Fermented feed based on various probiotics and preparation method thereof

Abstract

The invention belongs to the technical field of fermented feeds, and provides a preparation method of a fermented feed based on multiple probiotics, and the preparation method comprises the following steps: S1, adding water into 0.1-0.2 L of wood vinegar, 0.2-0.8 L of a microbial inoculum I, 9.45 kg of an auxiliary material and 9.45 kg of fresh pig manure, mixing and stirring uniformly, and controlling the water content to be 50%; heating the mixture, continuously heating the feed to 50-70 DEG C in a sealed manner, and fermenting for 12 hours to obtain an original fermented material; and S2, adding 1.2 L of a bacterial agent II into the original fermentation material, uniformly mixing, cooling the material to 10-40 DEG C, pressing, and carrying out anaerobic sealed fermentation for 30 hours, so as to obtain the fermented feed after the fermentation is finished. The pig manure and the auxiliary materials are used for synergistic fermentation, manure odor is effectively removed, harmful substances are degraded, the content of true protein and amino acid is increased, and the prepared feed is safe, nutritional and suitable for duck feeding and has dual benefits of environmental protection and economy.

Description

Technical Field

[0001] This invention relates to the field of fermented feed technology, and in particular to a fermented feed based on a variety of probiotics and its preparation method. Background Technology

[0002] Feces are not only pollutants, but also nutrients containing various proteins and amino acids. In particular, pig feces contain 20% dry matter because of the pig's digestive structure. If these nutrients are not recycled, it will result in a huge waste.

[0003] Ecological farming has been widely promoted in rural areas and livestock farms, mainly through the recycling of manure into organic fertilizer for vegetable irrigation. However, due to the limited variety of animals raised, animal manure is rarely used as feed. Pig manure, or manure processed through anaerobic fermentation, can be made into duck feed for recycling, and this cross-species utilization can be promoted in ecological farming areas or livestock farms.

[0004] Compound microbial preparations have a very promising application prospect in the aquaculture industry. Probiotics such as Bacillus subtilis can utilize the heat and acid produced during fermentation to inhibit and kill the growth of harmful bacteria, while also effectively passivating heavy metals. The acid production also improves the palatability of fermented feed, transforming foul-smelling manure into feed with a pleasant acidic aroma. Using compound microbial preparations and manure to make fermented feed can effectively reduce manure pollution and produce feed with high economic added value.

[0005] Therefore, how to provide a method for preparing fermented feed based on multiple probiotics that can effectively passivate heavy metals is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing fermented feed based on a variety of probiotics, in order to solve the technical problems in the prior art.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for preparing fermented feed based on multiple probiotics, comprising the following steps:

[0009] S1. Mix 0.1-0.2L of wood vinegar, 0.2-0.8L of microbial agent I, 9.45kg of auxiliary materials and 9.45kg of fresh pig manure with water and stir evenly, controlling the moisture content to 50%; heat the mixture, seal and continue heating the feed to 50-70℃ for 12 hours to obtain the original fermented material;

[0010] S2. Add 1.2L of bacterial agent II to the original fermentation material and mix evenly. Cool the material to 10-40℃, compress and seal for anaerobic fermentation for 30 hours. After fermentation is complete, fermented feed is obtained.

[0011] Preferably, the ratio of wood vinegar, microbial agent I, auxiliary material, and fresh pig manure is 0.1-0.2L: 0.2-0.8L: 9.45kg: 9.45kg.

[0012] Preferably, the bacterial agent I is composed of a mixture of fermentation broths of Lactobacillus acidophilus, Bacillus thermophilus, and Bacillus licheniformis in a volume ratio of 2:1:1.

[0013] Preferably, the bacterial agent II is composed of fermentation broths of Lactobacillus acidophilus and Bacillus megaterium in a volume ratio of 5:2.

[0014] Preferably, the effective viable count of the Lactobacillus acidophilus is ≥10. 9 CFU / mL; the effective viable count of the thermophilic Bacillus ≥10 9 CFU / mL; the effective viable count of the Bacillus licheniformis is ≥10. 9 CFU / mL; the effective viable count of the *Bacillus megaterium* is ≥10. 9 CFU / mL.

[0015] Preferably, the auxiliary material is composed of corn flour, wheat bran and soybean meal mixed in a mass ratio of 2:1:1; the pig manure is fresh pig manure with a moisture content between 60% and 70%.

[0016] Preferably, the wood vinegar is made from straw as raw material, using a continuous pyrolysis method. The initial temperature is 30°C, and the temperature is heated to 500°C. After pyrolysis for 2 hours, the generated gas is condensed to obtain a wood vinegar with a density of 0.97 kg / L and an acid content of 28%.

[0017] The present invention also provides a fermented feed based on multiple probiotics obtained by the preparation method described above.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) There have been many studies on the treatment of livestock and poultry manure, but there are few studies on the fermentation of pig manure. Moreover, most of the manure treatment is for large-scale livestock and poultry farms, and there are few studies on the treatment of manure for integrated and diversified breeding enterprises.

[0020] (2) Previously, most livestock and poultry manure treatment focused on producing organic fertilizer, with limited research on its use in feed production. Previous studies on manure treatment for feed primarily involved feeding livestock such as pigs, cattle, and sheep. In contrast, fermented pig manure feed is safer for ducks, mainly due to the significant species differences between pigs and ducks, resulting in a lower risk of cross-infection with pathogens. This invention uses manure as a partial substitute for auxiliary materials in the production of fermented feed. This process removes harmful substances and odors from the manure while simultaneously generating economically valuable feed, achieving two goals at once.

[0021] (3) Probiotics such as Bacillus subtilis can utilize the heat and acid produced during fermentation to inhibit and kill the growth of harmful bacteria, and can also effectively passivate heavy metals. Since acid production can also improve the palatability of fermented feed, it can transform foul-smelling manure into feed with a pleasant sour aroma. Using compound microbial preparations and manure to make fermented feed can effectively reduce manure pollution and produce feed with high economic added value, showing great promise for the livestock industry. Detailed Implementation

[0022] This invention provides a method for preparing fermented feed based on multiple probiotics, comprising the following steps:

[0023] S1. Mix 0.1-0.2L of wood vinegar, 0.2-0.8L of microbial agent I, 9.45kg of auxiliary materials and 9.45kg of fresh pig manure with water and stir evenly, controlling the moisture content to 50%; heat the mixture, seal and continue heating the feed to 50-70℃ for 12 hours to obtain the original fermented material;

[0024] S2. Add 1.2L of bacterial agent II to the original fermentation material and mix evenly. Cool the material to 10-40℃, compress and seal for anaerobic fermentation for 30 hours. After fermentation is complete, fermented feed is obtained.

[0025] In this invention, the ratio of wood vinegar, microbial agent I, excipients, and fresh pig manure is 0.1–0.2 L: 0.2–0.8 L: 9.45 kg: 9.45 kg, preferably 0.12–0.18 L: 0.25–0.75 L: 9.45 kg: 9.45 kg, more preferably 0.13–0.17 L: 0.30–0.70 L: 9.45 kg: 9.45 kg, and even more preferably 0.14–0.16 L: 0.35–0.65 L: 9.45 kg: 9.45 kg.

[0026] In this invention, in step S1, the fermentation temperature is 50-70°C, preferably 52-68°C, more preferably 55-65°C, and even more preferably 57-63°C.

[0027] In this invention, in step S2, the fermentation temperature is 10-40°C, preferably 15-35°C, more preferably 20-30°C, and even more preferably 22-28°C.

[0028] In this invention, the bacterial agent I is composed of a mixture of fermentation broths of Lactobacillus acidophilus, Bacillus thermophilus, and Bacillus licheniformis in a volume ratio of 2:1:1; the bacterial agent II is composed of a mixture of fermentation broths of Lactobacillus acidophilus and Bacillus megaterium in a volume ratio of 5:2.

[0029] In this invention, the effective viable count of the Lactobacillus acidophilus is ≥10. 9 CFU / mL; the effective viable count of the thermophilic Bacillus ≥10 9 CFU / mL; the effective viable count of the Bacillus licheniformis is ≥10. 9 CFU / mL; the effective viable count of the *Bacillus megaterium* is ≥10. 9 CFU / mL.

[0030] In this invention, the auxiliary material is composed of corn flour, wheat bran and soybean meal mixed in a mass ratio of 2:1:1; the pig manure is fresh pig manure with a moisture content between 60% and 70%.

[0031] In this invention, the wood vinegar is made from straw as raw material by continuous pyrolysis. The initial temperature is 30°C, and the temperature is heated to 500°C. After pyrolysis for 2 hours, the generated gas is condensed to obtain a wood vinegar with a density of 0.97 kg / L and an acid content of 28%.

[0032] The present invention also provides a fermented feed based on multiple probiotics obtained by the preparation method described above.

[0033] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1

[0035] The liquid microbial inoculant is divided into two groups and added in two separate processes. Overall, it consists of *Lactobacillus acidophilus*, *Bacillus thermophilum*, *Bacillus licheniformis*, and *Bacillus megaterium*, with a total viable count of ≥10⁻⁶ for the entire inoculum. 9CFU / mL. Inoculum agent I is composed of fermentation broths of Lactobacillus acidophilus, Bacillus thermophilus, and Bacillus licheniformis in a volume ratio of 2:1:1. Inoculum agent II is composed of fermentation broths of Lactobacillus acidophilus and Bacillus megaterium in a volume ratio of 5:2.

[0036] Wood vinegar is made from straw using a continuous pyrolysis method. The initial temperature is 30℃, and the temperature is increased to 500℃. After 2 hours of pyrolysis, the generated gas is condensed to obtain wood vinegar with a density of 0.97 kg / L and an acid content of 28%. The auxiliary materials consist of a mixture of corn flour, wheat bran, and soybean meal in a 2:1:1 ratio. The pig manure used is fresh pig manure with a moisture content between 60% and 70%.

[0037] 1.1 Test Methods:

[0038] 1.1.1 Experiment on fermented pig manure feed

[0039] The fermentation of pig manure feed consists of two steps: First, mix 0.2L of wood vinegar, 0.8L of microbial agent I, 9.45kg of auxiliary materials, and 9.45kg of fresh pig manure with water, ensuring the moisture content is controlled at 50%. Heat the mixture, seal it, and continue heating the feed to above 50℃ for 12 hours. After fermentation, proceed to the second step: add 1.2L of microbial agent II to the original fermented material, mix thoroughly, cool the material to below 40℃, compress it, and seal it for anaerobic fermentation for 30 hours. After fermentation, the feed can be fed directly.

[0040] 1.1.2 Safety and nutritional value testing of fermented pig manure feed

[0041] Safety testing: Heavy metal and aflatoxin content testing: After fermentation, the material is sent to the Guangxi Zhuang Autonomous Region Analysis and Testing Research Center for testing of total arsenic, lead, cadmium, fluorine, and aflatoxin B1 content.

[0042] Nutritional composition analysis: During the first 40 hours of fermentation, 5g of the fermented feed was sampled every 5 hours (0, 8, 16, 24, 32, and 40 hours), 10mL of sterile physiological saline was added, mixed well, and centrifuged at 1500r / min for 15min. The supernatant was then measured using a pH meter, and the results are expressed as pH. During the first 40 hours of fermentation, 100g of the fermented feed was sampled every 5 hours (0, 8, 16, 24, 32, and 40 hours), dried in a 105℃ drying oven for approximately 10 hours until constant weight was achieved, and then ground into powder. Crude protein (CP) was determined using the Kjeldahl method, true protein (TP) was determined after protein precipitation using the copper sulfate method, crude fiber (CF) was determined using the acid-base washing-gravimetric method, crude fat (CEE) was determined using the Soxhlet extraction method, and total amino acids (TAA) were determined using the formaldehyde method.

[0043] 1.1.3 Safety and nutritional value testing of fermented feed for feeding ducks

[0044] Forty healthy one-day-old Muscovy ducks were randomly divided into four groups. One group served as the control group, fed a basal diet. The other three groups received a basal diet supplemented with 20%, 30%, and 50% fermented feed, respectively. The preliminary experiment lasted 10 days, and the experimental period was 60 days. Each group had free access to feed and water. Immunization, deworming, and environmental disinfection were carried out according to the feeding management procedures. The basal diet conformed to NY / T2122—2012 "Standards for Meat Duck Feeding," and vaccinations were administered on time and according to the standard procedures.

[0045] (1) Detection of serum biochemical indicators in ducks: On the morning of day 60, blood was collected from the subwing vein on an empty stomach. The blood was centrifuged at 3500 r / min for 15 min, and the supernatant was separated and collected. The supernatant was then aliquoted into EP tubes, labeled, and stored at -20℃ for later testing. Eight indicators, namely glucose (GLU), total protein (TP), albumin (ALB), blood urea nitrogen (BUN), total cholesterol (TC), triglycerides (TG), alanine aminotransferase (ALT), and aspartate aminotransferase (AST), were detected in the serum of the experimental group and the control group using a kit from Nanjing Jiancheng Bioengineering Institute on a URIT-8000 fully automated biochemical analyzer. The changes in serum biochemical indicators were used to assess the metabolic level and degree of damage to functional organs such as the liver and kidneys, as well as the metabolism of nutrients.

[0046] (2) Detection of growth performance indicators of ducks: During the experiment, the total daily feed intake of each group of ducks was recorded, and they were weighed on an empty stomach every ten days in the morning. The average daily feed intake, average daily weight gain and feed conversion ratio of each group were calculated.

[0047] Average daily weight gain = (final weight - initial weight) / number of ducks per group × number of days

[0048] Average daily feed intake = Total feed intake per group / Number of ducks per group × Number of days

[0049] Feed conversion ratio = Average daily feed intake / Average daily weight gain

[0050] 2.1 Detection of heavy metals and aflatoxin in fermented feed

[0051] The results of heavy metal and aflatoxin tests in the fermented feed are shown in Table 1. Table 1 shows that aflatoxin levels are below the safe limit, meeting the national feed hygiene standards. Total arsenic and fluorine contents were 0.23 and 2.2%, respectively. Lead, arsenic, and cadmium were not detected because they were below the detection limit set by the testing agency, also meeting the national feed hygiene standards (GB13078—2017).

[0052] Table 1. Mass fractions of heavy metals and aflatoxin in fermented feed

[0053]

[0054] 2.2 Nutrient Component Detection of Fermented Feed

[0055] Table 2 shows the changes in crude protein, true protein, amino acids, crude fiber, crude fat content, and pH value of the fermented feed within 40 hours of fermentation. As can be seen from the table, crude protein, crude fiber, and crude fat all decreased. After 40 hours of fermentation, crude protein decreased by 56.75%, crude fiber by 43.75%, and crude fat by 62.34%, indicating that the probiotic strain consumed protein during fermentation. However, true protein increased by 55.29%, and amino acids increased by 87.18%, with significant differences (P < 0.05), indicating that the probiotics produced higher quality nutrients that were more easily digested and absorbed by the ducks. The pH eventually dropped below 4.0, indicating a good fermentation effect.

[0056] Table 2. Changes in major nutrient components of feed within 40 hours of fermentation.

[0057]

[0058]

[0059] 2.3 Detection of serum biochemical indicators in Muscovy ducks

[0060] Serum samples were collected from Muscovy ducks after 60 days of feeding and biochemical tests were performed. The results are shown in Table 3. The GLU index showed no significant difference compared to the control group (P>0.05). The TP index showed no significant difference compared to the control group (P>0.05), while the ALB index was significantly higher than the control group (P<0.05). The BUN, TC, TG, ALT, and AST indices showed no significant differences compared to the control group (P>0.05).

[0061] Table 3 Average indices of GLU, TP, ALB, BUN, TC, TG, ALT, and AST

[0062]

[0063] 2.4 Detection of growth performance indicators of Muscovy ducks

[0064] Table 4 shows the average daily feed intake and average daily weight gain of Muscovy ducks over 60 days. With the increase in fermented feed substitution, the average daily feed intake and average daily weight gain increased in all groups of Muscovy ducks. Compared with the control group, the 50% experimental group showed a 19.42% increase in final weight (P < 0.05), an 11.68% increase in average daily feed intake (P < 0.05), a 12.43% increase in average daily weight gain (P < 0.05), and a 0.87% decrease in feed conversion ratio (P < 0.05). No significant differences were observed in the other experimental groups (P > 0.05).

[0065] Table 4. Growth performance of Muscovy ducks after 60 days of feeding.

[0066]

[0067]

[0068] As can be seen from the above embodiments, the present invention provides a method for preparing fermented feed based on multiple probiotics, by partially replacing excipients with feces to produce fermented feed. This method removes harmful substances and odors from feces while simultaneously generating economically valuable feed, achieving two goals at once. Probiotics such as Bacillus subtilis can utilize the heat and acid produced during fermentation to inhibit and kill the growth of harmful bacteria, and can also effectively passivate heavy metals. The acid production also improves the palatability of the fermented feed, transforming foul-smelling manure into a feed with a pleasant acidic aroma. Using compound microbial preparations and feces to produce fermented feed can effectively reduce manure pollution and produce feed with high economic added value, showing great application potential in the livestock industry.

[0069] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a fermented feed based on a plurality of probiotic bacteria, characterized in that, The method comprises the following steps: S1, 0.1-0.2L wood vinegar, 0.2-0.8L bacteria agent I, 9.45kg auxiliary materials and 9.45kg fresh pig manure are mixed and stirred uniformly, and the water content is controlled at 50%; the mixture is heated, and the feed is continuously heated to 50-70℃ for fermentation for 12h to obtain the original fermented material; S2, 1.2L bacteria agent II is added to the original fermented material and mixed uniformly, the material is cooled to 10-40℃, and anaerobic sealing fermentation is carried out for 30h, and the fermented feed is obtained after fermentation.

2. The production method according to claim 1, characterized by, The ratio of the wood vinegar, bacteria agent I, auxiliary materials and fresh pig manure is 0.1-0.2L:0.2-0.8L:9.45kg:9.45kg.

3. The preparation method according to claim 1, characterized in that, The bacteria agent I is composed of Lactobacillus acidophilus, Bacillus thermophilus and Bacillus licheniformis fermentation broth mixed in a volume ratio of 2:1:

1. The bacteria agent II is composed of Lactobacillus acidophilus and Bacillus megaterium fermentation broth mixed in a volume ratio of 5:

2.

4. The production method according to claim 3, characterized by, The effective viable cell number of the Lactobacillus acidophilus is ≥10 9 CFU / mL; The effective viable cell number of the Bacillus thermophilus is ≥ 10 9 CFU / mL; The effective viable cell number of the said Bacillus licheniformis is ≥10 9 CFU / mL; The effective viable cell number of the Bacillus megaterium is ≥10 9 CFU / mL.

5. The preparation method according to claim 1, characterized in that, The auxiliary materials are composed of corn flour, bran and soybean meal mixed in a mass ratio of 2:1:

1. The pig manure is fresh pig manure with a water content of 60%-70%.

6. The method of claim 1, wherein, The wood vinegar is obtained by continuous pyrolysis of straw as raw material, starting at 30℃, heating to 500℃, and pyrolyzing for 2h, and the gas generated is condensed to obtain wood vinegar with a density of 0.97kg / L and an acid content of 28%.

7. A fermented feed based on multiple probiotics prepared by the method of any one of claims 1-6.

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