Silage bacterium enzyme compound preparation, preparation method thereof and application of silage bacterium enzyme compound preparation in preparation of space bamboo reed silage

Abstract

The invention belongs to the technical field of silage bacterium enzyme preparation preparation and silage processing, and particularly relates to a silage bacterium enzyme compound preparation, a preparation method thereof and application of the silage bacterium enzyme compound preparation in space bamboo reed silage. The compound bacteria and the compound enzyme in the ensiling bacteria enzyme compound preparation are precisely proportioned to form an efficient synergistic effect, soluble sugar generated by degrading crude fibers by the compound enzyme provides a sufficient carbon source for the compound bacteria components, and proliferation of beneficial bacteria such as lactic acid bacteria is promoted; the anaerobic acid environment created by the compound bacteria can improve the stability of the enzyme, prolong the action time of the compound enzyme, and synergistically strengthen the degradation efficiency and fermentation effect of crude fibers and lignin, so that the fermentation quality and nutritional value of the space bamboo reed silage are improved, and the space bamboo reed silage reaches the high-quality feed standard.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of silage enzyme preparation and silage feed processing, and particularly relates to a silage enzyme composite preparation, a preparation method thereof and application of the silage enzyme composite preparation in silage feed processing of space bamboo. BACKGROUND

[0002] With the development of the scale of the livestock breeding industry, the problem of the shortage of high-quality forage resources is increasingly prominent, and the development of new high-yield forage raw materials and supporting silage technology has become an urgent need in the industry. Space bamboo is a perennial herb with high biomass, wide adaptability and strong stress resistance, and has potential development value as forage raw material. However, the nutritional quality of the original space bamboo has obvious shortcomings, with a dry matter content of only 21.93%, a crude protein content of 10.75%, and a neutral detergent fiber content of 65.63% and an acid detergent fiber content of 48.03%, which are significantly higher than those of conventional high-quality forage; compared with GB / T 25882-2010 "Quality Classification of Silage Corn" and T / CAAA series hay quality classification standards, the forage quality of the original space bamboo is lower than the third level standard, and the fermentation is incomplete after direct silage, with a high pH value (≥4.48), which is easy to breed spoilage microorganisms, resulting in low forage value and seriously limiting the large-scale application of space bamboo in the field of livestock breeding.

[0003] In the existing silage technology, although a single bacterial agent (such as only containing lactic acid bacteria) can reduce the silage pH value to a certain extent, it is difficult to efficiently degrade the crude fiber and lignin components in the space bamboo; a single enzyme preparation can degrade part of the fiber, but lacks the regulation of the silage microecological environment, and cannot effectively inhibit the growth of spoilage bacteria, resulting in limited improvement of the silage quality. At the same time, there is a lack of special composite preparation for space bamboo characteristics, and the matching ratio of conventional preparation and auxiliary materials lacks targeted optimization, which further aggravates the technical bottleneck of space bamboo silage feed processing. Therefore, it is of great significance to develop a special composite preparation with synergistic bacteria and enzymes and a supporting optimized application process for promoting the large-scale and silage feed processing of space bamboo. SUMMARY

[0004] The purpose of the present application is to provide a silage enzyme composite preparation, a preparation method thereof and application of the silage enzyme composite preparation in silage feed processing of space bamboo, the composite bacteria and composite enzymes in the silage enzyme composite preparation have precise matching, can synergistically enhance the degradation efficiency of crude fiber and lignin and the fermentation effect, and further improve the fermentation quality and nutritional value of silage including space bamboo, so as to reach the standard of high-quality forage.

[0005] The application provides a silage enzyme complex preparation, which comprises a complex enzyme and a complex bacteria, and the mass ratio of the complex bacteria to the complex enzyme is (1-2):1; the complex enzyme comprises glucanase, cellulase and xylanase, and the enzyme activity ratio of the glucanase, the cellulase and the xylanase is 3-8:0-2:8-10; the complex bacteria comprises Lactobacillus plantarum GSICC 31247, Bacillus subtilis GSICC 30211 and Saccharomyces cerevisiae GSICC 51909; and the effective viable cell ratio of the Lactobacillus plantarum GSICC 31247, the Bacillus subtilis GSICC 30211 and the Saccharomyces cerevisiae GSICC 51909 is 8-10:3-8:0-2.

[0006] Preferably, the enzyme activity of the glucanase is greater than or equal to 50,000 U, the enzyme activity of the cellulase is greater than or equal to 10,000 U, and the enzyme activity of the xylanase is greater than or equal to 100,000 U.

[0007] Preferably, the effective viable cell number of the Lactobacillus plantarum GSICC 31247 is greater than or equal to 2 billion cfu / g, the effective viable cell number of the Bacillus subtilis GSICC 30211 is greater than or equal to 1 billion cfu / g, and the effective viable cell number of the Saccharomyces cerevisiae is greater than or equal to 200 million cfu / g.

[0008] The application further provides a preparation method of the silage enzyme complex preparation. The Lactobacillus plantarum GSICC 31247, the Bacillus subtilis GSICC 30211 and the Saccharomyces cerevisiae GSICC 51909 are inoculated into fermentation mediums respectively for expansion culture to obtain Lactobacillus plantarum bacterial liquid, Bacillus subtilis bacterial liquid and Saccharomyces cerevisiae bacterial liquid; The Lactobacillus plantarum bacterial liquid, the Bacillus subtilis bacterial liquid and the Saccharomyces cerevisiae bacterial liquid are subjected to solid-liquid separation respectively, and the precipitates are taken to obtain Lactobacillus plantarum bacterial bodies, Bacillus subtilis bacterial bodies and Saccharomyces cerevisiae bacterial bodies respectively; The Lactobacillus plantarum bacterial bodies, the Bacillus subtilis bacterial bodies and the Saccharomyces cerevisiae bacterial bodies are mixed according to the effective viable cell number ratio of 8-10:3-8:0-2 to obtain the complex bacteria; The glucanase, the cellulase and the xylanase are mixed according to the enzyme activity ratio of 3-8:0-2:8-10 to obtain the complex enzyme; The complex bacteria and the complex enzyme are mixed according to the mass ratio of (1-2):1, and then stirring is carried out to obtain the silage enzyme complex preparation.

[0009] The application further provides application of the silage enzyme complex preparation in the above technical solution or the silage enzyme complex preparation prepared by the preparation method in the above technical solution in silage feed preparation.

[0010] Preferably, the silage feed comprises a space bamboo reed silage feed.

[0011] The application further provides a preparation method of the space bamboo green silage, comprising the following steps: The space bamboo is subjected to rubbing treatment to obtain space bamboo fragments; The space bamboo fragments, the silage enzyme composite preparation and the corn powder are mixed and stirred to obtain mixed raw materials; The mixed raw materials are sealed and subjected to silage fermentation to obtain the space bamboo green silage; The silage enzyme composite preparation is the silage enzyme composite preparation in the above technical solution or the silage enzyme composite preparation prepared by the preparation method in the above technical solution.

[0012] Preferably, the silage enzyme composite preparation is used in an amount of 1-3 ‰ of the mass of the space bamboo fragments; and the silage enzyme composite preparation is used in an amount of 5-10 % of the mass of the space bamboo fragments.

[0013] Preferably, the silage fermentation is carried out at a temperature of 25-30 ℃, at a relative humidity of 50-60 %, in the dark, for 30-60 days.

[0014] The application further provides a space bamboo green silage, which is prepared by the preparation method in the above technical solution; the pH value of the space bamboo green silage is 3.80-3.86, the dry matter content is ≥ 35.87 %, the dry matter recovery rate is ≥ 163.57 %, the crude fat content is ≥ 3.05 %, the nitrogen-free extract content is ≥ 59.86 %, the total digestible nutrient content is ≥ 66.67 %, the neutral detergent fiber content is ≤ 31.41 %, the acid detergent fiber content is ≤ 23.85 %, and the crude fiber content is ≤ 20.52 %, which meets the third level standard of GB / T 25882-2010 "Quality Classification of Silage Corn".

[0015] Beneficial effects: The application provides a silage enzyme composite preparation, in which the composite bacteria and the composite enzyme form a high-efficiency synergistic effect, the soluble sugar produced by the composite enzyme degrading the crude fiber provides sufficient carbon source for the composite bacteria component, and promotes the proliferation of beneficial bacteria such as lactic acid bacteria; the anaerobic acidic environment created by the composite bacteria can improve the stability of the enzyme and prolong the action time of the composite enzyme, thereby being capable of synergistically enhancing the degradation efficiency of the crude fiber and lignin and the fermentation effect.

[0016] On this basis, the application provides a method for preparing space bamboo green silage feed based on the silage enzyme complex preparation, and finds that the nutritional quality of the space bamboo green silage feed is significantly improved, and the dry matter content of the space bamboo green silage is increased from 21.93% to 35.87%, the dry matter recovery rate reaches 163.57%, the crude fat content is increased from 1.93% to 3.05%, the nitrogen-free extract content is increased from 37.30% to 59.86%, and the total digestible nutrient content is increased from 48.26% to 66.67%; at the same time, the neutral detergent fiber content is reduced from 65.63% to 31.41%, the acid detergent fiber content is reduced from 48.03% to 23.85%, and the crude fiber content is reduced from 39.50% to 20.52%, and the nutritional indexes are comprehensively optimized.

[0017] Further, the quality of the space bamboo green silage feed prepared by using the silage enzyme complex preparation is significantly improved and the cost is controllable, and the specific performance is as follows: the indexes of the silage product completely meet the three-level standard of GB / T 25882-2010 “Quality Classification of Silage Corn”, and the silage product can be directly used to replace the conventional silage corn for livestock breeding such as cattle and sheep; the preparation adding amount is only 1.5‰ of the raw material quality, the corn powder adding ratio is 5%, the processing technology is simple, no special equipment is needed, and the production cost of the space bamboo green silage feed is significantly reduced.

[0018] Further, the silage stability of the space bamboo green silage using the silage enzyme complex preparation is strong, and the specific performance is as follows: after the silage, the microbial community is changed in a benign way, the aerobic bacteria Pseudomonadota are changed to anaerobic bacteria Bacillota on the door level, lactic acid bacteria ( Lactobacillus , Levilactobacillus ) become the dominant group on the genus level, the abundance of aerobic spoilage bacteria ( Agrobacterium ) is greatly reduced, the silage feed is not prone to spoilage, and the storage stability is significantly improved. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed to be used in the embodiments will be briefly introduced below.

[0020] Figure 1 It is a process diagram of the comparative example; Figure 2 It is a comparison result diagram of the bacterial species composition of different silage feeds measured in Example 4; Figure 3 It is a comparison result diagram of the fungal species composition of different silage feeds measured in Example 4; Figure 4For the three-factor model comprehensive analysis of corn meal, complex enzyme preparation, complex microbial agent and synergy on the quality of silage in Example 4, no microbial agent (A0), complex enzyme preparation (A1), complex microbial agent (A2), enzyme-microbial complex preparation (A3), no corn meal (B0), 5% corn meal (B1), 10% corn meal (B2) and 15% corn meal (B3) are added. DETAILED DESCRIPTION

[0021] The application provides a silage microbial enzyme complex preparation, which comprises complex enzymes and complex microorganisms, and the mass ratio of the complex microorganisms to the complex enzymes is (1-2):1; the complex enzymes comprise glucanase, cellulase and xylanase, and the enzyme activity ratio of the glucanase, the cellulase and the xylanase is 3-8:0-2:8-10; the complex microorganisms comprise Lactobacillus plantarum GSICC 31247, Bacillus subtilis GSICC 30211 and Saccharomyces cerevisiae GSICC 51909; and the effective viable cell ratio of the Lactobacillus plantarum GSICC 31247, the Bacillus subtilis GSICC 30211 and the Saccharomyces cerevisiae GSICC 51909 is 8-10:3-8:0-2.

[0022] As an implementation form, the mass ratio of the complex microorganisms to the complex enzymes is 1:1 or 2:1. As an implementation form, the enzyme activity ratio of the glucanase, the cellulase and the xylanase is 5:1:10. As an implementation form, the enzyme activity of the glucanase is greater than or equal to 500,000 U, the enzyme activity of the cellulase is greater than or equal to 10,000 U, and the xylanase is greater than or equal to 1,000,000 U.

[0023] As an implementation form, the effective viable cell ratio of the Lactobacillus plantarum GSICC 31247, the Bacillus subtilis GSICC 30211 and the Saccharomyces cerevisiae GSICC 51909 is 10:5:1. As an implementation form, the effective viable cell number of the Lactobacillus plantarum GSICC 31247 is greater than or equal to 2 billion cfu / g, the effective viable cell number of the Bacillus subtilis GSICC 30211 is greater than or equal to 1 billion cfu / g, and the effective viable cell number of the Saccharomyces cerevisiae is greater than or equal to 200 million cfu / g.

[0024] In the application, compared with other Lactobacillus plantarum, Bacillus subtilis and Saccharomyces cerevisiae, the Lactobacillus plantarum GSICC 31247, the Bacillus subtilis GSICC 30211 and the Saccharomyces cerevisiae GSICC 51909 have the advantages of dominant fermentation, rapid acid production, pH reduction, corruption inhibition, oxygen to anaerobic stage transition assistance, degradation assistance, fermentation quality optimization and palatability optimization.

[0025] The application further provides a preparation method of the silage microbial enzyme complex preparation. Lactobacillus plantarum GSICC 31247, Bacillus subtilis GSICC 30211 and Saccharomyces cerevisiae GSICC 51909 are inoculated into fermentation medium respectively for expansion culture to obtain Lactobacillus plantarum bacterial liquid, Bacillus subtilis bacterial liquid and Saccharomyces cerevisiae bacterial liquid; The Lactobacillus plantarum bacterial liquid, Bacillus subtilis bacterial liquid and Saccharomyces cerevisiae bacterial liquid are respectively solid-liquid separated, and the precipitates are taken to obtain Lactobacillus plantarum bacterial body, Bacillus subtilis bacterial body and Saccharomyces cerevisiae bacterial body respectively; The Lactobacillus plantarum bacterial body, Bacillus subtilis bacterial body and Saccharomyces cerevisiae bacterial body are mixed according to the effective viable count ratio of 10:5:1 to obtain a composite bacteria. The glucanase, cellulase and xylanase are mixed according to the enzyme activity ratio of 5:1:10 to obtain a composite enzyme. The composite bacteria and the composite enzyme are mixed according to the mass ratio of (1-2):1, and then the pH value of the mixed system is adjusted to 4.0, and then stirring is carried out to obtain the silage bacteria enzyme composite preparation.

[0026] The Lactobacillus plantarum GSICC 31247, Bacillus subtilis GSICC 30211 and Saccharomyces cerevisiae GSICC 51909 are inoculated into fermentation medium respectively for expansion culture to obtain Lactobacillus plantarum bacterial liquid, Bacillus subtilis bacterial liquid and Saccharomyces cerevisiae bacterial liquid. As an embodiment, the Lactobacillus plantarum GSICC 31247, Bacillus subtilis GSICC 30211 and Saccharomyces cerevisiae GSICC 51909 are inoculated into MRS medium, LB medium and YPD medium respectively for expansion culture to obtain Lactobacillus plantarum bacterial liquid, Bacillus subtilis bacterial liquid and Saccharomyces cerevisiae bacterial liquid. As an embodiment, the expansion culture temperature is 37℃, the time is 180r / min, and the time is 24h.

[0027] After obtaining the Lactobacillus plantarum bacterial liquid, Bacillus subtilis bacterial liquid and Saccharomyces cerevisiae bacterial liquid, the Lactobacillus plantarum bacterial liquid, Bacillus subtilis bacterial liquid and Saccharomyces cerevisiae bacterial liquid are respectively solid-liquid separated, and the precipitates are taken to obtain Lactobacillus plantarum bacterial body, Bacillus subtilis bacterial body and Saccharomyces cerevisiae bacterial body respectively. As an embodiment, the solid-liquid separation mode includes centrifugation, and the specific parameters of the centrifugation are not particularly limited, and the parameters of conventional bacterial liquid centrifugation in the art can be used.

[0028] After obtaining the Lactobacillus plantarum bacterial body, Bacillus subtilis bacterial body and Saccharomyces cerevisiae bacterial body, the Lactobacillus plantarum bacterial body, Bacillus subtilis bacterial body and Saccharomyces cerevisiae bacterial body are mixed according to the effective viable count ratio of 8-10:3-8:0-2 to obtain a composite bacteria.

[0029] The glucanase, cellulase and xylanase are mixed according to the enzyme activity ratio of 3-8:0-2:8-10 to obtain the composite enzyme.

[0030] After the composite bacteria and the composite enzyme are obtained, the composite bacteria and the composite enzyme are mixed according to the mass ratio of (1-2):1, and then stirring is performed to obtain the silage bacteria enzyme composite preparation. As an embodiment, the temperature of the stirring is 25 DEG C, the time is 30 min, and the rotating speed is 150-250 r / min. As an embodiment, after the composite bacteria and the composite enzyme are compounded, the silage bacteria enzyme composite preparation is in a liquid form, the pH value is 3.5-4.5, further 4.0, the shelf life is greater than or equal to 6 months under the condition of normal temperature and sealing, the Lactobacillus plantarum is the dominant bacteria, the color is green and uniform, the taste is sour and fragrant, and there is no moldy lump.

[0031] The application further provides application of the silage bacteria enzyme composite preparation in the silage feed preparation.

[0032] The application further provides a preparation method of the silage feed of the space bamboo, which comprises the following steps: The space bamboo is subjected to rubbing treatment to obtain space bamboo fragments. The space bamboo fragments, the silage bacteria enzyme composite preparation and the corn flour are mixed and stirred to obtain mixed raw materials. The mixed raw materials are sealed and subjected to silage fermentation to obtain the silage feed of the space bamboo. The silage bacteria enzyme composite preparation is the silage bacteria enzyme composite preparation in the above technical solution or the silage bacteria enzyme composite preparation prepared by the preparation method in the above technical solution.

[0033] The space bamboo is subjected to rubbing treatment to obtain space bamboo fragments. As an embodiment, the space bamboo in the application is two-year-old space bamboo with a height of 1.9-2.0 m, and the space bamboo has the advantages of large biomass, low lignification and fiberization, and relatively sufficient soluble carbohydrate content, which are beneficial to silage fermentation. As an embodiment, after the rubbing treatment, the obtained fragments are passed through a screen with a pore size of 1-2 cm to control the length of the space bamboo fragments.

[0034] After obtaining the space bamboo grass pieces, the space bamboo grass pieces, the silage fungus enzyme composite preparation and the corn flour are mixed and stirred to obtain mixed raw materials. As an embodiment, the silage fungus enzyme composite preparation is used in an amount of 1-3 ‰ of the mass of the space bamboo grass pieces, and further 1.5 ‰. The silage fungus enzyme composite preparation is used in an amount of 5%-10% of the mass of the space bamboo grass pieces, and further 5%. In the present application, the corn flour is selected as the auxiliary material for the space bamboo grass silage because adjusting the appropriate carbon-nitrogen ratio is conducive to starting fermentation. As an embodiment, the corn flour has a moisture content of 13%, a crude protein content of 8.5%, is crushed to pass through a 40-mesh sieve, and has the advantages of good dispersibility and the ability to simultaneously supplement carbon and nitrogen sources.

[0035] After obtaining the mixed raw materials, the mixed raw materials are sealed and subjected to silage fermentation to obtain the space bamboo grass silage feed. As an embodiment, the silage fermentation is carried out at a temperature of 25-30℃, and further 26-28℃. The relative humidity of the silage fermentation is 50%-60%, and further 55%. The silage fermentation is carried out in the dark. The silage fermentation is carried out for 30-60 days, and further 45 days. As an embodiment, the mixing step includes spraying the silage fungus enzyme composite preparation onto the space bamboo grass pieces and then mixing with the corn flour. As an embodiment, the stirring includes intermittent stirring, and the intermittent stirring includes stirring for 5 min, standing for 2 min, and then stirring for 10 min. As an embodiment, the sealing is carried out using a silage bag. The silage bag can be a polyethylene silage bag. As an embodiment, the sealing is checked every 10 days during the silage fermentation to prevent the silage bag from leaking.

[0036] The present application also provides a space bamboo grass silage feed prepared by the preparation method described above. The pH value of the space bamboo grass silage feed is 3.80-3.86, the dry matter content is ≥35.87%, the dry matter recovery rate is ≥163.57%, the crude fat content is ≥3.05%, the nitrogen-free extract content is ≥59.86%, the total digestible nutrient content is ≥66.67%, the neutral detergent fiber content is ≤31.41%, the acid detergent fiber content is ≤23.85%, and the crude fiber content is ≤20.52%, which meets the three-level standard of GB / T 25882-2010 "Silage Corn Quality Grading".

[0037] The present application realizes efficient conversion of space bamboo grass from low-value raw materials to high-adaptability forage grass through targeted enabling of special fungus enzyme composite preparations, significantly improves the nutritional value and economic value of the forage application of space bamboo grass, and provides efficient, stable and popularized special preparations and supporting technical solutions for the large-scale silage forage utilization of space bamboo grass.

[0038] In order to further illustrate the present application, the technical solutions provided by the present application are described in detail below in conjunction with the drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0039] The strains and sources used in the following examples are as follows: Lactobacillus plantarum was purchased from Gansu Provincial Microbial Culture Collection Center, with the number GSICC 31247, and the website is http: / / jzk.gsmsc.cn / web-j / article?id=503; Bacillus subtilis was purchased from Gansu Provincial Microbial Culture Collection Center, with the number GSICC 30211, and the website is http: / / jzk.gsmsc.cn / web-j / article?id=287; Saccharomyces cerevisiae was purchased from Gansu Provincial Microbial Culture Collection Center, with the number GSICC 51909, and the website is http: / / jzk.gsmsc.cn / web-j / article?id=22.

[0040] The sources of enzymes used in the following examples are as follows: Glucomylase, cellulase and xylanase were all provided by the cooperative unit Blue Bio.

[0041] The detection indexes of the nutritional quality of the silage in the following examples include pH, moisture, crude protein, crude fiber, crude fat, starch, crude ash, neutral detergent fiber, acid detergent fiber, acid detergent lignin and nitrogen-free extract.

[0042] High-throughput sequencing was used for microbial diversity analysis and bacterial and fungal rRNA gene detection of the silage in the following examples, and Origin Pro 7.0 was used to make bacterial and fungal distribution graphs at the door and genus levels.

[0043] Example 1 Preparation of silage enzyme complex preparation Preparation of complex bacteria component: Lactobacillus plantarum, Bacillus subtilis and Saccharomyces cerevisiae were inoculated into MRS medium, LB medium and YPD medium respectively, and cultured at 37℃, 180r / min for 24h; the bacterial bodies were collected by centrifugation, and the concentration was adjusted with sterile physiological saline, so that the viable bacterial count of Lactobacillus plantarum was 25.18 billion cfu / g, the viable bacterial count of Bacillus subtilis was 17.50 billion cfu / g, and the viable bacterial count of Saccharomyces cerevisiae was 2.16 billion cfu / g; the three strains were compounded according to the effective viable bacterial count ratio of 10:5:1.

[0044] Preparation of complex enzyme component: glucomylase (activity 50,000 U / g), cellulase (activity 10,000 U / g) and xylanase (activity 100,000 U / g) were mixed uniformly according to the mass ratio of 1:1:1.

[0045] Compound preparation of bacteria-enzyme complex: the complex bacteria component and the complex enzyme component were mixed uniformly at a mass ratio of 2:1, to obtain the silage bacteria-enzyme complex preparation.

[0046] Example 2 Application of the silage bacteria-enzyme complex preparation in the silage of space bamboo, the implementation process is shown in Figure 1 .

[0047] Raw material selection: two-year-old space bamboo with a height of 1.9-2.0 m was selected and transported to the processing site immediately after cutting; the key indicators of the raw material were detected: dry matter content 21.93%±0.56%, crude protein content 10.75%±0.14%, neutral detergent fiber content 65.63%±1.04%, and acid detergent fiber content 48.03%±0.62%.

[0048] Pretreatment: the space bamboo was crushed using a rubbing machine, and the crushing length was controlled by a 1-2 cm aperture screen. Weeds, stones and other impurities were removed for standby use.

[0049] Preparation of auxiliary materials: corn flour with a moisture content of 13% and a crude protein content of 8.5% was selected, ground through a 40 mesh sieve, and standby.

[0050] Mixing treatment: 100 kg of pretreated space bamboo raw material was taken, 150 mL of the silage bacteria-enzyme complex preparation prepared in Example 1 (pretreated space bamboo raw material mass 1.5‰ addition amount) was sprayed, and 5 kg of corn flour was added; put into a blender, adopt intermittent stirring mode (stirring 5 min→resting 2 min→stirring 10 min), to ensure uniform mixing.

[0051] Packaging and storage: 3 kg of mixed raw material was weighed and put into a 40×60 cm polyethylene silage bag, and a packaging machine was used to compact it; the silage bag was placed in a room at 25-30℃ and relative humidity 55%, and stored in the dark, and the sealing property was checked every 10 days during the period to prevent air leakage.

[0052] Product detection: after 45 days of storage, the silage feed nutritional quality was detected by opening the bag, the silage feed pH value was 3.83, the dry matter content was 35.87%, the dry matter recovery rate was 163.57%, the crude fat content was 3.05%, the nitrogen-free extract content was 59.86%, the total digestible nutrient content was 66.67%, the neutral detergent fiber content was 31.41%, the acid detergent fiber content was 23.85%, and the crude fiber content was 20.52%. All indicators met the three-level standard of GB / T 25882-2010 "Silage Corn Quality Grading".

[0053] According to the implementation process in Figure 1 , the preparation of silage feed in Comparative Examples 1-3 and Example 3 was carried out.

[0054] Comparative Example 1 Space-grown reed silage without added microbial enzymes Compared with the steps of space reed silage in Example 2, the difference is that no silage bacteria enzyme compound preparation is added, only 5% corn flour is added, and the rest of the steps are the same.

[0055] Finished product testing: The nutritional quality of the silage was tested after 45 days of storage. The results are shown in Table 1. The silage had a pH of 4.66, a dry matter content of 25.23%, a dry matter recovery rate of 115.03%, a crude fat content of 2.51%, a nitrogen-free extract content of 45.83%, a total digestible nutrient content of 59.34%, a neutral detergent fiber content of 52.12%, an acid detergent fiber content of 33.55%, and a crude fiber content of 31.29%. As shown in Table 1, all nutritional indicators were significantly lower than those in Example 2 and did not meet the Grade III standard of GB / T 25882-2010 "Grading of Silage Corn Quality". The silage had a slight moldy and off-flavor and poor palatability.

[0056] Comparative Example 2 Space-grown reed silage with added complex enzyme components Compared with the steps of silage of space-grown reed in Example 2, the difference is that only the compound enzyme component is added, and the amount of compound enzyme added is 1‰ of the mass of space-grown reed. No compound bacteria component is added, and the rest of the steps are the same.

[0057] Finished product testing: The nutritional quality of the silage was tested after 45 days of storage. The results are shown in Table 1. The silage had a pH of 3.94, a dry matter content of 29.25%, a dry matter recovery rate of 133.37%, a crude fat content of 2.79%, a nitrogen-free extract content of 54.08%, a total digestible nutrient content of 59.19%, a neutral detergent fiber content of 44.97%, an acid detergent fiber content of 33.49%, and a crude fiber content of 24.78%. Compared with Comparative Example 1, the dry matter recovery rate increased by 15.95%, the neutral detergent fiber content decreased by 13.72%, and the crude fiber content decreased by 20.74%, but it still did not meet the standard for grade III corn silage.

[0058] Comparative Example 3 Space-grown reed silage with only added compound microbial components Compared with the steps of space reed silage in Example 2, the difference is that only compound bacteria components are added, and the amount of compound bacteria added is 2‰ of the mass of space bamboo. No compound enzyme components are added, and the rest of the steps are the same.

[0059] Product detection: the nutritional quality of the silage was detected after 45 days of storage, and the results are shown in Table 1. The pH value of the silage was 4.44, the dry matter content was 31.48%, the dry matter recovery rate was 143.56%, the crude fat content was 2.77%, the nitrogen-free extract content was 53.46%, the total digestible nutrient content was 62.99%, the neutral detergent fiber content was 42.77%, the acid detergent fiber content was 28.81%, and the crude fiber content was 25.27%. Compared with Comparative Example 1, the dry matter recovery rate increased by 24.80%, the pH value decreased by 0.22, the neutral detergent fiber content decreased by 17.94%, and the crude fiber content decreased by 19.24%, but it did not completely meet the third level silage corn standard.

[0060] Example 3 Space bamboo silage with different proportions of bacteria and enzymes Compared with the steps of space bamboo silage in Example 2, the difference is that the complex bacteria component and the complex enzyme component in the silage bacteria and enzyme complex preparation are compounded at a mass ratio of 1:1, and the total addition amount is still 1.5 ‰ of the mass of the raw material, and the remaining steps are the same.

[0061] Product detection: the nutritional quality of the silage was detected after 45 days of storage, and the results are shown in Table 1. The pH value of the silage was 4.44, the dry matter content was 31.48%, the dry matter recovery rate was 143.56%, the crude fat content was 2.77%, the nitrogen-free extract content was 53.46%, the total digestible nutrient content was 62.99%, the neutral detergent fiber content was 42.77%, the acid detergent fiber content was 28.81%, and the crude fiber content was 25.27%. Compared with Comparative Example 1, the dry matter recovery rate increased by 24.80%, the pH value decreased by 0.22, the neutral detergent fiber content decreased by 17.94%, and the crude fiber content decreased by 19.24%, but it did not completely meet the third level silage corn standard.

[0062] Table 1 Effect of different bacteria and enzyme and complex treatment on the nutritional quality of silage

[0063] Example 4 Effect of bacteria and enzyme complex preparation on space bamboo silage quality (1) Optimization of space bamboo silage probiotic bacteria Lactic acid bacteria (Lactobacillus, Lactococcus, Pediococcus, Leuconostoc, Streptococcus, etc.) Lactic Acid BacteriaLactobacillus (LAB) is a general term for a class of Gram-positive bacteria that can ferment carbohydrates to produce large amounts of lactic acid. Their function is directly related to silage quality, and they are the core beneficial bacteria in silage (see Table 2). The family Lactobacillaceae is an important group of lactic acid bacteria, including the genus *Lactobacillus plantarum* (…). Lactiplantibacillus ), Weissella spp. Weissella ), Liquid Lactobacillus ( Liquorilactobacillus Lactobacillus spp. Levilactobacillus Lactococcus spp. Lactococcus Leuconostoc ( ) Leuconostoc Other lactic acid bacteria include Streptococcus genus of the Streptococcus family (Streptococcus). Streptococcus Enterococci (family Enterococci) Enterococcus ).

[0064] Table 2 Potential beneficial bacteria and their functions in silage

[0065] Bacterial species composition and diversity were analyzed on the initial space-bred Reed shoots, silage prepared from Comparative Examples 1-3, and Example 3. Six biological replicates were performed on each comparative example or example. The bacterial species composition results are as follows: Figure 2 As shown, in Figure 2 In the above, comparative examples 1, 2, 3 and 4 represent comparative examples 1, 2, 3 and 3, respectively.

[0066] like Figure 2 As shown in the bacterial species composition, the initial sample contained *Lactobacillus plantarum* and *Lactobacillus liquidus*. Liquorilactobacillus The relative abundance of the two types of lactobacilli was almost 0; however, in Comparative Examples 1-3 and Example 3, the abundance of both types of lactobacilli was significantly increased (accounting for more than 60%), indicating that silage can enrich beneficial lactobacilli; at the same time, the proportion of non-beneficial groups (such as norank_o_Chloroplast and norank_f_Mitochondria) that were highly abundant in the initial samples decreased significantly in the examples, indicating the optimization effect of silage on beneficial bacterial communities.

[0067] Compared with Comparative Examples 1, 2 and 3, the treatment with the bacterial enzyme compound preparation in Example 3 showed a more prominent regulatory effect on the high abundance of beneficial bacteria in the silage of Reed sphagnum moss from space. (1) Higher abundance of core beneficial bacteria: In Example 3, the relative abundance of Lactobacillus plantarum was the highest among all examples (accounting for over 70%). This bacterium is one of the highest quality beneficial bacteria in silage, with strong acid production capacity and good stress resistance, and can more efficiently dominate silage fermentation. This result also corroborates that the addition of Lactobacillus plantarum to the compound bacterial enzyme preparation colonizes and plays a key regulatory role in silage. (2) More concentrated functional flora: In Example 3, in addition to Lactobacillus plantarum, Coccidia spp. ( Kosakonia ), Weissella spp. Weissella ), Liquid Lactobacillus, Growth-promoting Lactobacillus ( Levilactobacillus Lactococcus spp. Lactococcus Leuconostoc ( ) Leuconostoc The presence of *Lactobacillus plantarum* indicates that it can synergistically enhance lactic acid accumulation efficiency and further strengthen the preservative ability of silage. (3) More thorough inhibition of miscellaneous bacteria: In Example 3, potentially harmful bacteria such as *Escherichia coli*-*Shigella* spp. were also present, indicating that it can synergistically enhance lactic acid accumulation efficiency and further strengthen the preservative ability of silage. Escherichia-Shigella ), Serratia ( Serratia ), Pseudomonas spp. Pseudomonas The abundance of bacteria such as ) was almost 0, while there were still a small amount of residues in other examples, indicating that the bacterial enzyme compound preparation more thoroughly inhibited harmful bacteria by enhancing the advantages of beneficial bacteria (see Table 3), improving the "beneficial purity" of silage microbiota, and reducing the risk of toxins.

[0068] Table 3 Common Potential Harmful Bacteria and Disease Risks in Silage

[0069] (II) Improving the fermentation quality of space-grown reed silage Yeasts can produce organic acids and alcohols to help lower the pH of silage, consume oxygen to improve aerobic stability, decompose complex carbohydrates to optimize feed digestibility, and inhibit the growth of harmful molds, making them key beneficial fungi in silage. These functional yeasts mainly include those in the genus *Pichia pastoris* (…). Pichia ), Yeast ( Saccharomyces ), Wickham yeast ( Vishniacozyma ), *Saccharomyces* genus ( Sporobolomyces ), Maye yeast genus ( Meyerozyma (See Table 2).

[0070] Fungal species composition and diversity were analyzed on the initial space-bred Reed shoots, silage prepared from Comparative Examples 1-3, and Example 3. Six biological replicates were performed on each comparative example or example. The fungal species composition results are as follows: Figure 3 As shown, in Figure 3 and the followingFigure 4 In the above, comparative examples 1, 2, 3 and 4 represent comparative examples 1, 2, 3 and 3, respectively.

[0071] like Figure 3 As shown in the fungal species composition, the initial sample contained beneficial yeast groups ( Saccharomyces , Vishniacozyma The relative abundance of fungi such as Fusarium is extremely low (close to 0), and the flora is dominated by harmful, non-functional fungi, including Fusarium species. Fusarium Aspergillus ( ) Aspergillus ), Penicillium ( Penicillium Alternaria ( ) Alternaria These fungi, such as , easily cause silage mold and toxin production, damaging feed quality. In all examples, the relative abundance of beneficial yeast groups was significantly increased (total proportion reaching 15%~30%), becoming the dominant functional group in the fungal community; meanwhile, the high abundance of harmful molds ( ) in the initial samples... Fusarium , Aspergillus The proportion of beneficial fungal flora decreased significantly (below 5% in most embodiments). This indicates the optimizing effect of silage on the beneficial fungal flora.

[0072] Compared with comparative examples 1-3, the treatment with the bacterial-enzyme compound preparation in Example 3 showed a more prominent regulatory effect on the high abundance of beneficial fungi in space-grown Reed silage. (1) The enrichment efficiency of the core beneficial yeast was higher: In Example 3 (bacterial-enzyme compound preparation group), the yeast genus ( Saccharomyces The relative abundance of *Saccharomyces cerevisiae* (such as *Saccharomyces cerevisiae*) was the highest in all examples (accounting for 12%–18%), far exceeding that of comparative examples 1–3 (mostly 5%–10%). This genus is the most potent beneficial yeast in silage, exhibiting high efficiency in acid and alcohol production and a significant effect on improving aerobic stability. Meanwhile, *Wickhamia spp.* (…) Vishniacozyma ), Maye yeast genus ( Meyerozyma The abundance of ) was also higher than in other examples. The synergistic effect of the three types of yeast further enhanced the beneficial functions, thereby improving the flavor and palatability of silage. (2) Harmful molds were more thoroughly inhibited: In Example 3, Fusarium spp. ( Fusarium Aspergillus ( ) Aspergillus ), Penicillium ( PenicilliumThe relative abundance of harmful molds such as Aspergillus was almost 0, while a small amount of residues (2%-8%) remained in Comparative Examples 1-3. Such harmful molds are the main producers of silage toxins (such as aflatoxin and vomitoxin), and the strong inhibition of Example 3 indicates that the safety risk of feed can be greatly reduced.

[0073] In summary, the enzyme complex preparation in Example 3 not only can more efficiently enrich core beneficial bacteria such as Lactobacillus plantarum and Lactobacillus liquidus (to produce acid, inhibit bacteria, and improve digestibility), and core beneficial fungi such as Saccharomyces and Williopsis (to strengthen acid production, inhibit bacteria, and improve aerobic stability and palatability), but also can effectively inhibit harmful bacteria such as Escherichia-Shigella, Serratia, Fusarium, and Aspergillus, while improving the stability and functional synergy of the microbial community structure, and is a better solution for regulating the nutrition and quality of space bamboo silage.

[0074] Example 5 Compared with the steps of space bamboo silage in Example 2, the difference is that the addition amount of corn powder is 10%, and the other steps are the same.

[0075] Product detection: After 45 days of storage, the silage feed was detected for nutritional quality, and the results are shown in Table 4.

[0076] Comparative Example 4 Compared with the steps of space bamboo silage in Example 2, the difference is that the addition amount of corn powder is 15%, and the other steps are the same.

[0077] Product detection: After 45 days of storage, the silage feed was detected for nutritional quality, and the results are shown in Table 4.

[0078] Comparative Example 5 Compared with the steps of space bamboo silage in Example 2, the difference is that the addition amount of corn powder is 0%, and the other steps are the same.

[0079] Product detection: After 45 days of storage, the silage feed was detected for nutritional quality, and the results are shown in Table 4.

[0080] Table 4 Nutritional quality detection results of silage feed prepared with different addition amounts of corn powder

[0081] From Table 4, it can be concluded that corn powder can improve the dry matter, crude protein and other nutrients of space bamboo green silage, reduce the pH value, and assist in improving fermentation and nutrient retention, and the effect of 5% addition is better. The additive effect of bacteria and enzymes is more significant: the compound bacterial agent and the enzyme-bacteria compound preparation can greatly reduce the fiber content, improve the total digestible nutrients, and rapidly reduce the pH value to build a stable fermentation environment, and the enzyme-bacteria compound preparation has the best effect. The synergy of the two (such as enzyme-bacteria compound preparation + 5% corn powder) can make the quality of the silage reach the third-grade silage corn standard. Figure 4 ).

[0082] Although the above embodiments make a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and people can also obtain other embodiments according to the present embodiments without creativity, which all belong to the protection scope of the present application.

Claims

1. A silage bacteria enzyme compound preparation, characterized in that, The mixture includes a complex enzyme and a complex microorganism, wherein the mass ratio of the complex microorganism to the complex enzyme is (1~2):1; the complex enzyme includes glucanase, cellulase, and xylanase, wherein the enzyme activity ratio of the glucanase, cellulase, and xylanase is 3~8:0~2:8~10; the complex microorganism includes *Lactobacillus plantarum* (…). Lactobacillus plantarum GSICC 31247, Bacillus subtilis ( Bacillus subtilis GSICC 30211 and brewer's yeast ( Saccharomyces cerevisiae The effective viable count ratio of *Lactobacillus plantarum* GSICC 31247, *Bacillus subtilis* GSICC 30211, and *Saccharomyces cerevisiae* GSICC 51909 is 8~10:3~8:0~2.

2. The silage bacteria enzyme compound preparation according to claim 1, characterized in that, The activity of the dextranase is ≥50,000 U, the activity of the cellulase is ≥10,000 U, and the activity of the xylanase is ≥100,000 U.

3. The silage bacteria enzyme compound preparation according to claim 1, characterized in that, The effective viable count of *Lactobacillus plantarum* GSICC31247 is ≥2 billion CFU / g, the effective viable count of *Bacillus subtilis* GSICC 30211 is ≥1 billion CFU / g, and the effective viable count of *Saccharomyces cerevisiae* GSICC 51909 is ≥200 million CFU / g.

4. The method for preparing the silage bacteria enzyme compound preparation according to any one of claims 1 to 3, characterized in that, Includes the following steps: Lactobacillus plantarum GSICC 31247, Bacillus subtilis GSICC 30211 and Saccharomyces cerevisiae GSICC 51909 were inoculated into fermentation medium for large-scale culture to obtain Lactobacillus plantarum inoculum, Bacillus subtilis inoculum and Saccharomyces cerevisiae inoculum. The liquid cultures of Lactobacillus plantarum, Bacillus subtilis, and Saccharomyces cerevisiae were separated into solid and liquid phases, and the precipitates were collected to obtain Lactobacillus plantarum cells, Bacillus subtilis cells, and Saccharomyces cerevisiae cells, respectively. The Lactobacillus plantarum cells, Bacillus subtilis cells, and Saccharomyces cerevisiae cells were mixed at an effective live cell ratio of 8~10:3~8:0~2 to obtain a compound bacteria; A complex enzyme was obtained by mixing glucanase, cellulase and xylanase at an enzyme activity ratio of 3~8:0~2:8~10. The compound bacteria and compound enzyme are mixed and stirred at a mass ratio of (1~2):1 to obtain the silage bacteria enzyme compound preparation.

5. The application of the silage bacteria enzyme compound preparation according to any one of claims 1 to 3 or the silage bacteria enzyme compound preparation prepared by the preparation method according to claim 4 in the preparation of silage feed.

6. The application according to claim 5, characterized in that, The silage includes space reed silage.

7. A method for preparing space-grown reed silage, characterized in that, Includes the following steps: The space reed is shredded to obtain space reed fragments; The space-grown reed flakes, silage bacteria enzyme compound preparation and corn flour are mixed and stirred to obtain mixed raw materials; The mixed raw materials are sealed and then fermented to obtain the space reed silage. The silage enzyme compound preparation is the silage enzyme compound preparation according to any one of claims 1 to 3 or the silage enzyme compound preparation prepared by the preparation method according to claim 4.

8. The preparation method according to claim 7, characterized in that, The dosage of the silage bacteria enzyme compound preparation is 1-3‰ of the mass of the space-grown reed shreds; the dosage of the silage bacteria enzyme compound preparation is 5%-10% of the mass of the space-grown reed shreds.

9. The preparation method according to claim 7, characterized in that, The silage fermentation temperature is 25~30℃, the relative humidity is 50%~60%, the silage fermentation is light-proof fermentation, and the silage fermentation time is 30~60 days.

10. A space-grade reed silage, characterized in that, The silage is prepared using the preparation method described in any one of claims 7 to 9; the space-grown reed silage has a pH value of 3.80 to 3.86, a dry matter content of ≥35.87%, a dry matter recovery rate of ≥163.57%, a crude fat content of ≥3.05%, a nitrogen-free extract content of ≥59.86%, a total digestible nutrient content of ≥66.67%, a neutral detergent fiber content of ≤31.41%, an acid detergent fiber content of ≤23.85%, and a crude fiber content of ≤20.52%, which meets the third-grade standard of GB / T25882-2010 "Grading of Silage Corn Quality".

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