CN111304112A - Composite microbial inoculum for ensiling sugarcane tail leaves - Google Patents
Composite microbial inoculum for ensiling sugarcane tail leaves Download PDFInfo
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- CN111304112A CN111304112A CN201911305751.3A CN201911305751A CN111304112A CN 111304112 A CN111304112 A CN 111304112A CN 201911305751 A CN201911305751 A CN 201911305751A CN 111304112 A CN111304112 A CN 111304112A
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- lactobacillus
- paracasei
- candida
- lactobacillus plantarum
- casei
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- 241000186605 Lactobacillus paracasei Species 0.000 claims abstract description 48
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K30/00—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs
- A23K30/10—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder
- A23K30/15—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging
- A23K30/18—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging using microorganisms or enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/125—Casei
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/165—Paracasei
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Mycology (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Engineering & Computer Science (AREA)
- Polymers & Plastics (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Animal Husbandry (AREA)
- Botany (AREA)
- Food Science & Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the field of microorganisms, and particularly relates to a composite microbial inoculum for ensiling sugarcane tail leaves, which is characterized by comprising the following components in parts by weight: its active ingredients include Candida parapsilosis (Candida humils), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus casei (Lactobacillus casei), and Lactobacillus paracasei (Lactobacillus paracasei). The inventor adopts the applicant to independently breed 4 varietiesThe method has the advantages that the advantageous bacterial strains are combined with the compound microbial inoculum form, and the silage quality of the fresh sugarcane tail leaves is further improved compared with the mode that any single bacterial strain is used as the microbial inoculum, such as the lactic acid and acetic acid content is obviously improved, the pH value is obviously reduced, and NH is reduced3-N content.
Description
Technical Field
The invention belongs to the field of biology, and particularly relates to a composite microbial inoculum for ensiling sugarcane tail leaves.
Background
In recent years, the demand for milk and meat of ruminants has been rapidly increased in many subtropical and tropical countries and regions, and the ruminant industry has also been rapidly developed, thereby causing a huge demand for roughage resources, and problems of shortage and unbalanced supply of high-quality forage are increasingly prominent.
Sugarcane is a gramineous plant widely grown in tropical and subtropical regions. The total national sugarcane yield in 2017 reaches 10440.4 ten thousand tons, wherein the total Guangxi yield reaches 7132.3 ten thousand tons and accounts for about 68.31 percent of the total national yield. The sugarcane tip is a general name of 2-3 tender knots and green leaves on the top of the sugarcane when the sugarcane is harvested, is an accessory product generated in the sugarcane planting process, and accounts for about 10% of the biomass of the sugarcane. The annual sugarcane tips produced in Guangxi areas are about 1400 million tons and huge in quantity, but less than 30 percent of sugarcane tips are used as feed to directly feed herbivorous animals, and most of sugarcane tips are burnt and discarded, so that the problems of great resource waste and serious environmental pollution are caused. The harvest season of the sugarcane is usually from 11 months to 5 months of the next year, and the forage application of the sugarcane tips can just make up the problem of the forage resource shortage caused by slow growth of the pasture in autumn and winter.
At present, the sugarcane tips are mainly directly and freshly fed or ensiled to feed in China, and more sugarcane tail ensiling methods are published. But the main method is to use lactic acid milk as a microbial additive. The invention discloses a method for preparing leaven with different addition proportions by using three dominant lactic acid bacteria and one yeast extracted from sugarcane tails to obtain better silage quality.
Disclosure of Invention
The invention aims to provide a compound microbial inoculum for ensiling sugarcane tail leaves. According to the invention, anaerobic ensiling is carried out by Candida parapsilosis (Candida humils), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus casei (Lactobacillus casei) and Lactobacillus paracasei (Lactobacillus paracasei) which are independently developed by the research laboratory, so that the fermentation speed can be effectively promoted, the pH value of ensiling feed can be reduced, and the quantity of lactic acid and organic acid can be increased, thereby improving the ensiling quality of sugarcane tail leaves.
The invention content of the invention is as follows:
a composite bacterial preparation for ensiling sugarcane tail leaves comprises active components of Candida parapsilosis (Candidahumis), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus casei (Lactobacillus casei) and Lactobacillus paracasei (Lactobacillus paracasei).
The Candida parapsilosis (Candida humils) GS2 is preserved in China, Wuhan university, China center for type culture Collection in 7 months and 02 days in 2018, and the preservation number is CCTCC M2018441; lactobacillus plantarum GS3 was deposited in China, Wuhan university, China center for type culture Collection in 2018, 08-03 month, with a collection number of CCTCC M2018522; lactobacillus casei (Lactobacillus casei) GS4 was deposited in China, Wuhan university, China center for type culture Collection in 2018, 08-03.month with a collection number of CCTCC M2018523; lactobacillus paracasei (Lactobacillus paracasei) GS5 was deposited in China, Wuhan university, China center for type culture Collection in 2018, 08-03.month with the collection number of CCTCC M2018524.
In the invention, the ratio of the Candida parapsilosis (Candida humils), the Lactobacillus plantarum (Lactobacillus plantarum), the Lactobacillus casei (Lactobacillus casei) and the Lactobacillus paracasei (Lactobacillus paracasei) is 0-100 percent, 0-10 percent and 0-10 percent in terms of viable count.
Preferably, the ratio of the Candida parapsilosis (Candida humils), the Lactobacillus plantarum (Lactobacillus plantarum), the Lactobacillus casei (Lactobacillus casei) and the Lactobacillus paracasei (Lactobacillus paracasei) is 20% -50%: 30% -60%: 10%: 10% in terms of viable count percentage.
The preparation method of the composite microbial inoculum for ensiling the sugarcane tail leaves comprises the following steps:
(1) and activating: respectively inoculating the extracted 4 strains into liquid culture medium according to 10% of the addition amount of the strains, culturing Candida parapsilosis (Candida humils) at 28 deg.C for 24h, and performing shake culture at 150 r/min; the temperature of Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus casei (Lactobacillus casei) and Lactobacillus paracasei (Lactobacillus paracasei) is 37 ℃ for 24 hours, and the Lactobacillus plantarum, the Lactobacillus plantarum and the Lactobacillus paracasei are statically cultured;
(2) and (3) expanding culture: then respectively inoculating the activated 4 strains into a liquid culture medium according to the addition of 5 percent, and carrying out shake culture at 150r/min at the culture temperature of 28 ℃ for 24h by using Candida parapsilosis; culturing Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus casei (Lactobacillus casei) and Lactobacillus paracasei (Lactobacillus paracasei) at 37 ℃ for 24h, statically culturing, and counting inoculated bacteria liquid by coating;
(3) and (3) freeze drying: wiping the inner wall of a centrifuge with 95% ethanol to perform sterilization treatment, mixing concentrated and cultured bacteria fermentation liquor according to the concentration proportion of 4 strains by using a sterilization centrifugal tube, centrifuging for 20min at 3000r/min, discarding supernatant to obtain bacteria mud, fully mixing the bacteria mud with a proper amount of sterile water to prepare bacteria suspension with uniform concentration, and adding a protective agent, namely the bacteria mud: and (3) transferring the protective agents into a sterile culture dish together with the protective agents with the thickness of 1:3, wrapping the protective agents with a preservative film, pre-freezing the protective agents in an ultralow temperature refrigerator for 5 hours to form small ice crystals, immediately transferring the protective agents into a freeze drying device for drying until no clear water exists, sampling to determine the viable count, immediately carrying out vacuum sealing packaging, and then preserving the protective agents with a 4-degree refrigerator.
In the present invention, further explained are: the protective agent comprises, by percentage, 80% of fat-free milk powder, 8% of trehalose, 8% of sodium glutamate and 4% of glycerol.
In the present invention, further explained are: in the steps (1) to (2), the MRS liquid medium is used for activating Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus casei (Lactobacillus casei) and Lactobacillus paracasei (Lactobacillus paracasei) and culturing bacterial liquid; MRS solid culture medium is used for coating and counting Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus casei (Lactobacillus casei) and Lactobacillus paracasei (Lactobacillus paracasei); YPD liquid culture medium is used for activating and culturing bacterial liquid of Candida parapsilosis (Candidahumils); YPD agar was used for plating and counting Candida parapsilosis (Candida humils).
The application of the composite microbial inoculum for silage of sugarcane tail leaves in preparation of silage sugarcane tail leaves.
In the invention, the use method of the composite bacteria for ensiling the sugarcane tail leaves further comprises the following steps: cutting fresh sugarcane tail leaves into 2-3cm, spraying the composite microbial inoculum for ensiling the sugarcane tail leaves on the fresh sugarcane tail leaves, uniformly mixing, loading into an ensiling bag, vacuumizing, and fermenting for 60 days.
Compared with the prior art, the beneficial effect of this application:
the applicant finds that the quality of the silage of the fresh sugarcane tail leaves is further improved by adopting a form of 4 dominant bacterial strains obtained by independent breeding of the applicant and combining the composite bacterial agents compared with a form of any single bacterial strain serving as the bacterial agent after the silage treatment of the fresh sugarcane tail leaves, such as the contents of lactic acid and acetic acid are obviously improved, the pH value is obviously reduced, and NH is reduced3-N content.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof will be described in detail with reference to the following examples. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Example 1
Candida parapsilosis (Candida humils), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus casei (Lactobacillus casei), Lactobacillus paracasei (Lactobacillus paracasei) strains were obtained and named:
picking out the silage sugarcane tails by an inoculating loop, and adding 0.5 percent of CaCO3And lactobacillus plantarum colonies growing on an MRS solid medium plate added with 0.001% (g/100ml) of natamycin, randomly selecting colonies with smooth surfaces, round, milky white or grey white shapes and obvious caldolytic rings, and then respectively carrying out catalase detection (taking a piece of glass slide cleaned in advance, dripping a drop of 3% hydrogen peroxide, dipping the colonies in hydrogen peroxide liquid by using an inoculating loop, observing whether bubbles are generated, and whether the bubbles are generated to be positive or negative or not) and gram staining microscopy; selecting bacterial colonies which are negative in catalase and positive in gram stain; and (3) selecting the implanted bacterial colony by using an inoculating loop, performing line drawing purification again on an MRS solid culture medium plate, performing purification twice, selecting the bacterial colony on the purified bacterial plate, inoculating the bacterial colony in an MRS liquid culture medium, performing amplification culture, and selecting the bacterial colony after culturing for 24 hours.
The bacteria are sent to the China center for type culture Collection of Wuhan university for strain preservation, and the strains are determined as follows:
the bacteria are sent to the China center for type culture Collection of Wuhan university for strain preservation, and the strains are determined as follows:
candida parapsilosis (Candida humils) GS2 was deposited in the China, Wuhan university, China center for type culture Collection in 2018, month 07, month 02, with a collection number of CCTCC M2018441;
lactobacillus plantarum GS3 was deposited in China, Wuhan university, China center for type culture Collection in 2018, 08-03 month, with a collection number of CCTCC M2018522;
lactobacillus casei (Lactobacillus casei) GS4 was deposited in China, Wuhan university, China center for type culture Collection in 2018, 08-03 month, with a collection number of CCTCC M2018523;
lactobacillus paracasei (Lactobacillus paracasei) GS5 was deposited in China, Wuhan university, China center for type culture Collection in 2018, 08-03.month with the collection number of CCTCC M2018524.
Example 2, preparation of complex microbial inoculum:
1. materials and methods
2.1 test materials
1.1.1 conventional materials: fresh sugarcane tails, elephant grass, trehalose, skimmed milk powder, glycerol, sodium glutamate, urea and the like.
1.1.2 microbial strains: candida parapsilosis (Candida humils), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus casei (Lactobacillus casei) and Lactobacillus paracasei (Lactobacillus paracasei).
1.1.3 Medium: MRS liquid culture medium is used for lactobacillus activation and bacterial liquid culture; MRS solid culture medium is used for lactobacillus plating counting; YPD liquid culture medium is used for yeast activation and bacterial liquid culture; YPD agar was used for yeast plating.
1.1.4 protective agent:
10% of skimmed milk powder, 8% of trehalose, 7.5% of sodium glutamate and 4% of glycerol.
1.2 test methods
1.2.1 formulation of Compound silage inoculant specially used for sugarcane tails
1.2.1.1 the addition of each strain is selected to determine different complex microbial inoculum formulas, and the dosage in the formula table is measured by viable count.
TABLE 1 sugarcane tail silage composite microbial inoculum formula
Unit: is based on
| Recipe number | Lactobacillus plantarum | Lactobacillus casei | Lactobacillus paracasei | Candida parapsilosis |
| 1 | 100 | 0 | 0 | 0 |
| 2 | 0 | 100 | 0 | 0 |
| 3 | 0 | 0 | 100 | 0 |
| 4 | 0 | 0 | 0 | 100 |
| 5 | 60 | 10 | 10 | 20 |
| 6 | 30 | 10 | 10 | 50 |
1.2.1.2 preparation of Complex microbial inoculum
The preparation process of the composite microbial inoculum comprises the following steps:
1.2.1.2.1 bacteria activation and culture
And (3) activation: inoculating 10% of the extracted strain into a liquid culture medium, and performing shake culture at 150r/min at 28 deg.C for 24 h; the lactobacillus culture temperature is 37 ℃ for 24h, and the lactobacillus is statically cultured.
And (3) amplification culture: inoculating 5% of the yeast into liquid culture medium, and performing shake culture at the culture temperature of 28 deg.C for 24 hr at 150 r/min; the lactobacillus culture temperature is 37 ℃ for 24h, and the lactobacillus is statically cultured.
The inoculated bacteria solution was counted with a smear.
1.2.1.2.2 Freeze drying
Mixing the yeast and the lactic acid bacteria after the concentrated culture according to the concentration of the bacteria and the proportion of an experimental scheme, and then carrying out a freeze drying test.
Preparation of bacterial suspension:
centrifuging the concentrated and cultured bacteria fermentation liquor at 3000r/min, sterilizing the centrifugal tube in advance, wiping the inner wall of the centrifugal machine with 95% ethanol, discarding supernatant, and mixing with appropriate amount of sterile water and bacteria mud to obtain bacteria suspension with uniform concentration.
Treating the protective agent:
pretreatment of a protective agent: and (4) ultraviolet sterilization is carried out for 2h, so that the sterility in the protective agent is ensured, and the influence on the test is avoided.
Pre-treatment of freeze drying:
adding a protective agent in the process of preparing the bacterial suspension, wherein the bacterial sludge: and (3) transferring the protective agents into a sterile culture dish together with a ratio of 1:3, wrapping the protective agents with a preservative film, placing the protective agents in an ultralow temperature refrigerator in parallel, pre-freezing the protective agents for 5 hours to form small ice crystals, immediately transferring the protective agents into a freeze drying device for drying until no clear water exists, taking out the protective agents, crushing the protective agents, sieving the protective agents with a 200-mesh sieve, sampling and measuring the number of viable bacteria, immediately performing vacuum sealing packaging, and then storing the protective agents with a 4-degree refrigerator.
1.2.2 sugarcane Tail silage fermentation test
The single comparison experiments using the bacterial agents of Lactobacillus brevis (Lactobacillus plantarum), Lactobacillus casei (Lactobacillus casei), Lactobacillus paracasei (Lactobacillus paracasei) and Candida albicans (Candida humils) as the leavening agents respectively and independently are formulas 1, 2, 3 and 4.
The blank control group is selected as a control group by adding normal saline and not adding any leavening agent.
In addition, on the basis of a self-made compound bacterial agent product, the addition amount of each bacterial liquid is 10ml, fresh sugarcane tail leaves cut into 2-3cm are respectively added, 3 times of each group are repeated, and all prepared ensilage bags are respectively marked as formulas 5 and 6.
The addition amount of the bacterial liquid or the normal saline corresponding to each silage bag is 10 ml.
2. Ensiling quality inspection
The bagged silage is stored in a shade place, and is subjected to unpacking inspection and evaluation at day 60, the smell, color and texture of the silage sample are observed and recorded (executed according to an attached table), and the sample is collected.
2.1 items and methods of measurement
(1) Sensory evaluation
According to the attached table item by item.
Sensory evaluation was performed with reference to "silage quality evaluation standards (trial) released in 1996 by the ministry of agriculture in China, and is specifically shown in table 3.
TABLE 2 sensory evaluation criteria
TABLE 3 sensory evaluation criteria
| Group of | Smell(s) | Color and luster | Texture of |
| Control group | 17 | 17 | 8 |
| Formulation 1 | 20.5 | 17.5 | 9 |
| Formulation 2 | 20 | 18 | 8.5 |
| Formulation 3 | 20.5 | 18 | 8.5 |
| Formulation 4 | 19 | 17.5 | 8 |
| Formulation 5 | 23.5 | 19.5 | 9.5 |
| Formulation 6 | 20 | 18 | 9 |
Therefore, after the sugarcane tail silage is finished, the bags are opened, sensory evaluation scoring is respectively carried out on the three aspects of color, smell and texture, and the sensory evaluation of each treatment group is superior in quality. The silage quality evaluation grades of the sugarcane tail silage are good or superior grades by various silage laboratory evaluation methods. The total score of other laboratory silage quality evaluation methods adopted by each test group is higher than that of a control group, and the results show that lactobacillus plantarum, lactobacillus casei, lactobacillus paracasei and candida parapsilosis have a certain improvement effect on fermented sugarcane tail leaves. The silage indexes evaluated by the silage quality evaluation methods are different, so that the total scores of the test groups are different.
(2) Conventional nutrient content determination
DM, CP, NDF, ADF, Total energy, and ash content in the silage sugarcane tails were determined.
(3) Analysis of pH and quality items
(1) Sample pretreatment
35g of sample per bag was placed in a 250mL jar, 150mL of deionized water was added, the mixture was extracted in a refrigerator at 4 ℃ for 24 hours, and then filtered through 2 layers of gauze, and the filtrate was used for determination of pH, lactic acid, ammoniacal nitrogen (NH3-N), and Volatile Fatty Acid (VFA). And (3) drying and crushing the residual sugarcane tail sample at 65 ℃ except the sugarcane tail used for the screening and culturing of the germ-removing seeds (sieving with a 40-mesh sieve) to prepare an air-dried sample for measuring the conventional nutrient components.
(2) Determination of pH
And (3) measuring by adopting a pH meter, wherein the type of the pH meter is as follows: HANNA HI 8424.
(3) The lactate determination principle takes NAD + as a hydrogen acceptor, and LDH catalyzes lactate dehydrogenation to generate pyruvic acid, so that NAD + is converted into NADH. Wherein the hydrogen transfer of PMS reduces NBT into purple color substance, and the absorbance of the color substance is in linear relation with the content of lactic acid when the absorbance is 530 nm.
(4) Determination of NH3-N
Measuring NH3-N by colorimetry, taking 5mL of filtrate obtained from ⑴, adding 5mL of 0.2mol/L HCL, centrifuging at 3500rpm for 10min, taking supernatant, using ammonium chloride as a standard substance, carrying out colorimetry by using an ultraviolet-visible spectrophotometer (PE Lambda 35 type, produced in USA) at 700nm, and obtaining the content of NH3-N according to an optical density value and a standard curve.
(5) Determination of VFA
0.5mL of the filtrate obtained from ⑴ was taken, 0.5mL of 8.2% metaphosphoric acid was added, centrifugation was carried out at 13000rpm/min for 10min, crotonic acid was added to the supernatant, and then measurement was carried out by Agilent 7890A type gas chromatograph with HP-INNOWAX (19091N-133) as a capillary column and 30 m.times.0.25 mm.times.0.25 um as a capillary column specification, and the contents of B, C and B in the sample were measured.
(6) Determination of ethanol
2mL of the filtrate is taken and centrifuged for 15min in a 2mL centrifuge tube 12000rpm/min, and the supernatant is taken and filtered through a 0.45um microporous filter membrane. Taking 0.9mL of the treated filtrate, adding 0.1mL of n-propanol into the GC bottle, uniformly mixing, measuring by an Agilent 7890A type gas chromatograph, and carrying out capillary column: HP-INNOWAX (19091N-133), column Specification of capillary: 30 m.times.0.25 mm.times.0.25 um, and the ethanol content of the sample was measured.
2.2 data analysis
Data were initially processed using EXCEL software, single factor analysis of variance using SPSS 16.0 software, and Duncan multiple comparisons.
2.3 results and analysis
TABLE 4 influence on fermentation quality of sugarcane Tail silage
From the table, the feed prepared by fully mixing silage with the compound agent of the formula 5 can effectively improve the indexes of lactic acid and acetic acid and obviously reduce the content of ammoniacal nitrogen in silage (P is less than 0.05).
TABLE 5 influence of composite micro-ecological agents on the quality of sugarcane tail mixed silage nutrition (absolutely dry basis)
As can be seen from the above table, the NDF and ADF of the mixed bacteria of formula 5 are reduced compared with those of the blank group and formulas 1-4.
Although the present invention has been described with respect to the preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (8)
1. The composite microbial inoculum for ensiling the sugarcane tail leaves is characterized in that: its active ingredients include Candida parapsilosis (Candida humils), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus casei (Lactobacillus casei), and Lactobacillus paracasei (Lactobacillus paracasei);
the Candida parapsilosis (Candida humils) GS2 is preserved in China, Wuhan university, China center for type culture Collection in 7 months and 02 days in 2018, and the preservation number is CCTCC M2018441; lactobacillus plantarum GS3 was deposited in China, Wuhan university, China center for type culture Collection in 2018, 08-03 month, with a collection number of CCTCC M2018522; lactobacillus casei (Lactobacillus casei) GS4 was deposited in China, Wuhan university, China center for type culture Collection in 2018, 08-03 month, with a collection number of CCTCC M2018523; lactobacillus paracasei (Lactobacillus paracasei) GS5 was deposited in China, Wuhan university, China center for type culture Collection in 2018, 08-03.month with the collection number of CCTCC M2018524.
2. The composite microbial inoculum for silage of sugarcane tail leaves as claimed in claim 1, which is characterized in that: the ratio of the Candida parapsilosis (Candida humils), the Lactobacillus plantarum (Lactobacillus plantarum), the Lactobacillus casei (Lactobacillus casei) and the Lactobacillus paracasei (Lactobacillus paracasei) is 20 percent to 30 percent to 60 percent to 10 percent in terms of viable count percentage.
3. The composite microbial inoculum for silage of sugarcane tail leaves as claimed in claim 1, which is characterized in that: the ratio of the Candida parapsilosis (Candida humils), the Lactobacillus plantarum (Lactobacillus plantarum), the Lactobacillus casei (Lactobacillus casei) and the Lactobacillus paracasei (Lactobacillus paracasei) is 20 percent to 60 percent to 10 percent in terms of viable count percentage.
4. The preparation method of the complex microbial inoculum for silage of sugarcane tail leaves according to any one of claims 1 to 3, characterized by comprising the following steps: the method specifically comprises the following steps:
(1) and activating: respectively inoculating the extracted 4 strains into liquid culture medium according to 10% of the addition amount of the strains, culturing Candida parapsilosis (Candida humils) at 28 deg.C for 24h, and performing shake culture at 150 r/min; the temperature of Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus casei (Lactobacillus casei) and Lactobacillus paracasei (Lactobacillus paracasei) is 37 ℃ for 24 hours, and the Lactobacillus plantarum and the Lactobacillus paracasei are statically cultured;
(2) and (3) expanding culture: then respectively inoculating the activated 4 strains into a liquid culture medium according to the addition of 5 percent, and carrying out shake culture at 150r/min at the culture temperature of 28 ℃ for 24h by using Candida parapsilosis; culturing Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus casei (Lactobacillus casei) and Lactobacillus paracasei (Lactobacillus paracasei) at 37 ℃ for 24h, statically culturing, and counting inoculated bacteria liquid by coating;
(3) and (3) freeze drying: wiping the inner wall of a centrifuge with 95% ethanol to perform sterilization treatment, mixing concentrated and cultured bacteria fermentation liquor according to the concentration proportion of 4 strains by using a sterilization centrifugal tube, centrifuging for 20min at 3000r/min, discarding supernatant to obtain bacteria mud, fully mixing the bacteria mud with a proper amount of sterile water to prepare bacteria suspension with uniform concentration, and adding a protective agent, namely the bacteria mud: and (3) transferring the protective agents into a sterile culture dish together with the protective agents with the thickness of 1:3, wrapping the protective agents with a preservative film, pre-freezing the protective agents in an ultralow temperature refrigerator for 5 hours to form small ice crystals, immediately transferring the protective agents into a freeze drying device for drying until no clear water exists, sampling to determine the viable count, immediately carrying out vacuum sealing packaging, and then preserving the protective agents with a 4-degree refrigerator.
5. The method of claim 4, wherein: the protective agent comprises, by percentage, 80% of fat-free milk powder, 8% of trehalose, 8% of sodium glutamate and 4% of glycerol.
6. The method of claim 4, wherein: in the steps (1) to (2), the MRS liquid medium is used for activating Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus casei (Lactobacillus casei) and Lactobacillus paracasei (Lactobacillus paracasei) and culturing bacterial liquid; MRS solid culture medium is used for coating and counting Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus casei (Lactobacillus casei) and Lactobacillus paracasei (Lactobacillus paracasei); YPD liquid culture medium is used for activating and culturing bacterial liquid of Candida parapsilosis (Candidahumils); YPD agar was used for plating and counting Candida parapsilosis (Candida humils).
7. The use of the complex microbial inoculant for the silage of sugarcane tail leaves according to any one of claims 1 to 6 in the preparation of the silage of sugarcane tail leaves.
8. The use method of the composite bacteria for the silage of sugarcane tail leaves as claimed in claim 1, characterized in that: comprises the following steps: cutting fresh sugarcane tail leaves into 2-3cm, spraying the composite microbial inoculum for ensiling the sugarcane tail leaves on the fresh sugarcane tail leaves, uniformly mixing, loading into an ensiling bag, vacuumizing, and fermenting for 60 days.
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| CN113995052A (en) * | 2021-07-12 | 2022-02-01 | 广西壮族自治区水牛研究所 | Grassiness sugarcane tail leaf silage and preparation method thereof |
| CN114276949A (en) * | 2021-11-10 | 2022-04-05 | 丰唐生态农业科技研发(山东)有限公司 | Lactobacillus paracasei and application thereof in preparation of broussonetia papyrifera silage |
| CN117778278A (en) * | 2024-02-27 | 2024-03-29 | 山东中科嘉亿生物工程有限公司 | Compound lactobacillus silage additive and preparation method and application thereof |
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| CN113995052A (en) * | 2021-07-12 | 2022-02-01 | 广西壮族自治区水牛研究所 | Grassiness sugarcane tail leaf silage and preparation method thereof |
| CN113995052B (en) * | 2021-07-12 | 2023-11-17 | 广西壮族自治区水牛研究所 | Ivory sugarcane tail leaf silage and preparation method thereof |
| CN114276949A (en) * | 2021-11-10 | 2022-04-05 | 丰唐生态农业科技研发(山东)有限公司 | Lactobacillus paracasei and application thereof in preparation of broussonetia papyrifera silage |
| CN117778278A (en) * | 2024-02-27 | 2024-03-29 | 山东中科嘉亿生物工程有限公司 | Compound lactobacillus silage additive and preparation method and application thereof |
| CN117778278B (en) * | 2024-02-27 | 2024-05-03 | 山东中科嘉亿生物工程有限公司 | Compound lactobacillus silage additive and preparation method and application thereof |
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Application publication date: 20200619 Assignee: Guangxi Yulin Yiliang Technology Co.,Ltd. Assignor: GUANGXI ZHUANG AUTONOMOUS REGION BUFFALO INSTITUTE Contract record no.: X2023980045755 Denomination of invention: A composite bacterial agent for sugarcane tail leaf silage Granted publication date: 20211001 License type: Common License Record date: 20231107 |