A composite microbial agent and its preparation method and application

Abstract

The present invention provides a composite microbial agent, a preparation method and application thereof, and relates to the technical field of composite microbial agents. The composite microbial agent of the present invention comprises, by weight, 3 to 6 parts of Lactobacillus plantarum, 3 to 6 parts of Pediococcus pentosaceus, 1 to 2 parts of Lactobacillus paracasei, 1 to 3 parts of Weissella mesenteroides, and 1 to 3 parts of Lactobacillus brevis. The composite microbial agent provided by the present invention has a fast growth rate and high acid production efficiency. Experiments in which the composite microbial agent provided by the present invention is used to prepare corncob silage show that the composite microbial agent can improve the smell, color and structure of corncob silage, reduce the crude fiber content and increase the crude fat content therein, and has a promoting effect on improving the quality of feed. The present invention verifies that adding corncob silage to the basic diet of pigs can improve the growth performance, slaughter performance and meat quality performance of pigs, reduce breeding costs and provide higher quality livestock products.

Description

Technical Field

[0001] The present invention belongs to the technical field of composite microbial agents, and specifically relates to a composite microbial agent, a preparation method thereof, and an application thereof. Background Art

[0002] Currently, pig farming is primarily based on fully enclosed, large-scale farming. Pig diets primarily consist of concentrated feed made from corn and soybean meal. However, this diet lacks crude fiber, which reduces the microbial diversity of the pig's intestinal flora and the number of probiotics, impacting pig health and leading to reduced pork quality. Corncobs contain 32%-36% cellulose, 35%-40% hemicellulose, and 17%-20% lignin. These large polysaccharides, such as cellulose, hemicellulose, and lignin, can only be broken down and absorbed by herbivores. Non-herbivores lack the intestinal flora that secrete cellulase, making them inaccessible. Existing technologies use microbial fermentation of corncobs, using enzymes produced during the fermentation process to degrade the cellulose, hemicellulose, and lignin in straw. However, improper microbial selection cannot completely degrade the polysaccharides in the corncobs, failing to provide a sufficient carbon source for animal growth. Furthermore, problems such as a longer fermentation cycle, reduced freshness of the silage, and a poorer taste can also occur. Therefore, there is an urgent need to provide a composite microbial agent to improve the quality of corn cob silage, thereby making it feasible to use corn cobs as ground-source feed in the fattening stage of local pigs. Summary of the Invention

[0003] In view of this, the object of the present invention is to provide a composite microbial agent, which has a fast growth rate, strong antibacterial and acid-producing properties, and can improve the silage quality of corncob mixtures.

[0004] Another object of the present invention is to provide a corn cob silage prepared by fermenting the composite microbial agent. The corn cob silage is added to the basic diet to improve the growth performance, slaughter performance and meat quality of experimental animals, reduce breeding costs and provide higher quality livestock products.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a composite microbial agent, which comprises, by weight, 3-6 parts of Lactobacillus plantarum, 3-6 parts of Pediococcus pentosaceus, 1-2 parts of Lactobacillus paracasei, 1-3 parts of Weissella mesenteroides, and 1-3 parts of Lactobacillus brevis.

[0007] Preferably, the plant lactobacillus is plant lactobacillus LL2022107, and the classification name of the plant lactobacillus LL2022107 is plant lactobacillus ( Lactobacillus plantarum ), the deposit number is CGMCC No.25862; the pentosaceae is pentosaceae LL2018368, and the classification name of the pentosaceae LL2018368 is pentosaceae ( Pediococcus pentosaceus ), the deposit number is CGMCC No.18709; the Lactobacillus paracasei is Lactobacillus paracasei LL2022059, and the classification name of the Lactobacillus paracasei LL2022059 is Lactobacillus paracasei ( Lactobacillus paracasei ), the deposit number is CGMCC No.25863; the Weissella mesenteroides is Weissella mesenteroides LL2018353, and the classification name of Weissella mesenteroides LL2018353 is Weissella mesenteroides ( Weissella paramesenteroides ), the deposit number is CGMCC No.18710; the Lactobacillus brevis is Lactobacillus brevis LL2022009, and the classification name of the Lactobacillus brevis LL2022009 is Lactobacillus brevis ( Lactobacillus brevis ), the deposit number is CGMCC No.25861.

[0008] Preferably, the effective viable count of each bacterial cell in the composite microbial agent is 10 10 ~10 11 CFU / g.

[0009] The present invention also provides a method for preparing the composite microbial agent, comprising the following steps: inoculating each bacterial strain into an MRS broth culture medium at a volume ratio of 0.05-0.2%, culturing at 35-37°C for 36-48 hours, and centrifuging at 5000-8000 rpm to obtain bacterial cells; freeze-drying the obtained bacterial cells and then mixing them by weight.

[0010] Preferably, the freeze-drying conditions are: freeze-drying at -60°C to -70°C and a pressure of 0.006 Pa for 10 to 15 hours.

[0011] The present invention also provides an application of the composite microbial agent in preparing feed.

[0012] The present invention also provides corn cob silage, which is prepared by mixing and fermenting the composite microbial agent and the corn cob mixture at a mass ratio of 1:8000-120000.

[0013] Preferably, the fermentation conditions are: fermentation at 25-28° C. for 20-25 days.

[0014] Preferably, the corncob mixture comprises corncobs and corn husks.

[0015] The present invention also provides a use of the corncob silage in improving animal growth performance, slaughter performance and / or meat quality performance.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The composite microbial agent of the present invention has a fast growth rate and strong antibacterial and acid-producing properties. After the composite microbial agent is co-fermented with a corncob mixture, the smell, color and structure of the fermented product can be improved, the crude fiber content can be reduced, and the crude fat content can be increased, thereby promoting the improvement of the fermentation quality and improving the silage quality of the corncob mixture.

[0018] The present invention utilizes composite microbial agent fermentation to obtain a corncob silage, which, when added to a basal diet, can improve the growth performance, slaughter performance, and meat quality performance of test animals, reduce breeding costs, and provide higher-quality livestock products. Specifically, compared with the control group, the present invention adds corncob silage to the basal diet to improve the terminal body weight, average daily weight gain, average daily feed intake, and gross income of test animals, resulting in better production and economic benefits. After adding corncob silage to the basal diet, the live weight before slaughter, slaughter rate, skin thickness, carcass straight length, carcass oblique length, pH value, marbling, and meat color can be increased, and drip loss, shear force, cooking loss, and water loss rate can be reduced. Compared with the control group, drip loss was reduced by 13.20%, shear force was reduced by 9.35%, cooking loss was reduced by 6.98%, and water loss rate was reduced by 53.77%. Adding corncob silage to the basal diet increased dry matter, crude protein, and crude fat in the longissimus dorsi muscle, with crude fat content increasing by 28.43%. Compared with the control group, adding corncob silage to the basal diet increased serum fasting blood glucose, total protein, globulin, phosphate, pancreatic amylase, and lipase, and decreased total bilirubin and cholesterol.

[0019] Biological Deposit Certificate

[0020] Lactobacillus plantarum LL2022107, classified as Lactobacillus plantarum, is deposited at the General Microbiology Center of China Culture Collection Administration (CGMCC), with the address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC No. 25862 and the deposit date: September 30, 2022.

[0021] Pediococcus pentosaceus LL2018368, classified as Pediococcus pentosaceus, deposited at the General Microbiology Center of China Culture Collection Administration (CGMCC), deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit number: CGMCC No. 18709, deposit date: October 21, 2019.

[0022] Lactobacillus paracasei LL2022059, classified as Lactobacillus paracasei, is deposited at the General Microbiology Center of China Culture Collection Administration (CGMCC), with the address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC No. 25863 and the deposit date: September 30, 2022.

[0023] Weissella paramesenteroides LL2018353, classified as Weissella paramesenteroides, is deposited at the General Microbiology Center of China Culture Collection Administration (CGMCC), with the address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC No. 18710 and the deposit date: October 21, 2019.

[0024] Lactobacillus brevis LL2022009, classified as Lactobacillus brevis, is deposited at the General Microbiology Center of China Culture Collection Administration (CGMCC), with the address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC No. 25861 and the deposit date: September 30, 2022. DETAILED DESCRIPTION

[0025] The present invention provides a composite microbial agent, which preferably comprises, by weight, 3-6 parts of Lactobacillus plantarum, 3-6 parts of Pediococcus pentosaceus, 1-2 parts of Lactobacillus paracasei, 1-3 parts of Weissella mesenteroides, and 1-3 parts of Lactobacillus brevis; more preferably, the composite microbial agent comprises, by weight, 4-5 parts of Lactobacillus plantarum, 4-5 parts of Pediococcus pentosaceus, 1.5-1.8 parts of Lactobacillus paracasei, 1.5-2.5 parts of Weissella mesenteroides, and 1.5-2.5 parts of Lactobacillus brevis; further preferably, the composite microbial agent is formed by mixing 5 parts of Lactobacillus plantarum, 4.5 parts of Pediococcus pentosaceus, 1.8 parts of Lactobacillus paracasei, 2 parts of Weissella mesenteroides, and 2.5 parts of Lactobacillus brevis.

[0026] In the present invention, the Lactobacillus plantarum is Lactobacillus plantarum LL2022107, with a deposit number of CGMCC No. 25862, the Pediococcus pentosaceus is Pediococcus pentosaceus LL2018368, with a deposit number of CGMCC No. 18709, the Lactobacillus paracasei is Lactobacillus paracasei LL2022059, with a deposit number of CGMCC No. 25863, the Weissella mesenteroides is Weissella mesenteroides LL2018353, with a deposit number of CGMCC No. 18710, and the Lactobacillus brevis is Lactobacillus brevis LL2022009, with a deposit number of CGMCC No. 25861. The above-mentioned microbial strains were all screened by the inventors and deposited in the General Microbiology Center of the China Culture Collection Administration Committee (abbreviated as: CGMCC). Among them, Pediococcus pentosaceus LL2018368 is disclosed in patent document ZL202110843467.2, and Weissella mesenteroides LL2018353 is disclosed in patent document ZL202010712678.8.

[0027] In the present invention, the effective viable count of each bacterial cell in the composite microbial agent is preferably 10 10 ~10 11 The composite microbial agent of the present invention has a fast growth rate, strong antibacterial and acid-producing properties, and can improve the silage quality of the corncob mixture.

[0028] The present invention also provides a method for preparing the composite microbial agent, preferably comprising the following steps: inoculating 0.05-0.2% of each bacterial species into MRS broth, culturing at 35-37°C for 36-48 hours, and centrifuging at 5000-8000 rpm to obtain bacterial cells; freeze-drying the obtained bacterial cells and then mixing them according to the above weight ratio to obtain the composite microbial agent. In the present invention, the culture conditions are adjusted according to the growth habits of each bacterial species. In the present invention, as an implementation method, the Lactobacillus plantarum is preferably cultured at 35°C for 36 hours, the Pediococcus pentosaceus is preferably cultured at 35°C for 48 hours, the Lactobacillus paracasei is preferably cultured at 35.5°C for 36 hours, the Weissella mesenteroides is preferably cultured at 35°C for 36 hours, and the Lactobacillus brevis is preferably cultured at 37°C for 35 hours. In the present invention, after centrifugation of the bacterial solution, a protective agent is preferably added, and the bacterial cells are freeze-dried using a freeze dryer. The present invention does not specifically limit the type of the protective agent, and protective agents well known in the art can be used. In the present invention, the freeze-drying conditions are preferably: freeze-drying at -60°C to -70°C and a pressure of 0.006 Pa for 10 to 15 hours; more preferably, the freeze-drying conditions are more preferably: freeze-drying at -65°C and a pressure of 0.006 Pa for 14 hours. In the present invention, the raw material ratio of the MRS broth medium is preferably as follows: 10.0g peptone; 5.0g beef extract; 4.0g yeast powder; 20.0g glucose; 2.0g dipotassium hydrogen phosphate; 2.0g triammonium citrate; 5.0g sodium acetate; 0.2g magnesium sulfate; 0.05g manganese sulfate; 1.0mL Tween 80; 1L water.

[0029] The present invention also provides an application of the composite microbial agent in preparing feed.

[0030] The present invention also provides corn cob silage, which is prepared by mixing and fermenting the composite microbial agent and the corn cob mixture preferably at a mass ratio of 1:8000-120000; more preferably at a mass ratio of 1:10000.

[0031] In the present invention, the corncob mixture preferably includes corncobs and corn husks.

[0032] In the present invention, the method for preparing corncob silage preferably comprises the following steps: mixing corncobs and corn husks in a weight ratio of 2-6:0-1, beating the mixture in a pulper to produce a corncob mixture; adjusting the moisture content of the corncob mixture with corn flour; and fermenting the mixture at 25-28°C for 20-25 days, more preferably at 25°C for 21 days. In the present invention, the particle size of the corncob mixture is preferably 0.5 cm to 1 cm. In the present invention, the corn flour is conventional feed corn flour, preferably purchased from Yunnan University Feed Co., Ltd. The corn flour is preferably added in an amount of 3% to 27.69% by volume.

[0033] The present invention also provides a use of the corncob silage in improving animal growth performance, slaughter performance and / or meat quality performance. Compared with the control group, the addition of corncob silage to the basal diet of the present invention can increase the terminal body weight, average daily weight gain, average daily feed intake and gross income of the test animals, bringing better production and economic benefits. After adding corncob silage to the basal diet, the live weight before slaughter, slaughter rate, skin thickness, carcass straight length, carcass oblique length, pH value, marbling and meat color can be increased, and drip loss, shear force, cooking loss and water loss rate can be reduced. Compared with the control group, drip loss was reduced by 13.20%, shear force was reduced by 9.35%, cooking loss was reduced by 6.98%, and water loss rate was reduced by 53.77%. After adding corncob silage to the basal diet, the dry matter, crude protein and crude fat in the longissimus dorsi muscle can be increased, among which the crude fat content can be increased by 28.43%. Compared with the control group, the addition of corncob silage to the basal diet increased serum fasting blood glucose, total protein, globulin, phosphate ions, pancreatic amylase, and lipase levels, and decreased total bilirubin and cholesterol levels. In summary, the present invention improves the growth performance, slaughter performance, and meat quality of experimental animals by adding corncob silage to the basal diet, reducing breeding costs and providing higher-quality livestock products.

[0034] The technical solutions provided by the present invention are 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.

[0035] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0036] Example 1

[0037] A composite microbial agent is prepared by mixing 5 parts of Lactobacillus plantarum, 4.5 parts of Pediococcus pentosaceus, 1.8 parts of Lactobacillus paracasei, 2 parts of Weissella mesenteroides, and 2.5 parts of Lactobacillus brevis;

[0038] In the above-mentioned composite microbial agent, the Lactobacillus plantarum is Lactobacillus plantarum LL2022107, with a preservation number of CGMCC No. 25862, the Pediococcus pentosaceus is Pediococcus pentosaceus LL2018368, with a preservation number of CGMCC No. 18709, the Lactobacillus paracasei is Lactobacillus paracasei LL2022059, with a preservation number of CGMCC No. 25863, the Weissella mesenteroides is Weissella mesenteroides LL2018353, with a preservation number of CGMCC No. 18710, and the Lactobacillus brevis is Lactobacillus brevis LL2022009, with a preservation number of CGMCC No. 25861.

[0039] The preparation method of the above-mentioned composite microbial agent is as follows: each bacterial species is inoculated into MRS broth culture medium at a volume ratio of 0.1%, Lactobacillus plantarum is cultured at 35°C for 36 hours, Pediococcus pentosaceus is cultured at 35°C for 48 hours, Lactobacillus paracasei is cultured at 35.5°C for 36 hours, Weissella mesenteroides is cultured at 35°C for 36 hours, and Lactobacillus brevis is cultured at 37°C for 35 hours; after the cultivation of each bacterial species is completed, the bacteria are centrifuged at 8000 rpm to obtain each bacterial cell; the obtained bacterial cells are freeze-dried at -65°C and a pressure of 0.006 Pa for 14 hours and then mixed in proportion to obtain the composite microbial agent.

[0040] Example 2

[0041] A composite microbial agent is prepared by mixing 3 parts of Lactobacillus plantarum, 3 parts of Pediococcus pentosaceus, 1 part of Lactobacillus paracasei, 3 parts of Weissella mesenteroides, and 3 parts of Lactobacillus brevis;

[0042] In the above-mentioned composite microbial agent, the Lactobacillus plantarum is Lactobacillus plantarum LL2022107, with a preservation number of CGMCC No. 25862, the Pediococcus pentosaceus is Pediococcus pentosaceus LL2018368, with a preservation number of CGMCC No. 18709, the Lactobacillus paracasei is Lactobacillus paracasei LL2022059, with a preservation number of CGMCC No. 25863, the Weissella mesenteroides is Weissella mesenteroides LL2018353, with a preservation number of CGMCC No. 18710, and the Lactobacillus brevis is Lactobacillus brevis LL2022009, with a preservation number of CGMCC No. 25861.

[0043] The preparation method of the composite microbial agent is the same as that in Example 1.

[0044] Example 3

[0045] A composite microbial agent is prepared by mixing 6 parts of Lactobacillus plantarum, 6 parts of Pediococcus pentosaceus, 2 parts of Lactobacillus paracasei, 3 parts of Weissella mesenteroides, and 3 parts of Lactobacillus brevis;

[0046] In the above-mentioned composite microbial agent, the Lactobacillus plantarum is Lactobacillus plantarum LL2022107, with a preservation number of CGMCC No. 25862, the Pediococcus pentosaceus is Pediococcus pentosaceus LL2018368, with a preservation number of CGMCC No. 18709, the Lactobacillus paracasei is Lactobacillus paracasei LL2022059, with a preservation number of CGMCC No. 25863, the Weissella mesenteroides is Weissella mesenteroides LL2018353, with a preservation number of CGMCC No. 18710, and the Lactobacillus brevis is Lactobacillus brevis LL2022009, with a preservation number of CGMCC No. 25861.

[0047] The preparation method of the composite microbial agent is the same as that in Example 1.

[0048] Example 4

[0049] A corncob silage is provided, wherein the corncob silage is obtained by mixing and fermenting the composite microbial agent of Example 1 and a corncob mixture at a mass ratio of 1:10000; the corncob mixture is obtained by mixing corncobs and corn husks at a weight ratio of 6:1.

[0050] The preparation method of the above-mentioned corn cob silage is as follows: corn cobs and corn husks are beaten with a pulper to obtain a corn cob mixture with a particle size of 0.5-1 cm, 27.69% corn flour is added by volume to adjust the moisture, and then fermented at 25°C for 21 days.

[0051] Example 5

[0052] A corncob silage is provided, wherein the corncob silage is obtained by mixing and fermenting the composite microbial agent of Example 1 and a corncob mixture at a mass ratio of 1:10000; the corncob mixture is obtained by mixing corncobs and corn husks at a weight ratio of 6:1.

[0053] The preparation method of the above-mentioned corn cob silage is as follows: corn cobs and corn husks are beaten with a pulper to obtain a corn cob mixture with a particle size of 0.5~1 cm, 22.96% corn flour is added by volume to adjust the moisture, and then fermented at 25°C for 21 days.

[0054] Example 6

[0055] A corncob silage is provided, wherein the corncob silage is obtained by mixing and fermenting the composite microbial agent of Example 1 and a corncob mixture at a mass ratio of 1:10000; the corncob mixture is obtained by mixing corncobs and corn husks at a weight ratio of 6:1.

[0056] The preparation method of the above-mentioned corn cob silage is as follows: corn cobs and corn husks are beaten with a pulper to obtain a corn cob mixture with a particle size of 0.5-1 cm, 18.57% corn flour is added by volume to adjust the moisture, and then fermented at 25°C for 21 days.

[0057] Example 7

[0058] A corncob silage is provided, wherein the corncob silage is obtained by mixing and fermenting the composite microbial agent of Example 1 and a corncob mixture at a mass ratio of 1:10000; the corncob mixture is obtained by mixing corncobs and corn husks at a weight ratio of 6:1.

[0059] The preparation method of the above-mentioned corn cob silage is as follows: corn cobs and corn husks are beaten with a pulper to obtain a corn cob mixture with a particle size of 0.5-1 cm, 14.48% corn flour is added by volume to adjust the moisture, and then fermented at 25°C for 21 days.

[0060] Example 8

[0061] A corncob silage is provided, wherein the corncob silage is obtained by mixing and fermenting the composite microbial agent of Example 1 and a corncob mixture at a mass ratio of 1:10000; the corncob mixture is obtained by mixing corncobs and corn husks at a weight ratio of 6:1.

[0062] The preparation method of the above-mentioned corn cob silage is as follows: corn cobs and corn husks are beaten with a beater to obtain a corn cob mixture with a particle size of 0.5~1 cm, 10.66% corn flour is added by volume to adjust the moisture, and then fermented at 25°C for 21 days.

[0063] Example 9

[0064] A corncob silage is provided, wherein the corncob silage is obtained by mixing and fermenting the composite microbial agent of Example 1 and a corncob mixture at a mass ratio of 1:10000; the corncob mixture is obtained by mixing corncobs and corn husks at a weight ratio of 6:1.

[0065] The preparation method of the above-mentioned corn cob silage is as follows: corn cobs and corn husks are beaten with a beater to obtain a corn cob mixture with a particle size of 0.5-1 cm, 7.10% corn flour is added by volume to adjust the moisture, and then fermented at 25°C for 21 days.

[0066] Example 10

[0067] A corncob silage is provided, wherein the corncob silage is obtained by mixing and fermenting the composite microbial agent of Example 1 and a corncob mixture at a mass ratio of 1:10000; the corncob mixture is obtained by mixing corncobs and corn husks at a weight ratio of 6:1.

[0068] The preparation method of the above-mentioned corn cob silage is as follows: corn cobs and corn husks are beaten with a pulper to obtain a corn cob mixture with a particle size of 0.5-1 cm, 3.75% corn flour is added by volume to adjust the moisture, and then fermented at 25°C for 21 days.

[0069] Test Example 1

[0070] Corn cobs and corn husks were pulped (0.5-1 cm) using a pulping machine. Corn flour was used for moisture adjustment (corn flour was added according to the ratios in Examples 4-10). The husks were randomly divided into 8 groups (7 experimental groups and 1 control group), with 6 replicates per group, and 3 barrels per replicate, each weighing approximately 50 kg. 100 g of the composite microbial agent prepared in Example 1 was added to each ton of corn cob mixture (100 g of composite microbial agent was made into 1500 mL of suspension). The trial lasted 21 days, from August 16 to September 6, 2024. The control group was treated with the same volume of water, while the experimental group was treated with the additive prepared in Example 1 (made into a suspension according to the ratios). Fermentation was performed at room temperature for a total of 21 days. The silage performance was evaluated at the end of the trial.

[0071] Table 1 Field evaluation scores of corncob mixtures with composite microbial fermentation agents for one day

[0072]

[0073] Table 2 Field evaluation scores of corncob mixtures with composite microbial fermentation agents for 21 days

[0074]

[0075] The effect of adding compound microbial agents on corn cob silage is shown in Tables 1 and 2: After adding compound microbial agents, the smell, color and structure of corn cob silage were improved, and ultimately the product quality was improved.

[0076] The effects of adding composite microbial agents on the conventional component indicators of corncob mixture are shown in Table 3:

[0077] Table 3 General component analysis

[0078]

[0079] The results in Table 3 show that adding the composite microbial agent to the corncob mixture can reduce the crude fiber content and increase the crude fat content, which has the potential to improve feed utilization.

[0080] Test Example 2

[0081] This study employed a single-factor experimental design. Twelve Yodian pigs (6 males and 6 females) weighing approximately 50 kg and approximately 117 days old were selected. Pre-experimental ear marks and weights were recorded. Based on the principle of similar weight and an equal distribution of males and females, the pigs were randomly divided into two groups, each containing one replicate of six pigs: a control group (3 males, 3 females) and an experimental group (3 males, 3 females). The control group was fed a basal diet; the experimental group was fed a basal diet supplemented with corncob silage prepared in Example 4. The experimental period for this study was 119 days, consisting of a 7-day pre-feeding period and a 112-day main experiment.

[0082] The experiment is divided into three phases:

[0083] 50kg~70kg: The control group was fed a basal diet; the experimental group was fed 80% of the basal diet (supplemented based on the control group's feed intake the day before the experiment), and corncob silage was available ad libitum.

[0084] 70kg~95kg: The control group was fed a basic diet; the experimental group was fed 70% of the basic diet (supplemented based on the control group's feed intake the day before the experiment), and corncob silage was available ad libitum.

[0085] 95kg~120kg: The control group was fed a basal diet; the experimental group was fed 60% of the basal diet (supplemented based on the control group's feed intake the day before the experiment), and corncob silage was available ad libitum.

[0086] The specific test plan is as follows:

[0087] 1. Experimental location: Pig Breeding Institute, Yunnan Academy of Animal Husbandry and Veterinary Sciences

[0088] 2. Test materials: 12 Dian pigs (6 males and 6 females) weighing approximately 50 kg; 2 sets of farmer-type breeding houses

[0089] 3. Test method:

[0090] (1) Twelve Dian pigs (6 males and 6 females) weighing about 50 kg (about 117 days old) were selected. Their ears were marked and their weights were recorded before the experiment. They were randomly divided into 4 groups based on the principle of close weight. There were 6 pigs in the control group (3 males and 3 females) and 6 pigs in the experimental group (3 males and 3 females).

[0091] (2) The experiment is divided into three stages:

[0092] ① During the 50kg-70kg stage, the control group was fed the basal diet, which was added and recorded at 8:30 every morning (based on the previous day's feeding situation, theoretically, 0.25kg of residual feed should be kept in the feed trough). The remaining feed was cleaned and weighed before feeding. At 8:30 every morning, the experimental group was fed a mixture of corncob silage and basal diet (8:2) according to the control group's basal diet intake the previous day. The feeding situation was observed in time, and the basal diet and corncob silage intake of the experimental group on that day were recorded.

[0093] ② During the 70kg-95kg stage, the control group was fed the basal diet, which was added and recorded at 8:30 every morning (based on the previous day's feeding situation, theoretically, 0.25kg of residual feed should be kept in the feed trough). The remaining feed was cleaned and weighed before feeding. At 8:30 every morning, the experimental group was fed a mixture of corncob silage and basal diet (7:3) according to the control group's basal diet intake the previous day. The feeding situation was observed in time, and the basal diet and corncob silage intake of the experimental group on that day were recorded.

[0094] ③ During the 95kg-120kg stage, the control group was fed the basal diet, which was added and recorded at 8:30 every morning (based on the previous day's feeding situation, theoretically, 0.25kg of residual feed should be kept in the feed trough). The remaining feed was cleaned and weighed before feeding. At 8:30 every morning, the experimental group was fed a mixture of corncob silage and basal diet (6:4) according to the control group's basal diet intake the previous day. The feeding situation was observed in time, and the basal diet and corncob silage intake of the experimental group on that day were recorded.

[0095] Different batches of processed feed must be sampled for analysis of conventional components, and a sample of no less than 500g must be retained. The basic diet formula is shown in Table 4:

[0096] Table 4 Basic diet formula

[0097]

[0098] (3) Adopt free feeding and free drinking methods; observe the diarrhea situation every day. At 09:00 and 16:00 every day, add feed and record the weight of added feed and the weight of leftover feed, and record the diarrhea situation. When recording the number of diarrheal animals, the severity of diarrhea should also be recorded. According to the method of Marquardt et al., diarrhea is divided into 4 levels.

[0099] 0 points: strips or granules; 1 point: soft feces, formed; 2 points: thick, unformed, feces and liquid are not separated; 3 points: liquid, unformed, feces and liquid are separated.

[0100] When the fecal score was 2 points or above, the pigs were considered to have diarrhea, and the diarrhea rate of each group was calculated at the end of the experiment.

[0101] (4) The experimental period was 112 days. Feed was plated and weighed at the beginning of the experiment and on days 28, 56, 84, and 112. Average daily weight gain, average daily feed intake, feed-to-weight ratio, and cost per kg of weight gain were calculated.

[0102] The gas quality at the height of the pigs in the experimental pigpen was observed. The gas composition (NH3, H2S, CO2 and CH4) and volatile organic compounds (VOCs) were measured at three points at each height. Fresh feces were collected and stored in liquid nitrogen (3 × 200 mg per group) and at -20°C (500 g per group).

[0103] Fresh fecal samples were collected from each pig's rectum by stimulating it with a cotton swab. Six samples were collected from each cycle using 5 mL cryovials.

[0104] After thoroughly mixing the fecal samples from each pen of pigs, 300 g was taken, and after removing the hair, 10 mL of 10% hydrochloric acid was added and mixed thoroughly. The feces were stored in a -20°C refrigerator for testing. Conventional components (crude protein, total nitrogen, moisture, crude fiber, crude fat) and metabolites (total biogenic amines, cadaverine, tryptamine, phenol, p-cresol, and skatole) were tested.

[0105] (5) During the experiment, the health of the experimental pigs was observed every day.

[0106] (6) After the experiment, three animals (two males and one female) were selected from each group for slaughter and slaughter performance evaluation. The longissimus dorsi muscle was collected for meat quality determination and routine component analysis. Volatile flavor compounds in the longissimus dorsi muscle were detected using two-dimensional gas chromatography-time of flight mass spectrometry (GC×GC-TOF-MS) analysis technology. 10 mL of anterior vena cava blood was collected and the blood was allowed to stand at low temperature for 30 minutes. The blood was then centrifuged at 3000 rpm for 10 minutes at 4°C to obtain serum. The serum was aliquoted and stored in a -20°C refrigerator for later use. Routine blood analysis was performed using an animal biochemical analyzer.

[0107] 4. Statistical analysis

[0108] The experimental data were analyzed using the one-way ANOVA module in the SPSS 20.0 software package, and Tukey's multiple comparisons were used for significant differences. All results are expressed as mean ± standard error. P < 0.01 indicates an extremely significant difference, and P < 0.05 indicates a significant difference.

[0109] 5. Measurement and analysis

[0110] (1) The dry matter and moisture contents were determined in accordance with the Determination of Moisture in Foods (GB 5009.3-2016), the ash content was determined in accordance with the Determination of Ash in Foods (GB 5009.4-2016), the crude protein content was determined in accordance with the Determination of Protein in Foods (GB 5009.5-2016), and the fat content was determined in accordance with the Determination of Fat in Foods (GB 5009.6-2016).

[0111] (2) Production performance measurement

[0112] ① Feed intake and weight: Daily records were kept of each group's feed intake and health status. Feed was collected and weighed (on an empty stomach after a morning feed) on days 1, 15, 29, 43, 57, 71, and 98 of the experimental period. The data were compiled. Feed intake and weight gain were calculated for each group. The effects of the diet on body weight and production performance were evaluated.

[0113] ②Calculate the average daily feed intake (ADFI), average daily weight gain and feed-to-weight ratio, and draw a growth curve.

[0114] (3) Slaughter test

[0115] On the day the experiment ended, three pigs (two males and one female) were selected from each group for slaughter and testing. The test indicators were as follows:

[0116] ① Slaughter performance indicators such as live weight before slaughter, carcass weight, lean meat rate, intramuscular fat, etc.

[0117] ② Meat quality (longissimus dorsi muscle): cooked meat rate, shear force, meat color, pH value, drip loss, marbling;

[0118] ③ Serum biochemical indicators: 5 mL of blood was collected and placed in a coagulant tube. After standing at room temperature for 30 min, it was centrifuged at 3000 rpm for 15 min to separate the serum. The serum was divided into EP tubes and stored at -20°C.

[0119] ④ Digestive tract contents: cecal contents (microbial diversity, non-targeted metabolomics, short-chain fatty acids)

[0120] ⑤ Chemical analysis (longissimus dorsi muscle): crude fat, crude protein, amino acids, fatty acids;

[0121] ⑥ Tissue sections: small intestine, subcutaneous fat, abdominal fat and subcutaneous fat

[0122] ⑦ Content of three nucleotides (inosinic acid, adenylic acid, guanylic acid)

[0123] ⑧ Volatile flavor detection: Two-dimensional gas chromatography-time of flight mass spectrometry (GC×GC-TOF-MS) analysis technology was used to detect volatile flavor compounds in the longissimus dorsi muscle.

[0124] 6. Test results

[0125] (1) Analysis of growth performance results

[0126] Table 5 Growth performance (50~70kg)

[0127]

[0128] Note: Different letters in a and b in the table indicate significant differences at the P<0.05 level

[0129] Table 5 shows that, compared with the control group, adding 20% corncob silage to the diet of Dian pigs weighing 50-70 kg increased the final body weight of the experimental animals and reduced the feed-to-gain ratio. Compared with the control group, the experimental group increased gross profit (daily weight gain per pig × 17 yuan / kg - daily feed cost per pig, with corncob silage calculated at 0.6 yuan / kg) by 55.81%, reduced the cost per kilogram of pig production by 17.49%, and reduced the feed-to-gain ratio by 16.04%. Therefore, adding 20% corncob silage to the diet at 50-70 kg has better production and economic benefits.

[0130] Table 6 Growth performance (70~95kg)

[0131]

[0132] Table 6 shows that adding 30% corncob silage to the diet of Dian pigs between 70 and 95 kg increased their final body weight, average daily gain (ADG), average daily feed intake (ADFI), and gross income (daily weight gain per pig × 17 yuan / kg - daily feed cost per pig) while reducing feed-to-weight ratio and cost. Compared with the control group, the 30% corncob supplementation group increased gross income (daily weight gain per pig × 17 yuan / kg - daily feed cost per pig) by 213.24% and ADG by 41.69%, while reducing feed-to-weight ratio by 25.64%. The cost per kilogram of pig produced was reduced by 27.05%. Therefore, adding 30% corncob silage to the diet between 70 and 95 kg can improve production and economic benefits.

[0133] Table 7 Growth performance (95~120kg)

[0134]

[0135] Table 7 shows that adding 40% corncob silage to the diet of Dian pigs between 90 and 120 kg increased final body weight, average daily gain, average daily feed intake, and gross income (daily weight gain per pig × 17 yuan / kg - daily feed cost per pig) while reducing feed-to-weight ratio and production costs compared to the control group. Compared to the control group, the 40% corncob silage supplementation group increased gross income by 153.09% and average daily gain by 38.34%, while reducing feed-to-weight ratio by 14.69% and production costs (per kilogram of pig) by 18.57%. Therefore, adding 40% corncob silage to the diet of Dian pigs between 90 and 120 kg can improve production and economic benefits.

[0136] Table 8 Slaughter performance and meat quality analysis

[0137]

[0138] Table 8 shows that the addition of corncob silage to the diet increased live weight before slaughter, dressing percentage, skin thickness, carcass straightness, carcass obliqueness, pH, marbling, and meat color, while reducing drip loss, shear force, cooking loss, and water loss. Compared with the control group, drip loss was reduced by 13.20%, shear force was reduced by 9.35%, cooking loss was reduced by 6.98%, and water loss was reduced by 53.77%. Compared with the control group, the addition of corncob silage to the diet significantly improved slaughter performance and meat quality in Dian pigs.

[0139] Table 9 Analysis of routine components of the longissimus dorsi muscle

[0140]

[0141] As shown in Table 9, compared with the control group, adding corncob silage to the diet can increase the dry matter, crude protein and crude fat in the longissimus dorsi muscle, among which the crude fat content can be increased by 28.43%.

[0142] Table 10 Serum biochemical analysis

[0143]

[0144] As shown in Table 10, compared with the control group, the addition of corncob fermentation products to the feed can increase the levels of fasting blood glucose, total protein, globulin, phosphorus ion, pancreatic amylase and lipase in serum, and reduce the levels of total bilirubin and cholesterol, among which the cholesterol content decreased significantly.

[0145] In summary, the corncob silage prepared by fermentation of the composite microbial agent of the present invention can be added to the basal diet to improve the growth performance, slaughter performance and meat quality performance of Dian pigs, reduce breeding costs and provide higher quality livestock products.

[0146] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A corncob silage, characterized in that: The composite microbial agent and corn cob mixture are mixed and fermented at a mass ratio of 1:10000; The corncob mixture is obtained by mixing corncobs and corn husks in a weight ratio of 6:1; The composite microbial agent comprises, by weight, 3 to 6 parts of Lactobacillus plantarum, 3 to 6 parts of Pediococcus pentosaceus, 1 to 2 parts of Lactobacillus paracasei, 1 to 3 parts of Weissella mesenteroides, and 1 to 3 parts of Lactobacillus brevis; The plant lactobacillus is plant lactobacillus LL2022107, and the classification name of the plant lactobacillus LL2022107 is plant lactobacillus ( Lactobacillus plantarum ), the deposit number is CGMCC No.25862; the pentosaceae is pentosaceae LL2018368, and the classification name of the pentosaceae LL2018368 is pentosaceae ( Pediococcus pentosaceus ), the deposit number is CGMCC No.18709; The Lactobacillus paracasei is Lactobacillus paracasei LL2022059, and the classification name of the Lactobacillus paracasei LL2022059 is Lactobacillus paracasei ( Lactobacillus paracasei ), the deposit number is CGMCC No.25863; the Weissella mesenteroides is Weissella mesenteroides LL2018353, and the classification name of Weissella mesenteroides LL2018353 is Weissella mesenteroides ( Weissella paramesenteroides ), the deposit number is CGMCC No.18710; the Lactobacillus brevis is Lactobacillus brevis LL2022009, and the classification name of the Lactobacillus brevis LL2022009 is Lactobacillus brevis ( Lactobacillus brevis ), the deposit number is CGMCC No.25861; The preparation method of the above-mentioned corn cob silage is as follows: corn cobs and corn husks are beaten with a pulper to obtain a corn cob mixture with a particle size of 0.5-1 cm, 27.69% corn flour is added by volume to adjust the moisture, and the mixture is fermented at 25°C for 21 days.

2. The corncob silage according to claim 1, characterized in that The effective viable bacteria count of each bacterial cell in the composite microbial agent is 10 10 ~10 11 CFU / g.

3. The corncob silage according to any one of claims 1 to 2, characterized in that The method comprises the following steps: inoculating each bacterial strain into an MRS broth culture medium at a volume ratio of 0.05-0.2%, culturing at 35-37°C for 36-48 hours, and centrifuging at 5000-8000 rpm to obtain bacterial bodies; and freeze-drying the obtained bacterial bodies and then mixing them according to weight.

4. The corncob silage according to claim 3, characterized in that The freeze-drying conditions are: freeze-drying at -60°C to -70°C and a pressure of 0.006 Pa for 10 to 15 hours.

5. Use of the corncob silage according to any one of claims 1 to 4 in improving pig growth performance, slaughter performance and / or meat quality performance.

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