Corn straw silage leavening agent and preparation method thereof

Abstract

The invention discloses a corn straw silage leavening agent and a preparation method thereof, and particularly relates to the technical field of corn straw silage leavening agent preparation.The corn straw silage leavening agent is prepared from, by weight, 15.9%-16.0% of corn straw, 79.7%-80.1% of distilled water, 1.0%-2.0% of molasses, 1.0% of lactic acid bacteria inoculum, 1.0% of aspergillus niger inoculum and 1.0% of lactobacillus plantarum DL3 inoculum. The corn straw is fresh corn straw with the water content of 60-70%. According to the method, the aspergillus niger inoculum is introduced, the aspergillus niger is used for decomposing lignin in the corn straw, the aspergillus niger can break a protective layer on the surface of the straw in a short time, degradation of lignin, cellulose, hemicellulose and other substances in the corn straw is achieved, the decomposition efficiency of the corn straw is improved, and the fermentation duration of the corn straw is shortened.

Description

Technical Field

[0001] The invention relates to the technical field of corn straw silage fermentation agent preparation, and more specifically to a corn straw silage fermentation agent and a preparation method thereof. Background Art

[0002] Silage starter is an additive used in the production of silage. Its main purpose is to improve the fermentation process and quality of silage by promoting the growth of beneficial microorganisms such as lactic acid bacteria. Silage starter can improve the nutritional value, palatability and preservation of feed, while reducing the growth of harmful microorganisms and preventing feed from mold and deterioration. Corn stalks, peanut vines, and sweet potato vines are all raw materials for the preparation of silage starter. Corn is the main agricultural crop, and corn stalks are more widely available. Therefore, corn stalks are often used as the main raw material for silage starter.

[0003] Usually, the silage fermentation agent using corn stalks as raw materials needs to use Bacillus subtilis used in a corn stalk biocomposite fermentation agent and a method for producing silage corn feed with prior art publication number CN106689669A to decompose cellulose and hemicellulose in corn stalks. However, in addition to cellulose and hemicellulose, corn stalks also contain about 15.42% lignin, which is one of the main components of plant cell walls and provides strength and hardness to plants. The presence of lignin will affect the decomposition of cellulose and hemicellulose by microorganisms. Although Bacillus subtilis has a certain ability to decompose lignin, the decomposition efficiency of Bacillus subtilis is low, which will affect the fermentation efficiency of the entire corn stalk. Summary of the invention

[0004] In order to overcome the above-mentioned defects of the prior art, the present invention provides a corn straw silage fermentation agent and a preparation method thereof. By introducing Aspergillus niger inoculum, Aspergillus niger is used to decompose lignin in corn straw. Aspergillus niger can break the protective layer on the surface of the straw in a short time, thereby achieving the degradation of lignin, cellulose, hemicellulose and other substances in the corn straw, improving the decomposition efficiency of corn straw, shortening the fermentation time of corn straw, so as to solve the problems arising from the above-mentioned background technology.

[0005] To achieve the above-mentioned purpose, the present invention provides the following technical scheme: a corn straw silage fermentation agent, wherein the raw materials used include, by weight, 15.9-16.0% corn straw, 79.7-80.1% distilled water, 1.0-2.0% molasses, 1.0% lactic acid bacteria inoculum, 1.0% Aspergillus niger inoculum, and 1.0% Lactobacillus plantarum DL3 inoculum.

[0006] In a preferred embodiment, the corn stalks are fresh corn stalks with a moisture content of 60-70%.

[0007] The present invention also provides a method for preparing the corn straw silage fermentation agent, comprising the following specific steps:

[0008] Step 1: pretreatment of corn stalks: cut corn stalks into small segments of 3-5 cm, then add the small segments of corn stalks and distilled water into a mixer, then add dilute acid, grind and stir, react for 3-4 minutes at a temperature of 150-190°C and a pH of 2.1-2.4, filter with 2 layers of gauze to obtain green juice;

[0009] Step 2, preparing lactic acid bacteria inoculum;

[0010] Step 3, preparing Aspergillus niger inoculum;

[0011] Step 4, preparing Lactobacillus plantarum DL3 inoculum;

[0012] Step 5: Mix the green juice with molasses, lactic acid bacteria inoculum, and Aspergillus niger inoculum, and ferment under anaerobic conditions at 30° C. for 3 days to obtain corn straw silage fermentation liquid;

[0013] Step 6: Ferment the corn straw silage fermentation liquid with the plant lactobacillus DL3 inoculum at 30°C and pH 6.0-6.5 for 1-2 days to obtain a corn straw silage fermentation agent.

[0014] In a preferred embodiment, the dilute acid added in step 1 is one of dilute sulfuric acid and dilute hydrochloric acid.

[0015] In a preferred embodiment, the steps of preparing the lactic acid bacteria inoculum in step 2 are as follows: S2.1, selecting and activating excellent lactic acid bacteria strains that have vigorous growth and can reach the logarithmic growth phase within 4-8 hours after inoculation; S2.2, inoculating the activated lactic acid bacteria strains into a lactic acid bacteria culture medium; S2.3, fermenting and culturing at 37°C. After the culture is completed, the bacteria are harvested to obtain the lactic acid bacteria inoculum.

[0016] In a preferred embodiment, the steps of preparing the Aspergillus niger inoculum in step three are as follows: S3.1, scraping the spores of Aspergillus niger from the slant culture medium, suspending them in sterile water, and preparing a spore suspension; S3.2, inoculating the spore suspension into Czapek medium at a culture temperature of 37° C., culturing, and harvesting the cells after the culture is completed to obtain the Aspergillus niger inoculum.

[0017] In a preferred embodiment, the steps of the plant lactobacillus DL3 inoculum in step 4 are as follows: S4.1, inoculating plant lactobacillus DL3 into MRS liquid culture medium, and culturing at 30-37°C for 18-30h to activate the bacteria; S4.2, inoculating the activated bacteria into a larger volume of MRS liquid culture medium for expansion culture until the required bacterial mass is reached; S4.3, collecting the bacteria by centrifugation, pouring out the supernatant, and washing the bacteria with sterile saline for 2-3 times to obtain bacterial mud; S4.4, mixing the bacterial mud with saline at a mass volume ratio of 1g:5ml to obtain a bacterial concentrate; S4.5, inoculating the bacterial concentrate into the fermentation medium according to the required inoculum amount, and fermenting and culturing. After the cultivation is completed, the bacteria are harvested to obtain the plant lactobacillus DL3 inoculum.

[0018] In a preferred embodiment, the centrifugation operation in step S4.3 needs to be performed under the conditions of a relative centrifugal force of 14136×g and a temperature of 4° C., and the centrifugation time is 10 min.

[0019] Technical effects and advantages of the present invention:

[0020] 1. In view of the problem that Bacillus subtilis in the prior art has a slow efficiency in decomposing lignin in corn stalks, the present invention specifically introduces Aspergillus niger inoculum and utilizes Aspergillus niger to decompose lignin in corn stalks. Aspergillus niger can break the protective layer on the surface of the stalks in a short time, thereby achieving the degradation of lignin, cellulose, hemicellulose and other substances in the corn stalks, improving the decomposition efficiency of the corn stalks and shortening the fermentation time of the corn stalks.

[0021] 2. The Aspergillus niger used in the present invention can interact with lactic acid bacteria to promote the growth and metabolism of each other. Moreover, the product obtained by mixed fermentation of Aspergillus niger and lactic acid bacteria not only has the sour aroma after lactic acid bacteria fermentation and rich lactic acid bacteria and a large amount of lactic acid, but also has rich enzyme substances in the metabolites of Aspergillus niger, which is helpful to improve the taste and nutritional value of corn straw silage.

[0022] 3. The present invention utilizes Lactobacillus plantarum DL3 in the Lactobacillus plantarum DL3 inoculum to destroy the integrity of the Aspergillus niger buns, so as to avoid the presence of pathogenic Aspergillus niger in the prepared corn straw silage fermentation agent, thereby improving the safety of the product. DETAILED DESCRIPTION

[0023] The technical solutions in the present invention will be described clearly and completely below in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of 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.

[0024] Example 1

[0025] The invention provides a corn straw silage fermentation agent. The raw materials used include, by weight, 15.9-16.0% of corn straw (the corn straw is fresh corn straw with a water content of 60-70%), 79.7-80.1% of distilled water, 1.0-2.0% of molasses, 1.0% of lactic acid bacteria inoculum, 1.0% of Aspergillus niger inoculum, and 1.0% of Lactobacillus plantarum DL3 inoculum.

[0026] Specifically in this embodiment, the raw materials used are 15.9% corn stalks, 80.1% distilled water, 1.0% molasses, 1.0% lactic acid bacteria inoculum, 1.0% Aspergillus niger inoculum, and 1.0% Lactobacillus plantarum DL3 inoculum.

[0027] The method for preparing corn straw silage fermentation agent using the above raw materials is as follows:

[0028] Step 1: pretreatment of corn stalks: cut corn stalks into 3 cm small segments, then add the small segments of corn stalks and distilled water into a mixer, then add dilute sulfuric acid, grind and stir, react for 3 minutes at a temperature of 150°C and a pH of 2.1, filter with 2 layers of gauze to obtain green juice;

[0029] Step 2: preparing lactic acid bacteria inoculum: S2.1: selecting and activating excellent lactic acid bacteria strains that have vigorous growth and can reach the logarithmic growth phase within 4 hours after inoculation;

[0030] S2.2, inoculating the activated lactic acid bacteria into a lactic acid bacteria culture medium;

[0031] S2.3, ferment and culture at 37°C. After the culture is completed, harvest the bacteria to obtain the lactic acid bacteria inoculum;

[0032] Step 3, preparing the Aspergillus niger inoculum: S3.1, scraping the spores of Aspergillus niger from the slant culture medium, suspending them in sterile water, and preparing a spore suspension; S3.2, inoculating the spore suspension into Czapek medium, culturing at a temperature of 37° C., and culturing. After the culturing is completed, harvesting the cells, and obtaining the Aspergillus niger inoculum;

[0033] Step 4, preparing Lactobacillus plantarum DL3 inoculum: S4.1, inoculating Lactobacillus plantarum DL3 into MRS liquid culture medium, and culturing at 37° C. for 18 h to activate the strain;

[0034] S4.2, inoculate the activated bacteria into a larger volume of MRS liquid culture medium for expansion culture until the required bacterial volume is reached;

[0035] S4.3. Collect the cells by centrifugation (the centrifugation operation needs to be carried out under the conditions of relative centrifugal force of 14136×g, temperature of 4°C, and centrifugation time of 10 min), pour out the supernatant, and wash the cells twice with sterile saline to obtain bacterial mud;

[0036] S4.4, mixing bacterial sludge with physiological saline at a mass volume ratio of 1 g:5 ml to obtain bacterial concentrate;

[0037] S4.5. Inoculate the bacterial concentrate into the fermentation medium according to the required inoculation amount for fermentation culture. After the culture is completed, harvest the bacteria to obtain the Lactobacillus plantarum DL3 inoculum.

[0038] Step 5: Mix the green juice with molasses, lactic acid bacteria inoculum, and Aspergillus niger inoculum, and ferment under anaerobic conditions at 30° C. for 3 days to obtain corn straw silage fermentation liquid;

[0039] Step 6: continue to ferment the corn straw silage fermentation liquid and the Lactobacillus plantarum DL3 inoculum at 30° C. and pH 6.0 for 2 days to obtain a corn straw silage fermentation agent.

[0040] The preparation time of the corn straw silage fermentation agent in this embodiment is 5 days, which is 2-5 days shorter than the preparation time of the traditional silage fermentation agent (7-10 days), thereby greatly improving the preparation efficiency of the corn straw silage fermentation agent.

[0041] Example 2

[0042] The invention provides a corn straw silage fermentation agent. The raw materials used include, by weight, 15.9-16.0% of corn straw (the corn straw is fresh corn straw with a water content of 60-70%), 79.7-80.1% of distilled water, 1.0-2.0% of molasses, 1.0% of lactic acid bacteria inoculum, 1.0% of Aspergillus niger inoculum, and 1.0% of Lactobacillus plantarum DL3 inoculum.

[0043] The raw materials used in this example are 16.0% corn stalks, 80.0% distilled water, 1.0% molasses, 1.0% lactic acid bacteria inoculum, 1.0% Aspergillus niger inoculum, and 1.0% Lactobacillus plantarum DL3 inoculum.

[0044] The method for preparing corn straw silage fermentation agent using the above raw materials is as follows:

[0045] Step 1: pretreatment of corn stalks: cut corn stalks into 4 cm small segments, then add the small segments of corn stalks and distilled water into a mixer, then add dilute sulfuric acid, grind and stir, react for 4 minutes at a temperature of 160°C and a pH of 202, filter with 2 layers of gauze to obtain green juice;

[0046] Step 2: preparing lactic acid bacteria inoculum: S2.1: selecting high-quality lactic acid bacteria strains that have vigorous growth and can reach the logarithmic growth phase within 6 hours after inoculation for activation;

[0047] S2.2, inoculating the activated lactic acid bacteria into a lactic acid bacteria culture medium;

[0048] S2.3, ferment and culture at 37°C. After the culture is completed, harvest the bacteria to obtain the lactic acid bacteria inoculum;

[0049] Step 3, preparing the Aspergillus niger inoculum: S3.1, scraping the spores of Aspergillus niger from the slant culture medium, suspending them in sterile water, and preparing a spore suspension; S3.2, inoculating the spore suspension into Czapek medium, culturing at a temperature of 37° C., and culturing. After the culturing is completed, harvesting the cells, and obtaining the Aspergillus niger inoculum;

[0050] Step 4, preparing Lactobacillus plantarum DL3 inoculum: S4.1, inoculating Lactobacillus plantarum DL3 into MRS liquid culture medium, and culturing at 33° C. for 22 hours to activate the strain;

[0051] S4.2, inoculate the activated bacteria into a larger volume of MRS liquid culture medium for expansion culture until the required bacterial volume is reached;

[0052] S4.3. Collect the cells by centrifugation (the centrifugation operation needs to be carried out under the conditions of relative centrifugal force of 14136×g, temperature of 4°C, and centrifugation time of 10 min), pour out the supernatant, and wash the cells twice with sterile saline to obtain bacterial mud;

[0053] S4.4, mixing bacterial sludge with physiological saline at a mass volume ratio of 1 g:5 ml to obtain bacterial concentrate;

[0054] S4.5. Inoculate the bacterial concentrate into the fermentation medium according to the required inoculation amount for fermentation culture. After the culture is completed, harvest the bacteria to obtain the Lactobacillus plantarum DL3 inoculum.

[0055] Step 5: Mix the green juice with molasses, lactic acid bacteria inoculum, and Aspergillus niger inoculum, and ferment under anaerobic conditions at 30° C. for 3 days to obtain corn straw silage fermentation liquid;

[0056] Step 6: continue to ferment the corn straw silage fermentation liquid and the Lactobacillus plantarum DL3 inoculum at 30° C. and pH 6.3 for 2 days to obtain a corn straw silage fermentation agent.

[0057] The preparation time of the corn straw silage fermentation agent in this embodiment is 5 days, which is 2-5 days shorter than the preparation time of the traditional silage fermentation agent (7-10 days), thereby greatly improving the preparation efficiency of the corn straw silage fermentation agent.

[0058] Example 3

[0059] The invention provides a corn straw silage fermentation agent. The raw materials used include, by weight, 15.9-16.0% of corn straw (the corn straw is fresh corn straw with a water content of 60-70%), 79.7-80.1% of distilled water, 1.0-2.0% of molasses, 1.0% of lactic acid bacteria inoculum, 1.0% of Aspergillus niger inoculum, and 1.0% of Lactobacillus plantarum DL3 inoculum.

[0060] The raw materials used in this example are 16.0% corn stalks, 79.7% distilled water, 1.3% molasses, 1.0% lactic acid bacteria inoculum, 1.0% Aspergillus niger inoculum, and 1.0% Lactobacillus plantarum DL3 inoculum.

[0061] The method for preparing corn straw silage fermentation agent using the above raw materials is as follows:

[0062] Step 1: pretreatment of corn stalks: cut corn stalks into 5 cm small segments, then add the small segments of corn stalks and distilled water into a mixer, then add dilute sulfuric acid, grind and stir, react for 4 minutes at a temperature of 190°C and a pH of 2.4, filter with 2 layers of gauze to obtain green juice;

[0063] Step 2: preparing lactic acid bacteria inoculum: S2.1: selecting and activating excellent lactic acid bacteria strains that have vigorous growth and can reach the logarithmic growth phase within 8 hours after inoculation;

[0064] S2.2, inoculating the activated lactic acid bacteria into a lactic acid bacteria culture medium;

[0065] S2.3, ferment and culture at 37°C. After the culture is completed, harvest the bacteria to obtain the lactic acid bacteria inoculum;

[0066] Step 3, preparing the Aspergillus niger inoculum: S3.1, scraping the spores of Aspergillus niger from the slant culture medium, suspending them in sterile water, and preparing a spore suspension; S3.2, inoculating the spore suspension into Czapek medium, culturing at a temperature of 37° C., and culturing. After the culturing is completed, harvesting the cells, and obtaining the Aspergillus niger inoculum;

[0067] Step 4, preparing Lactobacillus plantarum DL3 inoculum: S4.1, inoculating Lactobacillus plantarum DL3 into MRS liquid culture medium, and culturing at 37° C. for 30 h to activate the strain;

[0068] S4.2, inoculate the activated bacteria into a larger volume of MRS liquid culture medium for expansion culture until the required bacterial volume is reached;

[0069] S4.3. Collect the cells by centrifugation (the centrifugation operation needs to be carried out under the conditions of relative centrifugal force of 14136×g, temperature of 4°C, and centrifugation time of 10 min), pour out the supernatant, and wash the cells with sterile saline for 3 times to obtain bacterial mud;

[0070] S4.4, mixing bacterial sludge with physiological saline at a mass volume ratio of 1 g:5 ml to obtain bacterial concentrate;

[0071] S4.5. Inoculate the bacterial concentrate into the fermentation medium according to the required inoculation amount for fermentation culture. After the culture is completed, harvest the bacteria to obtain the Lactobacillus plantarum DL3 inoculum.

[0072] Step 5: Mix the green juice with molasses, lactic acid bacteria inoculum, and Aspergillus niger inoculum, and ferment under anaerobic conditions at 30° C. for 3 days to obtain corn straw silage fermentation liquid;

[0073] Step 6: continue to ferment the corn straw silage fermentation liquid and the Lactobacillus plantarum DL3 inoculum at 30° C. and pH 6.5 for 2 days to obtain a corn straw silage fermentation agent.

[0074] The preparation time of the corn straw silage fermentation agent in this embodiment is 5 days, which is 2-5 days shorter than the preparation time of the traditional silage fermentation agent (7-10 days), thereby greatly improving the preparation efficiency of the corn straw silage fermentation agent.

[0075] The corn straw silage fermentation agent prepared in the above Examples 1-3 was mixed with the crushed corn straw, and the fermented straw sample was inoculated into a potato dextrose agar medium. After culturing for a period of time, it was observed whether Aspergillus niger colonies grew, as shown in the following table:

[0076] Test sample source Whether there is Aspergillus niger colony growth on potato dextrose agar medium Example 1 none Example 2 none Example 3 none

[0077] The present invention can accelerate the fermentation efficiency of corn straw by adding Aspergillus niger. Compared with the prior art, the present invention shortens the time for preparing corn straw silage fermentation agent by 2-5 days, and the Aspergillus niger residues in the corn straw silage fermentation agent prepared by adding Lactobacillus plantarum DL3 can prevent the residual Aspergillus niger from multiplying in large quantities to produce toxins, which helps to improve the safety of subsequent corn straw silage.

[0078] Finally: The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A corn straw silage fermentation agent, characterized in that: The raw materials used include, by weight, 15.9-16.0% corn stalks, 79.7-80.1% distilled water, 1.0-2.0% molasses, 1.0% lactic acid bacteria inoculum, 1.0% Aspergillus niger inoculum, and 1.0% Lactobacillus plantarum DL3 inoculum.

2. The corn straw silage fermentation agent according to claim 1, characterized in that: The corn stalks are fresh corn stalks with a water content of 60-70%.

3. The method for preparing the corn straw silage fermentation agent according to claim 2, characterized in that: The specific steps include: Step 1: pretreatment of corn stalks: cut corn stalks into small segments of 3-5 cm, then add the small segments of corn stalks and distilled water into a mixer, then add dilute acid, grind and stir, react for 3-4 minutes at a temperature of 150-190°C and a pH of 2.1-2.4, filter with 2 layers of gauze to obtain green juice; Step 2, preparing lactic acid bacteria inoculum; Step 3, preparing Aspergillus niger inoculum; Step 4, preparing Lactobacillus plantarum DL3 inoculum; Step 5: Mix the green juice with molasses, lactic acid bacteria inoculum, and Aspergillus niger inoculum, and ferment under anaerobic conditions at 30° C. for 3 days to obtain corn straw silage fermentation liquid; Step 6: continue to ferment the corn straw silage fermentation liquid and the Lactobacillus plantarum DL3 inoculum at 30° C. and pH 6.0-6.5 for 1-2 days to obtain a corn straw silage fermentation agent.

4. The method for preparing the corn straw silage fermentation agent according to claim 3, characterized in that: The dilute acid added in step 1 is one of dilute sulfuric acid and dilute hydrochloric acid.

5. The method for preparing the corn straw silage fermentation agent according to claim 3, characterized in that: The steps of preparing the lactic acid bacteria inoculum in step 2 are as follows: S2.1, selecting and activating excellent lactic acid bacteria strains that have vigorous growth and can reach the logarithmic growth phase within 4-8 hours after inoculation; S2.2, inoculating the activated lactic acid bacteria into a lactic acid bacteria culture medium; S2.

3. Fermentation culture is carried out at 37°C. After the culture is completed, the bacteria are harvested to obtain the lactic acid bacteria inoculum.

6. The corn straw silage fermentation agent and preparation method thereof according to claim 3, characterized in that: The steps of preparing the Aspergillus niger inoculum in step 3 are as follows: S3.1, scraping the spores of Aspergillus niger from the slant culture medium, suspending them in sterile water to prepare a spore suspension; S3.

2. Inoculate the spore suspension into Czapek medium at a culture temperature of 37° C. and culture. After the culture is completed, harvest the bacteria to obtain the Aspergillus niger inoculum.

7. The corn straw silage fermentation agent and preparation method thereof according to claim 3, characterized in that: The steps of inoculating Lactobacillus plantarum DL3 in step 4 are as follows: S4.1, inoculating Lactobacillus plantarum DL3 into MRS liquid culture medium, and culturing at 30-37° C. for 18-30 hours to activate the strain; S4.2, inoculate the activated bacteria into a larger volume of MRS liquid culture medium for expansion culture until the required bacterial volume is reached; S4.

3. Collect the cells by centrifugation, discard the supernatant, and rinse the cells 2-3 times with sterile saline to obtain bacterial sludge; S4.4, mixing bacterial sludge with physiological saline at a mass volume ratio of 1 g:5 ml to obtain bacterial concentrate; S4.

5. Inoculate the bacterial concentrate into the fermentation medium according to the required inoculation amount for fermentation culture. After the culture is completed, harvest the bacteria to obtain the Lactobacillus plantarum DL3 inoculum.

8. The corn straw silage fermentation agent and preparation method thereof according to claim 7, characterized in that: The centrifugation operation in step S4.3 needs to be performed under the conditions of relative centrifugal force of 14136×g and temperature of 4°C, and the centrifugation time is 10 min.