High-temperature-resistant cronobacter sakazakii strain with cellulose degradation capability and application

Abstract

The invention belongs to the technical field of agricultural microorganism development and application industry, and particularly relates to a high-temperature-resistant cronobacter sakazakii strain with cellulose degradation capacity and application. According to the specific technical scheme, the high-temperature-resistant cronobacter sakazakii strain with the cellulose degradation capacity is named as cronobacter sakazakii, and the 16S rRNA nucleotide sequence of the cronobacter sakazakii strain is as shown in SEQ ID NO: 1; the strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.33826, and the preservation date is March 14, 2025. The Cronobacter sakazakii disclosed by the invention can be used for efficiently degrading cellulose, hemicellulose and acidic detergent lignin, and a new solution is provided for the feed pretreatment of straws and dregs, the solving of agricultural non-point source pollution caused by the straws and the acceleration of high-temperature composting. Moreover, the strain still keeps high enzymatic activity at a high temperature of 65 DEG C, so that the development of related products of a microbial agent of the strain is greatly facilitated, the application range of the strain is expanded, and the strain has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of agricultural microbial development and application industry, and particularly relates to a high-temperature-resistant Klebsiella quasipneumoniae strain with cellulose degradation capacity and application. BACKGROUND

[0002] A large amount of straw is produced in agricultural production every year, which is a huge renewable resource, but its utilization rate is less than 70%. A large amount of straw is abandoned or burned, which causes serious agricultural non-point source pollution. Cellulose, hemicellulose and lignin are the main components of straw, which are complex in structure and difficult to degrade, and are slowly decomposed under natural conditions. Long-term accumulation not only occupies land resources, but also breeds diseases and pests, pollutes water and soil; burning will produce a large amount of greenhouse gases and harmful particulate matter, which will aggravate environmental pollution, so it is urgent to seek an efficient and environmentally friendly method for harmless treatment of straw. At the same time, with the large-scale development of animal husbandry, the shortage of feed resources is increasingly prominent, especially the high dependence on external protein feed, so how to pretreat straw and dregs to convert them into palatable and easily digestible high-quality feed and realize the recycling of resources has become a hot issue in the current sustainable development of agriculture.

[0003] At present, the main treatment technologies of straw include physical method (such as chopping, briquetting), chemical method (such as ammoniation, alkali treatment) and biological method (such as microbial degradation). Among them, biological degradation has become the research focus due to its environmental protection and high efficiency, and through screening of cellulose-degrading bacteria and using bacterial strains to treat straw and dregs, cellulose can be fully degraded and utilized. In addition, dregs such as vinasse and vinegar dregs are rich in protein, sugar and trace elements, which are high-quality feed resources, but their resource recycling is affected due to poor palatability and low digestion rate. Therefore, screening of efficient and easy-to-culture cellulose-degrading bacteria has very important academic value and broad application prospect.

[0004] Bactrian camel (Camelus bactrianus) Camelus bactrianus lives in arid desert and semi-desert areas, and takes coarse feed such as desert plants rich in cellulose and hemicellulose as the main food source. Although cellulose and hemicellulose are difficult to digest, the digestion and utilization rate of cellulose by bactrian camel can reach 40% to 60%, which enables them to obtain sufficient energy and nutrients from coarse feed. Therefore, the patent screens strains capable of efficiently degrading cellulose from bactrian camel feces, studies the growth characteristics of the strains and the application of the strains in degrading straw and dregs rich in cellulose; the study has positive significance for pretreatment of straw and dregs for feed, solving agricultural non-point source pollution caused by straw and accelerating high-temperature composting process. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a high-temperature-resistant Klebsiella quasipneumoniae strain with cellulose degradation ability and an application thereof, which overcomes the shortcomings of the prior art Cronobacter sakazakii , can efficiently degrade cellulose, hemicellulose and acid washing lignin, has an optimal growth temperature range of 45-75 DEG C, and has the highest enzyme activity at 65 DEG C, and provides a new efficient degradation method for straw and residue feed pretreatment, solves the agricultural non-point source pollution caused by straw, and speeds up high-temperature composting To solve the above technical problems, the technical scheme adopted by the present application is: a high-temperature-resistant Klebsiella quasipneumoniae strain with cellulose degradation ability and an application thereof, the high-temperature-resistant Klebsiella quasipneumoniae strain with cellulose degradation ability is named Klebsiella quasipneumoniae Cronobacter sakazakii , which is isolated from the feces of a double-humped camel that can resist rough feeding, is preserved in the China General Microbiological Culture Collection Center, the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing, the preservation number is CGMCC No. 33826, and the preservation date is March 14, 2025; the 16S rRNA nucleotide sequence of the Klebsiella quasipneumoniae Cronobacter sakazakii is shown in SEQ ID NO: 1.

[0006] Preferably, the Klebsiella quasipneumoniae Cronobacter sakazakii has an optimal growth temperature range of 45-75 DEG C, and has the highest carboxymethyl cellulase enzyme activity at 65 DEG C, and has an optimal culture pH of 6.5.

[0007] The present application also provides an application of the Klebsiella quasipneumoniae strain described above, and the Klebsiella quasipneumoniae Cronobacter sakazakii is used for degrading cellulose, hemicellulose and acid washing lignin.

[0008] Preferably, the Klebsiella quasipneumoniae Cronobacter sakazakii is used for degrading cellulose, hemicellulose and acid washing lignin in wheat straw.

[0009] Preferably, the Klebsiella quasipneumoniae Cronobacter sakazakii is used for degrading cellulose, hemicellulose and acid washing lignin in wine lees.

[0010] Preferably, the Klebsiella quasipneumoniae Cronobacter sakazakii is used for degrading cellulose, hemicellulose and acid washing lignin in vinegar lees.

[0011] Preferably, the Klebsiella quasipneumoniae Cronobacter sakazakii is used for accelerating high-temperature composting, and the temperature of the high-temperature composting is 45 DEG C-75 DEG C.

[0012] Preferably, it is used for straw and residue feed pretreatment.

[0013] Compared with the prior art, the present invention has the following advantages: The present invention is based on the Cronobacter sakazakii. Cronobacter sakazakii It can efficiently degrade cellulose, hemicellulose, and acid-washing lignin, providing a new solution for the pretreatment of straw and dregs for feed production, addressing agricultural non-point source pollution caused by straw, and accelerating high-temperature composting. Furthermore, this strain can withstand high temperatures (45℃~75℃), greatly facilitating the development of related products and expanding its application scope, demonstrating broad application prospects.

[0014] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0015] Cronobacter sakazakii It is strain C8 (Cronobacter sakazakii) from Example 1 of this invention. Figure 1 The growth curve of ).

[0016] Cronobacter sakazakii It is strain C8 (Cronobacter sakazakii) from Example 1 of this invention. Figure 2 Hydrolysis zone (A) and colony morphology of strain C8 on sodium carboxymethyl cellulose solid medium plate (B).

[0017] Cronobacter sakazakii It is strain C8 (Cronobacter sakazakii) from Example 1 of this invention. Figure 3 Morphological identification of strain C8. (A) Gram staining results of strain C8; (B) Spore staining results of strain C8; (C) Scanning electron microscopy results of strain C8.

[0018] Cronobacter sakazakii It is strain C8 (Cronobacter sakazakii) from Example 1 of this invention. Figure 4 Agarose gel electrophoresis (A) and phylogenetic tree (B) of the 16S rRNA gene.

[0019] Cronobacter sakazakii It is strain C8 (Cronobacter sakazakii) from Example 2 of this invention. Figure 5 The glucose standard curve.

[0020] Cronobacter sakazakii It is strain C8 (Cronobacter sakazakii) from Example 3 of this invention. Figure 6 The enzymatic characteristics of carboxymethyl cellulase were analyzed. Among them, (A) is the effect of pH on the activity of carboxymethyl cellulase, (B) is the effect of pH on the stability of carboxymethyl cellulase, (C) is the effect of temperature on the activity of carboxymethyl cellulase, (D) is the effect of temperature on the stability of carboxymethyl cellulase, and (E) is the effect of time on the activity of carboxymethyl cellulase.

[0021] Cronobacter sakazakii In Example 3 of this invention, after 20 days of fermentation, the control group and strain C8 (Cronobacter sakazakii) were compared. Figure 7 The changes in cellulose (A), hemicellulose (B), and acid detergent lignin (C) content in wheat straw of the treatment groups are shown in the figure. ** indicates... P<0.01 .

[0022] Cronobacter sakazakii The control group and C8 group (Cronobacter sakazakii) in Example 3 of this invention. Figure 8 Cronobacter Scanning electron microscope image of cross-section of straw after 20 days of cultivation in the treatment group.

[0023] sakazakii The control group and C8 group (Cronobacter sakazakii) in Example 3 of this invention. Figure 9 Cronobacter Scanning electron microscope image of the outer surface of straw after 20 days of cultivation in the treatment group.

[0024] sakazakii The control group and C8 group (Cronobacter sakazakii) in Example 3 of this invention. Figure 10 Cronobacter Scanning electron microscope image of the inner surface of straw after 20 days of cultivation in the treatment group.

[0025] sakazakii Within 20 days of fermentation in Example 4 of this invention, the control group and strain C8 (Cronobacter sakazakii) showed... Figure 11 The changes in cellulose, hemicellulose, and acid detergent lignin content in the treated groups of distiller's grains and vinegar residues are shown in the figure. (A) represents the change in cellulose content in distiller's grains; (B) represents the change in hemicellulose content in distiller's grains; (C) represents the change in acid detergent lignin content in distiller's grains; (D) represents the change in cellulose content in vinegar residues; (E) represents the change in hemicellulose content in vinegar residues; and (F) represents the change in acid detergent lignin content in vinegar residues. ** in the figure indicate… P<0.01 .

[0026] Cronobacter sakazakii This refers to the control group and the control group and C8 group (Cronobacter sakazakii) in Example 4 of the present invention. Figure 12 Cronobacter Scanning electron microscope image of the fermented grains after 20 days of culture in the treatment group.

[0027] sakazakii This refers to the control group and the control group and C8 group (Cronobacter sakazakii) in Example 4 of the present invention. Figure 13 Cronobacter Scanning electron microscope image of vinegar residue after 20 days of culture in the treatment group. Detailed Implementation

[0028] Example 1 This example uses cellulose-degrading bacteria (Kronobacter sakazakii).sakazakii Screening and identification of ).

[0029] The culture medium used is as follows: (1) Enrichment medium: 10.0 g CMC-Na, 0.1 g sodium chloride, and distilled water to a final volume of 1000 mL (pH=7.0~7.5). Sterilize at 121℃ for 20 min.

[0030] (2) Sodium carboxymethyl cellulose solid culture medium plates: CMC-Na (sodium carboxymethyl cellulose) 5.0 g, (NH4)2SO4 2 g, K2HPO4 1.0 g, MgSO4·7H2O 0.5 g, sodium chloride 0.5 g, FeSO4·7H2O 0.1 g, agar powder 15.0 g, and distilled water to a final volume of 1000 mL (pH=7.0~7.2). Sterilize at 121℃ for 20 min.

[0031] (3) LB medium: 10.0 g tryptone extract, 5.0 g yeast extract, 10.0 g sodium chloride, distilled water to a final volume of 1000 mL (1.5% agar added to solid LB medium). Sterilize at 121℃ for 15 min.

[0032] (4) Shake flask fermentation medium: 1.0% CMC (carboxymethyl cellulose) was added to ordinary LB liquid medium for the fermentation culture of cellulose-degrading bacteria. Sterilize at 121℃ for 15 min.

[0033] (5) 1 g / L Congo Red staining solution: Weigh 0.1 g of Congo Red and dissolve it in 100 mL of distilled water.

[0034] (6) Preparation of 1 mol / L NaCl solution: Weigh 5.85 g NaCl, dissolve it in 100 mL of distilled water, mix thoroughly and autoclave.

[0035] (7) Glucose standard solution (10 mg / mL): Weigh 1.000 g glucose (AR) (dried at 105℃ to constant weight), dissolve it in acetate-sodium acetate buffer solution (pH 5.5), and make up to 100 mL. Store in the refrigerator for later use.

[0036] (8) Seed culture: The bacterial culture was added to LB liquid medium and cultured until OD was reached. 600 =1.0.

[0037] (9) CMC substrate solution: Weigh 1.0 g CMC and dissolve it in 80 mL of acetate-sodium acetate buffer solution (pH=5.5). While stirring, heat slowly until sodium carboxymethyl cellulose is completely dissolved. Then stop heating and continue stirring for 30 min. Make up to 100 mL with acetate-sodium acetate buffer solution. Shake well before use and store at 4℃ protected from light. Shelf life is 3 days.

[0038] (10) The culture medium composition is as follows: KH2PO4 2g, MgSO4 0.5g, CaCl2·6H2O 0.3g, FeCl3 0.01g, MnSO4·7H2O 0.0016g, ZnSO4·7H2O 0.0014g, yeast 0.5g, peptone 1g, distilled water 1000 mL.

[0039] (11) Preparation of buffer solutions with different pH values: A. Acetic acid-sodium acetate buffer solutions with different pH values: Acetic acid-sodium acetate buffer solutions with different pH values ​​were prepared according to the different ratios of 0.1 mol / L acetic acid solution and 0.1 mol / L sodium acetate solution in Table 1, and finally adjusted to 100 mL.

[0040] Table 1. Acetic acid-sodium acetate buffer solutions at different pH values B. Disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solutions at different pH values ​​(0.1 mol / L) Phosphate solutions of different pH values ​​can be mixed according to the following ratio of mother solution 1 and mother solution 2 (Table 2).

[0041] Mother liquor 1: 0.1 mol / L disodium hydrogen phosphate solution: Weigh 17.91 g of Na2HPO4·12H2O (molecular weight 358.14), dissolve in distilled water and bring the volume to 500 mL; Mother liquor 2: 0.1 mol / L sodium dihydrogen phosphate solution: Weigh 7.8 g of NaH2PO4·2H2O (molecular weight 156.01), dissolve in distilled water and bring the volume to 500 mL.

[0042] Table 2. Disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solutions at different pH values C. Glycine-sodium hydroxide buffer Glycine-sodium hydroxide buffer solutions of different pH values ​​can be mixed with water and diluted to 200 mL according to the following ratio of stock solution 1 and stock solution 2 (Table 3).

[0043] Mother liquor 1: 0.2 mol / L glycine: Weigh 7.5 g of glycine (molecular weight 75.07), dissolve in distilled water and bring the volume up to 500 mL.

[0044] Mother liquor 2: 0.2 mol / L NaOH: Weigh 4.0 g NaOH, dissolve it in distilled water, and bring the volume to 500 mL.

[0045] Table 3. Glycine-sodium hydroxide buffer solutions at different pH values 1. Screening of cellulose-degrading bacteria (1) Initial screening of cellulose-degrading bacteria Fecal samples were collected from Bactrian camels at Taiyuan Zoo. 1 g of fecal sample was weighed and placed in 100 mL of PBS buffer solution. The mixture was shaken at 30°C and 180 rpm for 10 min, then allowed to stand for 10 min. 5 mL of the bacterial suspension was added to 95 mL of enrichment medium and shaken at 30°C and 180 rpm for 3 days. The cultured bacterial solution was then serially diluted (10-10). -2 10 -3 10 -4 10 -5 10 -6 100 μL of the diluted solution was spread onto sodium carboxymethyl cellulose (CMC) solid medium plates and incubated at 30℃ for 3-6 days. A suitable amount of 1 g / L Congo red staining solution was added to the plates for 1 h of staining. After discarding the waste liquid, a suitable amount of 1 mol / L NaCl solution was added for washing 2-3 times. The diameter (D) of the hydrolysis zone around the colony and the colony diameter (d) on the CMC solid medium plates were measured using calipers, and the D / d ratio was used as a preliminary standard for judging the bacteria's ability to degrade cellulose. Colonies with a high D / d ratio were selected for expansion culture. Then, the colonies with high D / d ratios were repeatedly streaked on solid LB medium for isolation and purification culture for 4-5 generations. The purified cultures were stored at -80℃ with glycerol for later use. The isolation and screening of 9 cellulose-degrading strains are shown in Table 4.

[0046] (2) Preparation of seed culture of cellulose-degrading bacteria and secondary screening of cellulose-degrading bacteria Cellulose-degrading bacteria with a high D / d ratio were streaked onto solid LB medium for activation. A small number of single colonies were then picked up with an inoculation loop and transferred to liquid LB medium. The culture was incubated at 37°C and 220 rpm for 12 h with shaking. The culture was then transferred to LB medium at a 1.0% inoculation rate and activated twice more. The OD of the activated culture was then measured. 600 Adjusting it to 1.0 gives us the seed solution for the experiment.

[0047] Then, the seed culture (OD=1.0) was inoculated into 50 mL shake flask fermentation medium at an inoculation rate of 1.0%, and fermented at 37℃ and 220 rpm for 24 h. The fermentation broth was centrifuged at 5000 rpm and 4℃ for 15 min, and the supernatant was the seed culture. The cellulase activity of the seed culture was then measured, and strains with high cellulase activity were selected for subsequent experiments.

[0048] Table 4. Isolation and screening of 9 cellulose-degrading strains Note: D: Diameter of the clear zone, d: Colony diameter 2. Identification of cellulose-degrading bacteria Seed culture (OD=1.0) was inoculated at a rate of 1.0% into 100 mL of fresh LB medium and cultured in a shaker at 37°C for 6 h. Samples were taken every 2–4 h thereafter until 60 h. For measurement, the culture medium was diluted 10-fold with fresh LB medium, and the OD was determined using a spectrophotometer. 600 The values ​​were measured, growth was observed, and a growth curve for strain C8 was plotted. The results are as follows: Cronobacter sakazakii As shown, Figure 1 It grows rapidly. After 8–24 hours of fermentation, it enters the logarithmic phase; after 24–40 hours of culture, it is in a basically stable phase, which can last for about 16 hours; after 40 hours, it enters the decline phase.

[0049] Hydrolysis zones of strain C8 on sodium carboxymethyl cellulose solid medium plates are as follows Cronobacter sakazakii C8 As shown in (A), the morphology of strain C8 is as follows: Figure 2 As shown in (B).

[0050] Gram staining of the strain [ Figure 2 (A)], spore staining [ Figure 3 (B) and scanning electron microscopy observation [ Figure 3 (C) After that, it can be seen that the colony surface is smooth and opaque, often forming wrinkles; the strain is Gram-negative, and the bacterial cells are rod-shaped under the microscope; after the strain is stained with malachite green, it turns red and has no spores.

[0051] Homology analysis of the 16S rRNA sequencing results of strain C8 using the BLAST program on NCBI showed that the 16S rRNA gene of strain C8 was homologous to that of strain C8. Agarose gel electrophoresis results were as follows: Figure 3 As shown in (A), the 16S rRNA gene sequence length of the cellulose-degrading bacteria is 1498 bp; strain C8 shows 98% similarity to the 16S rRNA sequences of multiple strains of *Cronobacter*, as shown in the results. Figure 4As shown in (B). From the phylogenetic tree constructed using Mega11, this strain is related to... Figure 4 MDENA2-7 (JF690878.1) is the most closely related strain, with a sequence similarity of 98%. Based on the morphological and 16S rRNA molecular identification results, the cellulose-degrading bacterium C8 was finally identified as a strain of *Cronobacter sakazakii*, and named *Cronobacter sakazakii*. Cronobacter sakazakii strain .

[0052] Cronobacter sakazakii Cronobacter sakazakii The *Cronobacter sakazakii* strain described herein is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; accession number CGMCC No. 33826; deposit date March 14, 2025. Cronobacter sakazakii The 16S rRNA nucleotide sequence is shown in SEQ ID NO: 1.

[0053] Example 2 This example uses strain C8 (Cronobacter sakazakii) isolated in Example 1. Cronobacter sakazakii Cronobacter The cellulase activity of ).

[0054] The activity of carboxymethyl cellulase (CMCase) was determined according to the People's Republic of China Agricultural Industry Standard (NY / T912-2004), namely, the spectrophotometric method for determining cellulase activity in feed additives. The enzyme activity unit of CMCase is defined as the amount of enzyme required per minute to degrade CMC-Na to produce 1 μmol of reducing sugar per minute under the conditions of pH 5.5 and 40℃ for 30 min, expressed as U / mL.

[0055] 1. Preparation of glucose standard curve Standard blank sample: Pipette 4.0 mL of pH 5.5 acetate-sodium acetate buffer solution into a graduated test tube, add 5.0 mL of DNS reagent, heat in a boiling water bath for 5 min, cool to room temperature, and then dilute to 25.0 mL with distilled water.

[0056] Pipette 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, and 7.0 mL of glucose standard solution into corresponding 100 mL volumetric flasks, and dilute to 100 mL with buffer solution to prepare glucose standard solutions with concentrations ranging from 0.1 to 0.7 mg / μL. The specific experimental procedures are shown in Table 5. Plot glucose concentration on the x-axis and OD value on the y-axis. 540 Using the ordinate as the y-axis, plot the standard curve to obtain the linear regression equation. The plotting results are as follows: sakazakiiThe equation was measured to be y = 0.9729x - 0.0811, R0 2 It is 0.9992.

[0057] Table 5. Development of the glucose standard curve 2. Determination of carboxymethyl cellulase activity A B Add 5 mL of 3,5-dinitrosalicylic acid to 2 mL of enzyme solution in a graduated test tube, mix well, then add 2 mL of CMC substrate solution, mix well, react accurately at 40℃ for 30 min, boil in boiling water for 5 min, bring the volume to 25 mL, adjust to "0" with a standard blank sample, and measure the absorbance at a wavelength of 540 nm.

[0058] A E Add 2 mL of CMC substrate solution to 2 mL of enzyme solution in a graduated test tube, mix well, react accurately at 40℃ for 30 min, then add 5 mL of DNS, mix well, boil in boiling water for 5 min, bring the volume to 25 mL, adjust to "0" with a standard blank sample, and measure the absorbance at a wavelength of 540 nm.

[0059] The calculation formula for CMCase enzyme activity is: X D =[(A E -A B [(M×t)×K + Co] / (M×t)×1000 In the formula: X D CMCase activity in the sample dilution solution, U / mL; A E A represents the OD value of the enzyme reaction solution. B 1000 represents the OD value of the enzyme blank sample, K represents the slope of the glucose standard curve, Co represents the intercept of the glucose standard curve, M represents the molecular weight of glucose (180.2), t represents the enzymatic reaction time (min), and 1000 represents the transformants in mmol and μmol. If the OD value measured in the experiment is too high, the seed solution needs to be diluted to ensure the accuracy of the measurement.

[0060] The cellulase activity of strain C8 was calculated to be 0.89 U / mL.

[0061] Example 3 This example uses strain C8 (Cronobacter sakazakii) isolated in Example 1. Figure 5 Cronobacter Performance testing of strains.

[0062] (1) Optimal pH of carboxymethyl cellulase reaction 1.0% CMC-Na substrate was dissolved in buffer solutions ranging from pH 4.5 to 9.0, and the seed culture was also diluted with buffer solutions ranging from pH 4.5 to 9.0. The optimal pH for the CMCase reaction was determined at 40°C for 30 min, and each group was repeated 3 times.

[0063] (2) Optimal temperature of carboxymethyl cellulase reaction Under optimal pH conditions, a 1.0% CMC-Na substrate solution was mixed with an appropriately diluted seed solution and reacted at 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, and 80℃ for 30 min to determine the optimal temperature for the CMCase reaction. Each group was repeated 3 times.

[0064] (3) Thermal stability of carboxymethyl cellulase The seed culture was incubated in a water bath at 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃ and 80℃ for 1 h to evaluate the thermal stability of the CMCase. Each group was repeated 3 times.

[0065] (4) Acid-base stability of carboxymethyl cellulase The seed culture was mixed 1:1 with a buffer solution of pH 3.0–10.0 and incubated in a 40°C water bath for 1 h. The CMCase was then removed and evaluated for its acid and alkali tolerance under optimal pH conditions.

[0066] (5) Optimal reaction time of carboxymethyl cellulase The activity of CMCase was measured at different reaction times (5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, and 45 min) under optimal pH and temperature conditions to determine the optimal reaction time of the enzyme.

[0067] The measurement results are as follows sakazakii As shown, by Figure 6 (A) Observe that the optimal pH for the CMCase reaction is 6.5; when the seed culture of carboxymethyl cellulase is incubated with buffer solutions of different pH values ​​in a 40°C water bath for 1 h, its relative enzyme activity is between 70% and 80% in the pH range of 3 to 8. Figure 6 (B) indicates strain C8 (Cronobacter sakazakii) Figure 6 The CMCase secreted by it has strong acid and alkali resistance.

[0068] Under optimal pH conditions, the optimal temperature for the CMCase reaction is 65°C. Cronobacter sakazakii(C), its relative enzyme activity is between 70% and 90% in the range of 55℃ to 70℃, and between 60% and 90% in the range of 45℃ to 75℃. Figure 6 (D) indicates strain C8 (Cronobacter sakazakii) Figure 6 The CMCase secreted by it has strong thermal stability.

[0069] In summary, the aforementioned *Cronobacter sakazakii* Cronobacter sakazakii The optimal growth temperature range is 45℃~75℃, and the enzyme activity of carboxymethyl cellulase is the highest at 65℃. The optimal culture pH is 6.5.

[0070] The strain C8 of this invention (Cronobacter sakazakii) Cronobacter sakazakii It has high temperature resistance. When this strain is used to accelerate high-temperature composting, the temperature of high-temperature composting can be 45℃~75℃, and the relative enzyme activity of the strain can be maintained at more than 60%.

[0071] Furthermore, under optimal pH and temperature conditions, the CMCase enzyme activity is highest after 10 minutes of reaction. Cronobacter sakazakii (E) Subsequently, the enzyme activity of both the enzyme and the substrate gradually decreased with increasing reaction time.

[0072] Example 4 This example uses strain C8 (Cronobacter sakazakii) isolated in Example 1. Figure 6 Cronobacter The ability to degrade cellulose, hemicellulose and acid-washed lignin in wheat straw.

[0073] Straw powder was divided into several equal portions, each with a dry weight of 80g. After fermentation for 20 days, the samples were dried at 65℃ and weighed to calculate the dry matter content. A slightly modified Van der Waals fiber washing method was used, and the contents of neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined using a fully automated fiber analyzer, with three replicates per group. Sufficient 72% sulfuric acid was added to the ADF sample for soaking. Initially, the filter bag was stirred to ensure full contact with the sulfuric acid. After 3 hours, the sulfuric acid was discarded, and the sample was washed with hot water until neutral, then rinsed with acetone for 3 minutes. The sample was dried in an oven at 105℃ for approximately 3 hours, then cooled to room temperature in a desiccator and weighed (i.e., day 0). The dissolved portion during digestion was cellulose, while the undissolved residue consisted of acid detergent lignin and acid-insoluble ash. The contents of cellulose, hemicellulose, and acid detergent lignin were calculated for day 0 and day 20 of culture using the following formulas. Three replicates were performed for each group.

[0074] Hemicellulose content = NDF (%) - ADF (%); Cellulose content = ADF (%) - Residue after 72% sulfuric acid immersion (%); Acid washing lignin content = residue (%) - ash (%); The measurement results are as follows sakazakii As shown, the contents of cellulose, hemicellulose, and acid detergent lignin in wheat straw decreased significantly from 20.24%, 21.85%, and 3.74% before fermentation to 15.16%, 14.41%, and 2.13%, respectively (**). P<0.01 ).

[0075] 2. Electron microscopic observation of wheat straw Fresh, mold-free wheat straw was selected, washed, dried, and cut into 0.5cm long pieces. These pieces were placed in a 100mL Erlenmeyer flask, culture medium was added, and the flask was sterilized. The previously screened strain was then inoculated into the culture medium at a 10% inoculum size. The mixture was then shaken and cultured. This was used as strain C8 (Cronobacter sakazakii). Figure 7 The treatment group (C8 group) was used as a control group, in which wheat straw was cultured on a medium without bacterial solution for 20 days. The results are as follows: Cronobacter sakazakii , 9 As shown in Figure 10.

[0076] Looking at the cross-section of wheat straw ( Figure 8 After 20 days of fermentation, strain C8 (Cronobacter sakazakii) was added. Figure 8 The thin-walled tissue of wheat straw cultured continued to degrade significantly, the vascular tissue separated from the mechanical tissue, and the vascular bundle structure became loose.

[0077] From the outer surface of wheat straw ( Cronobacter sakazakii After 20 days of fermentation, strain C8 (Cronobacter sakazakii) was added. Figure 9 The wheat straw cultured by the experiment showed that the outer surface of the straw cracked, and the epidermal tissue under the waxy-silica layer gradually became visible.

[0078] Looking at the inner surface of wheat straw ( Cronobacter sakazakii After 20 days of fermentation, strain C8 (Cronobacter sakazakii) was added. Figure 10 The wheat straw cultivated in this way has its inner surface degraded and its basic internal structure destroyed.

[0079] Example 5 This example uses strain C8 (Cronobacter sakazakii) isolated in Example 1. Cronobacter sakazakii Cronobacter The ability to degrade cellulose, hemicellulose and acid-washed lignin in distillers' grains and vinegar residues.

[0080] 1. Changes in the content of main components in distiller's grains and vinegar lees Distillers' grains and vinegar residues were divided into several equal portions, each with a dry weight of 80g. After fermentation for 20 days, the portions were dried at 65℃ and weighed to calculate the dry matter content. The contents of cellulose, hemicellulose, and acid detergent lignin in the distillers' grains and vinegar residues after 0 days of cultivation (control group) and 20 days (C8 group) were determined. Each group had three replicates.

[0081] like sakazakii As shown in (A)-(C), the contents of cellulose, hemicellulose, and acid detergent lignin in the distiller's grains decreased significantly from 16.28%, 30.69%, and 4.89% before fermentation to 9.75%, 18.15%, and 2.22%, respectively. (**) P<0.01 ).like Figure 11 As shown in (D)-(F), the contents of cellulose, hemicellulose, and acid detergent lignin in vinegar residue decreased significantly from 22.89%, 37.86%, and 4.82% before fermentation to 13.58%, 26.75%, and 2.21%, respectively. P<0.01 ).

[0082] 2. Electron microscopic observation of distiller's grains and vinegar lees The culture medium composition is as follows: KH2PO4 2g, MgSO4 0.5g, CaCl2·6H2O 0.3g, FeCl3 0.01g, MnSO4·7H2O 0.0016g, ZnSO4·7H2O 0.0014g, yeast 0.5g, peptone 1g, and distilled water 1000mL.

[0083] Place the distiller's grains and vinegar residues separately into 100 mL Erlenmeyer flasks, add culture medium, sterilize, and then inoculate the previously screened strain into the culture medium at a 10% inoculum. Incubate with shaking for 20 days to obtain strain C8 (Cronobacter sakazakii). Figure 11 The treatment group (C8 group) was used as a control group, in which distiller's grains and vinegar residues were cultured on culture medium without bacterial culture for 20 days. The results are as follows: Cronobacter sakazakii As shown.

[0084] from Figures 12-13 It can be seen that after 20 days of fermentation and culture in the lees, the originally dense and regular fiber network structure was destroyed, the surface became loose and porous, and it presented an irregular fluffy state with obvious cracks and grooves. The surface was covered with holes of different sizes, which may be caused by the decomposition of cellulose and other components during the degradation process.

[0085] from Figure 12 Figure 13 It can be seen that the originally continuous cellulose areas in the vinegar residue have become fragmented and broken in large numbers, with numerous tiny fiber fragments scattered throughout. The surface is no longer smooth and flat, but is covered with densely packed holes and grooves of various sizes.

[0086] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A thermoresistant strain of *Cronobacter sakazakii* with cellulose degradation capabilities, characterized in that, The thermostable Cronos sakazakii strain with cellulose degradation capabilities was named Cronos sakazakii. Cronobacter sakazakii The *Cronobacter sakazakii* strain described herein is deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC No. 33826, on March 14, 2025. Cronobacter sakazakii The 16S rRNA nucleotide sequence is shown in SEQ ID NO:

1.

2. The thermoresistant Cronobacter sakazakii strain with cellulose degradation capability according to claim 1, wherein the Cronobacter sakazakii strain... Cronobacter sakazakii The optimal growth temperature range is 45~75℃, and the enzyme activity of carboxymethyl cellulase is the highest at 65℃. The optimal culture pH is 6.

5.

3. The application of a thermoresistant Cronobacter sakazakii strain with cellulose degradation capability as described in claim 1 or 2, characterized in that, The Cronobacter sakazakii Cronobacter sakazakii Used to degrade cellulose, hemicellulose, and acid-washing lignin.

4. The application according to claim 3, characterized in that, The Cronobacter sakazakii Cronobacter sakazakii Used to degrade cellulose, hemicellulose, and acid-washing lignin in wheat straw.

5. The application according to claim 3, characterized in that, The Cronobacter sakazakii Cronobacter sakazakii Used to degrade cellulose, hemicellulose, and acid-washing lignin in distiller's grains.

6. The application according to claim 3, characterized in that, The Cronobacter sakazakii Cronobacter sakazakii Used to degrade cellulose, hemicellulose, and acid detergent lignin in vinegar residue.

7. The application according to claim 3, characterized in that, The Cronobacter sakazakii Cronobacter sakazakii It is used to accelerate high-temperature composting, with the temperature of high-temperature composting being 45℃~75℃.

8. The application according to claim 3, wherein the *Cronobacter sakazakii* Cronobacter sakazakii Used for the pretreatment of straw and dregs for feed production.

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