Silage fermentation method using internal living substances of corn stalks

Abstract

The present application relates to a silage fermentation method using the active substances in corn straw, including four steps of low-temperature short-time steam explosion preservation, micro-oxygen pre-activation, three-microbe synergistic main fermentation and low-temperature after-ripening, the explosion is 105-135 DEG C / 0.5-1.2 MPa / 30-90 s, the control D_NDF is 12-22% and the endogenous cellulase residual activity is greater than or equal to 45%; the micro-oxygen pre-activation is carried out at pH 5.0-5.8, 28-34 DEG C for 18-30 h, the pre-activation time length is linked with D_NDF (D_NDF every +1%, time length-2~3 h), and the dissolved ferulic acid constitutes selective antibacterial; the inoculation is carried out by using Lactobacillus plantarum: Lactobacillus buchneri: Candida utilis = 4.5: 2.5: 1, the explosion of Lactobacillus buchneri is matched with ferulic acid, and Candida utilis is matched with 5-HMF, and the anaerobic main fermentation is carried out at 25-30 DEG C for 25-35 d, and the after-ripening is carried out at 10-15 DEG C for 15-25 d.The present application has the beneficial effects that the D_NDF-ER saddle point linkage is matched with by-products-microbe agents, which is different from the existing "high-temperature explosion + exogenous enzyme + general three-microbes" route, and the product has an oxygen stability of greater than or equal to 130 h in the opening pit, and the crude protein improvement rate is greater than or equal to 22%.

Description

Technical Field

[0001] This invention relates to the field of silage processing, specifically to a method for fermenting silage using living substances within corn stalks. Background Technology

[0002] Corn stalks are one of the most abundant crop straw resources in my country's agricultural production. Developing corn stalks into silage is an important way to realize the resource utilization of straw and ensure the feed supply for ruminants. In existing technologies, corn stalk silage fermentation generally adopts a process route of direct inoculation with exogenous microbial agents. To improve fiber degradation and fermentation stability, commercial cellulase preparations, hemicellulase preparations, and chemical antifungal and antimicrobial agents are usually added.

[0003] Existing conventional silage processes have the following significant drawbacks: First, corn stalks contain endogenous cellulase, endogenous hemicellulase, and various endogenous active substances such as flavonoids, phenols, and polysaccharides. However, existing processes do not activate and utilize these endogenous active substances, resulting in a waste of the stalk's biological resources. Furthermore, the extensive use of exogenous enzyme preparations and antibacterial agents significantly increases the production cost of silage. Second, to break down the dense fiber structure of stalks, existing pretreatment processes often employ high-temperature, high-pressure steam explosion. The high-temperature environment leads to the inactivation of endogenous enzyme systems and the degradation of endogenous active ingredients, failing to achieve the synergistic effect between endogenous substances and the exogenous fermentation system. Third, existing silage processes lack an endogenous pre-activation stage. Insufficient fermentable substrate at the start of fermentation results in slow lactic acid bacteria colonization, a high risk of contamination by other microorganisms, and ultimately, limited fiber degradation in the finished silage, lacking additional bioactive value.

[0004] Therefore, it is essential to provide a fermentation method for silage containing living substances from corn stalks to solve the aforementioned technical problems. Summary of the Invention

[0005] Based on the above description, the present invention provides a fermentation method for silage using living substances in corn stalks, in order to solve the problems of existing corn stalk silage processes that rely on exogenous enzyme preparations and chemical antibacterial agents, fail to effectively utilize endogenous active substances in the stalks, have insufficient fiber degradation, and have low biological added value in the feed.

[0006] The technical solution of this invention to solve the above-mentioned technical problems is as follows: A method for fermenting silage using living matter within corn stalks, comprising the following steps: Step 1: After cutting whole corn stalks into sections, low-temperature short-time steam explosion is performed at a temperature of 105-135℃, a pressure of 0.5-1.2MPa, a pressure holding time of 30-90s, and instantaneous pressure release, controlling the neutral detergent fiber degradation rate (D_NDF) to 12%-22% and the residual activity of endogenous cellulase to ≥45%, conditioning to a moisture content of 65-72% and a pH of 5.0-5.8; Step 2: After conditioning, the stalks are placed into a sealed container, leaving a headspace of 8-15% for... O2 partial pressure 3-8%, pre-activation at 28-34℃ for 18-30h, pre-activation time linked to D_NDF: for every 1% increase in D_NDF, pre-activation time decreases by 2-3h, O2 partial pressure decreases by 0.3-0.5 percentage points. At the end of pre-activation, reducing sugar production rate is 0.8-1.6 mg / g / h, phenolic compounds are dissolved at 120-280 mg / kg, and ferulic acid and p-hydroxycinnamic acid account for ≥60% of the HPLC phenolic fraction. Step 3: Inoculate with a compound bacterial agent of *Lactobacillus plantarum*: *Lactobacillus brunelli*: *Candida utilis* = 4.5:2.5:1, inoculation amount 1×10⁻⁶. 5 -5×10 5 CFU / g, after compaction and sealing, anaerobic primary fermentation at 25-30℃ for 25-35 days. Among them, Lactobacillus buchneri and 0.1-0.4 mmol / L ferulic acid released by explosion were matched to increase acetic acid by 18-27% on the 5th-10th day of primary fermentation compared with the plant + pentose control. Candida utilis and 8-22 mg / kg of 5-HMF by-product from explosion were matched to increase the 5-HMF conversion rate to >72%. Step 4, after primary fermentation, after ripening at 10-15℃ for 15-25 days.

[0007] Furthermore, with a holding time of 90s, D_NDF was 14-19% and the residual activity of endogenous cellulase was 45-50%; with a holding time of 120s, D_NDF was 22-26%, but the residual activity of endogenous cellulase dropped to 30-38%, and the rate of reducing sugar production decreased instead of increasing.

[0008] Furthermore, at a temperature of 135℃ and a holding pressure of 90s, 5-HMF was 15-22 mg / kg, and ferulic acid monomer accounted for 50-58% of the HPLC phenolic fraction; when the temperature rose above 140℃, 5-HMF was >25 mg / kg, the proportion of ferulic acid monomer was <45%, and the induction efficiency of Lactobacillus brevis ferulic esterase decreased.

[0009] Furthermore, the ferulic acid and p-hydroxycinnamic acid dissolved during the pre-activation stage have an effect on Enterobacteriaceae and Bacillus. The concentration was 0.18-0.35 mmol / L, effective against *Lactobacillus plantarum* and *Lactobacillus brunelli*. >1.2 mmol / L, forming a selective inhibition window, resulting in a lactic acid bacteria / miscellaneous bacteria ratio of 150 × 10⁻⁶ on day 30 of the main fermentation. 2 above.

[0010] Furthermore, when the pre-activated reducing sugar production rate is 0.8-1.1 mg / g / h, a temperature gradient of 25-30℃ is used for the first 5 days of primary fermentation, with a daily increase of +1.2-1.5℃, so that the peak nitrogen assimilation rate of Candida utilis falls on the 2nd-4th day of primary fermentation, and the crude protein increase rate of the finished product is ≥22%.

[0011] Furthermore, the pre-activation pH of 5.0-5.8 is regulated by food-grade citric acid, and this pH range is suitable for both the activation of endogenous cellulase and the stability of ferulic acid glycosidic bonds after bursting. When pH > 5.8, the oxidative polymerization of ferulic acid monomers is accelerated.

[0012] Furthermore, the method for determining the residual activity of endogenous cellulase is as follows: crude enzyme solution of straw before and after explosion is extracted with pH 5.4 acetate buffer in an ice bath, reacted at 50℃ with 1% CMC-Na as substrate, and reducing sugar is determined by DNS method. Residual activity = enzyme activity after explosion / enzyme activity before explosion × 100%.

[0013] Furthermore, among the compound bacterial agent, *Lactobacillus plantarum* is a homofermentative acid-producing strain, *Lactobacillus buchneri* is a heterofermentative acetic acid-producing strain, and *Candida utilis* has both aldehyde reductase detoxification and cell protein assimilation functions. The viable cell ratio of the three in the main fermentation was 4.5:2.5:1, which increased the lactic acid bacteria / miscellaneous bacteria ratio on the 3rd day of the main fermentation by 1.4-2.1 times compared with the control without *Candida utilis*.

[0014] Furthermore, during the post-ripening stage at 10-15℃, hemicellulose decreases by another 4-7%, lactic acid blends with endogenous flavonoids / polyphenols, and the pH of the fermentation system stabilizes at 3.8-4.2.

[0015] Furthermore, the finished silage has an aerobic stability of ≥130h after opening and a crude protein increase rate of ≥22%.

[0016] Compared with the prior art, the technical solution of this application has the following beneficial technical effects: 1. A parameter linkage of "D_NDF-ER saddle point" is formed between steps one and two, which differs from the conventional high-temperature steam explosion silage approach of "the higher the fiber breaking, the better." Conventional steam explosion silage follows a "high-temperature explosion + exogenous enzyme supplementary degradation" route, for which the residual activity of endogenous enzymes is not important, so there is no motivation to measure the residual activity of endogenous enzymes, let alone to link D_NDF with the residual activity of endogenous enzymes into a linkage window. This invention is designed in reverse: using the residual activity of endogenous cellulase ≥45% as a constraint to back-calculate the D_NDF window, a "low-temperature short-time" range of 105-135℃ / 0.5-1.2MPa / 30-90 seconds is obtained, so that D_NDF is controlled at 12%-22% while the residual activity of endogenous enzymes is maintained above 45%; 2. The 0.1-0.4 mmol / L ferulic acid left by the low-temperature short-time explosion forms an induced response, resulting in an acetic acid level 18-27% higher than the "plant + pentose" control on days 5-10 of the main fermentation. This increase drops to below 12% at temperatures above 135°C and ferulic acid monomer concentrations <45% (Example 4), indicating that the selection of the Bretcher and the explosion parameters are not arbitrary. The production of prion and the explosion byproduct 5-HMF at 8-22 mg / kg form a detoxification correspondence, pre-activating microaerobic tail-term aldehyde reductase upregulation and 5-HMF conversion rate >72%, thus relieving aldehyde inhibition on plant milk. Approximately 12 mg / L), which increased the ratio of lactic acid bacteria to miscellaneous bacteria on day 3 of primary fermentation by 1.4-2.1 times compared to the control group without Candida utilis; 3. When D_NDF is high, the fiber is loose and the enzyme substrate contact area is large, so the pre-activation time can be shortened accordingly; however, when D_NDF>22%, the residual activity of the endogenous enzyme has been lost, and the linkage formula automatically excludes this type of data. 4. Continuous sugar supply from endogenous enzymes (reducing sugar production rate 0.8-1.6 mg / g / h), combined with selective antibacterial activity from endogenous phenols (ferulic acid + p-hydroxycinnamic acid against miscellaneous bacteria). The concentration was 0.18-0.35 mmol / L, and it was effective against lactic acid bacteria. >1.2mmol / L), replacing exogenous enzymes and antibacterial agents; nitrogen assimilation of prion-producing candidiasis increases crude protein by ≥22%, and aerobic stability in fermentation pits is ≥130 hours, with synergistic improvement in fiber degradation, fermentation stability, and nutritional added value; 5. The process is compatible with existing silage equipment, with low industry barriers. Low-temperature short-time steam explosion can be operated with downgraded parameters in existing QBS-type explosion tanks. Pre-activation can be achieved by controlling the headspace ratio using existing bagging / cellar storage, without the need to add special equipment. Attached Figure Description

[0017] Figure 1 A flowchart of a silage fermentation method utilizing living substances in corn stalks, provided as an embodiment of the present invention. Detailed Implementation

[0018] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings, which illustrate embodiments of the present application. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the disclosure of this application will be thorough and complete.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0020] It is understood that spatial relation terms such as "below," "under," "below," "below," "above," "above," etc., can be used here to describe the relationship between one element or feature shown in the figure and other elements or features. It should be understood that, in addition to the orientation shown in the figure, spatial relation terms also include different orientations of the device in use and operation. For example, if the device in the figure is flipped, the element or feature described as "below" or "below" of the other element or feature will be oriented "above" the other element or feature. Therefore, the exemplary terms "below" and "below" can include both upper and lower orientations. Furthermore, the device may also include other orientations (e.g., rotated 90 degrees or other orientations), and the spatial descriptive terms used herein will be interpreted accordingly.

[0021] It should be noted that when one element is considered to be "connected" to another element, it can be directly connected to the other element or connected to the other element through an intermediary element. In the following embodiments, "connection" should be understood as "electrical connection," "communication connection," etc., if the connected circuits, modules, units, etc., have the transmission of electrical signals or data between them.

[0022] When used herein, the singular forms of “a,” “an,” and “the” may also include the plural forms unless the context clearly indicates otherwise. It should also be understood that the terms “comprising,” “including,” or “having,” etc., specify the presence of the stated feature, whole, step, operation, component, part, or combination thereof, but do not preclude the possibility of the presence or addition of one or more other features, wholes, steps, operations, components, parts, or combinations thereof.

[0023] Example 1: The raw material is whole-plant corn of "Zhengdan 958" at the late waxy maturity stage, with milk line of 80-90%, harvested with a stubble of 15cm, moldy and rotten sections removed, and cut to 2-3cm. The moisture content is adjusted to 68±2% by spraying with sterile water, and the pH is adjusted to 5.4±0.1 by spraying with food-grade citric acid (50g / L).

[0024] After conditioning, the straw was placed into a QBS-80 laboratory steam explosion vessel with a bulk density of approximately 0.15 t / m³. 3Saturated steam was introduced to 120℃ and 0.8MPa, and the pressure was maintained for 60 seconds. The pressure was then released instantaneously (<0.08 seconds). The material was discharged and spread out to air-dry to room temperature ≤30℃. The time from discharge to completion of air-drying was controlled within 15 minutes. The D_NDF of this batch was calculated as 16.3±0.8% using the formula D_NDF=(m0-m1) / m0×100%. The residual activity of endogenous cellulase ER_CMCase was calculated as 51±3% using the formula ER=A1 / A0×100% (A0 is the pre-burst enzyme activity, A1 is the post-burst enzyme activity, extraction was performed in an ice bath with an acetic acid buffer at pH 5.4, and CMCase was extracted using 1% CMC-Na as the substrate at 50℃ using the DNS method).

[0025] After blasting, the material is placed into a 500mL wide-mouth sealed container and lightly compacted to a bulk density of 0.55-0.65t / m³. 3 The headspace occupied 10% of the tank volume. After sealing, it was kept at a constant temperature of 30±1℃ for 24 hours to complete the pre-activation. The reducing sugar formation rate v_R at the end of the pre-activation was calculated as 1.06±0.09 mg / g / h according to the formula v_R=(c1-c0) / t (c0 and c1 are the 0-hour and 24-hour dry basis reducing sugar DNS method, t=24 hours), the total phenols were 188±12 mg GAE / kg DM, and the HPLC ferulic acid monomer accounted for 67±4% of the phenolic fraction.

[0026] After pre-activation, inoculate with a compound bacterial agent (Lactobacillus plantarum:Lactobacillus brunelli:Candida utilis = 4.5:2.5:1, total inoculation amount 3×10⁻⁶). 5 (CFU / g fresh weight, activated with sterile water at 30℃ for 30 minutes, sprayed and mixed), then transferred to a wrapping device and compacted in layers to 0.78t / m³. 3 After sealing, the primary fermentation was carried out anaerobically at 28±2℃ for 30 days, followed by a 20-day post-maturation at 12±1℃. The lactic acid bacteria / miscellaneous bacteria ratio R_B after 30 days of primary fermentation was calculated using the formula R_B=N_L / N_M (N_L was measured by MRS anaerobic assay, and N_M was measured by PDA+chloramphenicol at 37℃ aerobic assay) to be 186±15×10⁻⁶. 2 The aerobic stability after opening the pit is 142±9 hours, and the crude protein enhancement rate I_CP of the finished product is 26.4±1.8% according to the formula I_CP=(p1-p0) / p0×100% (Kjeldahl N×6.25).

[0027] Example 2: The only variable compared to Example 1 is that the holding time is extended from 60 seconds to 90 seconds, while all other parameters (120℃ / 0.8MPa, conditioning, pre-activation, microbial agent, fermentation curve) are the same.

[0028] This sample showed a DNDF of 18.7±1.0%, residual endogenous cellulase activity of 47±3% (just above the 45% threshold), reducing sugar production rate of 1.11±0.08 mg / g / h, HPLC ferulic acid monomer content of 63±3%, and lactic acid bacteria / mixed bacteria ratio of 178±12×10⁻⁶. 2 The aerobic stability after opening the cellar was 138±8 hours, and the crude protein increase rate was 25.7±1.5%. This indicates that the holding pressure of 90 seconds is still the self-verifying point within the essential window, the residual activity of endogenous enzymes has not fallen below 45%, and the linkage chain has not loosened.

[0029] Example 3: The only difference from Example 1 is that the pressure holding time is extended to 120 seconds; all other aspects are the same.

[0030] The DNDF in this sample was 24.7±1.2%, the residual activity of endogenous cellulase was 36±3% (falling below 45%), the reducing sugar production rate was 0.97±0.07 mg / g / h (the DNDF was higher but the reducing sugar production rate did not increase), the HPLC ferulic acid monomer content was 59±4%, and the lactic acid bacteria / mixed bacteria ratio was 134±11×10⁻⁶. 2 The aerobic stability after opening the pit was 118±7 hours, and the crude protein increase rate was 24.7±1.6%. Compared with Example 2, although the D_NDF increased by 5.9 percentage points when the holding time was increased from 90 seconds to 120 seconds, the residual activity of endogenous enzymes decreased by 11 percentage points, and the reducing sugar production rate, lactic acid bacteria / mixed bacteria ratio, stability, and crude protein all dropped, proving the rationality of the "holding time 30-90 seconds" upper limit.

[0031] Example 4: The difference from Example 1 is that the bursting parameters are changed to 135℃ / 1.0MPa / 90 seconds (the temperature limit should be 105-135℃, and the pressure holding time is 90 seconds), otherwise they are the same.

[0032] The DNDF in this sample was 21.9±1.1% (approaching 22%), the residual activity of endogenous cellulase was 39±3% (falling below 45%), the total phenols were 232±16 mg GAE / kg DM (increased but mostly due to oxidative polymerization), the HPLC ferulic acid monomer content was 52±3% (lower than the summary's "≥60%)", the acetic acid content on days 5-10 of the main fermentation was 12% higher than the "plant + pentose" control (lower than the summary's 18-27%), and the lactic acid bacteria / mixed bacteria ratio was 142±13×10⁻⁶. 2 The aerobic stability after opening the cellar was 126±8 hours, and the crude protein increase rate was 25.1±1.7%. Compared with Example 1, although the D_NDF still fell within 22% when the temperature was increased from 120℃ to 135℃, the residual activity of endogenous enzymes decreased, the proportion of ferulic acid monomers decreased, and the efficiency of the Bousley-ferulic acid induced response decreased, thus proving the rationality of the "upper limit of 135℃" and "ferulic acid monomers ≥60%".

[0033] Example 5: The only variable compared to Example 1 is that the burst temperature was changed to 180℃ / 1.8MPa / 240 seconds (typical window for conventional SE silage), and all other parameters are the same (including the same inoculant, the same pre-activation, and the same fermentation curve).

[0034] This sample contains: D_NDF 29.4±1.5%, endogenous cellulase residual activity 11±2%, reducing sugar production rate 0.28±0.04 mg / g / h (due to burst dissolution of background sugars, not enzymatic activation), total phenols 346±21 mg GAE / kg DM (mostly oxidative polymerization), ferulic acid monomer 31±3% by HPLC, and lactic acid bacteria / mixed bacteria ratio 47±6×10⁻⁶. 2 The aerobic stability after opening the cellar was 64±6 hours, and the crude protein increase rate was 7.3±1.1%.

[0035] A comparison of Examples 1 to 4 with existing technologies reveals that: the D_NDF of Comparative Example 1 is approximately 80% higher than that of Example 1, but the residual activity of endogenous enzymes is only one-fifth of that of Example 1, the reducing sugar production rate is only 26% of that of Example 1, the lactic acid bacteria / miscellaneous bacteria ratio is only 25% of that of Example 1, the aerobic stability is less than half that of Example 1, and the crude protein enhancement rate is only 28% of that of Example 1. This indicates that although conventional high-temperature SE has a higher D_NDF, due to endogenous enzyme collapse, ferulic acid spectrum deterioration, and excessive 5-HMF, even with the use of plant + Brucella + Candida utilis, the linkage and matching effect of this invention cannot be achieved. This is precisely the essential divergence point between the "D_NDF-ER saddle point linkage" and "byproduct-bacterial agent matching" of this invention and the existing "high temperature + exogenous enzyme + pan-three bacteria" approach. The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for fermenting silage using living matter in corn stalks, characterized by: Step 1: After cutting the whole corn stalks into sections, perform low-temperature short-time steam explosion at 105-135℃, pressure 0.5-1.2MPa, hold pressure for 30-90s, and then release pressure instantly. Control the neutral detergent fiber degradation rate (DNDF) to 12%-22% and the residual activity of endogenous cellulase to ≥45%. Condition the stalks to a moisture content of 65-72% and a pH of 5.0-5.

8. Step 2: After conditioning, pack the stalks into a sealed container, leaving 8-15% headspace to maintain an O2 partial pressure of 3-8%, and pre-activate them at 28-34℃ for 18-... 30h, pre-activation time is linked to D_NDF: for every 1% increase in D_NDF, the pre-activation time decreases by 2-3h, the O2 partial pressure decreases by 0.3-0.5 percentage points, the reducing sugar production rate at the end of pre-activation is 0.8-1.6mg / g / h, the dissolved phenols are 120-280mg / kg, and ferulic acid and p-hydroxycinnamic acid account for ≥60% of the HPLC phenolic fraction; Step 3, inoculate with a compound bacterial agent of *Lactobacillus plantarum*: *Lactobacillus brunelli*: *Candida utilis* = 4.5:2.5:1, with an inoculation amount of 1×10 5 -5×10 5 CFU / g, after compaction and sealing, anaerobic primary fermentation at 25-30℃ for 25-35 days. Among them, Lactobacillus buchneri and 0.1-0.4 mmol / L ferulic acid released by explosion were matched to increase acetic acid by 18-27% on the 5th-10th day of primary fermentation compared with the plant + pentose control. Candida utilis and 8-22 mg / kg of 5-HMF by-product from explosion were matched to increase the 5-HMF conversion rate to >72%. Step 4, after primary fermentation, after ripening at 10-15℃ for 15-25 days.

2. The method for fermenting silage using living matter in corn stalks according to claim 1, characterized in that, When the holding time was 90s, D_NDF was 14-19% and the residual activity of endogenous cellulase was 45-50%; when the holding time was 120s, D_NDF was 22-26%, but the residual activity of endogenous cellulase dropped to 30-38%, and the rate of reducing sugar production decreased instead of increasing.

3. The method for fermenting silage using living matter in corn stalks according to claim 1, characterized in that, At a temperature of 135℃ and a holding time of 90s, 5-HMF was 15-22 mg / kg, and ferulic acid monomer accounted for 50-58% of the phenolic fraction in HPLC. When the temperature was raised to above 140℃, 5-HMF was >25 mg / kg, the proportion of ferulic acid monomer was <45%, and the induction efficiency of Lactobacillus brevis ferulic esterase decreased.

4. The method for fermenting silage using living matter in corn stalks according to claim 1, characterized in that, The ferulic acid and p-hydroxycinnamic acid dissolved during the pre-activation stage have an effect on Enterobacteriaceae and Bacillus. The concentration was 0.18-0.35 mmol / L, effective against *Lactobacillus plantarum* and *Lactobacillus brunelli*. >1.2 mmol / L, forming a selective inhibition window, resulting in a lactic acid bacteria / miscellaneous bacteria ratio of 150 × 10⁻⁶ on day 30 of the main fermentation. 2 above.

5. The method for fermenting silage using living matter in corn stalks according to claim 1, characterized in that, When the pre-activated reducing sugar production rate is 0.8-1.1 mg / g / h, the temperature is increased by gradient from 25 to 30℃ and by +1.2-1.5℃ per day for the first 5 days of primary fermentation, so that the peak nitrogen assimilation rate of Candida utilis falls on the 2nd-4th day of primary fermentation, and the crude protein increase rate of the finished product is ≥22%.

6. The method for fermenting silage using living matter in corn stalks according to claim 1, characterized in that, The pre-activation pH of 5.0-5.8 is regulated by food-grade citric acid, and this pH range is suitable for both the activation of endogenous cellulase and the stability of ferulic acid glycosidic bonds after bursting. When pH > 5.8, the oxidative polymerization of ferulic acid monomers is accelerated.

7. The method for fermenting silage using living matter in corn stalks according to claim 1, characterized in that, The method for determining the residual activity of endogenous cellulase is as follows: crude enzyme solution of straw before and after explosion is extracted with pH 5.4 acetate buffer in an ice bath, reacted at 50℃ with 1% CMC-Na as substrate, and reducing sugar is determined by DNS method. Residual activity = enzyme activity after explosion / enzyme activity before explosion × 100%.

8. The method for fermenting silage using living matter in corn stalks according to claim 1, characterized in that, In the compound bacterial agent, *Lactobacillus plantarum* is a homofermentative acid-producing strain, *Lactobacillus buchneri* is a heterofermentative acetic acid-producing strain, and *Candida utilis* has both aldehyde reductase detoxification and cell protein assimilation functions. The viable cell ratio of the three in the main fermentation was 4.5:2.5:1, which increased the lactic acid bacteria / miscellaneous bacteria ratio on the 3rd day of the main fermentation by 1.4-2.1 times compared with the control without *Candida utilis*.

9. The method for fermenting silage using living matter in corn stalks according to claim 1, characterized in that, During the post-ripening stage at 10-15℃, hemicellulose decreases by another 4-7%, lactic acid blends with endogenous flavonoids / polyphenols, and the pH of the fermentation system stabilizes at 3.8-4.

2.

10. The method for fermenting silage using living matter in corn stalks according to claim 1, characterized in that, The finished silage has an aerobic stability of ≥130h after opening and a crude protein increase rate of ≥22%.

Images