A method of fermenting silage

Abstract

The present application relates to the silage preparation technical field, especially a kind of silage fermentation method.The present application provides a kind of silage fermentation method, including the following steps: (1) corn stalk is crushed;(2) the plant lactobacillus CICC24194 is mixed with the corn stalk after being crushed, and is wrapped and is fermented after being wrapped.The plant lactobacillus CICC24194 used in the present application can produce bacteriocin, can reduce the dry matter loss of raw material in the silage fermentation process, retains more nutrients.When used for producing high-quality silage, not only can the silage be fermented, but also bacteriocin with probiotic effect and no drug resistance can be produced, which is beneficial to produce high-quality whole plant corn silage with probiotic effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of silage preparation, and particularly relates to a fermentation method of silage. BACKGROUND

[0002] With the development of the livestock breeding industry, especially the dairy industry, large-scale and intensive breeding has been formed. In order to reduce costs, improve benefits and improve milk quality, breeders need to control each link, and silage is the key.

[0003] As one of the main components of the diet of ruminants, high-quality silage provides ruminants with rich nutrients such as crude protein, digestible fiber and trace nutrients. The fermentation quality of silage is usually determined by the silage raw material, attached bacteria, anaerobic conditions and additive types. Due to poor fermentation methods, silage is easily contaminated by undesirable microorganisms during fermentation, resulting in reduced silage quality, nutrient loss, affecting the production performance of ruminants, the safety of meat and dairy products, and even threatening human health.

[0004] Microorganisms related to silage contamination mainly include yeast, Escherichia coli, Listeria, Bacillus, Clostridium and Salmonella. These microorganisms can cause different degrees of diseases in ruminants, such as mild diarrhea and reduced feed intake in ruminants caused by Clostridium, and abortion and death in dairy cows caused by Listeria. Bacteriocins are a class of polypeptides or precursor polypeptides with antibacterial activity synthesized by some bacteria during metabolism, which can accelerate the death of related bacteria by destroying the lipid II metabolism of the cell membrane or inhibiting the replication of nucleic acids. Because bacteriocins have no drug resistance, no residue, and are environmentally friendly, they are considered as potential alternatives to antibiotics. Many lactic acid bacteria can produce bacteriocins.

[0005] Therefore, it is of great significance to explore the effects of bacteriocin-producing lactic acid bacteria on the nutritional components and harmful bacteria reproduction of whole corn wrapped silage for the research and application of new lactic acid bacteria feed additives for whole corn silage. SUMMARY

[0006] The present application aims to provide a fermentation method of silage.

[0007] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0008] The present application provides a fermentation method of silage, comprising the following steps:

[0009] (1) crushing corn stalks;

[0010] (2) mixing the Lactobacillus plantarum CICC24194 with the crushed corn stalks, wrapping and fermenting.

[0011] Preferably, the moisture content of the corn stalks in step (1) is 50-70%.

[0012] Preferably, the dried corn stalks in step (1) are crushed to a length of 1-2 cm.

[0013] Preferably, the viable bacterial count of the Lactobacillus plantarum CICC24194 in step (2) is (1-5) x 10 7 CFU / mL.

[0014] Preferably, the volume-mass ratio of the Lactobacillus plantarum CICC24194 to the crushed corn stalks in step (2) is 1 mL: 80-120 g.

[0015] Preferably, the density of the wrapping in step (2) is 0.91-0.93 g / cm 3 .

[0016] Preferably, the fermentation time in step (2) is 80-100 d.

[0017] Compared with the prior art, the present application has the following beneficial effects:

[0018] 1. The Lactobacillus plantarum CICC24194 used in the present application can produce bacteriocins (class IIa bacteriocins have been identified and purchased from CICC), which can reduce the dry matter loss of raw materials and retain more nutrients during the fermentation of silage feed. When used for producing high-quality silage feed, not only can the silage feed be fermented, but also bacteriocins with probiotic effects and no drug resistance can be produced, which is beneficial for producing high-quality whole corn silage feed with probiotic effects.

[0019] 2. According to the fermentation method of the present application, harmful bacteria inhibitors do not need to be added before the fermentation of whole corn silage, which saves the production cost and improves the fermentation effect of the silage feed. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the following embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0021] Figure 1 Whole corn silage feed species level bacterial community composition for Example 1;

[0022] Figure 2 Example 1: The rumen in vitro fermentation bacterial community composition of whole plant corn silage feed. DETAILED DESCRIPTION

[0023] The present application provides a fermentation method of silage feed, comprising the following steps:

[0024] (1) crushing corn stalks;

[0025] (2) mixing the Lactobacillus plantarum CICC24194 with the crushed corn stalks, and then wrapping and fermenting.

[0026] In the present application, the moisture content of the corn stalks in step (1) is 50-70%; preferably 60%.

[0027] In the present application, the dried corn stalks in step (1) are crushed to a length of 1-2 cm; preferably 1.5 cm.

[0028] In the present application, the viable bacterial count of the Lactobacillus plantarum CICC24194 in step (2) is (1-5) x 10 7 CFU / mL; preferably (2-4) x 10 7 CFU / mL; further preferably 3 x 10 7 CFU / mL.

[0029] In the present application, the volume to mass ratio of the Lactobacillus plantarum CICC24194 to the crushed corn stalks in step (2) is 1 mL: 80-120 g; preferably 1 mL: 90-110 g; further preferably 1 mL: 100 g.

[0030] In the present application, the density of the wrapping in step (2) is 0.91-0.93 g / cm 3 ; preferably 0.92 g / cm 3 .

[0031] In the present application, the fermentation time in step (2) is 80-100 d; preferably 84-96 d; further preferably 88-92 d; more preferably 90 d.

[0032] The technical solutions provided by the present application will be described in detail below in conjunction with examples, but they should not be understood as limiting the scope of protection of the present application.

[0033] Example 1

[0034] A method for fermenting silage feed, comprising the following steps:

[0035] (1) Artificially harvested whole corn (Dajingjiu ensilage 3876) was dried to a moisture content of 70% under natural ventilation, and then ground to a length of 1 cm;

[0036] (2) The Lactobacillus plantarum CICC24194 (live bacterial count of 1 x 10 7 CFU / mL) was mixed with the ground corn stalks at a volume to mass ratio of 1 mL: 80 g, and after packaging, a wrap (density of 0.91 g / cm 3 ) was obtained, which was fermented at room temperature (25°C) for 80 d.

[0037] Example 2

[0038] A method for fermenting silage, comprising the following steps:

[0039] (1) Artificially harvested whole corn (Dajingjiu ensilage 3876) was dried to a moisture content of 50% under natural ventilation, and then ground to a length of 2 cm;

[0040] (2) The Lactobacillus plantarum CICC24194 (live bacterial count of 5 x 10 7 CFU / mL) was mixed with the ground corn stalks at a volume to mass ratio of 1 mL: 120 g, and after packaging, a wrap (density of 0.93 g / cm 3 ) was obtained, which was fermented at room temperature (25°C) for 100 d.

[0041] Example 3

[0042] A method for fermenting silage, comprising the following steps:

[0043] (1) Artificially harvested whole corn (Dajingjiu ensilage 3876) was dried to a moisture content of 60% under natural ventilation, and then ground to a length of 1.5 cm;

[0044] (2) The Lactobacillus plantarum CICC24194 (live bacterial count of 3 x 10 7 CFU / mL) was mixed with the ground corn stalks at a volume to mass ratio of 1 mL: 100 g, and after packaging, a wrap (density of 0.92 g / cm 3 ) was obtained, which was fermented at room temperature (25°C) for 90 d.

[0045] Comparative Example 1

[0046] The other methods were identical to Example 3, except that the Lactobacillus plantarum CICC24194 was replaced by Lactobacillus plantarum (MTD / 1, NCIMB40027, purchased from Ecosyl Products Limited, UK).

[0047] Experimental Example 1

[0048] (1) The method of Example 3 was used as the CICC24194 group, and the method of Comparative Example 1 was used as the MTD / 1 group. The control group used an equal amount of distilled water instead of the addition of the bacterial strain. The fermentation characteristics of the whole-plant corn silage CICC24194 group, MTD / 1 group, and control group (after 90 days of fermentation) were statistically analyzed. The results are shown in Table 1.

[0049] Table 1 Fermentation characteristics of whole-plant corn silage

[0050]

[0051]

[0052] Note: LA, lactic acid; AA, acetic acid; PA, propionic acid; DM, dry matter; a-d Different lowercase letters indicate significant differences (P < 0.05) between treatments

[0053] The results show that the CICC24194 group significantly reduced the propionic acid concentration of the whole-plant corn silage compared to the control group and MTD / 1 group, indicating that the fermentation effect of Lactobacillus plantarum CICC24194 is better than that of the control group.

[0054] (2) The chemical composition of the whole-plant corn silage after 90 days of fermentation is shown in Table 2.

[0055] Table 2 Chemical composition of whole-plant corn silage after 90 days of fermentation

[0056]

[0057]

[0058] Note: DM loss, dry matter loss; FW, fresh weight; DM, dry matter; WSC, water-soluble carbohydrates; CP, crude protein; NPN, non-protein nitrogen; TN, total nitrogen; NH3-N, ammonia nitrogen; aNDF, acid detergent fiber; ADF, acid detergent fiber a-c Different lowercase letters indicate significant differences (P < 0.05) between treatment groups

[0059] The results show that both the CICC24194 group and the MTD / 1 group significantly increased the DM content of the whole-plant corn silage (P < 0.05) and significantly reduced the DM loss (P < 0.05). The CICC24194 group had the highest DM content (P < 0.05) and the lowest DM loss (P < 0.05).

[0060] The CP content in the CICC24194 group was significantly increased (P<0.05), while the NPN, NH3-N, ADF and aNDF contents were significantly decreased (P<0.05). The results showed that Lactobacillus plantarum CICC24194 reduced the DM loss of whole-plant corn silage and retained more nutrients.

[0061] (3) The bacterial community composition of whole-plant corn silage after 90 days of fermentation is shown in Table 3. Figure 1

[0062] The results showed that the relative abundance of the beneficial bacteria Lactobacillus buchneri in the CICC24194 group (producing bacteriocin LP) was significantly increased compared with the control group. In addition, the bacteriocin-producing Lactobacillus plantarum CICC24194 significantly reduced the relative abundance of the harmful bacteria Clostridium_sp._A18 and Uncultured_Bacillus_sp., as well as Uncultured_Bacterium. Among them, Clostridium and Bacillus are the main harmful bacteria that easily cause the corruption of silage, and there is no similar record in the prior art that silage fermentation can reduce the content of Clostridium_sp._A18.

[0063] Experimental Example 2

[0064] The fermentation products obtained from the MTD / 1 group, the CICC24194 group and the control group were subjected to in vitro rumen fermentation test.

[0065] (1) The donors of the rumen fluid were three artificial rumen lake sheep from the Gaolan Ecological and Agricultural Comprehensive Experimental Station of the Northwest Institute of Ecology and Environment, Chinese Academy of Sciences. They were fed twice a day (08:00 and 18:00) with mixed feed (TMR) containing 58% corn, 19% wheat bran, 18% soybean meal, 1% baking soda and 4% vitamins and a suitable amount of mineral supplements.

[0066] (2) The collected rumen fluid was filtered with four layers of sterile gauze and poured into a sterilized bottle (1500mL) preheated to 39℃. CO2 was immediately introduced into the sterilized bottle containing the filtrate to expel air.

[0067] (3) The artificial buffer was preheated to 39℃ and continuously introduced with CO2. The artificial buffer: filtrate obtained in step (2) was mixed at a ratio of 4:1 (v / v) to obtain artificial rumen fluid.

[0068] ​The configuration method of the artificial buffer solution is as follows: 237 mL of buffer solution (4.0 g / L NH4HCO3+ 35.0 g / L NaHCO3) per 1 L of artificial buffer solution (pH 7.0), 237 mL of constant elements (5.7 g / L Na2HPO4+ 6.2 g / L KH2PO4), 0.12 mL of trace elements (13.2 g / 100 mL CaCl2·2H2O+ 10.0 g / 100 mL MnCl2·2H2O+ 1.00 g / 100 mL CoCl2·6H2O+ 8.00 g / 100 mL FeCl3+ 6H2O), 1.22 mL of resazurin (100 mg / 100 mL), 50 mL of reducing agent (285 mg / 50 mL Na2S·7H2O+ 800 mg / 50 mL NaOH), and 474 mL of distilled water.

[0069] (4) 0.5 g of sample (fermented feed of different experimental groups, dried at 65°C for 72 h, and sieved through a 1 mm sieve) was weighed into a pre-dried fiber bag (F57, Ankom, USA) to a constant weight, and high-temperature sealing treatment was performed. Each experimental group was in quadruplicate.

[0070] Another 3 fiber bags were not placed into any sample as blank controls. Each fiber bag was separately placed into a 100 mL sterile sealed glass bottle (containing 50 mL of artificial rumen fluid).

[0071] (5) Two sterile sealed glass bottles of each experimental group in step (4) were connected to a microbial fermentation gas production automatic recorder (Beijing Boxiang Xingwang Technology Co., Ltd.), 90 mL of mixed in vitro fermentation liquid (70 mL of artificial buffer solution and 20 mL of filtrate) was added, and the total gas production was monitored. The remaining two sterile sealed glass bottles were connected to a gas collection bag (500 mL) for subsequent CH4 concentration analysis. All sterile sealed glass bottles were incubated at 39°C for 48 h.

[0072] (6) After 48 h of incubation, the fiber bag was washed with warm water until it was colorless, and dried at 105°C for 3 h to calculate the in vitro dry matter digestibility (IVDMD).

[0073] (7) 0.2 mL of 25% H3PO4 (containing 2-methyl butyric acid) was added to 1 mL of culture solution, and the concentration of volatile fatty acids (VFA) in the culture solution was determined by gas chromatography (trace1300, Thermo, Singapore; detector: conductivity detector, ECD; capillary column: 30 m x 0.32 x mm 0.50 x μm, Lanzhou Zhongke Kaide Chemical Industry New Technology Co., Ltd.).

[0074] The CH4 concentration was analyzed by gas chromatograph (trace 1300, Thermo Fisher, Singapore; detector: hydrogen flame ionization detector, FID; column, 19095P-QO3, 30 m x 0.53 mm x 40.00 pm; Agilent, USA).

[0075] The in vitro rumen fermentation characteristics and methane production of whole-plant corn silage are shown in Table 3.

[0076] Table 3 In vitro rumen fermentation characteristics of whole-plant corn silage

[0077]

[0078] Note: IDVMD, in vitro dry matter digestibility; NH3-N, ammonia nitrogen; Total gas, total gas production; CH4-to-total gas, CH4 / total gas ratio; SEM, standard error of mean. a-c Different lowercase letters represent significant differences between treatments (P < 0.05).

[0079] The results showed that compared with the control group, the MTD / 1 group and the CICC24194 group could significantly increase the IDVMD, NH3 and propionic acid (P < 0.05), while the total gas production was reduced and the concentration of valeric acid was increased (P < 0.05). Both treatment groups significantly reduced CH4 production, CH4 / total gas ratio, acetic / propionic acid ratio, acetic acid and butyric acid concentrations (P < 0.05); while CH4 production, CH4 / total gas ratio and butyric acid concentration were significantly lower in the CICC24194 group than in the MTD / 1 group (P < 0.05).

[0080] (2) The rumen in vitro fermentation bacterial community composition of whole-plant corn silage is shown in Table 4. Figure 2 In the Lactobacillus plantarum CICC24194 group (bacteriocin-producing LP), the relative abundance of Succinivibrionaceae_UCG-002 and Prevotella_1 was the lowest, while the relative abundance of Rikenellaceae_RC9_gut_group and Treponema_2 was the highest. The fermentation end products of Succinivibrionaceae were acetic acid and butyric acid, and its metabolic pathway tended to produce CH4. The main end product of Treponema fermentation was succinic acid, and its metabolic pathway tended to produce propionic acid rather than CH4. The results of microbial community composition were consistent with the results of reduced CH4 production and increased IDVMD in the CICC24194 group.

[0081] The above results show that, compared with the commercial Lactobacillus plantarum MTD / 1, Lactobacillus plantarum CICC24194 can effectively reduce the dry matter loss of whole corn silage, retain more nutrients, and promote the growth of beneficial bacteria in whole corn silage, while effectively inhibiting the growth of harmful bacteria. In addition, Lactobacillus plantarum CICC24194 can reduce CH4 production and improve digestibility by improving the composition of microorganisms in in vitro rumen fermentation.

[0082] The above merely describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.

Claims

1. A method of fermenting silage, characterized in that, It comprises the following steps: (1) crushing corn stalks; (2) mixing Lactobacillus plantarum CICC24194 with the crushed corn stalks, wrapping and fermenting; The fermentation time in step (2) is 80-100 days; The moisture content of the corn stalks in step (1) is 50-70%; The dried corn stalks in step (1) are crushed to a length of 1-2 cm; The viable cell number of the Lactobacillus plantarum CICC24194 in step (2) is (1-5) x 10 7 CFU / mL. The volume-mass ratio of Lactobacillus plantarum CICC24194 to the crushed corn stalks in step (2) is 1 mL: 80-100 g.

2. The fermentation process according to claim 1, characterized in that, The density of the wrapped package of step (2) is 0.91 to 0.93 g / cm3 3 .

Images