aspergillus oryzae and Lactobacillus plantarum; the Aspergillus oryzae is Aspergillus oryzae XMS01, with a preservation number of CCTCC: M2018425; the Lactobacillus plantarum is Lactobacillus plantarum XMS03, with a preservation number of CCTCC: M2019003; The additives include molasses and / or cellulase.

6. The method according to claim 5, characterized in that The added amount of the molasses is 4% of the substrate mass and the enzymatic activity of the cellulase is greater than or equal to 5000 U / g.

7. The method according to claim 5, characterized in that The viable count of Aspergillus oryzae is greater than or equal to 6.3×10 7 CFU / g; the viable count of the plant lactic acid bacteria is greater than or equal to 3.1×10 7 CFU / g.

8. The method according to claim 5, characterized in that The silage is stored for 45 days.

9. Use of the silage according to any one of claims 3 or 4 or the silage prepared by the method according to any one of claims 5 to 8 in raising chickens, wherein the use is: adding the silage in an amount of 6% by mass to the base feed to feed the chickens.

10. Use of the silage according to any one of claims 3 or 4 or the silage prepared by the method according to any one of claims 5 to 8 in reducing the abdominal fat content of chickens.

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