Method and application of fungus-enzyme synergistic fermentation of bagasse pith

Abstract

The invention relates to the technical field of biology, in particular to a method and application of fungus-enzyme synergistic fermentation of bagasse pith, and the method comprises the following steps: (1) adjusting the water content of the bagasse pith to 60-70%, and adjusting the pH to 4.0; (2) adding the following components according to dry matter mass: 3% of phanerochaete chrysosporium liquid with the concentration of 1.5 * 10 < 8 > CFU / ml, and uniformly mixing; 1% of sucrose; the cellulase liquid is 20000U / kg, and the cellulase liquid is prepared by dissolving crude enzyme obtained by centrifuging after trichoderma hedgehog is fermented to produce enzyme in a PBS (phosphate buffer solution); and (3) putting the mixture into a fermentation tank, and performing sealed low-oxygen fermentation for 30 days to obtain the bagasse pith feed. According to the bagasse pith feed obtained by the method, the nutritional value of the bagasse pith can be improved, the fermentation quality is improved, the digestion potential is improved, the storage time is prolonged, and high-quality feed application of the bagasse is realized.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a method for fermenting sugarcane pith in synergy with bacterial enzymes and its application. Background Art

[0002] Sugarcane is a globally important sugar and bioenergy crop. my country's sugarcane cultivation area ranks third in the world, and the country produces over 25 million tons of bagasse annually. Bagasse pith, the peeled bagasse, contains a large amount of crude fiber, which promotes rumination and salivation, thereby maintaining normal rumen function. However, bagasse pith is high in lignin, where digestible cellulose and hemicellulose combine with lignin to form a stable and complex lignin-hemicellulose-cellulose complex. Ruminants lack the microorganisms and enzymes that can degrade lignin in their rumens, resulting in this complex hindering the breakdown of roughage by proteases, cellulases, amylases, and rumen microorganisms. If the lignocellulose content can be reduced through bio-fermentation of sugarcane pith, lactic acid and beneficial microorganisms can be promoted to inhibit the growth of harmful bacteria, the fermentation quality will be improved, the storage time will be extended, and resource feed shortages will be alleviated. Chinese patent CN119632133A discloses a method for solid-state fermentation of sugarcane bagasse using white rot fungi, which can reduce the content of acid-insoluble lignin, hemicellulose, neutral detergent fiber, and acid detergent fiber. However, data show that the cellulose reduction effect is not significant enough. Therefore, the addition of cellulase to the white rot fungus fermentation can improve the poor cellulose degradation effect, further improve the utilization rate of sugarcane pith, and have great significance for the application of sugarcane pith as a dedicated bio-feed. Summary of the Invention

[0003] The present invention aims to solve the problem of sugarcane pith being high in fiber but low in feeding value, and provides a method and application for the synergistic fermentation of sugarcane pith with bacteria and enzymes. Compared with single bacteria treatment or enzyme treatment, the present invention synergistically ferments sugarcane pith with bacteria and enzymes, thereby increasing the digestible matter of the sugarcane pith and reducing the lignocellulose content, thereby improving the nutritional value, fermentation quality, digestion potential, etc.

[0004] To achieve the above object, the technical solution of the present invention is as follows: a method for fermenting sugarcane pith with bacterial enzymes, using Phanerochaete chrysosporium and cellulase to ferment sugarcane pith, comprising the following steps:

[0005] (1) Adjusting the water content of sugarcane pith to 60% to 70% and the pH to 4.0;

[0006] (2) Add the following components according to the dry matter mass and mix them evenly: 3% bacterial solution of Phanerochaete chrysosporium, concentration of bacterial solution:

[0007] 1.5×10 8CFU / ml; sucrose 1%; cellulase solution 20000U / kg, the cellulase solution is prepared by fermenting Trichoderma ergifer and then centrifuging the crude enzyme obtained and dissolving it in PBS buffer;

[0008] (3) The mixture is placed in a fermentation tank for sealed low-oxygen fermentation for 30 days to obtain sugarcane pith feed.

[0009] The preparation method of the Phanerochaete chrysosporium bacterial liquid comprises:

[0010] A. Activation and propagation of bacteria: Phanerochaete chrysosporium (CHCC336258) was purchased from Beijing Zhongjian Hangbiao Metrology Technology Co., Ltd. and was placed in PDA medium for activation and propagation. After 2-3 generations of vigorous propagation using the 4-zone streaking method, it was transferred to PDB medium. The shaker speed was set at 140 rpm and the temperature was 27°C. Glass beads were added to prevent the bacteria from agglomerating. The culture was completed after the bacterial solution became turbid for 2-3 days.

[0011] B. Strain Enumeration and Preservation: Count the cultured bacteria using the modified Martin's medium using the plate count method. Mix the resulting culture with 20% glycerol at a 1:1 ratio and store in a -80°C freezer until ready for use.

[0012] The preparation method of the cellulase solution comprises:

[0013] (1) Activate the Trichoderma ergifer preserved in the Guangdong Provincial Microbial Culture Collection Center. The activation steps are the same as step 1, part (1). After activation, rinse the plate with sterile water to obtain a suspension and place it in a 4°C refrigerator for later use.

[0014] (2) The suspension was inoculated into a seed culture medium at a ratio of 2% and cultured in a closed anaerobic manner for 2 days. The shaker speed was set at 180 r / min and the temperature was 27°C. The seed culture medium components were: 10 g / L sodium carboxymethyl cellulose, 3 g / L peptone, 4 g / L potassium dihydrogen phosphate, 0.3 g / L magnesium sulfate heptahydrate, and 0.3 g / L calcium chloride. The suspension was prepared with distilled water and placed in an autoclave for sterilization at 121°C for 20 min.

[0015] (3) The bacterial liquid obtained after the seed culture medium is cultured is inoculated into the enzyme production medium at a ratio of 6% and cultured in a closed anaerobic manner for 5 days. The shaker speed is set at 180 r / min and the temperature is 27°C. The enzyme production medium is composed of: 12 g / L sodium carboxymethyl cellulose, 3 g / L peptone, 0.2 g / L yeast powder, 2 g / L ammonium sulfate, 4 g / L potassium dihydrogen phosphate, 0.3 g / L magnesium sulfate heptahydrate, 0.3 g / L calcium chloride and 0.002 g / L zinc chloride. After being prepared with distilled water, it is placed in a high pressure sterilizer and sterilized at 121°C for 20 minutes;

[0016] (4) After the culture in the enzyme-producing medium is completed, the resulting bacterial liquid is centrifuged at 6000 rpm for 15 min to obtain a crude enzyme solution, which is then precipitated with 60% ethanol for 24 h and centrifuged at 6000 rpm for 15 min. The supernatant is discarded, and the precipitate is the crude enzyme obtained;

[0017] (5) The crude enzyme was dissolved in PBS buffer to obtain a cellulase solution, and the CMC activity of the cellulase solution was determined using the modified DNS method.

[0018] The feed has a neutral detergent fiber content of ≤83.75%, an acid detergent lignin content of ≤14.25%, a lactic acid content of ≥356.61 mg / L, and a pH of ≤3.68.

[0019] The steps for determining the CMC activity of cellulase solution using the DNS method are as follows:

[0020] (1) Add 0.25 ml of cellulase solution to 0.75 ml of sodium citrate buffer solution with a concentration of 10 g / L sodium carboxymethyl cellulose. The solution is divided into a control tube and a test tube. 0.75 ml of 3,5-dinitrosalicylic acid reagent is added to the control tube at the same time. No reagent is added to the test tube. The tubes are placed in a 50°C water bath for 30 min.

[0021] (2) Immediately after the water bath is completed, add 0.75 ml of 3,5-dinitrosalicylic acid reagent to the test tube. Then, place the control tube and the test tube in a boiling water bath for 5 minutes. After cooling, adjust the blank tube to zero at a wavelength of 540 nm and record the absorbance of the test tube.

[0022] (3) preparing a glucose standard solution and processing it according to steps (1) and (2) to establish a standard curve for the glucose standard solution;

[0023] (4) The absorbance obtained in step (2) was substituted into the glucose standard curve to calculate the glucose content in the crude enzyme solution, and the CMC enzyme activity of the crude enzyme solution was calculated according to the following formula:

[0024] CMC enzyme activity The beneficial effects of the present invention are:

[0025] 1. The simultaneous use of Phanerochaete chrysosporium and cellulase in the fermentation of sugarcane pith significantly reduced neutral detergent fiber (-3.62%) and acid detergent lignin (-23.22%), increased lactic acid (+66.48%) and acetic acid (+85.69%) contents, and improved fermentation quality.

[0026] 2. The gas production of rumen fermentation in vitro after 48 hours increased by 13.46%, and the gas production of the potentially degradable part increased by 45.58%, significantly improving the digestibility;

[0027] 3. Microbial diversity analysis showed that the abundance of beneficial bacteria (such as Lactobacillus) increased, while the abundance of harmful bacteria (such as Acinetobacter) decreased.

[0028] 4. The method of the present invention can be used to produce sugarcane pith feed with high digestibility on a large scale, thereby solving the problem of resource utilization of sugarcane bagasse and alleviating feed shortage. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The distribution of bacteria at the genus level in each component. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described below through examples.

[0031] Example 1

[0032] This example shows the results of conventional nutrient and fermentation index determinations of the method for the synergistic fermentation of sugarcane pith with fungi and enzymes according to the present invention. The fermented sugarcane pith feed was tested in the laboratory for changes in nutrient composition and fermentation indexes according to the methods for determination of neutral detergent fiber (NDF) in feed (GB / T 20806-2006), determination of acid detergent fiber (ADF) in feed (NY / T 1459-2007), determination of acid detergent lignin (ADL) in feed (GB / T 20805-2006), determination of crude ash in feed (GB / T 6438-2007), determination of cellulose (CL) in feed (Nandez method), pH in feed (pH meter model: FE28), determination of lactic acid in feed (p-hydroxybiphenyl method), and determination of volatile fatty acids in feed (gas chromatography). The test results are listed in Table 2.

[0033] Table 1 Specific contents of bacterial enzyme synergistic fermentation and control group

[0034]

[0035]

[0036] Table 2 Bacterial enzyme synergistic fermentation and control group conventional nutrients (%)

[0037]

[0038] Table 3 Fermentation indexes of bacterial enzyme synergistic fermentation and control group

[0039]

[0040] According to Tables 2 and 3, the neutral detergent fiber content of the bacteria-enzyme synergistic fermentation was 83.75%, and the neutral detergent fiber content of the enzyme fermentation was 86.90%, which was 3.62% lower than that of the enzyme fermentation. The neutral detergent fiber content of the bacteria-enzyme synergistic fermentation was 86.23%, which was 2.88% lower than that of the bacteria fermentation. The acid detergent fiber content of the bacteria-enzyme synergistic fermentation was 49.30%, and the acid detergent fiber content of the enzyme fermentation was 53.93%, which was 8.59% lower than that of the enzyme fermentation. The acid detergent fiber content of the bacteria-enzyme synergistic fermentation was 50.70%, which was 2.76% lower than that of the bacteria fermentation. The cellulose content of the bacteria-enzyme synergistic fermentation was 33.83%, and the cellulose content of the enzyme fermentation was 34.50%, which was 1.94% lower than that of the enzyme fermentation. The cellulose content of the bacteria-enzyme synergistic fermentation was 2.88% lower than that of the enzyme fermentation. The content of acidic washing lignin in the bacterial enzyme synergistic fermentation was 14.25%, and that in the enzyme fermentation was 18.56%, which was 23.22% lower than that in the enzyme fermentation. The content of acidic washing lignin in the bacterial fermentation was 15.01%, which was 5.06% lower than that in the bacterial fermentation. The crude ash content in the bacterial enzyme synergistic group was 2.62%, and that in the enzyme fermentation was 2.89%. The enzyme-cooperative fermentation was 9.34% lower than that of enzyme fermentation, the crude ash content of bacterial fermentation was 2.71%, and the bacterial-enzyme-cooperative fermentation was 3.32% lower than that of bacterial fermentation; the lactic acid content of bacterial-enzyme-cooperative fermentation was 356.61 mg / L, and the lactic acid content of enzyme fermentation was 321.99 mg / L, which was 10.75% higher than that of enzyme fermentation, and the lactic acid content of bacterial fermentation was 214.20 mg / L, which was 66.48% higher than that of bacterial fermentation; the pH of bacterial-enzyme-cooperative fermentation was 3 .68, the pH of enzyme fermentation was 3.63, which was 1.38% higher than that of enzyme fermentation; the pH of bacterial fermentation was 3.85, which was 4.42% lower than that of bacterial fermentation; the acetic acid content of bacterial fermentation was 9.86mmol / L, which was 8.33mmol / L, which was 18.37% higher than that of enzyme fermentation; the acetic acid content of bacterial fermentation was 5.31mmol / L, which was 85.69% higher than that of bacterial fermentation.

[0041] Example 2

[0042] This example shows the results of in vitro fermentation in the rumen using the method for fermenting sugarcane pith with bacterial enzymes according to the present invention. The results are shown in Table 6. The specific operation is as follows:

[0043] (1) The fermented feed was crushed and air-dried, and its absolute dry mass was determined. 200 mg of the air-dried sample was weighed and placed into a 50 mL glass syringe. Medical vaseline was evenly applied to 2 / 3 of the glass column. Five replicates were performed in each group.

[0044] (2) Experimental animals and diet formula are as follows:

[0045] Three Nili-Rafi buffaloes (530.26 ± 6.25 kg) of similar size, in good health, and dewormed from a location in Guangxi served as rumen fluid donors. All buffaloes were fitted with permanent rumen cannulas. The buffalo diet formula is shown in Table 4.

[0046] Table 4 Buffalo diet composition and nutritional level (air-dried matter basis)

[0047]

[0048]

[0049] 1) The premix is ​​composed of the following ingredients: VA 450,000 IU, VD 10,000 IU, VE 2,500 IU, Cu (as ascopper sulfate) 120 mg, Mn (as manganese sulfate) 40 mg, Fe (as ferrous sulfate) 50 mg, Se (as sodium selenite) 46 mg, Zn (as zinc sulfate) 50 mg, I (as potassium iodide) 15 mg, Co (as cobalt carbonate) 15 mg.

[0050] 2) Comprehensive net energy refers to the "China Feed Composition and Nutritional Value Table (2022 33rd Edition)"

[115] NEmf was a calculated value according to Tables of Feed Composition and Nutritive Values ​​in China (2022thirty-third edition),while the other nutrient levels were measured values.

[0051] (3) Artificial saliva was prepared as follows:

[0052] ① Start the constant temperature water bath shaker and multi-connected water bath;

[0053] ② Calculate the amount of artificial saliva required: During in vitro culture, each trachea requires 30 mL of culture medium (10 mL of rumen fluid and 20 mL of artificial saliva). Calculate the amount of artificial saliva and rumen fluid required based on the number of trachea designed for the experiment (reserve an additional 20% to account for operational errors).

[0054] ③Prepare reducing agent;

[0055] ④ Mix the stock solutions in order and proportion, place them in a water bath shaker or water bath, and introduce an appropriate amount of CO2 gas (avoid excessive airflow) to change the artificial saliva from blue to pink and finally to colorless. The artificial saliva configuration table is shown in Table 5.

[0056] Table 5 Preparation of artificial saliva stock solution

[0057]

[0058]

[0059] (1) Collection and processing of rumen fluid are as follows:

[0060] ① After arriving at the ranch, pour hot water into the bucket and adjust the water temperature to 39±0.5℃ to keep the rumen fluid warm (hot water should be used frequently to adjust the temperature);

[0061] ② Extract feed containing rumen fluid from three fistula buffaloes, filter it tightly with gauze, mix it thoroughly, and then pour it into a collection bottle (make sure to cover the bottle tightly to prevent oxygen from affecting microbial activity);

[0062] ③ After returning to the laboratory, place the collection bottle in a water bath and quickly filter it with four layers of gauze (to maintain the vitality of the microorganisms). The filtered liquid is saturated with CO2 gas;

[0063] ④ Use a measuring cylinder to measure the required amount of colorless artificial saliva and rumen fluid, mix them, and continuously introduce CO2 gas.

[0064] (2) Absorbing microbial culture fluid: Use an empty gas-producing tube to absorb 30 mL of microbial culture fluid and push it into the gas-producing tube containing the sample through a three-way tube.

[0065] (3) Exhaust: With the injection port facing upward and the clamp open, pull down the tube plug by hand to prevent the sample from rushing into the plastic tube; shake the gas-producing tube to mix the sample with the rumen fluid, then rotate the tube plug to exhaust the air in the tube.

[0066] (4) Reading: Hold the gas-producing tube upright with the injection port facing downwards and read the scale. After exhausting, the gas-producing tube should be randomly placed in a constant temperature water bath shaker for incubation (the shaker should be in a shaking state during operation to prevent the temperature from rising).

[0067] (5) Record gas production: At 2, 4, 8, 12, 24, 36, and 48 hours of culture, record the scale reading of the gas production tube (shake the gas production tube gently, rotate the tube plug, and read the reading. When reading, refer to the middle of the tube plug scale). If the gas production is too much and the gas production at the next time point may exceed the scale range, it is necessary to vent the gas. The operation is the same as exhausting.

[0068]

[0069] Where, GP t is the gas production of the sample at time t (mL); Vt is the scale reading of the gas production tube after the sample is fermented for t hours (mL); V0 is the scale reading of the gas production tube at the beginning of the culture (mL); W is the dry matter weight of the sample (mg); GP 空白 is the gas production of the blank control at time t, and its calculation method is the same as GP t consistent.

[0070] (6) The calculation formula of gas production parameters is: GP t =a+b×(1-e (-ct) )

[0071] Where, GP t is the gas production of the sample at time t (mL); a is the gas production of the rapidly degradable part (mL); b is the gas production of the slowly degradable part (m1); c is the gas production rate (% / h); a+b is the gas production of the potentially degradable part; t is the degradation time (h).

[0072] Table 6 Results of bacterial enzyme synergistic fermentation and control group rumen in vitro fermentation (mL)

[0073]

[0074] According to Table 4, the gas production of the bacterial enzyme synergistic fermentation for 48 hours was 40.19 ml, the gas production of the enzyme fermentation for 48 hours was 34.54 ml, the bacterial enzyme synergistic fermentation was 13.46% higher than the enzyme fermentation, the gas production of the bacterial fermentation for 48 hours was 38.52 ml, the bacterial enzyme synergistic fermentation was 4.34% higher than the bacterial fermentation; the rapid gas production part of the bacterial enzyme synergistic fermentation was 2.23 ml, the rapid gas production part of the enzyme fermentation was 1.89 ml, the bacterial enzyme synergistic fermentation was 17.99% higher than the enzyme fermentation, the rapid gas production part of the bacterial fermentation was 1.47 ml, the bacterial enzyme synergistic fermentation was 51.70% higher than the bacterial fermentation; The slow gas production part of the same fermentation was 87.22ml, the slow gas production part of the enzyme fermentation was 59.57ml, the bacterial-enzyme synergistic fermentation increased by 46.42% compared with the enzyme fermentation, the slow gas production part of the bacterial fermentation was 59.46ml, the bacterial-enzyme synergistic fermentation increased by 46.69% compared with the bacterial fermentation; the gas production of the potentially degradable part of the bacterial-enzyme synergistic fermentation was 89.46ml, the gas production of the potentially degradable part of the enzyme fermentation was 61.45ml, the bacterial-enzyme synergistic fermentation increased by 45.58% compared with the enzyme fermentation, the gas production of the potentially degradable part of the bacterial fermentation was 60.93ml, the bacterial-enzyme synergistic fermentation increased by 47.31% compared with the bacterial fermentation.

[0075] Example 3

[0076] This example is the 16S microbial diversity sequencing results of sugarcane pith fermented with bacterial enzymes according to the present invention. The results are shown in Table 6.

[0077] Table 7: Alpha diversity index analysis table

[0078]

[0079] According to Table 5, the ACE index, Chao1 index, Shannon index and Simpson index of bacterial-enzyme synergistic fermentation were all higher than those of enzyme fermentation and bacterial fermentation, which indicated that bacterial-enzyme synergistic fermentation could improve the bacterial richness and diversity of fermented sugarcane pith.

[0080] Depend on Figure 1 It can be seen that the synergistic fermentation of bacteria and enzymes can increase the relative abundance of beneficial bacteria represented by liquid Lactobacillus and Rhizobium, and reduce the relative abundance of harmful bacteria represented by Acinetobacter.

[0081] In summary, the present invention is better than single enzyme treatment or bacterial treatment in terms of conventional nutrients and fermentation indicators. Compared with the addition of chrysosporium or cellulase alone, the enzyme system of bacterial enzyme synergistic fermentation is more comprehensive, which can better degrade cellulose, hemicellulose and lignin, thereby producing more soluble substances, which are used by lactic acid bacteria carried by the sugarcane pith itself, and can be used to produce organic acids such as lactic acid and acetic acid, reduce feed pH, and improve fermentation quality. At the same time, through the synergistic treatment of multiple enzyme systems, the degradable part of the sugarcane pith itself can be increased, thereby increasing the 48h gas production of the sugarcane pith, and both the fast gas production part and the slow gas production part increase, which indicates that the sugarcane pith treated with bacterial enzymes will have a higher organic matter digestibility when fed, and has a higher digestion potential. At the same time, from the perspective of the 16s bacterial genus level, the relative abundance of beneficial bacteria increases and the relative abundance of harmful bacteria decreases, indicating that the safety of its feeding is also higher.

Claims

1. A method for fermenting sugarcane pith with bacterial enzymes, characterized in that: The method of using Phanerochaete chrysosporium and cellulase to synergistically ferment sugarcane pith includes the following steps: (1) Adjusting the water content of sugarcane pith to 60% to 70% and the pH to 4.0; (2) Add the following components according to dry matter mass and mix well: 3% bacterial solution of Phanerochaete chrysosporium, with a bacterial solution concentration of 1.5×10 8 CFU / ml; sucrose 1%; cellulase solution 20000U / kg, the cellulase solution is prepared by fermenting Trichoderma ergifer and then centrifuging the crude enzyme obtained and dissolving it in PBS buffer; (3) The mixture is placed in a fermentation tank for sealed low-oxygen fermentation for 30 days to obtain sugarcane pith feed.

2. The method according to claim 1, characterized in that The preparation method of the Phanerochaete chrysosporium bacterial liquid comprises: A. Activate and propagate Phanerochaete chrysosporium in PDA medium for 2-3 generations, transfer to PDB medium, and culture in a shaking incubator at 27°C and 140 rpm for 2-3 days. B. Count the cells using modified Martin's medium. Mix the bacterial solution with 20% glycerol at a ratio of 1:1 and store at -80°C.

3. The method according to claim 1, characterized in that The preparation method of the cellulase solution comprises: (1) Activate Trichoderma eringii and inoculate it into the seed culture medium, and culture it in a closed anaerobically manner at 27°C and 180 rpm for 2 days; The seed culture medium comprises: 10 g / L sodium carboxymethyl cellulose, 3 g / L peptone, 4 g / L potassium dihydrogen phosphate, 0.3 g / L magnesium sulfate heptahydrate, and 0.3 g / L calcium chloride, which is prepared with distilled water and placed in an autoclave for sterilization at 121° C. for 20 minutes. (2) Transfer the inoculum to the enzyme production medium at a rate of 6%, and culture under closed anaerobism at 27°C and 180 rpm for 5 days; The enzyme production culture medium is composed of: 12 g / L sodium carboxymethyl cellulose, 3 g / L peptone, 0.2 g / L yeast powder, 2 g / L ammonium sulfate, 4 g / L potassium dihydrogen phosphate, 0.3 g / L magnesium sulfate heptahydrate, 0.3 g / L calcium chloride, and 0.002 g / L zinc chloride; (3) The crude enzyme was collected by precipitation with 60% ethanol and centrifuged at 6000 r / min, and dissolved in PBS buffer. The enzyme activity was ≥20000 U / kg.

4. The sugarcane pith feed prepared by the method according to any one of claims 1 to 3, characterized in that The feed has a neutral detergent fiber content of ≤83.75%, an acid detergent lignin content of ≤14.25%, a lactic acid content of ≥356.61 mg / L, and a pH of ≤3.

68.

5. The method according to claim 3, characterized in that The steps for determining the CMC activity of cellulase solution using the DNS method are as follows: (1) Add 0.25 ml of cellulase solution to 0.75 ml of sodium citrate buffer solution with a concentration of 10 g / L sodium carboxymethyl cellulose. The solution is divided into a control tube and a test tube. 0.75 ml of 3,5-dinitrosalicylic acid reagent is added to the control tube at the same time. No reagent is added to the test tube. The tubes are placed in a 50°C water bath for 30 min. (2) Immediately after the water bath is completed, add 0.75 ml of 3,5-dinitrosalicylic acid reagent to the test tube. Then, place the control tube and the test tube in a boiling water bath for 5 minutes. After cooling, adjust the blank tube to zero at a wavelength of 540 nm and record the absorbance of the test tube. (3) preparing a glucose standard solution and processing it according to steps (1) and (2) to establish a standard curve for the glucose standard solution; (4) The absorbance obtained in step (2) was substituted into the glucose standard curve to calculate the glucose content in the crude enzyme solution, and the CMC enzyme activity of the crude enzyme solution was calculated according to the following formula:

6. Use of the sugarcane pith feed prepared according to the method according to any one of claims 1 to 4 in buffalo feed.

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