Compound bacterium enzyme preparation as well as preparation method and application thereof

Abstract

The invention discloses a compound bacterium enzyme preparation and a preparation method and application thereof, the bacterium enzyme preparation comprises a compound enzyme agent and a compound bacterium agent, the compound bacterium agent is bacillus subtilis and lactobacillus plantarum, and the compound enzyme preparation is xylanase, beta-mannase and cellulase. The compound bacterium enzyme preparation is proved to be capable of improving the fermentation quality of pennisetum sinese and the growth performance of livestock and poultry, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of bacterial enzyme preparations, and specifically relates to a composite bacterial enzyme preparation and a preparation method and application thereof. Background Art

[0002] Giant Juncao is rich in sugar, crude fiber and crude protein, and is easy to cultivate and has high yield. It is a high-yield, high-quality forage resource with market potential. However, because it contains rich crude fiber and lignin, it will affect its palatability and digestibility, hinder the digestion, absorption and metabolism of nutrients, and directly limit the use of giant Juncao to feed monogastric animals. The use of microbial fermentation technology can decompose difficult-to-decompose macromolecules into usable small molecules, improve nutritional value, and produce substances that are beneficial to animal growth. The production of organic acids gives giant Juncao a sour aroma, increases palatability, and improves feeding performance.

[0003] Currently, giant fungus grass fermented using microbial fermentation technology is available on the market. However, due to the low stability of the product, the giant fungus grass is not fermented sufficiently and is unstable, and cannot be fully digested and absorbed by animals. Summary of the invention

[0004] In view of the above problems, the present invention provides a composite bacterial enzyme preparation for improving the fermentation quality of giant grass. The bacteria and enzymes in the composite bacterial enzyme preparation can be fermented synergistically, and the giant grass fermented with the composite bacterial enzyme preparation can greatly improve the fermentation quality of the giant grass, decompose a large amount of fiber substances therein into usable reducing sugars, effectively improve the feed nutrition and its digestion and absorption rate, and help regulate the balance of gastrointestinal flora and enhance the body's immune function.

[0005] To achieve the above object, the present invention adopts the following technical solution: In a first aspect, the present invention provides a composite bacterial enzyme preparation for improving the fermentation quality of giant fungus grass, wherein the composite fermentation bacterial enzyme preparation is composed of a composite bacterial agent and a composite enzyme preparation.

[0006] According to the present invention, the composite bacterial agent is Bacillus subtilis and Lactobacillus plantarum.

[0007] According to the present invention, the complex enzyme preparation is xylanase, β-mannanase and cellulase.

[0008] According to the present invention, the mass ratio of the composite bacterial agent to the composite enzyme preparation is 1-3:2-5.

[0009] According to the present invention, the number of viable Bacillus subtilis in the composite fermentation bacteria enzyme preparation is ≥ 1.0×10 8 CFU / g.

[0010] According to the present invention, the viable count of Lactobacillus plantarum is ≥ 1.0×10 8CFU / g.

[0011] According to the present invention, the xylanase activity in the composite enzyme preparation is ≥20000 U / g.

[0012] According to the present invention, the β-mannanase activity is ≥10000U / g According to the present invention, the cellulase activity is ≥20000 U / g.

[0013] Wherein, the Bacillus subtilis, named Bacillus subtilis BFC1601, is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration (CGMCC), with a deposit number of CGMCC No.13131 and a deposit date of October 21, 2016; According to the present invention, the plant lactobacillus is named as plant lactobacillus BFC1611, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) with a deposit number of CGMCC No.13132 and a deposit date of October 21, 2016.

[0014] Bacillus subtilis and Lactobacillus plantarum are fermented and cultured respectively by a conventional method, and the fermentation is terminated at the late logarithmic stage to obtain bacterial liquids of Bacillus subtilis and Lactobacillus plantarum; the fermented liquids are centrifuged and precipitated respectively, wet bacterial bodies are collected, and the bacterial bodies are resuspended with physiological saline or a phosphate buffer with a pH of 6.8, and Bacillus subtilis and Lactobacillus plantarum are mixed according to a colony forming unit ratio of 2-5:1-3 to obtain a composite bacterial mixture.

[0015] According to the present invention, the composite bacterial mixture and the carrier are mixed in a ratio of 1-2:2-5 and dried at low temperature to obtain the composite fermentation bacterial agent.

[0016] Preferably, the carrier is a conventional carrier in the feed field, for example, rice husk powder or corn cob powder.

[0017] Preferably, the viable count of Bacillus subtilis is ≥ 1.0×10 8 CFU / g, for example, can be 2.0×10 8 cfu / g, 3.0×10 8 cfu / g, 4.0×10 8 cfu / g, 5.0×´10 8 cfu / g, 6.0×10 8 cfu / g or 7.0×10 8 cfu / g. Due to space limitations, we will not go into details one by one.

[0018] Preferably, the viable count of Lactobacillus plantarum is ≥ 1.0×10 8CFU / g, for example, can be 2.0×10 8 cfu / g, 3.0×10 8 cfu / g, 4.0×10 8 cfu / g, 5.0×10 8 cfu / g, 6.0×10 8 cfu / g or 7.0×10 8 cfu / g. Due to space limitations, we will not go into details one by one.

[0019] According to the present invention, the composite enzyme preparation is mixed with xylanase, β-mannanase and cellulase in a mass ratio of 2-3:1-2:2-3.

[0020] Preferably, the xylanase activity is ≥20000 U / g.

[0021] Preferably, the β-mannanase activity is ≥10000 U / g.

[0022] Preferably, the cellulase activity is ≥20000 U / g.

[0023] In a second aspect, the present invention provides use of the composite bacterial enzyme preparation described in the first aspect in preparing biological feed or feed supplementary materials.

[0024] In a third aspect, the present invention provides a method for fermenting giant fungus grass using the composite fermentation agent and the composite enzyme preparation as described in the first aspect, comprising the following steps: (1) After the fresh giant grass growing to about 2.5 m is harvested in batches, it is crushed with a crusher and passed through a 10-50 mesh sieve; (2) Preparation of a composite bacterial agent: Bacillus subtilis and Lactobacillus plantarum are mixed to obtain a composite fermentation bacterial agent, and the composite fermentation bacterial agent, brown sugar and water are mixed. The weight ratio of the mixture is: composite fermentation bacterial agent: brown sugar: water = 1:2-5:50-200. The mixture is dissolved, stirred evenly, and sealed. The mixture is activated in a bacterial activation tank at 35-37°C for 12-24 hours and is used as a bacterial liquid for standby use.

[0025] (3) Preparing a composite enzyme preparation: mixing xylanase, β-mannanase and cellulase to obtain a composite enzyme preparation; (4) Add the complex enzyme preparation of step (3) to the activated spare bacterial liquid, and mix evenly with the giant fungus grass of step (1) in a weight ratio of bacterial liquid: enzyme preparation: giant fungus grass = 50-200: 2-5: 1000, stir for 2-5 minutes, and ferment at room temperature for 3-7 days to obtain giant fungus grass fermented feed.

[0026] According to some specific embodiments of the present invention, in step (2), the mass ratio of Bacillus subtilis to Lactobacillus plantarum is 2-5:1-3, for example, it can be 2:3, 1:1, 2:1, 5:1, 5:2 or 5:3, as well as point values ​​between the above values, which are not described one by one due to space limitations.

[0027] According to some specific embodiments of the present invention, in the step (2), in the composite fermentation bacterial agent mixture, the composite bacterial mixture and the carrier are mixed in proportion and dried at low temperature to obtain the composite fermentation bacterial agent.

[0028] According to some preferred embodiments of the present invention, in step (2), the bacterial mixture is compounded with a carrier.

[0029] According to some specific embodiments of the present invention, the mass ratio of the bacterial mixture to the carrier is 1-2:2-5, for example, it can be 1:1, 1:2, 1:3, 1:4, 1:5, 2:3, 2:4 or 2:5, as well as point values ​​between the above values, which will not be described one by one due to space limitations.

[0030] Preferably, the mass ratio of the bacterial mixture to the carrier is 1:3.

[0031] According to some specific embodiments of the present invention, the activation time in step (2) is 12-24 h, for example, it can be 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h or 24 h.

[0032] According to some specific embodiments of the present invention, in the step (3), the mass ratio of xylanase, β-mannanase and cellulase is 2-4:1-3:2-5, for example, it can be 2:1:2, 2:1:3, 2:1:4, 2:1:5, 1:1:1, 2:2:3, 2:2:4, 2:2:5, 2:3:2, 2:3:3, 2:3:4, 2:3:5, 3:1:2, 3:1:3, 3:1:4, 3:1:5, 4:1:2, 4:2:3, 4:3:3, 4:3:4 or 4:3:5, as well as the point values ​​between the above values, which are not described one by one due to space limitations.

[0033] Preferably, the mass ratio of xylanase, β-mannanase and cellulase is 2:1:2.

[0034] According to some specific embodiments of the present invention, the stirring time in step (4) is 2-5 min, for example, 2 min, 3 min, 4 min or 5 min.

[0035] According to some specific embodiments of the present invention, the fermentation time in step (4) is 3-7 days, for example, 3 days, 4 days, 5 days, 6 days or 7 days.

[0036] In a fourth aspect, the present invention provides a giant fungus grass fermented feed prepared by the composite bacterial agent described in the first aspect of the present invention or the preparation method described in the third aspect.

[0037] The present invention has the following beneficial effects: The present invention adopts a composite fermentation agent, which contains composite bacterial agents Bacillus subtilis and Lactobacillus plantarum, and composite enzyme preparations xylanase, β-mannanase and cellulase. The bacteria and enzymes in the composite fermentation agent can be fermented synergistically. The giant fungus grass obtained by directional fermentation with the fermentation agent can not only effectively degrade macromolecular proteins in raw materials, increase protein content, and improve palatability, but also improve the growth performance and digestion performance of animals.

[0038] The present invention utilizes a composite fermentation agent to directionally ferment giant fungus grass. The obtained fungus grass fermented feed has obvious sour aroma and wine aroma, soft and fluffy texture, rich nutrition, good palatability, conducive to digestion and absorption, mellow sour and sweet taste, and unique flavor. Moreover, the giant fungus grass fermented feed gives the giant fungus grass feed a unique flavor and effectively improves the feed nutrition, and has broad market prospects. Implementation

[0039] To further explain the technical means and effects of the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without any creative work are within the scope of protection of the present invention.

[0040] The strains used in the following examples are: Bacillus subtilis, named Bacillus subtilis BFC1601, deposited in the General Microbiological Center of China Microorganism Culture Collection Administration (CGMCC), with a deposit number of CGMCC No.13131, and a deposit date of October 21, 2016; Lactobacillus plantarum, named Lactobacillus plantarum BFC1611, was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) with a deposit number of CGMCC No.13132 and a deposit date of October 21, 2016.

[0041] Example 1 Preparation of composite fermentation bacterial enzyme preparation Bacillus subtilis and Lactobacillus plantarum are fermented and cultured separately, and the fermentation is terminated in the late logarithmic period to obtain bacterial liquids of Bacillus subtilis and Lactobacillus plantarum; the fermentation liquids are centrifuged and precipitated separately, the wet bacteria are collected, and the bacteria are resuspended with physiological saline or a phosphate buffer with a pH of 6.8, and Bacillus subtilis and Lactobacillus plantarum are mixed according to a colony forming unit ratio (Bacillus subtilis: Lactobacillus plantarum = 2:1) to obtain a composite bacteria, and the composite bacteria mixture is mixed with rice husk powder according to a mass ratio (composite bacteria: carrier = 1:3), and low-temperature drying is performed to obtain a composite fermentation bacterial agent.

[0042] The composite enzyme preparation is prepared by mixing xylanase, β-mannanase and cellulase in a mass ratio of 2:1:2. Example 2

[0043] Bacillus subtilis and Lactobacillus plantarum are fermented and cultured separately, and the fermentation is terminated in the late logarithmic period to obtain bacterial liquids of Bacillus subtilis and Lactobacillus plantarum; the fermentation liquids are centrifuged and precipitated separately, the wet bacterial bodies are collected, and the bacterial bodies are resuspended with physiological saline or a phosphate buffer with a pH of 6.8, and Bacillus subtilis and Lactobacillus plantarum are mixed according to a colony forming unit ratio (Bacillus subtilis: Lactobacillus plantarum=2:1) ​​to obtain a composite bacterial body, and the composite bacterial body mixture is mixed with rice husk powder according to a mass ratio (composite bacterial body: carrier=1:3), and low-temperature drying is performed to obtain a composite fermentation bacterial agent.

[0044] The composite enzyme preparation is prepared by mixing xylanase, β-mannanase and cellulase in a mass ratio of 3:2:3. Example 3

[0045] Bacillus subtilis and Lactobacillus plantarum are fermented and cultured separately, and the fermentation is terminated in the late logarithmic period to obtain bacterial liquids of Bacillus subtilis and Lactobacillus plantarum; the fermentation liquids are centrifuged and precipitated separately, the wet bacterial bodies are collected, and the bacterial bodies are resuspended with physiological saline or a phosphate buffer with a pH of 6.8, and Bacillus subtilis and Lactobacillus plantarum are mixed according to a colony forming unit ratio (Bacillus subtilis: Lactobacillus plantarum=4:3) to obtain a composite bacterial body, and the composite bacterial body mixture is mixed with rice husk powder according to a mass ratio (composite bacterial body: carrier=1:3), and low-temperature drying is performed to obtain a composite fermentation bacterial agent.

[0046] The composite enzyme preparation is prepared by mixing xylanase, β-mannanase and cellulase in a mass ratio of 2:1:2.

[0047] Comparative Example 1 Bacillus licheniformis and Lactobacillus plantarum are fermented and cultured separately, and the fermentation is terminated in the late logarithmic period to obtain bacterial liquids of Bacillus licheniformis and Lactobacillus plantarum; the fermentation liquid is centrifuged to precipitate, the wet bacteria are collected, the bacteria are resuspended with physiological saline or a phosphate buffer with a pH of 6.8, Bacillus licheniformis and Lactobacillus plantarum are mixed according to a colony forming unit ratio (Bacillus licheniformis: Lactobacillus plantarum=2:1) ​​to obtain a composite bacteria, the composite bacteria mixture is mixed with rice husk powder according to a mass ratio (composite bacteria: carrier=1:3), and low-temperature drying is performed to obtain a composite fermentation bacterial agent.

[0048] The composite enzyme preparation is prepared by mixing xylanase, β-mannanase and cellulase in a mass ratio of 2:1:2.

[0049] Comparative Example 2 Bacillus subtilis and Lactobacillus plantarum are fermented and cultured separately, and the fermentation is terminated in the late logarithmic period to obtain bacterial liquids of Bacillus subtilis and Lactobacillus plantarum; the fermentation liquids are centrifuged and precipitated separately, the wet bacteria are collected, and the bacteria are resuspended with physiological saline or a phosphate buffer with a pH of 6.8, and Bacillus subtilis and Lactobacillus plantarum are mixed according to a colony forming unit ratio (Bacillus subtilis: Lactobacillus plantarum = 2:1) to obtain a composite bacteria, and the composite bacteria mixture is mixed with rice husk powder according to a mass ratio (composite bacteria: carrier = 1:3), and low-temperature drying is performed to obtain a composite fermentation bacterial agent.

[0050] The xylanase and the cellulase are mixed in a mass ratio of 1:1 to prepare a composite enzyme preparation.

[0051] Comparative Example 3 Bacillus subtilis and Lactobacillus plantarum are fermented and cultured separately, and the fermentation is terminated in the late logarithmic period to obtain bacterial liquids of Bacillus subtilis and Lactobacillus plantarum; the fermentation liquid is centrifuged to precipitate, the wet bacteria are collected, and the bacteria are resuspended with physiological saline or a phosphate buffer with a pH of 6.8, and Bacillus subtilis and Lactobacillus plantarum are mixed according to a colony forming unit ratio (Bacillus subtilis: Lactobacillus plantarum=2:1) ​​to obtain a composite bacteria, and the composite bacteria mixture is mixed with rice husk powder according to a mass ratio (composite bacteria: carrier=1:3), and low-temperature drying is performed to obtain a composite fermentation bacterial agent.

[0052] Comparative Example 4 The composite enzyme preparation is prepared by mixing xylanase, β-mannanase and cellulase in a mass ratio of 2:1:2.

[0053] Experiment 1 Preparation of Giant Juncao Fermentation Material (1) After the fresh giant grass growing to about 2.5 m is harvested in batches, it is crushed with a crusher and passed through a 10-mesh sieve; (2) Take Example 1-3 and Comparative Example 1-3, and add the obtained fermentation agent formula therein according to the following steps, respectively. Mix the fermentation agent, brown sugar and water to obtain a mixture. The weight ratio of the mixture is: fermentation bacteria: brown sugar: water = 1:5:100. Dissolve the mixture, stir it evenly, seal it, and activate it in a strain activation tank at 37°C for 24 hours to use as a bacterial liquid for later use.

[0054] (3) The activated bacterial liquid of Example 1-3 and Comparative Example 1-2 is mixed with the corresponding composite fermentation enzyme preparation and the giant fungus grass of step 1 in a weight ratio of: fermentation bacterial liquid: composite enzyme preparation: giant fungus grass = 100:5:1000, stirred for 2-5 minutes, and fermented at room temperature for 5 days to obtain giant fungus grass fermented feed.

[0055] The activated bacterial liquid of comparative example 3 was mixed with the giant fungus grass of step 1 in a weight ratio of: fermented bacterial liquid: giant fungus grass = 100:1000, stirred for 2-5 minutes, and fermented at room temperature for 5 days to obtain giant fungus grass fermented feed.

[0056] (5) The composite fermentation enzyme preparation prepared in comparative example 4 was mixed with the giant fungus grass prepared in step 1 in a weight ratio of composite enzyme preparation: giant fungus grass = 5:1000, stirred for 2-5 minutes, and fermented at room temperature for 5 days to obtain giant fungus grass fermented feed.

[0057] The sensory evaluation on site was conducted by referring to the sensory evaluation standard for fermented feed to score the quality of the fermented giant fungus grass obtained above. The sensory evaluation standard is shown in Table 1.

[0058] Table 1 Sensory evaluation criteria

[0059] From the results in Table 2, it can be seen that the sensory evaluation results of the fermented feed obtained in each embodiment are all excellent. Compared with comparative examples 1-4, the sensory evaluation total scores of the fermented giant fungus grass obtained in embodiments 1-3 are higher. Among them, the total sensory evaluation scores of the fermented giant fungus grass obtained in embodiments 1-3 are all above 90, which are all excellent. The color is light yellow-green or light yellow-green, with sour aroma and light sweet aroma, the texture is slightly moist, soft, and not easy to fall apart when held in the hand.

[0060] Table 2 Sensory evaluation results of giant fungus grass fermentation

[0061] Table 3 shows the changes in nutrients before and after fermentation of giant fungus grass with different ratios. As can be seen from the table, after the composite bacterial agent prepared in Examples 1-3 was combined with the composite enzyme preparation, the crude protein increased, and the contents of crude fiber, neutral detergent fiber, and acid detergent fiber were all reduced to a certain extent compared with the unfermented ones. The crude protein increased by 19.00%, and the crude fiber decreased by more than 13.35%. At the same time, the reducing sugar content increased by more than 52.32%. In terms of crude fiber and reducing sugar, there were significant differences compared with the control group ( P <0.05). After the comparative bacterial agent was fermented on giant grass, the crude fiber was also reduced to a certain extent, which was also a good improvement compared with the unfermented giant grass group, but compared with the composite bacterial fermentation, the overall effect was lower.

[0062] Table 3 Nutrients of giant fungus grass before and after fermentation

[0063] The results of the quality test of giant fungus grass fermented with different ratios are shown in Table 4.

[0064] pH value is an important indicator for measuring the quality of fermented feed. The pH value of good silage is lower than 3.7. As shown in the table, the pH values ​​of the fermented giant grass in Examples 1-3 by the compound probiotic fermentation group are all lower than 3.7, and are significantly lower than the four comparative examples.

[0065] The lactic acid content of Examples 1-3 is higher than that of the comparative example group, wherein the higher the lactic acid content, the lower the butyric acid content, indicating that the fermentation condition of giant fungus grass is better. As the pH value of the fermented giant fungus grass decreases, the activity of butyric acid bacteria and other harmful microorganisms can be effectively inhibited, butyric acid fermentation can be reduced, thereby improving the quality of fermented feed. High acetic acid and propionic acid can improve the silage quality of silage and increase aerobic stability.

[0066] Table 4 Quality results of giant grass fermentation

[0067] Experiment 2 Application of Giant Juncao Fermented Feed in Growing Pigs Experimental design: 135 healthy, roughly uniform-aged, 30kg Duroc Changda three-way pigs were selected and randomly divided into three groups. Each group was fed with three pens, 15 pigs in each pen, and the feeding cycle was 60 days. Harmful gases in the pig house were tested on the first, 25th, and 60th days of pig feeding.

[0068] Control group: basic diet + 10% unfermented giant fungus grass Experimental group 1: basic diet + 10% fermented giant fungus grass from comparative example 1, Experimental group 2: basic diet + 10% of the giant fungus grass fermented in Example 1.

[0069] The experiment was conducted at the pig breeding demonstration base of the National Engineering Research Center for Biofeed Development.

[0070] Feeding and management: The experimental pigs were managed according to the routine management methods of the pig farm, with free drinking water and feeding according to the daily feeding method of the pig farm. The feeding time was 8 am and 5 pm every day, and the feeding amount was carefully recorded each time. The feeding amount was fed until the pigs were full and there was no feed left in the feed trough; each experimental group was fed with the corresponding feed, and no changes were allowed during the experiment.

[0071] Growth performance indicators measured: average daily weight gain (kg / d), average daily feed intake (kg / d), feed-to-meat ratio, and survival rate.

[0072] Detection of harmful gases: Close the doors and windows of the pig house at 18:30 on the evening before sampling, and enter the pig house with a portable air detector for testing at 6:30, 10:30, and 12:30 the next day.

[0073] Data processing: SAS statistical software ANOVA procedure was used for statistical analysis of the results and Duncan's method was used for multiple comparisons. The results were expressed as mean ± standard deviation, and significant differences were judged at P < 0.05.

[0074] The growth performance results are shown in Table 5, and the results of the harmful gas content detection in the pig house are shown in Table 6.

[0075] Table 5 Effects of each test group on pig growth performance

[0076] The analysis in Table 5 shows that the daily weight gain, daily feed intake and feed-to-meat ratio of the two experimental groups were significantly different from those of the control group ( P <0.05). Compared with the control group, the daily weight gain of growing pigs fed with the giant fungus grass fermented feed prepared by the technical solution of the present invention increased by 15.38%, the daily feed intake increased by 6.25%, and the feed-to-meat ratio decreased by 8.37%. The daily weight gain and feed-to-meat ratio of fattening pigs in the second test group were better than those in the control group.

[0077] Table 6 Effects of each test group on the pig house environment

[0078] As shown in Table 6, after using giant fungus grass fermented feed, the ammonia concentration in the pig house was significantly reduced. On the 25th day, the ammonia concentration in the experimental group was 52.13% lower than that in the control group. After 60 days of feeding, the ammonia concentration in the pig house of the experimental group 2 on the 60th day was significantly lower than that in the control group ( P <0.05), compared with the first day of the experiment, the difference was significant ( P<0.05). Using giant fungus grass to ferment feed can effectively improve the digestibility and utilization rate of feed for pigs and reduce the organic matter in feces. At the same time, fermented probiotics can also inhibit the growth of some ammonia-producing bacteria, reduce the decomposition of organic matter itself, thereby reducing the ammonia concentration in the pig house and improving the air quality of the pig house.

[0079] The pork quality and nutritional value of each group of pigs were tested, and the test results are shown in Table 7-8.

[0080] Table 7 Effects of each test group on pork quality

[0081] As shown in Table 7, the meat color of test group 2 is bright red, the marble pattern is obvious, and the water holding capacity is strong, which is better than the control group ( P <0.05). The experimental results show that adding fermented giant fungus grass feed to the diet can improve the quality of pork.

[0082] In the protein of livestock and poultry meat, the higher the amino acid content and ratio, the higher the nutritional value of the protein. The higher the essential amino acid content in pork, the higher the nutritional value of pork protein. As shown in Table 8, the total amount of amino acids and the total amount of essential amino acids in pork in the experimental group 2 were the highest, and the control group was the lowest. There was a significant difference between the experimental group 2 and the control group ( P <0.05), the amino acid content and total essential amino acid content of the experimental group 2 were 23.65% and 18.71% higher than those of the control group, respectively. The unsaturated fatty acid content of the experimental group 2 was 3.1% higher than that of the control group, with a significant difference ( P <0.05).

[0083] Comprehensive comparison shows that feeding the fermented giant fungus grass feed to the two experimental groups can effectively improve the amino acid and fatty acid content of pork and enhance the quality of pork.

[0084] Table 8 Effects of each test group on the amino acid and fatty acid content of pork

[0085] Experiment 3 Application of giant fungus grass fermented feed in dairy cows Experimental design and feeding management: 30 lactating dairy cows with no significant differences in parity, body weight, milk yield, milk protein rate, milk fat rate, and milk somatic cell count were selected and randomly divided into 3 groups, with 10 replicates in each group and 1 cow in each replicate.

[0086] Cows in the control group were fed a basal diet.

[0087] Experimental group 1 used the giant fungus grass fermented feed prepared in comparative example 1 to replace all the silage in the basic diet.

[0088] The experimental group 2 used the giant fungus grass fermented feed prepared in Example 1 to replace all the silage in the basic diet. The experimental diet was prepared according to the NRC (2001) nutritional standard for dairy cows (the ratio of concentrate to roughage in each group of diets was 52:48). The pre-test period was 14 days and the formal test period was 42 days.

[0089] The daily milk production of each group of dairy cows was recorded, and milk samples were collected once a week in the morning, noon and evening. The samples were mixed in a ratio of 4:3:3, and the milk fat rate, milk protein rate, milk somatic cell count and total milk solids content were determined. The feed efficiency and 4% milk fat corrected milk were calculated.

[0090] As shown in Table 9, the DM, ADF and NDF contents of the fermented giant fungus grass in Example 1 and Comparative Example 1 were not significantly different from those of silage corn ( P >0.05), but the crude protein content of Example 1 is higher than that of silage corn, and the crude fiber content is lower than that of silage corn.

[0091] Table 9 Nutritional composition of giant fungus grass fermented feed and silage corn in each experimental group

[0092] As shown in Table 10, compared with the control group, the dry matter intake of dairy cows fed with fermented giant fungus grass feed increased slightly, but the difference was not significant ( P >0.05). The milk production of dairy cows in the experimental 2 group was higher than that in the control group and the experimental 1 group, and the difference was significant ( P <0.05), the 4% milk fat corrected milk of the experimental 2 groups of dairy cows increased by 11.79% compared with the control group ( P <0.05), feed efficiency increased by 8.85% ( P <0.05). Compared with the control group, the experimental group 2 fed with the giant fungus grass fermented feed of Example 1 can improve the milk quality of dairy cows, and the milk fat rate and milk protein rate are significantly increased ( P <0.05).

[0093] Table 10 Effects of using giant fungus grass fermented feed instead of silage corn on the production performance of dairy cows in each experimental group

Claims

1. A composite bacterial enzyme preparation, It is characterized in that The bacterial enzyme preparation comprises a composite enzyme agent and a composite bacterial agent, the composite bacterial agent is bacillus subtilis and lactobacillus plantarum, and the composite enzyme preparation is xylanase, beta-mannanase and cellulase.

2. The composite bacterial enzyme preparation according to claim 1, It is characterized in that The mass ratio of the composite bacterial agent to the composite enzyme preparation is 1-3:2-5; preferably, the viable count of the Bacillus subtilis is ≥1.0×10 8 CFU / g; Preferably, the viable count of Lactobacillus plantarum is ≥ 1.0×10 8 CFU / g; Preferably, the xylanase activity is ≥20000 U / g; Preferably, the β-mannanase activity is ≥10000 U / g; Preferably, the cellulase activity is ≥20000 U / g.

3. The composite bacterial enzyme preparation according to claim 1 or 2, It is characterized in that The Bacillus subtilis is named as Bacillus subtilis BFC1601, deposited in the General Microbiological Center of China National Microorganism Culture Collection Administration (CGMCC), with a deposit number of CGMCC No.13131, and a deposit date of October 21, 2016; the Lactobacillus plantarum is named as Lactobacillus plantarum BFC1611, deposited in the General Microbiological Center of China National Microorganism Culture Collection Administration (CGMCC), with a deposit number of CGMCC No.13132, and a deposit date of October 21, 2016.

4. The composite bacterial enzyme preparation according to any one of claims 1 to 3, It is characterized in that The mass ratio of xylanase, beta-mannanase and cellulase in the composite enzyme preparation is 2-3:1-2:2-3; the mass ratio of Bacillus subtilis and Lactobacillus plantarum is 2-5:1-3.

5. Use of the composite bacterial enzyme preparation according to any one of claims 1 to 4 for preparing feed and / or feed supplementary materials.

6. A biological fermented feed, It is characterized in that The feed is obtained by fermenting the composite bacterial enzyme preparation according to any one of claims 1 to 4.

7. The biological fermentation feed according to claim 6, It is characterized in that The fermented feed is obtained by fermenting giant fungus grass with the composite bacterial enzyme preparation described in any one of claims 1 to 4.

8. The bio-fermented feed according to claim 6 or 7, It is characterized in that The fermented feed is prepared by a method comprising the following steps: (1) Crush the giant fungus grass and pass it through a 10-50 mesh sieve; (2) preparing a composite bacterial agent: mixing Bacillus subtilis and Lactobacillus plantarum in a mass ratio of 2-5:1-3 to obtain a composite fermentation bacterial agent, mixing the composite fermentation bacterial agent, brown sugar and water, and activating the mixture; (3) Preparing a composite enzyme preparation: mixing xylanase, β-mannanase, and cellulase in a mass ratio of 2-4:1-3:2-5 to obtain a composite enzyme preparation; (4) Mix the bacterial solution: enzyme preparation: giant fungus grass in a weight ratio of 50-200: 2-5: 1000, stir for 2-5 minutes, and ferment at room temperature for 3-7 days.

9. The biological fermentation feed according to claim 8, It is characterized in that In the step (2), the ratio of composite fermentation agent: brown sugar: water is 1:2-5:50-200, and the mixture is activated at 35-37°C for 12-24h; preferably, the composite fermentation agent is prepared by mixing the agent mixture and the carrier in a ratio of 1-2:2-5 and drying at low temperature; Preferably, in step (4), the ratio of bacterial liquid: enzyme preparation: giant fungus grass is 100:5:1000.

10. A method for preparing giant fungus grass biological fermentation feed, It is characterized in that The following steps are involved: (1) Crush the giant fungus grass and pass it through a 10-50 mesh sieve; (2) Preparing a composite bacterial agent: mixing Bacillus subtilis and Lactobacillus plantarum in a mass ratio of 2-5:1-3 to obtain a composite fermentation bacterial agent, and mixing the composite fermentation bacterial agent, brown sugar and water for activation. (3) preparing a composite enzyme preparation: adding xylanase, β-mannanase and cellulase in a mass ratio of 2-4:1-3:2-5 to mix to obtain a composite enzyme preparation; (4) adding the composite enzyme preparation of step (3) to the activated standby bacterial solution, and mixing evenly with the giant fungus grass of step (1), mixing in a weight ratio of bacterial solution: enzyme preparation: giant fungus grass = 50-200: 2-5: 1000, stirring for 2-5 minutes, and fermenting at room temperature for 3-7 days; Preferably, in step (2), the ratio of composite fermentation agent: brown sugar: water is 1:2-5:50-200, and the mixture is activated at 35-37° C. for 12 to 24 hours; Preferably, the composite fermentation bacterial agent is prepared by mixing a bacterial agent mixture and a carrier in a ratio of 1-2:2-5 and drying at low temperature; Preferably, in step (4), the ratio of bacterial liquid: enzyme preparation: giant fungus grass is 100:5:1000.

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