A method for preparing fermented feed using bovine intestinal lactobacillus

Abstract

The present invention belongs to the technical field of fermented feed and relates to a method for preparing fermented feed by using bovine intestinal lactobacilli. The method specifically comprises the following steps: step 1, mixing intestinal contents with pre-sterilized physiological saline, filtering and adding amino acids; step 2, adjusting the pH of the intestinal solution, anaerobic culturing, and then mixing with an adsorption carrier to obtain a solidified bacterial agent; step 3, mixing the feed, the solidified bacterial agent and the filtrate of step 2, and fermenting in three stages; step 4, drying after the fermentation is completed to obtain the fermented feed. The present invention systematically solves the core problems of existing fermented feed, such as poor strain adaptability, low fermentation efficiency, easy contamination and high storage cost, by synergistically acting through multiple technologies such as specific strain screening, multi-stage dynamic control process and composite antibacterial.

Description

Technical Field

[0001] The invention belongs to the technical field of fermented feed and relates to a method for preparing fermented feed by utilizing bovine intestinal lactobacillus. Background Art

[0002] Conventional feeds currently contain large amounts of cellulose and non-starch polysaccharides, which are poorly digested and absorbed by livestock. Conventional feeds are typically made from raw materials such as corn, soybean meal, and wheat bran, which contain higher levels of cellulose and carbohydrates. To improve livestock's absorption of conventional feeds, fermentation is often performed using bacteria before feeding the feed to livestock. However, this has been ineffective due to the following drawbacks in existing fermentation methods:

[0003] 1) The selection of bacterial strains is single and has insufficient adaptability. Existing fermented feeds mostly use general lactic acid bacteria (such as Lactobacillus plantarum and Lactobacillus acidophilus) or yeast for fermentation. Although these strains can promote fermentation, they have limited adaptability to the animal intestine, which may lead to mismatch between metabolites and the host digestive system, reducing feed utilization. In addition, traditional strains are easily affected by the rumen environment in ruminant feed, resulting in decreased activity and difficulty in stabilization.

[0004] 2) The fermentation process is extensive and inefficient. Most existing technologies use single-stage fermentation (such as constant temperature and humidity), which lacks dynamic regulation, resulting in insufficient microbial metabolism. Due to insufficient oxygen and temperature control, single-stage fermentation is prone to excessive acidity or incomplete fermentation, resulting in nutrient loss (such as cellulose decomposition).

[0005] 3) The antibacterial measures are not perfect and are prone to contamination. Existing technologies mostly rely on fermentation to produce acid to inhibit miscellaneous bacteria, but in an open environment (such as turning the pile), mold or Bacillus is easily introduced. Fermented feed without adding antibacterial agents has a high mold rate during storage, affecting safety.

[0006] 4) Direct use of liquid bacterial agents has the problems of short shelf life and high transportation costs. If freeze-drying is used to preserve the strains, the process is complex and energy-intensive, and the cost of large-scale application is high.

[0007] Therefore, it is urgent to provide a method for fermenting feed with good fermentation effect, low mildew rate and low cost. Summary of the Invention

[0008] In order to solve the above problems, the present invention provides a method for preparing fermented feed using bovine intestinal lactobacilli, which specifically comprises the following steps:

[0009] Step 1: Take fresh bovine intestinal contents from a slaughterhouse and store them at 1-4°C for later use. Mix the intestinal contents with pre-sterilized saline and filter them through a filter with a pore size of 5-10 μm to remove the filter residue. Add amino acids to the filtrate and shake at 200-300 rpm for 5-10 minutes to obtain an intestinal solution.

[0010] Preferably, the mass ratio of the intestinal contents, pre-sterilized physiological saline and amino acids is 100:300:0.3. Most preferably, the bovine intestinal tract is bovine duodenum, and the bovine intestinal contents are bovine duodenum contents.

[0011] Preferably, the amino acid is one or more of cysteine, lysine, tryptophan, methionine, leucine, aspartic acid and glutamic acid.

[0012] Preferably, the physiological saline solution is sterilized by steam sterilization at 115-125° C. and 0.10-0.12 MPa for 15-20 minutes.

[0013] Step 2: Use lactic acid to adjust the pH of the intestinal solution to 3.8-4.2, culture at 4%-6% oxygen concentration and 30-40°C for 6-7 hours, then mix with the adsorption carrier, shake at 20-30°C and 300-400rpm for 2-3 hours, filter, and save the filtrate for later use. Dry the surface moisture of the filter residue to obtain a solidified bacterial agent.

[0014] Preferably, the mass ratio of the intestinal solution to the adsorption carrier is 5:1.

[0015] Preferably, the adsorption carrier is corn cob and crop straw, with a mass ratio of 1:1.

[0016] Preferably, the corn cobs and crop straws are mixed and crushed into 20-40 meshes, soaked in 0.5-0.6 mol / L citric acid solution for 10-12 hours, and then mixed with the intestinal solution.

[0017] Preferably, the mass ratio of the corn cob, crop straw and citric acid solution is 1:1:6.

[0018] Preferably, the crop straw is one or more of wheat straw, corn straw and rice straw.

[0019] Step 3: Mix the feed, solidified bacterial agent and the filtrate from step 2 in a mass ratio of (10-15): (6-8): (3-4) and ferment in stages.

[0020] In the first stage, the temperature is 30-35℃, the humidity is 60-70%, and oxygen is supplied for 5-8 minutes every 2-3 hours with an oxygen flow rate of 0.1m 3 / h, fermentation time 12-14h;

[0021] In the second stage, the temperature is adjusted every 7-8 hours in the order of (34-36)℃→(37-39)℃→(34-36)℃, the humidity is 65-75%, the oxygen concentration is 5-8%, the compost is turned every 1-2 hours, and the fermentation time is 23-26 hours;

[0022] In the third stage, the temperature is 25-28°C, the humidity is 55-65%, the oxygen concentration is <0.5%, the antibacterial agent is sprayed 1-3 times during the fermentation period, and the fermentation time is 23-26 hours.

[0023] Preferably, the feed is one or more of corn flour, soybean meal, wheat bran, forage grass, distiller's grains, bean curd residue, rice bran, fruit pomace, sweet potato residue, cassava residue, beet residue, and molasses. Most preferably, the feed is corn flour, forage grass, wheat bran, soybean meal, rice bran, and molasses in a mass ratio of 6:8:4:3:5:2.

[0024] Preferably, the mass ratio of the fermentation product to the antibacterial agent is 100:1.

[0025] Preferably, the antibacterial agent comprises tea polyphenols, chitosan, acetic acid and water in a ratio of 4g:2g:1mL:120mL.

[0026] Step 4: After fermentation is completed, the mixture is dried at 40-50° C. to a moisture content of ≤10% to obtain fermented feed.

[0027] The present invention has the following advantages:

[0028] (1) The present invention uses lactic acid to adjust the pH and inhibit other bacterial flora in the duodenal contents of cattle. It mainly utilizes the Lactobacillus flora whose metabolic characteristics are highly compatible with the host digestive system, which can produce more easily absorbed short-chain fatty acids (such as butyric acid) and improve the bioavailability of feed. In addition, this strain has strong tolerance to the rumen environment and is more stable than traditional strains, avoiding the problem of poor compatibility between general strains and the animal intestinal environment.

[0029] (2) The present invention adopts a three-stage fermentation method. In the first stage, facultative anaerobic bacteria are activated by intermittent oxygenation to quickly decompose large molecular substances (such as cellulose). In the second stage, gradient temperature control is used to promote the synergistic effect of different temperature-resistant bacterial groups, significantly shortening the fermentation cycle while reducing nutrient loss (such as increasing crude protein retention rate). In the third stage, anaerobic fermentation is combined with antibacterial agents to completely inhibit miscellaneous bacteria and avoid later contamination, thereby solving the problem of metabolic imbalance caused by single-stage fermentation.

[0030] (3) The present invention uses tea polyphenols, chitosan, acetic acid and water to prepare an antibacterial agent, which can specifically inhibit mold in the late fermentation period, significantly extend the shelf life, and avoid the problem of relying on natural acid production for antibacterial effect and unstable effect.

[0031] (4) The present invention uses corn cobs and straw as carriers, and after citric acid pretreatment, the bacterial cells are adsorbed. The shelf life of the solidified bacterial agent at room temperature is significantly extended, and it can be directly mixed with feed, which simplifies the process flow and avoids the problem of easy inactivation of liquid bacterial agents and high storage costs.

[0032] (5) The feed raw materials of the present invention include agricultural processing wastes such as distiller's grains, fruit residues, and cassava residues. Anti-nutritional factors (such as tannins) are degraded through fermentation, which significantly improves the utilization rate of waste materials, reduces production costs, and avoids the problems of high raw material costs and insufficient utilization of by-products. DETAILED DESCRIPTION

[0033] The following is a clear and complete description of the technical solutions in the embodiments of the invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0034] Example 1

[0035] Step 1: Collect duodenal contents from a slaughterhouse and store at 2°C until ready for use. Sterilize the duodenal contents with saline solution using steam at 120°C and 0.12 MPa for 20 minutes to obtain pre-sterilized saline solution. Mix the duodenal contents with the pre-sterilized saline solution and filter through a 10 μm pore size filter to remove the residue. Add cysteine ​​to the filtrate and shake at 250 rpm for 10 minutes to obtain an intestinal solution. The mass ratio of duodenal contents, pre-sterilized saline solution, and cysteine ​​is 100:300:0.3.

[0036] Step 2: The intestinal solution is adjusted to a pH of 4.0±0.2 with lactic acid, incubated at 5% oxygen and 35°C for 6 hours, then mixed with an adsorption carrier at a mass ratio of 5:1, shaken at 25°C and 400 rpm for 2 hours, filtered, and the filtrate stored for later use. The residue is then air-dried to obtain a solidified bacterial inoculum. The adsorption carrier is corn cobs and corn stalks in a mass ratio of 1:1. The corn cobs and corn stalks are mixed and pulverized to 30 mesh, mixed with a 0.5 mol / L citric acid solution at a solid-to-liquid ratio of 1:3, and soaked for 12 hours.

[0037] Step 3: Mix the feed, solidified bacterial agent and the filtrate from step 2 in a mass ratio of 12:7:4 and ferment in stages.

[0038] In the first stage, the temperature was 32°C, the humidity was 65%, oxygen was supplied for 5 minutes every 2 hours, and the oxygen flow rate was 0.1m 3 / h, fermentation time 12h;

[0039] In the second stage, the temperature is adjusted every 8 hours in the order of 35℃→38℃→35℃, the humidity is 70%, the oxygen concentration is 6%, the compost is turned every hour, and the fermentation time is 24 hours;

[0040] The third stage was conducted at a temperature of 27°C, a humidity of 60%, and an oxygen concentration of <0.5%. During fermentation, an antibacterial agent was sprayed twice, and the fermentation lasted for 24 hours. The feed consisted of cornmeal, oat straw, wheat bran, soybean meal, rice bran, and molasses in a mass ratio of 6:8:4:3:5:2. The fermentation product and antibacterial agent had a mass ratio of 100:1, and the antibacterial agent included tea polyphenols, chitosan, acetic acid, and water in a ratio of 4g:2g:1mL:120mL.

[0041] Step 4: After fermentation is completed, the mixture is dried at 45° C. to a moisture content of ≤10% to obtain fermented feed.

[0042] Test Example 1

[0043] The crude protein content, cellulose decomposition rate, short-chain fatty acids, mold inhibition rate, shelf life and feed conversion rate of the fermented feed prepared in Example 1 and the existing fermented cattle feed (fermented cattle feed produced by Shandong Xucheng Biological Feed Co., Ltd.) were tested respectively. The results are shown in Table 1.

[0044] Data detection method:

[0045] Crude protein: Kjeldahl method (GB / T 6432-2018).

[0046] Cellulose decomposition rate: The reducing sugar increment was determined by DNS method.

[0047] Short-chain fatty acids: gas chromatography (sum of acetic, propionic and butyric acids).

[0048] Mold inhibition rate: comparison of colony counts after 28 days of storage.

[0049] Shelf life: The effective time without mold or rancidity at room temperature.

[0050] Feed conversion rate: feeding experiment (daily weight gain / daily feed intake×100%, continuous measurement for 30 days, and average value).

[0051] Table 1

[0052] Traditional feed Feed of the present invention Improvement rate (%) Crude protein content (%) 18.5 22.3 20.5 Cellulose degradation rate (%) 45 68 51.1 Short-chain fatty acids (mg / g) 12.4 18.7 50.8 Mold inhibition rate (%) 70 95 35.7 Shelf life (months) 3 6 100 Feed conversion rate (%) 62 78 25.8

[0053] As can be seen from Table 1, the feed fermented by lactobacillus in the duodenum of cattle in the present invention can significantly improve the nutritional content, bioavailability and feed conversion rate of the feed compared with the feed fermented by general bacteria, and has a significant antibacterial effect. In addition, the present invention can use agricultural by-products as feed raw materials, which can also significantly reduce costs. Its effect is significantly better than that of existing feed.

[0054] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing fermented feed using bovine intestinal lactobacillus, characterized in that: The following steps are involved: Step 1: Mix the intestinal contents with pre-sterilized physiological saline, filter, remove the filter residue, add amino acids to the filtrate, and shake to obtain an intestinal solution; Step 2: adjusting the pH of the intestinal solution to 3.8-4.2 with lactic acid, culturing anaerobically, then mixing and shaking with the adsorption carrier, filtering, and storing the filtrate for later use. The surface moisture of the filter residue is dried to obtain a solidified bacterial agent; the mass ratio of the intestinal solution to the adsorption carrier is 5:1; Step 3: Mix the feed, solidified bacterial agent and the filtrate from step 2 in a mass ratio of (10-15): (6-8): (3-4) and ferment in stages. In the first stage, the temperature is 30-35℃, the humidity is 60-70%, and oxygen is supplied for 5-8 minutes every 2-3 hours with an oxygen flow rate of 0.1m 3 / h, fermentation time 12-14h; In the second stage, the temperature is adjusted every 7-8 hours in the order of (34-36)℃→(37-39)℃→(34-36)℃, the humidity is 65-75%, the oxygen concentration is 5-8%, the compost is turned every 1-2 hours, and the fermentation time is 23-26 hours; The third stage is at a temperature of 25-28°C, a humidity of 55-65%, an oxygen concentration of <0.5%, and an antibacterial agent is sprayed 1-3 times during the fermentation period. The fermentation time is 23-26 hours. Step 4: After fermentation is completed, the material is dried to a moisture content of ≤10% to obtain fermented feed.

2. The method for preparing fermented feed using bovine intestinal lactobacillus according to claim 1, characterized in that: The mass ratio of the intestinal contents, pre-sterilized physiological saline and amino acids in step 1 is 100:300:0.3, and the intestinal contents are bovine duodenum contents.

3. The method for preparing fermented feed using bovine intestinal lactobacillus according to claim 1, characterized in that: The amino acid in step 1 is one or more of cysteine, lysine, tryptophan, methionine, leucine, aspartic acid and glutamic acid.

4. The method for preparing fermented feed using bovine intestinal lactobacillus according to claim 1, characterized in that: The adsorption carrier in step 2 is corn cob and crop straw, with a mass ratio of 1:

1.

5. The method for preparing fermented feed using bovine intestinal lactobacillus according to claim 1, characterized in that: The feed in step 3 is one or more of corn flour, soybean meal, wheat bran, forage grass, distiller's grains, bean curd residue, rice bran, fruit residue, sweet potato residue, cassava residue, beet residue and molasses.

6. The method for preparing fermented feed using bovine intestinal lactobacillus according to claim 5, characterized in that: The feed comprises corn meal, forage grass, wheat bran, soybean meal, rice bran and molasses, with a mass ratio of 6:8:4:3:5:

2.

7. The method for preparing fermented feed using bovine intestinal lactobacillus according to claim 1, characterized in that: The mass ratio of the fermentation product to the antibacterial agent in step 3 is 100:

1.

8. The method for preparing fermented feed using bovine intestinal lactobacillus according to claim 1, characterized in that: The antibacterial agent in step three includes tea polyphenols, chitosan, acetic acid and water in a ratio of 4g:2g:1mL:120mL.

9. The fermented feed prepared by the method according to any one of claims 1 to 8.