A composite fermentation agent and its preparation and application in ruminant feed

Abstract

The present invention provides a composite fermentation agent, a preparation method thereof and an application thereof in feed for ruminants. The composite fermentation agent comprises Lactobacillus plantarum, Candida utilis and Aspergillus niger. The number of live bacteria of Lactobacillus plantarum per gram or per milliliter of the composite fermentation agent is 3.5×10 9 ‑6×10 9 CFU, the number of viable Candida utilis is 1×10 9 ‑3×10 9 CFU, the number of viable Aspergillus niger is 1×10 9 ‑3×10 9 CFU. The compound fermentation agent does not use other additives and feed additives, the formula is clean, the compound fermentation agent produces acid quickly during the feed fermentation process, the fermentation cycle is short, and the feed fermentation effect is good.

Description

Technical Field

[0001] The invention belongs to the technical field of fermented feed, and in particular relates to a composite fermentation agent and a preparation method thereof and an application thereof in feed for ruminants. Background Art

[0002] The part of fruit left after the juice is squeezed or the pulp is taken is called pomace. my country has rich pomace resources, with an annual output of up to 10 million tons, of which citrus pomace reaches 5 million tons. Tea residue refers to the tea waste left after the functional active substances such as tea polyphenols and caffeine are extracted or tea beverages are processed. In my country, the annual production of tea residue is as high as millions of tons. In addition, as the world's largest peanut producer, my country's annual output of its by-product peanut shell residue is as high as 5 million tons.

[0003] In fact, citrus residue and tea residue, as primary processed solid waste, still have high nutritional value. For example, citrus residue is rich in vitamins, and tea residue contains high content of tea polyphenols and caffeine. However, except for some citrus residue and tea residue used as raw materials for functional substance extraction, composting and feed processing, most of the citrus residue and tea residue are forced to be discarded due to high moisture content and easy to be contaminated by microorganisms, resulting in corruption and deterioration. Peanut shell residue is currently mainly used as solid fuel for power plants. It can be seen that the utilization rate of citrus residue, tea residue and peanut shell residue in my country is not high, which has caused serious waste of resources.

[0004] At present, there are still many problems in directly feeding animals with citrus residue, tea residue and peanut shell residue as feed raw materials. First, citrus residue and tea residue contain bitter flavor substances such as naringin, limonin and tea polyphenols, which make the raw materials less palatable and thus affect the animal's feeding rate; secondly, the moisture content of citrus residue and tea residue is high, and the drying energy consumption is high, the cost is high, and the output is large, so it is inconvenient to store and transport in time; finally, the anti-nutritional polysaccharide components such as pectin in the raw materials will also cause problems in animal digestion, absorption and utilization, which directly affects the feeding value of the pomace. Summary of the invention

[0005] The present invention aims to solve the problems that fruit residues, tea residues and peanut shell residues have poor palatability after being used to prepare fermented feed, are inconvenient to store and transport, and are easy to cause diarrhea in ruminants. A composite fermentation agent and its preparation and application in ruminant feed are proposed.

[0006] The above raw materials are fermented with a composite bacterial agent. After fermentation, the storage time of the fermented raw materials is extended, the conversion of plant protein into single-cell biological protein is more conducive to absorption, and anti-nutritional and poorly palatable substances such as phytic acid and tannin are degraded into small molecules with good palatability, thereby improving the nutritional value and utilization rate of the fermented raw materials, the palatability and quality of the feed, and achieving the effect of increasing the utilization rate of feed and promoting the digestion and absorption of nutrients by animals, thereby reducing the feed-to-weight ratio.

[0007] The technical solution of the present invention is:

[0008] In a first aspect, the present invention provides a composite fermentation agent, characterized in that it comprises Lactobacillus plantarum, Candida utilis and Aspergillus niger, and the number of live bacteria of Lactobacillus plantarum is 3.5×10 9 -6×10 9 CFU, the number of viable Candida utilis is 1×10 9 -3×10 9 CFU, the number of viable Aspergillus niger is 1×10 9 -3×10 9 CFU.

[0009] Preferably, the composite fermentation agent is characterized in that the number of viable Lactobacillus plantarum bacteria per gram or per milliliter of the composite fermentation agent is 4×10 9 -5×10 9 CFU, the number of viable Candida utilis bacteria was 1.5×10 9 -2.5×10 9 CFU, the number of viable Aspergillus niger is 1×10 9 -2.5×10 9 CFU.

[0010] Preferably, the composite fermentation agent is characterized in that the plant lactobacillus is a plant lactobacillus (Lactobacillus plantarum) S2 strain, the preservation number of which is CCTCC NO: M 2021340, and is preserved in the China Center for Type Culture Collection (CCTCC);

[0011] Alternatively, preferably, the Candida utilis is Candida utilis XZ-CU01 strain, with a deposit number of CCTCC NO: M 2023299, which is deposited in China Center for Type Culture Collection (CCTCC);

[0012] Alternatively, preferably, the Aspergillus niger is Aspergillus niger XZ-AN01 strain, with a deposit number of CCTCC NO: M 2023298, which is deposited in China Center for Type Culture Collection (CCTCC).

[0013] In a second aspect, the present invention provides a method for preparing the composite starter, wherein the Lactobacillus plantarum, the Candida utilis and the Aspergillus niger are mixed to obtain the composite starter.

[0014] Preferably, the method for preparing the composite starter comprises culturing the Lactobacillus plantarum, the Candida utilis and the Aspergillus niger separately, mixing the obtained bacterial liquids or bacterial powders to obtain the composite starter.

[0015] Preferably, the use of the composite starter or the composite starter obtained by the preparation method in fermented feed is characterized in that the use in fermented feed includes use in fermenting ruminant feed.

[0016] In a third aspect, the present invention provides a feed for ruminants, characterized in that the raw materials of the feed include a mixture of fruit residues, tea residues and peanut shell residues and the composite fermentation agent or a composite fermentation agent prepared by the preparation method of the composite fermentation agent.

[0017] Preferably, the feed for ruminants is characterized in that the pomace contains citrus pomace.

[0018] Preferably, the feed for ruminants is characterized in that, by weight, the feed comprises 60-70 parts of citrus residues, 15-25 parts of tea residues, 5-15 parts of peanut shell residues and a composite fermentation agent.

[0019] Preferably, the ruminant feed is characterized in that the water content of the ruminant feed is 65%-75%.

[0020] In a fourth aspect, the present invention provides a method for preparing the ruminant feed, comprising adding the composite fermentation agent or the composite fermentation agent prepared by the method for preparing the composite fermentation agent to a mixture containing fruit residues, tea residues and peanut shell residues, mixing and fermenting.

[0021] Preferably, in the method for preparing the ruminant feed, the fermentation method is anaerobic fermentation, and preferably, the fermentation temperature is 25-30°C.

[0022] Beneficial effects of the present invention:

[0023] The composite bacterial agent of the present invention has good feed fermentation ability, can increase the protein content in the fermented feed and decompose macromolecular substances such as fiber in the feed into usable small molecular substances, thereby increasing the flavor diversity and nutritional value of the fermented feed. The composite bacterial agent does not use other additives and feed additives, and the formula is clean. The composite fermentation agent produces acid quickly during the feed fermentation process, has a short fermentation cycle, and has a good feed fermentation effect.

[0024] The present invention mixes common solid waste fruit residues, tea residues and peanut shell residues in production to prepare a mixed material. The moisture content of the mixed material is controllable, and the nutritional composition ratio of the material can be adjusted according to the actual feeding differences and nutritional needs of the animals being fed. At the same time, the multi-material mixture is diverse in flavor composition and fragrant in sensory perception, which can solve the problem of poor palatability of the above-mentioned single material. The economic value of the fermented material is upgraded, that is, the high-value utilization of low-value materials is achieved, turning waste into treasure. At the same time, no undesirable substances are generated during the production process, and even if it is not used as feed or expires, it can still be reused as fertilizer, which is an environmentally friendly product.

[0025] The composite bacterial agent and fermented material of the present invention have low cost, simple fermentation process, low fermentation site requirements, and rapid accumulation of lactic acid during the fermentation process, thereby extending the shelf life of the material. The prepared fermented feed has good product stability, can increase the feeding rate of beef cattle and sheep during feeding, thereby reducing the feed-to-weight ratio, shortening the breeding cycle and reducing the breeding cost, and can effectively prevent diarrhea in feeding animals.

[0026] Strain deposit information

[0027] The Lactobacillus plantarum S2 strain of the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on April 8, 2021, with a deposit number of CCTCCNO: M 2021340, a deposit address: Wuhan University, Wuhan, China, postal code: 430072, telephone: (027)-68754052; and it is disclosed in a patent application with patent publication number CN113755360A.

[0028] The Candida utilis XZ-CU01 strain of the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on March 13, 2023, with a deposit number of CCTCC NO: M 2023299. The deposit address is: Wuhan University, Wuhan, China, postal code: 430072, telephone: (027)-68754052.

[0029] The Aspergillus niger XZ-AN01 strain of the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on March 13, 2023, with a deposit number of CCTCC NO: M2023298, and a deposit address of: Wuhan University, Wuhan, China, postal code: 430072, and telephone: (027)-68754052. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The kinetic curves of lactic acid production in the fermentation materials in the examples and comparative examples are shown. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings, but the present invention is not limited to the following technical solution.

[0032] Sources of biological materials

[0033] The Lactobacillus plantarum S2 strain was isolated from wild yak dung at an altitude of 5,400 meters in Tibet. The strain and its isolation and identification data are recorded in the prior art CN113755360A. It has good stress resistance, acid production performance, constipation improvement and intestinal repair function, which is beneficial to enhance the sour taste of fermented materials and play a role in regulating the intestinal flora of animals.

[0034] Candida utilis XZ-CU01 strain was collected from fermented feed in Gangjiu Village, Payang Town, Zhongba County, Shigatse City, Tibet. It has high protein production and good feed fermentation ability. The specific isolation and identification methods are as follows:

[0035] The collected feed sample was diluted to an appropriate gradient and then spread on a YPD solid medium, cultured at a constant temperature of 30°C, and a single colony was picked and continuously streaked for purification. The single colony obtained was sampled for molecular biological identification using the 16S rRNA gene and compared with the NCBI database. The strain numbered XZ-CU01 was finally identified as the Candida utilis XZ-CU01 strain, and the gene of the 16S rRNA of the strain was 16S rDNA, as shown in SEQ ID NO.1:

[0036]

[0037] Aspergillus niger XZ-AN01 strain was collected from Gangjiu Village, Payang Town, Zhongba County, Shigatse City, Tibet. The specific isolation and identification methods are as follows:

[0038] The fermented feed collected from the Tibetan Plateau was prepared into a suspension using physiological saline and diluted in gradients. It was then coated in PDA medium by glass bead coating for isolation and culture, and the plate was inverted at 30°C for 96 hours. According to the shape, size, color and surface characteristics of the colonies, the obtained strains were preliminarily identified in morphology in combination with the "Guide to Fermented Feed Production". The morphology of the culture was observed, and white dot-shaped colonies appeared after 24 hours of screening and culture; after continuing to culture for 48 hours, the white dot-shaped colonies further expanded and had a smooth surface. After culture for 72 hours, white hyphae began to appear on the edge, and the carpet-like tomb extended, showing concentric ring patterns: after 96 hours of culture, the front of the colony was black, the hyphae turned black, and the back was partially yellow and partially colorless. Under a microscope, the meristem spore stalk was transparent and spherical, and the molecular spores were spherical, black or colorless in color. Combined with the "Guide to Fermented Feed Production", the Aspergillus niger strains were preliminarily screened out for comparison. Finally, the strain initially identified as Aspergillus niger was subjected to molecular biological identification using the 26S rRNA gene and compared with the NCBI database to obtain an Aspergillus niger strain that met the requirements. The gene of the 26S rRNA was 26S rDNA, as shown in SEQ ID NO.2:

[0039]

[0040] EM bacteria were purchased from Henan Wobao Biotechnology Co., Ltd., and its main components are lactic acid bacteria, molds, yeasts, photosynthetic bacteria, spore-forming Bacillus, actinomycetes, etc.

[0041] Diyi bacteria was purchased from Zhengzhou Mutian Biotechnology Co., Ltd., and its main ingredients are Lactobacillus plantarum, Pediococcus acidilactici and Lactobacillus buchneri.

[0042] Haowangnong brand silage starter was purchased from Luoyang Oukebaik Biotechnology Co., Ltd., and its main ingredients are Lactobacillus plantarum, Pediococcus acidilactici and Lactobacillus buchneri.

[0043] Citrus residue and tea residue were provided by Angel Biotechnology Co., Ltd., and peanut shell residue was provided by Angel Bio-Agriculture Co., Ltd. The moisture content of citrus residue was 70%-80%, the moisture content of tea residue was 70%-80%, and the moisture content of peanut shell residue was 10%-15%.

[0044] In a specific embodiment of the present invention, the preparation method of the composite leavening agent comprises the following steps:

[0045] (1) Activation and expansion of strains: Take a glycerol storage tube of Lactobacillus plantarum S2 strain, inoculate it into MRS liquid culture medium, and incubate it at 37°C for 10-12 hours for activation. Take 1-2% of the S2 activated bacterial solution and inoculate it into MRS liquid culture medium under the same conditions for expansion, so that the final live bacterial concentration of Lactobacillus plantarum S2 reaches 10 10 CFU / mL, reserve;

[0046] Take the glycerol preservation tube of Candida utilis strain XZ-CU01, inoculate it into YPD liquid medium, and culture it at 30℃, 120r / min for 16-20h for activation. Take 1-2% of the XZ-CU01 activated bacterial solution and inoculate it into YPD liquid medium under the same conditions for expansion culture, so that the final live bacterial concentration of XZ-CU01 reaches 10 10 CFU / mL, reserve;

[0047] Take the Aspergillus niger XZ-AN01 strain glycerol storage tube, inoculate it into PDB liquid culture medium, and culture it at 30℃ for 24-36h for activation. Take 1-2% of the XZ-AN01 activated bacterial solution and inoculate it into PDB liquid culture medium containing glass beads, and culture it at 30℃, 120r / min for 24-36h for expansion, so that the final spore concentration of XZ-AN01 reaches 10 10 CFU / mL, for future use.

[0048] In a specific embodiment of the present invention, the three fermentation bacteria can be further freeze-dried, prepared into bacterial powders respectively, and mixed to obtain a composite starter, and the freeze-dried protective agent includes one or more of skim milk powder, whey protein powder, trehalose, and resistant starch.

[0049] (2) Preparation of composite starter: Microscopically count the culture fluids of the reserved Lactobacillus plantarum S2 strain, Candida utilis XZ-CU01 strain, and Aspergillus niger XZ-AN01 strain, or count the freeze-dried bacteria (spore) powders thereof, and calculate the number of viable bacteria per gram or per milliliter of composite starter, so that the number of viable Lactobacillus plantarum bacteria in the mixed composite starter is 3.5-6×10 9 CFU, the number of viable Candida utilis is 1-3×10 9 CFU, the number of viable Aspergillus niger is 1-3×10 9 CFU.

[0050] The source information of reagents and instruments is shown in Tables 1 and 2 below.

[0051] Table 1 Reagent information

[0052]

[0053] Table 2 Instrument information

[0054] instrument model Manufacturers for sale Spectrophotometer UVmini-1285 Shimadzu Corporation High-speed centrifuge 5430R Eppendorf, Germany Biochemical incubator HSP-160 Haier Clean bench HCB-1300V Haier PCR instrument T960 Likang Biomedical Technology Holding Group Phase contrast microscopy BH-2 Olympus Corporation Vacuum freeze dryer FD-1B-50 BoYiKang

[0055] Example 1

[0056] (I) Preparation of the mixed material: weigh 7 kg of citrus residue, 1.5 kg of tea residue and 1.5 kg of peanut shell residue and mix them evenly into a mixed material for later use, a total of 10 kg of the mixed material.

[0057] (II) Preparation of composite fermentation agent: The bacterial suspension of Lactobacillus plantarum S2 strain and Candida utilis XZ-CU01 strain and the spore liquid of Aspergillus niger XZ-AN01 strain after expansion were heated to 5×10 9 CFU / mL: 2×10 9 CFU / mL: 2×10 9 CFU / mL were mixed to prepare a composite fermentation culture for later use.

[0058] (III) Fermentation of mixed materials: 100 mL of composite fermentation agent was inoculated into the mixed materials, and the mixed materials to be fermented were transferred to an anaerobic fermentation bag (20 threads) with a one-way exhaust valve, and the bag was sealed and the air in the bag was removed as much as possible. The bag was then placed in a fermentation room at 30°C for static fermentation. During the fermentation process, the bloating of the bag was checked regularly, and exhaust was performed manually if necessary. During the fermentation process, the fermentation status of the materials was monitored in real time, and exhaust was performed manually if necessary. The fermentation cycle was 10 days.

[0059] (IV) Fermentation quality monitoring: During the fermentation process, the lactic acid production was tested every 1 day. When there was no significant change in the lactic acid content for 3 consecutive days, the fermentation was considered to be completed. After the fermentation was completed, the moisture content, crude ash, crude protein, true protein, neutral detergent fiber, pH value, bitter taste substance content of the fermented feed were tested and sensory index analysis was performed. The sensory indexes of the fermented feed are shown in Table 3 and the test results are shown in Table 3. Figure 1 and Table 4.

[0060] The determination of crude protein in feed adopts GB / T 6432-2018 Kjeldahl method, the determination of moisture content in feed adopts GB / T 6435-2014, the determination of crude ash in feed adopts GB / T 6438-2007, the determination of neutral detergent fiber (NDF) in feed adopts GB / T 20806-2022, the determination of pH in feed adopts DB15 / T 1458-2018, the determination of tea polyphenols in feed adopts GB / T 8313-2008, the determination of naringin in feed adopts GB 1886.262-2016, and the detection of limonin in feed adopts the method in "Spectrophotometric method for determination of limonin analogues in orange seeds" Tian Qingguo, Ding Xiaolin, 1999.

[0061] Table 3 Sensory indexes of fermented feed

[0062]

[0063] Example 2

[0064] (I) Preparation of mixed material: weigh 6 kg of citrus residue, 2.5 kg of tea residue and 1.5 kg of peanut shell residue, mix them evenly into a mixed material for later use, totaling 10 kg.

[0065] (II) Preparation of composite fermentation agent: The bacterial suspension of Lactobacillus plantarum S2 strain and Candida utilis XZ-CU01 strain and the spore liquid of Aspergillus niger XZ-AN01 strain after expansion were heated to 3.5×10 9 CFU / mL: 1×10 9 CFU / mL: 1×10 9 CFU / mL were mixed to prepare a composite fermentation agent for use.

[0066] (III) Fermentation of mixed materials: 100 mL of composite fermentation agent was inoculated into the mixed materials, and the mixed materials to be fermented were transferred to an anaerobic fermentation bag (20 threads) with a one-way exhaust valve, and the bag was sealed and the air in the bag was removed as much as possible. The bag was then placed in a fermentation room at 25°C for static fermentation. During the fermentation process, the bloating of the bag was checked regularly, and exhaust was performed manually if necessary. During the fermentation process, the fermentation status of the materials was monitored in real time, and exhaust was performed manually if necessary. The fermentation cycle was 10 days.

[0067] Step 4 remains unchanged, and the relevant results are shown in Figure 1 and Table 4.

[0068] Example 3

[0069] (I) Preparation of mixed material: same as in Example 1.

[0070] (ii) The bacterial suspensions of Lactobacillus plantarum S2 strain and Candida utilis XZ-CU01 strain and the spore liquid of Aspergillus niger XZ-AN01 strain after expansion were heated at 4×10 9 CFU / mL: 2×10 9 CFU / mL: 1×10 9 CFU / mL were mixed to prepare a composite fermentation agent for use.

[0071] (III) Fermentation of mixed materials: 100 mL of composite fermentation agent was inoculated into the mixed materials, and the mixed materials to be fermented were transferred to an anaerobic fermentation bag (20 threads) with a one-way exhaust valve, and the bag was sealed and the air in the bag was removed as much as possible. The bag was then placed in a fermentation room at 25°C for static fermentation. During the fermentation process, the bloating of the bag was checked regularly, and exhaust was performed manually if necessary. During the fermentation process, the fermentation status of the materials was monitored in real time, and exhaust was performed manually if necessary. The fermentation cycle was 10 days.

[0072] Step (IV) remains unchanged, and the relevant results are shown in Figure 1 and Table 4.

[0073] Example 4

[0074] (I) Preparation of mixed material: same as in Example 1.

[0075] (ii) The bacterial suspensions of Lactobacillus plantarum S2 strain and Candida utilis XZ-CU01 strain and the spore liquid of Aspergillus niger XZ-AN01 strain after expansion were heated at 4.5×10 9 CFU / mL: 2×10 9 CFU / mL: 1×10 9 CFU / mL were mixed to prepare a composite fermentation agent for use.

[0076] (III) Fermentation of mixed materials: 100 mL of composite fermentation agent was inoculated into the mixed materials, and the mixed materials to be fermented were transferred to an anaerobic fermentation bag (20 threads) with a one-way exhaust valve, and the bag was sealed and the air in the bag was removed as much as possible. The bag was then placed in a fermentation room at 25°C for static fermentation. During the fermentation process, the bloating of the bag was checked regularly, and exhaust was performed manually if necessary. During the fermentation process, the fermentation status of the materials was monitored in real time, and exhaust was performed manually if necessary. The fermentation cycle was 10 days.

[0077] Step (IV) remains unchanged, and the relevant results are shown in Figure 1 and Table 4.

[0078] Example 5

[0079] (I) Preparation of mixed material: same as in Example 1.

[0080] (ii) The bacterial suspensions of Lactobacillus plantarum S2 strain and Candida utilis XZ-CU01 strain and the spore liquid of Aspergillus niger XZ-AN01 strain after expansion were heated to 6×10 9 CFU / mL: 3×10 9 CFU / mL: 3×10 9 CFU / mL are mixed to prepare a composite fermentation agent for use.

[0081] (III) Fermentation of the mixed material: Same as in Example 1.

[0082] Step (iv) is the same as in Example 1, and the relevant results are shown in Figure 1 and Table 4.

[0083] Example 6

[0084] (I) Preparation of mixed material: same as in Example 1.

[0085] (ii) The bacterial suspensions of the expanded Lactobacillus plantarum S2 strain and the Candida utilis XZ-CU01 strain and the spore liquid of the Aspergillus niger XZ-AN01 strain were heated at 4×10 9 CFU / mL: 1.5×10 9 CFU / mL: 1×10 9 CFU / mL are mixed to prepare a composite fermentation agent for use.

[0086] (III) Fermentation of the mixed material: Same as in Example 1.

[0087] Step (iv) is also Example 1, and the relevant results are shown in Figure 1 and Table 4.

[0088] Example 7

[0089] (I) Preparation of mixed material: same as in Example 1.

[0090] (ii) The cultured Lactobacillus plantarum S2 strain and Candida utilis XZ-CU01 strain and the spore liquid of Aspergillus niger XZ-AN01 strain were heated at 5×10 9 CFU / mL: 2.5×10 9 CFU / mL: 2.5×10 9 The ratio of CFU / mL was determined, and skim milk powder as a freeze-drying protective agent was added respectively, and freeze-dried to prepare bacterial powders and mix them to obtain a composite starter.

[0091] (III) Fermentation of mixed materials: 100g of composite fermentation agent was inoculated into the mixed materials, and the mixed materials to be fermented were transferred to an anaerobic fermentation bag (20 silk) with a one-way exhaust valve, and the bag was sealed and the air in the bag was removed as much as possible. The bag was then placed in a fermentation room at 25°C for static fermentation. During the fermentation process, the bloating of the bag was checked regularly, and exhaust was performed manually if necessary. During the fermentation process, the fermentation status of the materials was monitored in real time, and exhaust was performed manually if necessary. The fermentation cycle was 10 days.

[0092] Step (iv) is the same as in Example 1, and the relevant results are shown in Figure 1 and Table 4.

[0093] Comparative Example 1

[0094] The mixed material in Example 1 (I) was inoculated with EM bacteria according to the product requirements, and steps (III) and (IV) were performed. The relevant results are shown in Figure 1 and Table 4.

[0095] Comparative Example 2

[0096] The mixed material in Example 1 (I) was inoculated with the silage fermentation agent "Diyi Bacteria" containing probiotics such as lactic acid bacteria, yeast and Bacillus subtilis according to the required proportion of the product, and steps (III) and (IV) were carried out. The relevant results are shown in Figure 1 and Table 4.

[0097] Comparative Example 3

[0098] The mixed material in Example 1 (I) was inoculated with the "Haowangnong" brand silage fermentation agent containing Lactobacillus plantarum, Pediococcus acidilactici and Lactobacillus buchneri in the required proportions of the product, and steps (III) and (IV) were carried out. The relevant results are shown in FIG. Figure 1 and Table 4.

[0099] Comparative Example 4

[0100] In Example 1 (I), the mixture was fermented naturally without inoculating the fermentation agent, that is, the implementation steps (III) and (IV) were directly carried out. The relevant results are shown in Figure 1 and Table 4.

[0101] Comparative Example 5

[0102] The mixed material in Example 1 (I) was not inoculated with a fermentation agent, and the mixed material was placed in a refrigerator at -20°C for sampling at any time. The implementation steps of (III) and (IV) remained unchanged. The relevant results are shown in Figure 1 and Table 4.

[0103] Table 4 Fermented feed indicators

[0104]

[0105]

[0106] As can be seen from Table 4, compared with natural fermentation without adding fermentation agents, the true protein content in the embodiment of inoculating the composite fermentation agent is significantly improved. It can be seen that the composite fermentation agent can effectively increase the true protein content in the mixed material and thus improve the nutritional value. The overall effect is better than the commercial EM bacteria fermentation agent and the commercial "Diyi Bacteria" and "Haowangnong" silage fermentation agents. After the fermentation is completed, the moisture content, crude ash, neutral detergent fiber, crude protein and pH value of the fermented feed all meet the standard limits (DB15 / T 2410-2021), and the contents of bitter flavor substances naringin, limonin and tea polyphenols all decrease with fermentation, and the sensory indicators meet the requirements of Table 3.

[0107] Depend on Figure 1 It can be seen that, compared with the natural fermentation without adding fermentation agent, the lactic acid in the examples inoculated with mixed fermentation agent began to accumulate rapidly on the 4th day. In the 5th to 8th day, the lactic acid production of Examples 1 to 7 reached a peak value, and the final lactic acid content was basically stable at about 19000ug / g, far exceeding 8000ug / g (the minimum bactericidal concentration of common pathogenic microorganisms such as golden wine bacteria, Bacillus cereus or Escherichia coli). This shows that the addition of composite fermentation agent helps the lactic acid production in the mixed material to inhibit the growth of other harmful bacteria, thereby achieving the purpose of extending the shelf life of the material.

[0108] Application Examples

[0109] (I) Experimental site and selection of experimental animals: The experimental site is the cooperative livestock farm of Angel Biotechnology Co., Ltd. 100 Limousin cattle weighing between 150-175kg were randomly selected and divided into 5 groups (experimental group A, experimental group B, experimental group C, experimental group D and experimental group E), 20 heads in each group, male and female are random. 100 Boer sheep weighing between 25-30kg were randomly selected and divided into 5 groups (experimental group F, experimental group G, experimental group H, experimental group I and experimental group J), 20 heads in each group, male and female are random.

[0110] (II) Feeding scheme for beef cattle and sheep: Test group A and test group F were fed with conventional diets for beef cattle and sheep respectively; test group B and test group G were fed with conventional diets for beef cattle and sheep respectively mixed with 10% fermented feed in Example 2; test group C and test group H were fed with conventional diets for beef cattle and sheep respectively mixed with 10% fermented feed in Example 3; test group D and test group I were fed with conventional diets for beef cattle and sheep respectively mixed with 10% fermented feed in Comparative Example 2; test group E and test group J were fed with conventional diets for beef cattle and sheep respectively mixed with 10% fermented feed in Comparative Example 3. The enclosure-type feeding mode was adopted, and conventional diets or conventional diets mixed with 10% fermented feed were fed three times a day in the morning, noon and evening, and the animals were fed freely, ensuring that the amount of residual feed was 3% to 5% per day, and drinking water was free. The feeding experiment period was 60 days.

[0111] (III) Feeding effect monitoring: The total feed intake per head, daily weight gain and feed-to-weight ratio of the experimental beef cattle and sheep within 60 days, as well as the diarrhea rate of each group of beef cattle and sheep were statistically analyzed, and the feeding effect of the fermented feed was determined after comprehensive analysis of the feeding data. The experimental results are shown in Table 5.

[0112] Table 5 Beef cattle and sheep feeding experimental results

[0113]

[0114]

[0115] As can be seen from Table 5, the feeding results of beef cattle and sheep are consistent, that is, compared with feeding only conventional daily feed, adding 10% of the fermented feed of Example 2 or Example 3 to the conventional daily feed helps to increase the daily feed intake of beef cattle and sheep, thereby reducing the feed-to-meat ratio. At the same time, feeding fermented feed also effectively reduces the diarrhea rate of beef cattle and sheep. It can be seen that fermented feed also has a certain improvement effect on the digestive system and intestinal microecology of animals. Overall, compared with the feeding effect of beef cattle and sheep fermented by commercial silage fermentation agents used in Comparative Examples 2 and 3, the fermented feeds of Examples 2 and 3 also show certain advantages.

[0116] In summary, inoculating the composite fermentation agent provided by the present invention into the mixed material can make the lactic acid in the material accumulate quickly to ensure the storage and transportability of the material, the true protein content in the fermented feed is improved to ensure the nutritional value of the feed, the bitter taste substance content is reduced and the sensory indicators meet the requirements, the flavor and palatability of the fermented feed are ensured, and other indicators meet the standard limits to ensure the quality of the fermented feed products. Overall, the composite fermentation agent is highly feasible in the use of citrus residue, tea residue and peanut shell residue mixed materials to ferment ruminant feed. Feeding beef cattle and sheep can increase the feeding rate of beef cattle and sheep and reduce the feed-to-weight ratio, shorten the breeding cycle and reduce the breeding cost, and can effectively prevent diarrhea in feeding animals.

Claims

1. A composite leavening agent, characterized in that: Including Lactobacillus plantarum, Candida utilis and Aspergillus niger, the number of live bacteria per gram or per milliliter of the composite fermentation agent is 3.5×10 9 -6×10 9 CFU, the number of viable Candida utilis is 1×10 9 -3×10 9 CFU, the number of viable Aspergillus niger is 1×10 9 -3×10 9 CFU; The plant lactobacillus is plant lactobacillus ( Lactobacillus plantarum )S2 strain, with the deposit number of CCTCCNO: M 2021340, deposited in China Center for Type Culture Collection (CCTCC); The Candida utilis is Candida utilis ( Candida utilis ) XZ-CU01 strain, with the deposit number of CCTCCNO: M 2023299, deposited in China Center for Type Culture Collection (CCTCC); The Aspergillus niger is Aspergillus niger ( Aspergillus niger )XZ-AN01 strain, with the deposit number of CCTCC NO: M2023298, is deposited in China Center for Type Culture Collection (CCTCC).

2. The composite leavening agent according to claim 1, characterized in that Calculated by the number of live bacteria per gram or milliliter of the composite fermentation agent, the number of live Lactobacillus plantarum is 4×10 9 -5×10 9 CFU, the number of viable Candida utilis bacteria was 1.5×10 9 -2.5×10 9 CFU, the number of viable Aspergillus niger is 1×10 9 -2.5×10 9 CFU.

3. A method for preparing the composite leavening agent according to claim 1 or 2, wherein: The plant lactobacillus, the utilis Candida utilis and the Aspergillus niger are mixed to obtain a composite starter.

4. The method for preparing the composite leavening agent according to claim 3, wherein: The preparation method comprises culturing the plant lactobacillus, the utilis Candida utilis and the Aspergillus niger respectively, mixing the obtained bacterial liquid or bacterial powder to obtain the composite starter.

5. Use of the composite starter according to claim 1 or 2 or the composite starter obtained by the preparation method according to claim 3 or 4 in fermented feed, characterized in that: The application in fermented feed includes application in fermented feed for ruminants.

6. A feed for ruminants, characterized in that: The raw materials of the feed include a mixture of fruit residues, tea residues and peanut shell residues and a composite leavening agent prepared by the composite leavening agent according to claim 1 or 2 or the composite leavening agent preparation method according to claim 3 or 4.

7. The feed for ruminants according to claim 6, characterized in that: The pomace contains citrus pulp.

8. The feed for ruminants according to claim 7, characterized in that: The feed comprises 60-70 parts of citrus residue, 15-25 parts of tea residue, 5-15 parts of peanut shell residue and a composite fermentation agent by weight.

9. The ruminant feed according to any one of claims 6 to 8, characterized in that: The water content of the ruminant feed is 65wt%-75wt%.

10. A method for preparing a feed for ruminants according to any one of claims 6 to 9, comprising adding the composite fermentation agent according to claim 1 or 2 or the composite fermentation agent prepared by the method for preparing the composite fermentation agent according to claim 3 or 4 to a mixture containing fruit residues, tea residues and peanut shell residues, mixing and fermenting.

11. The method for preparing ruminant feed according to claim 10, wherein the fermentation method is anaerobic fermentation.

12. The method for preparing ruminant feed according to claim 10, wherein the fermentation temperature is 25-30°C.

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