Micro-storage feed based on sea-buckthorn polysaccharide and compound enzyme preparation and preparation method of micro-storage feed

Abstract

The invention belongs to the technical field of micro-storage feeds, and provides a preparation method of a micro-storage feed based on sea-buckthorn polysaccharide and a complex enzyme preparation, which comprises the following steps: spraying 10-30mL of a mixed solution containing 2% of sea-buckthorn polysaccharide and 1% of bacillus licheniformis and purified water to 1-3Kg of sugarcane tail leaves containing 60% of water, and carrying out aerobic fermentation at room temperature for 2 days; the method comprises the following steps: spraying 10-30mL of a mixed solution of 1% lactobacillus plantarum and 1% lactobacillus acidophilus, 40-120mL of a compound enzyme preparation and 40-120mL of sterile water, uniformly mixing, spraying into sugarcane tail leaves containing 60% of water, loading into a silage bag with a one-way exhaust valve, fully extruding and exhausting, storing and fermenting, and taking out a fermented sample after 30 days, thereby obtaining the micro-storage feed. The sea-buckthorn polysaccharide, the probiotics and the compound enzyme are applied to the sugarcane tail leaf fermented feed, so that the nutritional value and the economic value of the sugarcane tail leaf fermented feed are improved.

Description

Technical Field

[0001] This invention relates to the field of micro-storage technology, and in particular to a micro-storage based on sea buckthorn polysaccharide and a compound enzyme preparation and its preparation method. Background Technology

[0002] Sugarcane is one of Guangxi's important specialty crops, with an annual planting area exceeding 1 million hectares. Sugarcane top leaves, consisting of the top 2-3 tender nodes and the attached leaves, account for approximately 20% of the total weight of the sugarcane stalk. Sugarcane top leaves, forage grass, and corn stalks are the main sources of roughage for livestock and poultry in Guangxi. Sugarcane top leaf production is seasonal, with the sugarcane harvest season from November to April of the following year. During this period, a large quantity of sugarcane top leaves is produced, meeting the roughage needs of livestock and poultry when other green fodder is insufficient in winter. The sugarcane harvest is relatively concentrated, and the sugarcane top leaves cannot be consumed directly through feeding in a short time. Fresh sugarcane top leaves have a high water content and are highly susceptible to mold and decay in high temperature and humidity environments. Therefore, how to efficiently and scientifically develop and utilize sugarcane top leaves to maximize their utilization value and efficiency has become the main direction for the current development and utilization of sugarcane top leaves.

[0003] The synergistic use of bacteria and enzymes in the production of fermented sugarcane tops and leaves has great potential in the livestock industry. Probiotics such as Bacillus licheniformis can utilize the heat and acid generated during fermentation to convert crude fiber into more nutritious crude protein, while compound enzymes can further transform crude fiber into more easily digestible substances. Using sea buckthorn polysaccharides and compound enzyme preparations to produce micro-silage can effectively reduce pollution and also create feed with high economic added value.

[0004] Therefore, how to provide an efficient and scientific method for preparing micro-silage based on sea buckthorn polysaccharides and compound enzyme preparations using sugarcane top leaves is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] The purpose of this invention is to provide a micro-storage feed based on sea buckthorn polysaccharide and compound enzyme preparation and its preparation method, so as to solve the technical problems in the prior art.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing micro-silage based on sea buckthorn polysaccharide and compound enzyme preparation, comprising the following steps:

[0008] S1. Aerobic fermentation: Spray 1-3 kg of sugarcane leaves with 60% water content with a mixture of 2% sea buckthorn polysaccharide and 1% Bacillus licheniformis A10-30 mL and 90-270 mL of purified water, and allow to ferment aerobically at room temperature for 2 days.

[0009] S2. Anaerobic silage: Spray 10-30 mL of a mixture of 1% Lactobacillus plantarum and 1% Lactobacillus acidophilus (B), 40-120 mL of compound enzyme preparation, and 40-120 mL of sterile water, mix well, and spray onto sugarcane top leaves with a water content of 60%. Pack the mixture into silage bags with one-way exhaust valves, squeeze out the air thoroughly, and store for fermentation. After 30 days, take out the fermented sample to obtain the micro-silage.

[0010] Preferably, the compound enzyme preparation is one or more of cellulase, protease, xylanase, and pectinase.

[0011] Preferably, the compound enzyme preparation is purchased from Nanning Pangbo Biotechnology Co., Ltd.; the compound enzyme preparation contains cellulase at a content of ≥150,000 U / g, protease at a content of ≥50,000 U / g, xylanase at a content of ≥200,000 U / g, and pectinase at a content of ≥30,000 U / g.

[0012] Preferably, the *Bacillus licheniformis* was purchased from the China Center for Type Culture Collection (CCTCCAB2010437); the *Lactobacillus plantarum* was purchased from the China Center for Type Culture Collection (CCTCC M2019002); and the *Lactobacillus acidophilus* was purchased from the China Center for Type Culture Collection (CCTCCAB 2010208).

[0013] Preferably, the viable count of the Bacillus licheniformis is ≥1×10⁻⁶. 10 CFU / g; The viable count of the *Lactobacillus plantarum* is ≥1×10⁻⁶. 10 CFU / g; The viable count of the Lactobacillus acidophilus is ≥1×10⁻⁶. 10 CFU / g.

[0014] Preferably, the sugarcane top leaves with a water content of 60% are 1-3 cm in length.

[0015] The present invention also provides a micro-storage feed obtained by the preparation method described in the claims.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) Sea buckthorn polysaccharide can be used as a feed additive because it has antibacterial, antioxidant and immunomodulatory biological activities. Using sea buckthorn polysaccharide as a feed additive is economical, environmentally friendly and has no side effects.

[0018] (2) Enzyme preparations and probiotics can work synergistically. Therefore, adding enzyme preparations to micro-storage can effectively degrade cellulose and hemicellulose in green fodder such as sugarcane leaves and convert them into monosaccharides and disaccharides, providing nutrition for probiotics in micro-storage. In addition to providing nutrition, probiotics can also improve animal immunity and reduce dependence on antibiotics, which is in line with the national policy of green ecological breeding development.

[0019] (3) Sugarcane top leaves are high in crude fiber and have a coarse texture. Silage fermentation can improve their palatability, enhance their nutritional quality, and extend their storage time. Silage is a complex fermentation process involving multiple microorganisms, each with different effects on silage quality. This invention applies sea buckthorn polysaccharides, probiotics, and compound enzymes to fermented sugarcane top leaves feed, thereby improving its nutritional and economic value. Detailed Implementation

[0020] This invention provides a method for preparing micro-silage based on sea buckthorn polysaccharide and compound enzyme preparation, comprising the following steps:

[0021] S1. Aerobic fermentation: Spray 1-3 kg of sugarcane leaves with 60% water content with a mixture of 2% sea buckthorn polysaccharide and 1% Bacillus licheniformis A10-30 mL and 90-270 mL of purified water, and allow to ferment aerobically at room temperature for 2 days.

[0022] S2. Anaerobic silage: Spray 10-30 mL of a mixture of 1% Lactobacillus plantarum and 1% Lactobacillus acidophilus (B), 40-120 mL of compound enzyme preparation, and 40-120 mL of sterile water, mix well, and spray onto sugarcane top leaves with a water content of 60%. Pack the mixture into silage bags with one-way exhaust valves, squeeze out the air thoroughly, and store for fermentation. After 30 days, take out the fermented sample to obtain the micro-silage.

[0023] In this invention, in S1, the amount of sugarcane top leaves with a water content of 60% is 1-3 kg, preferably 1.2-2.8 kg, more preferably 1.5-2.5 kg, and even more preferably 1.8-2.3 kg.

[0024] In this invention, in S1, the amount of the mixture A is 10-30 mL, preferably 12-28 mL, more preferably 15-25 mL, and even more preferably 18-23 mL.

[0025] In this invention, in S1, the amount of purified water used is 90-270 mL, preferably 100-250 mL, more preferably 120-230 mL, and even more preferably 150-200 mL.

[0026] In this invention, in S2, the amount of the mixture B is 10-30 mL, preferably 12-28 mL, more preferably 15-25 mL, and even more preferably 18-23 mL.

[0027] In this invention, in step S2, the amount of the compound enzyme preparation used is 40-120 mL, preferably 50-100 mL, more preferably 60-90 mL, and even more preferably 70-80 mL.

[0028] In this invention, in step S2, the amount of sterile water used is 40-120 mL, preferably 50-100 mL, more preferably 60-90 mL, and even more preferably 70-80 mL.

[0029] In this invention, the compound enzyme preparation is one or more of cellulase, protease, xylanase, and pectinase.

[0030] In this invention, the compound enzyme preparation was purchased from Nanning Pangbo Biotechnology Co., Ltd.; the compound enzyme preparation contains cellulase at a content of ≥150,000 U / g, protease at a content of ≥50,000 U / g, xylanase at a content of ≥200,000 U / g, and pectinase at a content of ≥30,000 U / g.

[0031] In this invention, the Bacillus licheniformis was purchased from the China Center for Type Culture Collection (CCTCCAB 2010437); the Lactobacillus plantarum was purchased from the China Center for Type Culture Collection (CCTCCM2019002); and the Lactobacillus acidophilus was purchased from the China Center for Type Culture Collection (CCTCCAB 2010208).

[0032] In this invention, the viable count of the Bacillus licheniformis is ≥1×10⁻⁶. 10 CFU / g; The viable count of the *Lactobacillus plantarum* is ≥1×10⁻⁶. 10 CFU / g; The viable count of the Lactobacillus acidophilus is ≥1×10⁻⁶. 10 CFU / g.

[0033] In this invention, the length of the sugarcane top leaf with a water content of 60% is 1-3 cm, preferably 1.2-2.8 cm, more preferably 1.5-2.5 cm, and even more preferably 1.8-2.3 cm.

[0034] The present invention also provides a micro-storage feed obtained by the preparation method described above.

[0035] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1

[0037] Each group consisted of 1 kg sugarcane top leaves with 60% moisture content. The first stage was aerobic fermentation: the sample was sprayed with 10 mL of a mixture of 2% sea buckthorn polysaccharide and 1% Bacillus licheniformis A and 90 mL of purified water, and fermented aerobically at room temperature for 2 days. The second stage was anaerobic silage: 10 mL of a mixture of 1% Lactobacillus plantarum and 1% Lactobacillus acidophilus B and 40 mL of compound enzyme preparation + 40 mL of sterile water were sprayed onto the sugarcane top leaves sample with 60% moisture content. The sample was divided into 3 equal parts, put into silage bags with one-way exhaust valves, each bag weighing about 330 g, and after fully squeezing out the air, it was stored for fermentation. After 30 days, the fermented sample was taken out for various tests to obtain micro-silage.

[0038] The compound enzyme preparation comprises cellulase, protease, xylanase, and pectinase. The compound enzyme preparation was purchased from Nanning Pangbo Biotechnology Co., Ltd.; the content of cellulase in the compound enzyme preparation is ≥150,000 U / g, the content of protease is ≥50,000 U / g, the content of xylanase is ≥200,000 U / g, and the content of pectinase is ≥30,000 U / g.

[0039] The *Bacillus licheniformis* was purchased from the China Center for Type Culture Collection (CCTCC), accession number CCTCC AB2010437; the *Lactobacillus plantarum* was purchased from the China Center for Type Culture Collection (CCTCC), accession number CCTCC M2019002; and the *Lactobacillus acidophilus* was purchased from the China Center for Type Culture Collection (CCTCCAB 2010208). The viable count of the *Bacillus licheniformis* was ≥1×10⁻⁶. 10 CFU / g; The viable count of the *Lactobacillus plantarum* is ≥1×10⁻⁶. 10 CFU / g; The viable count of the Lactobacillus acidophilus is ≥1×10⁻⁶. 10 CFU / g. The sugarcane top leaves with a moisture content of 60% are 1–3 cm in length.

[0040] Comparative Example 1

[0041] Each group consisted of 1 kg sugarcane leaf samples with 60% water content. In Group A, sea buckthorn polysaccharide was added to sterile water to prepare a 2% solution, which was then sprayed directly onto the sugarcane leaves for silage fermentation.

[0042] Comparative Example 2

[0043] Each group consisted of 1 kg sugarcane leaf samples with a moisture content of 60%. In Group B, the required 2% sea buckthorn polysaccharide and compound enzyme preparation were mixed and sprayed onto 2 cm long sugarcane leaves. The mixture was sprayed layer by layer and mixed evenly. The moisture content was adjusted to about 60% before normal silage fermentation was carried out.

[0044] Comparative Example 3

[0045] Each group consisted of 1 kg sugarcane leaf samples with 60% moisture content. Group C was the first stage of aerobic fermentation: the samples were sprayed with 10 mL of 2% seabuckthorn polysaccharide mixed with 1% Bacillus licheniformis solution and 90 mL of purified water, and fermented aerobically at room temperature for 2 days. The second stage was anaerobic silage: 10 mL of 1% Lactobacillus plantarum and 1% Lactobacillus acidophilus mixed solution + 80 mL of sterile water were sprayed. After the inoculum was mixed evenly, the sugarcane leaf samples were divided into 3 equal parts, packed into silage bags with one-way exhaust valves, each bag weighing about 330 g, and after being fully squeezed to remove air, they were stored and fermented for 30 days.

[0046] Performance testing:

[0047] 1.2 Experimental Animals

[0048] Seventy-five healthy Simmental cattle with similar initial weights were selected for the experiment and randomly divided into five groups, with three replicates per group and five cattle per replicate. Each group was fed fermented feed for 90 days.

[0049] 1.3 Experimental Content

[0050] Four experimental groups were set up. Comparative Example 1 consisted of a 2% sea buckthorn polysaccharide solution. Comparative Example 2 consisted of 0.15g cellulase, 0.15g protease, 0.1g xylanase, 0.1g pectinase, and 2% sea buckthorn polysaccharide added to 40mL of water. Comparative Example 3 consisted of 2% sea buckthorn polysaccharide, 1% *Lactobacillus plantarum*, 1% *Bacillus licheniformis*, and 1% *Lactobacillus acidophilus*. Example 1 consisted of 2% sea buckthorn polysaccharide, 1% *Lactobacillus plantarum*, 1% *Bacillus licheniformis*, 1% *Lactobacillus acidophilus*, and 40mL of a compound enzyme solution. The control group underwent natural fermentation and was sprayed with only purified water.

[0051] The control group of beef cattle was fed a naturally fermented sugarcane top leaf silage diet that was only sprayed with sterile water. Groups 1, 2, 3, and 4 of the experiment replaced the basal feed with equal amounts of the fermented sugarcane top leaf silage diets from Comparative Examples 1-3 and Example 1, respectively. The experiment lasted for 60 days. After 60 days, fasting blood samples were collected from each group of beef cattle in the morning. 15 ml of blood was collected from the jugular vein of each cattle, placed in a coagulation-promoting vacuum tube, allowed to stand for 1 hour, centrifuged at 3000 rpm for 15 minutes, and the supernatant serum was collected and stored in liquid nitrogen at -80℃ for later analysis.

[0052] 1.4 Indicator Evaluation

[0053] 1.4.1 Nutritional composition assessment

[0054] Moisture and dry matter were determined by drying method; crude protein (CP) was determined by Kjeldahl nitrogen determination method; crude fat (EE) was determined by Soxhlet extraction method; neutral detergent fiber (NDF) was determined according to international standard ISO 16472:2006 "Determination of neutral detergent fiber in feed"; acid detergent fiber (ADF) was determined according to international standard ISO 13906:2008 "Determination of acid detergent fiber in feed"; pH determination: after opening the silage, 5g of sample was added to 10mL of sterile physiological saline, mixed well, centrifuged at 1500r / min for 15min, and the supernatant was taken and measured with a pH meter. The result is expressed as pH.

[0055] 1.4.2 Assessment of Blood Biochemical Indicators

[0056] On the morning of day 90, fasting blood was collected from the jugular vein. The blood was centrifuged at 3500 rpm for 15 min, and the supernatant was separated and aliquoted into EP tubes, labeled, and stored at -20°C for later analysis. The activities of triglycerides (TG), cholesterol (TC), albumin (ALB), total protein (TP), glucose (GLU), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in the serum of the experimental and control groups were detected using a kit from Nanjing Jiancheng Bioengineering Institute on a URIT-8000 fully automated biochemical analyzer.

[0057] 1.4.3 Assessment of Blood Immune Indicators

[0058] According to the operating requirements of the enzyme-linked immunosorbent assay kit, the levels of interleukin-1β (IL-1β), interleukin-10 (IL-10), interleukin-2 (IL-2), interleukin-6 (IL-6), immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM) in the serum of beef cattle in each group were measured. All the above indicators were measured by the Ruigu Medical Laboratory of Guangxi Medical University.

[0059] 1.5 Data Statistics

[0060] After the experimental data were initially processed using Excel 2013, one-way ANOVA was performed on each group of data using SPSS 22.0. Duncan's method was used for multiple comparisons between groups. The data are expressed as mean ± standard error.

[0061] Table 1 Comparison of conventional nutrient components in feed within 40 hours of fermentation.

[0062]

[0063]

[0064] Note: Different letters in the same row indicate significant differences (P < 0.05), while identical letters or no letters indicate no significant differences;

[0065] Table 2. Amino acid composition and content in feed

[0066]

[0067]

[0068] Note: Different letters in the same row indicate significant differences (P < 0.05), while identical letters or no letters indicate no significant differences;

[0069] Table 3 Content of organic acids and their components in feed

[0070]

[0071]

[0072] Note: Different letters in the same row indicate significant differences (P < 0.05), while identical letters or no letters indicate no significant differences;

[0073] Table 4. Average indices of GLU, TP, ALB, TC, TG, ALT, and AST

[0074]

[0075] Note: Different letters in the same row indicate significant differences (P < 0.05), while identical letters or no letters indicate no significant differences;

[0076] Table 5. Average indices of IgA, IgG, IgM, IL-2, and IL-6

[0077]

[0078] Note: Different letters in the same row indicate significant differences (P < 0.05), while identical letters or no letters indicate no significant differences;

[0079] As shown in Table 1, the control group had the highest dry matter (DM) content, but the differences among the groups were not significant (P > 0.05). Crude protein (CP) was highest in the probiotic + compound enzyme group, and the difference was significant (P < 0.05). Crude fat (EE) content was also highest in the probiotic + compound enzyme group, and the difference was significant, followed by the probiotic group, which also had a relatively high content with a significant difference. The compound enzyme group showed no significant difference compared to the control group. Neutral detergent fiber (NDF) content decreased in all groups, with the probiotic + compound enzyme group having the lowest content, and the difference compared to the control group was significant. This was followed by the probiotic group and the compound enzyme group, where the differences between the two groups were not significant, but the differences compared to the control group were significant. Finally, acid detergent fiber (ADF) content was lowest in the probiotic + compound enzyme group, followed by the probiotic group, with significant differences compared to the control group.

[0080] Effects of different treatments of sugarcane tops silage on blood immune indicators in beef cattle. Serum samples were collected from beef cattle after 90 days of feeding and biochemical tests were performed. The results are shown in Table 4. In Comparative Examples 1 and 2, there were no significant differences in GLU, TP, and ALB indices compared to the control group (P>0.05). In Comparative Examples 3 and Example 1, GLU, TP, and ALB indices were significantly higher than those in the control group (P<0.05). The TG index in Example 1 was slightly higher than that in the control group, with a significant difference; however, there were no significant differences in TG indices between the other groups and the control group. There were no significant differences in TC, ALT, and AST indices between the experimental groups and the control group (P>0.05).

[0081] Serum samples were collected from beef cattle after 60 days of feeding, and immune indicators were tested. The results are shown in Table 5. The IgA, IgG, IgM, IL-2, and IL-6 indices of each experimental group were increased compared with the control group. Among them, the difference between Example 1 group and the control group was significant (P < 0.05). In Comparative Examples 1 to 3, except for Comparative Example 3 group, which showed a significant difference in IgG index compared with the control group (P < 0.05), the IgA, IgG, IgM, IL-2, and IL-6 indices of the other experimental groups were not significantly different from those of the control group (P > 0.05).

[0082] As can be seen from the above embodiments, this invention provides a method for preparing micro-silage based on sea buckthorn polysaccharide and compound enzyme preparations. Sea buckthorn polysaccharide can be used as a feed additive because it has antibacterial, antioxidant, and immunomodulatory biological activities. Using sea buckthorn polysaccharide as a feed additive is economical, environmentally friendly, and has no side effects. Enzyme preparations and probiotics can work synergistically. Therefore, adding enzyme preparations to micro-silage production can effectively degrade cellulose and hemicellulose in green fodder such as sugarcane tops and leaves, converting them into monosaccharides and disaccharides, providing nutrition for probiotics in the micro-silage. In addition to providing nutrition, probiotics can also improve animal immunity and reduce dependence on antibiotics, which is in line with the national policy of green and ecological farming development. Sugarcane tops and leaves have a high crude fiber content and a coarse texture. Ensilage fermentation can improve their palatability, enhance their nutritional quality, and extend their storage time. Silage is a complex fermentation process involving multiple microorganisms, and different microorganisms have different effects on silage quality. This invention applies sea buckthorn polysaccharide, probiotics, and compound enzymes to fermented sugarcane top leaves feed, thereby improving the nutritional and economic value of fermented sugarcane top leaves feed.

[0083] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing micro-storage feed based on sea buckthorn polysaccharide and compound enzyme preparation, characterized in that, Includes the following steps: S1. Aerobic fermentation: Spray 1-3 kg of sugarcane leaves with 60% water content with a mixture of 2% sea buckthorn polysaccharide and 1% Bacillus licheniformis A10-30 mL and 90-270 mL of purified water, and allow to ferment aerobically at room temperature for 2 days. S2. Anaerobic silage: Spray 10-30 mL of a mixture of 1% Lactobacillus plantarum and 1% Lactobacillus acidophilus (B), 40-120 mL of compound enzyme preparation, and 40-120 mL of sterile water, mix well, and spray onto sugarcane top leaves with a water content of 60%. Pack the mixture into silage bags with one-way exhaust valves, squeeze out the air thoroughly, and store for fermentation. After 30 days, take out the fermented sample to obtain the micro-silage.

2. The preparation method according to claim 1, characterized in that, The compound enzyme preparation is one or more of cellulase, protease, xylanase, and pectinase.

3. The preparation method according to claim 1, characterized in that, The compound enzyme preparation was purchased from Nanning Pangbo Biotechnology Co., Ltd. The compound enzyme preparation contains cellulase at a content of ≥150,000 U / g, protease at a content of ≥50,000 U / g, xylanase at a content of ≥200,000 U / g, and pectinase at a content of ≥30,000 U / g.

4. The preparation method according to claim 1, characterized in that, The Bacillus licheniformis was purchased from the China Center for Type Culture Collection, with accession number CCTCAB 2010437; The Lactobacillus plantarum was purchased from the China Center for Type Culture Collection, with accession number CCTCC M2019002. The Lactobacillus acidophilus was purchased from the China Center for Type Culture Collection, with accession number CCTCAB 2010208.

5. The preparation method according to claim 1, characterized in that, The viable count of Bacillus licheniformis is ≥1×10⁻⁶. 10 CFU / g; The viable count of *Lactobacillus plantarum* is ≥1×10⁻⁶. 10 CFU / g; The viable count of Lactobacillus acidophilus is ≥1×10⁻⁶. 10 CFU / g.

6. The preparation method according to claim 1, characterized in that, The length of the sugarcane top leaves with a water content of 60% is 1-3 cm.

7. A micro-storage feed obtained by the preparation method according to any one of claims 1 to 6.

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