Compound bacterium enzyme preparation for ensiling as well as preparation method and application of compound bacterium enzyme preparation

Abstract

The invention discloses a compound bacterium enzyme preparation for ensiling and application of the compound bacterium enzyme preparation, and belongs to the field of bacterium enzyme compound preparations. The bacterial enzyme preparation disclosed by the invention comprises lactobacillus plantarum, lactobacillus buchneri, cellulase and xylanase, has the effects of improving the fermentation quality of silage, promoting the improvement of the organism immunity and the anti-stress capability of ruminants and the like, and has wide market value.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation, and in particular relates to a composite bacterial enzyme preparation for silage, a preparation method and application thereof. Background Art

[0002] Whole-plant corn silage is a roughage with high water content. Under closed anaerobic conditions, the soluble sugar in the whole-plant corn silage raw material is converted into organic acid through the rapid and large-scale reproduction of lactic acid bacteria. When the pH drops to a certain level, the reproduction of most microorganisms is inhibited, and the nutrients in the whole-plant corn silage are preserved and finally made.

[0003] Whole-plant corn silage is soft and juicy, sour and delicious, and rich in nutrients. It is an important part of the ruminant diet. The quality of whole-plant corn silage determines the economic benefits of farmers. There are several points to note in the management of whole-plant corn silage: 1. The best mowing time and mowing height; 2. Cutting length and corn kernel fragmentation; 3. Compacting and sealing of the silage pit. However, even if the above points are paid attention to during silage management, whole-plant corn silage may still fail. The main reason is that the types and number of lactic acid bacteria naturally attached to the plant are reduced, and the number of putrefactive bacteria and other miscellaneous bacteria increases. They cannot quickly ferment the soluble sugars in the whole-plant corn silage raw materials to produce acetic acid, quickly reduce the pH, and inhibit the growth and reproduction of other miscellaneous bacteria, affecting the quality of whole-plant corn silage, and thus affecting the breeding benefits. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides a composite bacterial enzyme preparation for silage and application thereof, wherein the composite bacterial enzyme preparation can improve the quality of whole-plant corn silage and further improve the growth and production performance of livestock and poultry.

[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 silage, comprising Lactobacillus plantarum, Lactobacillus buchneri, cellulase and xylanase.

[0006] According to the composite bacterial enzyme preparation for silage of the present invention, the mass ratio of Lactobacillus plantarum, Lactobacillus buchneri, cellulase and xylanase is 1-2:1-2:1-3:1-3.

[0007] According to some specific embodiments of the present invention, the mass ratio of the Lactobacillus plantarum, Lactobacillus buchneri, cellulase and xylanase can be, for example, 1:1:1:1, 1:1:1:2, 1:1:1:3, 1:1:2:1, 1:1:2:1, 1:1:3:1, 1:1:2:2, 1:1:2:3, 1:1:3:1, 1:1:3:2, 1:1:3:3, 1:2:3:1, 1:2:3:3, 2:2:3:1, 2:2:3:2, 2:2:3:3, 2:2:1:1, which are not described one by one due to space limitations.

[0008] According to some preferred embodiments of the present invention, the mass ratio of Lactobacillus plantarum, Lactobacillus buchneri, cellulase and xylanase is 1:1:1:1.

[0009] According to some specific embodiments of the present invention, the number of viable Lactobacillus plantarum in the composite bacterial enzyme preparation is ≥ 2×10 10 CFU / g, for example, can be 2×10 10 CFU / g, 3×10 10 CFU / g, 4×10 10 CFU / g, 5×10 10 CFU / g, 6×10 10 CFU / g, 7×10 10 CFU / g, 8×10 10 CFU / g, 9×10 10 CFU / g.

[0010] According to some specific embodiments of the present invention, the viable count of Lactobacillus buchneri in the composite bacterial enzyme preparation is ≥ 4×10 10 CFU / g, for example, can be 4×10 10 CFU / g, 5×10 10 CFU / g, 6×10 10 CFU / g, 7×10 10 CFU / g, 8×10 10 CFU / g, 9×10 10 CFU / g, 10×10 10 CFU / g.

[0011] According to some specific embodiments of the present invention, the cellulase activity in the composite bacterial enzyme preparation is ≥ 2×10 3 U / g.

[0012] According to some specific embodiments of the present invention, the xylanase activity in the composite bacterial enzyme preparation is ≥4×10 4 U / g.

[0013] According to some specific embodiments of the present invention, the Lactobacillus plantarum is Lactobacillus plantarum BFC1602, which is deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee with a deposit number of CGMCC No.13132.

[0014] According to some specific embodiments of the present invention, the Lactobacillus buchneri is a commercially purchased strain.

[0015] According to the present invention, the Lactobacillus buchneri was purchased from Weikang Probiotics (Suzhou) Co., Ltd.

[0016] In a second aspect, the present invention provides a method for preparing the composite bacterial enzyme preparation as described in the first aspect, comprising the following steps: 1) After activating Lactobacillus plantarum, the culture is expanded to obtain a bacterial liquid of Lactobacillus plantarum; 2) centrifuging the bacterial solution of Lactobacillus plantarum to obtain a precipitate, collecting the precipitate, and freeze-drying the precipitate to obtain bacterial powder of Lactobacillus plantarum; 3) Mix Lactobacillus plantarum powder and Lactobacillus buchneri powder, cellulase and xylanase in a mass ratio of 1-2:1-2:1-3:1-3 to obtain a composite bacterial enzyme preparation.

[0017] According to some specific embodiments of the present invention, the mass ratio of the Lactobacillus plantarum, Lactobacillus buchneri, cellulase and xylanase can be, for example, 1:1:1:1, 1:1:1:2, 1:1:1:3, 1:1:2:1, 1:1:2:1, 1:1:3:1, 1:1:2:2, 1:1:2:3, 1:1:3:1, 1:1:3:2, 1:1:3:3, 1:2:3:1, 1:2:3:3, 2:2:3:1, 2:2:3:2, 2:2:3:3, 2:2:1:1, which are not described one by one due to space limitations.

[0018] According to some preferred embodiments of the present invention, the mass ratio of Lactobacillus plantarum, Lactobacillus buchneri, cellulase and xylanase is 1:1:1:1.

[0019] In a third aspect, the present invention provides a method for fermenting whole-plant corn silage using the composite bacterial enzyme preparation as described in the first aspect or the composite bacterial enzyme preparation prepared by the method described in the second aspect, comprising the following steps: (1) After harvesting the corn, adjust its moisture content to 65% to 70% and crush it into small pieces of 1.5-2 cm for later use; (2) Dissolving and diluting the composite bacterial enzyme preparation according to a mass volume ratio of the composite bacterial enzyme preparation to water of 1:500-2000; (3) Load the whole corn plant into the silo. After filling 4 to 6 cm, spray the surface of the whole corn plant with a diluted 0.4% to 0.5% bacterial enzyme solution at a ratio of 0.2% to 0.8%, compact it, and then continue filling; (4) Use film to seal.

[0020] According to some preferred embodiments of the present invention, the mass volume ratio of the composite bacterial enzyme preparation and water is 1:500-1000.

[0021] According to other preferred embodiments of the present invention, the mass volume ratio of the composite bacterial enzyme preparation to water is 1:500-1:800.

[0022] According to a further preferred embodiment of the present invention, the mass volume ratio of the composite bacterial enzyme preparation to water is 1:500-1:600.

[0023] According to some more preferred embodiments of the present invention, the mass ratio of the bacterial enzyme preparation solution to the silage corn is 1:125-500.

[0024] According to a further preferred embodiment of the present invention, the mass ratio of the bacterial enzyme preparation solution to silage corn is 1:125-1:250, for example, it can be 1:125, 1:126, 1:128, 1:130, 1:132, 1:135, 1:145, 1:150, 1:155, 1:158, 1:160, 1:180, 1:185, 1:190, 1:198, 1:200, 1:210, 1:220, 1:230, 1:240, 1:245, 1:250, and point values ​​between the above values, which will not be described one by one due to space limitations.

[0025] In a fourth aspect, the present invention provides the use of the composite bacterial enzyme preparation described in the first invention or the composite bacterial enzyme preparation prepared by the method described in the second aspect in the preparation of silage.

[0026] According to the present invention, the silage raw materials can be corn, ryegrass, alfalfa, sorghum, cabbage, sweet potato vine, sunflower and the like.

[0027] According to some specific embodiments of the present invention, the silage is whole-plant corn silage.

[0028] Compared with the prior art, the present invention has the following advantages: The composite bacterial enzyme preparation for silage provided by the present invention is based on the existing technology, and the bacterial strains are scientifically screened and the formula is further optimized to obtain a novel composite bacterial enzyme preparation. The whole corn silage fermented by the preparation has a mellow aroma, a fruity aroma, and a sour aroma, and the palatability is improved, which is conducive to eating. During the fermentation process, whole-plant corn silage promotes the improvement of ruminant animals' immunity and anti-stress ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Sensory evaluation chart of test groups 5-6.

[0030] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, and the technical solutions in the embodiments of the present invention are clearly and completely described. The described embodiments are only embodiments of a part of the present invention, not all of them. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Unless otherwise specified, the reagents and materials used in the present invention can be obtained from commercial sources.

[0032] 1. pH value and acid production of plant lactic acid bacteria BFC1602 at different culture times

[0033] Use a sterilized inoculating loop to take out Lactobacillus plantarum BFC1602 from the freezing tube, inoculate it on the MRS solid slope and streak it. Place the streaked MRS solid culture medium in a 35°C incubator to culture colonies. After activation twice, pick a single colony and inoculate it into MRS liquid culture medium at 35°C, 120rpm / min overnight to obtain Lactobacillus plantarum BFC1602 bacterial solution for use.

[0034] The obtained Lactobacillus plantarum BFC1602 bacterial solution was adjusted to a bacterial concentration of 2×10 10 CFU / g was then inoculated (the inoculum size was 10% of the weight of the liquid culture medium) into a liquid culture medium (12 g / L peptone, 12 g / L beef powder, 6 g / L yeast powder, 24 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 2 g / L diammonium hydrogen citrate, 5 g / L sodium acetate, 0.58 g / L magnesium sulfate, 0.25 g / L manganese sulfate, 5 mL Tween, and distilled water to 1 L; after the liquid culture medium was prepared according to the above formula, the pH value was adjusted to 5.9), and cultured at 35 ° C, 100 rpm / min for 24 h to obtain the fermentation broth of plant lactic acid bacteria BFC1602.

[0035] The control strain Lactobacillus plantarum (purchased from Weikang Probiotics (Suzhou) Co., Ltd.) was cultured in the same manner as above to a bacterial concentration of 2×10 10 CFU / g and then fermented to obtain the control strain plant lactic acid bacteria fermentation liquid.

[0036] 2. Experimental Methods Laboratory analysis Sample extraction: in accordance with the provisions of NY / T 2129.

[0037] pH: Measured according to GB 5009.237-2016.

[0038] Acid production: measured according to GB / T12456.

[0039] 3. Experimental results

[0040] It can be seen from the experimental data in the above table that after 24h of fermentation, the pH value of the plant lactobacillus BFC1602 of the present invention decreased from 6.77 at 0h to 3.16, and the acid production was as high as 1.96g / ml, which was higher than that of the control strain (the number of live Lactobacillus plantarum was 2×10 10 CFU / g) can reduce pH faster, produce acid faster and produce more acid. 2. Determination of fermentation quality of silage corn

[0041] 1. Experimental Materials 1) Test location: National Engineering Research Center for Biofeed Development 2) Silage raw material: Whole corn with a moisture content of 65% to 70% is used as silage raw material.

[0042] 3) Addition amount: 4 g of the composite bacterial enzyme preparation for silage of the present invention is added to one ton of whole-plant corn silage raw material.

[0043] 2. Experimental Design In this experiment, whole corn was used as silage raw material. After the whole corn was cut, it was crushed into 2 cm small pieces for use. The composite microbial agent for whole corn silage was dissolved and diluted with clean water, and the mass volume ratio of the additive to water was 1:1000.

[0044] Put the whole corn plant into the fermentation tank, and after filling 4-6cm, spray the surface with the diluted composite microbial fermentation agent solution and compact it, then continue filling. Finally, seal it with a film, and take samples for testing after 30 days of fermentation.

[0045] Experimental group 1: No silage fermentation agent was added.

[0046] Experimental group 2: Add a whole-plant corn silage fermentation agent containing only lactic acid bacteria sold on the market.

[0047] Experimental group 3: a starter culture was added in which Lactobacillus plantarum, Lactobacillus buchneri, cellulase and xylanase were mixed in a mass ratio of 1:1:2:2.

[0048] Experimental group 4: a starter culture in which Lactobacillus plantarum, Lactobacillus buchneri, cellulase and xylanase were mixed in a mass ratio of 2:2:3:3 was added.

[0049] Experimental group 5: a starter culture in which Lactobacillus plantarum, Lactobacillus buchneri, cellulase and xylanase were mixed in a mass ratio of 1:1:1:1 was added.

[0050] Experimental group 6: a starter culture prepared by adding Lactobacillus acidophilus, Lactobacillus buchneri, cellulase and xylanase in a mass ratio of 1:1:1:1.

[0051] Experimental group 7: a starter culture prepared by adding Lactobacillus plantarum, Leuconostoc mesenteroides, cellulase and xylanase in a mass ratio of 1:1:1:1.

[0052] Experimental group 8: a starter culture prepared by adding Lactobacillus acidophilus, Lactobacillus bukei, Leuconostoc mesenteroides, cellulase and xylanase in a mass ratio of 1:1:1:1:1.

[0053] All experimental groups were operated the same except for the addition of whole-plant corn silage fermentation agent.

[0054] 3. Experimental Methods The cutting height of whole corn silage is about 15cm, and the cutting length is about 2cm. The whole corn silage agent is evenly mixed in proportion and compacted in the tank. The compaction density is not less than 750kg / m 3 .

[0055] Laboratory analysis Sample extraction: in accordance with the provisions of NY / T 2129.

[0056] pH: Measured according to GB 5009.237-2016.

[0057] Moisture content: Measured according to GB / T 6435-2014.

[0058] Crude protein (CP) content: determined according to GB6432-86.

[0059] Ammoniacal nitrogen (TBN) content: determined by direct distillation method.

[0060] Neutral detergent fiber (NDF) content: measured according to GB / T 20806-2006.

[0061] Acid washed fiber (ADF) content: measured using NY / T 1459-2007.

[0062] Organic acid content: determined by high performance liquid chromatography.

[0063] The sensory evaluation system of the whole corn silage of the present invention adopts the German Agricultural Association (DLG) scoring method, and performs sensory scoring according to three aspects: smell, stems and leaves, and color, with a full score of 20. The collected gas components are sent to a testing agency for component determination.

[0064] 4. Data Processing and Analysis The statistical method used Excel software to process the basic data, and SPSS software to perform variance analysis on the data.

[0065] 5. Experimental Results The silage fermentation quality of each group is shown in Table 1, the chemical composition is shown in Table 2, the sensory score is shown in Table 3, and the gas composition is shown in Table 4. Some sensory evaluation samples are shown in the attached Figure 1 .

[0066] Table 1 Fermentation quality of whole-plant corn silage

[0067] Note: Data in the same column with the same letter or no letter in the shoulder indicate no significant difference ( P >0.05, adjacent letters indicate significant differences ( P <0.05).

[0068] Table 2 Chemical components of whole-plant corn silage

[0069] Table 3 Sensory scores of whole-plant corn silage

[0070] Table 4 Gas composition of whole-plant corn silage

[0071] 6. Experimental Conclusion From the data analysis of the above experiments, it can be seen that the comprehensive effects of the test groups 3-5 of the present invention are better, among which the various indicators of the test group 5 are better. In terms of the fermentation quality of silage, the pH and ammonia nitrogen content of the test group 5 are lower, and the lactic acid and acetic acid contents of the test group 5 are higher. In terms of the chemical composition of silage, the neutral detergent fiber and acid detergent fiber content of the test group 5 are lower. In terms of the sensory evaluation of silage, the color score and total score of the test group 5 are higher. In terms of the gas composition of whole-plant corn silage, the mellow aroma, fruity aroma and sour aroma of the test group 5 are higher. Therefore, the present invention can achieve the rapid reduction of the pH of the whole-plant corn silage and produce high-value, high-quality whole-plant corn silage.

[0072] Experimental plan three 1. Experimental design: 39 healthy dairy cows with parity (2-3), milk yield (30±1) kg / d, lactation days (45±5) d were selected and randomly divided into 3 groups. The control group was fed silage without additives. The supplier of silage starter for the experimental group 9 was Shenzhen Jinfuyuan Biotechnology Co., Ltd. The composition of the silage enzyme preparation for the experimental group 10 was: Lactobacillus plantarum: Lactobacillus buchneri: cellulase: xylanase = 1:1:1:1. The pre-test period was 7 days and the full test period was 60 days.

[0073] 2. Feeding management: The diet formula was prepared according to the NRC (2001) dairy cow feeding standard, and the cows were fed in the form of a total mixed ration (TMR), and sufficient clean drinking water was provided; except for feeding the whole corn silage with different treatments, the other feeding management of the three groups of cows was the same. The cows were housed and fed freely. The experimental cows were mechanically milked at 04:00 and 18:00 every day, and fed at 04:30 and 16:30.

[0074] 3. Measurement indicators and methods 1) Feed intake determination: Based on the experimental design and the actual situation of the pasture, the feed intake of the experimental animals was determined by weighing and recording once every 3 days. Every 12 days was a cycle. Data was selected once in each cycle for statistical analysis to determine the feed intake of the experimental animals.

[0075] 2) Milk sample collection and determination: During the trial period, milk samples were collected every 15 days for DHI (Dairy Herd Improve) testing. Milk samples were collected through a sampler connected to the milking machine. According to the milking time of the farm, milk samples were collected once in the morning and once in the evening, and mixed in a ratio of 4:6. The collected milk samples were tested for milk composition, and the milk yield was measured by the automatic metering facilities of the milking machine.

[0076] 3) Stress resistance test: On the 26th, 27th and 28th days, 15 ml of blood samples were collected from the tail vein of fasting dairy cows using a sterile vacuum blood collection needle before feeding every morning. The blood samples were placed in three 5 ml sodium heparin tubes. After centrifugation at 3000 r / min for 10 min, the serum samples were transferred to 10 1.5 ml clean centrifuge tubes and stored at -20°C until used for analysis of serum-related parameters.

[0077] 4. Data Processing and Analysis SPSS software was used to analyze the data univariately and Duncan's method was used for multiple comparisons.

[0078] 5. Experimental Results

[0079] Note: Data in the same row with the same letter or no letter in the shoulder indicate no significant difference (P>0.05), adjacent letters indicate significant difference (P<0.05), and alternate letters indicate extremely significant difference (P<0.01).

[0080] 6. Experimental Conclusion As can be seen from the table, both test group 9 and test group 10 can increase the feed intake of dairy cows, and the effect of test group 10 is better; the milk production of test group 9 and test group 10 is higher than that of the control group, among which the milk production of test group 10 is significantly higher than that of the control group; the milk protein and lactose contents of test group 10 are significantly higher than those of the other two groups; the MDA, GSH-Px and SOD contents in test group 102 are relatively high, indicating that the whole-plant corn silage prepared by the whole-plant corn silage composite bacterial enzyme preparation of the present invention can improve the anti-stress and antioxidant capacity of cattle.

[0081] In summary, the whole-plant corn silage composite bacterial enzyme preparation of the present invention can, under normal silage operation and production conditions, quickly reduce the pH value of whole-plant corn silage, improve the quality and chemical composition of whole-plant corn silage, improve the sensory quality of silage, and produce high-value, high-quality whole-plant corn silage.

Claims

1. A composite bacterial enzyme preparation for silage, It is characterized in that It comprises Lactobacillus plantarum, Lactobacillus buchneri, cellulase and xylanase. Preferably, the mass ratio of Lactobacillus plantarum, Lactobacillus buchneri, cellulase and xylanase is 1-2:1-2:1-3:1-3.

2. The bacterial enzyme preparation according to claim 1, It is characterized in that The plant lactobacillus is plant lactobacillus BFC1602, which is deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee with a deposit number of CGMCC No.13132.

3. The composite bacterial enzyme preparation for silage according to claim 1 or 2, It is characterized in that In the composite bacterial enzyme preparation, the number of viable Lactobacillus plantarum is ≥ 2×10 10 CFU / g, and / or the viable count of Lactobacillus buchneri ≥ 4×10 10 CFU / g, and / or the cellulase activity ≥2×10 3 U / g, and / or the xylanase activity is ≥4×10 4 U / g.

4. The composite bacterial enzyme preparation according to any one of claims 1 to 3, It is characterized in that The mass ratio of the Lactobacillus plantarum, Lactobacillus buchneri, cellulase and xylanase is 1:1:1:

1.

5. A method for preparing the composite bacterial enzyme preparation for silage according to any one of claims 1 to 4, It is characterized in that The following steps are involved: (1) activating the plant lactobacillus and then expanding the culture to obtain a bacterial liquid of the plant lactobacillus; (2) centrifuging the bacterial solution of Lactobacillus plantarum to obtain a precipitate, collecting the precipitate, and freeze-drying the precipitate to obtain bacterial powder of Lactobacillus plantarum; (3) Mix Lactobacillus plantarum powder and Lactobacillus buchneri powder, cellulase and xylanase in a mass ratio of 1-2:1-2:1-3:1-3 to obtain a composite bacterial enzyme preparation.

6. The method for preparing the composite bacterial enzyme preparation for silage according to claim 5, It is characterized in that The plant lactobacillus is plant lactobacillus BFC1602, which is deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee with a deposit number of CGMCC No.13132.

7. A method for fermenting whole-plant corn silage using the composite bacterial enzyme preparation according to any one of claims 1 to 4 or the composite bacterial enzyme preparation prepared by the method according to claims 5 to 6, It is characterized in that The steps include: (1) After the corn is harvested, its moisture content is adjusted to 65% to 70%, and the corn is crushed into small pieces of 1.5 to 2 cm for later use; (2) dissolving and diluting the composite bacterial enzyme preparation according to a mass volume ratio of the composite bacterial enzyme preparation to water of 1:500-2000; (3) Load the whole corn plant into the silo, and after filling 4 to 6 cm, spray the diluted bacterial enzyme preparation solution on the surface of the whole corn plant; (4) Use film to seal.

8. The method for fermenting whole-plant corn silage according to claim 7, It is characterized in that In the step (2), the mass ratio of the composite bacterial enzyme preparation to water is 1:500-1000.

9. The method for fermenting whole-plant corn silage according to claim 7 or 8, It is characterized in that The mass ratio of the bacterial enzyme preparation solution to the silage corn is 1:125-500.

10. Use of the composite bacterial enzyme preparation according to any one of claims 1 to 4 or the composite bacterial enzyme preparation prepared by the method according to claims 5 to 6 in preparing silage.

Images