Lactobacillus plantarum FD02 and application thereof in preparing liquor vinasse protein feed

Abstract

The present application relates to the field of microbial technology, and particularly relates to a lactobacillus plantarum FD02 and application thereof in preparation of liquor vinasse protein feed.The lactobacillus plantarum FD02 has the ability of simultaneously producing ferulic acid esterase, glucosidase and inhibiting mold, can be used for preparing the liquor vinasse protein feed with liquor vinasse as a substrate, significantly improves the crude protein and true protein content of the liquor vinasse protein feed, and also reduces the content of neutral detergent fiber (NDF) and acid detergent fiber (ADF), and improves the dry matter digestibility and protein digestibility.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a strain of Lactobacillus plantarum FD02 and its application in the preparation of baijiu (Chinese liquor) lees protein feed. Background Technology

[0002] Distillers' grains are a major byproduct of the brewing industry, rich in nutrients such as protein, carbohydrates, and cellulose. However, improper handling can lead to resource waste and environmental problems. Currently, single-strain fermentation of distillers' grains has limitations in increasing product added value and fully utilizing the grains. Mixed-strain fermentation technology can utilize the synergistic effects between different microorganisms to more effectively transform the components in the grains, enrich their nutritional content, and improve fermentation efficiency and product quality.

[0003] Chinese patent CN120442486A (A Method for Mixed-Cultural Fermentation of Distillers' Grains Feed) provides a method for mixed-culture fermentation of distillers' grains feed. This method involves separately culturing yeast with Bacillus subtilis, Bifidobacterium, Enterococcus faecalis, and Corynebacterium glutamicum before mixed-culture fermentation. The resulting product has the highest protein content at 22.47%, with a crude protein content increase of approximately 7%. However, it does not mention any improvement in true protein content. Although distillers' grains contain some crude protein, only about 70% of this crude protein is true protein, severely impacting the protein quality of the grains. Different mixed-culture fermentation methods have varying effects on crude and true protein content.

[0004] Chinese patent CN121400519A (A biological pretreatment method to improve the efficiency of true protein production from wet distillers' grains) enhances the efficiency of true protein production from wet distillers' grains by combining chemical and biological multi-stage pretreatment methods. However, chemical treatment may lead to poor palatability and reduced feed intake. Microbial fermentation of protein-producing feed can effectively reduce its anti-nutritional factors, improve its utilization efficiency, enhance palatability, and reduce toxicity.

[0005] Therefore, screening microbial strains, conducting research on compound strains and fermentation processes for baijiu lees protein feed, and utilizing microbial technology to improve the content of crude protein and true protein have important application value and practical significance. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a strain of *Lactobacillus plantarum* FD02 and its application in the preparation of baijiu (Chinese liquor) lees protein feed. The *Lactobacillus plantarum* FD02 of this invention possesses the ability to simultaneously produce ferulic acid esterase, glucosidase, and inhibit mold growth. It can be used to prepare baijiu lees protein feed, significantly increasing the crude protein and true protein content of the baijiu lees protein feed, and improving dry matter digestibility and protein digestibility.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a strain of Lactobacillus plantarum. plantarum Lactobacillus plantarum FD02 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252246.

[0008] This invention provides a microbial inoculant, the effective ingredient of which includes Lactobacillus plantarum FD02 as described in the above technical solution.

[0009] Preferably, the viable bacterial concentration of *Lactobacillus plantarum* FD02 in the microbial agent is ≥1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0010] This invention provides a compound fermentation agent, comprising separately packaged aerobic compound fermentation agent and anaerobic fermentation agent; the aerobic compound fermentation agent includes brewer's yeast (Saccharomyces cerevisiae). Saccharomyces cerevisiae Bacillus subtilis ( Bacillus subtilis Bacillus subtilis ( Bacillus subtilis ), Bacillus subtilis subsp. subtilis ( Bacillus subtilis subsp. Subtilis Aspergillus oryzae ( ) Aspergillus oryzae ) and Aspergillus niger ( Aspergillus niger The anaerobic fermentation agent is the microbial agent described in the above technical solution.

[0011] Preferably, the ratio of viable bacteria of the brewing yeast, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis subtilis subtilis, Aspergillus oryzae and Aspergillus niger is 1:1:1:1:1:1.

[0012] Preferably, the aerobic compound microbial agent includes Saccharomyces cerevisiae CICC 1421, Bacillus subtilis CICC 10260, Bacillus subtilis CICC 10089, Bacillus subtilis subtilis subtilis CICC 23830, Aspergillus oryzae CICC 2013, and Aspergillus niger CICC 2214.

[0013] This invention provides the application of Lactobacillus plantarum FD02, the microbial agent, or the compound fermentation agent described in the above-mentioned technical solutions in the preparation of animal feed.

[0014] Preferably, the fermentation substrate for the animal feed is liquor lees.

[0015] This invention provides a method for preparing animal feed, comprising the following steps: Using baijiu lees as the base, with 6×10 6The inoculum amount is CFU / g substrate, inoculated with aerobic compound microbial agent, and aerobic fermentation is carried out for 6-10 days, with the material turned over once every 3.5-4.5 hours; the aerobic compound microbial agent includes the aerobic compound microbial agent described in the above technical solution. After aerobic fermentation, at 1×10 6 Inoculate with the microbial agent described in the above technical solution at an inoculum amount of CFU / g substrate, and anaerobic ferment for 8-12 days to obtain the animal feed. Preferably, the substrate has a water content of 45%-55%, the aerobic fermentation temperature is 28-32℃, and ammonium sulfate is added to the substrate before aerobic fermentation; the mass ratio of ammonium sulfate to substrate is 1.5-2.5:100.

[0016] Beneficial effects: The *Lactobacillus plantarum* FD02 provided by this invention is a strain of *Lactobacillus* selected from the rumen fluid of beef cattle that simultaneously produces ferulic acid esterase and glucosidase and inhibits mold. It can be used to prepare *baijiu jiu* protein feed with *baijiu jiu* as the substrate, significantly improving the crude protein and true protein content of the *baijiu jiu* protein feed.

[0017] Furthermore, this invention, through a combination of aerobic and anaerobic fermentation, can increase the crude protein and true protein content in liquor lees feed, while reducing the content of neutral detergent fiber (NDF) and acid detergent fiber (ADF), thereby improving dry matter digestibility and protein digestibility.

[0018] Biological Preservation Instructions Lactobacillus plantarum FD02, classified as Lactiplantibacillus plantarum FD02 was deposited on October 17, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20252246. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0020] Figure 1 These are the screening results for some antibacterial lactic acid bacteria; Figure 2 The results of screening for the ability of antibacterial lactic acid bacteria to produce β-glucosidase; Figure 3 Results of ferulic acid esterase production capacity assay for Lactobacillus plantarum FD02; Figure 4 Phylogenetic tree of Lactobacillus plantarum FD02; Figure 5The effects of different treatments and different aerobic fermentation times on the crude protein content of distillers' grains feed. Figure 6 To investigate the effects of different treatments and different aerobic fermentation times on the true protein content of distillers' grains feed. Figure 7 The results show the biomimetic digestibility of broiler chickens fed with baijiu lees protein feed before fermentation. Different lowercase letters indicate significant differences between different groups under the same fermentation days, P<0.001. Detailed Implementation

[0021] This invention provides a strain of Lactobacillus plantarum FD02, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252246.

[0022] The *Lactobacillus plantarum* FD02 provided by this invention is a strain of *Lactobacillus* selected from the rumen fluid of beef cattle that simultaneously produces ferulic acid esterase and glucosidase and inhibits mold. It can be used to prepare *baijiu jiu* protein feed with *baijiu jiu* as the substrate, significantly increasing the crude protein and true protein content of *baijiu jiu* protein feed, while reducing the content of neutral detergent fiber (NDF) and acid detergent fiber (ADF), and improving dry matter digestibility and protein digestibility.

[0023] Based on the above advantages, the present invention provides a microbial inoculant, the effective ingredient of which includes Lactobacillus plantarum FD02 as described in the above technical solution.

[0024] As one embodiment, the viable bacterial concentration of *Lactobacillus plantarum* FD02 in the microbial agent is ≥1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0025] Based on the above advantages, the present invention provides a compound fermentation agent, comprising separately packaged aerobic compound fermentation agent and anaerobic fermentation agent; the aerobic compound fermentation agent includes Saccharomyces cerevisiae, Bacillus subtilis, Bacillus subtilis subtilis subsp. subtilis, Aspergillus oryzae, and Aspergillus niger; the anaerobic fermentation agent is the microbial agent described in the above technical solution.

[0026] In one embodiment, the ratio of viable bacteria of the brewing yeast, Bacillus subtilis, Bacillus subtilis var. subtilis, Bacillus subtilis subtilis subtilis var. subtilis, Aspergillus oryzae and Aspergillus niger is 1:1:1:1:1:1.

[0027] In one embodiment, the aerobic compound microbial agent includes Saccharomyces cerevisiae CICC 1421, Bacillus subtilis CICC 10260, Bacillus subtilis CICC 10089, Bacillus subtilis subtilis subtilis CICC 23830, Aspergillus oryzae CICC2013, and Aspergillus niger CICC 2214.

[0028] Based on the above advantages, the present invention provides the application of Lactobacillus plantarum FD02 or the microbial agent or the compound fermentation agent described in the above technical solution in the preparation of animal feed.

[0029] In one implementation, the fermentation substrate for the animal feed is liquor lees.

[0030] Based on the above advantages, the present invention provides a method for preparing animal feed, comprising the following steps: Using baijiu lees as the base, with 6×10 6 The inoculum amount is CFU / g substrate, inoculated with aerobic compound microbial agent, and aerobic fermentation is carried out for 6-10 days, with the material turned over once every 3.5-4.5 hours; the aerobic compound microbial agent includes the aerobic compound microbial agent described in the above technical solution. After aerobic fermentation, at 1×10 6 Inoculate with the microbial agent described in the above technical solution at an inoculum amount of CFU / g substrate, and anaerobic ferment for 8-12 days to obtain the animal feed. In one implementation method, the aerobic fermentation time is 8 days, and the anaerobic fermentation time is 10 days.

[0031] In one embodiment, the substrate has a moisture content of 45%-55%, the aerobic fermentation temperature is 28-32°C, and ammonium sulfate is added to the substrate before aerobic fermentation; the mass ratio of ammonium sulfate to substrate is 1.5-2.5:100. In another embodiment, the substrate has a moisture content of 50%, the aerobic fermentation temperature is 30°C, and ammonium sulfate is added to the substrate before aerobic fermentation; the mass ratio of ammonium sulfate to substrate is 2:100.

[0032] To further illustrate the present invention, the following detailed description, in conjunction with embodiments and accompanying drawings, describes a strain of *Lactobacillus plantarum* FD02 provided by the present invention and its application in the preparation of baijiu (Chinese liquor) lees protein feed. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1 Samples of straw silage, rumen fluid from beef cattle, feces from beef cattle, and rumen fluid from dairy cattle were collected from surrounding areas of Jiangsu Province and brought back to the laboratory for immediate screening of lactic acid bacteria.

[0034] 1. Screening of strains with anti-mold capabilities Take 10.00 g of sample and serially dilute with 90 mL of sterile physiological saline. Pour the diluted solution into MRS agar medium and incubate upside down at 37°C for 48 h. Count lactic acid bacteria. Select typical lactic acid bacteria colonies, purify them by streak plating twice, and store them at -80°C. Inoculate the stored lactic acid bacteria into MRS broth medium and subculture twice. Transfer the bacterial culture to fresh MRS medium at a 5% (v / v) inoculation rate and incubate statically at 37°C for 48 h.

[0035] Indicator strain Aspergillus flavus (CICC 2219) was inoculated onto PDA medium and cultured at 28°C for 7 days to obtain Aspergillus flavus spores. The spores were washed with sterile distilled water and filtered through sterile gauze into 50 mL sterile centrifuge tubes. The spore suspension concentration was adjusted to 5 × 10⁻⁶. 4 CFU / mL. The spore suspension was added to PDA medium and poured onto the top layer of MRS medium used to culture lactic acid bacteria. This double-layer culture method was used for initial screening of the lactic acid bacteria's ability to inhibit mold growth. Some screening results are shown below. Figure 1 .

[0036] The Oxford cup method was used to evaluate the antibacterial activity of fermentation supernatants of lactic acid bacteria with antifungal capabilities. After centrifugation of the lactic acid bacteria fermentation broth, the supernatant was collected and added to the wells of PDA plates inoculated with Aspergillus flavus CICC 2219. The plates were incubated at 30°C for 7 days, and the diameter of the inhibition zone and HC value were measured. Some results are shown in Table 1.

[0037] Table 1. Antibacterial effects of different strains against Aspergillus flavus under the same carbon source

[0038] Note: The Lactobacillus paracasei mentioned is Lactobacillus paracasei GRR in Chinese Patent CN112391325A, with accession number CCTCC NO: M2017777.

[0039] The results showed that strains L496, L4106 and L3101 all had antibacterial effects against Aspergillus flavus.

[0040] 2. Screening of strains possessing both β-glucosidase activity and fungal inhibitory ability The method described in the reference [Dai Shuang, Li He. Directed screening, recombinant expression and enzymatic properties of β-glucosidase Bgl747[J]. Bulletin of Microbiology, 2021, 48(8):2524-2533.] was used to preliminarily screen β-glucosidase using the substrate esculin functional screening method. Black hydrolysis zones were found on the screening plates, proving that they have β-glucosidase activity. Strains that produce β-glucosidase and have strong antibacterial ability were selected. The formula of the esculin selective medium is as follows: glucose 20g / L, peptone 10g / L, beef extract 10g / L, yeast extract 5g / L, anhydrous sodium acetate 5g / L, diammonium hydrogen citrate 2g / L, dipotassium hydrogen phosphate 2g / L, magnesium sulfate 0.58g / L, manganese sulfate 0.17g / L, agar 15g / L, esculin 5g / L and ferric citrate 0.25g / L.

[0041] Through primary and secondary screening, two strains of *Lactobacillus plantarum*, exhibiting both β-glucosidase activity and significant fungal inhibitory ability, were successfully obtained. These strains are designated L496 and L4106. Partial screening results are shown below. Figure 2 .

[0042] 3. Screening of lactic acid bacteria that simultaneously produce ferulic acid esterase and β-glucosidase and can inhibit molds. The screening method for ferulic acid esterase-producing strains is referenced in [Liu Beiyi, Wei Qing, Tian Jipeng, et al. Screening of ferulic acid esterase-producing lactic acid bacteria in sheep rumen fluid and their effect on the quality of rice straw silage [J]. Jiangsu Journal of Agricultural Sciences, 2022, 38(5): 1306-1314.]: Select three appropriate gradients (10 -4 10 -5 10 -6 100 μL of each diluted solution was added to MRS agar plates using the three-spot method, spread evenly, and then incubated anaerobically upside down at 37°C for 48 h. Colonies with different morphologies were selected and repeatedly streaked on MRS agar until purified single colonies were obtained. Single colonies were then picked up with a sterile toothpick and inoculated onto ferulic acid esterase selection medium, and incubated at 30°C for 12-72 h. The presence of a clear zone on the plate was observed. If a clear zone appeared, the strain was considered to have the ability to produce ferulic acid esterase. The formula of the ferulic acid esterase selection medium was as follows: peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, anhydrous sodium acetate 5 g / L, diammonium hydrogen citrate 2 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate 0.58 g / L, manganese sulfate 0.17 g / L, and agar 15 g / L. Results are shown below. Figure 3 And Table 2.

[0043] Table 2. Size of the clear zone of ferulic acid esterase-producing strains

[0044] The results showed that only strain 4106 had the ability to produce ferulic acid esterase.

[0045] 4. Identification and preservation of strain L4106 Genomic DNA was extracted from strain L4106, and 16S rDNA was amplified by PCR. The amplified product was then analyzed by gel electrophoresis and sent to a sequencing company for analysis. The obtained sequence (SEQ ID NO.1) was subjected to BLAST homology comparison in NCBI GenBank, and a phylogenetic tree was constructed. The results are shown below. Figure 4 .

[0046] SEQ ID NO.1:

[0047] Ultimately, it was found that only strain L4106 possessed β-glucosidase activity and the ability to produce ferulic acid esterase, and also exhibited significant antifungal activity. It was renamed Lactobacillus plantarum FD02 and sent to the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252246.

[0048] Example 2 A compound fermentation agent is composed of an aerobic fermentation compound agent and Lactobacillus plantarum FD02; the aerobic fermentation compound agent is composed of Saccharomyces cerevisiae CICC 1421, Bacillus subtilis CICC 10260 (producing β-mannanase), Bacillus subtilis CICC 10089 (producing hemicellulase), Bacillus subtilis subsp. subtilis CICC 23830 (producing bacterial laccase), Aspergillus oryzae CICC 2013, and Aspergillus niger CICC 2214.

[0049] Example 3 Each of the aerobic fermentation compound microbial agents in Example 2 was revived and cultured separately to obtain 6 revived bacterial solutions (all with a viable cell concentration of 1×10⁻⁶). 6 Six resuscitation bacterial solutions (CFU / mL) were mixed in equal volumes to obtain an aerobic fermentation compound bacterial solution; *Lactobacillus plantarum* FD02 was resuscitated and cultured to obtain a viable bacterial concentration of 1×10⁻⁶ CFU / mL. 6 CFU / mL of *Lactobacillus plantarum* FD02 bacterial suspension. The culture media and resuscitation temperatures used for different bacterial strains are as follows: Saccharomyces cerevisiae CICC 1421 was revived using YPD liquid medium at a revival temperature of 28°C. Bacillus subtilis CICC 10260, Bacillus subtilis CICC 10089, and Bacillus subtilis subtilis CICC 23830 were revived on LB medium at 37°C. Aspergillus oryzae CICC 2013 and Aspergillus niger CICC 2214 were revived using PDA medium at a temperature of 28-30℃. Lactobacillus plantarum FD02 was revived using MRS medium at a revival temperature of 37°C.

[0050] Using baijiu lees as the substrate, the moisture content of the substrate was adjusted to 50%, and 6×10 6 The inoculation amount was CFU / g of substrate. The aerobic fermentation compound bacterial solution was inoculated and aerobic fermentation was carried out at an ambient temperature of 30℃. The material was turned over once every 4 hours. The aerobic time was set to 4 days, 6 days and 8 days respectively.

[0051] After aerobic fermentation, at 1×10 6The inoculum was inoculated with the *Lactobacillus plantarum* FD02 culture at a substrate inoculation amount of CFU / g for anaerobic fermentation (at room temperature) for 10 days.

[0052] Three treatment groups were set up, and three parallel replicate experiments were conducted: CK group: The above method was used to first carry out aerobic fermentation and then anaerobic fermentation; Group 1: The above method is used to first carry out aerobic fermentation and then anaerobic fermentation. Ammonium sulfate is added at the same time as the aerobic fermentation compound bacterial solution is inoculated; the mass ratio of ammonium sulfate to substrate is 2:100. Group 2: The above method is used to first carry out aerobic fermentation and then anaerobic fermentation. Ammonium sulfate and molasses are added at the same time as the aerobic fermentation compound bacterial solution is inoculated; the mass ratio of ammonium sulfate, molasses and substrate is 2:1:100. The crude protein and true protein content of the baijiu mash after fermentation and before fermentation were detected, and the results are shown in Table 3. Figures 5-6 .

[0053] Crude protein was determined using a Kjeltec™ 2300 fully automated Kjeldahl nitrogen analyzer.

[0054] Determination of true protein: Mix the sample with copper sulfate solution and sodium hydroxide solution, and heat to near boiling. Copper sulfate forms copper hydroxide precipitate under alkaline conditions, co-precipitating the true protein. After precipitation, filter and wash to remove non-protein nitrogen from the filtrate. Place the precipitate (along with filter paper) in a Kjeldahl flask, add concentrated sulfuric acid and a catalyst (such as CuSO4 or K2SO4), and heat to digest, converting all organic nitrogen into ammonium sulfate. Then add excess sodium hydroxide and distill to remove ammonia, absorb with boric acid or a standard acid, and titrate with a standard acid or base. The final nitrogen content multiplied by the protein conversion factor of 6.25 gives the true protein content.

[0055] Table 3 Comparison of crude protein and true protein content of Baijiu lees after fermentation and before fermentation.

[0056] The results showed that, in group 1, after 8 days of aerobic fermentation followed by 10 days of anaerobic fermentation, the crude protein content of the baijiu lees increased by 8.83 percentage points compared to before fermentation; the true protein content increased by 4.96 percentage points. This demonstrates that the microbial agent and method provided by this invention can increase both the crude protein and true protein content of baijiu lees.

[0057] The dry matter digestibility and protein digestibility of broiler chickens were determined using a biomimetic digestion method for pre-fermentation baijiu (A), post-fermentation baijiu group 1 (B), and soybean meal (C). The biomimetic digestion method used a monogastric biomimetic digestion system. The method reference is [Han Mingxia, Deng Shengqing, Guo Chengnuo, et al. Nutrient content analysis of coconut meal and evaluation of its nutritional value in yellow-feathered broilers using in vitro biomimetic method [J]. Chinese Journal of Animal Nutrition, 2024, 36(8): 5376-5386.].

[0058] The results showed that, compared with the unfermented baijiu lees, the dry matter digestibility and protein digestibility of group 1 were significantly improved, increasing by 25.04% and 56.51% respectively compared with the unfermented baijiu lees. Figure 7 ).

[0059] Example 4 Three treatment groups were set up to ferment baijiu lees protein feed using baijiu lees as the substrate: Group CK: Aerobic fermentation for 8 days and anaerobic fermentation for 10 days were carried out using a similar method to Group 1 in Example 3. The difference was that no Lactobacillus plantarum FD02 bacterial solution was added during anaerobic fermentation.

[0060] FD group: Aerobic fermentation for 8 days and anaerobic fermentation for 10 days were carried out using the method of group 1 in Example 3.

[0061] Group LF: Aerobic fermentation for 8 days and anaerobic fermentation for 10 days were carried out using a method similar to Group 1 in Example 3, the difference being that *Lactobacillus sausageii* was inoculated during anaerobic fermentation. Lactobacillus farciminis SR2, added at a rate of 1×10 6 CFU / g substrate; the preservation number of the Lactobacillus sausageii SR2 is CCTCC NO: M 2022416, published in Chinese Patent CN114908011A.

[0062] The contents of crude protein, true protein, neutral detergent fiber (NDF), and acid detergent fiber (ADF) were determined after fermentation of Baijiu lees. The results are shown in Table 4. The contents of NDF and ADF were determined using ANKOM's fully automated filter bag technology. The methods for detecting crude protein and true protein were the same as in Example 3.

[0063] Table 4. Effects of inoculating with Lactobacillus plantarum FD02 on the fermentation of baijiu (Chinese liquor) residue protein feed.

[0064] Note: Different lowercase letters indicate that the same indicator has significant differences between different groups, P<0.001.

[0065] The results showed that the FD group had the highest crude protein and true protein content, significantly higher than the CK and LF groups, while the FD group had the lowest NDF and ADF content, significantly lower than the CK and LF groups. This means that inoculating with *Lactobacillus plantarum* FD02, which simultaneously produces ferulic acid esterase and glucosidase and inhibits mold, significantly increased the crude protein and true protein content of the liquor lees feed during anaerobic fermentation. Patent application CN120442486A discloses a method for mixed-culture fermentation of liquor lees feed. Before fermentation, the crude protein content of the liquor lees was 14.82%, and after mixed-culture fermentation, it reached 22.47%, an increase of 7.65 percentage points. In contrast, the crude protein content of this application increased by 9.39 percentage points after inoculation with *Lactobacillus plantarum* FD02, which simultaneously produces ferulic acid esterase and glucosidase and inhibits mold, during anaerobic fermentation. Therefore, the *Lactobacillus plantarum* FD02 described in this invention can significantly increase the crude protein and true protein content of liquor lees feed, and its effect is superior to existing technologies.

[0066] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Lactobacillus plantarum ( Lactiplantibacillus plantarum FD02, characterized in that, The Lactobacillus plantarum FD02 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252246.

2. A microbial inoculant, characterized in that, The active ingredient includes Lactobacillus plantarum FD02 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The viable bacterial concentration of *Lactobacillus plantarum* FD02 in the microbial agent is ≥1×10⁻⁶. 6 CFU / mL or ≥1×10 6 CFU / g.

4. A compound fermentation agent, characterized in that, Includes separately packaged aerobic compound microbial agents and anaerobic fermentation agents; the aerobic compound microbial agent includes brewer's yeast (Saccharomyces cerevisiae). Saccharomyces cerevisiae Bacillus subtilis ( Bacillus subtilis Bacillus subtilis ( Bacillus subtilis ), Bacillus subtilis subsp. subtilis ( Bacillus subtilis subsp. Subtilis Aspergillus oryzae ( ) Aspergillus oryzae ) and Aspergillus niger ( Aspergillus niger The anaerobic fermentation agent is the microbial agent described in claim 2 or 3.

5. The compound fermentation agent according to claim 4, characterized in that, The ratio of viable cells of the following bacteria is 1:1:1:1:1:1:

1.

6. The compound fermentation agent according to claim 4 or 5, characterized in that, The aerobic compound microbial agent includes Saccharomyces cerevisiae CICC 1421, Bacillus subtilis CICC 10260, Bacillus subtilis CICC 10089, Bacillus subtilis subtilis subtilis CICC 23830, Aspergillus oryzae CICC 2013, and Aspergillus niger CICC 2214.

7. The application of Lactobacillus plantarum FD02 as described in claim 1, or the microbial agent as described in claim 2 or 3, or the compound fermentation agent as described in any one of claims 4-6, in the preparation of animal feed.

8. The application according to claim 7, characterized in that, The fermentation substrate for the animal feed is baijiu lees.

9. A method for preparing animal feed, characterized in that, Includes the following steps: Using baijiu lees as the base, with 6×10 6 The inoculum amount is CFU / g substrate, inoculated with aerobic compound microbial agent, and aerobic fermentation is carried out for 6-10 days, with the material turned over once every 3.5-4.5 hours; the aerobic compound microbial agent includes the aerobic compound microbial agent described in any one of claims 4-6. After aerobic fermentation, at 1×10 6 The animal feed is obtained by inoculating the microbial agent according to claim 2 or 3 at an inoculum amount of CFU / g substrate and anaerobic fermenting for 8-12 days.

10. The method according to claim 9, characterized in that, The substrate has a moisture content of 45%-55%, the aerobic fermentation temperature is 28-32℃, and ammonium sulfate is added to the substrate before aerobic fermentation; the mass ratio of ammonium sulfate to substrate is 1.5-2.5:100.

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