Mixed strain for degrading palm meal fibers and feed

Abstract

The invention provides a mixed bacterial strain for degrading palm meal fibers. The mixed bacterial strain is prepared from Aspergillus niger, Trichoderma viride and Mucor racemosus, and the mixed bacterial strain is prepared from the following components: Aspergillus niger, Trichoderma viride and Mucor racemosus. The invention also provides a palm meal degradation method of the mixed strain, which comprises the following steps: crushing palm meal, adding konjac powder, and uniformly mixing; adjusting the moisture of the material to 50-70%, inoculating a mixed strain seed solution, and standing and fermenting at 25-37 DEG C for 48-72 hours; drying the fermented material, grinding to prepare crude enzyme powder, adding water for enzymolysis, and then freeze-drying and crushing. The physical and chemical detection method of the crushed sample comprises the following steps: detecting the content of reducing sugar according to GB 5009.7-2016 determination of reducing sugar in food in national food safety standard, and detecting neutral detergent fiber (NDF) according to GB / T 20806-2022 determination of NDF in feed. The dry matter content in the feed and the excrement is detected according to GB / T 6435-2014 determination of water in feed.

Description

Technical Field

[0001] This invention provides a mixed bacterial strain and feed for degrading palm meal fiber, belonging to the field of fermentation engineering technology. Background Technology

[0002] Palm oil meal, a major byproduct of palm oil processing, has a global annual production exceeding 22 million tons. Its price is only one-third to one-half that of soybean meal, and it is rich in protein, minerals, and other basic nutrients, making it a recognized high-quality feed substitute. However, the resource utilization of palm oil meal is limited by its unique fiber structure: the crude fiber content is as high as 13% to 22%, of which 58% to 78% is insoluble mannan. This component is not only hard and poorly palatable, but it also cannot be broken down by endogenous digestive enzymes in livestock and poultry, resulting in a dry matter digestibility of less than 60%. The proportion added to feed is usually limited to below 10%, greatly wasting the application value of this inexpensive raw material. Microbial degradation technology, which enables the targeted decomposition of fiber through enzyme production by the microorganisms themselves, has become a core means to overcome the bottleneck in the utilization of palm meal. Among them, fungi such as Aspergillus niger have been widely studied due to their broad enzyme production spectrum. However, existing strains and technical systems have significant shortcomings: First, the degradation targeting is weak. Most strains secrete insufficient mannanase activity, requiring the addition of exogenous enzymes such as β-mannanase and cellulase, which is costly. Second, the environmental tolerance is poor. The optimal growth temperature of existing strains is concentrated in 28~30℃, and the degradation efficiency drops sharply when the humidity fluctuates by more than 5%. Precise temperature and humidity control is required in industrial production, resulting in high equipment investment costs. Third, mannanase is the core enzyme for degrading palm meal NDF. It is divided into acidic and neutral categories according to the optimal pH: acidic enzymes (optimal pH 3.0-5.0) and neutral enzymes (optimal pH 6.0-7.0) are easily affected by the environmental pH, resulting in a sharp drop in activity. Moreover, they have weak specificity for mannan, which cannot meet the needs of industrialization.

[0003] This invention uses palm meal as the sole carbon source and obtains a target Aspergillus niger strain through ARTP mutagenesis and 10 generations of targeted domestication and selection. Its core advantages are significant: First, it improves degradation efficiency; the activity of the β-mannanase secreted by the strain reaches 2111.2 U / g, reducing the neutral detergent fiber content of palm meal to below 44.01% without the need for exogenous enzymes. Second, it has strong industrial adaptability, growing stably within a wide range of 25-37℃ and initial moisture content of 50%-70%, completing degradation in 48-72 hours, making it compatible with existing solid-state fermentation production lines. Third, it has low application cost; the dry matter digestibility (DM%) of palm meal after fermentation increases to over 55.28%, significantly reducing breeding costs and effectively solving the core pain points of poor targeting and high cost of existing technologies. Summary of the Invention

[0004] The purpose of this invention is to provide a highly efficient method for the synergistic degradation of palm meal fiber by bacteria and enzymes. The mixed bacterial strain for degrading palm meal fiber is characterized in that the mixed bacterial strain consists of *Aspergillus niger* (…). Aspergillus niger ), green Trichoderma ( Trichoderma viride ) and Mucor ( Mucor racemosus )composition.

[0005] The Aspergillus niger ( Aspergillus niger Separated from soil containing palm meal after long-term deep burial and natural degradation through gradient dilution, and obtained through primary screening using the clear zone method and secondary screening using shake flasks, the strain was identified as *Aspergillus niger* by colony morphology observation (black, fluffy colonies, spherical spores) and ITS sequence homology analysis (≥99% similarity to the *Aspergillus niger* type strain). It is deposited at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: M 20252141, deposit date: September 26, 2025, classified as *Aspergillus niger*, and located at Wuhan University, Wuhan, China. Its core function is the efficient secretion of β-mannanase for the targeted degradation of insoluble mannan.

[0006] The green Trichoderma ( Trichoderma viride (Purchased from the China Industrial Microbial Culture Collection Center, strain number CICC 13038). Its core function is to secrete cellulase, which helps to break down the cellulose components in palm meal.

[0007] The Mucor ( Mucor racemosus (Purchased from the China Industrial Microbial Culture Collection Center, strain number CICC 3116). Its core function is to secrete amylase and organic acids, improve the microenvironment of the fermentation system, and promote the growth and enzyme release of other strains.

[0008] The activation and expansion culture method of the strain is as follows: The preserved strain is inoculated onto PDA slant medium and cultured at 30℃ for 48-72 hours. After the spores mature, they are eluted with sterile physiological saline containing 0.1% Tween-80 to prepare 1×10⁻⁶ spores. 7 ~1×10 8 A spore suspension of spores / mL was prepared; the spore suspension was inoculated into seed culture medium (20 g / L wheat bran, 5 g / L glucose, 3 g / L yeast extract) at a 5% inoculation rate, and cultured at 31℃ and 220 r / min for 24 hours with shaking to obtain a high-density seed culture for later use.

[0009] Optimization of the synergistic ratio of the three bacteria: Through experimental verification, the optimal mass ratio of seed liquid of Aspergillus niger (A0-35), Trichoderma viride (T0-35), and Mucor (P0-35) was determined to be 2-3:3-5:1, with 2:3:1 being the preferred ratio. Under this ratio, the three bacteria can achieve enzyme complementarity, and the various bacterial enzymes can work synergistically, resulting in the highest fiber degradation efficiency.

[0010] The method for degrading palm meal using the mixed strains includes the following steps: (1) Pretreatment: After crushing the palm meal, add konjac flour and mix evenly; (2) Inoculation and fermentation: Adjust the moisture content of the material to 50%~70%, inoculate with mixed strain seed liquid, and let it ferment statically at 25~37℃ for 48~72 hours; (3) Enzymatic hydrolysis: The fermented material is dried, ground into crude enzyme powder, and then freeze-dried and pulverized after being hydrolyzed with water.

[0011] Adding konjac flour: Take palm meal that has been pulverized with a 40-mesh sieve and add konjac flour (particle size ≤ 0.1 mm) at 0.5%~1% of the palm meal mass, and mix evenly; Konjac flour has the functions of both a stabilizer and a gelling agent, which can improve the water retention and uniformity of the fermentation system, reduce material clumping during fermentation, and its gelling properties can encapsulate fiber degradation products, improving the palatability of the final product.

[0012] The palm meal fermentation employs a solid-state fermentation process. The specific steps are as follows: Palm meal, pulverized through a 40-mesh sieve, is inoculated with the aforementioned bacterial strain, and then allowed to ferment at a moisture content of 50%–70% and a temperature of 25°C–37°C for 48–72 days. During fermentation, konjac flour slowly releases glucomannan, providing an auxiliary carbon source for the bacterial strain and further promoting enzyme secretion. In a preferred embodiment, the initial moisture content is 60%, the inoculum size is 10%, the fermentation temperature is 31°C, and the fermentation time is 72 hours.

[0013] The strain was used in the degradation of palm meal using a crude enzyme hydrolysis method: The fermented palm meal substrate was removed and dried at 45℃ for 12 hours until the moisture content was ≤10%. The dried substrate was then ground into powder, passed through a 0.1mm sieve to remove coarse particles, and the powder was collected and sealed for storage; this is the crude enzyme product. Konjac flour forms a porous structure during the drying process, improving the stability of the crude enzyme product and reducing enzyme activity loss. 20g of palm meal was weighed and mixed with 80g of sterile water to form a substrate. This mixture was then placed in a 250mL Erlenmeyer flask (1 / 3 of the volume), and an appropriate amount of antibiotics (50mg / mL ampicillin + 50mg / mL chloramphenicol) was added to inhibit contamination. The crude enzyme product was added at a ratio of 0.5% of the hydrolysis substrate and mixed thoroughly. The mixture was then hydrolyzed on a shaker at 50℃ and 220rpm for 15 hours, observing the appearance of the hydrolysate during the process. After hydrolysis, the mixture was cooled to room temperature. The enzymatically hydrolyzed sample was frozen at -20°C for 24 hours until completely frozen, then freeze-dried at -50°C and 10 Pa for 24 hours until a loose powder was formed. The powder was then pulverized at 10,000 rpm for 1 minute, passed through a 0.1 mm sieve, collected, sealed, and stored for subsequent analysis. The animal experiment protocol for the strain is as follows: unfermented palm meal (control group), single-strain fermented palm meal, and multi-strain fermented palm meal are selected, and the digestion and metabolism of broilers are carried out using the full manure collection method.

[0014] The physicochemical testing methods for the pulverized samples are as follows: Reducing sugar content was tested according to GB 5009.7-2016 National Food Safety Standard - Determination of Reducing Sugars in Food; NDF was tested according to GB / T 20806-2022 Determination of Neutral Detergent Fiber (NDF) in Feed; and dry matter content in feed and feces was tested according to GB / T 6435-2014 Determination of Moisture in Feed.

[0015] Advantages of this invention: This invention provides a highly efficient method and application technology for the synergistic degradation of palm meal fiber by bacteria and enzymes. Its core advantages are as follows: 1. Synergistic innovation of three bacteria and enzymes: Breaking through the limitations of single strains or enzyme preparations in degradation, Aspergillus niger (mainly producing β-mannanase), Trichoderma viride (mainly producing cellulase), and Mucor (mainly producing amylase and organic acids) form a complementary enzyme system, which can simultaneously and efficiently degrade various fiber components in palm meal, such as insoluble mannan and cellulose. The β-mannanase activity reaches over 2111 U / g, and the neutral detergent fiber content can be reduced to below 44.01% without the need for exogenous enzymes. The degradation efficiency and overall performance are significantly superior to existing technologies.

[0016] 2. Innovative Dual-Function Application of Konjac Flour: For the first time, konjac flour is introduced into the palm meal fermentation system as both a stabilizer and a gelling agent. On the one hand, it can improve the water retention and uniformity of the fermented material, reduce the problems of clumping and uneven degradation caused by humidity fluctuations in industrial production, and reduce the pressure on equipment temperature and humidity control. On the other hand, its gelling properties can encapsulate fiber degradation products, improve the palatability of the fermented palm meal, and at the same time reduce liquid viscosity and improve crude enzyme stability during enzymatic hydrolysis, achieving full-chain optimization of "fermentation-enzymatic hydrolysis-product quality".

[0017] 3. Wide environmental adaptability and low cost advantages: The fermentation process can stably produce enzymes within a wide temperature range of 25~37℃ and a moisture range of 50%~70%, without the need for high-precision temperature control equipment. Degradation can be completed in 72 hours, making it compatible with existing solid-state fermentation production lines. The dry matter digestibility of the fermented palm meal is increased to over 55.28%, and it can be added to broiler diets at a ratio of 15%, significantly reducing the amount of corn and soybean meal used in feed. It is suitable for various breeding scenarios such as pigs, chickens, and ruminants, with outstanding resource value and economic benefits. Attached Figure Description

[0018] Figure 1: Colony morphology and spore micrographs of Aspergillus niger A0-35 (left: PDA plate colony, right: spores examined under a 40x microscope).

[0019] Figure 2: Effects of different fermentation times on the degradation of palm meal NDF and reducing sugar content by enzymes produced by the combined bacteria.

[0020] Figure 3: Effects of different fermentation temperatures on the degradation of palm meal NDF and reducing sugar content by combined microbial fermentation.

[0021] Figure 4: Effects of different inoculum amounts on the degradation of palm meal NDF and reducing sugar content by combined bacterial fermentation.

[0022] Figure 5: Effects of different initial moisture content during fermentation on the NDF and reducing sugar content of palm meal produced by the combined bacteria during fermentation.

[0023] Figure 6: Effects of different fermentation pH on the degradation of palm meal NDF and reducing sugar content by combined bacteria during fermentation. Detailed Implementation

[0024] Example 1: Screening and initial screening of strains 1.1 Sample Collection and Strain Isolation Ten 50g samples of soil (5-15cm depth) from long-term deep burial of palm meal were collected and placed in sterile sampling bags. 10g of soil sample was weighed and added to 90mL of sterile physiological saline. The mixture was shaken at 220rpm for 30min to prepare a bacterial suspension, which was then serially diluted to 10⁻⁶. -4 ~10 -6 times. Spread 100 μL of the diluted solution onto the selective medium (20 g / L palm meal, 3 g / L sodium nitrate, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 0.5 g / L potassium chloride, 20 g / L agar, pH 6.0, sterilized at 121℃ for 20 min), and incubate at 30℃ for 48–72 h. Single colonies with a black, fluffy morphology and spherical spores were selected and transferred to PDA slant medium for purification culture, yielding 30 suspected Aspergillus niger strains. Single colonies initially white and fluffy, gradually turning dark green, with dichotomously branched sporangiophores and oval, pale green conidia were selected and transferred to PDA slant medium for purification culture, yielding 30 suspected Trichoderma viride strains. Single colonies with white to pale yellow cottony appearance, well-developed aerial hyphae, no septa, spherical sporangia, and oval, colorless sporangiospores were selected and transferred to PDA slant medium for purification culture, yielding 30 suspected Mucor racemose strains.

[0025] 1.2 Initial screening and identification Ninety strains were inoculated into seed culture medium (20 g / L wheat bran, 5 g / L glucose, 3 g / L yeast extract, pH 6.0) and cultured at 30℃ and 220 rpm for 24 h to prepare seed liquid. A 5% inoculum was then transferred to fermentation medium (30 g / L palm meal, 5 g / L peptone, 3 g / L yeast extract, pH 6.0) and cultured at 30℃ for 72 h. The β-mannanase activity of the fermentation broth was then measured.

[0026] The strain with the highest enzyme activity (number A-01) was selected and sequenced by ITS.

[0027] and Aspergillus niger isolate MEBP0060 (MT597434.1) showed 99.83% homology and was identified as Aspergillus niger based on colony morphology. Aspergillus niger spores or mycelia were suspended in a sterile solution containing a cryoprotectant (15%-25% glycerol), aliquoted into cryovials, and subjected to gradient cooling (4℃→-20℃→-80℃) before being stored at -80℃ as the primary strain.

[0028] Example 2: ARTP mutagenesis and screening of strains 2.1 Preparations and Parameter Optimization Before Mutagenesis Preparation of bacterial suspension: Take the slant of the primary generation strain, wash away the spores with sterile physiological saline, and prepare a suspension with a concentration of 1×10⁻⁶. 8 Add 5% glycerol to a bacterial suspension at a final concentration of CFU / mL and mix well.

[0029] Equipment debugging: The helium pressure of the ARTP mutagen (Tianjin Jichuang, ARTP-Ⅱ) was adjusted to 0.6MPa and preheated for 10 minutes. The mutagenization power was set to 90W, 110W, and 130W, and the mutagenization time was set to 30s, 60s, 90s, and 120s, with 3 replicates per group. After mutagenesis, the bacterial culture was diluted and spread on PDA plates and incubated at 20℃ for 48 hours.

[0030] 2.2 Targeted screening of mutagenic strains The bacterial suspension after optimal parameter mutagenesis was spread onto a clear zone screening medium (15 g / L konjac flour, 20 g / L agar, pH 6.0) and incubated at 30°C for 48 h. The colony diameter (D) and the clear zone diameter (d) were measured, and 20 strains of each with d / D ≥ 2.5 were selected for shake-flask rescreening.

[0031] Sixty strains were inoculated into fermentation medium and cultured at 30℃ for 72 h. The β-mannanase activity and the degradation rate of insoluble mannan were then measured. The results showed that strain A0-35 had an enzyme activity of 2111.2 U / g, strain T0-35 had an enzyme activity of 1209.7 U / g, and strain P0-35 had an enzyme activity of 832.6 U / g. All strains exhibited significantly higher degradation rates than the initial generation strains and were thus identified as the target mutagenized strains.

[0032] Example 3: Verification of the synergistic effect of three-strain enzymes on the degradation of palm meal The mixing ratio (by mass) of seed culture of Aspergillus niger (A), Trichoderma viride (T), and Mucor (P) was set up in 28 groups, covering the full gradient from "single-strain → double-strain → triple-strain" to ensure the continuity and scientific validity of the ratio gradient. Single bacterial groups (3 groups): 6:0:0 (A pure bacteria), 0:6:0 (T pure bacteria), 0:0:6 (P pure bacteria); Double-bacterial groups (15 groups): 5:1:0, 4:2:0, 3:3:0, 2:4:0, 1:5:0 (AT double-bacterial); 5:0:1, 4:0:2, 3:0:3, 2:0:4, 1:0:5 (AP double-bacterial); 0:5:1, 0:4:2, 0:3:3, 0:2:4, 0:1:5 (TP double-bacterial); Triple-bacterial groups (10 groups): 4:1:1, 3:2:1, 3:1:2, 2:3:1, 2:2:2, 2:1:3, 1:4:1, 1:3:2, 1:2:3, 1:1:4 (ATP triple-bacterial group).

[0033] Fermentation process: (1) Material preparation: Take palm meal that has been crushed to 40 mesh, weigh 100g of each batch and put it into a 500mL fermentation bottle, add konjac flour (particle size ≤0.1mm) at 0.8%, and mix evenly; (2) Moisture adjustment: The initial moisture content of the material is uniformly adjusted to 50% by spray water addition method, and the moisture content is measured 3 times with a moisture meter to ensure that the error is ≤1%; (3) Seed liquid inoculation: 28 groups of mixed seed liquids with corresponding ratios were inoculated at an inoculation amount of 10% (V / V). The mixtures were stirred for 15 minutes at 80 r / min using a planetary mixer. Samples were taken to observe the uniformity of the materials to ensure that there was no seed liquid agglomeration. (4) Fermentation control: Place the fermentation bottle in a constant temperature incubator, set the temperature to 28℃, and let it ferment for 5 days; during the fermentation period, ventilate twice a day at 9:00 and 17:00, each time for 30 minutes (ventilation volume 0.5m). 3 / (h·kg)), use a thermometer inserted into the center of the material to check the temperature, ensuring it does not exceed 30℃; test results:

[0034] Note: In the table, A represents Aspergillus niger, T represents Trichoderma viride, and P represents Mucor. in conclusion: Single-strain degradation effect: The NDF content of groups 1-3 (single-strain groups) was between 64% and 66%, and the reducing sugar content was between 2.8% and 3.2%, which was significantly lower than that of other groups (P<0.05). This indicates that a single strain can only initially degrade palm meal fiber and cannot efficiently decompose the complex structure of insoluble mannan and cellulose.

[0035] Synergistic effect of dual bacteria: In groups 4-18 (dual bacteria groups), the NDF content decreased to 59%-63%, and the reducing sugar content increased to 4.8%-6.3%, which was 8%-10% lower than that of the single bacteria group and 60%-120% higher than that of the single bacteria group (P<0.05). Among them, the degradation effect of AT dual bacteria (group 6) and AP dual bacteria (group 11) was the best, which confirmed that there is a synergistic effect between β-mannanase of Aspergillus niger, cellulase of Trichoderma viride, and organic acid of Mucor. However, the dual bacteria combination still lacks a complete enzyme system and cannot completely break through the fiber barrier.

[0036] Synergistic effect of the three bacteria: In groups 19-28 (three bacteria groups), the NDF content further decreased to 50%-61%, and the reducing sugar content increased to 7.9%-11.4%, which was 8%-15% lower than that of the two bacteria groups and 40%-80% higher than that of the two bacteria groups (P<0.05); In particular, group 22 (A:T:P=2:3:1) had the lowest NDF (50.12%) and the highest reducing sugar (11.35%), indicating that under this ratio, Aspergillus niger (β-mannanase), Trichoderma viride (cellulase), and Mucor (amylase + organic acid) formed a complete complementary enzyme system and the microenvironment optimization effect was the best, achieving a synergistic degradation effect of "1+1+1>3".

[0037] Example 4: Effects of konjac flour on palm meal fermentation and product quality Experimental Design: Six konjac flour (KGP) addition treatment groups were set up, with three biological replicates per group and three technical replicates per replicate (n=9). The fermentation method was the same as in Example 3 (three-strain ratio 2:3:1). The specific groupings are as follows (Note: The concentration of added konjac flour (KGP) is 0.2%, which can be expressed as K...). 0.2 ): Control group (CK): Unfermented palm meal, no KGP added; Control group (K0): Fermented palm meal (A:T:P=2:3:1), no KGP added; Experimental group (K) 0.2 K 0.5 K 0.8 K 1.0 K 1.2 ): Fermented palm meal (A:T:P=2:3:1), with KGP added at 0.2%, 0.5%, 0.8%, 1.0%, and 1.2% of the dry weight of the palm meal, respectively. Each group was replicated 3 times. The fermentation process was as follows: initial moisture content 50%, inoculum 10%, temperature 28℃, and fermentation for 5 days.

[0038] Test results:

[0039] Conclusion: After adding konjac flour, the NDF content was further reduced to about 44%, and the reducing sugar content was increased to about 15%, which was significantly better than the three-strain group without added konjac flour. The NDF content was the lowest (44.89%) and the reducing sugar content was the highest (15.23%) at an addition of 1%, but considering the overall cost, the addition range of 0.5% to 1% is recommended.

[0040] Example 5: Combined enzymatic hydrolysis of palm meal 5.1 Optimization of crude enzyme fermentation process The effects of fermentation cycle, fermentation temperature, inoculum size, initial moisture content, and pH on the degradation of palm meal NDF by enzymes produced during fermentation of the combined molds A0-35, T0-35, and P0-35 (the optimal ratio was verified to be 2:3;1) were studied using a single-factor method.

[0041] Solid-state fermentation: Take palm meal that has been crushed to a fine 40 mesh.

[0042] The initial fermentation conditions were: inoculum size 5%, temperature 28 ℃, and initial moisture content 50%.

[0043] Under the initial fermentation medium and culture conditions described above, samples were taken every 12 hours for enzymatic hydrolysis experiments to study the effect of different fermentation times on the degradation of palm meal NDF by enzymes produced by the combined mold. The results are as follows:

[0044] Determining the optimal fermentation time: When fermentation lasts for 72 hours, the NDF content drops to its lowest level (50.12%), the reducing sugar content reaches its highest level (11.35%), the enzyme secretion of the strain reaches its peak, the fiber degradation and product accumulation reach equilibrium, and the material does not experience nutrient loss due to over-fermentation, which is the optimal fermentation time.

[0045] Based on the optimal results of the above experiments, samples were collected after fermentation at 19℃, 22℃, 25℃, 28℃, 31℃, 34℃, 37℃, 40℃, and 43℃ for enzymatic hydrolysis experiments. The effects of different fermentation times on the degradation of palm meal NDF by enzymes produced by the combined mold were investigated. The results are as follows:

[0046] The optimal fermentation temperature was determined as follows: Within the range of 25~37℃, the NDF content remained stable at 44~49%, and the reducing sugar content was 13~16%; when the temperature was below 25℃ (19℃, 22℃) or above 37℃ (40℃, 43℃), the NDF content increased sharply to over 60%, and the reducing sugar content dropped sharply to below 6%; the optimal temperature was 31℃, at which point the NDF content was the lowest (44.01%) and the reducing sugar content was the highest (16.12%).

[0047] Based on the optimal results of the above experiments, fermentation was carried out with different inoculum amounts (V / V) of 2%, 4%, 6%, 8%, 10%, 12%, and 14%, followed by enzymatic hydrolysis experiments. The effects of different inoculum amounts on the degradation of palm meal NDF by enzymes produced during fermentation by the combined mold were investigated. The results are as follows:

[0048] The optimal inoculum size for fermentation was determined: at an inoculum size of 10%, the NDF content was reduced to its lowest level (50.12%), and the reducing sugar content reached 11.35%. At this level, the ratio of strain concentration to substrate (palm meal fiber) showed the best match, avoiding both the slow cell proliferation and insufficient enzyme secretion leading to low degradation efficiency at low inoculum sizes (2-8%), and the resource waste seen at high inoculum sizes (12-20%). From an industrial production perspective, a 10% inoculum size does not require additional seed culture preparation costs and is compatible with existing solid-state fermentation production line inoculation equipment, meeting the requirements of "high efficiency + economy".

[0049] Based on the optimal results of the above experiments, samples were taken after fermentation culture at initial moisture levels of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% for enzymatic hydrolysis experiments. The effects of different initial moisture levels on the degradation of palm meal NDF by enzyme production during fermentation by the combined mold were studied. The results are as follows:

[0050] The optimal initial moisture content for fermentation was determined as follows: Within the moisture range of 50%–70%, the NDF content remained stable at 44–48%, and the reducing sugar content at 13–16%. Below 50% (30%, 40%) or above 70% (80%, 90%), NDF surged to over 63%, while reducing sugar plummeted to below 4%. The optimal initial moisture content was 60%, at which point NDF was lowest (44.01%) and reducing sugar was highest (16.12%). Note: Data in the table represents NDF content (%, dry basis); lower values ​​indicate better degradation.

[0051] Based on the optimal results of the above experiments, samples were taken after fermentation at pH values ​​of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0 for enzymatic hydrolysis experiments. The effects of different pH values ​​on the degradation of palm meal NDF by enzymes produced during fermentation by the combined mold were investigated. The results are as follows.

[0052] Conclusion: Within the pH range of 5.0-7.0, the NDF content remained stable at 44-49%, and the reducing sugar content was 12-16%. When the pH was below 5.0 (3.0, 4.0) or above 7.0 (8.0, 9.0), the NDF content increased sharply to over 65%, and the reducing sugar content dropped sharply to below 3%. The optimal pH was 6.0, at which the NDF content was the lowest (44.01%) and the reducing sugar content was the highest (16.12%).

[0053] The optimal process was determined as follows: initial moisture content 60%, inoculum size 10%, temperature 31℃, pH 6.0. Under these conditions, fermentation for 72 hours resulted in NDF content below 44.01%.

[0054] 5.2 Preparation of crude enzyme products and enzymatic hydrolysis experiments After fermentation, the material was dried at 45℃ until the moisture content was ≤10%, and then ground through a 0.1mm sieve to obtain crude enzyme powder. 20g of palm meal was taken, and water was added at a material-to-liquid ratio of 1:4. 0.1% crude enzyme powder was added, and the mixture was enzymatically hydrolyzed at 50℃ and 220r / min for 15h. A control group and a blank group were set up, and the results are shown in the table below:

[0055] Note: The enzyme activity (U / g) data in the table specifically refers to the enzyme activity of enzymes that can degrade mannan.

[0056] Conclusion: The optimized group (fermentation optimized with konjac flour and synergistic three-strain fermentation) had the lowest NDF (44.01%), the highest reducing sugar (16.12%), and the highest enzyme activity (2111.2 U / g), which were significantly better than the unoptimized group and commercial enzyme preparations, achieving a gradient degradation effect of "single strain < double strain < triple strain < konjac + triple strain < optimized group".

[0057] Example 6: Evaluation of the application of fermented palm meal 6.1 Animal Experiments Unfermented palm meal (control group), palm meal fermented with Aspergillus niger (Aspergillus niger group), palm meal fermented with Trichoderma viride (Trichoderma viride group), palm meal fermented with Mucor (Mucor group), palm meal fermented with both Aspergillus niger and Trichoderma viride (dual-strain group), palm meal fermented with mixed strains (tri-strain group), palm meal fermented with mixed strains and added konjac flour (konjac + tri-strain group), and palm meal fermented with mixed strains and added konjac flour after optimized fermentation (optimized group) were selected. After freeze-drying, the samples were pulverized and passed through a 40-mesh standard sieve for later use. The palm meal from each group was added to the basal diet of broilers at a ratio of 15% (corn 62%, soybean meal 15%, wheat bran 3%, premix 2%, metabolizable energy 2750 kPa / kg, crude protein 19.2%), while the control group was given an equal proportion of unfermented palm meal.

[0058] One hundred and twenty healthy AA broiler chicks aged one day were randomly divided into four groups, with six replicates per group and five chicks per replicate (half male and half female). The experiment lasted for 28 days (7 days for the pre-experiment and 21 days for the main trial). During the last three days of the main trial, the feces were collected daily at regular intervals using the full collection method (with 0.5% chromium trioxide added as an exogenous indicator). The feces were dried at 65°C to constant weight, pulverized, and passed through a 40-mesh sieve for analysis.

[0059] Calculation formula: Dry matter digestibility (%) = [(Total feed dry matter intake × Feed dry matter content) - (Total feces × Fecal dry matter content)] / (Total feed dry matter intake × Feed dry matter content) × 100% DM digestibility improvement rate (%) = (DM% of experimental strain group - DM% of control group) / DM% of control group × 100% Crude protein digestibility (%) = [(Total crude protein ingested from feed × Crude protein content in feed) - (Total crude protein in feces × Crude protein content in feces)] / (Total crude protein ingested from feed × Crude protein content in feed) × 100% The results are shown in the table below:

[0060] in conclusion: Dry matter digestibility and crude protein digestibility showed a "control group" effect.

Claims

1. A mixed bacterial strain for degrading palm meal fiber, characterized in that, The mixed strains are composed of Aspergillus niger ( Aspergillus niger ), green Trichoderma ( Trichoderma viride ) and Mucor ( Mucor racemosus )composition.

2. The mixed strain for degrading palm meal fiber according to claim 1, characterized in that, The seed liquid volume ratio of Aspergillus niger, Trichoderma viride and Mucor is 2-3:3-5:1; The accession number for Aspergillus niger is CCTCC NO: M 20252141, the accession date is September 26, 2025, and the classification name is Aspergillus niger.

3. The method for degrading palm meal using the mixed strains according to any one of claims 1-2, characterized in that, Includes the following steps: (1) Pretreatment: After crushing the palm meal, add konjac flour and mix evenly; (2) Inoculation and fermentation: Adjust the moisture content of the material to 50%~70%, inoculate with mixed strain seed liquid, and let it ferment statically at 25~37℃ for 48~72 hours; (3) Enzymatic hydrolysis: The fermented material is dried, ground into crude enzyme powder, and then freeze-dried and pulverized after being hydrolyzed with water.

4. The degradation method according to claim 3, characterized in that, The amount of konjac flour added is 0.5% to 1% of the dry weight of palm meal, and the particle size is not greater than 0.1 mm.

5. The degradation method according to claim 4, characterized in that, In the fermentation step, the initial moisture content is 60%, the inoculum amount is 10%, the fermentation temperature is 31℃, and the fermentation time is 72 hours.

6. The degradation method according to claim 3, characterized in that, After fermentation, the neutral detergent fiber content of palm meal decreased to below 44.01%, and the dry matter digestibility increased to over 55.28%.

7. A fermented palm meal prepared by the method according to any one of claims 3-6.

8. A feed, characterized in that, The fermented palm meal as described in claim 7 is added at a ratio of 10% to 15%.

9. The feed according to claim 8, characterized in that, The feed is suitable for pigs, chickens, or ruminants.

10. The use of a mixed strain according to any one of claims 1-2 in the preparation of palm meal degrading agent or feed additive.

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