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

Abstract

The present application 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 comprising: Lactobacillus plantarum R4-30, Lactobacillus acidophilus G32, Lactobacillus fermentum R6-11, Lactococcus lactis G3-1, Leuconostoc citreum G7-13, Candida boidinii G90-9, Pichia kudriavzevii G90-14, the preparation method of the compound microbial agent comprising: (1) activation and rejuvenation of the strain; (2) preparation of the compound microbial agent: uniformly mixing single-strain culture liquids according to mass ratio to obtain the microbial compound agent. After the compound microbial agent provided by the present application is applied to fermenting sugarcane yellow leaves for 8 days, the fermented feed is rich in nutrients and has a fragrant smell, the content of crude protein in the fermented feed is increased, the number of molds is reduced, and the contents of butyric acid, neutral detergent fiber and hemicellulose are all reduced.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a method for preparing and applying a compound microbial inoculant for fermenting yellow sugarcane leaves. Background Technology

[0002] Straw by-products themselves have certain nutritional value and great potential for development into feed resources. However, straw by-products also have certain limitations, such as high cellulose content and low protein content, leading to a significant reduction in utilization rate. Sugarcane harvesting produces a large amount of yellow leaves, which suffer from the common problem of straw lacking by-product resources: low protein, high fiber, and susceptibility to mold.

[0003] Microbial fermentation can be an important solution to the problem of straw by-products. However, natural fermentation or the arbitrary use of microbial agents has serious limitations, making it difficult to effectively address the multiple problems that coexist during straw fermentation. Compound microbial agents, formulated based on specific characteristics, can effectively compensate for these shortcomings after fermentation. Therefore, it is necessary to develop compound microbial agents containing lactic acid bacteria, yeast, and other microorganisms with synergistic effects to promote the degradation of sugarcane yellow leaf fiber, inhibit the growth of harmful microorganisms, suppress the production of harmful metabolites, and improve the quality of fermented sugarcane yellow leaf feed. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing and applying a compound microbial inoculant for fermenting yellow sugarcane leaves. This method enables the compound microbial inoculant, composed of lactic acid bacteria, yeast, and other microorganisms, to have a synergistic effect, promoting fiber degradation in yellow sugarcane leaves, inhibiting the growth of microorganisms, suppressing the production of harmful metabolites, and improving the quality of fermented yellow sugarcane leaf feed. Short-term fermentation increases crude protein content, reduces mold count, and lowers butyric acid, neutral detergent fiber, and hemicellulose content.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a compound microbial inoculant for fermenting yellow sugarcane leaves, comprising: *Lactobacillus plantarum* R4-30, *Lactobacillus acidophilus* G32, *Lactobacillus fermentum* R6-11, *Lactococcus lactis* G3-1, *Leuconostoc tumefaciens* G7-13, *Candida botrytis* G90-9, and *Pichia kudrica* G90-14.

[0006] The Lactobacillus plantarum R4-30 was deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO.M 2018437 on July 2, 2018.

[0007] The Lactobacillus acidophilus G32 strain is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO.65620, and the deposit date is December 11, 2024.

[0008] The Lactobacillus fermentum R6-11 was deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO.M 2018438 on July 2, 2018.

[0009] The Lactococcus lactis G3-1 strain is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO.65626, on December 11, 2024.

[0010] The Leuconostoc citreum G7-13 strain is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO.65621, on December 11, 2024.

[0011] The Candida boidinii G90-9 strain is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO.65527, on November 22, 2024.

[0012] The aforementioned Pichia kudriavzevii G90-14 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO.65528, on November 22, 2024.

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

[0014] (1) Activation and rejuvenation of strains: Take out the strains frozen in glycerol at -80℃, thaw them in a water bath at 37℃, use a sterile inoculation loop to take the bacterial solution from the glycerol cryopreservation tube, put it into sterile MRS or PDB liquid culture medium, and then culture it 2 to 3 times in a constant temperature air bath shaker at a suitable temperature and rotation speed to prepare a single strain culture solution.

[0015] Lactic acid bacteria strains were inoculated into 250 mL sterile Erlenmeyer flasks containing 150 mL of sterile MRS liquid culture medium and cultured at 35 °C and 200 rpm for 24 h on a shaker to achieve a viable bacterial concentration greater than 10⁻⁶. 8CFU / mL; yeast strains were inoculated into 250 mL sterile Erlenmeyer flasks containing 150 mL of sterile PDB liquid medium and cultured at 28 °C and 200 rpm for 24 h on a shaker to achieve a viable bacterial concentration greater than 10⁻⁶ CFU / mL. 8 cfu / mL;

[0016] (2) Preparation of compound microbial agent: The culture media of Lactobacillus plantarum R4-30, Lactobacillus acidophilus G32, Lactobacillus fermentum R6-11, Lactococcus lactis G3-1, Leuconostoc mesenteroides G7-13, Candida botrytis cinerea G90-9, and Pichia kudrica G90-14 were mixed evenly in a mass ratio of 1:1:1:(0-1.5):(0-1.5):(0-3):(0-3) to obtain the compound microbial agent.

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

[0018] The compound microbial agent prepared by the method is used in the production of fermented sugarcane yellow leaves feed. The compound microbial agent is added at a mass ratio of compound microbial agent to sugarcane yellow leaves of 0.3:1000. After mixing, the moisture content is adjusted to 50%, and the mixture is anaerobic and sealed at room temperature for ≥8 days.

[0019] The beneficial effects of this invention are as follows:

[0020] By utilizing lactic acid bacteria and yeast strains screened from high-quality sugarcane silage, and through different formulations, combining the functions of each strain effectively, a compound microbial agent is produced. The lactic acid bacteria play a role in fiber degradation and rapidly lower pH through acid production, effectively inhibiting the reproduction of harmful microorganisms and the generation of harmful metabolites; the yeast produces single-cell protein, effectively increasing the crude protein content in the feed.

[0021] After fermenting sugarcane yellow leaves for 8 days using the compound microbial agent prepared by this invention, the fermented feed is rich in nutrients and has a fragrant aroma. The crude protein content of the fermented feed increases, the number of molds decreases, and the content of butyric acid, neutral detergent fiber and hemicellulose all decrease. Detailed Implementation

[0022] The technical solution of the present invention will be further described in detail below through embodiments.

[0023] Example 1

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

[0025] (1) Activation and rejuvenation of bacterial strains: *Lactobacillus plantarum* R4-30, *Lactobacillus acidophilus* G32, *Lactobacillus fermentum* R6-11, *Lactococcus lactis* G3-1, and *Leuconostoc tartrate* G7-13 strains frozen at -80℃ in glycerol were taken out and thawed in a 37℃ water bath. Using a sterile inoculation loop, bacterial suspension was taken from the glycerol cryovials and placed into sterile MRS liquid culture medium. This medium was then inoculated into a 250mL sterile Erlenmeyer flask containing 150mL of sterile MRS liquid culture medium. The flasks were incubated at 35℃ and 200r / min for 24h on a shaker to ensure a viable bacterial concentration greater than 10%. 8 The concentration of cfu / mL was increased, and the mixture was repeated twice to prepare a single bacterial culture.

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

[0027] Example 2

[0028] This embodiment is another example of the preparation method and application of the compound microbial inoculant for fermenting yellow sugarcane leaves described in this invention, including the following steps:

[0029] (1) Activation and rejuvenation of bacterial strains: Lactic acid bacteria (Lactobacillus plantarum R4-30, Lactobacillus acidophilus G32, Lactobacillus fermentum R6-11) and yeast (Candida botrytis cinerea G90-9) strains frozen in glycerol at -80℃ were taken out and thawed in a 37℃ water bath. Bacterial solutions were taken from the glycerol cryovials using a sterile inoculation loop. The lactic acid bacteria and yeast were inoculated into 250mL sterile Erlenmeyer flasks containing 150mL of sterile MRS and PDB liquid culture media, respectively. The lactic acid bacteria and yeast were cultured in shakers at 35℃ and 200r / min and 28℃ and 200r / min, respectively, for 24h, to ensure that the viable bacterial concentration of the liquid bacterial agent was greater than 10%. 8 The concentration of cfu / mL was increased, and the mixture was repeated twice to prepare a single bacterial culture.

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

[0031] Example 3

[0032] This embodiment is another example of the preparation method and application of the compound microbial inoculant for fermenting yellow sugarcane leaves described in this invention, including the following steps:

[0033] (1) Activation and rejuvenation of bacterial strains: Lactic acid bacteria (Lactobacillus plantarum R4-30, Lactobacillus acidophilus G32, Lactobacillus fermentum R6-11) and yeast (Pichia pastoris G90-14) strains frozen in glycerol at -80℃ were taken out and thawed in a 37℃ water bath. Bacterial solutions were taken from the glycerol cryovials using a sterile inoculation loop. The lactic acid bacteria and yeast were inoculated into 250mL sterile Erlenmeyer flasks containing 150mL of sterile MRS and PDB liquid culture media, respectively. The lactic acid bacteria and yeast were inoculated at 35℃ and 200r / min and 28℃ and 200r / min, respectively, to ensure a viable bacterial concentration greater than 10%. 8 The concentration of cfu / mL was increased, and the mixture was repeated twice to prepare a single bacterial culture.

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

[0035] Example 4

[0036] This embodiment is another example of the preparation method and application of the compound microbial inoculant for fermenting yellow sugarcane leaves described in this invention, including the following steps:

[0037] (1) Activation and rejuvenation of bacterial strains: Lactic acid bacteria (Lactobacillus plantarum R4-30, Lactobacillus acidophilus G32, Lactobacillus fermentum R6-11, Lactococcus lactis G3-1, Leuconostoc tartrate G7-13) and yeast (Candida botrytis cinerea G90-9) strains frozen in glycerol at -80℃ were thawed in a 37℃ water bath. Bacterial solutions were taken from the glycerol cryovials using a sterile inoculation loop. The lactic acid bacteria and yeast were inoculated into 250mL sterile Erlenmeyer flasks containing 150mL of sterile MRS and PDB liquid culture media, respectively. The lactic acid bacteria and yeast were inoculated at 35℃ and 200r / min and 28℃ and 200r / min, respectively, to ensure a viable bacterial concentration greater than 10%. 8 The concentration of cfu / mL was increased, and the mixture was repeated twice to prepare a single bacterial culture.

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

[0039] Example 5

[0040] This embodiment is an application example of the composite microbial agent prepared by the method described in this invention, including the following steps:

[0041] (1) The compound microbial agent 1, compound microbial agent 2, compound microbial agent 3 and compound microbial agent 4 prepared in Examples 1 to 4 were added to the compound microbial agent at a mass ratio of 0.3:1000 between the compound microbial agent and the sugarcane yellow leaves. After mixing, the moisture content was adjusted to 50%, and the mixture was anaerobic and sealed for 8 days at room temperature.

[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 / T20806-2006), hemicellulose content (difference between neutral detergent fiber content and acid detergent fiber content), soluble sugar content (anthrone-sulfuric acid colorimetric method), and mold content (GB / T 13092-2006). The test results are listed in Table 1.

[0043] Table 1. Effects of compound microbial agents on fermented sugarcane yellow leaves in feed

[0044]

[0045]

[0046] As shown in Table 1, compared with the control group, the pH decreased by 0.42–0.52 after adding the compound microbial agent; the contents of butyric acid, ammonia nitrogen, and ethanol decreased by 0.64 g / kg DM–0.72 g / kg DM, 0.04 g / kg DM–0.23 g / kg DM, and 0.12 g / kg DM–0.40 g / kg DM, respectively; the number of molds decreased by 0.22 lg CFU / g–0.95 lg 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 experimental results show that the compound microbial agent played a role. Lactic acid bacteria degraded neutral detergent fiber and hemicellulose, rapidly lowered pH through acid production, reduced ammonia nitrogen content, inhibited the reproduction of harmful microorganisms such as mold, and limited the production of harmful metabolite butyric acid. Yeast produced single-cell protein through fermentation substrate, effectively increasing the crude protein content in feed. In particular, the synergistic effect of both resulted in lower levels of acetic acid and butyric acid. Therefore, the compound microbial agent prepared using this invention can increase the crude protein content in fermented feed, reduce the number of molds, and lower the content of butyric acid, neutral detergent fiber, and hemicellulose.

Claims

1. A compound microbial inoculant for fermenting yellow sugarcane leaves, characterized in that, include: Lactobacillus plantarum R4-30 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO. M 2018437. Lactobacillus acidophilus G32 is deposited at Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC NO.65620; Lactobacillus fermentum R6-11 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO. M 2018438. Lactococcus lactis G3-1 is deposited at Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO.65626; Leuconostoc citrinum G7-13 is deposited at Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC NO.65621; Candida boidinii G90-9 is deposited at the Guangdong Provincial Microbial Culture Collection Center, accession number GDMCC NO.65527; and Pichia kudriavzevii G90-14 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO.65528.

2. The method for preparing the compound microbial inoculant for fermenting yellow sugarcane leaves according to claim 1, characterized in that, Includes the following steps: (1) Activation and rejuvenation of strains: Take out the strains frozen in glycerol at -80℃, thaw them in a water bath at 37℃, use a sterile inoculation loop to take the bacterial solution from the glycerol cryopreservation tube, put it into sterile MRS or PDB liquid culture medium, and then culture it 2 to 3 times in a constant temperature air bath shaker at a suitable temperature and rotation speed to prepare a single strain culture solution. The *Lactobacillus plantarum* R4-30, *Lactobacillus acidophilus* G32, *Lactobacillus fermentum* R6-11, *Lactococcus lactis* G3-1, and *Leuconostoc tartrate* G7-13 were inoculated into 250 mL sterile Erlenmeyer flasks containing 150 mL of sterile MRS liquid culture medium and cultured at 35°C and 200 rpm for 24 h on a shaker to achieve a viable bacterial concentration greater than 10⁻⁶. 8 cfu / mL; the *Candida botrytis* G90-9 and *Pichia kudrica* G90-14 were inoculated into 250 mL sterile Erlenmeyer flasks containing 150 mL of sterile PDB liquid medium and cultured at 28 °C and 200 r / min for 24 h to achieve a viable cell concentration greater than 10 cfu / mL. 8 cfu / mL; (2) Preparation of the compound microbial agent: The culture media of the strains of Lactobacillus plantarum R4-30, Lactobacillus acidophilus G32, Lactobacillus fermentum R6-11, Lactococcus lactis G3-1, Leuconostoc mesenteroides G7-13, Candida botrytis cinerea G90-9, and Pichia kudrica G90-14 are mixed evenly in a mass ratio of 1:1:1:(0-1.5):(0-1.5):(0-3):(0-3) to obtain the compound microbial agent.

3. The preparation method according to claim 2, characterized in that, In step (1), the MRS liquid culture medium is used for the culture of *Lactobacillus plantarum* R4-30, *Lactobacillus acidophilus* G32, *Lactobacillus fermentum* R6-11, *Lactococcus lactis* G3-1, and *Leuconostoc tectorum* G7-13; the PDB liquid culture medium is used for the culture of *Candida botrytis* G90-9 and *Pichia kudrica* G90-14.

4. The compound microbial agent prepared by the method according to any one of claims 2-3 is used in the production of fermented sugarcane yellow leaves feed, characterized in that: Add the compound microbial agent to the sugarcane yellow leaves at a mass ratio of 0.3:1000, mix well, adjust the moisture content to 50%, and anaerobic ferment at room temperature in a sealed environment for ≥8 days.

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