CN113969243B - Lactobacillus plantarum capable of degrading tannin and application thereof - Google Patents
Lactobacillus plantarum capable of degrading tannin and application thereof Download PDFInfo
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- CN113969243B CN113969243B CN202010715508.5A CN202010715508A CN113969243B CN 113969243 B CN113969243 B CN 113969243B CN 202010715508 A CN202010715508 A CN 202010715508A CN 113969243 B CN113969243 B CN 113969243B
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- lactobacillus plantarum
- silage
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- tannin
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- 230000007017 scission Effects 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- NESLWCLHZZISNB-UHFFFAOYSA-M sodium phenolate Chemical compound [Na+].[O-]C1=CC=CC=C1 NESLWCLHZZISNB-UHFFFAOYSA-M 0.000 description 1
- 235000010339 sodium tetraborate Nutrition 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 description 1
- 239000001393 triammonium citrate Substances 0.000 description 1
- 235000011046 triammonium citrate Nutrition 0.000 description 1
- BSVBQGMMJUBVOD-UHFFFAOYSA-N trisodium borate Chemical compound [Na+].[Na+].[Na+].[O-]B([O-])[O-] BSVBQGMMJUBVOD-UHFFFAOYSA-N 0.000 description 1
- CMPGARWFYBADJI-UHFFFAOYSA-L tungstic acid Chemical compound O[W](O)(=O)=O CMPGARWFYBADJI-UHFFFAOYSA-L 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
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- 239000010457 zeolite Substances 0.000 description 1
Images
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- 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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- A23V2400/169—Plantarum
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- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
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Abstract
The invention discloses lactobacillus plantarum capable of degrading tannin and application thereof. The lactobacillus plantarum is lactobacillus plantarum (Lactobacillus plantarum) CAV-M6, and the registration number of the common microorganism center of the China Committee for culture Collection of microorganisms is CGMCC No. 17610. The strain can quickly reduce the tannin content of silage; effectively preserve Huang Liangmu nutrition; improving the dry matter digestibility and the crude protein digestibility of Huang Liangmu. The additive or silage prepared by using the lactobacillus plantarum CAV-M6 provided by the invention has the characteristics of good effect, low cost, safety and easiness in use.
Description
Technical Field
The invention relates to the technical field of silage processing, in particular to lactobacillus capable of degrading tannin plants and application thereof.
Background
Vegetable tannins are water-soluble phenolic compounds, which are secondary metabolites with self-protection function formed during the evolution of plants. The chemical structure and the property of the polyphenol hydroxyl of the tannin lead the tannin to have stronger antibacterial and antiviral effects, and a proper amount of tannin in the feed is beneficial to livestock and poultry, but the excessive concentration can seriously obstruct the digestion and utilization of nutrient substances by the livestock and poultry. The negative impact of high concentrations of tannins on animal growth and production is mainly due to: the plant condensed tannin can be combined or complexed with protein, digestive enzyme, alkaloid, polysaccharide, metal ion and the like to form precipitate, so that the digestion and absorption of animal organism to nutrients are reduced, and the palatability is reducedCauses low feed intake, adversely affects metabolism and causes toxicity. Condensed tannin in diet is more than 50 g.kg -1 DM or grass containing 60-120 g.kg -1 In DM, the feed intake of livestock and poultry is reduced, rumen fermentation is inhibited, and the digestibility of almost all nutrients including protein is obviously reduced. The hydrolyzed tannins have greater toxicity to animals, are easy to cause acute diseases of ruminants, and have gastrointestinal bleeding, liver necrosis, tubular necrosis and the like related to the toxicity of the hydrolyzed tannins, and a large amount of feeding of plants with the content of the hydrolyzed tannins of more than 20 percent can cause acute poisoning and death of the animals. Generally, it is not higher than 15 g.kg -1 DM tannin has no adverse effect on pig and broiler chicken growth, and cattle tolerance is 30-40g.kg -1 DM tannin; goat tolerating 30 g.kg -1 Tannins below DM; sheep tolerating 48 g.kg -1 Tannins below DM. Silage can effectively preserve nutrient substances in forage grass raw materials, is not influenced by weather, and is an important storage mode of feed. Methods for degrading feed tannins include physical methods, chemical methods, enzymatic methods, and microbial degradation methods. Among them, the physical method is difficult to realize in silage production, the cost of enzymolysis method is high, and the chemical polyethylene glycol method and the microbial degradation method are studied more, but the influence of polyethylene glycol on animals is not clear. Regarding the method of microbial degradation, most of previous researches are fungi, and the screened strains are not suitable for silage anaerobic fermentation.
Disclosure of Invention
The invention aims to solve the technical problems of reducing the tannin content of silage in the prior art to improve the utilization rate of the silage or improving the silage quality of the silage.
In order to solve the technical problems, the invention firstly provides a lactobacillus plantarum.
The lactobacillus plantarum provided by the invention is lactobacillus plantarum (Lactobacillus plantarum) CAV-M6, and the registration number of the lactobacillus plantarum is CGMCC No. 17610 in the common microorganism center of the China Committee for culture Collection of microorganisms. The strain is preserved in China general microbiological culture collection center (CGMCC) of China Committee for culture Collection of microorganisms (CGMCC) in the 4 th month of 2019. Hereinafter, lactobacillus plantarum CAV-M6 is abbreviated.
Lactobacillus plantarum CAV-M6 is gram-positive bacillus, glucose homotype fermentation, acid resistance (normal growth at pH 3.0), growth rate (MRS culture medium for 24 hr, OD) 620nm >2.00 High acid production speed (MRS culture medium is cultured for 24 hours, pH can be reduced to below 4.0), the growth temperature range is wide (normal growth can be carried out at 5-50 ℃), and lactic acid is produced under pure culture and silage conditions. The physiological and biochemical characteristics of lactobacillus plantarum CAV-M6 are shown in Table 1, and the carbon source fermentation experiments are shown in Table 2. The 16S rDNA sequence of the lactobacillus plantarum CAV-M6 is shown as SEQ ID No. 1.
Cultures of Lactobacillus plantarum CAV-M6 are also within the scope of the invention. The culture of Lactobacillus plantarum CAV-M6 is a culture obtained by culturing Lactobacillus plantarum CAV-M6 in a microorganism medium (e.g., a fermentation broth containing Lactobacillus plantarum CAV-M6 and a substance secreted into a liquid medium, or a culture broth containing Lactobacillus plantarum CAV-M6 and a substance secreted into a solid medium).
The lactobacillus plantarum CAV-M6 is separated from the yellow beam wood silage. The lactobacillus plantarum CAV-M6 contains tannin enzyme, and the enzyme activity of the strain tannin enzyme is 4.85 mu mol/min.mL, so that the tannin content of silage can be rapidly reduced.
The application of any one of the following A1-A5 and the product of A6-A7 of lactobacillus plantarum CAV-M6 also belongs to the protection scope of the invention:
a1, application of lactobacillus plantarum CAV-M6 in improving silage quality.
A2, lactobacillus plantarum CAV-M6 is applied to degrading tannin.
A3, application of lactobacillus plantarum CAV-M6 in preparation of silage.
A4, application of lactobacillus plantarum CAV-M6 in preparing silage products.
A5, application of lactobacillus plantarum CAV-M6 in preparing silage additive products.
A6, silage products prepared by using lactobacillus plantarum CAV-M6.
A7, silage additive product containing lactobacillus plantarum CAV-M6 as an active ingredient.
In order to solve the technical problems, the invention also provides a microbial inoculum.
The microbial inoculum provided by the invention contains lactobacillus plantarum CAV-M6 or/and a metabolite of lactobacillus plantarum CAV-M6 or/and a culture of lactobacillus plantarum CAV-M6.
The active ingredients of the microbial inoculum can be lactobacillus plantarum CAV-M6 or/and lactobacillus plantarum CAV-M6 metabolites, the active ingredients of the microbial inoculum can contain other biological ingredients or non-biological ingredients, and the other active ingredients of the microbial inoculum can be determined by one skilled in the art according to the effects of the microbial inoculum.
The microbial agent may also include a carrier. The carrier may be a solid carrier or a liquid carrier. The solid carrier is a mineral material and a biological material; the mineral material may be at least one of turf, clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the biological material is at least one of straws, pine shells, rice straws, peanut shells, corn flour, bean flour, starch, turf and animal excrement of various crops; the liquid carrier may be water; in the microbial inoculum, the lactobacillus plantarum CAV-M6 or/and the metabolites of the lactobacillus plantarum CAV-M6 can exist in the form of cultured living cells, fermentation liquid of the living cells, filtrate of cell culture or mixture of cells and filtrate. The dosage form of the microbial inoculum can be various dosage forms, such as liquid, emulsion, suspending agent, powder, granule, wettable powder or water dispersible granule.
Surfactants (such as Tween 20, tween 80, etc.), binders, stabilizers (such as antioxidants), pH regulators, etc. can also be added into the microbial inoculum according to the need.
In the above, the metabolite of Lactobacillus plantarum CAV-M6 may be a fermentation broth of Lactobacillus plantarum CAV-M6. The fermentation broth of lactobacillus plantarum CAV-M6 can be prepared according to the following method: culturing lactobacillus plantarum CAV-M6 in a liquid fermentation medium, and collecting fermentation liquor (containing lactobacillus plantarum CAV-M6 and substances secreted into the liquid culture medium), wherein the fermentation liquor is a metabolite of lactobacillus plantarum CAV-M6.
In the above, the microbial agent may be a microbial agent having at least one of the following properties:
1) Reduces the content of hydrolyzed tannin in silage,
2) Reduces the condensed tannin content of silage,
3) The total tannin content of the silage is reduced,
4) The pH value of the silage is reduced,
5) Reduces the ammonia nitrogen content of silage,
6) The lactic acid content of the silage is improved,
7) The lactic acid/acetic acid ratio of silage is improved,
8) The content of binding protein of silage is reduced,
9) The true protein content of the silage is improved,
10 Improving the in vitro crude protein digestibility of silage.
In the above, the silage is fermented from silage plants.
The invention also provides a method for preparing silage.
The method for preparing silage provided by the invention comprises the step of fermenting silage by using lactobacillus plantarum CAV-M6, or a culture of lactobacillus plantarum CAV-M6, or a microbial inoculum prepared by lactobacillus plantarum CAV-M6.
Herein, the silage plant may be any of the following:
b1 A) a woody plant,
b2 Dicotyledonous plants, a plant which is selected from the group consisting of,
b3 Plants of the order Rubiaceae,
b4 A plant of the family Rubiaceae,
b5 A plant of the genus Lemongrass,
b6 Yellow beam wood.
Silage (lactobacillus plantarum CAV-M6 fermented feed for short) prepared by the method has at least one characteristic of C1-C10:
c1 The content of hydrolyzed tannin of lactobacillus plantarum CAV-M6 fermented feed is lower than that of common fermented silage,
c2 The condensed tannin content of lactobacillus plantarum CAV-M6 fermented feed is lower than that of common fermented silage,
c3 The total tannin content of lactobacillus plantarum CAV-M6 fermented feed is lower than that of common fermented silage,
c4 The pH value of lactobacillus plantarum CAV-M6 fermented feed is lower than that of common fermented silage,
c5 The ammonia nitrogen content of lactobacillus plantarum CAV-M6 fermented feed is lower than that of common fermented silage,
c6 The lactic acid content of lactobacillus plantarum CAV-M6 fermented feed is higher than that of common fermented silage,
c7 Lactobacillus plantarum CAV-M6 fermented feed has a higher lactic acid/acetic acid ratio than that of the normal fermented silage,
c8 The content of the binding protein of lactobacillus plantarum CAV-M6 fermented feed is lower than that of the common fermented silage,
c9 The true protein content of lactobacillus plantarum CAV-M6 fermented feed is higher than that of common fermented silage,
c10 The in-vitro crude protein digestibility of lactobacillus plantarum CAV-M6 fermented feed is higher than that of common fermented silage.
Wherein, the common fermented silage is prepared by the following method: the conditions (silage and fermentation conditions) are the same as above except that the lactobacillus plantarum, or the culture, or the microbial inoculum is not added.
In the above method, the fermentation may be anaerobic fermentation at 15-45deg.C for 40-60 days.
The invention also provides a method for culturing the lactobacillus plantarum CAV-M6, which comprises the step of culturing the lactobacillus plantarum CAV-M6 on a culture medium.
The invention separates and screens lactobacillus plantarum CAV-M6 with high yield of lactic acid, acid resistance and high growth speed from the yellow beam wood, and adds the lactobacillus plantarum CAV-M6 into silage materials for fermentation. Experiments prove that after lactobacillus plantarum CAV-M6 fermentation is carried out on the yellow beam wood raw material, the contents of hydrolyzed tannin, condensed tannin and total tannin are obviously reduced, the pH value and ammonia nitrogen content of silage are obviously reduced, the content of binding protein is obviously reduced, the true protein content is obviously increased, and the in-vitro crude protein digestibility is obviously improved. After lactobacillus plantarum CAV-M6 is fermented, the hydrolyzed tannin content of the yellow beam wood is reduced by 34.7%, the condensed tannin content is reduced by 35.0%, the total tannin content is reduced by 34.9%, and the in-vitro crude protein digestibility is improved by 6.55%, which indicates that lactobacillus plantarum CAV-M6 can rapidly reduce the tannin content of silage; the pH value of silage is quickly reduced, the fermentation process is accelerated, and the silage rate is improved; effectively preserve Huang Liangmu nutrition; improving the dry matter digestibility and the crude protein digestibility of Huang Liangmu; can be widely used. The microbial inoculum additive prepared by using lactobacillus plantarum CAV-M6 has good silage effect, low cost, safety and easy utilization.
Description of biological Material preservation
Classification naming of biological materials: lactobacillus plantarum.
Latin Wen Xueming of biological material: lactobacillus plantarum.
Strain number of biological material: CAV-M6.
The preservation units are fully named: china general microbiological culture Collection center (China Committee for culture Collection).
The preservation unit is abbreviated as: CGMCC.
Address: no.1 and No.3 of the north cinquefoil of the morning sun area of beijing city.
Preservation date: 22 days of 2019, 04 month.
Preservation number: CGMCC No.17614.
Drawings
FIG. 1 shows the degradation of tannin transparent rings by Lactobacillus plantarum CAV-M6.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The configuration of the medium is described in the examples below:
the liquid MRS medium was prepared as follows: 10.0g of peptone, 5.0g of beef powder, 4.0g of yeast powder, 20.0g of glucose, 5.0g of sodium acetate, 2.0g of triammonium citrate, 1.0mL of tween 80, 2.0g of dipotassium hydrogen phosphate, 0.2g of magnesium sulfate and 0.05g of manganese sulfate, and using distilled water to fix the volume to 1000mL, wherein the pH value is 6.2. The prepared culture medium is sterilized in a high-pressure steam sterilizing pot at 121 ℃ for 15min.
The MRS solid medium is obtained by adding agar to the liquid MRS medium and sterilizing.
Example 1: isolation and identification of Lactobacillus plantarum CAV-M6
1. Isolation of Lactobacillus plantarum
The lactobacillus plantarum is separated by adopting a dilution plate method: under aseptic condition, weighing Huang Liangmu silage 10g, placing into a conical flask filled with 90mL of sterile distilled water, shaking uniformly to obtain 10-fold diluted sample suspension, and directly and continuously diluting according to a 10-fold dilution method. A suitable dilution was selected, 0.1mL was plated on MRS medium plates, incubated in an incubator at 30℃for 48h, and plate counts were performed. And (3) picking a typical colony according to the characteristics of the size, the shape, the color and the like of the colony, and carrying out gram staining microscopic examination and a catalase test. The strain which is gram-positive and negative in the catalase test can be preliminarily determined to be lactobacillus, and the lactobacillus is streaked and purified twice on an MRS culture medium, inoculated into the MRS solid slant culture medium and preserved in a refrigerator at the temperature of 4 ℃ for later use.
2. Acquisition of tannin-degrading lactobacillus plantarum CAV-M6
An MRS plate containing 1% tannin is prepared, after the culture medium is cooled and solidified, a sterilized oxford cup is placed on the culture medium by forceps, and bacterial liquid is inoculated in the oxford cup. After anaerobic cultivation at a constant temperature of 30℃for 48 hours, it was observed whether or not transparent rings were produced, and strains which could produce transparent rings were considered to have the ability to degrade tannins. The strain with the strain number of CAV-M6 (see figure 1) is obtained through screening, namely the lactobacillus plantarum (Lactobacillus plantarum) CAV-M6, CGMCC No. 17610 (hereinafter called lactobacillus plantarum CAV-M6) of the application, and the tannase activity of the strain is measured subsequently.
3. Physiological and biochemical characteristics of Lactobacillus plantarum CAV-M6
Gram staining of Lactobacillus plantarum CAV-M6, glucose gassing, catalase assay, growth temperature (5, 10, 45, 50 ℃) and growth pH (3.0, 3.5, 4.0, 4.5, 9.0), fermentation carbon source, pH and OD within 24h of fermentation 620nm And performing an isophysiologic and biochemical experiment.
The physiological and biochemical test method is as follows:
(1) Gram staining was described in "Manual for identification of common bacterial systems" by Toxiu beads.
(2) Lactobacillus plantarum growth pH was adjusted using 2mol/L NaOH and 2mol/L HCl.
(3) Glucose gassing test: the experimental strain is inoculated on MRS culture medium and cultured for 48 hours in an anaerobic incubator, single colony is selected and inoculated in a test tube containing 5mL MRS liquid culture medium (pH 6.5) (Du Shixiao tube is reversely buckled in the test tube), the test tube is placed in a common constant temperature incubator for culturing for 48 hours at 37 ℃, the result is observed and recorded, the record of the generated air bubble is positive, and the record of the generated air bubble is negative.
(4) Catalase assay: the experimental strain is inoculated on MRS culture medium and cultured in an anaerobic incubator for 48 hours, single colony is picked and coated on a culture dish to which 3% (w/w) hydrogen peroxide solution is dripped, whether bubbles are generated or not is observed, the generated bubbles are positive to catalase, and the generated bubbles are negative to the generated bubbles.
(5) The sugar fermentation was performed using a biochemical identification suite of lactobacillus produced by beijing road bridge technologies, inc. For gram-positive and catalase-negative experimental strains, a biochemical identification set containing 8 carbon sources is adopted, a Maillard turbidity tube is used as a reference, the experimental method is carried out according to the instruction, the temperature is kept constant at 37 ℃ for 48 hours, and the experimental result is recorded.
The results show that lactobacillus plantarum CAV-M6 is gram positive, homofermentative bacillus, can grow at the temperature of 5-50 ℃ and the pH value of 3.0-9.0, has stronger acid resistance, and can ferment most saccharides after the pH value is reduced to below 4.0 after 24 hours of fermentation (Table 1) and the lactobacillus plantarum CAV-M6 can produce acid quickly and grow quickly (Table 2).
TABLE 1 colony morphology and physiological and biochemical characteristics of Lactobacillus plantarum CAV-M6
Note that: -, no growth; and +, normal growth.
TABLE 2 sugar fermentation test results of Lactobacillus plantarum CAV-M6
Note that: -, no growth; and +, normal growth.
4. Determination of tannase
The bacterial liquid after overnight activation is inoculated into a basic culture medium containing 2g/L tannic acid (liquid is obtained by adding tannic acid into a liquid MRS culture medium until the tannic acid content is 2 g/L), shaking culture is carried out for 24 hours at 37 ℃ and then fermentation liquor is centrifuged, supernatant (crude enzyme liquid) is taken for measuring tannase activity of the strain, and the result shows that the tannase activity of the strain CAV-M6 is 4.85 mu mol gallic acid/min.mL fermentation liquor. The measuring principle and method of the tannase activity are as follows: propyl gallate is formed by esterifying gallic acid and n-propanol, can be hydrolyzed by tannin enzyme, and is often used as a substrate for researching the characteristics of the tannin enzyme. The tannase can catalyze the cleavage of ester bonds in propyl gallate to generate gallic acid, the gallic acid and a chromophore substance generated by the reaction around the tannin are developed under alkaline conditions, the absorbance value is measured at the wavelength of 520nm, the absorbance is in direct proportion to the content of the gallic acid, and the activity of the tannase is judged according to the absorbance.
4.1 preparation of solutions
(1) 0.1mol/L ph=5.0 in citric acid buffer:
0.1mol/L citric acid: 8.2ml+0.1mol/L sodium citrate: 11.8ml
(2) Gallic acid standard solution: prepared with 0.1mol/L pH=5.0 citrate buffer. 40, 60, 80, 100, 120, 140, 160, 180, 200, 240 and 260umol/L gallic acid standard solutions were prepared.
(3) Methanol Rao Danning solution (0.667% w/V): 0.0667g of rhodanine was weighed out to a volume of 10mL with methanol.
(4) KOH solution (0.5 mol/L): 1.4g KOH was weighed and the volume was set to 50ml.
(5) Propyl gallate solution (PG): 10mmol/L of propyl gallate as substrate was prepared.
4.2 measurement method
Blank tube: 0.25ml PG+0.25ml citrate buffer
Test tube: 0.25ml PG+0.25ml crude enzyme liquid
Control tube: 0.25ml PG+0.25ml inactivated crude enzyme liquid
Placing in 30deg.C water bath, and maintaining for 5min. All test tubes were separately added with 0.3mL of ethanol rhodanine (0.05 mol/L) solution, incubated in a water bath at 30℃for another 5min, 0.4mL of KOH aqueous solution at 0.5mol/L was separately added, incubated at 30℃for another 5min, then 4mL of distilled water was added to each test tube, incubated at 30℃for 5min, and the absorbance of the reaction mixture was measured at 520 nm. All tests were run in 3 replicates and arithmetic mean was taken.
Definition of enzyme activity unit: the amount of enzyme required to produce 1. Mu. Mol of gallic acid per minute at pH=5.0 at 30℃is defined as one unit of enzyme activity.
5. Identification of Lactobacillus plantarum CAV-M6
5.1 morphological identification
Lactobacillus plantarum CAV-M6 in the logarithmic growth phase and with stable colony size is subjected to single colony state description, and mainly comprises the size, color, transparency, colony surface state and colony edge state of the colony.
The results show that the bacterial strain lactobacillus plantarum CAV-M6 colony morphology is characterized by: round, smooth, fine, yellowish in color, and opaque.
5.2 16S rDNA sequence homology analysis
Lactobacillus plantarum CAV-M6 was inoculated into 0.5mL MRS liquid medium, cultured at 30℃and 180rmp overnight, and strain identification was performed by bacterial liquid PCR.
The primers were selected from the bacterial 16S rDNA gene amplification universal primers (available from Meji Biotechnology Co., ltd.):
27F:5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID No.2 of the sequence Listing)
1492R:5'-GGTTACCTTGTTACGACTT-3' (SEQ ID No.3 of the sequence Listing)
PCR reaction System (50. Mu.L):
| Premix Taq | 25μL |
| 27F(20μM) | 2.5μL |
| 1492R(20μM) | 2.5μL |
| bacterial liquid | 5μL |
| H 2 O | 15μL |
Reaction conditions:
sequencing the amplified product in the Meji biotechnology Co., ltd, and comparing the sequence of the 16S rDNA shown as SEQ ID No.1 in the sequence table with the sequence of NCBI, and combining the physiological and biochemical data of the strain to finally determine that the lactobacillus plantarum CAV-M6 belongs to lactobacillus plantarum.
In view of the above morphological, physiological and biochemical feature analysis and 16s rDNA sequence homology analysis results, lactobacillus plantarum CAV-M6 was identified as Lactobacillus plantarum (Lactobacillus plantarum) CAV-M6 CGMCC No. 17610. The lactobacillus plantarum (Lactobacillus plantarum) CAV-M6 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 17610 in the 4 th and 22 th days of 2019 at the address of 1 st national institute of advanced North Chen West Lu No.3 of the Beijing area.
Example 2: application effect of lactobacillus plantarum CAV-M6 added into silage
1. Preparation of silage (woody feed Huang Liangmu is taken as an example)
After the early flowering stage mowing, the un-aired Huang Liangmu leaves are shortened to about 2cm and uniformly mixed to obtain silage raw materials.
2. Preparation of lactobacillus plantarum CAV-M6 microbial inoculum
Inoculating Lactobacillus plantarum CAV-M6 into liquid MRS culture medium (pH 6.5), and shake culturing at 37deg.C and 150rpm for 24 hr to obtain lactobacillus plantarum CAV-M6 bacterial solution, wherein the lactobacillus plantarum CAV-M6 content in the lactobacillus plantarum CAV-M6 bacterial solution is 10 6 cfu/ml, the lactobacillus plantarum CAV-M6 bacterial liquid is the lactobacillus plantarum CAV-M6 bacterial agent.
3. Fermented silage raw material
4 experimental groups are respectively arranged for each silage raw material, namely a control group (CK) without silage additive bacterial agent, a control group (TA) with tannase, a bacterial agent group (LP) with plant lactobacillus bacterial agent without degradation tannin ability and a bacterial agent group (CAV-M6) with plant lactobacillus CAV-M6, and each experimental group is provided with 3 replicates. The control group and the microbial inoculum group were run in parallel.
Lactobacillus plantarum CAV-M6 was added to the microbial inoculum group (CAV-M6): the lactobacillus plantarum CAV-M6 bacterial agent in the step 2 is added into each kilogram of silage raw material, and the addition amount of the bacterial agent is 10 percent of that of each kilogram of silage raw material 6 cfu lactobacillus plantarum CAV-M6, uniformly mixing, bagging and ensiling. 150g of each material was packed in a polyethylene bag (30 cm. Times.20 cm) and evacuated by a vacuum sealer. Each treatment was repeated 3 times, 1 bag at a time, and stored at room temperature (20-25 ℃) for 30d.
Microbial inoculum group (LP) to which a plant lactic acid microbial inoculum having no ability to degrade tannin is added: the plant lactobacillus bacterial agent LP without tannin degradation capability is added into each kilogram of silage raw material (the bacterial agent preparation method is the same as the method in the step 2), and the addition amount of the bacterial agent is 10 percent of the addition amount of each kilogram of silage raw material 6 cfu plant lactobacillus, mixing uniformly, bagging and ensiling. 150g of each material was packed in a polyethylene bag (30 cm. Times.20 cm) and evacuated by a vacuum sealer. Each treatment was repeated 3 times, 1 bag at a time, and stored at room temperature (20-25 ℃) for 30d. The plant lactobacillus agent without degradation tannin ability is screened from feed mulberry, taken from Henan Luoyang national academy of sciences of agriculture and forestry experimental base (34.39, 112.12, 250m in east longitude and altitude, 24 days in 2016 year 06 month) and the screening method is a conventional screening method, and the screening method is tested to have no tannin degradation ability.
Control group (CK) without silage additive bacteria: adding liquid MRS culture medium (pH value is 6.5) (without bacteria) of step 2 in the same volume as lactobacillus plantarum CAV-M6 bacteria added in the bacteria group into each kilogram of silage raw material, uniformly mixing, and bagging silage. 150g of each material was packed in a polyethylene bag (30 cm. Times.20 cm) and evacuated by a vacuum sealer. Each treatment was repeated 3 times, 1 bag at a time, and stored at room temperature (20-25 ℃) for 30d.
Control group (TA) to which tannase was added: adding liquid MRS culture medium containing tannase (liquid obtained by adding tannase into the liquid MRS culture medium (pH value is 6.5) in the step 2) with the same volume as lactobacillus plantarum CAV-M6 microbial inoculum added in the microbial inoculum group into each kilogram of silage raw material, wherein the addition amount of the liquid MRS culture medium containing tannase is 2U tannase per kilogram of silage raw material, uniformly mixing, and bagging for silage. 150g of each material was packed in a polyethylene bag (30 cm. Times.20 cm) and evacuated by a vacuum sealer. Each treatment was repeated 3 times, 1 bag at a time, and stored at room temperature (20-25 ℃) for 30d.
4. Silage tannin content determination
After the silage is opened, a certain amount of silage samples are taken for freeze drying. Is used for measuring the content of hydrolyzed tannin and condensed tannin.
The hydrolyzed tannin content is the total phenol content minus the simple phenol content.
The method for measuring the total phenol content comprises the following steps: folin-Ciocalteu reagent colorimetry was used. 200mg of the crushed sample was weighed accurately, placed in a 25ml beaker, 5.00ml of acetone (70%) was added, and subjected to ultrasonic treatment at room temperature for 20 minutes. The treated centrifuge tube was centrifuged at 3000g for 10min at 4 ℃. The supernatant was collected, stored in an ice bath, the pellet was transferred to a beaker with 5ml acetone (70%), and after 20min of sonication at room temperature, centrifuged as described above and the supernatant was collected. Taking 0.2ml of tannin extract prepared by the method, placing into a test tube, adding distilled water, and adding distilled water to make up to 1.00ml. Adding 0.5ml Folin reagent and 2.5ml sodium carbonate solution (20%), mixing, shaking at constant temperature and constant speed at 25deg.C for 40min, measuring absorbance with 725nm ultraviolet spectrophotometer, using gallic acid as standard substance, and drawing standard curve.
Drawing a standard curve: 0.0248g of gallic acid is taken and dissolved by distilled water and is fixed to a volumetric flask of 1000ml to obtain a reference substance solution with the mass concentration of 0.0248 mg/ml. The controls were removed at 0.2,0.4,0.6,0.8,1ml, and distilled water was added to make up the total volume to 1.00ml. 0.5ml of Folin reagent and 2.5ml of sodium carbonate solution (20%) are added, the mixture is swirled and mixed uniformly, and after shaking at constant temperature and uniform speed for 40min at 25 ℃, the absorbance is measured by a 725nm ultraviolet spectrophotometer. And (5) drawing by taking the mass concentration of the gallic acid standard solution as an abscissa and the absorbance as an ordinate, and calculating a standard curve.
The method for measuring the content of the simple phenol comprises the following steps: 100.00mg of crosslinked insoluble polyvinylpyrrolidone is accurately weighed, placed in a test tube, added with 1ml of distilled water, then added with 1ml of tannin extract, swirled and mixed uniformly, and waited for 15min at 4 ℃. After swirling again and centrifugation at 3000g for 10min, 0.1ml of the supernatant was placed in a test tube, and distilled water was added to make up the total volume to 1ml. 0.5ml Folin reagent and 2.5ml sodium carbonate solution (20%) are added, the mixture is swirled and mixed uniformly, and after shaking for 40min at constant temperature and uniform speed at 25 ℃, the absorbance is measured by an ultraviolet spectrophotometer at 725nm to obtain the simple phenol content.
The method for measuring the content of condensed tannin comprises the following steps: the Vanilla-HCL method is adopted. 0.1g of crushed sample is accurately weighed, placed in a round bottom centrifuge tube, added with 5ml of methanol (containing 1% of hydrochloric acid), oscillated for 20 hours at normal temperature, and then the solution is replaced into a sharp bottom centrifuge tube, centrifuged to obtain 1ml of supernatant, and 5ml of vanillin color development liquid (8% of HCl:4% of vanillin=1:1) is added into the supernatant. After waiting for 20min at normal temperature, the absorbance was measured at 495 nm. Catechin is selected as a standard substance, and a standard curve is drawn.
Total tannin content = hydrolyzed tannin + condensed tannin.
The data from the experiments were processed with Excel software and analyzed by one-way anova program in SAS9.0 software. Huang Liangmu the content of hydrolyzed tannin is 33.59 g.kg before silage -1 DM, condensed tannin 45.63 g.kg -1 DM, total tannin is 79.22 g.kg -1 And DM. The results of tannin content after silage are shown in Table 3, and after the Huang Liangmu silage raw material is fermented by lactobacillus plantarum CAV-M6, the CK result shows that silage can reduce the content of Huang Liangmu hydrolyzed tannin and increase the content of condensed tannin and total tannin. Compared with CK, the addition of CAV-M6 obviously reduces the content of Huang Liangmu hydrolyzed tannin and condensed tannin; and the total tannin content of the treatment added with CAV-M6 is lower than that of the raw materials. After lactobacillus plantarum CAV-M6 fermentation, the hydrolyzed tannin content of the yellow beam wood is reduced by 34.7%, the condensed tannin content is reduced by 35.0%, and the total tannin content is reduced by 34.9%. The explanation shows that lactobacillus plantarum CAV-M6 can effectively promote silage fermentation and reduce the tannin content of silage.
Table 3 Huang Liangmu tannin content after silage (g.kg) -1 DM)
Note that: CK, control; TA, tannase; LP, non-degradable tannin lactic acid bacteria; the representation of each column of data with the same letter suffix is not significantly different, and the data without the same letter is significantly different
5. Silage fermentation quality determination
The fermentation quality analysis method of Huang Liangmu silage is as follows:
the ammonia nitrogen content is determined by using a phenol-sodium hypochlorite colorimetric method; the organic acid was measured by using an Shimadzu GC-14 type high performance liquid chromatograph (column: KC-81column, shimadzu, japan), detector: SPD-M10AVP, mobile phase: 3mmol L -1 Perchloric acid aqueous solution, flow rate 1mL min -1 The column temperature is 50 ℃, the detection wavelength is 210nm, and the sample injection amount is 5 mu L. Lactic acid, acetic acid and propionic acid are used as standard substances respectively for quantitative analysis according to the retention time qualitative property of the standard substances and by adopting a standard curve method (external standard method).
The data from the experiments were processed with Excel software and analyzed by one-way anova program in SAS9.0 software. The post silage fermentation quality of Huang Liangmu is shown in table 4. Compared with CK, the addition of lactobacillus plantarum CAV-M6 can obviously reduce Huang Liangmu silage pH and ammonia nitrogen content (p is less than 0.05), and obviously improve lactic acid content and lactic acid/acetic acid ratio (p is less than 0.05), namely, the silage quality is obviously improved Huang Liangmu. Wherein after lactobacillus plantarum CAV-M6 fermentation, the pH value of the yellow beam wood is reduced by 12.5%, the ammonia nitrogen content is reduced by 98.0%, the lactic acid content is increased by 71.4%, and the lactic acid/acetic acid ratio is increased by 44.7%.
Table 4 Huang Liangmu post silage fermentation quality (g.kg) -1 DM)
Note that: CK, control; TA, tannase; LP, non-degradable tannin lactic acid bacteria; the representation of each column of data with the same letter suffix is not significantly different, and the data without the same letter is significantly different
6. Silage protein component analysis
Huang Liangmu silage is dried for 48 hours at 65 ℃ through an oven after being unpacked, is crushed through a 40-mesh sieve, and is subjected to protein component measurement and calculation according to the following steps.
(1) Crude Protein (CP)
The Kjeldahl method is adopted, and a FOSS KJ2300 full-automatic azotometer is used for measurement.
(2) Soluble protein (SOLP)
The soluble protein fraction is defined as the true protein in the rumen dissolved in buffer, non-protein nitrogen (NPN) is not included, and the assay is performed according to the modified boric acid-phosphate buffer procedure of Licitra et al (1996).
Preparation of boric acid-phosphate buffer: 12.2g of sodium dihydrogen phosphate (NaH) was weighed out 2 PO 4 ·H 2 O), 8.91g sodium borate (Na 2 B 4 O 7 ·10H 2 O), and measuring 100mL of tertiary butanol in a 1000mL volumetric flask to volume with distilled water.
The measurement process is as follows:
1) 0.5g of the crushed air-dried sample was weighed into a 125mL conical flask, 50mL of boric acid-phosphoric acid buffer was added, 1mL of sodium azide (10 g of sodium azide was dissolved in 100mL of water) was added, and the mixture was left at room temperature for 3 hours.
2) Suction filtration was performed using quantitative filter paper.
3) The residue was rinsed with 150mL of buffer.
4) The filter paper and the residue were dried at 60℃under low temperature.
5) The nitrogen content of the filter paper and the residue was measured by Kjeldahl method, and the measurement was made with the filter paper as a blank.
6) The soluble crude protein is obtained by subtracting the insoluble protein content of the residue from the crude protein content of the sample.
(3) Non-protein nitrogen (NPN)
The assay method is described in Licitra et al (1996). The total protein minus the fraction of true protein solubilized by tungstic acid. Reagent preparation:
1) 10% sodium tungstate solution (Na 2 WO 4 ·H 2 O，100g/L)；
2) 0.5mol/L sulfuric acid (7 mL of concentrated sulfuric acid (95% -98%) was dissolved in 1L).
The measuring step comprises the following steps:
3) 0.5g of the crushed feed sample is weighed and placed in a 125mL conical flask, and 50mL of cold distilled water is added;
4) Adding 8mL sodium tungstate solution, and culturing for 30min at 20-25 ℃;
5) Adding about 10mL of 0.5M sulfuric acid to adjust the pH value to 2.0, and standing at room temperature overnight;
6) Filtering with quantitative filter paper, and washing twice with cold distilled water;
7) The filter paper and the residue were dried at 60℃under low temperature.
8) The nitrogen content of the filter paper and the residue was measured by Kjeldahl method, and the measurement was made with the filter paper as a blank.
9) The nitrogen of the residue is subtracted from the crude protein content of the sample to obtain non-protein nitrogen.
(4) Neutral wash insoluble Nitrogen (NDIP)
And measuring the nitrogen content of the filter bag and residues after the measurement of the neutral washing fiber, taking the filter bag which is not added with feed and is subjected to the measurement procedure of the neutral washing fiber as a blank value for measuring the protein content, and calculating to obtain the nitrogen content in the NDF, namely the NDIP.
(5) Acidic wash insoluble nitrogen (ADIP)
After the acid washing fiber is measured by adopting the filter bag technology, the filter bag and residues are placed in a Kjeldahl nitrogen determination instrument to measure the nitrogen content, the filter bag which is not added with feed and is subjected to the acid washing fiber measurement procedure is used as a blank value for measuring the protein content, and the nitrogen content in the ADF is calculated to be ADIP.
(6) The calculation method of the CNCPS protein component is as follows:
non-protein nitrogen FA (% CP) =npn (% CP)
Fast degrading protein FB1 (% CP) =solp (% CP) -FA (% CP)
Slow degrading protein FB3 (% CP) =ndip (% CP) -ADIP (% CP)
Binding protein FC (% CP) =adip (% CP)
Medium-speed degradation protein FB2 (% CP) =100-FA (% CP) -FB1 (% CP) -FB3 (% CP) -FC (% CP)
The content of the protein components before Huang Liangmu silage is respectively as follows: non-protein nitrogen FA is 3.36% CP, binding protein FC is 15.50% CP, and protein FB is rapidly degraded 1 0.39% CP, moderate rate degradationProtein FB 2 47.70% CP, slow degradation protein FB 3 31.01% CP, true protein FB is 79.1% CP.
The protein component content after Huang Liangmu silage is shown in table 5. The data from the experiments were processed with Excel software and analyzed by variance and multiple comparisons using the one-way analysis of variance program in SPSS17.0 software. As can be seen from the CK results, the content of non-protein nitrogen and binding protein is increased after silage, and the content of true protein is reduced. Compared with the CK result, the treatment of adding lactobacillus plantarum CAV-M6 obviously reduces the binding protein content of silage materials and improves the true protein content, thereby effectively preserving silage nutrient substances. Wherein, after lactobacillus plantarum CAV-M6 fermentation, the binding protein (FC) content of the yellow beam wood is reduced by 34.3%, and the true protein (FB) content is improved by 11.0%.
Table 5 Huang Liangmu post silage protein component content (% CP)
Note that: each column of data has no significant difference in the representation of the same letter suffix and no significant difference in the representation of the same letter suffix (P < 0.05). "%" is mass percent.
7. Silage protein digestibility
The in vitro digestibility of Huang Liangmu feed before and after silage was determined by means of in vitro digestion.
Before Huang Liangmu ensiling, the digestibility of crude protein is 58.99%; the results after silage are shown in table 6, and the data obtained from the test were processed with Excel software and analyzed by a one-way anova program in SAS9.0 software. As can be seen from the CK results, the Huang Liangmu crude protein digestibility is reduced; compared with CK, the treatment of adding lactobacillus plantarum CAV-M6 obviously improves the in-vitro crude protein digestion rate of Huang Liangmu, so that the silage digestion rate is improved. After lactobacillus plantarum CAV-M6 fermentation, the in-vitro crude protein digestibility of the yellow beam wood is improved by 13.0 percent.
Table 6 digestibility of crude protein in vitro after silage 6 Huang Liangmu
Note that: CK, control; TA, tannase; LP, non-degradable tannin lactic acid bacteria; IVCPD, in vitro protein digestibility; each column of data has no significant difference in the representation of the same letter suffix and no significant difference in the representation of the same letter suffix (P < 0.05). "%" is mass percent.
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Claims (6)
1. Lactobacillus plantarum, its characterized in that: the lactobacillus plantarum is [ ]Lactobacillus plantarum) The strain number is CAV-M6, and the registration number of the strain in the China general microbiological culture Collection center is CGMCC No. 17610.
2. Use of the lactobacillus plantarum of claim 1 for any of the following:
a1, the application of the lactobacillus plantarum in silage quality improvement,
a2, the application of the lactobacillus plantarum in degrading tannin according to claim 1,
a3, the application of the lactobacillus plantarum in preparing silage according to claim 1,
use of the Lactobacillus plantarum according to claim 1 for the preparation of silage products,
use of lactobacillus plantarum according to claim 1 for the preparation of silage additive products.
3. A product prepared using the lactobacillus plantarum of claim 1, any of the following:
b1, silage product prepared using the Lactobacillus plantarum of claim 1,
b2, silage additive product prepared using the Lactobacillus plantarum of claim 1 containing the Lactobacillus plantarum of claim 1 as an active ingredient.
4. The microbial inoculum is characterized in that: the microbial inoculum comprises the lactobacillus plantarum of claim 1.
5. A method of preparing silage comprising the step of fermenting silage plants with the lactobacillus plantarum of claim 1 or the microbial inoculum of claim 4 to obtain silage.
6. The method according to claim 5, wherein: the silage plant is yellow beam wood.
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| CN109536416A (en) * | 2018-12-27 | 2019-03-29 | 南昌大学 | It is a kind of degrade feed with paper-mulberry leaf tannin silage inoculants bacteria preparation and its application method |
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