A bacterial enzyme composite preparation for improving straw digestion rate and application thereof

Abstract

The application discloses a bacteria-enzyme composite preparation for improving straw digestion rate and application thereof. The bacteria-enzyme composite preparation is combined by a Neocallimastix sp. fermentation liquor and a cellulase-xylanase bifunctional enzyme liquor in a volume ratio of 1:1-3, wherein the cellulase-xylanase bifunctional enzyme liquor is prepared from a cellulase-xylanase bifunctional enzyme mutant CelXyn2-S343 with an amino acid sequence shown in SEQ ID No. 2, and the Neocallimastix sp. fermentation liquor is prepared from Neocallimastix sp. LGM-Z A7 with a preservation number of CGMCC No. 17591. The bacteria-enzyme composite preparation can be used for producing biological fermentation straw with high digestion rate, and the production process is simple and operable, and can increase the dry matter degradation rate of straw feed by 30%, effectively improves the straw digestion rate, and further promotes the digestion and utilization of the straw by ruminants.

Description

Technical Field

[0001] This invention belongs to the field of biology, specifically relating to a bacterial enzyme compound preparation for improving the digestibility of straw and its application. Background Technology

[0002] Crop straw is a rich and renewable lignocellulosic biomass resource that can be used as feed for ruminants. my country is a major agricultural producer, with an annual straw output of 700-800 million tons, but the utilization rate is less than 50%. Only about 22-28% of the straw is used as feed, while over 30% is discarded or burned, resulting in resource waste and environmental pollution. Given the current limitations of feed supply in China's livestock industry, this represents a huge waste. However, the high fiber content, poor palatability, and low digestibility of crop straw also limit its use in the feed industry. Therefore, how to develop the application potential of straw resources in the feed industry is an urgent problem to be solved.

[0003] Bio-fermentation, a traditional method of fresh forage processing based on anaerobic microbial fermentation, is widely used due to its advantages such as minimal nutrient loss, good palatability, and high silage content. However, it also has limitations, such as the potential for silage failure if the processing is not done properly. *Neopyramidalis*, the only obligate anaerobic microorganism in the fungal community, plays a crucial role in the degradation of roughage. *Neopyramidalis* not only degrades difficult-to-degrade fibrous tissue through rhizome growth but also secretes a series of highly active plant cell wall degrading enzymes, synergistically degrading the complex structure of plant cell walls. This ability makes *Neopyramidalis* and its plant cell wall degrading enzymes increasingly promising for alleviating feed shortages in livestock farming and improving the utilization rate of animal roughage. Therefore, developing *Neopyramidalis* and its corresponding enzymes into a microbial-enzyme compound preparation is a promising strategy, but further research is needed to refine it. Summary of the Invention

[0004] This invention provides a microbial-enzyme compound preparation for improving the digestibility of straw and its application. The preparation method of the microbial-enzyme compound preparation is simple and can be used to produce bio-fermented straw with high digestibility, thereby improving its digestibility in the rumen of ruminants.

[0005] To achieve the above-mentioned objectives, the present invention employs the following technical solution: This invention provides a bacterial-enzyme compound preparation for improving the digestibility of straw. The bacterial-enzyme compound preparation is composed of a mixture of *Eriocaulon buergerianum* fermentation broth and a cellulase-xylanase bifunctional enzyme solution.

[0006] Furthermore, the volume ratio of the *Eriocaulon buergerianum* fermentation broth to the cellulase-xylanase bifunctional enzyme solution is 1:1~3.

[0007] Furthermore, the concentration of the *Neotrichum candida* fermentation broth is 1×10⁻⁶. 7 -1×10 8 cfu / ml.

[0008] Furthermore, the concentration of the cellulase-xylanase bifunctional enzyme solution is 0.2-0.5 mg / mL.

[0009] Furthermore, the cellulase-xylanase bifunctional enzyme solution is prepared from the cellulase-xylanase bifunctional enzyme mutant CelXyn2-S343, whose amino acid sequence is shown in SEQ ID No. 2.

[0010] Furthermore, the cellulase-xylanase bifunctional enzyme mutant CelXyn2-S343 is obtained by mutating cysteine ​​at position 343 of the cellulase-xylanase bifunctional enzyme with the amino acid sequence SEQ ID No.1 to serine.

[0011] Furthermore, the fermentation broth of *Euphorbia milii* was prepared from *Euphorbia milii* LGM-ZA7, which has the preservation number CGMCC No. 17591.

[0012] The present invention also provides the application of the aforementioned bacterial enzyme compound preparation in the production of bio-fermented straw with high digestibility.

[0013] Furthermore, the steps for producing bio-fermented straw are as follows: (1) After chopping the straw raw materials into 2-3 cm pieces, add them to the anaerobic reactor as fermentation material; (2) Add fermentation buffer and the bacterial enzyme compound preparation to the fermentation material, mix well, and ferment at 22℃~28℃ for 40~45 days to obtain bio-fermented straw with high digestibility.

[0014] Furthermore, the mass-to-volume ratio of the fermentation product to the fermentation buffer is 1:1.5~2.

[0015] Furthermore, the fermentation buffer includes basal culture medium, buffer A, buffer B, cell-free rumen fluid, reducing agent, and redox indicator.

[0016] Furthermore, the reducing agent is L-cysteine ​​hydrochloride, and the redox indicator is 0.1 w / v % resazurin.

[0017] Furthermore, the mass-to-volume ratio of the fermented product to the bacterial enzyme compound preparation is 1:1 to 1.5.

[0018] Furthermore, the straw-based raw materials include wheat straw and rice straw.

[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects: This invention utilizes fermentation broth and enzyme solution obtained through fermentation or purification of *E. nigra* LGM-ZA7 and the cellulase-xylanase bifunctional enzyme mutant CelXyn2-S343 in a scientifically proportioned manner to prepare a microbial-enzyme composite preparation. This microbial-enzyme composite preparation can be used to produce bio-fermented straw with high digestibility. The production process is simple and highly operable; the fermentation buffer components are simple and the raw materials are readily available; the amount of microbial-enzyme composite preparation used is small; and this process significantly reduces the content of dry matter, neutral detergent fiber, and hemicellulose in straw, while increasing the content of lactic acid, acetic acid, and ammonia nitrogen. Simultaneously, it increases the degradation rates of dry matter, neutral detergent fiber, acid detergent fiber, and cellulose in straw during in vitro fermentation, effectively improving the digestibility of straw and thus promoting its digestion and utilization by ruminants. This provides a new method for developing unconventional feeds in animal husbandry. Attached Figure Description

[0020] Figure 1 This is a process flow diagram of the present invention; Figure 2 The dry matter degradation rate of bio-fermented straw during in vitro fermentation; Figure 3 The degradation rate of neutral detergent fiber in bio-fermented straw during in vitro fermentation; Figure 4 The degradation rate of acid-washed fiber from bio-fermented straw during in vitro fermentation; Figure 5 The cellulose degradation rate of bio-fermented straw during in vitro fermentation. Detailed Implementation

[0021] The technical solution of the present invention will be further described in detail below with reference to specific examples. However, those skilled in the art will readily understand that the content described in the embodiments is only for illustrating the present invention and should not and will not limit the present invention as described in detail in the claims. In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0022] Example 1: Isolation, Identification and Culture of Bio-fermentation Strains Rumen contents were obtained from the goat rumen using a fistula method and cultured in fermentation buffer containing a specific antibiotic (penicillin / streptomycin) as shown in Table 1. The enriched cultures were serially diluted 10-fold to reduce bacterial and protozoan contamination. Bacterial-free anaerobic fungal enriched cultures were obtained through continuous subculturing. The enriched cultures were inoculated into agar-containing roller tubes, and pure *Flammulina neonsis* LGM-ZA7 was obtained through continuous roller tube culture.

[0023] The depositary institution for *Neocomonas nigra* LGM-ZA7 is the China General Microbiological Culture Collection Center (CGMCC); address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; deposit date: April 12, 2019; *Neocomonas nigra* Neocallimastix sp The accession number is CGMCC No. 17591.

[0024] The new strain of *Euphorbia milii* LGM-ZA7 requires a strictly anaerobic environment to grow, and its growth viability is maintained by continuous subculturing in fermentation buffer; it can grow well under anaerobic conditions at 39°C and pH 6.8.

[0025] Table 1. Fermentation Buffer Preparation Table (1 L)

[0026] Example 2: Obtaining and purifying a cellulase-xylanase bifunctional enzyme mutant Using the protein domain of the cellulase-xylanase bifunctional enzyme with the amino acid sequence shown in SEQ ID No. 1, the substrate-binding pocket site that determines the cellulase-xylanase bifunctional enzyme was predicted and identified through homology comparison. Mutation of the active site yielded a cellulase-xylanase bifunctional enzyme mutant, named CelXyn2-S343, with its amino acid sequence shown in SEQ ID No. 2 and the nucleotide sequence of the encoding gene shown in SEQ ID No. 3. The cellulase-xylanase bifunctional enzyme mutant CelXyn2-S343 was obtained by mutating cysteine ​​at position 343 of the cellulase-xylanase bifunctional enzyme with the amino acid sequence in SEQ ID No. 1 to serine.

[0027] Using the gene of the cellulase-xylanase bifunctional enzyme mutant CelXyn2-S343 and pET28a as expression vectors, a recombinant expression vector containing the cellulase-xylanase bifunctional enzyme mutant CelXyn2-S343 was constructed, and then transformed into Escherichia coli (DE3) to obtain a recombinant strain containing the cellulase-xylanase bifunctional enzyme mutant CelXyn2-S343.

[0028] After activation, the recombinant bacterial strain was inoculated into 6 mL of LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C and 200 rpm / min. Subsequently, 6 mL of the bacterial culture was transferred to narrow-necked flasks containing 600 mL of LB liquid medium containing 50 μg / mL kanamycin and cultured with shaking at 37°C and 200 rpm / min. When the OD600 value of the liquid medium in the narrow-necked flasks reached 0.5–0.6, IPTG (final concentration 0.5 mM) was added to each flask, and the culture was continued for 24 h at 15°C and 150 rpm / min. The cells were centrifuged at 8000 rpm / min and 4°C for 20 min, the supernatant was removed, and the cells were resuspended in buffer. The cell walls were then disrupted, and the cells were centrifuged at 12000 g and 4°C for 30 min. The supernatant was collected. The supernatant obtained above was purified by His purification using an AKTA Pure protein purifier via a nickel column. The resulting purified solution is the purified cellulase-xylanase bifunctional enzyme mutant enzyme solution.

[0029] Example 3: Study on the treatment of rice straw using a bacterial-enzyme compound preparation 1. Use a chaff cutter to cut the rice straw into pieces about 2-3 centimeters in length; 2. *Neotrichum candida* was cultured in the fermentation buffer shown in Table 1 for 72 hours to obtain a pure culture of *Neotrichum candida* (1×10⁻⁶). 7 -1×10 8 The prepared microbial enzyme complex is obtained by mixing (cfu / ml) and cellulase-xylanase bifunctional enzyme mutant enzyme solution (0.2-0.5 mg / mL) at a volume ratio of 1:1, which can be used for the next step of bio-fermentation treatment.

[0030] 3. Thoroughly mix 30 g of rice straw fragments with 40 mL of fermentation buffer, add 30 mL of bacterial enzyme compound preparation, and anaerobic ferment at room temperature (25 ºC) for 42 days to obtain bio-fermented straw. The above treatment is the treatment group.

[0031] Meanwhile, a control group was set up under the same conditions, except that 30 mL of bacterial enzyme compound preparation was replaced with 30 mL of distilled water.

[0032] After 42 days of fermentation, compared with the control group, the treatment group showed a significant reduction in the content of dry matter, neutral detergent fiber, and hemicellulose in rice straw (Table 2). It was also found that the addition of the bacterial enzyme compound significantly altered the fermentation type of the anaerobic fermentation system, thereby changing its metabolites. The results are shown in Table 2. The various metabolites in the treatment group were significantly increased: lactic acid production increased from 5.22 g / kg DM to 12.86 g / kg DM, an increase of approximately 2.5 times; acetic acid production increased from 2.46 g / kg DM to 9.98 g / kg DM, an increase of approximately 4 times; and ammonia nitrogen production increased from 1.84 mg / kg DM to 7.76 mg / kg DM, an increase of approximately 4.8 times.

[0033] Table 2 Results of treatment of rice straw with bio-fermentation inoculants

[0034] Example 4: In vitro digestibility study of bio-fermented straw 1. The bio-fermented straw from Example 3 was dried at 65°C and then pulverized into 0.3 cm length pieces using a pulverizer; 2. Prepare the in vitro fermentation buffer solution according to the formula in Table 3; 3. Add 1 g of crushed straw to the anaerobic reactor as the fermentation substrate; add 90 mL of in vitro fermentation buffer and 10 mL of fresh Hu sheep rumen fluid filtered through four layers of gauze to the anaerobic reactor to make the final concentration (mass-volume ratio) of the fermentation substrate 1% and start the reaction; the reaction temperature is 39℃ and the reaction time is 24 hours. The above is set as the treatment group.

[0035] A control group was set up, in which the bio-fermented straw was replaced with 1 g of untreated rice straw as the fermentation substrate.

[0036] Table 3. Preparation of in vitro fermentation buffer solution (1 L)

[0037] After 24 hours of in vitro fermentation, the dry matter degradation rate of the treatment group ( Figure 2 ), Neutral detergent fiber degradation rate ( Figure 3 ), degradation rate of acid detergent fibers ( Figure 4 ) and cellulose degradation rate ( Figure 5 Both were significantly improved, with the dry matter degradation rate increasing by approximately 30%. Simultaneously, the metabolites of rice straw after in vitro fermentation were altered after treatment with the bacterial-enzyme compound preparation. The results are shown in Table 4. In the bio-fermented straw group, acetic acid production increased from 38.9 mM to 48.2 mM, an increase of approximately 1.2 times; ammonia nitrogen production increased from 17.5 mg / dL to 22.1 mg / dL, an increase of approximately 1.3 times.

[0038] Table 4. Results of in vitro fermentation of straw.

[0039] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. The application of a microbial-enzyme compound preparation for improving straw digestibility in the production of bio-fermented straw with high digestibility, characterized in that, The bacterial enzyme compound preparation is made by mixing the fermentation broth of *Eriocaulon buergerianum* with a bifunctional enzyme solution of cellulase and xylanase. The volume ratio of the *Euphorbia milii* fermentation broth to the cellulase-xylanase bifunctional enzyme solution is 1:1~3. The cellulase-xylanase bifunctional enzyme solution was prepared from the cellulase-xylanase bifunctional enzyme mutant CelXyn2-S343, whose amino acid sequence is shown in SEQ ID No.

2. The cellulase-xylanase bifunctional enzyme mutant CelXyn2-S343 was obtained by mutating cysteine ​​at position 343 of the cellulase-xylanase bifunctional enzyme with the amino acid sequence SEQ ID No.1 to serine. The fermentation broth of *Euphorbia milii* was prepared from *Euphorbia milii* LGM-ZA7, which has the preservation number CGMCC No. 17591.

2. The application according to claim 1, characterized in that, The steps for producing bio-fermented straw are as follows: (1) After chopping the straw raw materials into 2-3 cm pieces, add them to the anaerobic reactor as fermentation material; (2) Add fermentation buffer and the bacterial enzyme compound preparation to the fermentation material, mix well, and ferment at 22℃~28℃ for 40~45 days to obtain bio-fermented straw with high digestibility.

3. The application according to claim 2, characterized in that, The mass-to-volume ratio of the fermentation product to the fermentation buffer is 1:1.5~2.

4. The application according to claim 2, characterized in that, The mass-to-volume ratio of the fermented product to the bacterial enzyme compound preparation is 1:1 to 1.

5.

5. The application according to claim 2, characterized in that, The straw-based raw materials include wheat straw and rice straw.

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