Compound microbial agent as well as preparation method and application thereof

Abstract

The invention provides a compound microbial agent as well as a preparation method and application thereof, and relates to the technical field of compound microbial agents. The compound microbial agent disclosed by the invention is prepared from the following components in parts 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 compound microbial agent provided by the invention is high in growth speed and high in acid production efficiency. Experiments of using the compound microbial agent provided by the invention for preparing the corncob silage show that the compound microbial agent can improve the smell, color and structure of the corncob silage, can reduce the content of crude fibers in the corncob silage and increase the content of crude fat in the corncob silage, and has a promoting effect on improvement of feed quality. The invention verifies that after the corncob silage is added into the basic ration of the pigs, the growth performance, the slaughter performance and the meat quality performance of the pigs can be improved, the breeding cost is reduced, and animal products with higher quality are provided.

Description

Technical Field

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

[0002] At present, the pig breeding mode is mainly large-scale closed breeding, and the pig diet is mainly composed of concentrated feed processed from corn and soybean meal. However, the pig diet lacks crude fiber, which reduces the microbial diversity of the pig intestinal flora and the number of probiotics, affecting the health status of pigs and thus leading to a decline in pork quality. Corncobs contain 32% - 36% cellulose, 35% - 40% hemicellulose, and 17% - 20% lignin. Macromolecular polysaccharides such as cellulose, hemicellulose, and lignin can only be decomposed and absorbed by herbivores. The intestinal wall of non-herbivorous animals lacks the flora that secretes cellulase, so it cannot be absorbed and utilized. In the prior art, microbial fermentation of corncobs is used to degrade cellulose, hemicellulose, and lignin in straw through the biological enzymes produced during the fermentation process. However, improper selection of microorganisms cannot completely degrade the polysaccharides in corncobs and cannot provide sufficient carbon sources for animal growth. In addition, problems such as a longer fermentation period, a decrease in the freshness of silage feed, and a worse taste will also occur. Therefore, there is an urgent need to provide a compound microbial agent to improve the quality of corncob silage feed, and thus make it feasible to use corncobs as a local feed in the fattening stage of local pigs. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a compound 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 purpose of the present invention is to provide a corncob silage feed fermented by using the compound microbial agent. Adding this corncob silage feed to the basal diet can improve the growth performance, slaughter performance, and meat quality performance of experimental animals, reduce the breeding cost, and provide higher-quality livestock products.

[0005] In order to achieve the above invention purposes, the present invention provides the following technical solutions: The present invention provides a compound microbial agent, which includes 3 - 6 parts by weight of Lactobacillus plantarum, 3 - 6 parts by weight of Pediococcus pentosaceus, 1 - 2 parts by weight of Lactobacillus paracasei, 1 - 3 parts by weight of Weissella paramesenteroides, and 1 - 3 parts by weight of Lactobacillus brevis.

[0006] Preferably, the Lactobacillus plantarum is Lactobacillus plantarum LL2022107, and the taxonomic name of the Lactobacillus plantarum LL2022107 is Lactobacillus plantarum ( Lactobacillus plantarum), with the preservation number of CGMCC No. 25862; the Pediococcus pentosaceus is Pediococcus pentosaceus LL2018368, and the taxonomic naming of the Pediococcus pentosaceus LL2018368 is Pediococcus pentosaceus ( Pediococcus pentosaceus ), with the preservation number of CGMCC No. 18709; the Lactobacillus paracasei is Lactobacillus paracasei LL2022059, and the taxonomic naming of the Lactobacillus paracasei LL2022059 is Lactobacillus paracasei ( Lactobacillus paracasei ), with the preservation number of CGMCC No. 25863; the Weissella paramesenteroides is Weissella paramesenteroides LL2018353, and the taxonomic naming of the Weissella paramesenteroides LL2018353 is Weissella paramesenteroides ( Weissella paramesenteroides ), with the preservation number of CGMCC No. 18710; the Lactobacillus brevis is Lactobacillus brevis LL2022009, and the taxonomic naming of the Lactobacillus brevis LL2022009 is Lactobacillus brevis ( Lactobacillus brevis ), with the preservation number of CGMCC No. 25861.

[0007] Preferably, the effective viable count of each bacterium in the compound microbial inoculant is 10 10 ~10 11 CFU / g.

[0008] The present invention also provides a preparation method of the compound microbial inoculant, comprising the following steps: inoculating each strain into MRS broth medium according to a volume ratio of 0.05~0.2%, culturing at 35~37°C for 36~48 h, and centrifuging at 5000~8000 rpm to obtain bacteria; freeze-drying the obtained bacteria and mixing them according to parts by weight.

[0009] Preferably, the conditions for freeze-drying are: freeze-drying at -60°C to -70°C and a pressure of 0.006 Pa for 10~15 h.

[0010] The present invention also provides an application of the compound microbial inoculant in the preparation of feed.

[0011] The present invention also provides a corn cob silage feed, which is prepared by fermenting the mixture of the compound microbial inoculant and corn cob according to a mass ratio of 1:8000~120000.

[0012] Preferably, the conditions for fermentation are: fermenting at 25~28°C for 20~25 days.

[0013] Preferably, the corn cob mixture includes corn cob and corn husk.

[0014] The present invention also provides an application of the corncob silage feed in improving the growth performance, slaughter performance and / or meat quality performance of animals.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The compound microbial inoculum of the present invention has a fast growth rate, strong antibacterial and acid-producing properties; after co-fermenting the compound microbial inoculum with the corncob mixture, it can improve the odor, color and structure of the fermented product, reduce the content of crude fiber therein, increase the content of crude fat therein, promote the improvement of the fermentation quality, and improve the silage quality of the corncob mixture.

[0016] The present invention obtains a corncob silage feed by fermenting with a compound microbial inoculum. Adding this corncob silage feed to the basal diet can improve the growth performance, slaughter performance and meat quality performance of experimental animals, reduce the breeding cost and provide higher-quality livestock products. Specifically, compared with the control group, adding the corncob silage feed to the basal diet in the present invention can increase the final body weight, average daily gain, average daily feed intake and gross income of experimental animals, bringing better production and economic benefits. After adding the corncob silage feed to the basal diet, it can increase the live weight before slaughter, slaughter rate, skin thickness, carcass straight length, carcass diagonal length, pH value, marbling and meat color, and reduce the drip loss, shear force, cooking loss and water loss rate. Compared with the control group, the drip loss is reduced by 13.20%, the shear force is reduced by 9.35%, the cooking loss is reduced by 6.98%, and the water loss rate is reduced by 53.77%. After adding the corncob silage feed to the basal diet, it can increase the dry matter, crude protein and crude fat in the longissimus dorsi muscle, and the content of crude fat can be increased by 28.43%. Compared with the control group, after adding the corncob silage feed to the basal diet, it can increase the contents of fasting blood glucose, total protein, globulin, phosphate ion, pancreatic amylase and lipase in the serum, and reduce the contents of total bilirubin and cholesterol.

[0017] Biological preservation certificate Lactobacillus plantarum LL2022107, taxonomically named Lactobacillus plantarum, preservation unit: China General Microbiological Culture Collection Center (abbreviated as CGMCC), address of the preservation unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, preservation number: CGMCC No. 25862, preservation date: September 30, 2022.

[0018] Pediococcus pentosaceus LL2018368, classified and named as Pediococcus pentosaceus, depositary institution: General Microbiology Center of China Committee for Culture Collection of Microorganisms (abbreviated as CGMCC), address of the depositary institution: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, deposit number: CGMCC No. 18709, deposit date: October 21, 2019.

[0019] Lactobacillus paracasei LL2022059, classified and named as Lactobacillus paracasei, depositary institution: General Microbiology Center of China Committee for Culture Collection of Microorganisms (abbreviated as CGMCC), address of the depositary institution: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, deposit number: CGMCC No. 25863, deposit date: September 30, 2022.

[0020] Weissella paramesenteroides LL2018353, classified and named as Weissella paramesenteroides, depositary institution: General Microbiology Center of China Committee for Culture Collection of Microorganisms (abbreviated as CGMCC), address of the depositary institution: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, deposit number: CGMCC No. 18710, deposit date: October 21, 2019.

[0021] Lactobacillus brevis LL2022009, classified and named as Lactobacillus brevis, depositary institution: General Microbiology Center of China Committee for Culture Collection of Microorganisms (abbreviated as CGMCC), address of the depositary institution: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, deposit number: CGMCC No. 25861, deposit date: September 30, 2022. Detailed implementation mode

[0022] The present invention provides a compound microbial inoculant. Preferably, the compound microbial inoculant comprises 3 - 6 parts by weight of Lactobacillus plantarum, 3 - 6 parts by weight of Pediococcus pentosaceus, 1 - 2 parts by weight of Lactobacillus paracasei, 1 - 3 parts by weight of Weissella paramesenteroides, and 1 - 3 parts by weight of Lactobacillus brevis; more preferably, the compound microbial inoculant comprises 4 - 5 parts by weight of Lactobacillus plantarum, 4 - 5 parts by weight of Pediococcus pentosaceus, 1.5 - 1.8 parts by weight of Lactobacillus paracasei, 1.5 - 2.5 parts by weight of Weissella paramesenteroides, and 1.5 - 2.5 parts by weight of Lactobacillus brevis; further preferably, the compound microbial inoculant is formed by mixing 5 parts by weight of Lactobacillus plantarum, 4.5 parts by weight of Pediococcus pentosaceus, 1.8 parts by weight of Lactobacillus paracasei, 2 parts by weight of Weissella paramesenteroides, and 2.5 parts by weight of Lactobacillus brevis.

[0023] In the present invention, the Lactobacillus plantarum is Lactobacillus plantarum LL2022107, with the deposit number of CGMCC No.25862; the Pediococcus pentosaceus is Pediococcus pentosaceus LL2018368, with the deposit number of CGMCC No.18709; the Lactobacillus paracasei is Lactobacillus paracasei LL2022059, with the deposit number of CGMCC No.25863; the Weissella paramesenteroides is Weissella paramesenteroides LL2018353, with the deposit number of CGMCC No.18710; the Lactobacillus brevis is Lactobacillus brevis LL2022009, with the deposit number of CGMCC No.25861. The above microbial strains were all screened by the inventors and deposited in the China General Microbiological Culture Collection Center (abbreviation: CGMCC). Among them, Pediococcus pentosaceus LL2018368 was disclosed in the patent document ZL202110843467.2, and Weissella paramesenteroides LL2018353 was disclosed in the patent document ZL202010712678.8.

[0024] In the present invention, the effective viable count of each bacterial cell in the compound microbial inoculum is preferably 10 10 ~10 11 CFU / g. The compound microbial inoculum 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.

[0025] The present invention also provides a method for preparing the composite microbial inoculum, preferably including the following steps: inoculating each strain into MRS broth medium at a volume ratio of 0.05 - 0.2%, culturing at 35 - 37°C for 36 - 48 h, and centrifuging at 5000 - 8000 rpm to obtain bacterial cells; freeze-drying the obtained bacterial cells and mixing them according to the above weight ratio to obtain the composite microbial inoculum. In the present invention, the culture conditions are adjusted according to the growth habits of each strain. In the present invention, as an implementable method, Lactobacillus plantarum is preferably cultured at 35°C for 36 h, Pediococcus pentosaceus is preferably cultured at 35°C for 48 h, Lactobacillus paracasei is preferably cultured at 35.5°C for 36 h, Weissella paramesenteroides is preferably cultured at 35°C for 36 h, and Lactobacillus brevis is preferably cultured at 37°C for 35 h. In the present invention, after centrifuging the bacterial liquid, a cryoprotectant is preferably added and the bacterial cells are freeze-dried using a freeze dryer. The present invention does not make special limitations on the type of the cryoprotectant, and a cryoprotectant well-known in the art can be used. In the present invention, the conditions for freeze-drying are preferably: freeze-drying at -60°C to -70°C and a pressure of 0.006 Pa for 10 - 15 h; more preferably, the conditions for freeze-drying are more preferably: freeze-drying at -65°C and a pressure of 0.006 Pa for 14 h. In the present invention, the raw material ratio of the MRS broth medium is preferably as follows: peptone 10.0 g; beef extract 5.0 g; yeast powder 4.0 g; glucose 20.0 g; dipotassium hydrogen phosphate 2.0 g; ammonium citrate 2.0 g; sodium acetate 5.0 g; magnesium sulfate 0.2 g; manganese sulfate 0.05 g; Tween 80 1.0 mL; water 1 L.

[0026] The present invention also provides an application of the composite microbial inoculum in preparing feed.

[0027] The present invention also provides a corn cob silage feed, which is preferably fermented from a mixture of the composite microbial inoculum and corn cobs at a mass ratio of 1:8000 - 120000; more preferably, it is fermented at a mass ratio of 1:10000.

[0028] In the present invention, the corn cob mixture preferably includes corn cobs and corn husks.

[0029] In the present invention, the preparation method of the corncob silage preferably includes the following steps: mixing corncobs and corn husks in a weight ratio of 2 - 6:0 - 1, placing them in a pulper to obtain a corncob mixture, adjusting the moisture of the corncob mixture with corn flour, and then fermenting it at 25 - 28 °C for 20 - 25 days; more preferably, fermenting it at 25 °C for 21 days. In the present invention, the particle size of the corncob mixture is preferably 0.5 cm - 1 cm. In the present invention, the corn flour is ordinary corn flour for feed, preferably purchased from Yunda Feed Co., Ltd. The addition amount of the corn flour by volume ratio is preferably 3% - 27.69%.

[0030] The present invention also provides an application of the above-mentioned corncob silage in improving the growth performance, slaughter performance and / or meat quality performance of animals. Compared with the control group, adding corncob silage to the basal diet in the present invention can increase the final body weight, average daily gain, average daily feed intake and gross income of the experimental animals, bringing better production and economic benefits. After adding corncob silage to the basal diet, it can increase the live weight before slaughter, slaughter rate, skin thickness, carcass straight length, carcass diagonal length, pH value, marbling and meat color, and reduce drip loss, shear force, cooking loss and water loss rate. Compared with the control group, the drip loss is reduced by 13.20%, the shear force is reduced by 9.35%, the cooking loss is reduced by 6.98%, and the water loss rate is reduced by 53.77%. After adding corncob silage to the basal diet, it can increase the dry matter, crude protein and crude fat in the longissimus dorsi muscle, and the crude fat content can be increased by 28.43%. Compared with the control group, after adding corncob silage to the basal diet, it can increase the contents of fasting blood glucose, total protein, globulin, phosphate ion, pancreatic amylase and lipase in the serum, and reduce the contents of total bilirubin and cholesterol. In summary, adding corncob silage to the basal diet in the present invention can improve the growth performance, slaughter performance and meat quality performance of the experimental animals, reduce the breeding cost and provide higher-quality livestock products.

[0031] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0032] In the following experimental methods of the examples, unless otherwise specified, they are all conventional methods. The test materials used in the following examples, unless otherwise specified, are all commercially available products.

[0033] Example 1 A compound microbial inoculum 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 paramesenteroides, and 2.5 parts of Lactobacillus brevis; In the above compound microbial inoculum, Lactobacillus plantarum is Lactobacillus plantarum LL2022107 with the deposit number of CGMCC No.25862, Pediococcus pentosaceus is Pediococcus pentosaceus LL2018368 with the deposit number of CGMCC No.18709, Lactobacillus paracasei is Lactobacillus paracasei LL2022059 with the deposit number of CGMCC No.25863, Weissella paramesenteroides is Weissella paramesenteroides LL2018353 with the deposit number of CGMCC No.18710, and Lactobacillus brevis is Lactobacillus brevis LL2022009 with the deposit number of CGMCC No.25861.

[0034] The preparation method of the above compound microbial inoculum is as follows: inoculate each strain into MRS broth medium at a volume ratio of 0.1%. Lactobacillus plantarum is cultured at 35°C for 36 h, Pediococcus pentosaceus is cultured at 35°C for 48 h, Lactobacillus paracasei is cultured at 35.5°C for 36 h, Weissella paramesenteroides is cultured at 35°C for 36 h, and Lactobacillus brevis is cultured at 37°C for 35 h. After each strain is cultured, centrifuge at 8000 rpm to obtain each bacterial cell. Freeze-dry the obtained bacterial cells at -65°C and a pressure of 0.006 Pa for 14 h and then mix them in proportion to obtain the compound microbial inoculum.

[0035] Example 2 A compound microbial inoculum is prepared by mixing 3 parts of Lactobacillus plantarum, 3 parts of Pediococcus pentosaceus, 1 part of Lactobacillus paracasei, 3 parts of Weissella paramesenteroides, and 3 parts of Lactobacillus brevis. In the above compound microbial inoculum, Lactobacillus plantarum is Lactobacillus plantarum LL2022107 with the deposit number of CGMCC No.25862, Pediococcus pentosaceus is Pediococcus pentosaceus LL2018368 with the deposit number of CGMCC No.18709, Lactobacillus paracasei is Lactobacillus paracasei LL2022059 with the deposit number of CGMCC No.25863, Weissella paramesenteroides is Weissella paramesenteroides LL2018353 with the deposit number of CGMCC No.18710, and Lactobacillus brevis is Lactobacillus brevis LL2022009 with the deposit number of CGMCC No.25861.

[0036] The preparation method of the above compound microbial inoculum is the same as that in Example 1.

[0037] Example 3 A compound microbial inoculum is prepared by mixing 6 parts of Lactobacillus plantarum, 6 parts of Pediococcus pentosaceus, 2 parts of Lactobacillus paracasei, 3 parts of Weissella paramesenteroides, and 3 parts of Lactobacillus brevis. In the above compound microbial inoculum, Lactobacillus plantarum is Lactobacillus plantarum LL2022107 with the preservation number of CGMCC No.25862, Pediococcus pentosaceus is Pediococcus pentosaceus LL2018368 with the preservation number of CGMCC No.18709, Lactobacillus paracasei is Lactobacillus paracasei LL2022059 with the preservation number of CGMCC No.25863, Weissella paramesenteroides is Weissella paramesenteroides LL2018353 with the preservation number of CGMCC No.18710, and Lactobacillus brevis is Lactobacillus brevis LL2022009 with the preservation number of CGMCC No.25861.

[0038] The preparation method of the above compound microbial inoculum is the same as that in Example 1.

[0039] Example 4 A corn cob silage feed, which is prepared by mixing and fermenting the compound microbial inoculum of Example 1 with a corn cob mixture in a mass ratio of 1:10000; the corn cob mixture is obtained by mixing corn cobs and corn husks in a weight ratio of 6:1.

[0040] The preparation method of the above corn cob silage feed is as follows: The corn cobs and corn husks are pulped by a pulper to obtain a corn cob mixture with a particle size of 0.5 - 1 cm. After adjusting the moisture content by adding 27.69% corn flour by volume ratio, it is fermented at 25°C for 21 days.

[0041] Example 5 A corn cob silage feed, which is prepared by mixing and fermenting the compound microbial inoculum of Example 1 with a corn cob mixture in a mass ratio of 1:10000; the corn cob mixture is obtained by mixing corn cobs and corn husks in a weight ratio of 6:1.

[0042] The preparation method of the above corn cob silage feed is as follows: The corn cobs and corn husks are pulped by a pulper to obtain a corn cob mixture with a particle size of 0.5 - 1 cm. After adjusting the moisture content by adding 22.96% corn flour by volume ratio, it is fermented at 25°C for 21 days.

[0043] Example 6 A corn cob silage feed, which is prepared by mixing and fermenting the compound microbial inoculum of Example 1 with a corn cob mixture in a mass ratio of 1:10000; the corn cob mixture is obtained by mixing corn cobs and corn husks in a weight ratio of 6:1.

[0044] The preparation method of the above corn cob silage feed is as follows: The corn cobs and corn husks are pulped by a pulper to obtain a corn cob mixture with a particle size of 0.5 - 1 cm. After adjusting the moisture content by adding 18.57% corn flour by volume ratio, it is fermented at 25°C for 21 days.

[0045] Example 7 A kind of corncob silage feed, which is prepared by mixing and fermenting the compound microbial inoculum of Example 1 and the corncob mixture in a mass ratio of 1:10000; the corncob mixture is obtained by mixing corncobs and corn husks in a weight ratio of 6:1.

[0046] The preparation method of the above corncob silage feed is as follows: The corncobs and corn husks are pulped by a pulping machine to obtain a corncob mixture with a particle size of 0.5 - 1 cm. After adjusting the moisture content by adding 14.48% of corn flour by volume ratio, it is fermented at 25°C for 21 days.

[0047] Example 8 A kind of corncob silage feed, which is prepared by mixing and fermenting the compound microbial inoculum of Example 1 and the corncob mixture in a mass ratio of 1:10000; the corncob mixture is obtained by mixing corncobs and corn husks in a weight ratio of 6:1.

[0048] The preparation method of the above corncob silage feed is as follows: The corncobs and corn husks are pulped by a pulping machine to obtain a corncob mixture with a particle size of 0.5 - 1 cm. After adjusting the moisture content by adding 10.66% of corn flour by volume ratio, it is fermented at 25°C for 21 days.

[0049] Example 9 A kind of corncob silage feed, which is prepared by mixing and fermenting the compound microbial inoculum of Example 1 and the corncob mixture in a mass ratio of 1:10000; the corncob mixture is obtained by mixing corncobs and corn husks in a weight ratio of 6:1.

[0050] The preparation method of the above corncob silage feed is as follows: The corncobs and corn husks are pulped by a pulping machine to obtain a corncob mixture with a particle size of 0.5 - 1 cm. After adjusting the moisture content by adding 7.10% of corn flour by volume ratio, it is fermented at 25°C for 21 days.

[0051] Example 10 A kind of corncob silage feed, which is prepared by mixing and fermenting the compound microbial inoculum of Example 1 and the corncob mixture in a mass ratio of 1:10000; the corncob mixture is obtained by mixing corncobs and corn husks in a weight ratio of 6:1.

[0052] The preparation method of the above corncob silage feed is as follows: The corncobs and corn husks are pulped by a pulping machine to obtain a corncob mixture with a particle size of 0.5 - 1 cm. After adjusting the moisture content by adding 3.75% of corn flour by volume ratio, it is fermented at 25°C for 21 days.

[0053] Test Example 1 Select corncobs and corn husks and beat them with a beater (0.5 - 1 cm). Adjust the moisture content with corn flour (add corn flour according to the ratio of Examples 4 - 10), and randomly divide them into 8 groups (7 experimental groups and 1 control group), with 6 replicates in each group, 3 barrels in each replicate, and each barrel weighing about 50 kg. According to the ratio of adding 100 g of the compound microbial inoculant prepared in Example 1 per ton of the corncob mixture (100 g of the compound microbial inoculant is made into 1500 mL of suspension), the test period is from August 16th to September 6th, 2024, and the test period is 21 days. The control group adds the same volume of water, and the experimental groups add the additive prepared in Example 1 (made into suspension according to the ratio), and ferment at room temperature. The total test period is 21 days, and the silage effect is evaluated at the end of the test.

[0054] Table 1 On-site evaluation scores of the corncob mixture added with compound microbial fermentation inoculant after 1-day fermentation

[0055] Table 2 On-site evaluation scores of the corncob mixture added with compound microbial fermentation inoculant after 21-day fermentation

[0056] The effects of adding compound microbial inoculant on the silage effect of corncobs are shown in Tables 1 - 2: After adding the compound microbial inoculant, the odor, color, and structure of the corncob silage feed are improved, and finally the product quality is improved.

[0057] The effects of adding compound microbial inoculant on the conventional component indexes of the corncob mixture are shown in Table 3: Table 3 Conventional component analysis

[0058] According to the results shown in Table 3, after adding compound microbial inoculant to the corncob mixture, the content of crude fiber can be reduced and the content of crude fat can be increased, which has the potential to improve the utilization rate of the feed.

[0059] Test Example 2 This test example adopts a single-factor experimental design. Select 12 approximately Dian pigs (6 males, 6 females) with a body weight of about 50 kg and an age of about 117 days. Punch ear tags and record the weight before the experiment. According to the principle of close body weight and half male and half female, randomly divide them into 2 groups, with 1 replicate in each group, 6 pigs in one replicate, namely the control group (3 males, 3 females) and the experimental group (3 males, 3 females). Among them, the control group is fed with the basal diet; the experimental group is fed with the basal diet plus the corncob silage feed prepared in Example 4. The total test period of this test example is 119 days, the pre-feeding period is 7 days, and the formal test is 112 days.

[0060] The experiment is divided into three stages: 50 kg to 70 kg: The control group was fed a basal diet; the experimental group was fed 80% of the basal diet (added according to the feed intake of the control group the day before the experiment), and the corncob silage was available for free intake.

[0061] 70 kg to 95 kg: The control group was fed a basal diet; the experimental group was fed 70% of the basal diet (added according to the feed intake of the control group the day before the experiment), and the corncob silage was available for free intake.

[0062] 95 kg to 120 kg: The control group was fed a basal diet; the experimental group was fed 60% of the basal diet (added according to the feed intake of the control group the day before the experiment), and the corncob silage was available for free intake.

[0063] The specific experimental plan is as follows: 1. Test location: Pig Research Institute of Yunnan Academy of Animal Husbandry and Veterinary Sciences 2. Test materials: 12 Yuedian pigs (6 males and 6 females) weighing about 50 kg; 2 sets of farmer-type breeding houses 3. Test methods: (1) Select 12 Yuedian pigs (6 males and 6 females) weighing about 50 kg (about 117 days old). Before the experiment, ear tags were punched and the weights were recorded. According to the principle of similar body weight, they were randomly divided into 4 groups. Among them, the control group had 6 pigs (3 males and 3 females), and the experimental group had 6 pigs (3 males and 3 females), which were the control group and the experimental group respectively.

[0064] (2) The experiment was divided into 3 stages: ① In the 50 kg to 70 kg stage, the diet of the control group was the basal diet. Feeding was carried out at 8:30 every morning and recorded (fed according to the previous day's feeding situation, and theoretically, 0.25 kg of leftover feed was kept in the feed trough). Before feeding, the remaining feed was cleaned and weighed and recorded. The experimental group was fed a mixture of corncob silage and basal diet (8:2) with the same weight as the basal diet intake of the control group the day before at 8:30 every morning, and the feeding situation was observed in a timely manner. The basal diet intake and corncob silage intake of the experimental group on the same day were recorded; ② In the 70 kg to 95 kg stage, the diet of the control group was the basal diet. Feeding was carried out at 8:30 every morning and recorded (fed according to the previous day's feeding situation, and theoretically, 0.25 kg of leftover feed was kept in the feed trough). Before feeding, the remaining feed was cleaned and weighed and recorded. The experimental group was fed a mixture of corncob silage and basal diet (7:3) with the same weight as the basal diet intake of the control group the day before at 8:30 every morning, and the feeding situation was observed in a timely manner. The basal diet intake and corncob silage intake of the experimental group on the same day were recorded; ③In the stage of 95 kg - 120 kg, the control group was fed with the basal diet. Feed was delivered at 8:30 every morning and recorded (fed according to the previous day's feed intake, and theoretically, 0.25 kg of leftover feed was kept in the feeder). Before feeding, the remaining feed was cleaned and weighed and recorded. The experimental group was fed with a mixture of corn cob silage and basal diet (6:4) of the same weight as the control group's basal diet intake the previous day at 8:30 every morning, and the feeding situation was observed in a timely manner. Record the basal diet intake and corn cob silage intake of the experimental group on the same day; For feed processed in different batches, samples of no less than 1000 g should be taken for analysis of conventional components, and samples of no less than 500 g should be reserved. The formula of the basal diet is shown in Table 4: Table 4 Formula of the basal diet

[0065] (3)The methods of free access to feed and water were adopted; the diarrhea situation was observed every day. At 09:00 and 16:00 every day, record the weight of added feed and leftover feed when adding feed, and record the diarrhea situation. When recording the number of diarrhea pigs, it should also be recorded according to the severity level of diarrhea. Diarrhea was divided into 4 grades according to the method of Marquardt et al.

[0066] 0 points: strip or granular; 1 point: soft feces, formed; 2 points: thick, unformed, feces and water not separated; 3 points: liquid, unformed, feces and water separated.

[0067] When the fecal score is 2 points or above, it is considered that the pigs have diarrhea. At the end of the experiment, calculate the diarrhea rate of each group.

[0068] (4)The experimental period was 112 d. At the beginning of the experiment and on days 28, 56, 84, and 112, the feed was inventoried and weighed, and the average daily gain, average daily feed intake, feed-to-gain ratio were calculated, and the cost per kg of weight gain was calculated.

[0069] Observe the gas quality at the height of the pigs in the experimental pigsty. Three points were measured at each height. The gas components (NH 3 , H 2 S, CO 2 and CH 4 ) and volatile organic compounds VOC were collected, and fresh feces were collected in liquid nitrogen (3×200 mg per group) and stored at -20 °C (500 g per group); Fresh fecal samples were collected by stimulating the rectum of each pig with a cotton swab, and 6 samples were collected per pen using 5 mL cryogenic storage tubes; After thoroughly mixing the fecal samples of each pen of pigs, 300 g was taken, and after removing the hair and dander, 10 mL of 10% hydrochloric acid was added and mixed thoroughly, and then stored in a -20 °C refrigerator for testing. Detect the conventional components (crude protein, total nitrogen, moisture, crude fiber, crude fat) and metabolites (total biogenic amines, cadaverine, tryptamine, phenol, p-cresol, and skatole).

[0070] During the experiment period, observe the health status of the experimental pigs every day.

[0071] (6) After the experiment, select 3 pigs (2 males and 1 female) from each group for slaughter to evaluate the slaughter performance. Collect the longissimus dorsi muscle for meat quality determination and analyze the conventional components. Use two-dimensional gas chromatography-time of flight mass spectrometry (GC×GC-TOF-MS) analysis technology to detect volatile flavor substances in the longissimus dorsi muscle. Collect 10 mL of blood from the anterior vena cava. After standing the blood at low temperature for 30 min, centrifuge it at 3000 r / min for 10 min at 4 °C to obtain serum. After aliquoting, store it in a -20 °C refrigerator for later use. Use an animal biochemical analyzer to perform a blood routine analysis.

[0072] 4. Statistical Analysis The experimental data were analyzed by one-way ANOVA module in the SPSS 20.0 software package for one-way analysis of variance. For those with significant differences, Tukey's multiple comparison was used. All result data were expressed as mean ± standard error. P < 0.01 indicated extremely significant differences, and P < 0.05 indicated significant differences.

[0073] 5. Determination and Analysis (1) Refer to "Determination of Moisture in Foods GB5009.3-2016" to determine the content of dry matter and moisture, refer to "Determination of Ash in Foods GB5009.4-2016" to determine the content of ash, refer to "Determination of Protein in Foods GB5009.5-2016" to determine the content of crude protein, and refer to "Determination of Fat in Foods GB5009.6-2016" to determine the content of fat.

[0074] (2) Production Performance Determination ① Feed intake and weighing: Record the feed intake and health status of each group every day. On the 1st, 15th, 29th, 43rd, 57th, 71st, and 98th days of the formal trial period, conduct tray feeding and weighing (fasting after morning feeding), and collect data. Calculate the feed intake, weight gain, etc. of each group. Evaluate the effect of the feed on body weight and production performance.

[0075] ② Calculate the average daily feed intake (ADFI), average daily weight gain, and feed to weight ratio, and draw a growth curve.

[0076] (3) Slaughter Determination On the day when the experiment ends, select 3 pigs (2 males and 1 female) from each group for slaughter determination. The determination indexes are as follows: ① Slaughter performance indexes such as live weight before slaughter, carcass weight, lean meat rate, intramuscular fat, etc.; ② Meat quality (longissimus dorsi muscle): cooked meat rate, shear force, meat color, pH value, drip loss, marbling; ③ Serum biochemical indexes: 5 mL of blood was collected and placed in a coagulation-promoting tube. After standing at room temperature for 30 min, it was centrifuged at 3000 rpm for 15 min using a centrifuge to separate the serum, which was then aliquoted into EP tubes and stored at -20°C.

[0077] ④ Gastrointestinal tract contents: Cecal contents (microbial diversity, untargeted metabolomics, short-chain fatty acids) ⑤ Chemical routine (longissimus dorsi muscle): Crude fat, crude protein, amino acids, fatty acids; ⑥ Tissue sections: Small intestine, subcutaneous fat, abdominal fat and subcutaneous fat ⑦ Contents of 3 kinds of nucleotides (inosinic acid, adenylic acid, guanylic acid) ⑧ Volatile flavor detection: The longissimus dorsi muscle was analyzed for volatile flavor substances using two-dimensional gas chromatography-time of flight mass spectrometry (GC×GC-TOF-MS) analysis technology.

[0078] 6. Test results (1) Analysis of growth performance results Table 5 Growth performance (50 - 70 kg)

[0079] Note: Different letters a and b in the table indicate significant differences at the P < 0.05 level. As can be seen from Table 5, in the stage of about 50 kg - 70 kg of Yuedian pigs, compared with the control group, adding 20% corn cob silage to the diet could increase the final weight of the experimental animals and reduce the feed-to-weight ratio; compared with the control group, the experimental group increased the gross income by 55.81% (weight gain per head per day of live pigs × 17 yuan / kg - feed cost per head per day of pigs, with corn cob silage calculated at 0.6 yuan / kg), the cost of producing per kilogram of live pigs was reduced by 17.49%, and the feed-to-weight ratio was reduced by 16.04%. Therefore, adding 20% corn cob silage to the diet had better production and economic benefits in the 50 kg - 70 kg stage.

[0080] Table 6 Growth performance (70 - 95 kg)

[0081] As can be seen from Table 6, in the stage of about 70 kg - 95 kg of Yunnan pigs, adding 30% corn cob silage to the diet can increase the final weight, average daily gain, average daily feed intake and gross income (weight gain per live pig per day × 17 yuan / kg - feed cost per pig per day) of the experimental animals, and reduce the feed-to-weight ratio and cost. Compared with the control group, the 30% corn cob addition group increased the gross income by 213.24% (weight gain per live pig per day × 17 yuan / kg - feed cost per pig per day) and the average daily gain by 41.69%, and reduced the feed-to-weight ratio by 25.64%. The cost of producing per kilogram of live pig was reduced by 27.05%. Therefore, in the 70 kg - 95 kg stage, adding 30% corn cob silage to the diet can improve production and economic benefits.

[0082] Table 7 Growth Performance (95 - 120 kg)

[0083] As can be seen from Table 7, in the stage of about 90 kg - 120 kg of Yunnan pigs, adding 40% corn cob silage to the diet can increase the final weight, average daily gain, average daily feed intake and gross income (weight gain per live pig per day × 17 yuan / kg - feed cost per pig per day) of the experimental animals, while the feed-to-weight ratio and cost decreased compared with the control group. Compared with the control group, the 40% corn cob silage addition group increased the gross income by 153.09% and the average daily gain by 38.34%, and reduced the feed-to-weight ratio by 14.69% and the production cost (per kilogram of live pig) by 18.57%. Therefore, in the settlement of about 90 kg - 120 kg of Yunnan pigs, adding 40% corn cob silage to the diet can bring better production and economic benefits.

[0084] Table 8 Determination and Analysis of Slaughter Performance and Meat Quality

[0085] As can be seen from Table 8, after adding corn cobs to the diet, the live weight before slaughter, slaughter rate, skin thickness, carcass straight length, carcass diagonal length, pH value, marbling and meat color can be increased, while the drip loss, shear force, cooking loss and water loss rate can be reduced. Compared with the control group, the drip loss decreased by 13.20%, the shear force decreased by 9.35%, the cooking loss decreased by 6.98%, and the water loss rate decreased by 53.77%. Compared with the control group, adding corn cob silage to the diet can significantly improve the slaughter performance and meat quality performance of Yunnan pigs.

[0086] Table 9 Determination and Analysis of Conventional Components of Longissimus Dorsi Muscle

[0087] As can be seen from 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, and the crude fat content can be increased by 28.43%.

[0088] Table 10 Serum biochemical determination and analysis

[0089] As can be seen from Table 10, compared with the control group, adding corncob fermentate to the diet can increase the contents of fasting blood glucose, total protein, globulin, phosphate ion, pancreatic amylase, and lipase in the serum, and decrease the contents of total bilirubin and cholesterol, and the cholesterol content decreases significantly.

[0090] In summary, after adding the corncob silage prepared by fermenting with the compound microbial inoculant of the present invention to the basal diet, it can improve the growth performance, slaughter performance, and meat quality performance of Yunnan pigs, reduce the breeding cost, and provide higher-quality livestock products.

[0091] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A composite microbial agent, characterized in that: The composite microbial agent comprises 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 by weight.

2. The composite microbial agent according to claim 1, characterized in that: 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 Pediococcus is pentosaceae Pediococcus LL2018368, and the classification name of the pentosaceae Pediococcus LL2018368 is Pediococcus 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 short lactobacillus is short lactobacillus LL2022009, and the classification name of the short lactobacillus LL2022009 is short lactobacillus ( Lactobacillus brevis ), the deposit number is CGMCC No.25861.

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

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

5. The preparation method according to claim 4, 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.

6. Use of the composite microbial agent according to any one of claims 1 to 3 in preparing feed.

7. A corncob silage, characterized in that: The composite microbial agent according to any one of claims 1 to 3 is mixed and fermented with a corncob mixture at a mass ratio of 1:8000 to 120000.

8. The corncob silage according to claim 7, characterized in that: The fermentation conditions are: fermentation at 25-28° C. for 20-25 days.

9. The corncob silage according to claim 7, characterized in that: The corn cob mixture includes corn cobs and corn husks.

10. Use of the corncob silage according to any one of claims 7 to 9 in improving animal growth performance, slaughter performance and / or meat quality performance.

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