Brewer's grain type fermented total mixed ration as well as preparation method and application thereof

Abstract

The invention relates to the technical field of agriculture, in particular to a brewer's grain type fermented total mixed ration and a preparation method and application thereof, and the preparation method specifically comprises the following steps: weighing 10-20 parts by weight of brewer's grain, 26.35-30 parts by weight of whole corn silage, 14.2-19.05 parts by weight of straw, 4.9-10 parts by weight of soybean meal, 28.9-32.6 parts by weight of corn flour, 1.2 parts by weight of premix, 0.2 part by weight of salt and 0.6 part by weight of stone powder; mixing the raw materials in parts by weight to obtain a mixed feed; uniformly spraying a microbial additive into the mixed feed to obtain the brewer's grain type fermented total mixed ration, the microbial additive is lactobacillus plantarum or high-temperature bacterial powder JF prepared from the lactobacillus plantarum. The quality of the fermented total mixed ration prepared by the preparation method provided by the invention is remarkably improved, the digestion and absorption of beef cattle on the brewer's grain type fermented total mixed ration can be improved, and the fermented total mixed ration is suitable for popularization and application in feed processing.

Description

Technical Field

[0001] This invention relates to the field of agricultural technology, specifically to a beer lees-based fermented total mixed ration (TMR) and its preparation method and application. Background Technology

[0002] Brewer's grains are the most abundant byproduct of the brewing industry. They contain a variety of nutrients, including protein, vitamins, minerals, and amino acids, with a protein content as high as 30% and containing up to 17 different amino acids. Furthermore, brewer's grains contain arabinoxylan and β-glucan, which can promote the activity of probiotics, regulate the balance of gut microbiota, and increase feed palatability. However, brewer's grains have a high water content (up to 80%) and are also high in crude fiber and anti-nutritional factors. Direct feeding can easily cause animal health problems, and storage or transportation is prone to contamination and costly, limiting their usability in livestock farming. Traditional disposal methods mainly involve landfilling or selling at low prices locally, resulting in resource waste and environmental pollution.

[0003] Fermented Total Mixed Ration (FTMR) using brewer's grains is an emerging technology that rationally utilizes agricultural byproducts to formulate complete diets that meet the nutritional needs of ruminants at different growth and development stages. However, due to the large number of miscellaneous bacteria attached to brewer's grains, probiotics are unable to dominate during the natural fermentation of FTMR, resulting in significant nutrient loss.

[0004] Therefore, there is an urgent need to provide a method for preparing a total mixed diet based on brewer's grains fermentation to improve its feed value. Summary of the Invention

[0005] To address the above problems, this invention provides a brewer's grains-based fermented total mixed diet (FTMR) and its preparation method and application. It effectively increases the content of lactic acid, acetic acid, soluble carbohydrates, and crude fat in the fermented FTMR, while lowering its pH value. The addition of exogenous thermophilic microbial powder (JF) increases the lactic acid content of the brewer's grains-based FTMR, while decreasing the pH value, ammonia nitrogen, and acetic acid content. In summary, the addition of fresh brewer's grains and thermophilic microbial powder (JF) improves the quality of FTMR and inhibits the growth of harmful microorganisms.

[0006] This invention is achieved through the following technical solution: A method for preparing a brewer's grains-based fermented total mixed ration (TMR) specifically includes the following steps: Weigh out 10-20 parts by weight of brewer's grains, 26.35-30 parts of whole-plant corn silage, 14.2-19.05 parts of rice straw, 4.9-10 parts of soybean meal, 28.9-32.6 parts of corn flour, 1.2 parts of premix, 0.2 parts of salt, and 0.6 parts of limestone powder.

[0007] The raw materials in the specified weight proportions are mixed, and the moisture content is adjusted to 45%~55% by weight to obtain a mixed feed.

[0008] After adding microbial additives to the mixed feed, it was sealed and stored at 25℃~30℃ for ≥7 days to obtain a brewer's grains-based fermented total mixed diet.

[0009] The microbial additive is *Lactobacillus plantarum* (… Lactobacillus plantarum (or high-temperature bacterial powder JF prepared from Lactobacillus plantarum; each gram of the mixed feed contains 1×10 Lactobacillus plantarum) 6 CFU~5×10 6 CFU; the amount of high-temperature bacterial powder JF added per ton of mixed feed is 2 g to 5 g.

[0010] Preferably, the sealed storage time is 7 days to 30 days.

[0011] Preferably, the viable count of the high-temperature bacterial powder JF is ≥2.0 × 10⁻⁶. 10 CFU / g.

[0012] The brewer's grains fermented total mixed diet prepared by the method described above.

[0013] The application of the brewer's grains-based fermented total mixed diet in ruminant feeding.

[0014] Preferably, the ruminant is a cow.

[0015] Preferably, brewer's grains fermented total mixed diet can improve the rumen fermentation characteristics of ruminants.

[0016] Preferably, the improvement of rumen fermentation characteristics in ruminants refers to increasing one or more of the following: rumen dry matter digestibility, crude protein digestibility, neutral detergent fiber digestibility, and acid detergent fiber digestibility in beef cattle.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention achieves efficient and high-value resource utilization of brewer's grains, solving the problems of storage and feeding. It successfully integrates fresh brewer's grains, which have high moisture content and are easily perishable, into a fermented total mixed ration (TMR) system at a ratio of 10% to 20%. Through fermentation, not only are the animal health risks from direct feeding of brewer's grains completely avoided, but the problems of high storage and transportation costs and easy contamination associated with separate storage are also overcome. This provides a reliable pathway for the large-scale and safe application of by-products from bulk agricultural product processing as feed.

[0018] (2) Significantly improved the fermentation and nutritional quality of total mixed rations. Experiments showed that diets with 20% brewer's grains added and treated by the method of this invention could form a stable acidic environment (pH≤4.5) after fermentation, with a significant increase in lactic acid content (≥3.5% DM). This environment effectively inhibited the activity of harmful microorganisms, as evidenced by controlled ammonia nitrogen content (≤6.5% TN) and reduced protein degradation losses. At the same time, the crude fat content of the fermented diet was significantly increased, providing higher energy supply for ruminants.

[0019] (3) Precise control of the fermentation process was achieved through specific microbial additives, ensuring product stability. Addressing the industry pain points of numerous contaminating bacteria in brewer's lees and unstable quality during natural fermentation, this invention preferentially uses *Lactobacillus plantarum* or its prepared high-temperature bacterial powder JF as an inoculum. Experiments have shown that this additive can rapidly establish dominant bacterial communities (such as *Lactiplantibacillus*), significantly optimize bacterial community structure, and directionally promote lactic acid fermentation, thereby ensuring successful fermentation and uniform quality across different batches.

[0020] (4) Effectively improves the utilization efficiency of the diet in ruminants. In vitro rumen fermentation experiments confirmed that the brewer's grains-based fermented total mixed diet prepared in this invention, especially the group treated with high-temperature microbial powder JF, can significantly improve in vitro dry matter digestibility (IVDMD) and crude protein digestibility (IVCPD), and increase the yield of total volatile fatty acids (TVFAs). This indicates that the diet can be more fully decomposed and utilized by rumen microorganisms, which helps to improve the production performance of animals.

[0021] In summary, this invention successfully transforms brewer's grains into a valuable resource, producing brewer's grains-based FTMR of excellent quality. Furthermore, the use of microbial technology ensures the stability and efficiency of the fermentation process, ultimately achieving comprehensive benefits such as improving rumen digestion efficiency, reducing feeding costs, and promoting the sustainable development of animal husbandry. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a diagram showing the relative abundance of bacterial communities in FTMR under different brewer's grain ratios regulated by the microbial additives of this invention. Figure 1 In the diagram, A represents the relative abundance at the genus level; B represents the relative abundance at the species level.

[0024] Figure 2Correlation analysis of FTMR-level bacteria and fermentation quality in this invention; Note: Red indicates positive correlation, blue indicates negative correlation; * P <0.05;** P <0.01; ***, P <0.001.

[0025] Figure 3 Correlation analysis of FTMR-level bacteria and fermentation quality in this invention; Note: Red indicates positive correlation, blue indicates negative correlation; * P <0.05;** P <0.01; ***, P <0.001.

[0026] Figure 4 The cumulative gas production curve of FTMR in vitro fermentation with the microbial additive of the present invention after 48 h; Figure 4 In the figure, A represents the in vitro gas production results with different proportions of brewer's grains added; B represents the in vitro gas production results with different microorganisms added.

[0027] Figure 5 The pH value and ammonia nitrogen content of rumen fluid under the treatment of the microbial additives of this invention; Figure 5 In the figure, A is the result of FTMR rumen fluid pH value under additive treatment; B is the result of FTMR ammonia nitrogen content under additive treatment.

[0028] Figure 6 Correlation analysis of FTMR quality and rumen in vitro fermentation characteristics of this invention; Note: * P <0.05;** P <0.01; Note: * P <0.05;** P <0.01. Detailed Implementation

[0029] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0030] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0031] The inventive concept of this invention is as follows: Brewer's grains, a major byproduct of the beer industry, are produced in large quantities annually and are rich in nutrients such as protein and fiber. However, due to their high moisture content (up to 80%) and the presence of numerous attached microorganisms, they are extremely prone to spoilage. Direct feeding poses health risks to animals, and storage and transportation costs are high. Traditional processing methods not only result in severe resource waste but also cause environmental pollution. Although preparing them into fermented total mixed rations (FTMRs) is an ideal way to utilize resources, the difficulty in controlling the attached microorganisms during natural fermentation often leads to a lack of dominance by beneficial bacteria, resulting in unstable fermentation quality and significant nutrient loss, thus limiting their application in high-quality ruminant feeds.

[0032] Based on this, the present invention proposes a systematic solution: First, by scientifically combining brewer's grains with whole-plant corn silage, rice straw, soybean meal, corn flour, and other raw materials, a nutritionally balanced and suitable substrate for fermentation is constructed. Second, and most importantly, a specific functional microbial additive—Lactobacillus plantarum or its high-temperature inoculum powder JF—is introduced for targeted inoculation of the mixture. This is not simply adding microbial strains, but rather, through the introduction of exogenous dominant microbial agents, the microbial community succession is dominated in the early stages of fermentation, effectively inhibiting the growth of miscellaneous bacteria in the raw materials, thereby transforming uncontrollable natural fermentation into a controllable, targeted lactic acid fermentation process. Finally, by controlling appropriate moisture, temperature, and sealed fermentation time, a high-quality brewer's grains-based fermented total mixed ration (TMR) with low pH, high lactic acid content, good nutrient preservation, and high digestibility is obtained.

[0033] The beneficial effects of the present invention will be illustrated below through specific embodiments.

[0034] FTMR raw materials: The brewer's grains, whole-plant corn silage, rice straw, soybean meal, corn flour, premix, salt, and limestone powder used in this invention were all purchased from Hongya Zhiyuan Livestock Breeding Co., Ltd., Hongya County, Sichuan Province. The premix provided the following nutrients: Vitamin A: 50,000 IU; Vitamin E: 18,000 IU; Vitamin D3: 5,000 IU; Cu: 100 mg; Fe: 1200 mg; Mn: 800 mg; I: 0.5 mg; Zn: 30 mg; Se: 0.15 mg; Co: 0.1 mg.

[0035] The microorganisms used in this invention are as follows: Pediococcus acidilactici ( Pediococcus acidilactici ), designated PA 068; Bacillus subtilis ( Bacillus subtilis ), denoted as B; Lactobacillus plantarum ( Lactobacillus plantarum ), denoted as LP.

[0036] Among them, Pediococcus lactis ( Pediococcus acidilacticiGenBank: PP972223) PA 068 and Bacillus subtilis ( Bacillus subtilis B was donated by Dr. Wu Qifeng of the Forage Cultivation and Processing Team of the College of Grassland Science and Technology, Sichuan Agricultural University (contact: 18795333406); Lactobacillus plantarum ( Lactobacillus plantarum LP was purchased from Sichuan Gaofuji Biotechnology Co., Ltd.

[0037] The high-temperature bacterial powder JF used in this invention is composed of Lactobacillus plantarum ( Lactobacillus plantarum LP was prepared.

[0038] The high-temperature bacterial powder JF is disclosed in the literature “[1]Tahir M. Effects of planting density and lactic acid bacteria inoculation on oat silage quality and microbial community [D]. Sichuan Agricultural University, 2022.DOI:10.27345 / d.cnki.gsnyu.2022.001067. and [2]Li Xiaoling, Guan Hao, Shuai Yang, et al. Effects of single and compound lactic acid bacteria additives on the silage quality of flat-spike oxtail grass [J]. Journal of Grassland Science, 2019, 28(06):119-127.”

[0039] The standardized preparation process for high-temperature bacterial powder JF is as follows: Strain activation: First, the original bacterial culture is pretreated to activate and restore its physiological activity.

[0040] Culture medium preparation: Prepare a suitable culture medium to provide a nutritional basis for subsequent fermentation.

[0041] Inoculation and fermentation: The activated strain is inoculated into the culture medium and fermented under controlled conditions.

[0042] Centrifugation collection: After fermentation, the bacterial cells are separated and collected by centrifugation.

[0043] Protectant addition and freeze-drying: A special protectant is added to the obtained mycelial sludge, followed by vacuum freeze-drying.

[0044] Grinding and preparation of semi-finished products: The freeze-dried material is ground into fine powder to obtain the semi-finished bacterial powder.

[0045] Repackaging: The bacterial powder is accurately repackaged according to the predetermined specifications.

[0046] Packaging: Seal and externally package the repackaged products.

[0047] Quality inspection: Strict quality inspection is carried out on the packaged finished products.

[0048] Warehousing and Storage: Products that pass inspection are placed in the warehouse and stored under specified conditions. The viable count of this high-temperature bacterial powder JF is ≥2.0 × 10⁻⁶. 10 CFU / g.

[0049] Example 1: A method for preparing a beer lees-based fermented total mixed diet (BW10+7d+LP) Specifically, the steps include the following: Weigh out 10 parts by weight of brewer's grains, 26.35 parts of whole-plant corn silage, 19.05 parts of rice straw, 10 parts of soybean meal, 32.6 parts of corn flour, 1.2 parts of premix, 0.2 parts of salt, and 0.6 parts of limestone powder.

[0050] The raw materials in the specified weight proportions are mixed, and the moisture content is adjusted to 45% FM to obtain a mixed feed.

[0051] After evenly spraying the microbial additive into the mixed feed, compact it and fill it into a sealed polyethylene tank. Then, put the sealed polyethylene tank into a polyethylene bag, vacuum it, and seal it again.

[0052] The total mixed diet (FTMR) was obtained by sealing and storing at 25℃ for 7 days. The microbial additive was *Lactobacillus plantarum* LP, and the dosage of *Lactobacillus plantarum* LP was 1×10⁻⁶. 6 CFU / g mixed feed.

[0053] Example 2: A method for preparing a beer lees-based fermented total mixed diet (BW10+7 d+JF) High-temperature bacterial powder JF was used as a microbial additive, with an addition amount of 2 g / t of mixed feed. The remaining steps were exactly the same as in Example 1.

[0054] Comparative Example 1: A method for preparing a brewer's grains-based fermented total mixed diet (BW10+7 d+PA 068) Pediococcus lactis PA 068 was used as a microbial additive, and the remaining steps were exactly the same as in Example 1.

[0055] Comparative Example 2: A method for preparing a brewer's grains-based fermented total mixed diet (BW10+7 d+B). Bacillus subtilis B was used as a microbial additive, and the remaining steps were exactly the same as in Example 1.

[0056] Comparative Example 3: A method for preparing a brewer's grains-based fermented total mixed diet (BW10+7 d+0) No microbial additives were added, and the remaining steps were exactly the same as in Example 1.

[0057] Example 3: A method for preparing a brewer's grains-based fermented total mixed diet (BW10+30 d+LP) Specifically, the steps include the following: Weigh out 10 parts by weight of brewer's grains, 26.35 parts of whole-plant corn silage, 19.05 parts of rice straw, 10 parts of soybean meal, 32.6 parts of corn flour, 1.2 parts of premix, 0.2 parts of salt, and 0.6 parts of limestone powder.

[0058] The raw materials in the specified weight proportions are mixed, and the moisture content is adjusted to 55% FM to obtain a mixed feed.

[0059] After evenly spraying the microbial additive into the mixed feed, compact it and fill it into a sealed polyethylene tank. Then, put the sealed polyethylene tank into a polyethylene bag, vacuum it, and seal it again.

[0060] The total mixed diet (TMR) was obtained by sealing and storing at 30℃ for 30 days, and denoted as FTMR. The microbial additive was *Lactobacillus plantarum* LP, and the dosage of *Lactobacillus plantarum* LP was 5 × 10⁻⁶. 6 CFU / g mixed feed.

[0061] Example 4: A method for preparing a brewer's grains-based fermented total mixed ration (BW10+30 d+JF). High-temperature bacterial powder JF was used as a microbial additive, with an addition amount of 5 g / t of mixed feed. The remaining steps were exactly the same as in Example 3.

[0062] Comparative Example 4: A method for preparing a brewer's grains-based fermented total mixed diet (BW10+30 d+PA 068) Pediococcus lactis PA 068 was used as a microbial additive, and the remaining steps were exactly the same as in Example 3.

[0063] Comparative Example 5: A method for preparing a brewer's grains-based fermented total mixed diet (BW10+30 d+B). Bacillus subtilis B was used as a microbial additive, and the remaining steps were exactly the same as in Example 3.

[0064] Comparative Example 6: A method for preparing a brewer's grains-based fermented total mixed diet (BW10+30 d+0) No microbial additives were added, and the remaining steps were exactly the same as in Example 3.

[0065] Example 5: A method for preparing a beer lees-based fermented total mixed diet (BW20+7 d+LP) Specifically, the steps include the following: Weigh out 20 parts by weight of brewer's grains, 30 parts of whole-plant corn silage, 14.2 parts of rice straw, 4.9 parts of soybean meal, 28.9 parts of corn flour, 1.2 parts of premix, 0.2 parts of salt, and 0.6 parts of limestone powder.

[0066] The remaining steps are exactly the same as in Example 1.

[0067] Example 6: A method for preparing a brewer's grains-based fermented total mixed diet (BW20+7 d+JF) High-temperature bacterial powder JF was used as a microbial additive, with an addition amount of 2 g / t of mixed feed. The remaining steps were exactly the same as in Example 5.

[0068] Comparative Example 7: A method for preparing a brewer's grains-based fermented total mixed diet (BW20+7 d+PA 068) Pediococcus lactis PA 068 was used as a microbial additive, and the remaining steps were exactly the same as in Example 5.

[0069] Comparative Example 8: A method for preparing a brewer's grains-based fermented total mixed diet (BW20+7 d+B). Bacillus subtilis B was used as a microbial additive, and the remaining steps were exactly the same as in Example 5.

[0070] Comparative Example 9: A method for preparing a brewer's grains-based fermented total mixed diet (BW20+7d+0) No microbial additives were added, and the remaining steps were exactly the same as in Example 5.

[0071] Example 7: A method for preparing a brewer's grains-based fermented total mixed diet (BW20+30 d+LP) Specifically, the steps include the following: Weigh out 20 parts by weight of brewer's grains, 30 parts of whole-plant corn silage, 14.2 parts of rice straw, 4.9 parts of soybean meal, 28.9 parts of corn flour, 1.2 parts of premix, 0.2 parts of salt, and 0.6 parts of limestone powder.

[0072] The remaining steps are exactly the same as in Example 3.

[0073] Example 8: A method for preparing a beer lees-based fermented total mixed ration (BW20+30 d+JF) High-temperature bacterial powder JF was used as a microbial additive, with an addition amount of 5 g / t of mixed feed. The remaining steps were exactly the same as in Example 7.

[0074] Comparative Example 10: A method for preparing a brewer's grains-based fermented total mixed diet (BW20+30 d+PA 068) Pediococcus lactis PA 068 was used as a microbial additive, and the remaining steps were exactly the same as in Example 7.

[0075] Comparative Example 11: A method for preparing a brewer's grains-based fermented total mixed diet (BW20+30 d+B). Using Bacillus subtilis B as a microbial additive, the remaining steps are exactly the same as in Example 7.

[0076] Comparative Example 12: A method for preparing a brewer's grains-based fermented total mixed diet (BW20+30 d+0) No microbial additives were added, and the remaining steps were exactly the same as in Example 7.

[0077] Comparative Example 13: A method for preparing a brewer's grains-based fermented total mixed diet (BW0+7 d+LP). Weigh out the following ingredients by weight: 0 parts brewer's grains, 25.24 parts whole-plant corn silage, 22.77 parts rice straw, 14.94 parts soybean meal, 35.05 parts corn flour, 1.2 parts premix, 0.2 parts salt, and 0.6 parts limestone powder.

[0078] The remaining steps are exactly the same as in Example 1.

[0079] Comparative Example 14: A method for preparing a brewer's grains-based fermented total mixed diet (BW0+7 d+JF) High-temperature bacterial powder JF was used as a microbial additive at a dosage of 2 g / t of mixed feed. The remaining steps were exactly the same as those in Comparative Example 13.

[0080] Comparative Example 15: A method for preparing a brewer's grains-based fermented total mixed diet (BW0+7 d+PA 068) Pediococcus lactis PA 068 was used as a microbial additive, and the remaining steps were exactly the same as in Example 13.

[0081] Comparative Example 16: A method for preparing a brewer's grains-based fermented total mixed diet (BW0+7 d+B). Bacillus subtilis B was used as a microbial additive, and the remaining steps were exactly the same as in Example 13.

[0082] Comparative Example 17: A method for preparing a brewer's grains-based fermented total mixed diet (BW0+7 d+0) No microbial additives were added, and the remaining steps were exactly the same as in Example 13.

[0083] Comparative Example 18: A method for preparing a brewer's grains-based fermented total mixed diet (BW0+30 d+LP). Weigh out the following ingredients by weight: 0 parts brewer's grains, 25.24 parts whole-plant corn silage, 22.77 parts rice straw, 14.94 parts soybean meal, 35.05 parts corn flour, 1.2 parts premix, 0.2 parts salt, and 0.6 parts limestone powder.

[0084] The remaining steps are exactly the same as in Example 3.

[0085] Comparative Example 19: A method for preparing a brewer's grains-based fermented total mixed diet (BW0+30 d+JF) High-temperature bacterial powder JF was used as a microbial additive at a dosage of 5 g / t of mixed feed. The remaining steps were exactly the same as those in Comparative Example 18.

[0086] Comparative Example 20: A method for preparing a brewer's grains-based fermented total mixed diet (BW0+30 d+PA 068) Pediococcus lactis PA 068 was used as a microbial additive, and the remaining steps were exactly the same as in Example 18.

[0087] Comparative Example 21: A method for preparing a brewer's grains-based fermented total mixed diet (BW0+30 d+B). Bacillus subtilis B was used as a microbial additive, and the remaining steps were exactly the same as in Example 18.

[0088] Comparative Example 22: A method for preparing a brewer's grains-based fermented total mixed diet (BW0+30 d+0) No microbial additives were added, and the remaining steps were exactly the same as in Example 18.

[0089] Experimental Example 1 The raw materials (the amount of beer lees added is 10%) were weighed according to the weight ratio described in Example 1, and after mixing, they were recorded as BW10. The subsequent steps in Example 1 were not performed.

[0090] The raw materials (the amount of beer lees added is 20%) were weighed according to the weight ratio described in Example 5, and after mixing, they were labeled as BW20. The subsequent steps in Example 5 were not performed.

[0091] Weigh the raw materials according to the weight ratio described in Comparative Example 13 (the amount of beer lees added is 0%), mix them and record the mixture as BW0, and do not perform the subsequent steps in Comparative Example 13.

[0092] Subsequently, BW0, BW10, and BW20 were tightly packed into 1L polyethylene tanks, the tank openings were sealed with four layers of plastic wrap, and the inner and outer lids were tightened. The tanks were then placed into 30×40 cm polyethylene silage bags and vacuum-sealed. Each silage bag was labeled with its treatment, and its fermentation quality and nutritional components were analyzed at 3 days, 7 days, and 30 days.

[0093] (1) Effect of brewer's grain ratio on FTMR fermentation quality As shown in Table 1, the proportion of beer lees added and the fermentation time both had a highly significant impact on the fermentation quality of FTMR. P<0.01). With increasing proportion of brewer's grains, the pH value at each fermentation time point significantly decreased, while the contents of lactic acid (LA), acetic acid (AA), and ammonia nitrogen (NH3-N) significantly increased. P <0.05).

[0094] After 30 days of fermentation, the pH (4.52), lactic acid content (3.13% DM), and acetic acid content (1.34% DM) of group BW10 (10% brewer's grains) were significantly better than those of group BW0. P The value <0.05 indicates that 10% brewer's grains significantly improved fermentation quality. The BW20 group (20% brewer's grains) exhibited the best fermentation state at 30 days, with the lowest pH (4.41), highest lactic acid content (3.64% DM), highest acetic acid content (1.53% DM), and an ammonia nitrogen content of 5.66% TN, all significantly better than the BW10 and BW0 groups (P<0.05). Propionic acid and butyric acid remained at low levels in all groups (<0.08% DM).

[0095] The results above indicate that the fermentation quality of FTMR is significantly improved within the range of 10% to 20% of brewer's grains added, and the overall fermentation effect is optimal with an addition of 20%.

[0096] Table 1. Fermentation quality of FTMR with different brewer's grain ratios Note: BW0, FTMR with 0% lees; BW10, FTMR with 10% lees; BW20, FTMR with 20% lees; P: lees addition percentage; F: fermentation days. SEM, standard error. Different lowercase letters in the same column indicate significant differences between different recipes for the same number of days; different uppercase letters in the same row indicate significant differences between different recipes for the same number of days; * P <0.05;** P <0.01, ***, P <0.001; ND, not detected. NS, no significant difference.

[0097] (2) Effect of brewer's grains ratio on the nutritional quality of FTMR Table 2 shows that the proportion of brewer's grains significantly affects the nutritional composition of FTMR (French Flour Mixture). P <0.001). As the proportion of brewer's grains increases, the dry matter (DM) and crude protein (CP) content decrease slightly, while the crude fat (EE) content increases significantly.

[0098] At 30 days of fermentation, the crude fat content of the BW10 and BW20 groups was 2.95% DM and 4.29% DM, respectively, both significantly higher than that of the BW0 group (3.29% DM).P <0.05). In particular, the EE content of the BW20 group continued to rise during fermentation, indicating that a high proportion of brewer's grains is beneficial to improving the energy level of the diet.

[0099] Furthermore, the content of neutral detergent fiber (NDF) and acid detergent fiber (ADF) in each group did not change significantly during fermentation, indicating that the addition of brewer's grains did not have a negative impact on the fiber structure.

[0100] In summary, when the proportion of brewer's grains added is 10% to 20%, the crude fat content of the feed can be significantly increased while maintaining the stability of the fiber structure. Among them, the 20% addition is more prominent in terms of increasing the energy value.

[0101] Table 2 Nutritional quality of FTMR with different brewer's grains ratios Note: BW0, FTMR with 0% lees; BW10, FTMR with 10% lees; BW20, FTMR with 20% lees; P: lees addition percentage; F: fermentation days. SEM, standard error. Different lowercase letters in the same column indicate significant differences between different recipes for the same number of days; different uppercase letters in the same row indicate significant differences between different recipes for the same number of days; * P <0.05;** P <0.01, ***, P <0.001; NS, no significant difference; " / " indicates this item is not present.

[0102] Experiment Example 2 The following experimental analysis was conducted on the beer lees fermented total mixed diets (FTMR) obtained in Examples 5 to 8 and Comparative Examples 7 to 22.

[0103] (1) Effects of microbial additives on the fermentation quality of FTMR Table 3 shows that microbial additives can regulate the fermentation quality of FTMR to varying degrees. Overall, in the early fermentation stage, adding B to the BW0 group was more effective in reducing pH, NH3-N, and AA content. However, in the later fermentation stage, except for PA068 which significantly increased the NH3-N content in the BW0 group, the other microbial additive treatments improved the fermentation quality of the BW0 group. In the BW20 group, adding LP in the early fermentation stage showed better performance in reducing pH and AA content and increasing LA content, while the JF treatment was most effective in reducing NH3-N. In the later fermentation stage, the PA068 and JF treatments had the best effects on the fermentation quality of the BW20 group.

[0104] Table 3. Effects of microbial additives on the fermentation quality of FTMR Note: BW0, FTMR with 0% lees; BW20, FTMR with 20% lees; P: lees addition ratio; T: additive; F: fermentation days. SEM, standard error. Different lowercase letters in the same column indicate statistically significant differences for the same treatment on the same day. * P <0.05;** P <0.01, ***, P <0.001; ND, not detected; NS, no significant difference; " / " indicates this item is not present.

[0105] (2) Effects of microbial additives on the nutritional quality of FTMR Table 4 shows that throughout the fermentation process, the addition of treatment B to group BW0 had a better effect on increasing CP content, while the addition of treatment JF had the most significant effect on increasing WSC content in group BW0. The addition of treatment JF had a better effect on improving CP and WSC content in group BW20 during fermentation 0-30 days.

[0106] Table 4. Effects of microbial additives on the nutritional quality of FTMR Note: BW0, FTMR with 0% lees; BW20, FTMR with 20% lees; P: lees addition ratio; T: additive; F: fermentation days. SEM, standard error; different lowercase letters in the same column indicate statistically significant differences for the same treatment on the same day. * P <0.05;** P <0.01, ***, P <0.001; NS, no significant difference; " / " indicates this item is not present.

[0107] It should be noted that the experiments of Examples 1 to 4 and Comparative Examples 1 to 6 of the present invention were also conducted in the same manner, and their experimental results were consistent with the changing trends and effect patterns of Examples 5 to 8 and the corresponding comparative groups, so they will not be repeated here.

[0108] Experimental Example 3 Taking Examples 5 to 8 and Comparative Examples 7 to 12 as examples, the effects of microbial additives on the bacterial community of FTMR (BW20 group) with a beer lees ratio of 20% were analyzed by high-throughput sequencing.

[0109] (1) Population and abundance of microbial additive-regulated FTMR bacteria Microbial additives regulate the relative abundance of bacterial communities in FTMR at different brewer's grain ratios, such as... Figure 1 As shown, at the genus level (such as...) Figure 1As shown in A), in the unfermented state (0d), the dominant genus in group BW20 was... Prevotella (28.91%). However, after 7 days of fermentation, the dominant genus in group BW20 was gradually replaced. Limosilactobacillus , Lactiplantibacillus The first dominant genus of group BW20 under PA068 and B treatments was replaced. Limosilactobacillus (55.60%, 56.50%). After 30 days of fermentation, the dominant genus in group BW20 all transformed into... Limosilactobacillus At this level (e.g.) Figure 1 (As shown in B in the figure), the dominant species in the unfermented BW20 group is Prevotella_cerevisiae (28.91%). At 7 days of fermentation, the dominant species in group BW20 was [missing information] under both the LP and JF treatments. Lactiplantibacillus_pentosus However, after 30 days of fermentation, the dominant species in group BW20 under the JF treatment remained […]. Lactiplantibacillus_pentosus (53.28%), the dominant species in all other treatments changed. Among them, in the BW20 group, the absolute dominant species in the LP and PA068 treatments changed to Limosilactobacillus_panis (21.00%, 26.35%), while the first dominant species in group BW20 under CK (no microorganisms added) and B treatments changed to Lactobacillus_xujianguonis (40.30%, 19.99%).

[0110] (2) Correlation analysis between FTMR bacterial community and fermentation quality Correlation analysis between dominant bacterial communities and fermentation quality during FTMR fermentation, as follows: Figure 2 and Figure 3 As shown. At the bacterial community genus level ( Figure 2 In Group BW20, undefined_Chloroplast , Ligilactobacillus It is significantly negatively correlated with LA content ( P <0.05). Ligilactobacillus , Lactococcus and Lactiplantibacillus It showed a highly significant positive correlation with pH value, and a significant negative correlation with NH3-N and AA content. P <0.05), Liquorilactobacillus , Aeriscardovia , Lactobacillus, Weissella , Streptococcus , Limosilactobacillus It showed a significant negative correlation with pH value, while NH3-N and AA content showed a significant positive correlation. P <0.05).

[0111] At the bacterial community level ( Figure 3 In Group BW20, Limosilactobacillus_pontis , Lactobacillus_xujianguonis , Lactobacillus_acetotolerans ,Lactobacillus_ timonensis , Liquorilactobacillus_vini It showed a highly significant positive correlation with AA and NH3-N content, and a highly significant negative correlation with pH value. P <0.001). Lactiplantibacillus_pentosus , Lactobacillus_ johnsonii , Ligilactobacillus_murinus It showed a highly significant negative correlation with the contents of AA and NH3-N, and a highly significant positive correlation with pH value. P <0.001). Furthermore, Lactobacillus_amylovorus It showed a significant negative correlation with the contents of LA, AA, and NH3-N. P <0.01). LA content Limosilactobacillus_mucosae , Limosilactobacillus_ secaliphilus Significant negative correlation ( P <0.05). Limosilactobacillus_pontis , Weissella_cibaria It is significantly negatively correlated with pH value. Limosilactobacillus_pontis It is significantly positively correlated with NH3-N. Weissella_ cibaria It is significantly positively correlated with AA ( P <0.05).

[0112] Examples 1 to 4 and Comparative Examples 1 to 6 of this invention also underwent the experiments described in Example 3. The experimental results were the same as those of Examples 5 to 8 and the corresponding comparative groups, so they will not be repeated here.

[0113] Experiment Example 4 This experiment used FTMR obtained after 30 days of fermentation in Examples 7-8, Comparative Examples 10-12, and Comparative Examples 18-22 for rumen in vitro fermentation experiments (conducted in rumen fluid) to study the rumen in vitro fermentation characteristics of FTMR treated with microbial additives. A total of 30 samples were collected (2 formulations × 5 additives × 1 day × 3 replicates). Group BW0 represents FTMR with 0% brewer's grains; Group BW20 represents FTMR with 20% brewer's grains; CK represents FTMR without any added microorganisms. The rumen fluid used was obtained from the rumen of 3 healthy Angus cattle with fistulas, sourced from Beijing Fangshan Hengsheng Animal Husbandry Technology Co., Ltd.

[0114] (1) FTMR in vitro fermentation parameters treated with microbial additives The results of in vitro digestibility analysis of FTMR treated with microbial additives are shown in Table 5. Except for the fact that the microbial additives had no significant effect on the IVNDFD content of FTMR, the proportion of brewer's grains added, the microbial additives, and their interaction significantly affected the IVDMD, IVCPD, IVNDFD, and IVADFD of FTMR.P <0.05). With increasing proportions of brewer's grains added, the IVDMD of FTMR significantly decreased, with a minimum value of 41.93% ( P <0.05). The IVCPD in the BW20 group was higher than that in the BW0 group under JF treatment. Both JF and PA068 treatments significantly increased the IVCPD in the BW20 group compared to the control group (CK), with the JF treatment showing a higher increase in IVCPD (7.00% vs 14.96%). P <0.05. LP treatment significantly reduced IVNDFD in the BW20 group ( P <0.05. All treatments resulted in IVADFD ranging from 38.98% to 45.50%. Compared to the CK treatment, the JF treatment significantly improved IVADFD in the BW20 group ( P <0.05).

[0115] Overall, the addition of JF improved the in vitro digestibility of the BW20 group.

[0116] Table 5. In vitro digestibility of FTMR under microbial additive treatment Note: BW0, FTMR with 0% lees; BW20, FTMR with 20% lees; P: lees addition percentage; T: additive. SEM, standard error; different lowercase letters in the same column indicate significant differences between different treatments for the same additive; different uppercase letters in the same row indicate statistically significant differences between different additives for the same treatment; * P <0.05;** P <0.01, ***, P <0.001; NS, no significant difference.

[0117] The gas production results of FTMR in vitro culture with microbial additives for 48 h are shown in Table 6 and Figure 4 As shown. The proportion of brewer's grains added, additives, and the interaction between the two significantly affect the in vitro gas production of FTMR (as shown). P <0.01). With prolonged in vitro culture time, the BW0 group ( Figure 4 A) and BW20 group ( Figure 4 The in vitro gas production of B) showed an increasing trend, and the gas production of the BW0 group was higher than that of the BW20 group. Throughout the fermentation process, compared with the CK treatment, the addition of B significantly increased the in vitro gas production of the BW0 group (B). P<0.05). All microbial additives increased the in vitro gas production of the BW20 group. Among them, the highest in vitro gas production of the BW20 group was 36.80 mL when JF treatment was added after 12 h of in vitro culture. During the period of 24 h to 48 h of culture, the in vitro gas production of the BW20 group under PA068 treatment was the largest increase compared with the CK treatment, with the increases at each culture time point being 5.54%, 3.84%, and 3.73%, respectively.

[0118] Table 6. In vitro gas production of FTMR under microbial additive treatment Note: BW0, FTMR with 0% brewer's grains; BW20, FTMR with 20% brewer's grains; P: brewer's grains addition percentage; T: additive. SEM, standard error; different lowercase letters in the same column indicate significant differences between different treatments for the same additive; different uppercase letters in the same row indicate statistically significant differences between different additives for the same treatment. P <0.01, ***, P <0.001.

[0119] The effects of microbial additive treatment on the fermentation parameters of the fermentation broth are shown in Table 7 and... Figure 5 As shown. The proportion of brewer's grains added significantly affects the pH, NH3-N, BA, IAV, and AV content of FTMR. P <0.05); Microbial additives significantly affected the pH, NH3-N, AA, PA, BA, AA / PA, and TVFAs content of FTMR ( P <0.05); the interaction between the two significantly affected the pH, NH3-N, AA, AA / PA and TVFAs content of FTMR ( P <0.05). The rumen fluid pH values ​​of all treatments ranged from 6.87 to 6.94, and the rumen pH values ​​of the BW20 group were all higher than those of the BW0 group. Compared with the CK treatment, both PA068 and JF treatments significantly reduced the pH value of the BW20 group, with the PA068 treatment showing a greater reduction (0.89% vs 0.72%). P <0.05. All microbial additive treatments significantly increased the rumen NH3-N and AA content in the BW20 group ( P <0.05), and the highest content was observed with the addition of B, with the two being 30.13 mg / 100 mL and 45.32 mmol / L, respectively. The addition of JF and B significantly increased the PA content in the BW20 group, and the increase was greater with the addition of B compared to the CK treatment (4.22% vs 5.12%). P <0.05%. Adding B significantly increased the BA content in the BW20 group ( P<0.05. Both LP and PA068 treatments significantly increased the AA / PA ratio in the BW20 group ( P <0.05). Compared with the CK treatment, JF and B treatments significantly increased the TVFAs content in the BW20 group, with the highest TVFAs content in the BW20 group under the B treatment, reaching 70.08 mmol / L ( P <0.05).

[0120] Table 7. Effects of FTMR treatment with microbial additives on fermentation parameters of in vitro fermentation broth. Table 7 (continued) shows the effects of microbial additive treatment on fermentation parameters of in vitro fermentation broth. Table 7 (continued) shows the effects of microbial additive treatment on fermentation parameters of in vitro fermentation broth. Table 7 and its continuation: BW0, FTMR with 0% brewer's grains; BW20, FTMR with 20% brewer's grains; P: brewer's grains addition ratio; T: additive. SEM, standard error. Different lowercase letters in the same column indicate significant differences between different treatments for the same additive; different uppercase letters in the same row indicate statistically significant differences between different additives for the same treatment. * P <0.05;** P <0.01, ***, P <0.001; NS, no significant difference.

[0121] (2) Correlation analysis between FTMR quality and rumen fermentation parameters Correlation analysis of FTMR quality and rumen in vitro fermentation characteristics, such as... Figure 6 As shown. IVDMD,

[0122] The ratio of ammonia nitrogen to total nitrogen in IVCPD and FTMR were significantly negatively correlated, and their correlation coefficients were respectively -0.60 and -0.76, and in addition, IVCPD showed a significant positive correlation with IVCMD and CP levels, with correlation coefficients of 0.59 and 0.39, respectively. P <0.05). Rumen fluid pH was significantly negatively correlated with EE in FTMR, with a correlation coefficient of -0.8 ( ). P <0.05). Rumen fluid NH3-N was significantly positively correlated with pH value of FTMR, while rumen fluid AA / PA was significantly negatively correlated with rumen fluid pH value and 48-hour cumulative gas production (V48 h), with correlation coefficients of -0.62 and -0.58, respectively. P <0.05).

[0123] Rumen digestibility and gas production are strongly positively correlated. Gas production is a direct product of rumen microorganisms' digestion and decomposition of feed dry matter and organic matter. Digestibility determines the baseline level of gas production, and gas production can also serve as an important indirect indicator for assessing digestibility. The relationship between the two is essentially a reflection of the material and energy conversion patterns in rumen microbial fermentation metabolism. Under JF treatment, the in vitro digestibility of both diet groups increased. This may be because the main microorganisms in JF can alter the composition and structural basis of the silage cell wall in the early stages by disrupting the connections between lignin and structural polysaccharides, thereby making it easier for rumen microorganisms to utilize the substrate during fermentation, thus accelerating the digestion and absorption of the diet by ruminants. Furthermore, the addition of brewer's grains reduced the in vitro digestibility of the diet, but the dry matter digestibility and crude protein digestibility of the diet with added brewer's grains under JF treatment were significantly increased, and higher than the digestibility of the control group without added brewer's grains. This indicates that JF treatment has a positive effect on improving the digestibility of the diet with 20% brewer's grains.

[0124] It should be noted that the experiments in Example 4 were conducted using the FTMR prepared in the other examples. The results showed that the JF treatment had a positive effect on improving the digestibility of the diet with 10% distillers' grains added, so it will not be repeated here.

[0125] The research of this invention shows that: (1) The application of brewer's grains (10% and 20%) promoted the fermentation of FTMR, and the FTMR with 20% brewer's grains had the highest content of ammonia nitrogen, lactic acid, acetic acid and crude fat, the lowest pH value and the best fermentation quality. (2) Microbial additives improved the quality of FTMR in different ways: high-temperature microbial powder JF significantly increased the content of ammonia nitrogen, lactic acid, acetic acid and crude fat in the FTMR of the 20% brewer's grains group. Limosilactobacillus and Lactiplantibacillus The relative abundance of promotes lactic acid fermentation, reduces its ammonia nitrogen content, and improves the quality of FTMR. (3) The beer lees FTMR inoculated with high-temperature bacterial powder JF significantly increased the in vitro gas production in the rumen of beef cattle, reduced the acetate-propionic acid ratio, and improved the in vitro digestibility of FTMR.

[0126] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0127] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this invention should be determined by the appended claims.

Claims

1. A method for preparing a total mixed ration (TMR) fermented from brewer's grains, characterized in that, Specifically, the following steps are included: Weigh out 10-20 parts by weight of brewer's grains, 26.35-30 parts of whole-plant corn silage, 14.2-19.05 parts of rice straw, 4.9-10 parts of soybean meal, 28.9-32.6 parts of corn flour, 1.2 parts of premix, 0.2 parts of salt, and 0.6 parts of limestone powder. The raw materials in the specified weight proportions are mixed, and the moisture content is adjusted to 45%~55% by weight to obtain a mixed feed. After adding microbial additives to the mixed feed, it was sealed and stored at 25℃~30℃ for more than 6 days to obtain brewer's grain fermented total mixed diet. The microbial additive is *Lactobacillus plantarum* (… Lactobacillus plantarum (or high-temperature bacterial powder JF prepared from Lactobacillus plantarum); Each gram of mixed feed contains 1×10 Bacillus plantarum. 6 CFU~5×10 6 CFU; the amount of high-temperature bacterial powder JF added per ton of mixed feed is 2 g to 5 g.

2. The preparation method according to claim 1, characterized in that, The sealed storage time is 7 days to 30 days.

3. The preparation method according to claim 1, characterized in that, The viable count of the high-temperature bacterial powder JF is ≥2.0×10⁻⁶. 10 CFU / g.

4. The brewer's grains fermented total mixed diet prepared by the preparation method according to claim 1.

5. The application of the beer lees-based fermented total mixed ration (TMR) according to claim 4 in the feeding of ruminants.

6. The application according to claim 5, characterized in that, The ruminant animal is a cow.

7. The application according to claim 5, characterized in that, Brewer's grains fermented total mixed diets can improve rumen fermentation characteristics in ruminants.

8. The application according to claim 7, characterized in that, The improvement of rumen fermentation characteristics in ruminants refers to increasing one or more of the following: rumen dry matter digestibility, crude protein digestibility, neutral detergent fiber digestibility, and acid detergent fiber digestibility in beef cattle.

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