CN113980858A - Lactobacillus plantarum YL399 for producing high-activity tannase and application thereof in preparation of codonopsis pilosula fermented feed - Google Patents
Lactobacillus plantarum YL399 for producing high-activity tannase and application thereof in preparation of codonopsis pilosula fermented feed Download PDFInfo
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- CN113980858A CN113980858A CN202111368072.8A CN202111368072A CN113980858A CN 113980858 A CN113980858 A CN 113980858A CN 202111368072 A CN202111368072 A CN 202111368072A CN 113980858 A CN113980858 A CN 113980858A
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- Prior art keywords
- lactobacillus plantarum
- codonopsis pilosula
- tannase
- activity
- codonopsis
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- 108010038851 tannase Proteins 0.000 title claims abstract description 35
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- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 description 6
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- XJRPTMORGOIMMI-UHFFFAOYSA-N ethyl 2-amino-4-(trifluoromethyl)-1,3-thiazole-5-carboxylate Chemical compound CCOC(=O)C=1SC(N)=NC=1C(F)(F)F XJRPTMORGOIMMI-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
-
- 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
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/116—Heterocyclic compounds
- A23K20/121—Heterocyclic compounds containing oxygen or sulfur as hetero atom
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/163—Sugars; Polysaccharides
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/20—Inorganic substances, e.g. oligoelements
-
- 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
- C12N1/04—Preserving or maintaining viable microorganisms
-
- 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
- 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)
- Polymers & Plastics (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Animal Husbandry (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Physiology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Inorganic Chemistry (AREA)
- Mycology (AREA)
- Botany (AREA)
- Sustainable Development (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A lactobacillus plantarum YL399 for producing high-activity tannase and application thereof in preparing fermented feed relate to the field of fermented feed processing, the lactobacillus plantarum YL399 for producing the high-activity tannase is preserved in China center for type culture collection (CCTCC NO) at 9 months and 27 months of 2021: m20211228. The invention obtains a Lactobacillus plantarum (Lactobacillus plantarum) YL399 for producing high-activity tannase by separation, identification and screening, and the Lactobacillus plantarum YL399 is applied to codonopsis pilosula fermented feed, and experiments prove that the reduction rate of tannin content in the fermented codonopsis pilosula feed is 83.92%. The invention expands the sources of tannase and provides a new way for preparing microbial fermentation feed.
Description
Technical Field
The invention relates to the technical field of fermented feed processing, and particularly relates to lactobacillus plantarum YL399 for producing high-activity tannase and application of lactobacillus plantarum YL399 in preparation of codonopsis pilosula fermented feed.
Background
Tannin is a plant secondary metabolite, is a polyphenol compound and widely exists in various plant feed raw materials. Tannins are generally considered nutritionally undesirable and the presence of high mass concentrations of tannins in feed has an anti-nutritional effect which can affect the digestion of protein, cellulose, starch and fat by the animal, reducing the nutritional value of the food or feed. It is necessary to take appropriate measures to reduce or remove the tannin content of the feed.
The anti-nutritional property of tannin can be reduced or even eliminated by physical degradation method, chemical degradation method and microbial degradation method in the feed processing. The microbial degradation method is a long-term and significant method, and can improve the nutritional value of tannin-containing plants as feed. The microbial degradation of tannins is achieved by means of tannase enzymes produced by it. Tannase is known as tannin acyl hydrolase (Tannase, ec3.1.1.20), which hydrolyzes ester and dephenolic carboxyl bonds in gallic acid tannin to produce gallic acid and glucose. The research and application of tannase have been deeply carried out in the processes of food processing, feed processing, cosmetic production and leather production. Microorganisms such as molds, bacteria, yeasts and filamentous fungi are the main producers of tannase. Ever, people are looking for new sources of tannase to obtain tannase with more commercial application value.
Lactobacillus plantarum is an important microorganism species that is clearly specified in the feed additives catalog (Ministry of agriculture, No. 318) in China and allowed to be used. The tannase-producing lactobacillus plantarum is applied to feed fermentation, so that the tannin content in the feed can be reduced, and the feed is endowed with new nutrition and functions. However, the activity of producing tannase by some lactobacillus plantarum is not high, so that screening lactobacillus plantarum with high tannase activity has very important practical significance for the application of feed raw materials with high tannin content.
Disclosure of Invention
The invention aims to provide a lactobacillus plantarum YL399 for producing high-activity tannase and application thereof in preparation of codonopsis pilosula fermented feed.
The technical scheme adopted by the invention for solving the technical problem is as follows:
the Lactobacillus plantarum (L399) for producing the high-activity tannase is preserved in China Center for Type Culture Collection (CCTCC) at 9/27 of 2021, and the preservation number is CCTCC NO: m20211228.
The invention discloses application of Lactobacillus plantarum (Lactobacillus plantarum) YL399 for producing high-activity tannase in preparation of codonopsis pilosula fermented feed.
As a preferred embodiment, the preparation method of the codonopsis pilosula fermented feed comprises the following steps:
step one, preparation of lactobacillus plantarum YL399 freeze-dried leaven
Continuously carrying out passage activation on a lactobacillus plantarum YL399 cryopreserved strain in a liquid MRS culture medium for 3 times, inoculating the lactobacillus plantarum YL399 cryopreserved strain in the liquid MRS culture medium according to the inoculation amount of 1-3%, carrying out static culture at 37-42 ℃ for 16-20 hours, centrifuging to obtain a thallus precipitate, adding a lyophilization protectant, carrying out freeze drying, packaging to obtain a lactobacillus plantarum YL399 lyophilized starter, and storing at 4 ℃;
step two, preparation of codonopsis pilosula fermented feed
(1) Soaking dried traditional Chinese medicine codonopsis pilosula in 0.5-1% of salt solution for 2 times, cleaning with clear water, putting terahertz ore into the bottom of a fermentation tank, putting codonopsis pilosula, covering terahertz ore on the upper surface of codonopsis pilosula, wherein the total weight of the added terahertz ore is 1-2% of the total weight of codonopsis pilosula;
(2) adding a lactobacillus plantarum YL399 freeze-dried starter in an amount which is 0.01-0.05% of the mass of the codonopsis pilosula, covering a plastic film, sealing, fermenting for 10-20 days at a constant temperature of 30-35 ℃, and performing solid-liquid separation after fermentation is finished;
(3) and (3) repeatedly fermenting the codonopsis pilosula once under the same condition, combining fermentation liquor obtained in two times, filtering and packaging to obtain the codonopsis pilosula fermented feed, wherein the pH value is 3.0-3.5.
In a preferred embodiment, in step one, the strain is inoculated in the liquid MRS medium in an inoculation amount of 2%.
As a preferred embodiment, in the first step, the culture is carried out at 37 ℃ for 16 hours.
In a preferred embodiment, in the first step, the bacterial cells are centrifuged at 3000-6000 rpm at 4 ℃ for 10min to obtain bacterial cell precipitates.
As a preferred embodiment, in step one, the lyoprotectant comprises: 40g/L glucose, 5g/L sodium ascorbate, 80g/L fructo-oligosaccharide and 30g/L trehalose.
As a preferred embodiment, the specific steps in step two (1) are as follows: soaking dried root of radix Codonopsis with 0.5% saline solution for 2 times, cleaning with clear water, adding terahertz ore into the bottom of the fermentation tank, adding radix Codonopsis, covering terahertz ore on the upper surface of radix Codonopsis, wherein the total weight of the added terahertz ore is 1.5% of the total weight of radix Codonopsis.
In a preferred embodiment, in the second step (2), the lactobacillus plantarum YL399 freeze-dried starter is added in an amount of 0.03% by mass of codonopsis pilosula.
In a preferred embodiment, in the second step (2), the fermentation is carried out at a constant temperature of 30 ℃ for 10 days.
The invention has the beneficial effects that:
the invention obtains a Lactobacillus plantarum (Lactobacillus plantarum) YL399 for producing high-activity tannase by separation, identification and screening, and the Lactobacillus plantarum YL399 is applied to producing fermented codonopsis pilosula feed, and experiments prove that the reduction rate of tannin content in the unfermented codonopsis pilosula feed is 83.92%.
The Lactobacillus plantarum (Lactobacillus plantarum) YL399 for producing high-activity tannase screened by the invention is used for removing tannin in tannin-containing feeds such as radix codonopsis pilosulae, etc., so that the source of the tannase is expanded, and a new way is provided for preparing the traditional Chinese medicine feed by microbial fermentation.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless otherwise specified, all PBS buffers in the examples were PBS buffer of 0.1M at pH 7.0.
Solid BCP medium: contains 2.5g/L yeast extract, 5g/L peptone, 5g/L glucose, 0.04g/L bromocresol purple, 15g/L agar and the balance of water; unless otherwise specified, pH 7.0.
Liquid BCP media differs from solid BCP media only in that no agar is added.
Solid MRS medium: contains 10g/L of peptone, 10g/L of beef extract and 5g/L, KH of yeast extract2PO42g/L, sodium acetate 5g/L, sodium citrate 5g/L, MgSO4·7H2O 0.2g/L、MnSO4·4H2O0.05 g/L, Tween-801 mL/L, agar 15g/L, glucose 20g/L and the balance of water; unless otherwise specified, pH 6.6.
Liquid MRS medium differs from solid MRS medium only in that no agar is added.
Example 1 isolation, identification and preservation of Lactobacillus plantarum strains
Separation of Lactobacillus plantarum strains
Taking a sample: the corn silage is naturally fermented.
And repeatedly carrying out streak culture by using a BCP (brain-cell culture) medium plate, selecting a single colony producing a yellow circle, inoculating the single colony on a solid MRS (MRS) medium plate for continuous culture, and repeatedly carrying out streak culture and purification by using the solid MRS medium plate to obtain multiple strains of pure culture strains.
Inoculating the pure cultured strain to liquid MRS culture medium, culturing, adding 20% glycerol, and storing in refrigerator at-80 deg.C.
Screening out a plurality of strains with gram stain positive, catalase test negative, indole reaction negative, hydrogen sulfide production test negative, gelatin liquefaction test negative, starch hydrolysis test negative and nitrate reduction test negative from pure cultured strains. Among them, 1 strain was designated as YL 399.
II, identification of the strains
Physiological and biochemical identification results of YL 399: gram staining is positive, catalase test is negative, benzidine test is negative, indole test is negative, and acetyl methyl methanol test is positive; starch is not hydrolyzed, gelatin is not liquefied, hydrogen sulfide is not generated, and acid and gas are not generated by fermenting glucose; immotile bacilli; the growth can be carried out at the temperature of 15 ℃ and 45 ℃, and the optimal growth temperature is 37-42 ℃; the pH is suitably 5.0 to 7.0; tolerates 6.5% NaCl; YL399 is grown in a uniform turbid way in a liquid MRS culture medium, and the thalli are white and precipitated after being placed for a long time.
TABLE 1 Lactobacillus plantarum YL399API50 CHL identification results
16S rDNA sequence homology analysis: extracting genome DNA, carrying out PCR amplification by adopting a primer pair (16 sF: 5 '-AGAGTTTGATCCTGGCTCCAG-3'; 16 sR: 5 '-AGAAAGGAGGTGATCCAGC-3') consisting of 16sR and 16sR, and then sequencing an amplification product, wherein the 16s rDNA sequence is shown as a sequence 1 in a sequence table. Homology of 99% with Lactobacillus plantarum RI-113(GenBank: CP017406.1) was determined by homology alignment of 16s rDNA sequence in GenBank database using BLAST program.
According to the above identification result, the strain YL399 was identified as Lactobacillus plantarum (Lactobacillus plantarum).
Third, preservation of the Strain
The Lactobacillus plantarum (Lactobacillus plantarum) YL399 has been preserved in China Center for Type Culture Collection (CCTCC) at 9 months and 27 days 2021, and the addresses are as follows: eight-path Wuhan university school (Wuhan university collection center) 299 in Wuchang area of Wuhan city, Hubei province, with the collection number of CCTCC NO: m20211228.
Example 2 measurement of tannase Activity produced by Lactobacillus plantarum (Lactobacillus plantarum) YL399
First, strain culture and preparation of cell-free culture solution
Continuously passaging and activating a Lactobacillus plantarum (Lactobacillus plantarum) YL399 frozen strain in a liquid MRS culture medium for 3 times, inoculating the strain in the liquid MRS culture medium according to the inoculation amount of 2 percent, standing and culturing for 16h at 37 ℃, centrifuging for 8min at 3000r/min and 4 ℃, and removing supernatant to obtain a thallus precipitate; the obtained thallus sediment is subjected to cell wall cracking by an ultrasonic crushing method: adding equal volume of sterile normal saline, adding lysozyme to the concentration of 1mg/ml, performing constant temperature water bath at 37 ℃ for 30min, performing ultrasonic disruption of thallus cells (800w, 2s, 2s, 40 ℃, 15min), centrifuging at 4000r/min at 4 ℃ for 10min, and collecting supernatant, which is cell-free extract and contains high-activity tannase.
Secondly, determination of tannase activity
The activity of tannase contained in the cell-free extract was determined by spectrophotometry. Taking 3 test tubes, namely a blank tube, a sample tube and a control tube, and marking in sequence. Respectively adding 0.25mL and 1mmol/L methyl gallate solution into each test tube, preheating in 30 deg.C water bath for 5min, adding 0.25mL citric acid buffer solution into blank tube, respectively adding 0.25mL preheated cell-free extract solution and boiling water bath inactivated enzyme solution into sample tube and control tube, and placing in 30 deg.C water bath for 5 min. 0.3mL and 0.05mol/L rhodanine ethanol solution is respectively added into each test tube, the temperature of the test tube is kept for 5min in water bath at 30 ℃, 0.2mL and 0.5mol/L KOH solution are respectively added, the temperature is kept for 5min at 30 ℃, 4mL of distilled water is then added into each test tube, the temperature is kept for 5min at 30 ℃, and the absorbance of the reaction mixture is measured at the wavelength of 520 nm. All measurements were averaged over 3 replicates. Definition of enzyme activity unit: the amount of enzyme required to produce 1pmol of gallic acid per minute under the above reaction conditions was defined as one unit of enzyme activity (U).
As can be seen from the above experiments, tannase activity was studied by using gallic acid methyl ester as a reaction substrate for the enzyme, and the tannase contained in the cell-free extract had the highest activity of 358U/mL under the conditions of a temperature of 30 ℃ and a pH of 7.
Example 3 application of Lactobacillus plantarum (L.plantarum) YL399 for producing high-activity tannase in preparation of radix codonopsis pilosulae fermented feed
Preparation of lactobacillus plantarum YL399 freeze-dried starter
Continuously carrying out passage activation on a Lactobacillus plantarum (Lactobacillus plantarum) YL399 frozen strain in a liquid MRS culture medium for 3 times, inoculating the strain into the liquid MRS culture medium according to the inoculation amount of 2%, standing and culturing at 37 ℃ for 16h, centrifuging (3000-6000 rpm, 10min and 4 ℃) to obtain thallus precipitate, adding a freeze-drying protective agent (40g/L glucose, 5g/L sodium ascorbate, 80g/L fructo-oligosaccharide and 30g/L trehalose), freeze-drying, packaging to obtain the Lactobacillus plantarum YL399 freeze-dried starter, and storing at 4 ℃. The viable count of the obtained lactobacillus plantarum YL399 freeze-dried starter is more than 1000 hundred million/g.
Preparation of codonopsis pilosula fermented feed
Taking dried roots of traditional Chinese medicine codonopsis pilosula, cleaning and slicing the dried roots, soaking the dried roots in 0.5% of salt solution for 2 times, cleaning the dried roots with clear water, putting terahertz ore into the bottom of a fermentation tank, putting codonopsis pilosula into the fermentation tank, covering the upper surface of the codonopsis pilosula with the terahertz ore, wherein the total weight of the added terahertz ore is 1.5% of the total weight of the codonopsis pilosula; adding lactobacillus plantarum YL399 lyophilized starter culture in an amount of 0.03% of the weight of radix Codonopsis, sealing with plastic film, fermenting at constant temperature of 30 deg.C for 10 days, and performing solid-liquid separation after fermentation; and fermenting the codonopsis pilosula again under the same condition, combining fermentation liquor obtained in two times, filtering and packaging to obtain the codonopsis pilosula fermented feed.
After fermentation is finished, the content of fermentation thalli in the obtained traditional Chinese medicine fermentation product is not less than 2.5 hundred million/ml, and the pH value is 3.0-3.5. The codonopsis pilosula fermented feed obtained in the embodiment can be directly used as a traditional Chinese medicine feed additive. In addition, the codonopsis pilosula fermented feed obtained in the embodiment can be prepared into various dosage forms, such as aqueous solutions, powders, tablets and the like, but is not limited thereto.
Example 4 measurement of tannin content in Codonopsis pilosula fermented feed
Preparation of first, Standard Curve
Preparing 1mg/mL tannin standard substance stock solution, respectively sucking tannin standard solutions of 0.4mL, 0.8mL, 1.2mL, 1.6mL and 2.0mL, and diluting with distilled water to a constant volume of 10mL to prepare a gradient standard solution. Respectively sucking 0.5mL of each gradient standard solution, mixing with 3mL of methanol solution containing 4% vanillin and 1.5mL of concentrated hydrochloric acid, standing for 40min, measuring absorbance at 510nm wavelength, and replacing blank group with distilled water. The regression equation of the standard curve of the tannin is obtained through experiments and is Y-698.15476X +0.98745, wherein Y is the concentration of the tannin in the solution and has the unit of mg/L; x is the absorbance of tannin at 510nm, R is 0.99651, and the linearity is good.
Secondly, measuring the content of the sample
Diluting fermented radix Codonopsis feed with 5-10 times of distilled water, mixing, centrifuging (4000rpm/min, 4 deg.C, 10min), collecting supernatant 0.5mL, adding 3mL of methanol solution containing 4% vanillin and 1.5mL of concentrated hydrochloric acid, mixing, standing for 40min, measuring absorbance at 510nm wavelength, and substituting distilled water for blank group. And (4) substituting the obtained absorbance into a standard curve to calculate the tannin content in the codonopsis pilosula fermented feed.
Thirdly, result display
The tannin content in the unfermented radix Codonopsis feed is 0.187g/kg, and the tannin content in the fermented radix Codonopsis feed is 0.03g/kg, and the reduction rate of tannin content is 83.92%.
The invention discloses a lactobacillus plantarum YL399 for producing high-activity tannase and application thereof in preparation of fermented traditional Chinese medicine feed. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the technology can be practiced and applied by modifying or appropriately combining the products described herein without departing from the spirit and scope of the invention.
Sequence listing
<110> lactobacillus plantarum YL399 for producing high-activity tannase and application thereof in preparation of codonopsis pilosula fermented feed
<120> Shanghai plant fermentation biological science Co., Ltd
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1519
<212> DNA
<213> Artificial (Objective gene)
<400> 1
gacgaacgct gscggcgtgc ctaatacatg caagtcgaac gaactctggt attgattggt 60
gcttgcatca tgatttacat ttgagtgagt ggcgaactgg tgagtaacac gtgggaaacc 120
tgcccagaag cgggggataa cacctggaaa cagatgctaa taccgcataa caacttggac 180
cgcatggtcc gagcttgaaa gatggcttcg gctatcactt ttggatggtc ccgcggcgta 240
ttagctagat ggtggggtaa cggctcacca tggcaatgat acgtagccga cctgagaggg 300
taatcggcca cattgggact gagacacggc ccaaactcct acgggaggca gcagtaggga 360
atcttccaca atggacgaaa gtctgatgga gcaacgccgc gtgagtgaag aagggtttcg 420
gctcgtaaaa ctctgttgtt aaagaagaac atatctgaga gtaactgttc aggtattgac 480
ggtatttaac cagaaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg 540
gcaagcgttg tccggattta ttgggcgtaa agcgagcgca ggcggttttt taagtctgat 600
gtgaaagcct tcggctcaac cgaagaagtg catcggaaac tgggaaactt gagtgcagaa 660
gaggacagtg gaactccatg tgtagcggtg aaatgcgtag atatatggaa gaacaccagt 720
ggcgaaggcg gctgtctggt ctgtaactga cgctgaggct cgaaagtatg ggtagcaaac 780
aggattagat accctggtag tccataccgt aaacgatgaa tgctaagtgt tggagggttt 840
ccgcccttca gtgctgcagc taacgcatta agcattccgc ctggggagta cggccgcaag 900
gctgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc 960
gaagctacgc gaagaacctt accaggtctt gacatactat gcaaatctaa gagattagac 1020
gttcccttcg gggacatgga tacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga 1080
tgttgggtta agtcccgcaa cgagcgcaac ccttattatc agttgccagc attaagttgg 1140
gcactctggt gagactgccg gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc 1200
atgcccctta tgacctgggc tacacacgtg ctacaatgga tggtacaacg agttgcgaac 1260
tcgcgagagt aagctaatct cttaaagcca ttctcagttc ggattgtagg ctgcaactcg 1320
cctacatgaa gtcggaatcg ctagtaatcg cggatcagca tgccgcggtg aatacgttcc 1380
cgggccttgt acacaccgcc cgtcacacca tgagagtttg taacacccaa agtcggtggg 1440
gtaacctttt aggaaccagc cgcctaaggt gggacagatg attagggtga agtcgtaaca 1500
aggtagccgt agggagaac 1519
Claims (10)
1. A lactobacillus plantarum (Lactobacillus plantarum) YL399 for producing high-activity tannase is characterized in that the strain is preserved in China center for type culture collection (CCTCC NO) at 9 months and 27 days 2021: m20211228.
2. The use of a Lactobacillus plantarum (YL 399) for producing high-activity tannase according to claim 1 in the preparation of fermented codonopsis pilosula feed.
3. The use of claim 2, wherein the preparation method of the radix codonopsis fermented feed comprises the following steps:
step one, preparation of lactobacillus plantarum YL399 freeze-dried leaven
Continuously carrying out passage activation on a lactobacillus plantarum YL399 cryopreserved strain in a liquid MRS culture medium for 3 times, inoculating the lactobacillus plantarum YL399 cryopreserved strain in the liquid MRS culture medium according to the inoculation amount of 1-3%, carrying out static culture at 37-42 ℃ for 16-20 hours, centrifuging to obtain a thallus precipitate, adding a lyophilization protectant, carrying out freeze drying, packaging to obtain a lactobacillus plantarum YL399 lyophilized starter, and storing at 4 ℃;
step two, preparation of codonopsis pilosula fermented feed
(1) Soaking dried traditional Chinese medicine codonopsis pilosula in 0.5-1% of salt solution for 2 times, cleaning with clear water, putting terahertz ore into the bottom of a fermentation tank, putting codonopsis pilosula, covering terahertz ore on the upper surface of codonopsis pilosula, wherein the total weight of the added terahertz ore is 1-2% of the total weight of codonopsis pilosula;
(2) adding a lactobacillus plantarum YL399 freeze-dried starter in an amount which is 0.01-0.05% of the mass of the codonopsis pilosula, covering a plastic film, sealing, fermenting for 10-20 days at a constant temperature of 30-35 ℃, and performing solid-liquid separation after fermentation is finished;
(3) and (3) repeatedly fermenting the codonopsis pilosula once under the same condition, combining fermentation liquor obtained in two times, filtering and packaging to obtain the codonopsis pilosula fermented feed, wherein the pH value is 3.0-3.5.
4. The use according to claim 3, wherein in step one, the MRS culture medium is inoculated in an amount of 2%.
5. The use according to claim 3, wherein in step one, the stationary culture is carried out at 37 ℃ for 16 hours.
6. The use of claim 3, wherein in the first step, the bacterial pellet is obtained after centrifugation at 3000-6000 rpm and 4 ℃ for 10 min.
7. The use of claim 3, wherein in step one, the lyoprotectant comprises: 40g/L glucose, 5g/L sodium ascorbate, 80g/L fructo-oligosaccharide and 30g/L trehalose.
8. The use of claim 3, wherein the specific steps in step two (1) are as follows: soaking dried root of radix Codonopsis with 0.5% saline solution for 2 times, cleaning with clear water, adding terahertz ore into the bottom of the fermentation tank, adding radix Codonopsis, covering terahertz ore on the upper surface of radix Codonopsis, wherein the total weight of the added terahertz ore is 1.5% of the total weight of radix Codonopsis.
9. The use according to claim 3, wherein in step two (2), the lyophilized starter culture of Lactobacillus plantarum YL399 is added in an amount of 0.03% by mass of Codonopsis pilosula.
10. The use according to claim 3, wherein in step two (2), the fermentation is carried out at 30 ℃ for 10 days.
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