Preparation method and application of Bacillus licheniformis and composite bacterial agent for improving corn gluten powder quality

Abstract

The present invention provides a preparation method and application of Bacillus licheniformis and a composite bacterial agent for improving the quality of corn gluten powder, and belongs to the field of microbial engineering technology. The Bacillus licheniformis CGMCCNO.29918 of the present invention has the function of secreting protease and cellulase, and can be used as a feed additive for poultry animals to promote growth performance and improve the intestinal barrier function of poultry; it is used with Aspergillus niger and Lactobacillus plantarum as composite bacterial agents to carry out two-stage solid-state fermentation on corn gluten powder, which can effectively degrade high molecular weight proteins and alcohol-soluble proteins in corn gluten powder, improve the characteristics of corn gluten powder being difficult to absorb and low in utilization, and improve the growth performance and intestinal barrier function of poultry; it can also increase the number of fermented live bacteria in the product and the yield of corresponding beneficial metabolites, and affect the yield of metabolites such as zeaxanthin (such as carotenoids) and functional amino acids and polypeptides in corn gluten powder.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial engineering, and in particular relates to a preparation method and application of Bacillus licheniformis and a composite bacterial agent for improving the quality of corn protein powder. Background Art

[0002] In order to solve the problems of protein feed resource shortage and low utilization of corn gluten meal, improve the nutritional value of corn gluten meal, develop fermented feed using the function of probiotics, and develop biological feed resources, it has become a hot topic in current research. Solid-state fermentation can improve the nutritional value of protein feed by improving its digestibility, amino acid composition and flavor, and at the same time improve the body's antioxidant capacity and immunity to a certain extent, which is beneficial to the growth of animals.

[0003] Corn gluten meal (CGM), a byproduct of corn starch processing, is extremely abundant in production. In addition to containing a large amount of protein nutrients, CGM also contains rich carotenoids (zeaxanthin, β-carotene, lutein), etc., and is often used as animal feed to meet the animal's protein needs. However, the protein in CGM is mainly alcohol-soluble protein (60%), gluten (22%) and globulin (1.2%). Animals have low absorption and utilization of most alcohol-soluble proteins, and alcohol-soluble proteins have a poor taste, which reduces their value as feed and leads to a waste of corn gluten meal resources. At the same time, CGM lacks essential amino acids (Lys, Trp) after hydrolysis, which limits its application in animal feed and still needs further development.

[0004] In view of the shortage of protein feed resources in animal husbandry and the insufficient utilization of corn gluten meal resources, it is particularly urgent to provide a high-quality corn gluten meal and use it as a protein source in feed to improve the productivity of poultry. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a preparation method and application of Bacillus licheniformis and its composite bacterial agent for improving the quality of corn gluten meal. The quality of corn gluten meal can be improved by carrying out two-stage solid-state fermentation with corn gluten meal as a substrate. As a feed additive, it can improve the intestinal barrier function of poultry and promote poultry growth.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] A Bacillus licheniformis for improving the quality of corn gluten meal, which is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC NO.29918 and a deposit date of February 29, 2024.

[0008] The present invention also provides a composite bacterial agent for improving the quality of corn protein powder, comprising a first-stage fermentation culture solution and a second-stage fermentation culture solution, wherein the first-stage fermentation culture solution is a Bacillus licheniformis CGMCC NO.29918 culture solution and an Aspergillus niger CICC 40048 culture solution, and the second-stage fermentation culture solution is a Lactobacillus plantarum CICC 22134 culture solution.

[0009] Preferably, in the first stage fermentation culture liquid, the bacterial liquid of Bacillus licheniformis CGMCC NO.29918 and the bacterial liquid of Aspergillus niger CICC 40048 are mixed in a volume ratio of 1-2:1-2; the number of viable bacteria of the bacterial liquid of Bacillus licheniformis CGMCC NO.29918 is ≥10 9 CFU / mL, the spore count of Aspergillus niger CICC 40048 bacterial liquid is ≥10 8 spores / mL.

[0010] Preferably, in the second stage fermentation culture liquid, the number of viable bacteria of Lactobacillus plantarum CICC 22134 liquid is ≥10 9 CFU / mL.

[0011] The invention also provides application of the composite bacterial agent in fermenting corn protein powder.

[0012] Preferably, the method comprises the following steps: using corn protein meal and bran as fermentation substrates, inoculating the first stage fermentation culture solution for aerobic fermentation, inoculating the second stage fermentation culture solution for anaerobic fermentation after the fermentation is completed, and performing drying and crushing after the fermentation is completed.

[0013] Preferably, the corn gluten meal and bran are mixed in a mass ratio of 4-8:2-6 as a fermentation substrate.

[0014] Preferably, the inoculation amount of the first stage fermentation culture liquid is ≥10 9 CFU / g fermentation substrate, the inoculation amount of the second stage fermentation culture liquid is ≥10 9 CFU / g fermentation substrate.

[0015] Preferably, the fermentation temperature in the first stage is 28-35°C, and the aerobic fermentation lasts for 3-5 days, and the fermentation temperature in the second stage is 30-40°C, and the anaerobic fermentation lasts for 3-5 days, and the solid-liquid ratio of the fermentation material is maintained at 2:1 during the fermentation period.

[0016] The invention also provides a feed additive prepared by fermenting corn protein powder with the composite bacterial agent.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The invention provides a Bacillus licheniformis CGMCC NO.29918 for improving the quality of corn protein powder. The strain has the function of secreting protease and cellulase, can be used for preparing feed additives for poultry animals, promotes growth performance, and improves the intestinal barrier function of poultry.

[0019] The present invention also provides a composite bacterial agent prepared by Bacillus licheniformis CGMCC NO.29918 and its application in fermenting corn gluten powder. Through two-stage solid-state fermentation, the composite bacterial agent can effectively degrade high molecular weight protein and alcohol-soluble protein in corn gluten powder, improve the characteristics of corn gluten powder being difficult to absorb and having low utilization, and improve the growth performance and intestinal barrier function of poultry; it can also increase the number of fermented live bacteria in the product and the yield of corresponding beneficial metabolites, and affect the yield of metabolites such as zeaxanthin (such as carotenoids) and functional amino acids, polypeptides, etc. in corn gluten powder.

[0020] The two-stage solid-state fermentation process of the present invention is easy to operate, has simple culture medium components, and has low solid-state culture cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the colony morphology of Bacillus licheniformis CGMCC NO.29918;

[0022] Figure 2 This is the phylogenetic tree of Bacillus licheniformis CGMCC NO.29918;

[0023] Figure 3 The content change of high molecular weight protein in corn gluten meal after aerobic and anaerobic two-stage solid-state fermentation;

[0024] Figure 4 The content changes of carotenoids in corn gluten meal after aerobic and anaerobic solid-state fermentation;

[0025] Figure 5 The changes in the contents of amino acids and peptides in corn gluten meal after aerobic and anaerobic solid-state fermentation;

[0026] Figure 6 The invention discloses the effects of corn gluten powder fermented with the composite bacterial agent on the growth performance and intestinal barrier function of poultry.

[0027] The biological material involved in the present invention includes Bacillus licheniformis, which is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is February 29, 2024, and the deposit number is CGMCC NO.29918.

[0028] The biological materials involved in the present invention also include Aspergillus niger CICC 40048 and Lactobacillus plantarum CICC 22134, which are purchased from the China Industrial Microbiological Culture Collection Management Center. DETAILED DESCRIPTION

[0029] The present invention provides a Bacillus licheniformis for improving the quality of corn protein powder, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee, with a deposit number of CGMCC NO.29918 and a deposit date of February 29, 2024. The Bacillus licheniformis has the function of secreting protease and cellulase, and the pathogenicity and toxicity tests on mice prove that the strain has no toxic side effects, and can be used for the preparation of feed additives for poultry animals, promote growth performance, and improve the intestinal barrier function of poultry.

[0030] The present invention also provides a composite bacterial agent containing the above-mentioned Bacillus licheniformis, comprising a first-stage fermentation culture bacterial liquid and a second-stage fermentation culture bacterial liquid, wherein the first-stage fermentation culture bacterial liquid is Bacillus licheniformis CGMCC NO.29918 bacterial liquid and Aspergillus niger CICC 40048 bacterial liquid, and the second-stage fermentation culture bacterial liquid is Lactobacillus plantarum CICC 22134 bacterial liquid.

[0031] In the first stage of the fermentation culture, the bacterial solution of Bacillus licheniformis CGMCC NO.29918 and the bacterial solution of Aspergillus niger CICC 40048 are preferably mixed in a volume ratio of 1-2:1-2, more preferably 1:2 or 2:1; preferably, the number of live bacteria in the bacterial solution of Bacillus licheniformis CGMCC NO.29918 is ≥10 9 CFU / mL, more preferably 10 9 CFU / mL, preferably Aspergillus niger CICC 40048 spore count ≥ 10 8 spores / mL, more preferably 10 8 spores / mL.

[0032] The preferred method of culturing the bacterial solution of Bacillus licheniformis CGMCC NO.29918 of the present invention comprises the following steps:

[0033] (1) Seed cultivation stage:

[0034] The seed culture medium preferably comprises LB culture medium: 2wt% peptone, 1wt% yeast powder, 0.5wt% NaCl and the balance water, sterilized by high pressure at 121°C for 20min;

[0035] Pick up a loop of Bacillus licheniformis CGMCC NO.29918 and inoculate it into a seed culture medium. The preferred culture temperature is 30-37°C, more preferably 35-36°C; the preferred rotation speed is 160-240rpm, more preferably 180-220rpm, more preferably 200rpm; the preferred culture time is 10-20h, more preferably 12-18h, more preferably 15-16h;

[0036] (2) Spore culture stage:

[0037] The spore culture medium preferably comprises LB medium: 2wt% peptone, 1wt% yeast powder, 1wt% glucose, 0.5wt% NaCl and the balance water, sterilized by high pressure at 121°C for 20min;

[0038] The seed culture fermentation liquid is inoculated into the spore culture medium, preferably the inoculation amount is 2%-8% (v / v), more preferably 5%; the preferred culture temperature is 30-37°C, more preferably 35°C; the preferred rotation speed is 160-240rpm, more preferably 190rpm; the preferred culture time is 10-20h, more preferably 16h; the viable cell count is ≥10 9 CFU / mL.

[0039] The preferred culture method of Aspergillus niger CICC 40048 bacterial liquid of the present invention comprises the following steps:

[0040] (1) Seed cultivation stage:

[0041] The seed culture medium preferably comprises PDA culture medium: 5wt% potato extract, 2wt% glucose, 1wt% agar and the balance water, sterilized by high pressure at 121°C for 20min;

[0042] Pick up a ring of Aspergillus niger CICC 40048 and inoculate it into a seed culture medium. The culture temperature is preferably 25-35° C., more preferably 30° C.; culture is statically allowed to stand; the culture time is preferably 5-8 days, more preferably 5 days;

[0043] (2) Spore culture stage:

[0044] The spore culture medium preferably comprises PDA culture medium: 5wt% potato extract, 2wt% glucose, 1wt% agar and the balance water, sterilized by high pressure at 121°C for 20min;

[0045] The seed culture fermentation liquid is inoculated into the spore culture medium, preferably the inoculation amount is 2%-5% (v / v), more preferably 4%; the preferred culture temperature is 25-35°C, more preferably 30°C; static culture; the preferred culture time is 3-5 days, more preferably 3 days; the spore number is ≥10 8 spores / mL.

[0046] After obtaining the bacterial solution of Bacillus licheniformis CGMCC NO.29918 and the bacterial solution of Aspergillus niger CICC 40048, they are mixed in a volume ratio of 1-2:1-2 to obtain a composite bacterial solution, which is used as the first-stage fermentation culture bacterial solution.

[0047] In the second stage of fermentation culture, the number of viable bacteria of Lactobacillus plantarum CICC 22134 is preferably ≥ 10 9 CFU / mL, the culture method includes the following steps:

[0048] (1) Seed cultivation stage:

[0049] The seed culture medium preferably comprises MRS culture medium: casein peptone 2.5wt%, beef extract 2.5wt%, yeast powder 1.25wt%, glucose 1.25wt%, sodium acetate 1.25wt%, disodium citrate 1wt%, Tween 80 0.5wt%, K2HPO4 1wt%, MgSO4 0.5wt%, MnSO4·H2O 0.2wt% and the balance water, and is sterilized by high pressure at 121°C for 20min;

[0050] Pick a Lactobacillus plantarum CICC 22134 ring and inoculate it into a seed culture medium. The preferred culture temperature is 35-40° C., more preferably 37° C.; the preferred rotation speed is 160-240 rpm, more preferably 190 rpm; the preferred culture time is 3-5 days, more preferably 3 days;

[0051] (2) Spore culture stage:

[0052] The spore culture medium preferably comprises MRS culture medium: casein peptone 2.5wt%, beef extract 2.5wt%, yeast powder 1.25wt%, glucose 1.25wt%, sodium acetate 1.25wt%, disodium citrate 1wt%, Tween800.5wt%, K2HPO41wt%, MgSO40.5wt%, MnSO4·H2O 0.2wt% and the balance water, and is sterilized by high pressure at 121°C for 20min;

[0053] The seed culture fermentation liquid is inoculated into the spore culture medium, preferably the inoculation amount is 0.5%-2% (v / v), more preferably 1.5%; the preferred culture temperature is 35-40°C, more preferably 37°C; the preferred rotation speed is 160-240rpm, more preferably 190rpm; the preferred culture time is 3-5 days, more preferably 3 days, to obtain the plant lactobacillus CICC 22134 bacterial liquid, the number of viable bacteria is ≥10 9 CFU / mL, used as the second stage fermentation culture broth.

[0054] The present invention also provides an application of the composite bacterial agent in fermenting corn gluten powder. The corn gluten powder is used as a substrate and the composite bacterial agent undergoes two stages of solid-state fermentation to improve the quality of the corn gluten powder, facilitate poultry absorption, and promote poultry growth and development.

[0055] The invention preferably comprises the following steps: using corn protein powder and bran as fermentation substrates, inoculating the first stage fermentation culture solution for aerobic fermentation, inoculating the second stage fermentation culture solution for anaerobic fermentation after the aerobic fermentation, and drying and crushing after the anaerobic fermentation.

[0056] The present invention further preferably mixes corn gluten meal and bran in a mass ratio of 4-8:2-6 as a fermentation substrate, more preferably 6:4.

[0057] The present invention further preferably has a first stage fermentation culture liquid inoculation amount of viable bacteria ≥ 10 9 CFU / g fermentation substrate, the inoculation amount of the second stage fermentation culture liquid is ≥10 9 CFU / g fermentation substrate.

[0058] The present invention further preferably has a first stage fermentation culture temperature of 28-35°C, more preferably 30°C, and aerobic fermentation for 3-5 days, more preferably 3 days; the second stage fermentation culture temperature is further preferably 30-40°C, more preferably 37°C, and anaerobic fermentation for 3-5 days, more preferably 3 days; and the solid-liquid ratio of the fermentation material is further preferably maintained at 2:1 during fermentation. The present invention preferably has two stages of solid-state fermentation culture medium in a natural state before use, without the need for high-pressure sterilization.

[0059] The present invention further preferably has a drying temperature of 40-70°C, more preferably 55°C, to reduce the water content of the solid fermentation product to 10wt%-18wt%; further preferably, the particle size of the solid fermentation product is reduced to 40-60 mesh, and as an implementable method, a pulverizer is used for pulverization.

[0060] The present invention uses a composite bacterial agent to ferment corn protein powder, and the solid fermentation product obtained by drying and crushing is a feed additive, which is added to poultry feed as a feed nitrogen source, preferably 5%-15% of the solid fermentation product is added according to the mass of the poultry diet feed, and more preferably 10%. The solid fermentation product of the present invention is rich in carotenoids as a feed additive, can enhance immunity, promote poultry growth and development, and improve the intestinal barrier function of poultry.

[0061] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0062] Example 1

[0063] Isolation and Identification of Bacillus licheniformis

[0064] 15 samples were collected from corn stalks at Jilin Agricultural University in Changchun, Jilin Province. 5 g of each sample was weighed, 45 mL of sterile saline was added, and the culture was cultured at 37 ° C. After shaking, the culture solution was gradient diluted and spread on a modified LB separation plate. The culture was cultured at 37 ° C for 48 h to obtain 30 strains of Bacillus. A single colony was isolated and purified, and a fermentation enzyme production experiment and enzyme activity determination were performed. After further screening, a strain of Bacillus licheniformis was obtained.

[0065] The modified LB medium formula is 10g CBM (60wt% corn gluten meal, 40wt% bran), 5g yeast extract, 3g glucose, 5g NaCl, and 1L H2O.

[0066] Identification of Bacillus licheniformis:

[0067] The strain was identified by colony morphology, physiological and biochemical analysis, and molecular biology. The specific identification results are as follows:

[0068] Morphological identification: The colony of this strain is milky white, with irregular edges, raised and wrinkled. After Gram staining, the cell morphology was observed under an optical microscope (100×) and it was Gram-positive. The bacteria were short rod-shaped with blunt ends and oval spores. Figure 1 shown.

[0069] Physiological and biochemical identification: The results are shown in Table 1.

[0070] Table 1 Physiological and biochemical experiments of Bacillus licheniformis

[0071]

[0072]

[0073] Note: In Table 1, + indicates that the feature or reaction is positive, and - indicates that the feature or reaction is negative.

[0074] 16S rDNA molecular biological identification: The total DNA of the Bacillus licheniformis was extracted and used as a template to perform PCR amplification using bacterial 16S rDNA universal primers to obtain an amplified product of about 1.4 kb in length. The amplified product was recovered and sequenced by Shanghai Shenggong Bioengineering Technology Service Co., Ltd. Sequencing Company. The measured sequence is shown in the sequence table SEQ ID NO: 1. By using Blast for comparison, the highest similarity sequence provided by GenBank was constructed as shown in the following Figure 2The phylogenetic tree shown showed that the homology with Bacillus licheniformis (GenBank Accession No. MT642945.1) was 99.92%.

[0075] Example 2

[0076] A method for preparing a composite microbial agent for improving the quality of corn gluten powder comprises the following steps:

[0077] (1) Preparation of the first stage fermentation culture liquid:

[0078] (1.1) Preparation of Bacillus licheniformis CGMCC NO.29918 bacterial solution:

[0079] Seed cultivation stage:

[0080] Pick a loop of Bacillus licheniformis CGMCC NO.29918 and inoculate it into a seed culture medium (LB culture medium: 2wt% peptone, 1wt% yeast powder, 0.5wt% NaCl and the balance water, autoclaved at 121°C for 20min), the culture temperature is 35°C, the rotation speed is 200rpm, and the culture time is 15h;

[0081] Spore culture stage:

[0082] The seed culture fermentation liquid was inoculated into spore medium (LB medium: 2wt% peptone, 1wt% yeast powder, 1wt% glucose, 0.5wt% NaCl and the balance water, autoclaved at 121℃ for 20min) with an inoculum volume of 5% (v / v), a culture temperature of 35℃, a rotation speed of 190rpm, and a culture time of 16h until the viable count was ≥10 9 CFU / mL.

[0083] (1.2) Preparation of Aspergillus niger CICC 40048 bacterial solution:

[0084] Seed cultivation stage:

[0085] Pick a loop of Aspergillus niger CICC 40048 and inoculate it into a seed culture medium (PDA culture medium: 5wt% potato extract, 2wt% glucose, 1wt% agar and the balance water, sterilized by high pressure at 121°C for 20min), the culture temperature is 30°C, the culture is statically cultured, and the culture time is 5 days;

[0086] Spore culture stage:

[0087] The seed culture fermentation liquid was inoculated into spore medium (PDA medium: 5wt% potato extract, 2wt% glucose, 1wt% agar and the balance water, autoclaved at 121℃ for 20min) with an inoculum volume of 4% (v / v), a culture temperature of 30℃, and static culture for 3 days until the spore count was ≥10. 8 spores / mL.

[0088] (1.3) The Bacillus licheniformis solution prepared in step (1.1) and the Aspergillus niger solution prepared in step (1.2) are mixed in a volume ratio of 1:2 to obtain a first-stage fermentation culture solution.

[0089] (2) Preparation of the second stage fermentation culture liquid:

[0090] Preparation of Lactobacillus plantarum CICC 22134 bacterial solution:

[0091] Seed cultivation stage:

[0092] Pick a loop of Lactobacillus plantarum CICC 22134 and inoculate it into a seed culture medium (MRS culture medium: casein peptone 2.5wt%, beef extract 2.5wt%, yeast powder 1.25wt%, glucose 1.25wt%, sodium acetate 1.25wt%, disodium citrate 1wt%, Tween800.5wt%, K2HPO41wt%, MgSO40.5wt%, MnSO4·H2O 0.2wt% and the balance of water, autoclaved at 121°C for 20min), the culture temperature is 37°C, the rotation speed is 190rpm, and the culture time is 3 days;

[0093] Spore culture stage:

[0094] The seed culture fermentation liquid was inoculated into spore medium (MRS medium: casein peptone 2.5wt%, beef extract 2.5wt%, yeast powder 1.25wt%, glucose 1.25wt%, sodium acetate 1.25wt%, disodium citrate 1wt%, Tween800.5wt%, K2HPO41wt%, MgSO40.5wt%, MnSO4·H2O0.2wt% and the balance of water, autoclaved at 121℃ for 20min), the inoculation amount was 1.5% (v / v), the culture temperature was 37℃, the rotation speed was 190rpm, the culture time was 3 days, until the number of viable bacteria was ≥10 9 CFU / mL, used as the second stage fermentation culture broth.

[0095] Example 3

[0096] A method for preparing a composite microbial agent for improving the quality of corn gluten powder comprises the following steps:

[0097] (1) Preparation of the first stage fermentation culture liquid:

[0098] (1.1) Preparation of Bacillus licheniformis CGMCC NO.29918 bacterial solution:

[0099] Seed cultivation stage:

[0100] Pick a loop of Bacillus licheniformis CGMCC NO.29918 and inoculate it into the seed culture medium. The culture temperature is 30°C, the rotation speed is 160 rpm, and the culture time is 20 hours.

[0101] Spore culture stage:

[0102] The seed culture fermentation liquid was inoculated into the spore medium at an inoculation volume of 2% (v / v), the culture temperature was 30°C, the rotation speed was 160 rpm, and the culture time was 20 h until the number of viable bacteria was ≥10 9 CFU / mL.

[0103] (1.2) Preparation of Aspergillus niger CICC 40048 bacterial solution:

[0104] Seed cultivation stage:

[0105] Pick a ring of Aspergillus niger CICC 40048 and inoculate it into the seed culture medium. The culture temperature is 25°C and the culture time is 8 days.

[0106] Spore culture stage:

[0107] The seed culture fermentation liquid was inoculated into the spore medium at an inoculation volume of 2% (v / v), the culture temperature was 25°C, and the culture time was 5 days until the spore count was ≥10 8 spores / mL.

[0108] (1.3) The Bacillus licheniformis solution prepared in step (1.1) and the Aspergillus niger solution prepared in step (1.2) are mixed in a volume ratio of 2:1 to obtain a first-stage fermentation culture solution.

[0109] (2) Preparation of the second stage fermentation culture liquid:

[0110] Preparation of Lactobacillus plantarum CICC 22134 bacterial solution:

[0111] Seed cultivation stage:

[0112] Pick a loop of Lactobacillus plantarum CICC 22134 and inoculate it into the seed culture medium. The culture temperature is 35°C, the rotation speed is 160 rpm, and the culture time is 5 days.

[0113] Spore culture stage:

[0114] The seed culture fermentation liquid was inoculated into the spore medium at an inoculation volume of 0.5% (v / v), the culture temperature was 35°C, the rotation speed was 160 rpm, and the culture time was 5 days until the number of viable bacteria was ≥10 9 CFU / mL, used as the second stage fermentation culture broth.

[0115] Example 4

[0116] A method for preparing a composite microbial agent for improving the quality of corn gluten powder comprises the following steps:

[0117] (1) Preparation of the first stage fermentation culture liquid:

[0118] (1.1) Preparation of Bacillus licheniformis CGMCC NO.29918 bacterial solution:

[0119] Seed cultivation stage:

[0120] Pick a loop of Bacillus licheniformis CGMCC NO.29918 and inoculate it into the seed culture medium. The culture temperature is 37°C, the rotation speed is 240 rpm, and the culture time is 10 h.

[0121] Spore culture stage:

[0122] The seed culture fermentation liquid was inoculated into the spore medium at an inoculation volume of 8% (v / v), the culture temperature was 37°C, the rotation speed was 240 rpm, and the culture time was 10 h, until the number of viable bacteria was ≥10 9 CFU / mL.

[0123] (1.2) Preparation of Aspergillus niger CICC 40048 bacterial solution:

[0124] Seed cultivation stage:

[0125] Pick a ring of Aspergillus niger CICC 40048 and inoculate it into the seed culture medium. The culture temperature is 35°C and the culture time is 6 days.

[0126] Spore culture stage:

[0127] The seed culture fermentation liquid was inoculated into the spore medium at an inoculation volume of 5% (v / v), the culture temperature was 35°C, and the culture time was 4 days until the spore count was ≥10 8 spores / mL.

[0128] (1.3) The Bacillus licheniformis solution prepared in step (1.1) and the Aspergillus niger solution prepared in step (1.2) are mixed in a volume ratio of 1:1 to obtain a first-stage fermentation culture solution.

[0129] (2) Preparation of the second stage fermentation culture liquid:

[0130] Preparation of Lactobacillus plantarum CICC 22134 bacterial solution:

[0131] Seed cultivation stage:

[0132] Pick a ring of Lactobacillus plantarum CICC 22134 and inoculate it into the seed culture medium. The culture temperature is 40°C, the rotation speed is 240 rpm, and the culture time is 4 days.

[0133] Spore culture stage:

[0134] The seed culture fermentation liquid was inoculated into the spore medium at an inoculation volume of 2% (v / v), the culture temperature was 40°C, the rotation speed was 240 rpm, and the culture time was 4 days until the viable count was ≥10 9 CFU / mL, used as the second stage fermentation culture broth.

[0135] Example 5

[0136] A method for fermenting corn gluten meal to improve its quality comprises the following steps:

[0137] Corn gluten meal and bran were mixed in a mass ratio of 6:4 as fermentation substrate; the inoculation amount was ≥10 9 CFU / g Fermentation substrate inoculation Example 2 The first stage of fermentation culture liquid was subjected to solid aerobic fermentation, the fermentation culture temperature was 30°C, and the aerobic fermentation was carried out for 3 days; after the aerobic fermentation was completed, the inoculation amount was set to a viable count of ≥10 9 CFU / g fermentation substrate inoculation example 2 second stage fermentation culture liquid solid anaerobic fermentation, fermentation culture temperature is 37 ℃, anaerobic fermentation is 3-5 days, during the fermentation period the fermentation material solid-liquid ratio is maintained at 2:1; after the anaerobic fermentation is completed, the solid fermentation product is dried at 55 ℃ until the water content is reduced to 15wt%, and then crushed to a particle size of 50 mesh by a pulverizer.

[0138] Example 6

[0139] A method for fermenting corn gluten meal to improve its quality comprises the following steps:

[0140] Corn gluten meal and bran were mixed in a mass ratio of 8:2 as fermentation substrate; the inoculation amount was ≥10 9 CFU / g Fermentation substrate inoculation Example 3 The first stage of fermentation culture liquid was subjected to solid aerobic fermentation, the fermentation culture temperature was 28°C, and the aerobic fermentation was carried out for 5 days; after the aerobic fermentation was completed, the inoculation amount was set to a viable count of ≥10 9CFU / g fermentation substrate inoculation example 3 second stage fermentation culture liquid solid anaerobic fermentation, fermentation culture temperature is 30 ℃, anaerobic fermentation is 5 days, during the fermentation period the fermentation material solid-liquid ratio is maintained at 2:1; after the anaerobic fermentation is completed, the solid fermentation product is dried at 40 ℃ until the water content is reduced to 18wt%, and then crushed to a particle size of 40 mesh by a pulverizer.

[0141] Example 7

[0142] A method for fermenting corn gluten meal to improve its quality comprises the following steps:

[0143] Corn gluten meal and bran were mixed in a mass ratio of 4:6 as fermentation substrate; the inoculation amount was ≥10 9 CFU / g Fermentation substrate inoculation Example 4 The first stage of fermentation culture liquid was subjected to solid aerobic fermentation, the fermentation culture temperature was 35°C, and the aerobic fermentation was carried out for 4 days; after the aerobic fermentation was completed, the inoculation amount was set to a viable count of ≥10 9 CFU / g fermentation substrate inoculation example 4 second stage fermentation culture liquid solid anaerobic fermentation, fermentation culture temperature is 40 ℃, anaerobic fermentation is 4 days, during the fermentation period the fermentation material solid-liquid ratio is maintained at 2:1; after the anaerobic fermentation is completed, the solid fermentation product is dried at 70 ℃ until the water content is reduced to 10wt%, and then crushed to a particle size of 60 mesh by a pulverizer.

[0144] Example 8

[0145] The fermented corn gluten meal of Example 5 is added to poultry diets as a feed nitrogen source, and the added amount is 10%-15%.

[0146] Example 9

[0147] Effect of the ratio of Bacillus licheniformis to Aspergillus niger on the first stage of solid fermentation

[0148] According to the method of steps (1.1) and (1.2) of Example 2, a bacterial solution of Bacillus licheniformis CGMCC NO.29918 and a bacterial solution of Aspergillus niger CICC 40048 were prepared, and the mixtures were mixed in volume ratios of 1:2, 1:1, 1:3, 3:1, and 2:1, respectively. The mixed bacterial solution (inoculation amount 50 wt%) was inoculated with corn gluten meal and bran (mass ratio 6:4) as fermentation substrates for aerobic fermentation. The fermentation temperature was 30° C. and the moisture ratio of the fermentation material was maintained at 1:1 by ventilation. After culturing for 3 days, the total viable count and carotenoid content in the fermentation substrate were determined.

[0149] The total viable bacterial count was determined by the plate colony counting method.

[0150] Determination of carotenoid content: A 1 g aliquot of each sample was homogenized with 5 mL of extraction solvent (hexane / acetone / methanol, 50:25:25, V / V). The mixture was placed in a thermostatic oscillator set at 100 r / min for 30 min and then centrifuged at 6000 r / min for 10 min at 4°C. The top colored hexane layer was recovered and transferred to a 2.5 mL volumetric flask and the volume was adjusted to 2.5 mL with hexane. The absorbance of the sample was measured at 450 nm. The results were calculated using the extinction coefficient of carotene, i.e., E 1％ =2505

[0151] Table 2 Effect of the ratio of Bacillus licheniformis-Aspergillus niger on the fermentation substrate of corn gluten meal

[0152]

[0153] The results are shown in Table 2. According to Table 2, it can be seen that the compound ratio of Bacillus licheniformis and Aspergillus niger affects the aerobic fermentation effect of the fermentation substrate. Considering the total viable bacteria count and carotenoid content, the aerobic fermentation effect is best when Bacillus licheniformis and Aspergillus niger liquid are mixed in a volume ratio of 1:2 or 2:1. At this time, the number of viable bacteria in the fermentation substrate is large and the carotenoid content is high.

[0154] Example 10

[0155] Effects of aerobic-anaerobic two-stage solid-state fermentation on the quality of corn gluten meal

[0156] The composite bacterial agent was prepared according to the method of Example 2, and the two-stage solid-state fermentation of corn gluten powder was carried out according to the method of Example 5. The contents of high molecular weight proteins, carotenoids, amino acids and polypeptides in the fermentation substrate were measured before fermentation, after the first stage of solid-state aerobic fermentation and the second stage of solid-state anaerobic fermentation.

[0157] Large molecular weight protein: SDS-PAGE; Dissolve the sample (5 mg) in 1 mL 2× protein loading buffer and boil for 5 min. Centrifuge at 4°C, 10,000×g for 5 min, and take the supernatant (10 μL) for analysis. Use 4% stacking gel and 12% separation gel, with the voltage of the stacking gel and separation gel being 80 V and 120 V, respectively. After electrophoresis, stain the gel with Coomassie Brilliant Blue R250 and then decolorize with a decolorizing solution consisting of acetic acid and ethanol.

[0158] The method for determining the carotenoid content is the same as in Example 9.

[0159] Amino acids: GC-MS (non-targeted metabolomics); peptides: accurately weigh 3.00g of sample in a 50mL centrifuge tube, then add 15mL of 15% trichloroacetic acid solution (v / v), mix well and let stand for 5min, then continue to add 25% trichloroacetic acid 15mL to the centrifuge tube, mix well and let stand for 20min. Use a centrifuge to centrifuge the mixture at 4000rmp for 10min, remove the supernatant and filter it with filter paper to obtain the filtrate. Use a pipette to accurately transfer 10mL of filtrate into the digestion tube, add 10mL of sulfuric acid and let stand for 4h, then use a digestion furnace for digestion, then use a Kjeldahl nitrogen analyzer to determine the protein content in the filtrate, and finally calculate the corn peptide content based on the measured value.

[0160] The results are as follows Figure 3-5 As shown. Figure 3-5 It can be seen that the two-stage solid-state fermentation of the composite microbial agent can effectively degrade the high molecular weight protein in corn gluten powder ( Figure 3 ), improve the characteristics of corn gluten meal that is difficult to absorb and has low utilization; it can also affect the important parameters of the production of zeaxanthin (such as carotenoids) and functional amino acids, peptides and other metabolites in corn gluten meal, and the production of carotenoids has increased by 84.4% ( Figure 4 ), functional amino acids include L-lysine, L-norvaline, L-histidine, L-glutamine, L-phenylalanine, L-methionine, L-isoleucine, L-tyrosine and L-arginine, etc., and the content of dipeptides and tetrapeptides is also increased ( Figure 5 ).

[0161] Embodiment 11

[0162] Application effect of solid-state fermented corn gluten meal in poultry feeding

[0163] A total of 450 healthy 7-day-old chicks with a body weight range of 51.36±0.74 were selected and the experiment was conducted at the Animal Breeding Center of Jilin Agricultural University.

[0164] According to the principle of no significant difference in body weight, the chickens were randomly divided into three groups, namely the control group and two experimental groups (10 replicates in each group, 15 chickens in each replicate). The control group was fed with a normal basal diet, the L-FCBM experimental group was fed with a basal diet + 10% compound bacterial fermentation (where the mass percentage of the compound bacterial fermentation was 10%), and the H-FCBM experimental group was fed with a basal diet + 15% compound bacterial fermentation. The experimental period was 42 days, the pre-adaptation period was 7 days, and the formal period was 35 days. The poultry growth performance, feed intake and feed-to-weight ratio were measured, and the levels of ZO-1, Occluding and β-actin in the chicks were measured.

[0165] The average daily gain (ADG), average daily feed intake (ADFI) and feed-to-weight ratio were calculated by recording the initial and final body weights and feed intake. Jejunal samples were collected in enzyme-free tubes and frozen at -80°C for further Western blotting. Total protein from jejunal tissue was extracted and stored at -80°C for subsequent analysis. Total protein concentration was quantified using the BCA assay (Beyotime, P0010), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred to a membrane for immunoblotting. ZO-1 antibody (Abcam, ab307799) and Occludin antibody (Abcam, ab216327) were used as primary antibodies and incubated with the membrane overnight at 4°C. The membrane was then incubated with a secondary antibody. The experiment was repeated three times and analyzed using ImageJ software.

[0166] The specific results are shown in Table 3 and Figure 6 shown.

[0167] Table 3 Effects of solid-state fermented corn gluten meal on poultry growth performance, daily feed intake and feed-to-weight ratio

[0168]

[0169] It can be seen from Table 3 that at the end of the feeding experiment, the final weight, weight gain and average daily weight gain of the poultry in the experimental group were significantly improved, and the feed-to-weight ratio was reduced, indicating that adding the solid fermentation product of the present invention to the ordinary basic diet can significantly improve the growth performance of chicks.

[0170] according to Figure 6 It can be seen that the solid fermentation product of the present invention can improve the characteristics of corn protein powder that is difficult to absorb and has low utilization, and improve the intestinal barrier function of poultry.

[0171] 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composite bacterial agent for improving the quality of corn gluten meal, characterized in that: The method comprises a first-stage fermentation culture liquid and a second-stage fermentation culture liquid, wherein the first-stage fermentation culture liquid is a liquid of Bacillus licheniformis CGMCC NO.29918 and a liquid of Aspergillus niger CICC 40048, and the second-stage fermentation culture liquid is a liquid of Lactobacillus plantarum CICC 22134; In the first stage of fermentation culture, Bacillus licheniformis CGMCC NO.29918 culture liquid and Aspergillus niger CICC40048 culture liquid are mixed in a volume ratio of (1-2):(1-2); the number of live bacteria in the Bacillus licheniformis CGMCC NO.29918 culture liquid is ≥10 9 CFU / mL, the spore count of Aspergillus niger CICC 40048 bacterial liquid is ≥10 8 spores / mL; In the second stage fermentation culture liquid, the number of viable bacteria of Lactobacillus plantarum CICC 22134 liquid is ≥10 9 CFU / mL.

2. Use of the composite bacterial agent according to claim 1 in fermenting corn gluten meal.

3. The use according to claim 2, characterized in that: The following steps are involved: Corn protein meal and bran are used as fermentation substrates, and the first stage fermentation culture solution is inoculated for aerobic fermentation. After the fermentation is completed, the second stage fermentation culture solution is inoculated for anaerobic fermentation. After the fermentation is completed, drying and crushing are carried out.

4. The use according to claim 3, characterized in that: The corn protein powder and bran are mixed in a mass ratio of (4-8):(2-6) as a fermentation substrate.

5. The use according to claim 3, characterized in that: The inoculation amount of the first stage fermentation culture liquid is ≥10 9 CFU / g fermentation substrate, the inoculation amount of the second stage fermentation culture liquid is ≥10 9 CFU / g fermentation substrate.

6. The use according to claim 3, characterized in that: The first stage fermentation culture temperature is 28-35°C, and the aerobic fermentation lasts 3-5 days. The second stage fermentation culture temperature is 30-40°C, and the anaerobic fermentation lasts 3-5 days. During the fermentation period, the solid-liquid ratio of the fermented material is maintained at 2:

1.

7. A feed additive prepared by fermenting corn protein powder with the composite bacterial agent according to claim 1.

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