Preparation method and application of compound microbial agent for fermenting sugarcane yellow leaves

Abstract

The invention relates to the technical field of microbial fermentation, in particular to a preparation method and application of a compound microbial agent for fermenting sugarcane yellow leaves. The compound microbial agent is prepared from lactobacillus plantarum R4-30, lactobacillus acidophilus G32, lactobacillus fermentum R6-11, lactococcus lactis G3-1, leuconostoc citreum G7-13, candida boidinii G90-9 and pichia kudriavzevii G90-14, and the compound microbial agent is prepared from lactobacillus plantarum R4-30, lactobacillus acidophilus G32, lactobacillus fermentum R6-11, lactococcus lactis G3-1, leuconostoc citreum G7-13 The preparation method of the composite microbial agent comprises the following steps: (1) activation and rejuvenation of strains; and (2) preparation of the composite microbial agent: uniformly mixing single strain culture solutions according to a mass ratio to obtain the composite microbial agent. After the compound microbial agent provided by the invention is used for fermenting the sugarcane yellow leaves for 8 days, the fermented feed is rich in nutrition and fragrant in smell, the content of crude protein in the fermented feed is increased, the number of moulds is reduced, and the content of butyric acid, neutral detergent fibers and hemicellulose is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and in particular to a preparation method and application of a composite microbial agent for fermenting yellow sugarcane leaves. Background Art

[0002] While straw byproducts possess significant nutritional value, they hold great potential as a feed resource. However, straw byproducts have limitations, such as high cellulose content and low protein content, which significantly reduces their utilization. Harvesting sugarcane produces a large amount of yellow sugarcane leaves, which exhibit the common drawbacks of straw as a non-productive resource: low protein, high fiber, and susceptibility to mold.

[0003] Microbial fermentation can be an important solution to the problems associated with straw byproducts. However, natural fermentation or the arbitrary use of microbial agents presents significant limitations, making it difficult to effectively address the multiple challenges associated with straw fermentation. Composite microbial agents tailored to their specific characteristics can effectively address these deficiencies after fermentation. Therefore, it is necessary to develop synergistic composite microbial agents composed of lactic acid bacteria, lactic acid bacteria, and yeasts to promote the degradation of sugarcane leaf fiber, inhibit the production of harmful microorganisms and harmful metabolites, and improve the quality of fermented sugarcane leaf feed. Summary of the Invention

[0004] The present invention aims to provide a method for preparing and applying a composite microbial inoculant for fermenting yellow sugarcane leaves. This composite inoculant, comprised of lactic acid bacteria, yeast, and lactic acid bacteria, achieves a synergistic effect, promoting fiber degradation in yellow sugarcane leaves, inhibiting the growth of harmful microorganisms and the production of harmful metabolites, and improving the quality of fermented sugarcane leaf feed. Short-term fermentation increases crude protein content, reduces mold counts, and reduces butyric acid, neutral detergent fiber, and hemicellulose content.

[0005] To achieve the above object, the present invention provides the following technical solution: a composite microbial agent for fermenting yellow sugarcane leaves, comprising: Lactobacillus plantarum R4-30, Lactobacillus acidophilus G32, Lactobacillus fermentum R6-11, Lactococcus lactis G3-1, Leuconostoc citrinum G7-13, Candida boidinii G90-9, Pichia kudriavzevii G90-14,

[0006] The Lactobacillus plantarum R4-30 was deposited in the China Center for Type Microorganism Collection with the deposit number CCTCC NO.M 2018437 on July 2, 2018.

[0007] The Lactobacillus acidophilus G32 was deposited in Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC NO.65620 and the deposit date was December 11, 2024.

[0008] The Lactobacillus fermentum R6-11 was deposited in the China Center for Type Microorganism Collection with the deposit number CCTCC NO.M 2018438 on July 2, 2018.

[0009] The Lactococcus lactis G3-1 was deposited in Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC NO.65626 and the deposit date was December 11, 2024.

[0010] The Leuconostoc citreum G7-13 was deposited in Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC NO.65621 and the deposit date was December 11, 2024.

[0011] The Candida boidinii G90-9 was deposited in Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC NO.65527 and the deposit date was November 22, 2024.

[0012] The Pichia kudriavzevii G90-14 is deposited in the Guangdong Provincial Microbial Culture Collection Center with a deposit number of GDMCC NO.65528 and a deposit date of November 22, 2024.

[0013] The preparation method of the composite microbial agent for fermenting yellow sugarcane leaves comprises the following steps:

[0014] (1) Activation and rejuvenation of the strain: Take out the strain frozen in -80℃ glycerol, thaw it in a 37℃ water bath, use a sterile inoculating loop to dip the bacterial solution from the glycerol cryopreservation tube, place it in sterile MRS or PDB liquid culture medium, and then culture it 2-3 times in a constant temperature air bath shaker at an appropriate temperature and speed to prepare a single strain culture solution;

[0015] The lactic acid bacteria strains were inoculated into 250 mL sterile conical flasks containing 150 mL sterile MRS liquid culture medium and cultured in a shaking incubator at 35°C and 200 rpm for 24 h until the viable bacterial concentration of the liquid inoculum was greater than 10 8cfu / mL; yeast strains were inoculated into 250 mL sterile conical flasks containing 150 mL sterile PDB liquid culture medium and cultured in a shaking incubator at 28 ° C and 200 r / min for 24 h, so that the live bacterial concentration of the liquid inoculum was greater than 10 8 cfu / mL;

[0016] (2) Preparation of composite microbial agent: The culture broth of strains of Lactobacillus plantarum R4-30, Lactobacillus acidophilus G32, Lactobacillus fermentum R6-11, Lactococcus lactis G3-1, Leuconostoc citri G7-13, Candida boidinii G90-9 and Pichia kudriavzevii G90-14 were mixed uniformly in a mass ratio of 1:1:1:(0-1.5):(0-1.5):(0-3):(0-3) to obtain the composite microbial agent.

[0017] The MRS liquid culture medium in step (1) is used for culturing lactic acid bacteria (Lactobacillus plantarum R4-30, Lactobacillus acidophilus G32, Lactobacillus fermentum R6-11, Lactococcus lactis G3-1, and Leuconostoc citriodora G7-13); and the PDB liquid culture medium is used for culturing yeast (Candida boidinii G90-9 and Pichia kudriavzevii G90-14).

[0018] The composite microbial agent prepared by the method is used in the production of fermented feed of yellow sugarcane leaves, which comprises adding the composite microbial agent to yellow sugarcane leaves at a mass ratio of 0.3:1000, mixing well, adjusting the moisture content to 50%, and performing anaerobically closed fermentation at room temperature for ≥8 days.

[0019] The beneficial effects of the present invention are:

[0020] This composite microbial inoculant utilizes lactic acid bacteria and yeast strains selected from high-quality sugarcane tail silage, blended in different ratios to effectively combine the functions of the individual strains. The lactic acid bacteria degrade fiber, producing acid that rapidly lowers pH, effectively inhibiting the growth of harmful microorganisms and the production of harmful metabolites. The yeast produces single-cell protein, effectively increasing the crude protein content in the feed.

[0021] After yellow sugarcane leaves are fermented with the composite microbial agent prepared by the present invention for 8 days, the fermented feed is nutritious and fragrant, and the crude protein content in the fermented feed is increased, the number of molds is reduced, and the butyric acid content, neutral detergent fiber and hemicellulose contents are all reduced. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further described in detail below through examples.

[0023] Example 1

[0024] This embodiment is an example of a preparation method and application of a composite microbial agent for fermenting yellow sugarcane leaves according to the present invention, comprising the following steps:

[0025] (1) Activation and rejuvenation of strains: Take out Lactobacillus plantarum R4-30, Lactobacillus acidophilus G32, Lactobacillus fermentum R6-11, Lactococcus lactis G3-1, and Leuconostoc citri G7-13 strains frozen in glycerol at -80°C, thaw in a 37°C water bath, use a sterile inoculating loop to dip the bacterial solution from the glycerol cryopreservation tube, put it into sterile MRS liquid culture medium, and then inoculate it into a 250mL sterile conical flask containing 150mL sterile MRS liquid culture medium, and culture at 35°C, 200r / min shaking table for 24h, so that the live bacterial concentration of the liquid inoculum is greater than 10 8 cfu / mL, and repeated twice to prepare a single bacterial culture solution.

[0026] (2) Preparation of composite microbial agent: The culture solutions of Lactobacillus plantarum R4-30, Lactobacillus acidophilus G32, Lactobacillus fermentum R6-11, Lactococcus lactis G3-1 and Leuconostoc citri G7-13 were mixed uniformly in a mass ratio of 1:1:1:1.5:1.5 to obtain composite microbial agent 1.

[0027] Example 2

[0028] This embodiment is another example of the preparation method and application of a composite microbial agent for fermenting yellow sugarcane leaves according to the present invention, comprising the following steps:

[0029] (1) Activation and rejuvenation of strains: lactic acid bacteria (Lactobacillus plantarum R4-30, Lactobacillus acidophilus G32, Lactobacillus fermentum R6-11) and yeast (Candida boidinii G90-9) strains frozen in glycerol at -80°C were taken out and thawed in a 37°C water bath. The bacterial solution was dipped from the glycerol cryopreservation tube using a sterile inoculating loop. The lactic acid bacteria and yeast were inoculated into 250 mL sterilized conical flasks containing 150 mL sterilized MRS and PDB liquid culture media, respectively. The lactic acid bacteria and yeast were shaken at 35°C, 200 r / min and 28°C, 200 r / min for 24 h, respectively, so that the live bacterial concentration of the liquid inoculum was greater than 10 8 cfu / mL, and repeated twice to prepare a single bacterial culture solution.

[0030] (2) Preparation of composite microbial agent: The culture solutions of Lactobacillus plantarum R4-30, Lactobacillus acidophilus G32, Lactobacillus fermentum R6-11, and Candida boidinii G90-9 were mixed uniformly in a mass ratio of 1:1:1:3 to obtain composite microbial agent 2.

[0031] Example 3

[0032] This embodiment is another example of the preparation method and application of a composite microbial agent for fermenting yellow sugarcane leaves according to the present invention, comprising the following steps:

[0033] (1) Activation and rejuvenation of strains: lactic acid bacteria (Lactobacillus plantarum R4-30, Lactobacillus acidophilus G32, Lactobacillus fermentum R6-11) and yeast (Pichia kudriavzevii G90-14) strains frozen in glycerol at -80°C were taken out and thawed in a 37°C water bath. The bacterial solution was dipped from the glycerol cryopreservation tube using a sterilized inoculating loop. The lactic acid bacteria and yeast were inoculated into 250 mL sterilized conical flasks containing 150 mL sterilized MRS and PDB liquid culture media, respectively. The lactic acid bacteria and yeast were heated at 35°C, 200 r / min, and 28°C, 200 r / min, respectively, so that the live bacterial concentration of the liquid inoculum was greater than 10 8 cfu / mL, and repeated twice to prepare a single bacterial culture solution.

[0034] (2) Preparation of composite microbial agent: The culture solutions of Lactobacillus plantarum R4-30, Lactobacillus acidophilus G32, Lactobacillus fermentum R6-11, and Pichia kudriavzevii G90-14 strains were mixed uniformly in a mass ratio of 1:1:1:3 to obtain composite microbial agent 3.

[0035] Example 4

[0036] This embodiment is another example of the preparation method and application of a composite microbial agent for fermenting yellow sugarcane leaves according to the present invention, comprising the following steps:

[0037] (1) Activation and rejuvenation of strains: lactic acid bacteria (Lactobacillus plantarum R4-30, Lactobacillus acidophilus G32, Lactobacillus fermentum R6-11, Lactococcus lactis G3-1, Leuconostoc citrinum G7-13) and yeast (Candida boidinii G90-9) strains frozen in glycerol at -80°C were taken out and thawed in a 37°C water bath. The bacterial solution was dipped from the glycerol cryopreservation tube using a sterilized inoculating loop. The lactic acid bacteria and yeast were inoculated into 250 mL sterilized conical flasks containing 150 mL sterilized MRS and PDB liquid culture media, respectively. The lactic acid bacteria and yeast were incubated at 35°C, 200 r / min and 28°C, 200 r / min, respectively, so that the live bacterial concentration of the liquid inoculum was greater than 10 8 cfu / mL, and repeated twice to prepare a single bacterial culture solution.

[0038] (2) Preparation of composite microbial agent: The culture solutions of Lactobacillus plantarum R4-30, Lactobacillus acidophilus G32, Lactobacillus fermentum R6-11, Lactococcus lactis G3-1, Leuconostoc citriodora G7-13 and Candida boidinii G90-9 were mixed uniformly in a mass ratio of 1:1:1:1.5:1.5:3 to obtain composite microbial agent 4.

[0039] Example 5

[0040] This embodiment is an application example of the composite microbial agent prepared by the method of the present invention, comprising the following steps:

[0041] (1) The composite microbial agents 1, 2, 3, and 4 prepared in Examples 1 to 4 were added to the yellow sugarcane leaves at a mass ratio of 0.3:1000, mixed well, and the moisture content was adjusted to 50%. Anaerobically, the mixture was fermented in a closed manner at room temperature for 8 days.

[0042] (2) The feed was tested for crude protein content (GB / T 6432-2018), pH value (pH meter model: FE28), ethanol content (gas chromatography), ammonia nitrogen content (phenol-sodium hypochlorite colorimetric method), organic matter content (GB / T 6438-2007), neutral detergent fiber content (GB / T 20806-2006), hemicellulose content (the difference between neutral detergent fiber content and acid detergent fiber content), soluble sugar content (anthrone-sulfuric acid colorimetric method), and mold (GB / T 13092-2006). The test results are listed in Table 1.

[0043] Table 1 Effect of compound microbial agent on fermentation feed of yellow sugarcane leaves

[0044]

[0045]

[0046] As shown in Table 1, compared with the control group, the pH value decreased by 0.42-0.52 after the addition of the composite microbial agent, the contents of butyric acid, ammonia nitrogen and ethanol decreased by 0.64g / kg DM-0.72g / kg DM, 0.04g / kg DM-0.23g / kg DM and 0.12g / kg DM-0.40g / kg DM, the number of molds decreased by 0.22lg CFU / g-0.95lg CFU / g, the contents of crude protein, soluble sugar and organic matter increased by 0.22%-0.95%, 6.24%-35.09% and 0.15%-2.02% respectively, and the contents of neutral detergent fiber and hemicellulose decreased by 1.81%-3.03% and 2.30%-5.55% respectively.

[0047] The above test results show that the composite microbial agent has played a role. Lactic acid bacteria degrade the neutral detergent fiber and hemicellulose content, rapidly produce acid to reduce pH, reduce ammonia nitrogen content, inhibit the growth of harmful microorganisms such as mold, and limit the production of harmful metabolites such as butyric acid. Yeast produces single-cell protein by fermenting the substrate, effectively increasing the crude protein content in the feed. In particular, the synergistic effect of the two produces lower acetic acid and butyric acid content. Therefore, the composite microbial agent prepared by the present invention can increase the crude protein content in fermented feed, reduce the number of molds, and reduce butyric acid content, neutral detergent fiber, and hemicellulose content.

Claims

1. A composite microbial agent for fermenting yellow sugarcane leaves, characterized in that: include: Lactobacillus plantarum R4-30, Lactobacillus acidophilus G32, Lactobacillus fermentum R6-11, Lactococcus lactis G3-1, Leuconostoc citrinum G7-13, Candida boidinii G90-9, Pichia kudriavzevii G90-14, The Lactobacillus plantarum R4-30 was deposited in the China Center for Type Microorganism Collection with the deposit number CCTCC NO.M 2018437 on July 2, 2018. The Lactobacillus acidophilus G32 was deposited in Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC NO.65620 and the deposit date was December 11, 2024. The Lactobacillus fermentum R6-11 was deposited in the China Center for Type Microorganism Collection with the deposit number CCTCC NO.M 2018438 on July 2, 2018. The Lactococcus lactis G3-1 was deposited in Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC NO.65626 and the deposit date was December 11, 2024. The Leuconostoc citrinum G7-13 was deposited in Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC NO.65621 and the deposit date was December 11, 2024. The Candida boidinii G90-9 was deposited in Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC NO.65527 and the deposit date was November 22, 2024. The Pichia kudriavzevii G90-14 is deposited in the Guangdong Provincial Microbial Culture Collection Center with a deposit number of GDMCC NO.65528 and a deposit date of November 22, 2024.

2. The method for preparing the composite microbial agent for fermenting yellow sugarcane leaves according to claim 1, characterized in that: The compound microbial agent includes: Lactobacillus plantarum R4-30, Lactobacillus acidophilus G32, Lactobacillus fermentum R6-11, Lactococcus lactis G3-1, Leuconostoc citrinum G7-13, Candida boidinii G90-9, Pichia kudriavzevii G90-14, The preparation method of the composite microbial agent comprises the following steps: (1) Activation and rejuvenation of the strain: Take out the strain frozen in -80℃ glycerol, thaw it in a 37℃ water bath, use a sterile inoculating loop to dip the bacterial solution from the glycerol cryopreservation tube, place it in sterile MRS or PDB liquid culture medium, and then culture it 2-3 times in a constant temperature air bath shaker at an appropriate temperature and speed to prepare a single strain culture solution; The lactic acid bacteria strains were inoculated into 250 mL sterile conical flasks containing 150 mL sterile MRS liquid culture medium and cultured in a shaking incubator at 35°C and 200 rpm for 24 h until the viable bacterial concentration of the liquid inoculum was greater than 10 8 cfu / mL; yeast strains were inoculated into 250 mL sterile conical flasks containing 150 mL sterile PDB liquid culture medium and cultured in a shaking incubator at 28 ° C and 200 r / min for 24 h, so that the live bacterial concentration of the liquid inoculum was greater than 10 8 cfu / mL; (2) Preparation of composite microbial agent: The culture broth of strains of Lactobacillus plantarum R4-30, Lactobacillus acidophilus G32, Lactobacillus fermentum R6-11, Lactococcus lactis G3-1, Leuconostoc citri G7-13, Candida boidinii G90-9 and Pichia kudriavzevii G90-14 were mixed uniformly in a mass ratio of 1:1:1:(0-1.5):(0-1.5):(0-3):(0-3) to obtain the composite microbial agent.

3. The method according to claim 1, characterized in that The MRS liquid culture medium in step (1) is used for culturing lactic acid bacteria (Lactobacillus plantarum R4-30, Lactobacillus acidophilus G32, Lactobacillus fermentum R6-11, Lactococcus lactis G3-1, and Leuconostoc citriodora G7-13); and the PDB liquid culture medium is used for culturing yeast (Candida boidinii G90-9 and Pichia kudriavzevii G90-14).

4. The composite microbial agent prepared by the method according to any one of claims 1 to 2 is used in the production of sugarcane yellow leaf fermented feed, characterized in that: The compound microbial agent is added according to the mass ratio of the compound microbial agent to the yellow sugarcane leaves of 0.3:1000. After mixing, the moisture content is adjusted to 50%, and anaerobically closed fermentation is carried out at room temperature for ≥8 days.

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