阅读说明：本技术 一种提高青贮饲料有氧稳定性的方法 (Method for improving aerobic stability of silage ) 是由 王加友 王远 孙丽娟 杨德玉 李小明 焦姣 董常鹏 于 2020-12-21 设计创作，主要内容包括：本发明属于青贮饲料制备技术领域,涉及一种提高青贮饲料有氧稳定性的方法。将青贮饲料发酵剂种于培养基中在厌氧条件下培养至体系pH为3.95-4.45,且体系内乳酸浓度/乙酸浓度比值为9.69～13.25,将所得培养液离心,收集沉淀将其接种至青贮原料发酵成青贮饲料,即可提高青贮饲料有氧稳定性。本发明通过控制青贮饲料接种培养物的产酸量,乳酸、乙酸的含量及比例,使乳酸浓度/乙酸浓度比值在的比例在9.69-13.25之间,进而在制备青贮饲料过程中可以有效抑制霉菌、酵母菌的生长,大幅度的提高青贮饲料的有氧稳定性,可将开窖后青贮饲料的储存时间延长至40d以上,而且可以提高青贮饲料感官评价评分,改善青贮饲料的品质。(The invention belongs to the technical field of silage preparation, and relates to a method for improving aerobic stability of silage. The silage starter is cultured in a culture medium under an anaerobic condition until the pH value of a system is 3.95-4.45 and the ratio of the concentration of lactic acid to the concentration of acetic acid in the system is 9.69-13.25, the obtained culture solution is centrifuged, and the obtained culture solution is collected, precipitated and inoculated to silage raw materials to be fermented into silage, so that the aerobic stability of the silage can be improved. The invention controls the acid yield of the silage inoculation culture, the content and the proportion of the lactic acid and the acetic acid, so that the ratio of the lactic acid concentration to the acetic acid concentration is 9.69-13.25, the growth of mould and yeast can be effectively inhibited in the silage preparation process, the aerobic stability of the silage is greatly improved, the storage time of the silage after opening the pit can be prolonged to more than 40d, the sensory evaluation score of the silage can be improved, and the quality of the silage is improved.)

1. A method for improving the aerobic stability of silage, which is characterized by comprising the following steps: the silage starter is cultured in a culture medium under an anaerobic condition until the pH value of a system is 3.95-4.45 and the ratio of the concentration of lactic acid to the concentration of acetic acid in the system is 9.69-13.25, the obtained culture solution is centrifuged, and the obtained culture solution is collected, precipitated and inoculated to silage raw materials to be fermented into silage, so that the aerobic stability of the silage can be improved.

2. A method of improving the aerobic stability of silage as claimed in claim 1, wherein: inoculating the silage starter in a culture medium, culturing under an anaerobic condition until the pH value of a system is 3.95-4.45, the ratio of the concentration of lactic acid to the concentration of acetic acid in the system is 9.69-13.25, the concentration of propionic acid is less than or equal to 0.92g/L, centrifuging the obtained culture solution, collecting precipitates, and carrying out precipitation on the precipitates according to the ratio of 1 × 10⁵cfu/g～5×10⁵cfu/g (silage) is inoculated into silage raw materials and fermented into silage, so that the aerobic stability of the silage can be improved.

3. A method of improving the aerobic stability of silage as claimed in claim 1 or 2, characterized in that: the culture medium is an improved MRS culture medium.

4. The method of improving aerobic stability of silage according to claim 3, wherein the modified MRS medium formulation is: 10.0-20g/L of peptone, 10.0-16g/L of beef extract, 5.0-8.8g/L of yeast powder, 20.0-30g/L of glucose, 2.8-8.6g/L of sodium acetate, 2.0g/L of dipotassium hydrogen phosphate, 2.0g/L of diammonium hydrogen citrate, 0.2g/L of MgSO4 & 7H2O 0.2, 0.05g/L of MnSO4 & 4H2O 0.05, Tween-801 mL and pH 6.2-6.4.

5. A method of improving the aerobic stability of silage as claimed in claim 2, wherein: after the silage starter culture is cultured, the cell number of a culture reaches 5 multiplied by 10⁸cfu/mL-4×10⁹cfu/mL, the pH value of the fermentation liquid reaches 3.95-4.45, the ratio of the concentration of lactic acid to the concentration of acetic acid is 9.69-13.25, and the concentration of propionic acid is less than or equal to 0.92 g/L.

6. A method of improving the aerobic stability of silage as claimed in claim 1 or 2, characterized in that: the silage raw materials are whole corn, corn straw without ears and alfalfa, and are also suitable for other straw plants which can be ensiled.

7. A method of improving the aerobic stability of silage as claimed in claim 1 or 2, characterized in that: the silage starter is lactobacillus and/or silage culture.

8. The method of improving the aerobic stability of silage as claimed in claim 7, wherein: the lactobacillus is one or more of Lactobacillus buchneri, Lactobacillus brevis, Lactobacillus coryniformis, Lactobacillus hilgardii and Leuconostoc cremoris.

Technical Field

The invention belongs to the technical field of silage preparation, and relates to a method for improving aerobic stability of silage.

Background

Ensiling is a storage mode for storing feed for a long time by utilizing the anaerobic fermentation of lactic acid bacteria in ensiling materials to enable the raw materials to generate organic acid, reduce the pH value and inhibit and kill the multiplication of various harmful microorganisms. The silage biological feed is an important feed source for ruminants, can effectively preserve nutrient components of silage plants, ensures fresh and tender juice of the raw materials when the raw materials are green, has the advantages of soft texture, good palatability, high digestibility and the like, and is an important feed source for the ruminants (such as cattle, sheep and the like). Ensiling may occur in silos, silage heaps, silage silos, silage bales, or any other method suitable for ensiling the selected plant material.

Ensiling is an effective and economical method to solve the problem of difficult storage of green fodder. However, ensiling is difficult in a natural state, so that the microbial inoculum is generally added for ensiling at present, and strains such as lactobacillus plantarum, lactobacillus buchneri, lactobacillus brevis, pediococcus pentosaceus and the like are added into the ensiling to accelerate the fermentation speed of ensiling, so that the quality of the ensiling is improved. CN106615609B discloses that the provided lactobacillus buchneri can increase the silage fermentation quality of alfalfa and japonica rice straw, reduce protein loss and increase the aerobic stability of silage; the patent describes that the aerobic stability time is 422h at the longest, and the aerobic stability time is shorter, which is not beneficial to the storage of silage.

CN 106103697B discloses a method for treating silage to enhance aerobic stability by inhibiting the growth of microorganisms selected from the group consisting of yeast, mold and spore forming bacteria, improving the fermentation and stability of the silage and allowing for earlier aerobic exposure, the invention aerobic stability time being 30 d. The bacterial used in the patent is Lactobacillus buchneri or Lactobacillus brevis, the patent does not mention the strain screening and evaluating method,

CN 106028829B provides a method for treating silage, comprising adding a silage inoculant to the silage, the silage inoculant being effective to prevent or reduce aerobic spoilage. The silage provided by the invention has the aerobic stability for 73.05h, and the aerobic stability is poor, so that the quality of the silage after opening the cellar is influenced.

Although the methods can improve the corresponding aerobic stability to a certain extent, the aerobic stability of the silage is short, the quality of the silage after opening the pit is influenced, the long-term storage of the silage is not facilitated, and a method for prolonging the aerobic stability is further needed.

Disclosure of Invention

The invention aims to provide a method for improving the aerobic stability of silage.

In order to achieve the purpose, the invention adopts the technical scheme that:

a method for improving the aerobic stability of silage comprises the steps of culturing silage starter in a culture medium under an anaerobic condition until the pH value of a system is 3.95-4.45 and the ratio of the concentration of lactic acid to the concentration of acetic acid in the system is 9.69-13.25, centrifuging the obtained culture solution, collecting precipitates, inoculating the precipitates to silage raw materials, and fermenting the silage raw materials into the silage, so that the aerobic stability of the silage can be improved.

Inoculating the silage starter in a culture medium, culturing under an anaerobic condition until the pH value of a system is 3.95-4.45, the ratio of the concentration of lactic acid to the concentration of acetic acid in the system is 9.69-13.25, the concentration of propionic acid is less than or equal to 0.92g/L, centrifuging the obtained culture solution, collecting precipitates, and carrying out precipitation on the precipitates according to the ratio of 1 × 10⁵cfu/g～5×10⁵cfu/g (silage) toThe aerobic stability of the silage can be improved by fermenting the silage raw materials into silage.

The culture medium is an improved MRS culture medium.

The improved MRS culture medium comprises the following components in percentage by weight: 10.0-20g/L of peptone, 10.0-16g/L of beef extract, 5.0-8.8g/L of yeast powder, 20.0-30g/L of glucose, 2.8-8.6g/L of sodium acetate, 2.0g/L of dipotassium hydrogen phosphate, 2.0g/L of diammonium hydrogen citrate, 0.2g/L of MgSO4 & 7H2O 0.2, 0.05g/L of MnSO4 & 4H2O 0.05, Tween-801 mL and pH 6.2-6.4.

After the silage starter culture is cultured, the cell number of a culture reaches 5 multiplied by 10⁸cfu/mL-4×10⁹cfu/mL, the pH value of the fermentation liquid reaches 3.95-4.45, the ratio of the concentration of lactic acid to the concentration of acetic acid is 9.69-13.25, and the concentration of propionic acid is less than or equal to 0.92 g/L.

The silage raw materials are whole corn, corn straw without ears and alfalfa, and are also suitable for other straw plants which can be ensiled.

The silage starter is lactobacillus and/or silage culture.

The lactobacillus is one or more of Lactobacillus buchneri, Lactobacillus brevis, Lactobacillus coryniformis, Lactobacillus hilgardii and Leuconostoc cremoris.

The invention has the advantages and positive effects that:

the invention controls the acid yield of the silage inoculation culture, the content and the proportion of the lactic acid and the acetic acid, so that the ratio of the lactic acid concentration to the acetic acid concentration is 9.69-13.25, the growth of mould and yeast can be effectively inhibited in the silage preparation process, the aerobic stability of the silage is greatly improved, the storage time of the silage after opening the pit can be prolonged to more than 40d, the sensory evaluation score of the silage can be improved, and the quality of the silage is improved.

Detailed Description

The following examples are presented to further illustrate embodiments of the present invention, and it should be understood that the embodiments described herein are for purposes of illustration and explanation only and are not intended to limit the invention.

The following silage aerobic stability determinations: after silage fermentation is finished, 3 parts of silage which is well stored are randomly taken by each treatment group, the silage is uniformly mixed and sampled, 800-1000 g of the uniformly mixed silage is placed in a large plastic sealing bag until the plastic bag is full, a plurality of small holes are pricked by toothpicks, a loose and unsealed plastic bag is sleeved again, cross contamination is prevented, moisture loss is reduced, and temperature change is measured by a common thermometer at 50 ℃. The samples were placed in the dark, the ambient temperature was determined according to the actual conditions, the temperature change was recorded 1 time every 4h, until the temperature of the silage material exceeded the ambient temperature by 2 ℃. In the test, the tools such as thermometers, toothpicks and the like need to be sterilized by ultraviolet before use.

And (3) pH value measurement: after silage is finished, opening the sealing bags, respectively and uniformly mixing the silage of each group, accurately weighing 20g of silage, adding 180ml of distilled water, uniformly stirring, crushing by using a tissue crushing machine for 1min, standing for precipitation, filtering by using four layers of gauze and qualitative filter paper, filtering out grass residues to obtain leachate, and directly measuring the pH value of the leachate, namely the pH value of the silage by using a pH meter.

The method for measuring the content of the organic acid by the high performance liquid phase comprises the following steps: equipment model Agilent 1260, chromatographic column Agilent SB-C18; liquid phase conditions: mobile phase a was acetonitrile, mobile phase B was 0.08% phosphoric acid solution, a: B ═ 5: 95; the detection wavelength is 200nm, and the flow rate is 0.7 mL/min; the loading amount was 10. mu.L.

Example 1

Placing 20g of commercially available silage corn straw into a 500mL triangular flask (containing 180mL of sterile water), sealing, placing on a shaker, shaking at 150r/min for 2h, sucking 1mL of supernatant, adding into 9mL of sterile water, performing vortex oscillation, diluting with sterile water, and diluting to 10%^-4、10^-5、10^-6、10^-7Respectively taking 100 mu L of bacterial liquid with 4 gradients, coating the bacterial liquid on an improved solid MRS plate culture medium, carrying out anaerobic culture at 30 ℃ for 48-72h, selecting a single bacterial colony according to the color, the size and the luster of the bacterial colony, whether a transparent ring exists or not, carrying out streaking on the selected single bacterial colony on the MRS solid culture medium, carrying out anaerobic culture at the constant temperature of 30 ℃ for 24-60h, and repeating streaking separation culture for 3 times according to the mode until the culture is single in shape, namelyPure cultures were obtained.

The pure culture is transferred to an improved MRS culture medium to be cultured for 24h, then the pH value of the fermentation liquor is measured to be 4.45, and the organic acid in the fermentation liquor is measured by utilizing a high performance liquid chromatography, wherein the lactic acid content of the culture is 6.89g/L, the acetic acid content is 0.52g/L, the propionic acid content is 0.36g/L, no butyric acid is generated, and the ratio of the lactic acid concentration to the acetic acid concentration is 13.25. The method for measuring the content of the organic acid by the high performance liquid phase comprises the following steps: equipment model Agilent 1260, chromatographic column Agilent SB-C18; liquid phase conditions: mobile phase a was acetonitrile, mobile phase B was 0.08% phosphoric acid solution, a: B ═ 5: 95; the detection wavelength is 200nm, and the flow rate is 0.7 mL/min; the loading amount was 10. mu.L.

The culture is picked up and cultured for 24h by using an improved MRS liquid culture medium, and the colony count can reach 1.5 multiplied by 10⁹cfu/mL (counted by plating plate colonies) and the cells were collected by centrifugation and used for corn silage experiments.

Preparing silage: inoculating the pure culture into corn straws, which comprises the following steps: harvesting whole corn in wax ripeness stage, cutting into 1-3cm long, centrifuging to obtain pure culture with experimental design dosage of 2 × 10⁵cfu/g of straws are dissolved in 500g of deionized water, evenly sprayed on 50kg of chopped whole corn plants by a sprayer, and evenly stirred; placing in ensiling bag, vacuumizing, sealing, storing in room temperature environment at 22-28 deg.C for 45d, testing, measuring pH value and aerobic stability of ensiling straw, and performing sensory evaluation. The experiments were done in 3 replicates. After ensiling is finished, the ensiled corn straws are yellow-green, have aromatic sour taste, wet and compact appearance and better sensory image; the pH value of the silage corn is measured to be 3.89, the aerobic stability time reaches 45d, and the storage period of the silage is greatly prolonged.

Example 2

Placing 20g of commercially available ensilage alfalfa into a 500mL triangular flask (containing 180mL of sterile water), sealing, placing on a shaker, shaking at 150r/min for 2h, sucking 1mL of supernatant, adding into 9mL of sterile water, vortexing, diluting with sterile water, and diluting to 10%^-4、10^-5、10^-6、10^-7The 4 gradients are equal to each other and,respectively taking 100 mu L of 4 gradient bacterial liquids, coating the bacterial liquids on an improved solid MRS plate culture medium, carrying out anaerobic culture at 30 ℃ for 48-72h, selecting single bacterial colonies according to the color, the size and the luster of the bacterial colonies, whether transparent rings exist or not, and the like, streaking the selected single bacterial colonies on the MRS solid culture medium, carrying out anaerobic culture at the constant temperature of 30 ℃ for 24-60h, repeating streaking separation culture for 3 times according to the mode until the culture is single in shape, and obtaining a pure culture.

The pure culture is transferred to an improved MRS culture medium to be cultured for 24h, then the pH value in the fermentation liquor is determined to be 4.21, and the organic acid in the fermentation liquor is determined by utilizing a high performance liquid chromatography, wherein the lactic acid content is 19.16g/L, the acetic acid content is 1.98g/L, no propionic acid or butyric acid is generated, and the ratio of the lactic acid concentration to the acetic acid concentration is 9.69. The method for measuring the content of the organic acid by the high performance liquid phase comprises the following steps: equipment model Agilent 1260, chromatographic column Agilent SB-C18; liquid phase conditions: mobile phase a was acetonitrile, mobile phase B was 0.08% phosphoric acid solution, a: B ═ 5: 95; the detection wavelength is 200nm, and the flow rate is 0.7 mL/min; the loading amount was 10. mu.L. Selecting the culture, and culturing with improved MRS liquid culture medium at 30 deg.C for 24 hr to obtain 2.5 × 10 colonies⁹cfu/mL (by plating plate colony count), centrifugation to collect thalli, for use in preparing alfalfa ensilage test.

Preparing silage: taking 2-year-old alfalfa mown 2 nd time (8 months) in the current year as a silage raw material, after mowing, wilting the plants for 4h in the sun, and cutting the plants to 1.0-1.5 cm. Inoculating the pure culture into alfalfa straw, and using 5 × 10 of the pure culture according to the design of experiments⁵Dissolving cfu/g alfalfa in 500g deionized water, uniformly spraying onto 30kg of chopped alfalfa by using a sprayer, and uniformly stirring; placing into silage bags, vacuumizing, sealing, storing in room temperature environment at 22-28 deg.C for silage period of 60d, testing pH and aerobic stability of silage alfalfa, and performing sensory evaluation. The experiments were done in 3 replicates. The pH value of the ensilaged alfalfa is 3.98 after 60 days, the aerobic stability time reaches 42 days, and the storage period of the ensilaged feed is greatly prolonged.

Example 3

Commercially available strain Lactobacillus corynebacterium was cultured in MRS medium for 20h and then assayedThe pH value of the fermentation liquor is 3.95, and the organic acid in the fermentation liquor is measured by adopting a high performance liquid chromatography, wherein the content of lactic acid is 28.65g/L, the content of acetic acid is 2.61g/L, the content of propionic acid is 0.92g/L, no butyric acid is generated, and the ratio of the concentration of lactic acid to the concentration of acetic acid is 10.98. The strain is cultured for 20-24h by using MRS liquid culture medium, thalli are collected by centrifugation, and bacterial colony count of a coating plate can reach 3.0 multiplied by 10⁹cfu/mL。

Ensiling test: harvesting whole corn in wax ripeness stage, cutting into 1-3cm long, and using 1 × 10 of centrifuged Lactobacillus corynebacterium thallus according to experimental design⁵cfu/g of straws are dissolved in 500g of deionized water, evenly sprayed on 50kg of chopped whole corn plants by a sprayer, and evenly stirred; placing in ensiling bag, vacuumizing, sealing, storing in room temperature environment at 22-28 deg.C for ensiling period of 45d, testing, measuring pH value and aerobic stability of ensiling straw, and performing sensory evaluation. The experiments were done in 3 replicates. 45d, the pH value of the silage corn is 3.93, the silage corn is yellow-green in appearance and has aromatic sour taste, and sensory evaluation is better; the aerobic stability time is 41.3d, and the shelf life of the silage is prolonged.

Comparative example

The pH value of fermentation liquor is measured to be 4.41 after a commercial strain Lactobacillus buchneri (preservation number: CGMCC1.15607) is cultured for 24 hours in an MRS culture medium under anaerobic condition, and organic acid in the fermentation liquor is measured by high performance liquid chromatography, wherein the ratio of lactic acid concentration to acetic acid concentration is 13.26, the lactic acid content is 8.57g/L, the acetic acid content is 0.65g/L, the propionic acid content is 0.34g/L, and no butyric acid is generated. The strain is cultured for 24h by using an improved MRS liquid culture medium, and the colony count of the strain coated with a flat plate can reach 1.3 multiplied by 10⁹cfu/mL, and centrifugally collecting the thalli for preparing a corn silage test.

Preparing silage:

harvesting whole corn in wax ripeness stage, cutting into 1-3cm long, and centrifuging to obtain thallus at 2 × 10⁵cfu/g of straws are dissolved in 500g of deionized water, evenly sprayed on 50kg of chopped whole corn plants by a sprayer, and evenly stirred; placing in silage bag, vacuumizing, sealing, storing in roomIn a warm environment, the environment temperature is 22-28 ℃, the ensiling period is 45d, the pH value and the aerobic stability of the ensiled straws are measured after the test, and sensory evaluation is carried out. The experiments were done in 3 replicates. After 45 days, determining the pH value of the ensiled straws to be 3.83, and the aerobic stability time to be 15.5 days; the ensiled straw is yellow brown in color, has strong sour taste, is moist in appearance and has better sensory evaluation.