CN117070427B - Lactobacillus buchneri and silage starter thereof - Google Patents
Lactobacillus buchneri and silage starter thereof Download PDFInfo
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- CN117070427B CN117070427B CN202311321511.9A CN202311321511A CN117070427B CN 117070427 B CN117070427 B CN 117070427B CN 202311321511 A CN202311321511 A CN 202311321511A CN 117070427 B CN117070427 B CN 117070427B
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- silage
- cfu
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- PYKYMHQGRFAEBM-UHFFFAOYSA-N anthraquinone Natural products CCC(=O)c1c(O)c2C(=O)C3C(C=CC=C3O)C(=O)c2cc1CC(=O)OC PYKYMHQGRFAEBM-UHFFFAOYSA-N 0.000 description 1
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- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 1
- 239000004462 maize silage Substances 0.000 description 1
- CDUFCUKTJFSWPL-UHFFFAOYSA-L manganese(II) sulfate tetrahydrate Chemical compound O.O.O.O.[Mn+2].[O-]S([O-])(=O)=O CDUFCUKTJFSWPL-UHFFFAOYSA-L 0.000 description 1
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- 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
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
-
- 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
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
- A61K36/8969—Polygonatum (Solomon's seal)
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- 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
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C12R2001/225—Lactobacillus
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
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Abstract
The invention discloses a Lactobacillus buchneri and silage starter thereof, belonging to the technical field of microorganism application, which screens out a Lactobacillus buchneri JYZC-LB06 from silage and belongs to the field ofLactobacillus buchneriThe strain has been deposited in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on the 8 th month 23 th year 2023, and has a biological deposit number of: the strain has high acid production efficiency and strong acid resistance, has antibacterial effect on various pathogenic bacteria and saccharomycetes, and has better development potential as silage microbial inoculum.
Description
Technical Field
The invention relates to the technical field of microorganism application, in particular to a lactobacillus buchneri and silage starter thereof.
Background
Silage is one of the most important daily ration compositions of ruminants at present, and the preparation principle is mainly that the lactobacillus is used for rapidly converting soluble carbohydrate in a substrate into organic acid in an anaerobic environment, so that the pH of the silage is rapidly reduced, other nutrients causing decomposition of the substrate by the corrosive microorganism are inhibited, and the aim of long-term storage is finally achieved. Lactic acid bacteria are the most commonly used fermentation promoters, and they play an important role in the silage process and can be classified into homofermentative lactic acid bacteria and heterofermentative lactic acid bacteria according to the metabolic products produced. Common homotype fermentation lactobacillus includes lactobacillus plantarum, pediococcus pentosaceus, streptococcus and the like, only lactic acid is produced during metabolism, the silage fermentation speed can be accelerated, and the loss of nutrient substances is reduced. The abnormal fermentation lactobacillus has the lactobacillus buchneri, lactobacillus brevis and the like, the metabolites of the abnormal fermentation lactobacillus are acetic acid or ethanol and the like besides lactic acid, and the generated acetic acid has good inhibition effect on the growth and propagation of harmful microorganisms such as saccharomycetes and mould in silage, and when the silage is unsealed, the time for recovering the activity of the harmful bacteria is relieved, the aerobic stability of the silage is effectively improved, the preservation time of the silage is prolonged, but the acid production efficiency is low, and the abnormal fermentation lactobacillus is unfavorable for commercial application. Therefore, the development of more excellent lactic acid bacteria resources has important value for the production of high-quality silage.
In recent years, the safety incidents of domestic and foreign animal products frequently occur, and people increasingly favor safe and pollution-free green animal products. The Chinese herbal medicine additive has the dual functions of nutrition and medicine, and is an ideal additive for ensuring the health of the human body from feed and animal products. The main functional components of the rhizoma polygonati comprise polysaccharides, saponins, flavone and anthraquinone compounds, and besides alkaloids, lignans, vitamins, various essential amino acids and trace elements of organisms, and the rhizoma polygonati has wide pharmacological activities, including antioxidation, blood sugar regulation, blood fat reduction, anti-inflammatory, anti-tumor and the like. There is no report on the use of Polygonatum sibiricum extract as silage starter.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide lactobacillus buchneri and silage starter thereof.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a strain of Lactobacillus buchneriLactobacillus buchneri）JYZC-LB06：
The strain is preserved in China general microbiological culture collection center (CGMCC) for 8 months and 23 days in 2023, and has the biological preservation number of: CGMCC No. 28233.
The Lactobacillus buchneri is @Lactobacillus buchneri) JYZC-LB06, isolated from silage, had the following characteristics:
on MRS culture medium, the colony is white, round, moist in surface, opaque and neat in edge; the bacterial cells are in the shape of rod, 0.4-0.5 μm×0.7-1.6 μm, and are arranged singly or in pairs, and are gram positive.
In a second aspect of the present invention, there is provided a silage starter comprising the above Lactobacillus buchneri (Lactobacillus buchneri) JYZC-LB06.
Preferably, the silage starter comprises the following raw materials by mass:
5-9g of composite microbial inoculum and 15-19g of rhizoma polygonati extract;
the composite microbial inoculum is prepared from lactobacillus buchneri JYZC-LB06, pediococcus pentosaceus and enterococcus faecalis according to a mass ratio of 3:1:2, preparing the product.
The invention firstly separates and screens a strain of Lactobacillus buchneri JYZC-LB06 from corn silage, the strain has high acid production efficiency and acid resistance, and has bacteriostasis to a plurality of pathogenic bacteria and saccharomycetes; because the homotype/heterotype lactic acid bacteria have advantages and disadvantages in the fermentation process, besides the heterotype lactic acid bacteria lactobacillus buchneri, the silage starter culture also comprises homotype lactic acid bacteria pediococcus pentosaceus and enterococcus faecalis, and the fermentation quality of the silage is improved due to the synergistic effect among various lactic acid bacteria;
in addition, as the survival rate of the strain in the silage is not high by directly putting the microbial inoculum into the silage, the rhizoma polygonati extract is taken as a base material, the rhizoma polygonati extract can promote the growth of the strain, can improve the fermentation quality and the nutrition components of the silage, is beneficial to improving the organism immunity of ruminants and prevents common diseases.
Preferably, the pediococcus pentosaceus is ]Lactobacillus pentosus) The preservation number of the strain is CGMCC 1.7665, and the strain is purchased from the China general microbiological culture Collection center; the enterococcus faecalis is [ ]Enterococcus faecalis) Is available from China industry microbiological culture collection center with a deposit number of CICC 20423.
Preferably, in the composite microbial inoculum, the viable count of the Lactobacillus buchneri JYZC-LB06 is more than or equal to 3 multiplied by 10 8 cfu/g, the viable count of Pediococcus pentosaceus is more than or equal to 1 multiplied by 10 8 cfu/g; the viable count of the enterococcus faecalis is more than or equal to 2 multiplied by 10 8 cfu/g。
Preferably, the rhizoma polygonati extract is prepared by the following method:
(1) Cleaning fresh rhizoma Polygonati, pulverizing, and adding water as fermentation medium;
(2) Inoculating mixed seed liquid into a fermentation culture medium, wherein the inoculation amount of the mixed seed liquid is 10% of the volume of the fermentation culture medium, and the mixed seed liquid is formed by mixing Aspergillus niger, lactobacillus plantarum and Bifidobacterium adolescentis; fermenting at 35-38deg.C for 6-10d to obtain fermentation broth;
(3) Filtering and sterilizing the fermentation liquor, and performing vacuum freeze drying to obtain the rhizoma polygonati extract.
According to the invention, aspergillus niger, lactobacillus plantarum and bifidobacterium adolescentis are selected to ferment the rhizoma polygonati, so that the nutritional ingredients of the rhizoma polygonati can be fully extracted, and the nutritional efficacy of the rhizoma polygonati can be fully exerted; the aspergillus niger has two functions, namely, the aspergillus niger is firstly used for fermenting the polygonatum kingianum, and the fermentation products of the aspergillus niger comprise cellulase, xylanase and the like; the straw coarse fodder contains a large amount of plant polysaccharide which is difficult to degrade and has complex structure, so that the digestibility of ruminants is low, and the cellulose and xylanase can damage plant cell walls and hydrolyze polysaccharide which is difficult to degrade into monosaccharide which is easy to be utilized by animals, so that the nutrition quality of the straw fodder is improved, and the fermentation characteristic of silage and the production performance of animals are improved.
Preferably, the aspergillus niger is [ ]Aspergillus niger) The deposit number is CICC 40273, and is purchased from China center for type culture Collection; the lactobacillus plantarum is [ (] plant bacillus ]Lactiplantibacillus plantarum) Is purchased from China industry microbiological culture collection center (CICC) 22712; the bifidobacterium adolescentis isBifidobacterium adolensentis) The deposit number is CICC 6179, and is purchased from China industry microbiological culture Collection center.
Preferably, in the step (1), the mass ratio of the fresh Polygonatum kingianum to the water is 1:10-15.
Preferably, in the step (2), the concentration of Aspergillus niger in the mixed seed solution is 1.0X10 7 ~1.0×10 8 cfu/mL, the concentration of lactobacillus plantarum is 1.0X10 6 ~1.0×10 7 cfu/mL, the concentration of bifidobacterium adolescentis is 1.0X10 7 ~1.0×10 8 cfu/mL。
Preferably, in the step (3), the conditions of vacuum freeze-drying are: the vacuum degree is 15-20 Pa, and the temperature is-40 to-45 ℃.
The invention has the beneficial effects that:
the invention separates and screens a strain of Lactobacillus buchneri which has high acid production efficiency and strong acid resistance from silageLactobacillus buchneri) JYZC-LB06, and has bacteriostasis to a plurality of pathogenic bacteria and saccharomycetes, and has better development potential as silage bacterial agent.
The silage starter provided by the invention comprises the composite microbial inoculum and the rhizoma polygonati extract, wherein the rhizoma polygonati extract is used as a traditional Chinese medicine extract, so that not only can the fermentation quality and the nutrition components of the silage be improved, but also the immunity of ruminants can be improved, and common diseases can be prevented; by the synergistic effect between the composite microbial inoculum and the rhizoma polygonati extract, the fermentation quality and the nutritional ingredients of the silage are greatly improved, and the aerobic stabilization time of the corn silage is remarkably improved.
Drawings
FIG. 1 is a flow chart of the separation and screening of strain JYZC-LB 06;
FIG. 2 is a photograph of a macroscopic morphology of strain JYZC-LB 06;
FIG. 3 is a photomicrograph of strain JYZC-LB 06;
FIG. 4 is a phylogenetic tree of the 16S rDNA sequence of strain JYZC-LB 06;
FIG. 5 is a phylogenetic tree of the pheS gene sequence of strain H38.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As described in the background art, the development of more excellent lactobacillus resources has important value for the production of high-quality silage, and the current Lactobacillus buchneri is unfavorable for commercial application due to low acid production efficiency. The invention separates a strain of bacteria from silage, named JYZC-LB06, according to morphology,Physiological and biochemical characteristics are detected to belong to the genus Lactobacillus, 16S rDNA is sequenced, and a phylogenetic tree is constructed to identify the bacterium as Lactobacillus buchneriLactobacillus buchneri). The strain has high acid production efficiency and strong acid resistance, has antibacterial effect on various pathogenic bacteria and saccharomycetes, and has better development potential as silage microbial inoculum. In addition, as people pay more attention to animal product safety, the invention applies the extract of the traditional Chinese medicine rhizoma polygonati to the silage starter for the first time, and uses the rhizoma polygonati extract and the compound microbial inoculum as silage additives and silage raw materials for co-fermentation, thereby promoting the growth and propagation of zymophytes and improving the fermentation quality of silage.
In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention are all conventional in the art and are commercially available.
Wherein Pediococcus pentosaceus is used for treating the skin diseaseLactobacillus pentosus) The preservation number of the strain is CGMCC 1.7665, and the strain is purchased from the China general microbiological culture Collection center; enterococcus faecalisEnterococcus faecalis) Is purchased from China industry microbiological culture collection center (CICC) 20423; aspergillus nigerAspergillus niger) The deposit number is CICC 40273, and is purchased from China center for type culture Collection; lactobacillus plantarumLactiplantibacillus plantarum) Is purchased from China industry microbiological culture collection center (CICC) 22712; bifidobacterium adolescentisBifidobacterium adolensentis) The deposit number is CICC 6179, and is purchased from China industry microbiological culture Collection center.
Staphylococcus aureus [ (S.aureus ]Staphylococcus aureus) The preservation number of the strain is CGMCC 1.6722, and the strain is purchased from the China general microbiological culture Collection center; bacillus cereus @Bacillus cereus) Is available from China industry microbiological culture collection center with a deposit number of CICC 21290; single-core hyperplasia LissTerylella (Thunb.) KuntzeListeria monocytogenes) The deposit number is CICC 21639, and is purchased from China center for type culture Collection; escherichia coli @Escherichia coli) The preservation number of the strain is CGMCC 1.12873, and the strain is purchased from the China general microbiological culture Collection center; salmonella (Salmonella)Salmonella) The preservation number of (2) is CCTCC CB 20082515, and is purchased from China center for type culture Collection; saccharomyces cerevisiaeSaccharomyces cerevisiae) Is available from China center for type culture Collection (CICC) 1423.
The composition of the medium used was as follows:
MRS liquid medium: beef extract 10 g/L, yeast extract 5 g/L, peptone 10 g/L, glucose 20 g/L, dipotassium hydrogen phosphate 2 g/L, sodium acetate 5 g/L, magnesium sulfate heptahydrate 0.2 g/L, manganese sulfate tetrahydrate 0.05 g/L, tween-80 1 g/L, triammonium citrate 2 g/L, and pH6.2.
MRS solid medium: agar 15 g/L and bromocresol purple 0.8 g/L, pH6.2 were added to MRS broth.
Example 1: isolation and identification of strains:
1. isolation and purification of strains:
corn silage samples of silage 3, 7, 15, 30, 45 and 60d are taken, uniformly mixed, 10g samples are weighed and added into 90 mL sterile water, and 150 r/min oscillation is 2 h. Taking 100 mu L of bacterial suspension, properly diluting, selecting 3 bacterial suspensions with proper concentration, and uniformly coating the bacterial suspensions on an MRS solid culture medium. Under anaerobic conditions, placing the MRS solid culture medium at 37 ℃ for culturing for 48-72 hours, selecting single colonies generating a calcium dissolving ring, and carrying out repeated streak purification on the fresh MRS solid culture medium until microscopic examination is free of infectious microbe. Single colony is selected and inoculated in 10 mL MRS liquid culture medium, shaking culture is carried out at 30 ℃ and 200 rpm for 24 h, bacterial liquid is added with 15 percent glycerol (v/v) and then is sub-packaged in a freezing tube, and the frozen tube is preserved at-80 ℃ for standby.
Gram staining test is carried out on the separated strain, and the lactobacillus is gram positive.
The inspection method comprises the following steps: culturing pure bacteria with MRS liquid culture medium, shaking bottle for overnight culture at 180 rpm and 28deg.C; a small amount of bacterial liquid is dipped on the glass slide by an inoculating loop, smeared evenly along the middle of the glass slide and dried naturally in the air; after the bacterial liquid is dried, placing the glass slide in an alcohol lamp to slightly burn and fix with the outer flame, then dripping a crystal violet solution to uniformly disperse the glass slide, dyeing for 1 min, and washing with clear water; adding iodine solution, dyeing for 1 min, and washing with clear water to remove floating liquid; adding 95% ethanol solution, continuously shaking the glass slide 30 s until the purple color falls off, and washing with clear water to remove floating liquid; adding a double-dyeing liquid, dyeing for 1 min, and washing with clear water to remove floating liquid; after the glass slide is dried, performing 1000 times of microscopic examination under a microscope; gram positive bacteria appear purple; gram negative bacteria appear red.
3. Screening of strains:
regulating the concentration of the purified bacterial strain solution to 10 8 cfu/mL was shaken and inoculated into 100. Mu.L of a centrifuge tube containing MRS liquid medium, and the mixture was placed in a constant temperature incubator at 37℃for 36 hours to determine the pH value of the fermentation broth. OD (Optical density) value at 600nm is measured, and the strain with pH less than or equal to 4 and OD less than or equal to 2 is selected as the primary screening representative strain.
The strain bacterial liquid of the primary screening representative strain is used for adjusting the OD thereof by MRS liquid culture medium 600 Adding into MRS liquid culture medium with pH of2, 3, 4, 5 at an inoculation amount of 6%, mixing, packaging into 2 mL centrifuge tube, standing at 25deg.C under anaerobic condition for culturing 72 h, and determining OD of each strain culture solution 600 As a result, the strain JYZC-LB06 had the best acid tolerance and could grow at pH 2-5. The separating and screening flow chart of JYZC-LB06 strain is shown in figure 1.
2. Identification of strains:
1. morphological observation
JYZC-LB06 strain was inoculated on MRS solid medium plate, and colony morphology was observed. The macroscopic morphology photograph of JYZC-LB06 strain is shown in figure 2.JYZC-LB06 colony is white, round, wet on the surface, opaque and neat in edge. The microscopic morphology of the strain is shown in FIG. 3, the bacterial cells are in the shape of rods, 0.4-0.5 μm×0.7-1.6 μm, and are arranged singly or in pairs, and gram-positive.
2. Biochemical identification
Physiological and biochemical identification of strains using VITEK ANC identification cards
Physiological and biochemical results of the strain JYZC-LB06 are shown in Table 1.
TABLE 1 physiological and biochemical characteristics of JYZC-LB06 strain
Note that: "+", positive; "w", weak positive; "-", negative.
3. Molecular biological identification
Identification of 16S rDNA gene sequence and pheS gene sequence is carried out by sending JYZC-LB06 strain to China industry microbiological culture collection center, the 16S rDNA gene sequence is shown as SEQ ID NO.1, the pheS gene sequence is shown as SEQ ID NO.2, and the identification result shows that the strain belongs to the Lactobacillus chrous of BuhennaLentilactobacillus buchneri）。
Based on the homology of the 16S rDNA and pheS gene sequences, phylogenetic tree as shown in FIG. 4 and FIG. 5 was obtained by performing systematic evolution analysis using MEGA software.
The JYZC-LB06 strain is identified as Lactobacillus buchneri and named Lactobacillus buchneri by combining the results of morphological identification, biochemical identification and molecular biological identificationLactobacillus buchneri) JYZC-LB06. And performing biological preservation on the strain, wherein the preservation information is as follows:
strain name: lactobacillus buchneri JYZC-LB06
Latin name:Lactobacillus buchneri
preservation mechanism: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)
The preservation organization is abbreviated as: CGMCC
Address: beijing city, chaoyang area, north Chenxi Lu No.1 and 3
Preservation date: 2023.8.23
Preservation number: CGMCC No.28233
It should be noted that: lactobacillus buchneriLentilactobacillus buchneri) The great-usage name isLactobacillus buchneri @Lactobacillus buchneri). (reference: zheng J, wittouck S, salvetti E, et al A taxonomic note on the genus Lactobacillus: description of23 novel genera, emended descriptionof the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae [ J ]]. Int J Syst Evol Microbiol, 2020, 70:2782-2858.）。
Example 2: determination of broad-spectrum antibacterial effect of strain JYZC-LB 06:
the bacterial inhibition rate of the strain JYZC-LB06 on staphylococcus aureus, bacillus cereus, listeria monocytogenes, escherichia coli, salmonella and saccharomyces cerevisiae is evaluated by adopting a plate counter experiment.
Antibacterial ratio = [ (control colony diameter-treated colony diameter)/control colony diameter ] ×100%
Table 2 determination of broad-spectrum antibacterial effect of JYZC-LB06 strain
As shown in Table 2, the strain JYZC-LB06 has remarkable inhibition effect on a plurality of pathogenic bacteria such as staphylococcus aureus, bacillus cereus, listeria monocytogenes, escherichia coli, salmonella and the like, can inhibit the proliferation of saccharomycetes, and is beneficial to improving the aerobic stability of silage.
Example 3: preparation of silage starter:
1. the raw materials comprise: 5g of composite microbial inoculum and 15g of rhizoma polygonati extract;
2. the preparation method comprises the following steps:
(1) Preparation of rhizoma polygonati extract:
1) Cleaning fresh Polygonatum kingianum, pulverizing, and adding water as fermentation medium, wherein the mass ratio of fresh Polygonatum kingianum to water is 1:12;
2) Inoculating mixed seed liquid into the fermentation medium, wherein the inoculation amount of the mixed seed liquid is 10% of the volume of the fermentation medium, and the mixed seed liquid is prepared from aspergillus niger, lactobacillus plantarum and bifidobacterium adolescentisMixing the above mixed seed solution, wherein the concentration of Aspergillus niger is 1.0X10% 8 cfu/mL, the concentration of lactobacillus plantarum is 1.0X10 7 cfu/mL, the concentration of bifidobacterium adolescentis is 1.0X10 8 cfu/mL; fermenting at 37 ℃ for 8d to obtain fermentation liquor;
3) Filtering the fermentation broth with a 0.22 μm sterilization filter membrane for sterilization, and vacuum freeze-drying to obtain rhizoma Polygonati extract; the conditions for vacuum freeze drying are: the vacuum degree is 15Pa, and the temperature is-40 ℃;
(2) Preparing a composite microbial inoculum: the method comprises the steps of (1) mixing lactobacillus buchneri JYZC-LB06, pediococcus pentosaceus and enterococcus faecalis according to a mass ratio of 3:1:2, preparing;
wherein the active ingredients of the composite bacterial agent are Lactobacillus buchneri JYZC-LB06, pediococcus pentosaceus and enterococcus faecalis, and the viable count of the Lactobacillus buchneri JYZC-LB06 is 3 multiplied by 10 8 cfu/g, pediococcus pentosaceus viable count of 1×10 8 cfu/g; the viable count of enterococcus faecalis is 2×10 8 cfu/g；
(3) And weighing the composite microbial inoculum and the rhizoma polygonati extract according to weight, and uniformly mixing to obtain the silage starter.
Example 4: preparation of silage starter:
the raw materials comprise: 7g of composite microbial inoculum and 17g of rhizoma polygonati extract;
the preparation method is the same as that of example 3, and silage starter is prepared.
Example 5: preparation of silage starter:
the raw materials comprise: 9g of composite microbial inoculum and 19g of rhizoma polygonati extract;
the preparation method is the same as that of example 3, and silage starter is prepared.
Comparative example 1
The difference between comparative example 1 and example 3 is that:
the silage starter was prepared by omitting the Polygonatum sibiricum extract from the raw material composition and otherwise performing the same as that of example 3.
Comparative example 2
The difference between comparative example 2 and example 3 is that:
the composite microbial inoculum in the raw material composition is omitted, and the silage starter is prepared in the same manner as in example 3.
Comparative example 3
The raw materials comprise: lactobacillus buchneri JYZC-LB06 (5 g) and 15g of Polygonatum sibiricum extract;
wherein the viable count of the Lactobacillus buchneri JYZC-LB06 is 3×10 8 cfu/g；
The preparation method is the same as that of example 3, and silage starter is prepared.
Comparative example 4
The raw materials comprise: 5g of pediococcus pentosaceus and 15g of rhizoma polygonati extract;
wherein the viable count of Pediococcus pentosaceus is 1×10 8 cfu/g；
The preparation method is the same as that of example 3, and silage starter is prepared.
Comparative example 5
The raw materials comprise: 5g of enterococcus faecalis and 15g of rhizoma polygonati extract;
wherein the viable count of enterococcus faecalis is 2×10 8 cfu/g ；
The preparation method is the same as that of example 3, and silage starter is prepared.
Comparative example 6
The Lactobacillus buchneri JYZC-LB06 is selected as silage ferment, wherein the viable count of the Lactobacillus buchneri JYZC-LB06 is 3 multiplied by 10 8 cfu/g。
Test example:
1. preparation of corn silage:
harvesting corn stalks when silage corn enters the mature period of the ears, and cutting the corn stalks into small pieces of about 3 cm by a grinder on the spot to prepare silage raw materials.
24 parts of silage raw materials of 10Kg each are weighed, and the following eight groups of treatments are respectively carried out:
treatment 1 (control): no treatment is performed; treatment 2: 0.05g of silage starter prepared in example 3 was added per kg of silage raw material; treatment 3-treatment 8: 0.05g of silage starter prepared in comparative examples 1-6 is added per kilogram of silage raw material; each treatment group was repeated 3 times.
All additives were dissolved in 100 mL sterile water before treatment, thoroughly mixed and then sprayed evenly on the weighed corn silage material using a hand-held sprayer, and the control group sprayed only 100 mL sterile water. And (3) fully and uniformly mixing the additive and the raw materials, filling the mixture into a plastic package bag (95 cm multiplied by 55 cm) special for silage, vacuumizing and sealing the mixture, and storing the mixture at room temperature (18-28 ℃) for 60 days.
2. Fermentation quality measurement of corn silage:
after 60d silage, unsealing, immediately weighing 10g of fresh silage, cutting the silage into pieces, placing the silage into a conical flask, adding 90 mL ultrapure water, and sealing. Standing in a refrigerator at 4deg.C for 24. 24 h, centrifuging the supernatant in a 50 mL centrifuge tube, taking out the centrifuged supernatant, and measuring the pH of one part and the organic acid content of the other part by ProminenceLC high performance liquid chromatograph (Shimadzu product of Japan), as shown in Table 3.
3. Determination of nutrient content of corn silage
3.1 determination of dry matter:
taking fresh silage with certain mass, measuring the weight of the silage to be W, putting the silage into a constant-temperature drying oven, and measuring the weight of the silage at intervals until the weight of the silage is constant, and recording the weight of the silage as W1, wherein W1/W is the dry matter content of the silage.
3.2 determination of ammoniacal nitrogen/total nitrogen value:
the ammoniacal nitrogen/total nitrogen values of the four treated corn silage were determined using phenol-sodium hypochlorite colorimetry.
3.3 measurement of crude protein:
four treatments of corn silage nitrogen were performed to determine crude protein content using kjeldahl.
4. Determination of aerobic stability of maize silage
After the silage was unsealed, a 100 g sample was taken out and placed in a 250 mL beaker, the beaker was left open, a thermometer was inserted inside the beaker, the ambient temperature was kept at 25 ℃, the temperature of the feed and the room temperature were recorded every day at intervals, and the time when the temperature of the sample exceeded the room temperature by 2 ℃.
TABLE 3 fermentation quality of corn silage
As can be seen from table 3, the corn silage with the silage starter of the invention added had the lowest pH and the organic acid content was significantly higher than in the control group and the other treatment groups and the control group.
Table 4 corn silage nutritional ingredients and aerobic stability time
As can be seen from table 4, treatment 2 groups added the silage starter prepared in example 3 to the corn silage raw material at the highest dry matter content and the corn silage using the silage starter according to the invention had significantly lower ammonia nitrogen/total nitrogen values. In addition, in terms of crude protein content, the crude protein content of the corn silage adopting the silage starter is greatly improved. The silage starter of the invention also greatly improves the aerobic stabilization time of the corn silage.
The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the same, but rather, various modifications and variations may be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.
Claims (4)
1. The silage starter is characterized by comprising the following raw materials in parts by mass:
5-9g of composite microbial inoculum and 15-19g of rhizoma polygonati extract;
the composite microbial inoculum is prepared from lactobacillus buchneri JYZC-LB06, pediococcus pentosaceus and enterococcus faecalis according to a mass ratio of 3:1:2, preparing the mixture;
the preservation number of the Lactobacillus buchneri JYZC-LB06 is CGMCC No. 28233; the preservation number of the pediococcus pentosaceus is CGMCC 1.7665; the preservation number of the enterococcus faecalis is CICC 20423;
in the composite microbial inoculum, the viable count of the Lactobacillus buchneri JYZC-LB06 is 3 multiplied by 10 8 cfu/g, the viable count of Pediococcus pentosaceus is 1×10 8 cfu/g; the viable count of the enterococcus faecalis is 2 multiplied by 10 8 cfu/g；
The rhizoma polygonati extract is prepared by the following steps:
(1) Cleaning fresh rhizoma Polygonati, pulverizing, and adding water as fermentation medium;
(2) Inoculating mixed seed liquid into a fermentation culture medium, wherein the inoculation amount of the mixed seed liquid is 10% of the volume of the fermentation culture medium, and the mixed seed liquid is formed by mixing Aspergillus niger, lactobacillus plantarum and Bifidobacterium adolescentis; fermenting at 35-38deg.C for 6-10d to obtain fermentation broth;
(3) Filtering and sterilizing the fermentation liquor, and performing vacuum freeze drying to obtain the rhizoma polygonati extract.
2. The silage starter culture according to claim 1, wherein in the step (1), the mass ratio of fresh polygonatum kingianum to water is 1:10-15.
3. The silage starter culture according to claim 1, wherein in step (2), the concentration of Aspergillus niger in the mixed seed liquid is 1.0X10% 7 ~1.0×10 8 cfu/mL, the concentration of lactobacillus plantarum is 1.0X10 6 ~1.0×10 7 cfu/mL, the concentration of bifidobacterium adolescentis is 1.0X10 7 ~1.0×10 8 cfu/mL。
4. Silage ferment according to claim 1, characterized in that in step (3), the conditions of vacuum freeze-drying are: the vacuum degree is 15-20 Pa, and the temperature is-40 to-45 ℃.
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| CN115669809A (en) * | 2022-10-17 | 2023-02-03 | 湖南农业大学 | Corn silage additive |
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